Case 3

Path 417 Cases
Instructor
	David Harris Agatha Jassem Ramon KleinGeltink Inna Sekirov
Case Projects
	Case 1
	Case 2
	Case 3
	Case 4

10-year-old Ronnie has developed abdominal cramps, bloody diarrhea and a low grade fever. His parents take him to see the family doctor. The doctor asks about what Ronnie has eaten in the past week. His parents recall that last weekend at a neighbor’s barbecue they were concerned that the hamburgers may not have been cooked thoroughly and Ronnie ate two burgers. The doctor performs a physical examination noting no rebound tenderness just some mild periumbilical tenderness. He asks the parents to collect a stool sample for the Microbiology Laboratory and also issues a requisition for routine bloodwork (to be performed at the local laboratory). The Microbiology Laboratory report comes back positive for E.coli 0157:H7.

The Body System

i) Describe the signs and symptoms presented in the case. Are there any other signs or symptoms that could have been commented on but are not presented in the case? What are the key History of Presenting Illness elements presented? What laboratory samples are taken and why? What are the meanings of the laboratory results reported? (No need to describe physiology of the signs and symptoms and no need to describe the laboratory testing itself as these are the basis of other questions).

Figure 1: Abdominal examination- checking for Blumberg’s sign or rebound tenderness (10).

In clinical medicine, a sign refers to an abnormal characteristic that is observed upon physical examination. In Ronnie’s case, the clinical signs were having mild periumbilical tenderness and a low-grade fever. Periumbilical tenderness refers to pain in the periumbilical region of the abdomen which is located around the navel (1). It was also noted during the physical examination that there was no rebound tenderness. Rebound tenderness, also known as Blumberg’s sign, is assessed by a physician applying pressure with their hand to the patient’s abdomen, then subsequently removing their hand and asking the patient to report whether they feel any pain (2). In terms of laboratory signs, Ronnie’s stool sample tested positive for E. coli 0157:H7.

A symptom is defined as a characteristic a person experiences. Ronnie’s symptoms include abdominal cramps, bloody diarrhea, and fever. Other symptoms known to be associated with E. coli 0157:H7 infection that were not reported in this case include fatigue and/or nausea (3). In severe cases, E. coli 0157:H7 may result in hemolytic uremic syndrome (HUS) which results in additional symptoms such as pale skin tone, irritability, unexplained bruises, decreased urination, bleeding from nose and mouth, and losing pink colour in cheeks and inside lower eyelids (3). Clinical signs of HUS that were not documented in this case include: microangiopathic hemolytic anemia and thrombocytopenia (4). Microangiopathic hemolytic anemia can be diagnosed if there is presence of schistocytes (fragmented red blood cell pieces) on a peripheral blood smear (4). Thrombocytopenia can be confirmed by a blood test showing a platelet count of less than 150 × 103 platelets per μL (4).

The History of Present Illness (HPI) is a description of a patient’s illness that can help a physician reach a diagnosis and design a treatment plan (5). While the exact elements that need to be included in an HPI may differ based on the source consulted, elements may include location, quality, severity, duration, timing, context, modifying factors, and associated signs and symptoms (5, 6). In Ronnie’s case, several of these elements were present. First, Ronnie indicated that his cramps were located in his abdomen, which is further supported by the sign of periumbilical tenderness the physician observed. Severity was indicated for Ronnie’s fever, which was described as low-grade. Ronnie attended a barbeque the weekend prior to visiting the clinic at which he consumed two burgers that his parents noted may have been undercooked. A common route of transmission of E. coli 0157:H7 to humans is via consumption of undercooked meat, because cattle are major reservoirs of E. coli 0157:H7 (7). The duration of Ronnie’s symptoms was not provided, but they presumably developed after the neighbor’s barbeque. Timing (when the sign or symptom happens), context (what accompanies a sign or symptom), modifying factors (how Ronnie can reduce or increase the signs or symptoms) were not presented in the case (6). However, several associated symptoms were presented, such as bloody diarrhea and low-grade fever associated with his abdominal cramps.

Figure 2: Sequences of events leading to disease by E. coli O157:H7 (8).

A stool sample and blood sample were taken from Ronnie for laboratory testing, as the physician likely suspected enterohemorrhagic E. coli (EHEC) infection based on the presence of blood in the diarrhea (7). The stool sample was taken to identify the presence of E. coli in the diarrheal sample, which would indicate infection by the bacteria (7). In this case, the stool sample was positive for E. coli O157:H7, indicating that this bacterial serotype was present in Ronnie and may be the source of his illness (7). The blood work may have been ordered to obtain a complete blood count examining the number and features of erythrocytes, leukocytes, and platelets (7). This is useful in ruling out leukocytosis, hemolysis, and thrombocytopenia (7). A comprehensive metabolic panel would also allow the physician to rule out dehydration, electrolyte disturbance, and uremia as the causes of Ronnie’s illness (7). The combination of stool and blood tests is useful to help the physician reach a diagnosis and proceed with a treatment plan.

Figure 3: Signs and symptoms associated with hemolytic uremic syndrome (HUS) (9).

References:

1. Abdominal mass [Internet]. Mount Sinai Health System. [cited 2022 Mar 12]. Available from: https://www.mountsinai.org/health-library/symptoms/abdominal-mass

2. Bell D, Iqbal S. Blumberg sign. In: Radiopaedia.org. Radiopaedia.org; 2021.

3. Signs & symptoms [Internet]. Cdc.gov. 2019 [cited 2022 Mar 12]. Available from: https://www.cdc.gov/ecoli/2014/o157h7-05-14/signs-symptoms.html

4. Gülhan B, Özaltın F. Hemolytic uremic syndrome in children. Turk Arch Pediatr [Internet]. 2021;56(5):415–22. Available from: http://dx.doi.org/10.5152/TurkArchPediatr.2021.21128

5. Bird L. History of present illness. Pharos Alpha Omega Alpha Honor Med Soc [Internet]. 2016 [cited 2022 Mar 18];79(4):22–5. Available from: https://www.acc.org/tools-and-practice-support/practice-solutions/coding-and-reimbursement/documentation/evaluation-and-management/history-of-present-illness

6. HPI-Explanation-1.pdf [Internet]. [cited 2022 Mar 9]. Available from: https://tennriverderm.com/wp-content/uploads/2017/02/HPI-Explanation-1.pdf

7. Ameer MA, Wasey A, Salen P. Escherichia Coli (E Coli 0157 H7). StatPearls Publishing; 2021.

8. Pepper IL, Gerba CP, Gentry TJ, Maier RM, editors. Environmental microbiology. Academic press; 2011 Oct 13

9. Haemolytic Uraemic Syndrome [Internet]. Youtube.com. 2022 [cited 18 March 2022]. Available from: https://www.youtube.com/watch?v=KA1vtqBtKeo

10. Rebound tenderness. [Internet]. Tumblr. 2022 [cited 18 March 2022]. Available from: https://lulabelleismyshihtzu.tumblr.com/post/28015881904/rightatrium-rebound-tenderness-is-a-clinical

ii) Which body system is affected? In what way has the normal physiological functioning of this body system been disturbed by the infection (specifically looking at the physiological changes without detailing the bacterial mechanism of this disturbance as that is the basis of another question). Representing this diagrammatically is helpful to demonstrate understanding.

Figure 4: Mechanism of action of E. coli O157:H7 (8).

The digestive system is the principal body system affected, as much of the pathogenesis of E. coli O157:H7 occurs in the digestive tract. After ingestion through the fecal-oral route, E. coli O157:H7 binds intestinal epithelial cells in the distal ileum and colon, which flattens microvilli as the actin cytoskeleton of the cells is rearranged by the bacteria (1,2). This loss of microvilli disrupts intestinal epithelial cell functions such as the absorption of luminal contents, causing malabsorption and osmotic diarrhea (3). Additionally, E. coli O157:H7 releases large amounts of Shiga toxin (also known as Verotoxin) upon binding to the intestinal mucosa, causing hemorrhagic colitis (3). Hemorrhagic colitis is a result of Shiga toxin which damages the intestines by destroying intestinal mucosa cells (1). Shiga toxin does this by 1) disrupting ribosomal peptide elongation and protein synthesis and 2) eliciting an intense inflammatory response from the host which can cause cell death (1,4). The destruction of the mucosal intestinal lining causes malabsorption and often blood loss into the lumen, resulting in bloody diarrhea (5).

The effects of the Shiga toxin are not limited to the digestive system, as the toxins cause intestinal lesions by damaging the microvasculature of the cell wall (2). Following toxin-mediated vascular damage, bacterial products can access the circulatory system and disrupt its normal function (2). Lipopolysaccharides associated with E. coli O157:H7 affect the circulatory system by damaging endothelial cells, increasing TNF levels, activating platelets, inducing the coagulation cascade, and increasing interleukin expression (2). Shiga toxin affects the circulatory system by inducing chemokine synthesis to promote inflammation, causing damage to blood vessels (2).

Figure 5: Organ systems that can be affected by HUS (8).

After gaining access to the bloodstream, E. coli O157:H7 Shiga-like toxin can disseminate throughout the body to disturb other body systems – such as the kidney in the excretory system (2). A complication from E. coli infection progression is hemolytic uremic syndrome (HUS), which leads to kidney failure, death, as well as additional complications in the central nervous system which may lead to strokes and death (6,7). HUS is a severe, life-threatening complication characterized by three primary conditions: microangiopathic hemolytic anemia (low red blood cell counts), thrombocytopenia (low platelet counts) and kidney failure (8). This condition is caused by the Shiga toxins, which damage blood vessels, break down red blood cells prematurely, form blood clots, and ultimately clog the host’s bloodstream and kidneys’ filtering systems (10). Some signs and symptoms of HUS include pale colouring, fatigue, decreased urination or blood in urine, among others (10). In addition, 25% of HUS patients develop complications in the central nervous system, underscoring the necessity of identifying and treating E. coli infections quickly (9,10).

E. coli O157:H7’s ability to affect other body systems is mediated by thrombocytopenia, a condition induced by platelets accumulating in damaged blood vessels to form small blood clots that impair blood flow to various organs (11). While E. Coli O157:H7 initially only affects the digestive and circulatory systems, the bacteria can have significant detrimental effects on nearly every body system because of the effects of its Shiga toxin on blood vessels (11). For example, the central nervous system is affected in 30-60% of cases, which can result in seizures, coma, and stroke (8). Long-term deficits to the gastrointestinal tract, brain, liver, heart, adrenal glands, spleen, and pancreas can also occur, including hypertension (occurs in 8-12% of children who survive HUS), cardiovascular disease, diabetes and Irritable Bowel Syndrome (8).

References:

Ameer MA, Wasey A, Salen P. Escherichia Coli (E Coli 0157 H7). In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Mar 8]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK507845/
Rahal E, Kazzi N, Nassar F, Matar G. Escherichia coli O157:H7—Clinical aspects and novel treatment approaches. Front Cell Infect Microbiol [Internet]. 2012 [cited 2022 Mar 9];2. Available from: https://www.frontiersin.org/article/10.3389/fcimb.2012.00138
Evans DJ, Evans DG. Escherichia Coli in Diarrheal Disease. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2022 Mar 9]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK7710/
Pathogenic E. coli [Internet]. [cited 2022 Mar 9]. Available from: http://textbookofbacteriology.net/E.coli_4.html
Pathophysiology of diarrhea [Internet]. Colostate.edu. [cited 2022 Mar 12]. Available from: http://www.vivo.colostate.edu/hbooks/pathphys/digestion/smallgut/diarrhea.html
Na taro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev [Internet]. 1998 Jan [cited 2022 Mar 10];11(1):142–201. Available from: https://journals.asm.org/doi/10.1128/CMR.11.1.142
Griffin PM, Ostroff SM, Tauxe RV, Greene KD, Wells JG, Lewis JH, et al. Illnesses associated with escherichia coli 0157:h7 infections. Ann Intern Med [Internet]. 1988 Nov 1 [cited 2022 Mar 10];109(9):705–12. Available from: https://www.acpjournals.org/doi/abs/10.7326/0003-4819-109-9-705
About E. coli O157:H7 [Internet]. The food safety law firm. Available from: https://about-ecoli.com/ecoli_o157
E. coli [Internet]. [cited 2022 Mar 9]. Available from: https://www.who.int/news-room/fact-sheets/detail/e-coli
Hemolytic uremic syndrome (Hus) - Symptoms and causes [Internet]. Mayo Clinic. [cited 2022 Mar 10]. Available from: https://www.mayoclinic.org/diseases-conditions/hemolytic-uremic-syndrome/symptoms-causes/syc-20352399
STEC Hemolytic Uremic Syndrome [Internet]. NORD (National Organization for Rare Disorders). [cited 2022 Mar 11]. Available from: https://rarediseases.org/rare-diseases/stec-hemolytic-uremic-syndrome

iii) What antibiotics might be given (i.e., what are antibacterial treatments and how do these antibiotics work to help the body clear the organism)? Representing mechanisms diagrammatically is helpful to demonstrate understanding (No need to include pictures of chemical structures). Is there any potential harm in giving antibiotics?

Image of intracellular mechanism of action by quinolone antibiotics in their role of inhibiting bacterial DNA replication to result in cell death.

To treat the bacterial infection, some antibiotics that may be used or have been used are ciprofloxacin, azithromycin, and rifaximin (1, 2). All of these antibiotics are administered orally, with doses ranging, where common side effects include nausea, diarrhea, headache, vomiting, and abdominal pain (1). Fosfomycin have also been used in clinical studies, and shown to reduce complication risk from E. coli infection, as well as streptomycin (15). Mechanism of the antibiotics varies. For example, ciprofloxacin is a type of quinolone antibiotics. These antibiotics work by inhibiting the DNA gyrase and DNA topoisomerase to inhibit the relaxation of supercoiled DNA to ultimately inhibit DNA replication of E. coli (3, 4). This means the bacteria will be unable to replicate and grow. Ciprofloxacin antibiotics are also known to be especially effective against gram-negative bacilli bacteria, which is what E. coli is (3). However, many E. coli strains have grown resistant to ciprofloxacin, at about a rate of 40% (5). This calls for caution when prescribing ciprofloxacin as a treatment for E. coli infections.

Depiction of mechanism of action by macrolide antibiotics and their role in inhibiting bacterial protein synthesis.

For Azithromycin, it is a type of macrolide antibiotic. They work by inhibiting bacterial protein synthesis by binding reversibly to the 23S rRNA of the 50S subunit of bacterial ribosomes (6, 7). This means azithromycin will inhibit the assembly of the 50S ribosomal subunit, which means transpeptidation and consequently, translocation step in E. coli protein synthesis will be inhibited (7). This can happen because azithromycin has stronger affinity to bacterial ribosomes than their other protein subunit counterparts. Unfortunately, E. coli resistance to azithromycin have also developed over the years, with increasingly decreased bactericidal activity (8).

Rifaximin is a bactericidal rifamycin derivative, which inhibits bacterial protein synthesis by irreversible binding to the beta-subunit of bacterial DNA-dependent RNA polymerase (9). By preventing bacterial growth by killing it, it stops toxin production, infection, and damage, also stopping diarrhea (10). Rifaximin was at one point a popular bactericidal antibiotic for many types of bacteria, including both gram-positive and gram-negative, as well as aerobic and anaerobic bacteria (9). This is because it had low risk for toxicity and unexpected drug-drug interactions as it is not absorbed by the gastrointestinal tract. It also has minimal impact on the human’s natural intestinal microflora, and specifically targets bacterial polymerase to inhibit protein synthesis. Unfortunately, resistance for rifaximin has also been noted in E. coli (11). In the laboratory, E. coli was seen developing this resistance in just a single step, particularly if it has previously been exposed to rifaximin (11). It may also happen with efflux pumps and amino acid substitutions that pump out the antibiotic and change the dynamic of the interactions required for antibiotic to associate properly to carry out its antimicrobial activity (11).

Image of mechanism of action by aminoglycoside antibiotics to inhibit bacterial protein synthesis.

Fosfomycin inhibits the MurA enzyme that catalyzes a key step in peptidoglycan synthesis, the reaction of UDP-N-acetylglucosamine with phosphoenolpyruvate to form UDP N-acetylmuramic acid (16). This prevents the synthesis of peptidoglycan, a key component of the E. coli cell wall, resulting in bacterial cell death (16). Streptomycin is an aminoglycoside antibiotic that prevents translation initiation by binding to the 16S rRNA and S12 protein in the 30S ribosomal subunit of E. coli (17).

Some potential harm in administering these antibiotics is increasing the amount of toxins produced by the bacteria; thus, increasing Shiga toxin activity and the damage it causes (1). This may lead to higher risk of patient developing hemolytic urema syndrome (HUS) (1, 12). Furthermore, anti-diarrheal medications such as loperamide are also at times not recommended, and instead, patients are guided towards home recovery over 5-10 days (12, 13). Taking anti-diarrheal medication can slow down the digestive system and interfere with the body flushing out the toxins (14). Taking multiple drugs to treat symptoms can also induce potentially harmful drug-drug interactions, so it is important to follow instructions of physicians and not make changes or additions.

The best recommended treatment is to prevent dehydration and electrolyte loss by drinking clear fluids and replacing electrolytes, such as water, soups, and eating bananas (1, 13). It is also advised to avoid certain foods, such as alcohol or high-fiber foods that may encourage bacterial growth and interfere with the body’s digestive system in getting rid of the toxins (14). Resting is important to support the body’s immune system, and practicing good hygiene will help prevent re-infection, such as ensuring you don’t come in contact with your feces, disinfecting, and using separate bathrooms from family members and roommates (1). It is also important to visit the hospital if symptoms persist or worsen (13).

References

1. E. Coli treatments & medications | Singlecare [Internet]. [cited 2022 Mar 11]. Available from: https://www.singlecare.com/conditions/e-coli-treatment-and-medications

2. Escherichia coli infections - infections [Internet]. Merck Manuals Consumer Version. [cited 2022 Mar 11]. Available from: https://www.merckmanuals.com/en-ca/home/infections/bacterial-infections-gram-negative-bacteria/escherichia-coli-infections

3. Thai T, Salisbury BH, Zito PM. Ciprofloxacin. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Mar 11]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK535454/

4. Webber M, Piddock LJ. Quinolone resistance in Escherichia coli. Vet Res. 2001 Aug;32(3–4):275–84

5. Jadoon RJ, Jalal-ud-Din M, Khan SA. E. Coli resistance to ciprofloxacin and common associated factors. J Coll Physicians Surg Pak. 2015 Nov;25(11):824–7.

6. Kirst HA. Macrolide antibiotics. In: Marinelli F, Genilloud O, editors. Antimicrobials: new and old molecules in the fight against multi-resistant Bacteria [Internet]. Berlin, Heidelberg: Springer; 2014 [cited 2022 Mar 11]. p. 211–30. Available from: https://doi.org/10.1007/978-3-642-39968-8_11

7. Azithromycin [Internet]. [cited 2022 Mar 11]. Available from: https://go.drugbank.com/drugs/DB00207

8. Gomes C, Ruiz-Roldán L, Mateu J, Ochoa TJ, Ruiz J. Azithromycin resistance levels and mechanisms in Escherichia coli. Sci Rep [Internet]. 2019 Apr 15 [cited 2022 Mar 11];9:6089. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6465286/

9. Koo HL, DuPont HL. Rifaximin: a unique gastrointestinal-selective antibiotic for enteric diseases. Curr Opin Gastroenterol [Internet]. 2010 Jan [cited 2022 Mar 11];26(1):17–25. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4737517/

10. Rifaximin: medlineplus drug information [Internet]. [cited 2022 Mar 11]. Available from: https://medlineplus.gov/druginfo/meds/a604027.html

11. Kothary V, Scherl EJ, Bosworth B, Jiang Z-D, DuPont HL, Harel J, et al. Rifaximin resistance in Escherichia coli associated with inflammatory bowel disease correlates with prior rifaximin use, mutations in rpob, and activity of phe-arg-β-naphthylamide-inhibitable efflux pumps. Antimicrob Agents Chemother [Internet]. 2013 Feb [cited 2022 Mar 11];57(2):811–7. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3553721/

12. Escherichia coli o157:h7 [Internet]. [cited 2022 Mar 11]. Available from: https://www.hopkinsmedicine.org/health/conditions-and-diseases/escherichia-coli-o157-h7

13. E. coli: what is it, how does it cause infection, symptoms & causes [Internet]. Cleveland Clinic. [cited 2022 Mar 11]. Available from: https://my.clevelandclinic.org/health/diseases/16638-e-coli-infection

14. E. coli - diagnosis and treatment - Mayo Clinic [Internet]. [cited 2022 Mar 11]. Available from: https://www.mayoclinic.org/diseases-conditions/e-coli/diagnosis-treatment/drc-20372064

15. Panos GZ, Betsi GI, Falagas ME. Systematic review: are antibiotics detrimental or beneficial for the treatment of patients with Escherichia coli O157:H7 infection? Aliment Pharmacol Ther. 2006 Sep;24(5):731–42

16. Silver LL. Fosfomycin: Mechanism and Resistance. Cold Spring Harb Perspect Med. 2017 Feb;7(2):a025262

17. PubChem. Streptomycin [Internet]. Nih.gov. [cited 2022 Mar 12]. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Streptomycin

iv) Is this a (Public Health) reportable disease? Why?

The Centre for Disease Control (CDC) defines a ‘reportable’ or ‘notifiable’ disease as “one for which regular, frequent, and timely information regarding individual cases is considered necessary for the prevention and control of the disease” (1). Reporting of such diseases by healthcare professionals protects public health by aiding public health officials in measuring disease trends, assessing the effectiveness of control and prevention measures, recognizing certain populations or areas at risk, and preparing specific resources. While different health authorities may implement varying rules and regulations regarding reportable diseases, most health authorities, including the British Columbia Centre for Disease Control and Prevention (BC CDC), does include Escherichia coli as a reportable disease (2). In fact, disease from E. coli is a public health reportable disease in Canada across the country in all provinces and territories (3). The CDC protocol for physicians is also to report all patients with Shiga toxin-positive diarrheal illness or HUS to the health department (4).

Rates of Shigatoxigenic E. coli by Year, 2009-2018, in BC vs Canada (14).

E. coli O157 may be transmitted through consumption of contaminated and undercooked (or uncooked) foods and liquids, and person to person via fecal shedding (5). Transmission of the bacteria from the intestines of healthy cattle animals, like cows, to humans is especially typical in ground meats, thus underlying the importance of properly and thoroughly cooking meat (6). Other preventative measures for avoiding E. coli O157:H7 infection include keeping food at safe temperatures and protecting water sources from animal wastes (5). In addition, prompt reporting of food-borne illnesses allows for the faster identification of its source, and plays a vital role in preventing or limiting outbreaks.

In Canada, it is noted that E. coli infections have been under-reported because testing for E. coli was previously not a part of routine screening at laboratories (7). Now, when presented with a stool sample in screening for bacterial gastroenteritis, the Canadian Public Health Laboratory Network recommends including screening for E. coli (8). Health departments that receive such reports from physicians may coordinate with local health departments and the CDC to identify outbreaks. Identifying sources and potential outbreaks is especially important, as, though it may spontaneously resolve without medical attention for some, food-borne illnesses can be life-threatening for vulnerable populations, such as children, older people, immunocompromised individuals, or pregnant people (9). To help combat E. coli infections and under-reporting, Health Canada has also put forward industry guidelines and regulatory authorities to help identify and mitigate E. coli in Canadian foods (7). They have also asked Canadians for additional data, reports, and help in identifying E. coli in foods to support future strategies in reducing E. coli incidence in the production, distribution, and consumption of foods (7). Furthermore, the number of E. coli O157 cases remained low in 2018 (35 cases only in BC), the reason for this low number of cases is unclear but could be due to improved meat processing practices (10).

Rates of Shigatoxigenic E. coli by Health Service Delivery Area, 2018 (14).

After the stool sample is analyzed by the laboratory and E. coli O157:H7 is identified, the sample is sent to a public health laboratory for whole genome sequencing and entered into a database to identify whether the bacteria is genetically similar to E. coli from other samples (11). Cases are reported to provincial or territorial departments of health to keep surveillance on the disease. Public health laboratories will send reports to keep surveillance and monitoring data updated. Since illnesses caused by bacteria that are genetically similar are likely to have a common source, public health officials use this to identify related cases to investigate potential outbreaks and their sources (11). This can limit the severity of outbreaks by preventing people from being infected by the same source and preventing similar outbreaks from occurring in the future (12). Patients who have been infected are advised to stay at home away from work and school until they have been completely free of symptoms for 48 hours (13).

References

CDC, 2014. Summary of notifiable diseases – United States, 2012. Morbidity and Mortality Weekly Report 61, 1–23.
List of reportable communicable diseases in BC January 2018 [Internet]. 2018 [cited 2022Mar12]. Available from: http://www.bccdc.ca/Documents/BC%20Reportable%20Disease%20List.pdf
Canada PHA of Surveillance of E. coli (Escherichia coli) infection [Internet]. 2017 [cited 2022 Mar 11]. Available from: https://www.canada.ca/en/public-health/services/diseases/e-coli/surveillance-e-coli.html
Resources for clinicians and laboratories [Internet]. Cdc.gov. 2019 [cited 2022 Mar 12]. Available from: https://www.cdc.gov/ecoli/clinicians.html
Ameer MA, Wasey A, Salen P. Escherichia Coli (E Coli 0157 H7). In: StatPearls [Internet]. StatPearls Publishing; 2021.
Escherichia coli O157:H7 [Internet]. Johns Hopkins Medicine. [cited 2022Mar11]. Available from: https://www.hopkinsmedicine.org/health/conditions-and-diseases/escherichia-coli-o157-h7
Canada H. Call for data: Request for scientific data and information on Shiga toxin-producing Escherichia coli in various food commodities [Internet]. 2021 [cited 2022 Mar 11]. Available from: https://www.canada.ca/en/health-canada/services/food-nutrition/public-involvement-partnerships/call-for-data-scientific-data-information-shiga-toxin-producing-escherichia-coli-food-commodities.html
Chui L, Christianson S, Alexander DC, Arseneau V, Bekal S, et al. CPHLN recommendations for the laboratory detection of Shiga toxin-producing Escherichia coli (O157 and non-O157) [Internet]. 2018 [cited 2022 Mar 11]. Available from: https://www.canada.ca/content/dam/phac-aspc/documents/services/reports-publications/canada-communicable-disease-report-ccdr/monthly-issue/2018-44/issue-11-november-1-2018/ccdrv44i11a06-eng.pdf
Ministry of Health. Food Safety & Security [Internet]. Province of British Columbia. Province of British Columbia; 2021 [cited 2022Mar11]. Available from: https://www2.gov.bc.ca/gov/content/health/keeping-bc-healthy-safe/food-safety
Annual Summaries of Reportable Diseases [Internet]. BCCDC. 2022. Available from: http://www.bccdc.ca/health-professionals/data-reports/communicable-diseases/annual-summaries-of-reportable-diseases
CDC. Reporting Timeline for Foodborne Outbreaks [Internet]. Centers for Disease Control and Prevention. 2021 [cited 2022 Mar 11]. Available from: https://www.cdc.gov/foodsafety/outbreaks/investigating-outbreaks/reporting-timeline.html
Reports of Selected E. coli Outbreak Investigations | E. coli | CDC [Internet]. 2021 [cited 2022 Mar 11]. Available from: https://www.cdc.gov/ecoli/outbreaks.html
Escherichia coli (E. coli) O157 symptoms and treatment [Internet]. [cited 2022 Mar 11]. Available from: https://www.nhsinform.scot/illnesses-and-conditions/infections-and-poisoning/escherichia-coli-e-coli-o157
BC Centre for Disease Control. British Columbia Centre for Disease Control [Internet]. BC Centre for Disease Control. 2019 [cited 2022Mar18]. Available from: http://www.bccdc.ca/resource-gallery/Documents/Statistics%20and%20Research/Statistics%20and%20Reports/Epid/Annual%20Reports/AR2014FinalSmall.pdf

The Microbiology Laboratory

i) Including any stated bacteria, what are the most common bacterial pathogens associated with this type of clinical presentation?

Table 1. Epidemiologic settings and clinical features of infection of selected diarrheal pathogens (8)

Table 1 (8) introduces some potential infectious causes and their epidemiological settings of Ronnie’s symptoms, and a select few most likely of the organisms are further discussed below.

Escherichia Coli 0157:H7

Figure 1. Molecular mechanisms that mediate colonization of STEC strains (12)

It has been determined in Ronnie’s case that E. coli 0157:H7 is the culprit of his illness.The bacteria Escherichia Coli 0157:H7 is a Gram-negative, enterohemorrhagic E.coli (EHEC), Shiga-toxin producing strain that causes diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (HUS) (1). It is usually transmissible through the fecal-oral route by consuming contaminated liquids, raw foods, and fecal shedding (1). It infects the alimentary tract and causes abdominal cramps with hemorrhagic diarrhea (1). The Shiga-toxin it produces may cause the development of HUS, watery diarrhea, and hemorrhagic colitis (1). Production of the Shiga toxins in particular is important in contributing to the development of the gastrointestinal illnesses and particularly the hemorrhagic colitis (9). E. coli O157:H7 can cause systemic illness by hemolytic uremic syndrome by inducing enterohemorrhagic disease, manifesting as acute renal failure, hemolytic anemia, and thrombocytopenia (9). These can result in potentially life-threatening chronic illness if left untreated (9). In the United States, about 63 000 infected cases are reported annually and around 2.8 million cases are reported globally (1). Farm animals, like cattle, are usually the main reservoir for this bacteria. The infectious dose for this bacteria is small, only requiring as little as 10 bacteria to cause disease in humans (1). After entering the host, it binds to the intestinal mucosa and starts to release Shiga toxin; hemorrhagic diarrhea develops after three days of infection (1). Hemorrhagic diarrhea will usually go away by itself within seven days. However, children may sometimes develop systemic manifestations such as HUS. Other symptoms that may develop include nausea, vomiting, dehydration, a lack of energy, and possibly a fever (1). Treatment requires sufficient water intake to maintain fluid balance; antibiotics seem to be ineffective and may even worsen the current outcome (1). Other bacterial species that might present similar symptoms are E. coli O104:H4, Shigella dysenteriae, Campylobacter, Clostridioides difficile, and Salmonella (1).

Other Shiga toxin-producing E.coli

Escherichia Coli O104:H7 is a Gram-negative, enteroaggregative E.coli (EAEC), Shiga-toxin producing bacteria that was the cause of the outbreak in Germany during 2011 (2). A total of 3842 cases had been reported during this time with 2987 of confirmed Shiga-toxin producing E.coli, making it the largest outbreak in Germany yet (2). It’s main reservoir is also cattle, just like strain 0157:H7. It is known for its expression of Shiga-toxin 2 and Extended Spectrum β-Lactamase (3). Its phenotype combines characteristics from EHEC and EAEC bacteria (3). Some symptoms include bloody diarrhea, abdominal cramps, fever, HUS, and even neurological symptoms (3). It is also transmitted through the fecal-oral route, including consumption of contaminated/raw foods and liquids. Treatment for this bacteria involves IV-administered fluids and antibiotics (3).

There are many other strains of shiga toxin-producing E. coli that cause illness in people, referred to as non-O157 Shiga toxin-producing E.coli (STEC). (10) Non-O157 STEC strains have been causing sporadic outbreaks of illness worldwide. (10) Illnesses linked to STEC serotypes other than O157:H7 appear to be on the rise in the United States and worldwide, suggesting that some of these organisms may be emerging pathogens. (10) Some cases of non-O157 STEC illness appear to be as severe as cases caused by E.coli O157:H7. (10) There is much variation in virulence potential within STEC serotypes, and many may not be pathogenic. (10) Of more than 400 serotypes isolated, fewer than 10 serotypes cause the majority of STEC-related human illnesses. Different types of virulence factors are involved in non-O157 STEC pathogenicity. (10) Many laboratories have attempted to develop methods for detection and identification, however, a practical method of STEC detection has yet to be justified. (10) Worldwide, foods associated with non-O157 STEC illness include sausage, ice cream, milk, and lettuce. (10) Results from several studies suggest that control measures for O157 may be effective for non-O157 STEC. (10)

Shigella Dysenteriae

Shigella Dysenteriae is a Gram-negative, nonmotile, facultatively anaerobic rod bacteria belonging to serotype A (4). There are usually about 188 million cases per year globally, with 1 million of them resulting in death (4). It is transmitted through the fecal-oral route, water-borne, or food-borne. It is common in developing countries and can infect all age groups (4). However, children, elderly, and immunocompromised people are at high risk (4). When ingested, the bacteria will colonize in the small intestine and enter the colon, where it produces toxins that result in bloody diarrhea (4). The clinical manifestations of shigellosis, the illness caused by shigella bacteria, includes tenesmus, abdominal pain, watery diarrhea and/or dysentery (bloody and mucoid stools) (8). Other symptoms include vomiting, abdominal pain, and high fevers (unlike E. coli 0157:H7 presented in the case) (4). Treatments include antibiotics and oral rehydration therapy (4). However, symptoms will usually resolve itself without 5 to 7 days (4).

Campylobacter

Campylobacter is a Gram-negative, motile, corkscrew-shaped rod with a single flagellum (5). This bacterial species is one of the most common causes of diarrhea illness, and can also cause gastroenteritis which presents with abdominal pain and vomiting (5). It is usually transmitted by consuming raw poultry, raw milk, and contaminated water (5). In the United States, there are about 1.3 million reported cases annually (5). The elderly and immunocompromised people are at high risk for infection. Some symptoms observed are excessive diarrhea (may be bloody), abdominal cramps, rigors, dizziness, body aches, and high fevers (unlike E. coli 0157:H7 presented in the case) (5). While the symptoms are usually mild, treatment involves fluid intake/electrolyte repletion and macrolide antibiotics for high-risk patients (5).

In rare cases, Campylobacter infection can lead to the potentially fatal Guillain-Barré syndrome (GBS). Symptoms of GBS include muscle weakness persisting for several weeks or even paralysis and permanent nerve damage. Studies report an estimated 40% of GBS cases in the United States as being associated with Campylobacter infection, making Campylobacter the leading cause of GBS development (13).

Since Campylobacter exposure is normally associated with raw or undercooked poultry, it is unlikely that Ronnie’s symptoms are caused by C. jejuni infection – though without microbiological testing, it would be remiss to eliminate it from the list of possible pathogens.

Clostridioides Difficile

C. difficile is a gram-positive, pore-forming, drumstick-shaped bacillus (6). It is known to produce toxins and is usually found in water, air, human, and animal feces (6). It is usually transmitted through the fecal-oral route; it produces two types of toxins called toxin A and toxin B. However, only toxin B has been reported globally (6). There are about half a million cases annually in the United States, with about 29 000 of them being fatal (6). One major risk factor for this bacteria is antibiotic usage. Some of them include penicillins, cephalosporins, fluoroquinolone, and clindamycin (6). This is due to antibiotics altering the gut microbiome, which makes the host more susceptible to infection (6). Adults with a robust immune system will be asymptomatic carriers (6). Some observed symptoms include watery/bloody diarrhea, anorexia, vomiting, nausea, a low fever, and abdominal pain (6). Treatment includes administering the correct antibiotics such as vancomycin and fidaxomicin. Metronidazole may be administered intravenously for patients with ileus (6).

Salmonella

Salmonella are a gram-negative, motile, bacilli (7). It usually manifests into gastroenteritis and enterocolitis; most strains may also cause typhoid fever (7). However, some serotypes of Salmonella may be nontyphoidal (NTS). There are about 200 million cases annually, with about 200 000 resulting in death (7). It is more prominent in south Asia and sub-saharan Africa (7). It may be acquired by ingesting raw eggs, meat, and dairy products (7). Children under the age of one and the immunocompromised are at high risk for infection. Individuals with chronic steroid use or malignancy may also be infected with Salmonella (7). NTS symptoms include diarrhea, fever, and abdominal cramps (7). For individuals who develop enteric fever, some observed symptoms are fevers, abdominal cramps, diarrhea (may be bloody), headache, shock, and lethargy (7). Treatment should focus on hydration and maintaining electrolyte balance (7). Patients with NTS gastroenteritis are usually not given antibiotics unless they are immunocompromised or under the age of three (7). If NTS bacteremia develops, some antibiotics administered are ceftriaxone, azithromycin, fluoroquinolone (7). Patients with enteric fever will be given fluoroquinolone first. Cephalosporins or azithromycin may be given as an alternative if it is a multidrug-resistant enteric fever (7).

Yersinia species

Yersinia are a group of Gram-negative and facultative anaerobic coccobacilli. (11) They can be transmitted by the consumption of contaminated food products including vegetables, milk products, and meat. (11) Optimum growth temperature is about 30 °C, and they can grow at refrigeration temperatures. (11) They can also survive the freezing process. (11) Temperature and calcium concentrations play important roles in regulating the expression of virulence factors, dissemination, and survival of Yersinia enterocolitica. The enteropathogenic strains produce heat-stable enterotoxins that resemble the toxins made by E. coli and allow the bacterium to penetrate the mucus barrier and eventually attach to intestinal cells, with a particular preference for the M cells in Peyer’s patches. Once the pathogen is expelled on the basolateral side of the epithelial barrier and internalized by phagocytes, it will replicate in these phagocytes and cause an inflammatory response (Fabrega, 2012).Symptoms of infection caused by Yersinia enterocolitica, which is the most well-known heterogenous group of strains, include diarrhea which may be bloody in severe cases, mild fever, abdominal pains, and possibly vomiting. (11)

References:

1. Ameer MA, Wasey A, Salen P. Escherichia Coli (E Coli 0157 H7). In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Mar 9]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK507845/

2. Piérard D, De Greve H, Haesebrouck F, Mainil J. O157:H7 and O104:H4 Vero/Shiga toxin-producing Escherichia coli outbreaks: respective role of cattle and humans. Vet Res. 2012;43(1):13.

3. Ullrich S, Bremer P, Neumann-Grutzeck C, Otto H, Rüther C, von Seydewitz CU, et al. Symptoms and Clinical Course of EHEC O104 Infection in Hospitalized Patients: A Prospective Single Center Study. PLoS ONE. 2013 Feb 27;8(2):e55278.

4. Aslam A, Okafor CN. Shigella. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Mar 9]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK482337/

5. Fischer GH, Paterek E. Campylobacter. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Mar 9]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK537033/

6. Mada PK, Alam MU. Clostridioides Difficile. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Mar 9]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK431054/

7. Ajmera A, Shabbir N. Salmonella. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Mar 9]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK555892/

8. Hale TL, Keusch GT. Shigella. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 22. Available from: https://www.ncbi.nlm.nih.gov/books/NBK8038/

9. Erickson M, Liao J, Payton A, Cook P, Ortega Y. Survival and internalization of Salmonella and Escherichia coli O157:H7 sprayed onto different cabbage cultivars during cultivation in growth chambers. Journal of the Science of Food and Agriculture. 2019;99(7):3530-3537.

10. L;, M. E. C. C. Y. E. E. H. (n.d.). Non-o157 shiga toxin-producing escherichia coli in foods. Journal of food protection. Retrieved March 9, 2022, from https://pubmed.ncbi.nlm.nih.gov/20828483/

11. D’Agostino, M., & Cook, N. (2015, September 22). Foodborne pathogens. Encyclopedia of Food and Health. Retrieved March 9, 2022, from https://www.sciencedirect.com/science/article/pii/B9780123849472003263

12. Journals.asm.org. (n.d.). Retrieved March 10, 2022, from https://journals.asm.org/doi/10.1128/IAI.05907-11

13. Questions and Answers | Campylobacter | CDC [Internet]. 2019 [cited 2022 Mar 10]. Available from: https://www.cdc.gov/campylobacter/faq.html

ii) What samples could be taken for laboratory testing and how does the Microbiology Laboratory help in the diagnosis of this infectious disease?

Stool sample

Figure 2. Various procedures conducted for common pathogens associated with Ronnie’s symptoms (4)

A stool (feces) sample can provide doctors with valuable information about what is going on when a child has a problem in the stomach, intestines, or other part of the gastrointestinal (GI) system. (7) A stool culture helps the doctor see if there is a bacterial infection in the GI tract. (7) Evaluation of a stool sample for bacterial pathogens is indicated for patients with fever, bloody or mucoid stool, severe abdominal cramps, or periumbilical tenderness, or for those who are more vulnerable to complications (3). Routine stool cultures are used to identify common enteric pathogens, such as Shigella, Campylobacter, and Shigella toxin-producing E. coli. Fecal inflammatory markers are good indicators for the presence of invasive enteric pathogens, such as Shigella, Salmonella, Campylobacter, or the non-invasive but inflammatory C. difficile. In particular, stool cultures for routine testing in the case of acute community-acquired diarrhea should all be simultaneously cultured for E. coli O157:H7 and tested for Shiga toxins, according to the Center for Disease Control and Prevention (1). As well, additional tests for leukocytes in stool samples should be done when patients present with bloody diarrhea (8).

A sample of the patient’s diarrheal stool sample within the first few days after onset, usually about 5ml, should be collected (1). If stool is solid, 0.5g - 2g is enough to make a sufficient culture (9). It should be put in a clean, dry container with a lid to avoid contamination (9). Rectal swabs, though less sensitive than stool samples for culture purposes, may be an acceptable alternative for infants and young children, and can be useful in recovering Shigella specifically. The swab must be inserted about an inch into the rectum to properly collect the sample (9). The sample itself must not be contaminated with fluids such as urine or water (11). It is then transported to the lab right away or refrigerated before transport. The transport media that is used for a stool sample are Stuart’s, Aimes, or Cary-Blair (9). These transport media are specially formulated to contain minimal nutrients to prevent bacterial replication while minimizing oxidation and loss of culture viability via acid formation (11,12). It is important to process the samples as soon as possible, as the viability of specific enteric pathogens (such as Shigella and Campylobacter) quickly decreases once out of the host environment. In addition, failure to properly store the sample once collected can allow for growth of non-pathogenic commensals to overtake the culture, potentially resulting in false negatives (13). Stool specimens should be processed within 2 hours of collection, which is particularly important for ensuring the survival of Shigella or Campylobacter. It would be difficult to detect the presence of this bacterial strain after a week of illness, so it is important to get the timing of onset correct (8). The stool sample would be cultured on special media that will allow it to grow and inhibit the growth of bacteria that are usually present in the digestive tract (8).

To do a stool test, a technician places small stool samples in sterile plastic dishes with nutrients that encourage the growth of certain bacteria. (7) The targeted bacteria will only grow if they are already in the stool sample. (7) If bacterial colonies form, the technician evaluates them using a microscope and chemical tests to identify the organism. (7) Rapid transport to the laboratory is necessary to prevent production of acid by normal fecal bacteria. (7)

The stool culture might be ordered if an individual has diarrhea for several days or has bloody diarrhea, especially if there has been an outbreak of foodborne illness in the individual’s local community or an individual has recently consumed undercooked meat or unpasteurized milk. (7) When the sample arrives at the laboratory, a technician smears stool samples on a growth-encouraging substance inside sterile plates. (7) These plates are kept at a temperature that ensures the most efficient growth of targeted bacteria. (7) If no bacterial colonies form, the test is negative, meaning that there is no sign of a bacterial infection. However, if bacterial colonies do form, the technician then examines them under a microscope and may perform chemical tests to identify them more specifically. (7)

The presence of Shiga toxins in the fresh stool specimen can be identified using immunoassay such as Biostar OIA SHIGATOX. This test can lead to a qualitative result in 0.5 to 2 hours and can provide helpful information for the clinician before a culture analysis yields additional information. Detection of Shiga toxin genes can also be done using DNA amplification, which also provides preliminary information before a confirmatory culture result becomes available.

Blood sample

The culture of blood is one of the most important procedures performed in the clinical microbiology laboratory. The accuracy of the test is directly related to the methods conducted to collect the blood sample. The most important factor that determines the success of a blood culture is the volume of blood processed. (10) Approximately 20 ml of blood should be collected from an adult for each blood culture, and proportionally smaller volumes should be collected from children and neonates. (10) Most importantly, because many hospitalized patients are susceptible to infections with organisms colonizing their skin, the patient’s skin should be always carefully disinfected. Blood samples should be obtained for cases of acute bacterial gastroenteritis, particularly for patients with high fever or severe symptoms such as bloody diarrhea. Although a complete blood count does not identify specific bacterial pathogens, it is helpful as an indicator for potential complications. A needle would be inserted into the patient’s arm for sample collection (9). This is performed so that other possible diseases or disorders such as leukocytosis, hemolysis, and thrombocytopenia can be ruled out (1). It can also rule out dehydration, electrolyte imbalances, and uremia (1). However, because not all STEC infections can be detected via a blood test, it is usually not considered standard of care (10).

Most blood samples are inoculated directly into bottles filled with enriched nutrient broths. (10) To ensure the maximal recovery of important organisms, two bottles of media should be inoculated for each culture (10 ml of blood per bottle). (10) When these inoculated bottles are received in the laboratory, they are incubated at 37°C and inspected at regular intervals for evidence of microbial growth. (10) In most laboratories, this is accomplished using automated blood culture instruments. (10) When growth is detected, the broths are subcultured to isolate the organism for identification and antimicrobial susceptibility testing. (10) Most clinically significant isolates are detected within the first 1 to 2 days of incubation. (10) However, all cultures should be incubated for a minimum of 5 to 7 days. (10) More prolonged incubation is generally unnecessary. It is worth noting that according to Figure 1, since E.coli O157:H7 produces Shiga toxin (Stx) that can travel into the bloodstream more easily than the pathogen, a laboratory blood test will be helpful in terms of confirming the presence of pathogenic E.coli by detecting the presence of Shiga toxins in the bloodstream. In this case, the doctor ordered routine blood work that tests things like white blood cell counts, levels of electrolytes, glucose, and other clinically relevant molecules to detect infections. For instance, a high number of white blood cells or inflammatory markers is an indicator of invasive bacteria or pseudomembranous colitis, whereas a low platelets count can be used to detect early manifestations of hemolytic-uremic syndrome. Blood urea nitrogen and creatinine levels should also be obtained for patients infected with E. coli O157:H7, or other strains of E. coli that produce Shiga toxins. Finally, a serum electrolyte panel is also indicated if a patient experiences severe volume depletion due to vomiting or diarrhea (8).

Infected tissue sample / urine sample

In addition to stool and blood samples, tissues from the infected areas or urine samples may also be taken for detecting the presence of pathogens or changes in fluid composition.

Food sample

Another possible sample that could be collected and sent to the laboratory for analysis is food samples. If possible, a sample of the burger that Ronnie had eaten would be helpful to confirm the source of E. coli infection. However, seeing as the burger was ingested around a week ago, it seems unlikely that Ronnie’s family would have access to the contaminated food source as a sample to be tested in the Microbiology Lab.

References:

Center for Disease Control and Prevention. 2014. E. coli (Escheria coli). https://www.cdc.gov/ecoli/general/index.html
Complete blood count (CBC) - Mayo Clinic [Internet]. [cited 2022 Mar 11]. Available from: https://www.mayoclinic.org/tests-procedures/complete-blood-count/about/pac-20384919
Elsevier Point of Care. 2022. Bacteria Gastroenteritis. https://www-clinicalkey-com.ezproxy.library.ubc.ca/#!/content/clinical_overview/67-s2.0-3aba746e-854e-40e3-bc68-8f9c0322c509
Google. (n.d.). Clinical bacteriology. Google Books. Retrieved March 9, 2022, from https://books.google.ca/books?id=KxA3gfbO-C0C&lpg=PA89&ots=Ttgvm6SFH9&dq=%22when+a+stool+specimen+is+submitted+to+the+laboratory%2C+various+procedures+are+conducted%22&pg=PA88#v=twopage&q&f=false
Humphries RM, Linscott AJ. Practical Guidance for Clinical Microbiology Laboratories: Diagnosis of Bacterial Gastroenteritis. Clin Microbiol Rev. 2015 Jan;28(1):3–31.
Mueller M, Tainter CR. Escherichia Coli. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Mar 11]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK564298/
Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2021). Medical microbiology. Elsevier.
Sattar, S., Singh, S. 2021. Bacterial Gastroenteritis. NCBI.https://www.ncbi.nlm.nih.gov/books/NBK513295/
Shiga toxin-producing Escherichia coli [Internet]. Testing.com. 2020 [cited 2022 Mar 11]. Available from: https://www.testing.com/tests/shiga-toxin-producing-escherichia-coli/
The Nemours Foundation. (n.d.). Stool test: Bacteria culture (for parents) - nemours kidshealth. KidsHealth. Retrieved March 9, 2022, from https://kidshealth.org/en/parents/test-bac-culture.html#:~:text=A%20stool%20(feces)%20sample%20can,bacterial%20infection%20in%20the%20intestines
Infection by Escherichia coli O157:H7 and Other Enterohemorrhagic E. coli (EHEC) - Infectious Diseases [Internet]. Merck Manuals Professional Edition. [cited 2022 Mar 10]. Available from: https://www.merckmanuals.com/en-ca/professional/infectious-diseases/gram-negative-bacilli/infection-by-escherichia-coli-o157-h7-and-other-enterohemorrhagic-e-coli-ehec
Stool Culture [Internet]. Testing.com. 2021 [cited 2022 Mar 11]. Available from: https://www.testing.com/tests/stool-culture/
eLab Handbook [Internet]. [cited 2022 Mar 11]. Available from: http://www.elabhandbook.info/PHSA/Test/PrintPageWithMaster.aspx

iii) Explain the tests that can be performed on the samples in order to detect any of the potential bacterial pathogens causing this infectious disease.

The pathogen Escherichia coli O157:H7 is a Gram-negative, rod-shaped, facultative anaerobe (1). Rapid fermentation of lactose resulting in gas production, indole production from tryptophan, and lack of D-sorbitol fermentation are features which typically allow strains of E. coli such as O157 to be distinguished from other faecal coliforms (1,2). Bacterial culture using stool or blood samples is currently the gold standard diagnostic test for isolating bacterial pathogens or observing them under a direct smear. However, culture from stool samples is a time-consuming and labour-intensive process, with frequent sample contamination from commensals resulting in culture results being positive in less than 40% of cases (3). As such, culture independent tests should also be performed on these samples as adjuncts to traditional isolation methods. In Ronnie’s case, a wide variety of biochemical, culturing, and non culture detection methods can be used to confirm the identity of the pathogen (a sample protocol for confirmation of E. coli O157 is shown below, in figure 3).

Figure 3. Overview of clinical laboratory recommendations for the isolation and characterization of Shiga Toxin-producing E. coli from clinical specimens. Reprinted from (4).

Bacterial stool culture

Generally speaking, in a bacterial culture test, the stool sample is taken to the laboratory and a special medium is used to deliberately encourage the growth of any micro-organisms in the stool sample. The sample is then examined under a microscope to identify specific bacteria which will grow in colonies, defined as a visible mass of genetically alike micro-organisms.

In order to identify the causative agent in Ronnie’s case of infectious diarrhea, it is important to obtain a pure culture from the collected stool sample. This is important as the intestine is colonized by a wide variety of enteric commensals, and as such the stool which passes through the digestive tract will yield a mixed culture containing a multitude of organisms. After isolating a pure culture, the culture can be plated on differential or selective media or agars, allowed to grow under specific conditions, and then observed for colony morphology or chromogenic changes on the plate.

The most basic faecal culture setup is described below by Humphries and Linscott (2):

MacConkey (MAC) agar
Selective/differential media for recovery of Salmonella and Shigella
Media for recovery of Campylobacter
Media designed for recovery of E. coli O157 or enrichment broth for testing against Shiga toxins

Selective media is typically used to ‘select’ for growth of a specific microorganism, inhibiting the growth of all other microorganisms. For example, addition of an antibiotic to broth will prevent the growth of antibiotic-sensitive organisms while allowing for growth of antibiotic-resistant organisms. On the other hand, differential media may allow many species of bacteria to grow, but may contain additives which allows for different microbes to be visually differentiated from each other. These two types of media can also be combined to create selective/differential media.

MacConkey agar

MacConkey agar is a selective/differential medium for the isolation of Gram-negative rods and identification of lactose-fermenting organisms. It contains crystal violet dye and bile salts to inhibit the growth of Gram-positive bacteria, while presence of a pH indicator and lactose differentiates lactose-fermenting Gram-negatives (5). If an organism is capable of utilizing lactose as an energy source, the process of fermentation will produce organic acids, causing the pH indicator to turn pink. As such, Gram-negative organisms which ferment lactose will form pink colonies, while non-lactose-fermenting Gram-negatives will form colonies that are off-white in colour (5).

There are variations of MacConkey agar that exist to be used in the identification of E. coli O157. These include MacConkey agar with sorbitol (SMAC) and SMAC supplemented with cefixime and tellurite (CT-SMAC) (2,4). In SMAC, the lactose in traditional MacConkey agar is replaced with sorbitol. Sorbitol-fermenting strains of Gram-negative bacteria (such as E. coli O157) will then form pink colonies. Addition of cefixime and tellurite to SMAC also inhibits the growth of many enteric commensals which can grow on SMAC and MAC agar, such as non-pathogenic E. coli (2).

Several other selective/differential agars would also be used to screen for other enteric pathogens, such as xylose-lysin-deoxycholate (XLD), salmonella-shigella (SS), Hektoen enteric (HE), brilliant green (BG), or bismuth sulfite (BS) medium (2). In addition, the use of Campylobacter selective media – blood-free charcoal-cefoperazone-deoxycholate agar (CCDA), charcoal-based selective agar; and blood-based Campy-CVA (cefoperazone, vancomycin, and amphotericin) – and growth under microaerophilic conditions would be important to rule it out as a possible causative agent. Negative results from these tests would allow the Microbiology Laboratory to exclude Salmonella, Shigella, and Campylobacter from the list of possible pathogens and narrow it down to a strain of E. coli.

In addition to observing the presence or absence of growth on selective and differential media, the lab can also discern colony morphology and growth characteristics from stool cultures. Colony characteristics of interest would include colour (though this can differ depending on the growth medium), texture, growth pattern, height, shape, and size (6). Colonies can also be transferred and spread on a glass slide to be observed underneath a brightfield microscope after staining, so that microscopic characteristics can be noted (such as size, cell shape, cluster formation, and presence or absence of spores).

Rainbow agar

The inability of E. coli O157 to produce β-glucuronidase is used to differentiate it from other organisms by Rainbow agar (7). Rainbow agar is a chromogenic agar, meaning that the culture medium changes colour in response to the presence of a particular protein or enzyme. The medium contains chromogenic substrates that are specific for two E. coli-associated enzymes: β-galactosidase (a blue-black chromogenic substrate) and β-glucuronidase (a red chromogenic substrate). If E. coli O157 is the causative bacteria, one would expect the colour associated with the β-glucuronidase substrate to be maintained, as there is no enzyme to interact with it. Rainbow agar tests also exist to detect Salmonella strains and Shigella strains (8). Rainbow agar tests are not meant to be a diagnostic test to discriminate between different causative strains, necessarily, rather, they are meant to be used to discriminate between different strains of a selected bacteria. This is because the substrates selected must be exclusive to, or rather, must be able to discriminate, between the bacterial strains one is testing for. The substrates/measurements that allow for the discrimination between E.coli and Salmonella strains, for instance, are not the same. Therefore, this test usually accompanies other tests, such as PCR, in the confirmation of a bacterial causative agent.

Deoxycholate Citrate Agar (DCA)

DCA is commonly used for the isolation of Gram-negative enteric pathogens (9). Similar to other bacterial growth culture tests, this medium uses the metabolic features of the bacteria to allow for the identification of the bacterial species. Varying metabolic activity between bacterial species results in the production of different metabolic by-products, which alters the pH of the medium. Changes in pH of the medium are visible through colour changes in the pH indicator present in the sample. The key ingredients in the medium are neutral red (pH indicator), ferric ammonium citrate, lactose, and proteose peptone (9). In general, this test cannot be used to identify specific strains of a bacteria within a species, as intra-species metabolism is often not strain-specific.

Xylose Lysine Deoxycholate Agar (XLD)

XLD is a selective growth medium used in the isolation of Shigella, Salmonella and E.coli species from food and clinical samples (10). The growth medium used in this bacterial culture relies on the metabolic characteristics of the bacteria to identify bacteria, similar to MAC and SMAC. Xylose fermentation lowers the pH of the culture, and the phenol red pH indicator reflects this change by changing colours from red to yellow (10). XLD cultures contain xylose, lactose, phenol red, agar, various sodium by-products, and yeast extract (10). This test, like other bacterial growth culture tests discussed, cannot be used to identify specific strains of the bacteria, only the bacterial species.

Mueller-Hinton Agar (MHA):

MHA has been included in this discussion as it is used for the isolation and growth of Campylobacter (11), however, it is rarely used for diagnostic purposes. Campylobacter is difficult to identify, isolate and grow, therefore, cultures are now rarely used to diagnose and confirm Campylobacter infections (12). MHA will grow almost any bacteria which is plated on it, which makes it excellent for antibiotic susceptibility testing (29). Various antibiotic concentrations can be included in the medium to only permit the growth of Campylobacter. Kanamycin, Trimethoprim, Cefoperazone, Tetracycline, Streptomycin, and Nalidixic acid are antibiotics used in the MHA medium to limit bacterial growth to just Campylobacter species (11). Genetic Culture-Independent Diagnostic Tests (CIDTs), such as PCR screening, are far more commonly used in a clinical setting to diagnose Campylobacter infections (12). Furthermore, the wide majority of Campylobacter infections are self-resolving, and hence rarely require laboratory diagnostic confirmation (12). The laboratory diagnosis of Campylobacter mainly serves epidemiological purposes, rather than a clinical purpose.

Gram stain

A Gram stain differentiates Gram-positive and Gram-negative bacteria based on cell wall composition (13). However, with many of the potential pathogens involved in causing hemorrhagic colitis being Gram-negative and rod-shaped, the usefulness of this technique can be limited. As such, Gram staining is not typically performed on stool specimens (2). However, in the case of Campylobacter infections, Gram staining can allow for visualization and identification of the characteristic curved campylobacters under a microscope, up to a sensitivity of 94% in patients with acute enteritis (2).

To perform a Gram stain, a slide must first be prepared by spreading the culture in a thin film and allowing it to air-dry or be heat-fixed. Alternatively, a methanol fixation step may also be performed. The addition of crystal violet dye results in all bacteria becoming stained purple due to the presence of peptidoglycan in the cell wall preventing the dye from diffusing out (13). The mordant (iodine) is added in a subsequent fixation step, where the crystal violet and iodine form complexes which prevents the dye from being easily removed. Ethanol is typically used as a decolorizer, and causes Gram-negative cells, which lack a thick peptidoglycan layer, to become colourless. This is due to the decolorizer causing disruption of the lipids in the outer membrane of Gram-negative pathogens, causing the crystal violet-iodine complexes to be washed away (13). Finally, a counterstain such as carbol-fuchsin or safranin is added to the slide, staining the colourless Gram-negatives a bright pink and allowing for them to be differentiated from the still-purple Gram-positives. E. coli O157 (and other Gram-negative pathogens mentioned in question i) would appear pink and rod-shaped when viewed under a brightfield microscope.

Biochemical tests

Traditionally, in order to verify a presumptive identification from culture, a variety of biochemical tests will be done. These can include tests to confirm that the pathogen exhibits the phenotypic properties that are consistent with the suspected organism. For enteric pathogens, this would typically include a test against lactose fermentation (most enteric pathogens would test negative) and H2S production (some enteric pathogens produce H2S) (2). Other tests which may be done to confirm the identity of an enteric pathogen may include motility testing, indole production, a catalase test, and Voges-Proskauer.

These biochemical tests were previously the gold standard in identifying bacterial pathogens due to their high sensitivity allowing for precise identification. However, they are now considered to be time-consuming and labour-intensive (2). Fortunately, commercial kits (such as API 20E and ID 32E) are now available for the simultaneous testing of an organism in up to 32 different biochemical tests, and studies have been done to develop streamlined biochemical testing regimens for confirmation of pathogenic E. coli (14,15). Commercial kits such as ID 32E contain enzymatic, biochemical, and sugar utilization tests in individual wells, and require minimal colony material, plus decreased handling time and reagents.

Most laboratories will proceed with a secondary screen following an initial selective culturing step (2). In a secondary screen, suspicious colonies (i.e. those that are able to grow on MacConkey agar or those positive for H2S production) are inoculated and subcultured on a series of tubed or slanted media. In the traditional three-tube system, bacteria are inoculated onto a triple sugar iron (TSI) slant, a lysine iron agar (LIA), and a Christensen urea agar – though other media such as motility-indole-ornithine (MIO), motility-indole-lysine (MIL), or motility-indole-lysine-sulfide (MILS) can also be used (2).

Figure 4. Schematic of TSI reaction. Reprinted from (16).

Following secondary screening, if results are consistent with an enteric pathogen, a commercial testing kit such as API 20E may be used to further identify the organism. However, these kits may be limited in their ability to identify Salmonella, Shigella, or Yersinia beyond the genus level (2).

Antigenic testing

In order to identify organisms past the genus level, antigenic testing may be employed. These tests are based on the principles of antigen-antibody interactions, where antigens present in a sample are detected using highly specific antibodies through the formation of antigen-antibody complexes, which can be detected via a colour change or fluorescence. Immunoassays can be performed after, or in parallel with the bacterial culture (17).

In the case of E. coli O157: H7, following selection on SMAC agar to encourage the growth of E. coli, a latex agglutination test can be performed as a confirmatory test. This can be done with latex agglutination kits (where serotype-specific antibodies are coated on latex beads, and a clumping reaction occurs when the specific serotype of the organism is added) (2,4). For E. coli O157:H7, an agglutination test for the presence of Shiga toxins in stool samples is often performed. Alternative antigenic testing strategies may employ the use of enzyme-linked immunosorbent assays (ELISAs) or antiserum (serotype specific, e.g. O157- and H7-specific antisera) (2,4).

ELISA is a plate-based assay technique designed for detecting and quantifying the presence of soluble substances, such as proteins. There are various forms of ELISA (direct assay, indirect assay, sandwich assay), but fundamentally, all of them involve the presence of an antibody (or antibodies) associated with a fluorochrome. The antibody will be specific to the epitope of a particular target antigen. If the target antigen is present, the antibody will bind and release an organized, quantifiable colour change or fluorescent signal . ELISA can be used to detect E.coli O157 (or any bacteria) if an antibody (or antibodies) are used which target an antigen specific to that bacteria. ELISA is often used in tandem with SMAC to confirm the presence of E. coli in particular (18).

Vero-cell Cytotoxicity Assay

Vero cells are a line of cells used in cell cultures. Their lineage was isolated from the epithelial lining of a kidney from the African Green Monkey. Vero cells exhibit cytotoxicity in response to Shiga-toxins exclusively (Stx 1 and Stx 2). Therefore, Vero cell cultures can be used to test for the presence of Stx, and by extension, the presence of STEC/EHEC E.coli in a sample. The Vero cell assay is considered to be the ‘gold standard’ for screening Stx-positive samples in 48-72 hours by examining the morphology of Vero cells under a microscope, observing for cytotoxicity (19). Often, dyes, such as Trypan blue, are added into the Vero cell culture. These dyes are taken up by cells undergoing a cytotoxic reaction, allowing for cytotoxicity to be observed under the microscope. The assay has since been modified, such that the detection of Lactate Dehydrogenase (LDH) from Vero cells can now be detected and used as a proxy measure for cytotoxicity in 12-16 hours (20). Amongst the potential bacterial causes of Ronnie’s clinical presentation, only two groups of bacteria produce Shiga-toxin: STEC and Shigella, namely S. dysenteriae. To & Bhunia demonstrated that a 3D Vero cell platform is specific for (i.e., only produces cell cytotoxicity for) Stx-positive STEC bacterial isolates (19), however, they did not test the 3D cell platform for Shigella.

Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS)

Due to the low cost and convenience of MALDI-TOF mass spectrometry, laboratories have begun to replace traditional biochemical assays with proteomic profiling (2). In MALDI-TOF MS, microbes can be identified by fragmenting peptides into ions using a laser, then detecting ionized fragments using mass spectrometry (21). These ionized fragments will scatter based on mass and charge in predictable and known fashion, creating a characteristic spectrum called a peptide mass fingerprint (PMF) and allowing specific proteins to be identified (21). Microbes can then be identified by comparing the PMF of the organism with known PMFs in a database.

MALDI-TOF MS has been shown to be able to identify Salmonella spp. down to the genus level and Campylobacter, though current systems and databases are not as useful in differentiating E. coli from Shigella, or pinpointing E. coli and Salmonella down to the serotype (2).

Figure 5. Schematic diagram of MALDI-TOF MS workflow. Reprinted from (21).

Culture-independent methods

In addition to culture-based testing methods, culture-independent methods can be useful in identification of enteric pathogens. These are especially useful for STEC and C. difficile infections, the latter of which may be difficult to identify using traditional culturing methods (2). In fact, the CDC recommends that stool samples collected from patients with suspected STEC infections be cultured in selective/differential media for E. coli O157 in parallel with inoculation in enrichment media for detection of Shiga toxin 1 (Stx1) and Stx2 (4).

Figure 6. Nonculture testing options for STEC. NAAT, Nucleic Acid Amplification Test. Reprinted from (2).

Nucleic Acid Amplification Test (NAAT), or Polymerase Chain Reaction (PCR)

Nucleic acid-based identification methods provide an advantage over traditional culture-dependent methods in that they are more rapid, sensitive, and specific (22). In fact, compared to the multiple days to weeks that culture-based methods require, nucleic acid-based methods can provide results in as little as one day.

These methods employ the use of the polymerase chain reaction (PCR) to detect and amplify pathogen-specific genes (4). Traditional PCR is a technique that relies on the binding of specific primers to DNA, allowing for amplification of the region of interest through subsequent heat cycling steps. Amplified products can then be detected using agarose gel electrophoresis. In the case of E. coli O157, virulence genes of interest to be targeted with PCR include stx1 and stx2, though studies have used genes for eae, fimA, rfbE, Z3276, and uidA (4,23).

Multiplex real-time PCR is an alternative to traditional PCR and involves a variety of serotype-specific primers conjugated to fluorescent probes (23). In contrast with traditional PCR, multiplex PCR allows for the detection and amplification of multiple genes simultaneously from the same template. Recognition and amplification of a pathogen-specific sequence can then be detected via accumulation of a fluorescent signal. PCR as a means to identify enteric pathogens is highly specific, with a sensitivity of 95% to 100% for enterovirus (24).

However, fecal specimens are among the most complex specimens for direct PCR testing due to the presence of inherent PCR inhibitors that are often co-extracted along with bacterial DNA (25). Potential PCR inhibitors include heme, bile, salts, and complex carbohydrates. Therefore, despite the conventional use, accuracy and speed of PCR tests, many Microbiology Laboratories still use SMAC followed by serological testing with O157 antisera to confirm the presence of E. coli O157 (25).

Finally, there are a few more culture-independent tests that can be done directly on the stool sample.

The first one is a faecal white blood cell (WBC) test, which involves checking for the presence of white blood cells in the stool sample. This should be done if the case suggests an invasive bacterial cause. If the symptoms of a patient were not caused by a bacterial infection, then there should be no white blood cells in the sample. This test, however, has a relatively low sensitivity of 70% and specificity of 50% for inflammatory processes (27).

The second test is a fecal occult blood test, which is used to identify hidden blood in stool samples, and a positive guaiac test result is an indicator of bacterial infection (28). A guaiac test involves placing the stool sample on guaiac paper and applying hydrogen peroxide; if blood is present in the sample, the reaction between the compounds will yield a blue colour within a few seconds (27).

Finally, a faecal lactoferrin test can also be performed. Faecal lactoferrin is a neutrophil marker, and therefore its presence in stool samples can support the diagnosis of inflammatory diarrhea. This test has a relatively high sensitivity of 90%+ and specificity of 70%+, when compared to a faecal WBC test (28).

Blood sample

A complete blood count test should be performed to detect leukocytosis, hemolysis, and thrombocytopenia. In invasive bacterial gastroenteritis, an elevated white blood cell count is typically seen. A basic metabolic panel that measures the level of electrolyte, creatinine, glucose, and blood urea nitrogen should be taken – especially for patients infected with E. coli O157:H7 or other strains of Shiga toxin-producing E. coli – to identify early manifestations of hemolytic uremic syndrome (26). Concerning findings that suggest the presence of bacterial pathogens may include hypernatremic dehydration (serum sodium level greater than 150 mEq/L), a serum glucose level that is lower than 50 mg/dL, or a serum bicarbonate level that is lower than 17 mEq/L. Furthermore, abnormally high creatinine level suggests the possibility of more significant prerenal dehydration or intrinsic renal damage from hemolytic uremic syndrome.

References:

Baylis CL, Penn CW, Thielman NM, Guerrant RL, Jenkins C, Gillespie SH. Escherichia coli and Shigella spp. In: Principles and Practice of Clinical Bacteriology [Internet]. John Wiley & Sons, Ltd; 2006 [cited 2022 Mar 11]. p. 347–65. Available from: https://onlinelibrary.wiley.com/doi/abs/10.1002/9780470017968.ch28
Humphries RM, Linscott AJ. Practical Guidance for Clinical Microbiology Laboratories: Diagnosis of Bacterial Gastroenteritis. Clin Microbiol Rev [Internet]. 2015 Jan [cited 2022 Mar 10];28(1):3–31. Available from: https://journals.asm.org/doi/10.1128/CMR.00073-14
Hatchette TF, Farina D. Infectious diarrhea: when to test and when to treat. CMAJ Can Med Assoc J [Internet]. 2011 Feb 22 [cited 2022 Mar 11];183(3):339. Available from: https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC3042443/
Guidance for public health laboratories : isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) from clinical specimens [Internet]. [cited 2022 Mar 11]. Available from: https://stacks.cdc.gov/view/cdc/21592
Jung B, Hoilat GJ. MacConkey Medium. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Mar 11]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK557394/
Colony Characteristics of E.Coli [Internet]. Sciencing. [cited 2022 Mar 11]. Available from: https://sciencing.com/colony-characteristics-ecoli-8507841.html
Kehl SC. Role of the laboratory in the diagnosis of enterohemorrhagic escherichia coli infections. Journal of Clinical Microbiology. 2002;40(8):2711–5.
Novicki TJ, Daly JA, Mottice SL, Carroll KC. Comparison of sorbitol MacConkey Agar and a two-step method which utilizes enzyme-linked immunosorbent assay toxin testing and a chromogenic agar to detect and isolate enterohemorrhagic escherichia coli. Journal of Clinical Microbiology. 2000;38(2):547–51.
Aryal S. Deoxycholate citrate agar (DCA)- composition, principle, preparation, results, uses [Internet]. Microbe Notes. 2022 [cited 2022Mar11]. Available from: https://microbenotes.com/deoxycholate-citrate-agar-dca/
Rollender W, Beckford O, Belsky RD, Kostroff B. Comparison of xylose lysine deoxycholate agar and Macconkey agar for the isolation of salmonella and Shigella from clinical specimens. American Journal of Clinical Pathology. 1968;51(2_ts):284–6.
Davis L, DiRita V. Growth and laboratory maintenance of campylobacter jejuni. Current Protocols in Microbiology. 2008;10(1).
Hill D, Ryan E, Endtz HP. Campylobacter Infections. In: Hunter's Tropical Medicine and Emerging Infectious Diseases (tenth edition). Elsevier; 2020. p. 507–11.
Tripathi N, Sapra A. Gram Staining. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Mar 11]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK562156/
Flores Abuxapqui JJ, Suárez Hoil GJ, Heredia Navarrete MR, Puc Franco MA, Vivas Rosel ML. Four biochemical tests for identification of probable enteroinvasive Escherichia coli strains. Rev Latinoam Microbiol. 1999 Dec;41(4):259–61.
Leclercq A, Lambert B, Pierard D, Mahillon J. Particular Biochemical Profiles for Enterohemorrhagic Escherichia coli O157:H7 Isolates on the ID 32E System. J Clin Microbiol [Internet]. 2001 Mar [cited 2022 Mar 11];39(3):1161. Available from: https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC87895/
Triple sugar iron (TSI) slant - ppt download [Internet]. [cited 2022 Mar 11]. Available from: https://slideplayer.com/slide/17423304/
Elsevier Point of Care. 2022. Bacteria Gastroenteritis. https://www-clinicalkey-com.ezproxy.library.ubc.ca/#!/content/clinical_overview/67-s2.0-3aba746e-854e-40e3-bc68-8f9c0322c509
Novicki TJ, Daly JA, Mottice SL, Carroll KC. Comparison of sorbitol MacConkey Agar and a two-step method which utilizes enzyme-linked immunosorbent assay toxin testing and a chromogenic agar to detect and isolate enterohemorrhagic escherichia coli. Journal of Clinical Microbiology. 2000;38(2):547–51.
To CZ, Bhunia AK. Three dimensional vero cell-platform for rapid and sensitive screening of shiga-toxin producing escherichia coli. Frontiers in Microbiology. 2019;10.
Roberts PH, Davis KC, Garstka WR, Bhunia AK. Lactate dehydrogenase release assay from vero cells to distinguish verotoxin producing escherichia coli from non-verotoxin producing strains. Journal of Microbiological Methods. 2001;43(3):171–81.
Singhal N, Kumar M, Kanaujia PK, Virdi JS. MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis. Front Microbiol [Internet]. 2015 [cited 2022 Mar 11];6. Available from: https://www.frontiersin.org/article/10.3389/fmicb.2015.00791
Holland JL, Louie L, Simor AE, Louie M. PCR Detection of Escherichia coli O157:H7 Directly from Stools: Evaluation of Commercial Extraction Methods for Purifying Fecal DNA. J Clin Microbiol [Internet]. 2000 Nov [cited 2022 Mar 11];38(11):4108. Available from: https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC87549/
Li B, Liu H, Wang W. Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E. coli. BMC Microbiol [Internet]. 2017 Nov 9 [cited 2022 Mar 11];17(1):215. Available from: https://doi.org/10.1186/s12866-017-1123-2
Botkin, D.J. 2012. Development of a multiplex PCR assay for detection of Shiga toxin-producing Escherichia coli, enterohemorrhagic E. coli, and enteropathogenic E. coli strains. https://www.frontiersin.org/articles/10.3389/fcimb.2012.00008/full
Bej AK, Atlas RM, Haff L, DiCesare JL. Detection of escherichia coli and shigella spp. in water by using the polymerase chain reaction and gene probes for uid. Applied and Environmental Microbiology. 1991;57(8):2445–.
Elsevier Point of Care. 2021. Bacteria Gastroenteritis in children. https://www-clinicalkey-com.ezproxy.library.ubc.ca/#!/content/clinical_overview/67-s2.0-ded09d71-0ba6-47c1-a571-77fe753f3316
Kaur, K., Adamski, J.J. 2021. Fecal Occult Blood Test. NCBI. https://www.ncbi.nlm.nih.gov/books/NBK537138/
Acute Diarrhea in Adults. 2022. https://www-clinicalkey-com.ezproxy.library.ubc.ca/#!/content/clinical_overview/67-s2.0-07521bcc-c986-4b07-9344-cfe989a1ce25

iv) What are the results expected from the tests that might allow the identification of different bacteria causing these symptoms, including any stated bacteria above.

Bacterial stool culture

E. coli O157 exhibits specific growth patterns and characteristics on conventional media used for the isolation and identification of enteric pathogens. On non-differential and selective agar (such as tryptic soy agar), E. coli colonies are typically off-white or grey, shiny in texture, slightly raised, and with an entire/fixed margin (1).

MacConkey agar (MAC)

MacConkey agar is a selective/differential media for isolating Gram-negative enteric microorganisms, differentiating lactose fermenters from lactose non-fermenters. Its components include lactose and a pH indicator, in addition to inhibitors such as bile salts and crystal violet to inhibit the growth of non-enteric organisms (2). These inhibitors function by degrading the thick peptidoglycan layer of Gram-positive bacteria, which is protected by the outer membrane of Gram-negative bacteria. Organisms capable of fermenting lactose will produce secondary metabolites and wastes that are acidic in nature, resulting in the pH indicator turning red (2). In the case of E. coli O157, which is capable of fermenting lactose, colonies that grow on MacConkey agar will appear pink in colour, in contrast to other enteric pathogens such as Salmonella, Shigella, and Yersinia (which are able to grow on MacConkey agar but are unable to ferment lactose, producing colourless colonies). In the case of Campylobacter, which is a relatively fastidious organism with complex nutrient requirements for growth, colony formation on MacConkey agar is rare (3). In fact, one study found that only 33% of Campylobacter spp. tested could grow on MacConkey medium, even though Campylobacter is a Gram-negative organism (3).

Figure 7. Growth of various microorganisms on MacConkey agar. Reprinted from (4).

Sorbitol-MacConkey agar differs from traditional MacConkey agar in that lactose is replaced by sorbitol (5). In contrast with most strains of E. coli, E. coli O157 is unable to ferment sorbitol and so colonies that grow on SMAC will appear colourless rather than pink (6). An additional variant of sorbitol-MacConkey agar includes cefixime and tellurite, which inhibit the growth of many enteric microorganisms, including commensal strains of E. coli (5).

Rainbow agar

In rainbow agar, if E.coli O157 is the causative bacteria, one would expect the colony colour to be Blue/Black, as the bacteria has the necessary enzymes to breakdown the red substrate (β-glucuronidase), but not the Blue/Black substrate (β-galactosidase). Other strains of STEC/EHEC, such as E.coli O48:H21, E.coli O26:H11, and E.coli O111:H8, possess enzymes to breakdown both substrates present in the culture, hence they will produce varying shades of purple/violet. This test is not able to discriminate between different bacterial causes, rather, it is used to discriminate between different strains within a bacterial species.

Xylose Lysine Deoxycholate Agar (XLD)

Most gut bacteria, including Salmonellae, can ferment the sugar xylose to produce acid; Shigella colonies cannot do this and therefore, the culture remains red. Salmonellae strains will decarboxylate Lysine after exhausting the Xylose supply, increasing the pH of the culture once again, mimicking the red Shigella colonies. In this metabolic process, however, Salmonellae will produce hydrogen sulfide, resulting in the formation of black-coloured centers in the colonies (7). This characteristic allows them to be differentiated from Shigella colonies. E. coli, like Salmonellae, also ferments the Xylose present in the medium, however, it does so to an extent that prevents decarboxylation, resulting in the medium remaining acidified and the culture turning yellow. This variability in colour can allow the lab technician to identify what species of bacteria is contained in the sample.

Deoxycholate Citrate Agar (DCA)

Lactose non-fermenters produce transparent, colourless to light pink or tan-coloured colonies with or without black centres (8). Examples of bacteria that would fit into this category include S. enteritidis, S. typhimurium, Shigella flexneri, and Shigella sonnei. Similar to the growth pattern observed in XLD agar, some lactose non-fermenters, namely Salmonellae, will produce hydrogen sulfide, leading to black-centered colonies forming. Shigella flexneri and Shigella sonnei are differentiated by the late lactose fermentation of S. sonnei, leading to a delayed pink colour change in the medium (8). Lactose fermenters, like E. coli, produce a red colony with or without a bile precipitate. E. coli has poor growth on this medium, hence it produces a colour shift to pink, rather than red.

Mueller-Hinton Agar (MHA)

If appropriate antibiotics are used in the medium, the growth of bacteria should be limited to Campylobacter species. If these antibiotics are used and no growth is observed after 48 hours under specific conditions (microaerophilic and 42C), then the sample is negative for Campylobacter infection.

Gram stain

Figure 8. Gram stain of Escherichia coli. Reprinted from (9).

As mentioned in the previous section, performing a Gram stain in this case likely does not provide much identifying information, as most enteric pathogens are Gram-negative and rod-shaped (5). As such, they will appear pink when examined under a brightfield microscope. A Gram-stained sample of E. coli is shown below (Figure 8).

Figure 9. Gram-negative Campylobacter organisms in a mixed culture (indicated with arrows). Reprinted from (10).

One instance in which performing a Gram stain would be useful for identification would be if the causative agent of Ronnie’s disease was Campylobacter spp (10). In fact, a recent study has shown that Gram stain identification of the characteristic Gram-negative, gull-wing shaped campylobacters in patient stool samples can provide specific and accurate verification of Campylobacter infection (10).

Biochemical tests

Table 2. Select secondary screening test results for typical enteric pathogens. Adapted from (5).

Following microbiological culture on selective and differential media to obtain a preliminary diagnosis, secondary biochemical screening may be performed.

Some of the secondary screening test results for typical enteric pathogens are shown below (adapted from (5)):

Abbreviations: SMAC, sorbitol MacConkey agar; HE, Hektoen enteric agar; MAC, MacConkey agar; XLD, xylose-lysine-deoxycholate agar; SS, Salmonella-shigella agar; CIN, cefsulodin-Irgasan-novobiocin agar. Test results (e.g. K/A) indicate pH of slant over pH of the medium in the butt of the tube (K, alkaline; A, acidic).

Figure 10. TSI results for various enteric pathogens. Reprinted from (11).

When inoculated onto TSI agar, the following results may be observed depending on the organism:

If E. coli O157 (or any E. coli strain) is the causative agent of disease, the TSI tube will appear yellow in the slant and the butt of the tube (11), indicating glucose and lactose and/or sucrose fermentation. Production of gas will also be observed.
If Salmonella spp. is the causative agent, the TSI tube will appear red in the slant and yellow in the butt, indicating glucose fermentation in the butt and acid oxidation in the slant (11). Some strains of Salmonella are able to produce H2S, which can cause a blackening in the butt of the tube due to the reaction between H2S and ferric ammonium citrate.
If Shigella spp. is the causative agent, the TSI tube will appear red in the slant and yellow in the butt, indicating glucose fermentation in the butt and inability to utilize lactose or sucrose.

Antigenic testing

Figure 11. Agglutination of E. coli O157 and H7 latex reagent. Reprinted from (13).

A latex agglutination test for identification of E. coli O157 may also be performed. If E. coli O157 is present in the stool sample, clumping will occur within 15 minutes to 1 hour following inoculation of the organism into the reagent (Figure 11) (12). Due to the specificity of the antibodies conjugated to the latex beads, other enteric pathogens will not produce a clumping reaction.

Binding of an antibody to the target antigen in an ELISA produces a quantifiable fluorescent or chromogenic signal. As such, a sufficient degree of signal can be established to confirm the presence of the target antigen or bacteria within a patient’s stool sample. Antibodies against various antigens specific to a bacterial species can be used to confirm, or reject, the presence of the targeted bacteria within a sample. For example, Tong et al., demonstrated that OmpT, a highly conserved antigen amongst E.coli strains, can be used in indirect ELISA to confirm the presence of E.coli within a sample (14). Many bioscience companies sell and distribute ELISA kits which use anti-O157 antibodies for the detection of E. coli O157 (15).

Vero cell cytotoxicity assay

If a STEC is present in the centrifuged sample that is introduced to the Vero cell assay, one would expect to observe cytotoxicity in the Vero cells, either by way of microscopic analysis or using another assay to test for the presence of chemical by-products of cytotoxicity (i.e., Lactate Dehydrogenase). Theoretically, the Vero cell cytotoxicity assay would also detect S. dysenteriae, which is another potential cause of Ronnie’s symptoms. Other potential causes of Ronnie’s symptoms, such as Salmonella, non-STEC E.coli, and Campylobacter, would not cause cytotoxicity in Vero cells. This test is of the greatest use when determining if Shiga-toxin is a virulence factor contributing to the clinical presentation, as it directly tests for the presence of Shiga-toxin. Given the limited number of bacteria which produce Shiga-toxin, this test can be used to significantly narrow down the potential bacterial causes if cytotoxicity of Vero cells is observed.

Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS)

Figure 12. Typical mass spectra of four biomarker proteins in E. coli O157. Reprinted from (16).

The specificity of the MALDI-TOF MS method can allow for strains of E. coli O157 to be discerned from non-O157 strains (16). The presence or absence of specific biomarker peaks in the PMF generated is key in this identification (shown below in Figure 12). Other strains of enteric pathogens will produce entirely different PMFs, allowing for precise identification of the causative agent in Ronnie’s case.

Nucleic Acid Amplification Test (NAAT)

Figure 13. Agarose gel electrophoresis of PCR products generated from an E. coli O157:H7 detection kit. Reprinted from (17).

Commercial kits specific to the detection of E. coli O157:H7 exist from various manufacturers. These contain primers which target and bind to the regions encoding Shiga toxins 1 and 2 (stx1 and stx2) (17). Running of the amplified product on an agarose gel will separate amplified products based on size of the DNA fragment amplified, with the fragments produced by Norgen’s E. coli O157:H7 detection kit (Cat: EP41300) yielding fragments of approximately 580 and 350 base pairs, corresponding to stx2 and stx1 respectively (Figure 13). The absence of stx1 and stx2 in other strains of E. coli and enteric pathogens (although some strains of Shigella also produce Stx, E. coli O157’s Stx1 and Stx2 are functionally similar yet genetically distinct, allowing for detection of only Stx1 and 2 expressed by E. coli O157) means that no band corresponding to those amplified products will be seen.

Similarly, the sdiA gene is one of many genetic sequences which can be identified with a PCR test that would confirm the presence of Salmonellae (18). For the identification of the Campylobacter species, the omp50 gene is one of the genes that can be screened for (and detected) by the PCR (19). If a Shigella species is the causative bacteria, the ipaH gene could be targeted and detected by PCR (20). There are an extensive battery of genes unique to each bacteria, and therefore, numerous gene combinations can be targeted by oligonucleotide primers for the detection of a particular bacteria in PCR.

References:

Colony Characteristics of E.Coli [Internet]. Sciencing. [cited 2022 Mar 11]. Available from: https://sciencing.com/colony-characteristics-ecoli-8507841.html
Jung B, Hoilat GJ. MacConkey Medium. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Mar 11]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK557394/
Baserisalehi M, Bahador N, Kapadnis B p. A novel method for isolation of Campylobacter spp. from environmental samples, involving sample processing, and blood- and antibiotic-free medium. J Appl Microbiol [Internet]. 2004 [cited 2022 Mar 11];97(4):853–60. Available from: https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1365-2672.2004.02375.x
Aryal S. MacConkey Agar- Composition, Principle, Uses, Preparation and Colony Morphology [Internet]. Microbiology Info.com. 2015 [cited 2022 Mar 11]. Available from: https://microbiologyinfo.com/macconkey-agar-composition-principle-uses-preparation-and-colony-morphology/
Humphries RM, Linscott AJ. Practical Guidance for Clinical Microbiology Laboratories: Diagnosis of Bacterial Gastroenteritis. Clin Microbiol Rev [Internet]. 2015 Jan [cited 2022 Mar 10];28(1):3–31. Available from: https://journals.asm.org/doi/10.1128/CMR.00073-14
Schmidt H, Scheef J, Huppertz HI, Frosch M, Karch H. Escherichia coli O157:H7 and O157:H− Strains That Do Not Produce Shiga Toxin: Phenotypic and Genetic Characterization of Isolates Associated with Diarrhea and Hemolytic-Uremic Syndrome. J Clin Microbiol [Internet]. 1999 Nov [cited 2022 Mar 11];37(11):3491. Available from: https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC85676/
Rollender W, Beckford O, Belsky RD, Kostroff B. Comparison of xylose lysine deoxycholate agar and Macconkey agar for the isolation of salmonella and Shigella from clinical specimens. American Journal of Clinical Pathology. 1968;51(2_ts):284–6.
Aryal S. Deoxycholate citrate agar (DCA)- composition, principle, preparation, results, uses [Internet]. Microbe Notes. 2022 [cited 2022Mar11]. Available from: https://microbenotes.com/deoxycholate-citrate-agar-dca/
Beards DG. English: Escherichia coli Gram Stain [Internet]. 2021 [cited 2022 Mar 11]. Available from: https://commons.wikimedia.org/wiki/File:Escherichia_coli_Gram_Stain.jpg
Kamata K, Tokuda Y. Rapid diagnosis of Campylobacter jejuni by stool Gram stain examination. Case Rep [Internet]. 2014 Jan 28 [cited 2022 Mar 11];2014:bcr2013202876. Available from: https://casereports.bmj.com/content/2014/bcr-2013-202876
Aryal S. TSIA Test- Principle, Media, Procedure, Results, Uses, Limitations [Internet]. Microbe Notes. 2022 [cited 2022 Mar 11]. Available from: https://microbenotes.com/triple-sugar-iron-agar-tsia-test/
Escherichia coli O157 Latex Test [Internet]. [cited 2022 Mar 11]. Available from: https://www.thermofisher.com/order/catalog/product/DR0620M
Mohammed Hamzah A, Mohammed Hussein A, Mahmoud Khalef J. Isolation of Escherichia coli 0157:H7 strain from fecal samples of zoo animal. ScientificWorldJournal. 2013;2013:843968.
Tong C, Wu Z, Yu L, Fan Z, Chen L, Hu R, et al. Development of an indirect ELISA for detection ofe. Coliantibodies in cow serum using a recombinant Ompt as antigen. Journal of Immunoassay and Immunochemistry. 2014;35(3):241–55.
E. coli O157 ELISA assay kit [Internet]. Eagle Biosciences. 2021 [cited 2022Mar11]. Available from: https://eaglebio.com/product/e-coli-0157-elisa-assay-kit/
Ojima-Kato T, Yamamoto N, Suzuki M, Fukunaga T, Tamura H. Discrimination of Escherichia coli O157, O26 and O111 from Other Serovars by MALDI-TOF MS Based on the S10-GERMS Method. PLoS ONE [Internet]. 2014 [cited 2022 Mar 11];9(11). Available from: https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC4239071/
E.coli O157:H7 Detection Kits (Cat. TM41300, TM41310, EP41300) | Norgen Biotek Corp. [Internet]. [cited 2022 Mar 11]. Available from: https://norgenbiotek.com/product/ecoli-o157h7-detection-kits
Halatsi K, Oikonomou I, Lambiri M, Mandilara G, Vatopoulos A, Kyriacou A. PCR detection OFSALMONELLASPP. using primers targeting the Quorum Sensing Genesdia. FEMS Microbiology Letters. 2006;259(2):201–7.
Dedieu L, Pagès Jean-Marie, Bolla J-M. Use of the omp50 gene for identification of campylobacter species by PCR. Journal of Clinical Microbiology. 2004;42(5):2301–5.
Thiem VD, Sethabutr O, von Seidlein L, Van Tung T, Canh DG, Chien BT, et al. Detection of shigella by a PCR assay targeting the ipah gene suggests increased prevalence of shigellosis in Nha Trang, Vietnam. Journal of Clinical Microbiology. 2004;42(5):2031–5.

The Bacterial Pathogen Using the following pathogenic steps, outline the pathogenesis of the bacteria named as being responsible for this infection.

i) Encounter: where does the organism normally reside, geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence. How might our patient have come in contact with this bacteria?

Geographic location:

Bacterium strains like Shiga toxin-producing E. coli (STEC) may cause foodborne illness within humans and is primarily transmitted via consumption of food that has been contaminated (1). This may include ground meat products that are undercooked or raw, raw milk, contaminated raw fruits and vegetables as well as through cross-contamination during the preparation of food (1). Other contaminated foods that may contain E. coli O157:H7 include unpasteurized milk, drinking water, salami, beef jerky, and fresh produce such as lettuce, radish sprouts, fresh spinach, and apple cider (9). In addition, ruminant animals on farms and contaminated water within lakes, pools, or various drinking sources may act as a natural reservoir for the bacterium, E. coli O157:H7 (2).

Figure 1: E. coli O157:H7 on contaminated beef

STEC infections are most reported within industrialized countries such as Canada, the United States, the United Kingdom, Japan, and Europe compared to developing countries (4). Reports have found that E. coli O157:H7 are more common in the western provinces of Canada compared to the east in Canada and are more common in the northern states of the United States, compared to southern states (5). Within large geographic areas, STEC infections can result in a large number of cases for a long duration of time due to outbreaks from waterborne or food origin within a community (6). In the northwest of the United States, it was found that E. coli O157:H7 can commonly be found within cattle as they act as asymptomatic carriers for the organism (5). Fecal shedding within cattle is intermittent and seasonal, where more shedding occurs in months of the summer (5). Furthermore, animals such as sheep, pigs, horses, dogs, deer, and feces of birds may also carry E. coli O157:H7 and thus act as vehicles for transmission of the bacteria. However, they cause severe diarrheal disease only when accidentally introduced into the human intestinal tract.

Environmental locations such as those of farms, ponds, dams, wells, barns, water and water troughs, farm equipment, ground and pasture can also act as potential habitats for E. coli O157:H7 (5). E. coli O157:H7 can survive for up to a year in manure-treated soil and up to 21 months in raw manure that had not been composted (10). Additionally it has a large presence within water sources which may be due to the fact that E. coli O157:H7 can survive for long periods of time within water at low temperatures (7). Water trough sediments contaminated with cattle feces act as a reservoir of E. coli O157:H7 and is a source of infection (11). Thus, natural events such as through rainwater, wind or the movement of animals and humans can contribute to the spreading and transmission of this bacterium (5). From this, it can be understood why animals such as cattle may carry the bacterium as they may come into contact with it through drinking contaminated water sources. Similarly, flies that then encounter contaminated cattle may consequently be contaminated with the bacterium and thus transmit it onto food which may be left out. A study had been conducted which found that flies can be potential effective vectors for the spread of E. coli O157:H7 within the environment, as well as to humans (5).

Thus, to be persistent in various environments, it is essential for E. coli O157:H7 to adapt to variations in temperature, pH, and osmolarity conditions. E. coli O157:H7 is able to survive for up to 10 months in aquatic environments that are nutrient deficient. During growth under nutrient-sufficient conditions (i.e. animal feces or manure), the bacteria accumulate reserve carbon sources that are stored for use in nutrient-poor environments (13). When nutrient conditions in aquatic habitats are unfavorable, E. coli O157:H7 can reduce cell size, thus increasing its surface/volume ratio to allow more efficient uptake of poorly available nutrients. This physiological state resulting from an insufficient amount of nutrients is known as the starvation-survival state (14). In this state, E. coli activates enzymes required to catabolize available nutrients (15). Additionally, E. coli may increase the production of toxins or antibiotics to promote killing other cells in the environment in order to decrease competition (16, 17). Finally, the bacterium switches to a survival state, which enhances its resistance to many stresses and its ability to remain viable during long periods without nutrients (18).

The exopolysaccharide (EPS) of E. coli O157:H7 is associated with heat and acid tolerance. The lipid composition of membranes can also be altered in response to heat stress to better adapt to the changes in temperature (12). These all contribute to its ability to survive in harsh environments.

Host location:

E. coli O157:H7 resides within the intestinal cellular walls and gut of the host as a commensal or pathogenic bacteria. The characteristics of E. coli O157:H7 allows it to effectively utilizes the fimbriae of intestinal cellular walls and attaches onto the microvilli of intestinal epithelial cells, via producing attaching and effacing (AE) lesions (2, 5). Once ingested, the bacteria will bind to intestinal mucosa and begin shedding or releasing Shiga toxin (2).

Once E. coli O157:H7 enters the host through digestion, one of the first antibacterial barriers the bacteria will encounter is the low pH of the stomach and stomach acid of the host. Thus, the bacterium must be highly resistant to acidic environments. The bacterium expresses an acid resistance (AR) system 1, which utilizes a rpoS sigma factor and F1F0 ATPase, within cells to allow them to survive in acidic environments (8). It’s been found that rpoS plays a critical role for regulating biofilm formation so that it can better adhere to surfaces and contribute towards transition of motile cells during environmental stress (8). Other AR mechanisms consist of decarboxylase and antiporter systems to assist with the exportation of protons out of cells so that intracellular pH increases (8). Ultimately, these characteristics of E. coli O157:H7 allows it to effectively reside within the gastrointestinal tract of hosts.

Figure 2: E. coli O157:H7 within the digestive tract of cattle, acquired from the environment

In the cattle intestine, E. coli acquires nutrients from the intestinal mucus. It upregulates genes that encode enzymes involved in the catabolism of N-acetylglucosamine, sialic acid, glucosamine, gluconate, arabinose and fucose (19). Decreased ability to colonize the intestine are observed when there are mutations in pathways that utilize galactose, hexuronates, mannose, and ribose (20). It has also been observed that multiple mutations in a single strain had an additive effect on colonization levels, suggesting that some E. coli strains depend on the simultaneous metabolism of up to six sugars to ensure colonization of the intestine (20). These findings suggest that E. coli uses multiple limiting sugars for growth in the intestine and suggest that E. coli grows in the intestine using simple sugars released upon breakdown of complex polysaccharides by anaerobic gut residents (21).

During the butchering process, E. coli sometimes gets onto the surface of the meat. Whole cuts of meat such as steaks or roasts usually only have E. coli on the surface, which makes the E. coli easier to kill by cooking. When the meat is ground or mechanically tenderized, E. coli on the surface can be transferred to the inside of the meat. This is why ground meat and mechanically tenderized meat are more likely to cause illness than whole cuts of meat. E. coli can be killed if the meat is cooked thoroughly (22). In this particular case and according to information provided by Ronnie's parents, our patient Ronnie most likely may have come into contact with E. coli O157:H7 through the contaminated, undercooked, or raw burger meat which he had at his neighbor's barbecue. This being said, it may be possible that contamination of other food via improper handling or hand washing is responsible for Ronnie's symptoms.

References: 1. E. coli [Internet]. Who.int. [cited 2022 Mar 12]. Available from: https://www.who.int/news-room/fact-sheets/detail/e-coli

2. Ameer MA, Wasey A, Salen P. Escherichia Coli (E Coli 0157 H7). In: StatPearls [Internet]. StatPearls Publishing; 2021.

3. Questions and answers [Internet]. Cdc.gov. 2019 [cited 2022 Mar 12]. Available from: https://www.cdc.gov/ecoli/general/index.html

4. Escherichia coli, Diarrheagenic - Chapter 4 - 2020 Yellow Book [Internet]. Cdc.gov. [cited 2022 Mar 12]. Available from: https://wwwnc.cdc.gov/travel/yellowbook/2020/travel-related-infectious-diseases/escherichia-coli-diarrheagenic

5. Bach SJ, McAllister TA, Veira DM, Gannon VPJ, Holley RA. Transmission and control of Escherichia coli O157:H7 — A review. Can J Anim Sci [Internet]. 2002;82(4):475–90. Available from: http://dx.doi.org/10.4141/a02-021

6. Belk KE. Managing pathogen contamination on the farm. In: Improving the Safety of Fresh Meat. Elsevier; 2005. p. 214–27.

7. Rahal EA, Kazzi N, Nassar FJ, Matar GM. Escherichia coli O157:H7-Clinical aspects and novel treatment approaches. Front Cell Infect Microbiol [Internet]. 2012;2:138. Available from: http://dx.doi.org/10.3389/fcimb.2012.00138

8. Kay KL, Breidt F, Fratamico PM, Baranzoni GM, Kim G-H, Grunden AM, et al. Escherichia coli O157:H7 Acid Sensitivity Correlates with Flocculation Phenotype during Nutrient Limitation. Front Microbiol [Internet]. 2017;8:1404. Available from: http://dx.doi.org/10.3389/fmicb.2017.01404

9. Dunn J, Keen J, Thompson R. Prevalence of shiga-toxigenicEscherichia coliO157:H7 in adult dairy cattle. Journal of the American Veterinary Medical Association. 2004;224(7):1151-1158.

10. Jiang X, Morgan J, Doyle M. Fate of Escherichia coli O157:H7 in Manure-Amended Soil. Applied and Environmental Microbiology. 2002;68(5):2605-2609.

11. LeJeune J, Besser T, Hancock D. Cattle Water Troughs as Reservoirs of Escherichia coli O157. Applied and Environmental Microbiology. 2001;67(7):3053-3057.

12. Jiang X, Morgan J, Doyle MP. Fate of Escherichia coli O157:H7 in manure-amended soil. Appl Environ Microbiol [Internet]. 2002 [cited 2022 Mar 12];68(5):2605–9. Available from: https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC127522/

13. Morita R. Bacteria in oligotrophic environments. New York: Chapman & Hall; 1997.

14. Burgess G. Bacteria in Oligotrophic Environments: Starvation Survival Lifestyle. World Journal of Microbiology and Biotechnology. 1997;14(2):305-305.

15. Tao H, Bausch C, Richmond C, Blattner F, Conway T. Functional Genomics: Expression Analysis of Escherichia coli Growing on Minimal and Rich Media. Journal of Bacteriology. 1999;181(20):6425-6440.

16. Miller J, Mekalanos J, Falkow S. Coordinate Regulation and Sensory Transduction in the Control of Bacterial Virulence. Science. 1989;243(4893):916-922.

17. Martín J. Phosphate Control of the Biosynthesis of Antibiotics and Other Secondary Metabolites Is Mediated by the PhoR-PhoP System: an Unfinished Story. Journal of Bacteriology. 2004;186(16):5197-5201.

18. Siegele D, Kolter R. Life after log. Journal of Bacteriology. 1992;174(2):345-348.

19. Chang DE, Smalley DJ, Tucker DL, Leatham MP, Norris WE, Stevenson SJ, Anderson AB, Grissom JE, Laux DC, Cohen PS, et al. Carbon nutrition of Escherichia coli in the mouse intestine. Proc Natl Acad Sci U S A. 2004;101:7427–7432

20. Fabich AJ, Jones SA, Chowdhury FZ, Cernosek A, Anderson A, Smalley D, McHargue JW, Hightower GA, Smith JT, Autieri SM, et al. Comparison of carbon nutrition for pathogenic and commensal Escherichia coli strains in the mouse intestine. Infect Immun. 2008;76:1143–1152.

21. Martens EC, Roth R, Heuser JE, Gordon JI. Coordinate regulation of glycan degradation and polysaccharide capsule biosynthesis by a prominent human gut symbiont. J Biol Chem. 2009;284:18445–18457.

22. HealthLink BC. E. coli Infection. BC Center for Disease Control. 2022. Retrieved 16 March 2022, from https://www.healthlinkbc.ca/healthlinkbc-files/e-coli-infection

ii) Entry: how does the bacteria enter into the human host and take up residence. What are the molecular, cellular and/or physiological factors at play in this site specificity, and in the initial adherence step, (referencing both the bacteria and the host).

After consuming the uncooked burgers, the food travelled down Ronnie’s digestive tract and entered his intestine where it began to produce the toxins. Prior to the intestinal adhesion, E. coli entered the stomach, which has a median pH of around 2.0. Acid resistance (AR) is an important property of E. coli, allowing the organism to survive gastric acidity and volatile fatty acids produced as a result of fermentation in the intestine. The ability to resist these acid stresses is likely one of the reasons this organism can colonize and establish a commensal relationship with mammalian hosts (1). There are three systems that protect the bacterial cells against pH 2 to 2.5 (1). The first is dependent on the alternative sigma factor ςS, encoded by the gene rpoS, and the second and third are clearly defined systems. One system requires glutamic acid in an acidic environment to survive pH 2.0 and is assumed to use an inducible glutamate decarboxylase, and the other requires arginine and an inducible arginine decarboxylase encoded by 6adiA (1). These decarboxylase systems are believed to consume protons during the decarboxylation of glutamate or arginine. The end products, γ-aminobutyric acid (GABA, formed from glutamate decarboxylase [GAD]) and agmatine (formed from arginine decarboxylase), are then transported out of the cell in exchange for the new substrate. This transport process is catalyzed by specific antiporter systems, GadC for glutamate and an unknown antiporter for arginine (1). The result is that protons leaking into the cell during acid stress are consumed and excreted from the cell, thereby preventing the internal pH from decreasing to lethal levels (1).

Another reason why E. coli resists acidic conditions is through colonic acid secretion (CA). E. coli secretes a variety of EPS, including CA. E. coli O157:H7 cells are inactivated during acidic conditions (low pH) due to proton accumulation. CA is negatively charged, which may serve as a buffer by neutralizing protons at the cell surface. This prevents the positively charged protons from accumulating and penetrating cells. Without CA, cell surfaces carry a less negative charge and are less able to buffer the acidic environment. The protons will accumulate and can enter the bacteria, causing an imbalance of intracellular charge and ultimately causing cell death (2).

At the level of the intestines, another host barrier the bacteria will need to face is bile produced by the gallbladder, released into the intestine at the duodenum, which acts to break down the cell membrane of bacterial cells (3). Bile can also cause oxidative stress and result in the damage of DNA and proteins (3). Thus, it is important that bacteria such as E. coli O157:H7 has developed adaptations involving membrane structures to reduce the membrane permeability so that the influx of bile is reduced, and additional structures such as efflux pump to remove bile out of the cell (3). When bile is introduced, genes including arcA, arcB, arcR, micF, marA, marB, and marR, which are all involved with the regulation of ompF expression and bile efflux were shown to increase two-fold in E. coli O157:H7 cells (3). Additionally, the availability of oxygen may continuously change as the bacteria travels through the gastrointestinal tract and thus aerobic respiration is essential for the successful colonization of E. coli O157:H7 (3). Aerobic respiration essentially allows the bacteria to continue providing energy so that it can successfully grow and colonize within the host.

Since iron is a nutrient within the human host, which is not readily available for the bacteria, it is important for E. coli O157:H7 to obtain iron. Interestingly, it is found that the bacteria increase mRNA for 17 genes which play a role in the acquisition of iron once the bacteria are exposed to bile (3). These iron acquisition genes may play a beneficial role in allowing for the bacteria to grow when in the small intestine (3).

Luo, 2000. The bacterium/host-cell interface via proteins intimin and Tir (7).

Genetic studies have shown that the genes responsible for A/E lesions are due to the locus of enterocyte effacement (LEE) (4). The LEE is a pathogenicity island required for A/E lesions produced on epithelial cells of humans as it contains all the genes necessary for inducing the A/E lesions (5). The LEE is composed of at least 41 different genes organized into three major regions, a type III secretion system that exports effector molecules; an adhesion called intimin and its translocated receptor, Tir, which is translocated into the host cell membrane by the TTSS; and several secreted proteins (Esp) as a part of TTSS, which are important in modification of host cell signal transduction during the formation of A/E lesions (6). Once translocated, the Tir protein spans the host cell membrane, taking on a hairpin loop structure with both its N and C termini in the host cytoplasm and a central extracellular domain that binds intimin. In addition to serving as a receptor for intimin, the Tir protein is capable of interacting with host cytoskeletal and signalling components using its N- and C-terminal domains located in the host cell cytoplasm (6).

Castanie-Cornet M-P, Penfound TA, Smith D, Elliott JF, Foster JW. Control of acid resistance in Escherichia coli. J Bacteriol [Internet]. 1999 [cited 2022 Mar 17];181(11):3525–35. Available from: https://pubmed.ncbi.nlm.nih.gov/10348866/
Jordan KN, Oxford L, O’Byrne CP. Survival of low-pH stress by Escherichia coli O157:H7: correlation between alterations in the cell envelope and increased acid tolerance. Appl Environ Microbiol [Internet]. 1999 [cited 2022 Mar 17];65(7):3048–55. Available from: https://pubmed.ncbi.nlm.nih.gov/10388702/
Hamner S, McInnerney K, Williamson K, Franklin MJ, Ford TE. Bile salts affect expression of Escherichia coli O157:H7 genes for virulence and iron acquisition, and promote growth under iron limiting conditions. PLoS One [Internet]. 2013 [cited 2022 Mar 17];8(9):e74647. Available from: https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC3769235/
Lim JY, Yoon J, Hovde CJ. A brief overview of Escherichia coli O157:H7 and its plasmid O157. J Microbiol Biotechnol [Internet]. 2010 [cited 2022 Mar 17];20(1):5–14. Available from: https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC3645889/
DeVinney R, Stein M, Reinscheid D, Abe A, Ruschkowski S, Finlay BB. Enterohemorrhagic Escherichia coli O157:H7 produces Tir, which is translocated to the host cell membrane but is not tyrosine phosphorylated. Infect Immun [Internet]. 1999 [cited 2022 Mar 17];67(5):2389–98. Available from: https://pubmed.ncbi.nlm.nih.gov/10225900/
McWilliams BD, Torres AG. Enterohemorrhagic Escherichia coli adhesins. Microbiol Spectr [Internet]. 2014 [cited 2022 Mar 17];2(3). Available from: https://pubmed.ncbi.nlm.nih.gov/26103974/
Luo Y, Frey EA, Pfuetzner RA, Creagh AL, Knoechel DG, Haynes CA, et al. Crystal structure of enteropathogenic Escherichia coli intimin-receptor complex. Nature [Internet]. 2000 [cited 2022 Mar 18];405(6790):1073–7. Available from: https://www.nature.com/articles/35016618

iii) Multiplication and Spread: does the organism remain extracellular or do they enter into cells and what are the molecular and cellular determinants of these events. Do the bacteria remain at the entry site or do they spread beyond the initial site i.e. are there secondary sites of infection and why do the bacteria hone in on these particular secondary sites.

After E. coli O157:H7 establishes itself as a colony on the intestine surface, multiplication and spread begins. Further colonization along the large intestine is the first location of spread (1). After further activation, the Shiga toxins produced can also be absorbed through the intestinal epithelium and join the circulatory system. The pathogen does not only remain extracellularly, but actively invades the intestinal epithelial cells, pumping proteins and toxins into the body’s circulatory system (1).Though the organism does not appear to reproduce intracellularly, proteins secreted by the bacteria can be taken up by host cells [2]. E. coli has the ability to disrupt host cell processes after attachment, which is commonly done through these secreted proteins. Hijacking and altering host cell signaling pathways can result in coordinated host cell invasion, evasion of host immune responses, and efficient colonization, leading to illness [3]. In animals, especially in cattle, initial colonization of the large intestine often spreads to the rectum.

Shiga toxins (Stx) have a large role in the propagation process of E. coli . Shiga toxin components aid in cell binding, and after adhesion, Shiga toxins Stx 1 and Stx2 bind to enterocytes, the absorptive epithelial cells facing the lumen in small and large intestines (4, 1). The pentameric B portion of the Shiga toxin binds to the cellular glycolipid receptor globotriaosylceramide (Gb3) and globotriaosylceramide (Gb4) that is found on the plasma membrane of enterocytes, as well as various other cell types such as renal, aortic, and brain endothelial cells, mesangial cells, renal tubular and lung epithelial cells, cells of the monocytic lineage, polymorphonuclear cells, in addition to platelets and erythrocytes (5, 6). The monomeric A subunit of the Shiga toxin is endocytosed into the enterocyte and is transported to the rough endoplasmic reticulum by the Golgi apparatus (4). In the trans golgi network (TGN), the toxin is cleaved by the enzyme furin into the A1 and A2 subunits. From the TGN, the toxin is transported to the endoplasmic reticulum and then translocated into the cytoplasm. If toxin was not cleaved by furin, then the cytosolic enzyme caplain may cleave the molecule (7). A1 is a glycosidase that hydrolyzes a specific adenine-ribose bond in the ribosomal 28S RNA. The cleaved ribosomal 28S RNA is unable to allow aminoacyl-tRNA binding, ultimately inhibiting protein synthesis, resulting in cell death (8). Stx induces chemokine synthesis from intestinal epithelial cells. This produces interleukins including IL-8 and IL-1 as well as TNF. TNF and IL-1 activate the endothelium which leads to an increase in toxin receptor expression and thus increased sensitivity of the cell to Stx (6). Stx then enters the bloodstream as the intestinal epithelium has been breached. The capillary endothelial cells are exposed to the Shiga toxin and killed by the same mechanism as described above. Endothelial cell death initiates the activation and aggregation of platelets in an attempt to recover the damaged endothelium. This also initiates leukocyte adherence, cytokine secretion, and vasoconstriction. These responses all contribute to fibrin deposition and clot formation in the capillary and in the subendothelial tissue (8, 9). Evidence indicates that individuals or animals who lack Gb3 are unaffected by the Shiga toxin (10). Consequently, these toxins have been determined to also cause apoptosis of supporting cells such as inflammatory mediators (10).

In addition to immediate death, the uptaken Shiga toxins can also move across the intestinal epithelium without affecting cell function for further propagation into the circulatory system (11). It is through this proposed mechanism that researchers have hypothesized how Shiga toxins are able to affect the brain and kidneys beyond the initial location of infection (11). A secondary pathway that also causes circular uptake involves Shiga toxins causing CXC chemokine production in human endothelium and intestinal mucosal epithelial cells (11). This further promotes intestinal epithelial injury and increased Stx absorption into the systemic circulation by inducing PMN infiltration into the intestinal epithelium (12). Once the toxin reaches the bloodstream, it is common for the pathogen to head to either the kidneys or to the brain. Along the blood vessels, and into the kidney, the toxin starts to cause damage breaking down red blood cells and the vessels (11). In very rare cases does the E. coli continue to spread to the brain.

In addition to internal damage, sloughing caused by the Shiga toxins is also a form of bacterial propagation especially amongst animals (1). Infected animals who experience high amounts of diarrhea also expel a substantial amount of E. coli in their stool. This enables other organisms that may reside in close proximity to the infected host to also become infected with E. coli. In human hosts, hygiene is an important measure in place to ensure infected individuals do not contaminate communal apparatuses or expose other individuals.

Aside from Stx, E. coli O157:H7 also has the pO157 plasmid, which contains the hly operon that encodes enterohemolysin (13). This hemolysin allows E. coli O157:H7 to utilize the blood released into the intestine as a source of iron (14). Some strains of E. coli O157:H7 also have EspP, which is a protease from the family of serine protease autotransporters of Enterobacteriaceae (SPATE). EspP cleaves pepsin A and human coagulation factor V, contributing to hemorrhage into the intestinal tract (15). Additionally, EspP cleaves several complement system components. This protects the bacteria from immune system-mediated elimination (16).

Secondary Sites of Infection

The Shiga toxin (Stx) has systemic effects on vascular endothelial cells, resulting in vasculitis, and manifests most commonly as hemolytic uremic syndrome (HUS), then abdominal pain, and rarely, thrombotic thrombocytopenic purpura (17). HUS comprises acute renal failure and its consequential perturbation of fluid and electrolyte balance, hemolysis, disruption of the clotting cascade with thrombocytopenia, with the risk of stroke (18). Shiga toxin effects manifest not only in the kidneys, but in its most severe manifestations, can result in a diffuse vasculitic injury that affects multiple organ systems and result in organ failures (17).

Colonic vascular damage by Stx may allow lipopolysaccharide and other inflammatory mediators to gain access to the circulation, thus initiating the hemolytic–uremic syndrome (19). The bacteria, by transcytosis, can get through microfold cells to reach the submucosa within the colon to cause infection (20). Stx that are released by E. coli O157:H7 can also play a role in giving access to the submucosa due to disruptions of tight junctions (20). It is here in which the bacteria may be able to cause infection to the colon as Stx are uptake into resident macrophages and then released from dead macrophages as they escape phagocytosis to invade the basolateral side of colonocytes (20). T3SS effectors, such as IpaC, that work to cause actin polymerization and ruffle formation as well as recruit ARP2/3 complex by activating SRC kinases are secreted to aid in the process of bacterial entry (20). Additional effectors, such as IpaA, VirA, and IpgD, are also involved with destabilizing actin and microtubules so that the bacteria can invade phagosome and effectors such as IpaB, PiaC, IpaD, and IpaH allow the bacteria to escape phagosomes (20). Effectors such as IpgD allow the bacteria to avoid apoptosis, IpaB effector plays a role in mediating cell cycle rest through the targeting of an inhibitor of anaphase, while MAD2L2 and OspE are involved with preventing epithelial cell attachments via interacting with integrin-linked kinase (ILK) (20). Furthermore, there are four effectors which play an important role in allowing the evasion of innate immune responses so that the bacteria can persist within colonocytes. OspF and IpaH targets the nucleus to irreversibly dephosphorylate protein kinases important for nuclear factor-KB (NF-KB) regulated transcription genes and interferes with the expression of inflammatory cytokines by interacting with splicing factors respectively (20). OspG inhibits NF-KB activation and OspB plays a role in reducing interleukin-8 (IL-8) through recruitment of host factors to help remodel chromatin (20). Ultimately, it is with these mechanisms that allow the bacteria to persist, survive and maintain infection by reducing the host's inflammatory responses.

Polymorphonuclear leukocytes (PMN) have been suggested to be involved in the spread of Shiga-toxin-related injury by delivering Stx to critical sites, such as the kidneys (18). Though Stx would travel through the lungs prior to the kidneys, research shows that the damage to the lungs is not as severe as in the kidneys. This is likely due to the fact that many kidney cells express the Stx receptor and contain Stx-sensitive cells. Likewise, the high volume of blood flow and filtration rate of blood in kidneys increases the chance of Stx interaction with cells of the renal microvasculature and the filtration barrier. Finally, if the renal filtration barrier is damaged for any reason, Stx would then have access to interact with tubular epithelial cells of the nephron, thereby leading to further damage (21). Globotriaosylceramide (Gb3), the glycolipid that acts as the cellular receptor for Stx has been detected in various glomerular cell types, including endothelial, podocyte, and mesangial (21). Human podocytes have been shown to be very sensitive to Stx2, as well as renal microvascular endothelial cells, the latter of which also expresses a large amount of Gb3. The primary role of podocytes is to participate in blood filtration as it functions in the barrier along with the glomerular endothelium and basement membrane. Podocytes also provide VEGF in support of glomerular endothelium, but Stx2 has been shown to decrease VEGF production by human podocytes (21). Evidence also indicates that Gb3 is expressed by the proximal tubules (21).

Urinary tract infection may also occur, causing cystitis or eventually cause pyelonephritis in the kidneys when the bacteria is left untreated and spreads to these sites (20). This can occur through adhesion to the uroepithelium, in which most adhesins generate separate morphological features termed pili (or fimbriae), which are rod-like structures with a diameter of 5–10 nm that are not flagella [2]. Outer-membrane proteins like intimin (a bacterial adhesion molecule involved in intimate attachment of enteropathogenic and enterohemorrhagic E. coli to mammalian host cells [2]. The adhesion to the uroepithelium is mediated by fimbrial adhesin H (FimH) binding to glycosylated uroplakin Ia in the bladder (20). FimH can also bind to alpha-3 and beta-1 integrins at the site of invasion, and destabilization of microtubule can also occur to further assist with invasion into the urinary tract (420). Prostatitis in men and pelvic inflammatory disease (PID) in women may be secondary outcomes of UTI’s [22]. Finally, the bacteria may eventually reach the bloodstream and make its way further to other tissues of the host (23).

References

1. CDC. E. coli (Escherichia coli) [internet]. Centers for Disease Control and Prevention.2022.; Available from: https://www.cdc.gov/ecoli/general/index.html

2. Kaper JB, Nataro JP, Mobley HL. Pathogenic Escherichia coli. Nat Rev Microbiol [Internet]. 2004 [cited 2022 Mar 12];2(2):123–40. Available from: https://www.nature.com/articles/nrmicro818

3. Bhavsar AP, Guttman JA, Finlay BB. Manipulation of host-cell pathways by bacterial pathogens. Nature [Internet]. 2007 [cited 2022 Mar 12];449(7164):827–34. Available from: https://www.nature.com/articles/nature06247

4. Bhavsar AP, Guttman JA, Finlay BB. Manipulation of host-cell pathways by bacterial pathogens. Nature [Internet]. 2007 [cited 2022 Mar 12];449(7164):827–34. Available from: https://www.nature.com/articles/nature06247

5. Betz J, Bielaszewska M, Thies A, Humpf H-U, Dreisewerd K, Karch H, et al. Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: Differential Association with membrane lipid raft microdomains. Journal of Lipid Research. 2011;52(4):618–34.

6. Meyers KEC, Kaplan BS. Many cell types are shiga toxin targets. Kidney International. 2000;57(6):2650–1.

7. Hofmann, S. L. (1993). Southwestern Internal Medicine Conference: Shiga-like toxins in hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura. Am. J. Med. Sci. 306, 398–406.

8. Karmali M.A. Host and pathogen determinants of verocytotoxin-producing Escherichia coli-associated hemolytic uremic syndrome. Kidney Int Suppl. 2009;75(Suppl 112):S4–S7.

9. Alpers C.E. The kidney. In: Kumar V., Abbas A.K., Fausto N., editors. Robbins and Cotran pathologic basis of disease. 7th edition. Elsevier Saunders; Philadelphia: 2005. pp. 955–1021.

10. van de Kar NC, Monnens LA, Karmali MA, van Hinsbergh VW. Tumor necrosis factor and interleukin-1 induce expression of the verocytotoxin receptor globotriaosylceramide on human endothelial cells: implications for the pathogenesis of the hemolytic uremic syndrome. Blood. 1992 Dec 1;80(11):2755-64. PMID: 1333300.

11. Acheson DWK, Moore R, De Breucker S, Lincicome L, Jacewicz M, Skutelsky E and Keusch GT (1996). Translocation of Shiga toxin across polarized intestinal cells in tissue culture. Infection and Immunity 64: 3294–3300

12. Thorpe CM, Smith WE, Hurley BP and Acheson DWK (2001). Shiga toxins induce, superinduce and stabilize a variety of C-X-C chemokine mRNAs in intestinal epithelial cells, resulting in increased chemokine expression. Infection and Immunity 69: 6140–6147.

13. Schmidt H, Kernbach C, Karch H. Analysis of the EHEC hly operon and its location in the physical map of the large plasmid of enterohaemorrhagic escherichia coli O157:H7. Microbiology. 1996;142(4):907–14.

14. Mead PS, Griffin PM. Escherichia coli O157:H7. The Lancet. 1998;352(9135):1207–12.

15. Brunder W, Schmidt H, Karch H. ESPP, a novel extracellular serine protease of enterohaemorrhagic escherichia coli O157:H7 cleaves human coagulation factor V. Molecular Microbiology. 1997;24(4):767–78.

16. Orth D, Ehrlenbach S, Brockmeyer J, Khan AB, Huber G, Karch H, et al. ESPP, a serine protease of enterohemorrhagic escherichia coli , impairs complement activation by cleaving complement factors C3/C3b and C5. Infection and Immunity. 2010;78(10):4294–301.

17. Ameer MA, Wasey A, Salen P. Escherichia Coli (E Coli 0157 H7) [Updated 2021 Dec 29]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK507845/

18. Goldwater, P.N., Bettelheim, K.A. Treatment of enterohemorrhagic Escherichia coli (EHEC) infection and hemolytic uremic syndrome (HUS). BMC Med 10, 12 (2012). https://doi.org/10.1186/1741-7015-10-12

19. Boyce TG, Swerdlow DL, Griffin PM. Escherichia coli O157: H7 and the hemolytic–uremic syndrome. New England Journal of Medicine. 1995 Aug 10;333(6):364-8.

20. Croxen MA, Finlay BB. Molecular mechanisms of Escherichia coli pathogenicity. Nat Rev Microbiol [Internet]. 2010 [cited 2022 Mar 12];8(1):26–38. Available from: https://www.nature.com/articles/nrmicro2265

21. Obrig TG. Escherichia coli Shiga Toxin Mechanisms of Action in Renal Disease. Toxins. 2010; 2(12):2769-2794. https://doi.org/10.3390/toxins2122769

22. Urinary Tract Infections [Internet]. ucsfhealth.org. [cited 2022 Mar 12]. Available from: https://www.ucsfhealth.org/conditions/urinary-tract-infections

23. Abreu AG, Barbosa AS. How Escherichia coli Circumvent Complement-Mediated Killing. Front Immunol [Internet]. 2017;8:452. Available from: http://dx.doi.org/10.3389/fimmu.2017.00452

iv) Bacterial Damage: do the bacteria cause any direct damage to the host (or is the damage fully attributable to the host response, as indicated below) and, if so, what is the nature of the bacterial damage. Can it be linked to any of the signs and symptoms in this case? Can bacterial damage be paradoxically enhanced by giving antibiotics?

The clinical presentation of Escherichia coli O157:H7 infection is diarrhea that is with or without blood (1). Other symptoms often associated with diarrhea include abdominal cramping and fever, which are observed in Ronnie, as well as nausea, headache, and vomiting (1). Hemorrhagic diarrheal symptoms usually resolve after seven days. 85% of patients will have a spontaneous resolution. 15% of patients, often children, will develop systemic manifestations, usually hemolytic uremic syndrome (HUS) (2).

Most of the damage to the host is direct, attributed to the Shiga toxins of the E. coli O157:H7. Shiga toxin causes damage to the intestine by killing cells, as described above. Stx enters enterocytes expressing globotriaosylceramide (Gb3) and globotriaosylceramide (Gb4) via endocytosis (3). Within the Trans golgi network, the enzyme furin cleaves the toxin into its A1 and A2 subunits. The processed A1 fragment cleaves one adenine residue from the 28S RNA of the 60S ribosomal subunit, thus inhibiting protein translation and triggering the ribotoxic and endoplasmic reticulum stress responses (4). Ultimately, this leads to cell apoptosis through p38 mitogen-activated protein kinase (p38 MAPK) activation and other apoptotic pathways (4).

This leads to the sloughing off of intestinal mucosa cells, which results in hemorrhagic diarrhea observed (2). The Shiga toxin also has systemic effects on vascular endothelial cells, resulting in vasculitis, and manifests in HUS, abdominal pain, and in rare cases, thrombotic thrombocytopenic purpura (2).

Shiga toxins induce an increase in chemokine synthesis from intestinal epithelial cells. This augments host mucosal inflammatory responses with release of interleukins, such as IL-8 and IL-1, as well as Tumor Necrosis Factor (TNF), as known as pyrogens (5). These pyrogens can enter the organum vasculosum of the lamina terminalis (OVLT) within the anterior hypothalamus (3). Upon entry into the perivascular space of the OVL, cells are then stimulated to produce prostaglandin E2 which diffuses into the adjacent preoptic area to upturn the temperature set point and cause fever. Ultimately, the increased shift of the thermostatic set point will signal efferent nerves, especially sympathetic fibers innervating peripheral blood vessels, to initiate heat conservation (3). This is responsible for the fever observed.

The inflammatory response initiation due to the Shiga toxins also may lead to abdominal tenderness (2), as in the case of Ronnie. The abdominal discomfort may be more severe if there is hemorrhagic vasculitis. The inflammatory response also leads to leukocyte aggregation, apoptosis of the affected cells, platelet aggregation, microthrombi formation, hemolysis, and renal dysfunction. In more severe cases, E. coli O157:H7 infection can affect multiple organ systems and cause multiple organ failures (6).

HUS displays the classical triad of microangiopathic hemolytic anemia (fragmented RBCs on blood film), thrombocytopenia, and renal failure (7). Thrombotic thrombocytopenia purpura (TTP) is even rarer than HUS and mainly affects the adult population. In TTP, there is less renal damage and there are fewer diarrhea cases. Both HUS and TTP are associated with neurological abnormalities such as seizures, coma, and hemiparesis. These two conditions are not always differentiated and may be referred to as HUS/TTP (8). The colon and kidney are usually affected but other organs such as the brain, pancreas, heart, and lungs may also be affected (9). The brain may present with CNS dysfunction in around 33% of the HUS cases and seizures are observed in <20% of pediatric patients. The pancreas may present with abnormalities that lead to diabetes mellitus, pancreatitis, and rarely, exocrine dysfunction (7).

HUS affects the kidneys and affects blood clotting capabilities of infection individuals by destroying red blood cells and platelets (10). The toxin is also able to destroy and deteriorate existing blood vessels. The breakdown of these structures creates additional filtrate load on the glomeruli which become clogged with platelets and damaged red blood cells (11). This then leads to filtration problems within renal cells causing build up of waste products. If prolonged and developed to an extreme degree, this phenomenon can lead to kidney failure (11). The usual symptoms associated with these conditions include bloody diarrhea/stool, stomach pain, fever and vomiting. Once the Kidney is severely affected, individuals may experience edema, albuminuria, decreased urine output, hypoalbuminemia and blood in the urine (12). These symptoms can also be related to those experienced by Ronnie (specifically abdominal cramps and fever) however, in most cases further testing is needed to confirm diagnosis of HUS. Treatment options for HUS usually involve hospitalization and in extreme cases dialysis (12). Blood transfusions and special diets may also be employed. Children under the age of 5 are particularly susceptible for developing HUS due to a weak immune system, but due to his older age, Ronnie does not seem to be at high risk (11).

Apart from Shiga toxins, other bacterial products like lipopolysaccharide (LPS) can cause both direct and indirect damage. LPS can by itself also damage endothelial cells, increase TNF levels, activate platelets, and induce the blood coagulation cascade. It may also lead to an increase in the levels of interleukins such as IL-8, which can activate white blood cells (WBCs). The host WBCs can then cause damage to the host by producing tissue-damaging enzymes such as elastase (13).

Since the infection causes prolific diarrhea, E. coli treatment mostly consists of rest and plenty of hydration. It is not recommended to treat E. coli O157:H7 infections with antibiotics. Antibiotics may trigger an SOS-response and initiate the lytic cycle of bacteriophages (14). Antibiotic-induced injury to the bacterial membrane would lead to the release of large amounts of toxins. Antibiotic use is also observed to lead to more HUS cases as the bacterial motility is decreased due to antibiotics, which would expose the intestines to the toxins for a longer time. This would increase the symptoms observed and the risk of HUS is increased by 17-fold (15). Antibiotic use is only recommended in cases of sepsis.

References

Nataro J.P., Bopp C.S., Fields P.I. Escherichia, Shigella, and Salmonella. In: Murray P.R., Baron E.J., Jorgensen J.H., editors. Manual of clinical microbiology. 9th edition. American Society for Microbiology; Washington, DC: 2007. pp. 670–687.
Ameer MA, Wasey A, Salen P. Escherichia Coli (E Coli 0157 H7) [Updated 2021 Dec 29]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK507845/
Rahal EA, Kazzi N, Nassar FJ, Matar GM. Escherichia coli O157: H7—Clinical aspects and novel treatment approaches. Frontiers in Cellular and Infection Microbiology. 2012 Nov 15;2:138.
Joseph A, Cointe A, Mariani Kurkdjian P, Rafat C, Hertig A. Shiga Toxin-Associated Hemolytic Uremic Syndrome: A Narrative Review. Toxins. 2020; 12(2):67. https://doi.org/10.3390/toxins12020067
El-Radhi A. S. (2019). Pathogenesis of Fever. Clinical Manual of Fever in Children, 53–68. https://doi.org/10.1007/978-3-319-92336-9_3
Raji MA, Jiwa SF, Minga MU, Gwakisa PS. Escherichia coli 0157: H7 reservoir, transmission, diagnosis and the African situation: a review. East Afr Med J. 2003 May;80(5):271-6.
Gianantonio CA. Hemolytic uremic syndrome. Acute Renal Failure. 1984;327–39.
Nauschuetz, W. Emerging foodborne pathogens: enterohemorrhagic Escherichia coli. Clin. Lab. Sci. 1998; 11:298–304.
Siegler, R. L. The hemolytic uremic syndrome. Pediatr. Clin. North Am. 1995; 42: 1505–1529.
Melton-Celsa A., Shiga Toxin (Stx) Classification, Structure, and Function. American Society For Microbiology. 2014.; Available from: https://journals.asm.org/doi/10.1128/microbiolspec.EHEC-0024-2013
Hemolytic Uremic Syndrome (HUS). National Kidney Foundation. 2015.; Available from: https://www.kidney.org/atoz/content/hemolytic
Hemolytic Uremic Syndrome in Children. National Institute of Diabetes and Digestive and Kidney Diseases. 2015.; Available from: https://www.niddk.nih.gov/health-information/kidney-disease/children/hemolytic-uremic-syndrome
Meyers KEC, Kaplan BS. Many cell types are shiga toxin targets. Kidney International. 2000;57(6):2650–1.
Corogeanu D, Willmes R, Wolke M, Plum G, Utermöhlen O, Krönke M. Therapeutic concentrations of antibiotics inhibit shiga toxin release from enterohemorrhagic E. coli O104:H4 from the 2011 German outbreak. BMC Microbiology. 2012;12(1).
Audrey J Tan DO. Hemolytic uremic syndrome in emergency medicine treatment & management: Emergency department care, consultations [Internet]. Hemolytic Uremic Syndrome in Emergency Medicine Treatment & Management: Emergency Department Care, Consultations. Medscape; 2021 [cited 2022Mar11]. Available from: https://emedicine.medscape.com/article/779218-treatment

The Host Immune Response

i) Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection.

Fig 1. TLR 4 and TLR5 binding to EHEC flagellin and activating a downstream signalling pathway Hajam IA, Dar PA, Shahnawaz I, Jaume JC, Lee JH. Bacterial flagellin—a potent immunomodulatory agent. Exp Mol Med. 2017 Sep;49(9):e373–e373.

The strain of bacteria the patient is infected with is known as enterohemorrhagic Escherichia coli (EHEC), which can be ingested orally where it will infiltrate the host in the GI tract, specifically the small intestine. Escherichia coli is a gram-negative bacillus that belongs to a family of bacteria known as Enterobacteriaceae (1). E. coli 0157:H7, in particular, is a strain of E. coli that produces Shiga-like toxin and results in an infection that is associated with a variety of clinical illnesses, including hemorrhagic colitis (2). Therefore, the first line of defence is gastric acidity; ideally, the pH of the gastric acid will denature and kill the bacteria(1). As well, the small intestinal peristalsis and population of normal flora bacteria in the large intestine contribute as nonspecific defences against infection (1). If the infection persists, there will be an immune response divided into two categories: i) innate or nonspecific immunity, which consists of pre-existent mechanisms including the natural barriers and secretions; and ii) adaptive or specific immunity, which is targeted against a previously recognized specific microorganism or antigen (3). Innate immunity is the host’s first line of defence and is intended to prevent infection and attack the invading pathogens(3). This nonspecific mechanism is fast (minutes to hours) while the adaptive response takes longer (days to weeks) (3).

Innate Immune Response:

The primary site of infection is the attachment of the E.coli to the mucosal surface epithelium lining of the gastrointestinal (GI) tract. Once the bacterium begins to invade the lining, the innate immune system recognizes the pathogen (4). The innate immune response is the first line of host defence against invading microbial pathogens after the nonspecific anatomical defences. The innate response will recognize pathogen-associated molecular patterns (PAMPs) by their pathogen recognition receptors (PRRs) expressed on the surface or within immune cells (4). The first PRR to recognize the pathogen is Toll-like-receptor 5 (TLR5), which recognizes the flagellin of the EHEC, TLR4 also recognizes the liposaccharide (LPS) on the bacterial capsule as well PRRs could also recognize peptidoglycan or CpG DNA to initiate an inflammatory response(4). Upon binding to a PAMP, TLRs will dimerize and undergo a conformational change to recruit TIR-domain-containing adaptor protein-inducing IFN-beta (TRIF) and myD88. This activates signalling pathways which initiate the production of pro-inflammatory cytokines, particularly Type 1 interferon (IFN), to activate the innate immune inflammatory response(4).

Chemokines and Cytokines

The cytokines and chemokines produced during the innate immune response include TNF-alpha, IL-1beta, IL-4, IL-6, IL-8, IL-12(5). The inflammatory cytokines TNF-alpha, IL-1, IL-6 released will induce the low-grade fever that the patient, Ronnie, is experiencing as a side effect of the pro-inflammatory response to the pathogen (6). This is due to the cytokines having an impact on the thermoreceptors in the thalamus that will increase temperature (6). The aforementioned cytokines can also be used to produce more complement, C-reactive protein, and to induce the expression of adhesion molecules on the vascular epithelium to promote diapedesis of immune effector cells to the area of inflammation (7).

The TNF-alpha is produced rapidly during infection and is a potent activator of macrophages and neutrophils, which was found in a study to play a significant role in limiting bacterial infection and spread(5).

The chemokine IL-8 is produced by the epithelial cells experiencing the infection. IL-8 acts as a chemoattractant for granulocytes, specifically neutrophils, that will be recruited to the site of infection with the corresponding CXCL8. The IL-8 will cause a conformational change in the LFA-1 ligand and increase its affinity to bind to ICAM-1 on the granulocytes(5)

The intestinal epithelial cells can also release antimicrobial peptides during innate immune response, including defensins and cathelicidins. Defensins and cathelicidins will bind to the LPS detected on the gram-negative bacterial surface of EHEC and inactivate its biological function (5).

The EHEC will adhere to the intestinal epithelial cells and induce the formation of rearranging the cytoskeleton, which will trigger the inflammatory response and the secretion of IL-8 from the intestinal epithelial cells, which will recruit the neutrophils into the area which are then followed by macrophages (5).

Fig 2: Neutrophil effector functions to fight off bacteria Source: Lu T, Kobayashi S, Quinn M, Deleo F. A NET outcome. Front Immunol. 2012 Dec 5;3:365.

Phagocytic Cells: Macrophage + Neutrophils

Phagocytic cells are essential to innate immunity and will be recruited to the area of infection to eliminate the pathogen before it begins to spread. Resident macrophages already in the tissue will be the first to sense the pathogen with their surface and endosomal TLRs that recognize the bacterial PAMP. The cytokines, IL-1Beta, TNF-alpha, G-CSF and chemokines IL-8, MIP-2 are secreted by the epithelial cells and macrophages, which recruit the neutrophils. Both macrophages and neutrophils have the ability to eliminate pathogens either directly by their PRRs or indirectly via opsonization. Direct recognition is done by phagocytosing the pathogen into a phagosome that fuses with an endosome which will eventually fuse with a lysosome making a phagolysosome. In the phagolysosome, there are oxidative and non-oxidative mechanisms to kill the microbe. Neutrophils kill the pathogen in the phagolysosome by assembling and activating the NADPH oxidase species that will generate reactive oxygen species (ROS), which are lethal. An example of a reactive species is hypochlorite which is a bactericidal oxidant that can also be released in the extracellular milieu by neutrophil extensions to kill bacteria, which can cause host damage.

However, non-oxidative mechanisms are used in macrophages, where they will acidify the phagolysosome by using antimicrobial peptides and various enzymes which will also kill the bacteria (5). IL-8 is made from NF-kB and MAPK signalling which is triggered by the release of pro-inflammatory cytokines and TLR Ligands, which will cause downstream signalling of promoting expression of IL-8 and multiple other cytokine genes(4)

Macrophages are also known the sense type III secretion system (T3SS) activity, which is a virulence factor of the bacteria, to activate the NLRC4 inflammasome to release cytokines IL-1B, Il-18 to cause pyroptosis, cell death that causes inflammation (8).

Mast Cells

In the tissues of the intestinal wall there are resident mast cells that interface between the environment and the host. Mast cells will secrete heparin, histamine, and serotonin when they sense there is a pathogenic invasion into the tissues (5). The release of histamine will stimulate the release of TNF-alpha and IL-10 from the resident macrophages as well as proinflammatory lipids produced by mast cells which will increase vascular permeability (5). This allows immune effector cells to squeeze through the endothelial lining to enter the site of inflammation to fight off the pathogen.

NF-κB Pathway

Fig 3. NF-kB and MAPK pathway signalling diagram. Source: Wu X, Schauss A. Mitigation of Inflammation with Foods. J Agric Food Chem. 2012 Apr 2;60.

The most crucial factor for initiation of IL-8 gene expression in the host is activation of the key transcriptional regulator of innate immune signaling, nuclear factor-κB (NF-κB) (4). NF-κB proteins are transcription factors that control gene expression during inflammation and are activated rapidly in response to various stimuli, including pathogens, stress signals, and proinflammatory cytokines such as TNF and IL-1β (4). There are five members of the NF-κB/Rel family of proteins: p65 (RelA), p50 (NF-κB1), p52 (NF-κB2), c-Rel, and RelB, which all share an N-terminal Rel homology domain that mediates DNA binding, dimerization, and nuclear translocation (4).

NF-κB activation is generally categorized as canonical, noncanonical, or alternative, depending on the stimuli (4). Canonical NF-κB signaling is representative of the general scheme of NF-κB signaling and is triggered by TLR ligands, proinflammatory cytokines, pathogens, and engagement of the T-cell receptor by antigen(4). Upon ligand recognition, a receptor such as TNFR1, IL-1R, or PRR triggers signaling events that result in activation of the IκB kinase (IKK) complex consisting of catalytic kinase subunits (IKKα and/or IKKβ) (4). Activated IKK phosphorylates NF-κB, followed by subsequent ubiquitylation and degradation of IκB by the host cell proteasome (4). In a resting cell IκB is bound to p50/p65 dimers, which are released upon proteasomal degradation of IκB and transported into the nucleus through the nuclear pore complex where they can upregulate expression of multiple cytokine genes, including IL8(4).

MAPK Pathway

In addition to NF-κB, the IL-8 promoter region contains binding sites for several other transcription factors, including NF-IL-6, AP-1, and AP-3 (4). In particular, AP-1-dependent IL-8 production occurs during EHEC infection in intestinal epithelial cells (4). AP-1 activation is regulated by mitogen-activated protein kinases (MAPK), which are highly conserved host proteins that play a central role in a number of cell responses, including regulation of cytokine expression, stress responses, and cytoskeletal reorganization (4).

The MAPKs comprise three subfamilies of serine/threonine kinases, including the extracellular signal-related protein kinases ERK and two stress-activated protein kinases, p38 and c-Jun amino-terminal kinase (JNK) (4,9). These kinases regulate cellular processes through phosphorylation of target protein substrates, including other protein kinases, phospholipases, transcription factors, and cytoskeletal proteins (4,9).

Complement

The complement system is activated by 3 pathways: classical, alternative, and lectin pathways (5). All the pathways will converge when the C3 convertase generation which will lead to the membrane attack complex (MAC)(9). The main goals of complement activation is the recruitment of inflammatory cells, opsonization, and the phagocytosis of the foreign particles (9). The activation of the complement pathway leads to generation of TCC that may result in either cell lysis or the production of C3a and C5a, which further contribute to the inflammatory response of accumulating leukocytes at the site of infection(9). There is also deposition of C3b from the convertase onto the bacterial pathogen and this results in the activation of the alternative pathway. In patients suffering from Hemolytic-Uremic Syndrome (HUS), there has been evidence showing elevated levels of complement products in the patient serum during the acute phase of disease(9). It has been shown that complement is not only a secondary effect due to the cell damage caused by shiga toxin, but that complement is induced by shiga toxin to have a more pronounced inflammatory response to promote the development of EHEC infection into HUS(9). It has been suggested that the shiga toxin promotes complement activation primarily through the alternative pathway.

Adaptive Immune System

The adaptive immune system is the last line of defence when the innate system cannot clear the infection. It can be split into two: humoral and cellular immune response. Immature dendritic cells are highly phagocytic and will phagocytose the bacteria in the site of infection and process it to present it on their major histocompatibility complex (MHC) I and MHC II proteins in order to activate a CD4+ T cell (10). The naïve CD4+ T cells are found in the draining secondary lymphatic tissues, which include mesenteric lymph nodes and Peyer’s patches (11). These responses lead to a robust antibody response to clear infection and reduce the severity of EHEC disease (11). Antigen-presenting cells (APC), including dendritic cells, will internalize the target via phagocytosis, pinocytosis or clathrin-mediated endocytosis. The manner in which the antigen is phagocytosed will determine which major histocompatibility complex (MHC) it will be displayed on for T cells to recognize (10). The dendritic cells will then migrate to the lymph nodes to activate the B and T cells.

MHC II receptors will display the exogenous antigens recognized and will activate CD4+ T helper cells. MHC I receptors will display endogenous antigens to activate CD8+ cytotoxic T cells(10). Some APCs, like the dendritic cells, can do cross-presentation, which is the presentation of exogenous antigens on the MHC I receptor to activate CD8+ T cells(10). The activation of the T cells is mediated through their TCR interacting with the MHC protein and scanning to see what the receptor is presenting.

In this type of infection, a model organism study was done with C.rodentium where it shares clinical pathogenic mechanisms to EHEC (10). It was found that the CD4+ T cells would differentiate into effector forms Th1, Th17, Th22 which are specific to the gastrointestinal tract (10).

Humoral Adaptive Immune Response

Fig 4. Portrayal of how dendritic cell (DC) activates naive CD4+ T cell to differentiate into effectors to release cytokines Source: Lepenies B, Lee J, Sonkaria S. Targeting C-type lectin receptors with mu... [Adv Drug Deliv Rev. 2013] - PubMed - NCBI. Adv Drug Deliv Rev. 2013 Aug 1;65:1271–81.

The humoral adaptive immune system is also known as the antibody-mediated immunity (12). Using the help from an activated helper T cell, the B cell can differentiate into plasma B cells that will produce a secreted version of their BCR, antibodies, that are specific against an antigen. This response is usually developed in response to a circulating pathogen, like EHEC, and can be present outside the infected cell (10). The antibody can bind to an epitope of the antigen and neutralize them or mark them for opsonization by a phagocytic cell (10). It was found this infection, EHEC O157:H7, stimulates IgA class of antibody (13). This type of antibody inhibits the adherence of the bacteria to cells and the secreted versions of this antibody, S-IgA, are transported to the mucosal surface. This is done by using receptor-mediated transport to get through the intestinal epithelial cells that can function to eliminate the pathogen in the lumen of the intestines preventing EHEC bacterial colonization (13).

IgA is a type of antibody that is commonly found in mucus, saliva, tears, blood and even breast milk(14). Its presence in breast milk provides neonates with passive immunity against certain bacteria(15). IgA is the most abundant antibody found in the human body, and it is a key component of mucosal immunity IgA exhibits antibacterial activity by forming dimers and neutralizing pro-inflammatory PAMPs of bacteria(14). Activated LPDCs promote the differentiation of T cells into effector T cells, such as CD4 helper T cells (Tfh) or CD8 cytotoxic T cells. CD4 helper T cells induce the differentiation of naïve B cells into IgA-secreting plasma cells and memory B cells(16). In fact, given the constant exposure of the gastrointestinal tract to foreign substances, the largest population of IgA-secreting plasma cells is found in the mucosa of the small intestine. Aside from isolated lymphoid follicles, IgA induction is commonly associated with other types of gut-associated lymphoid tissues (GALT) as well, such as Peyer’s patches (PPs), mesenteric lymph nodes (mLNs), and the cecal patch (17).

Cellular Adaptive Immune response

This aspect of adaptive immunity depicts the mechanism for the CD4+ T cells to differentiate into their effector forms after recognizing an MHC II presenting an antigen on the APC(10). Infection with this particular bacterium elicits the differentiation of the CD4+ T cells into Th1, Th17, Th22 (10). Th1 will increase expression of IFN-gamma (4). Th17 will secrete IL-17A, IL-17F, IL-22, IL-21 in mucosal infections(18). Th22 will secrete IL-22, IL-13 and TNF-a to assist in infection(19). The release of IL-17 cytokines will lead to further recruitment of neutrophils, and inflammation (18). The TNF-a released will contribute to Ronnie’s low-grade fever. The IL-22 produced will specifically act on the intestinal epithelial cells causing fucosylation and release of more antimicrobial peptides to help fight the bacteria (19).

References

1. Evans DJ, Evans DG. Escherichia Coli in Diarrheal Disease. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2022 Mar 11]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK7710/

2. Rahal E, Kazzi N, Nassar F, Matar G. Escherichia coli O157:H7—Clinical aspects and novel treatment approaches. Front Cell Infect Microbiol [Internet]. 2012 [cited 2022 Mar 17];2. Available from: https://www.frontiersin.org/article/10.3389/fcimb.2012.00138

3. Aristizábal B, González Á. Innate immune system [Internet]. Autoimmunity: From Bench to Bedside [Internet]. El Rosario University Press; 2013 [cited 2022 Mar 17]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK459455/

4. Pearson JS, Hartland EL. The Inflammatory Response during Enterohemorrhagic Escherichia coli Infection. Microbiol Spectr. 2014 Aug 15;2(4):2.4.11.

5. Basset C, Holton J, O’Mahony R, Roitt I. Innate immunity and pathogen–host interaction. Vaccine. 2003 Jun 1;21:S12–23.

6. Evans SS, Repasky EA, Fisher DT. Fever and the thermal regulation of immunity: the immune system feels the heat. Nat Rev Immunol. 2015 Jun;15(6):335–49.

7. Innate immunity and pathogen–host interaction - ScienceDirect [Internet]. [cited 2022 Mar 11]. Available from: https://www.sciencedirect.com/science/article/pii/S0264410X03001956?casa_token=sCu4WnBJ9XUAAAAA:GPFopdOIKMeY63A5RGhtftMJrBkG8lgnwRCIHPusXXpjmE_WnKiwlc9yTnyMhbKKL1FEoSqG

8. Puhar A, Sansonetti PJ. Type III secretion system. Curr Biol. 2014 Sep 8;24(17):R784–91.

9. Orth-Höller D, Würzner R. Role of Complement in Enterohemorrhagic Escherichia coli–Induced Hemolytic Uremic Syndrome. Semin Thromb Hemost. 2014 Jun;40(04):503–7.

10. Gaudino SJ, Kumar P. Cross-Talk Between Antigen Presenting Cells and T Cells Impacts Intestinal Homeostasis, Bacterial Infections, and Tumorigenesis. Front Immunol [Internet]. 2019 [cited 2022 Mar 11];10. Available from: https://www.frontiersin.org/article/10.3389/fimmu.2019.00360

11. Lebeis SL, Sherman MA, Kalman D. Protective and destructive innate immune responses to enteropathogenic Escherichia coli and related A/E pathogens. Future Microbiol. 2008 Jun;3(3):315–28.

12. Adaptive Immunity – Humoral and Cellular Immunity [Internet]. [cited 2022 Mar 11]. Available from: https://www.healio.com/hematology-oncology/learn-immuno-oncology/the-immune-system/adaptive-immunity-humoral-and-cellular-immunity

13. Nagano K, Taguchi K, Tokoro S, Tatsuno I, Mori H. Adhesion of Enterohemorrhagic Escherichia coli O157:H7 to the Intestinal Epithelia Is Essential for Inducing Secretory IgA Antibody Production in the Intestine of Mice. Biol Pharm Bull. 2014;37(3):409–16.

14. Dynamic interactions between bacteria and immune cells leading to intestinal IgA synthesis - ScienceDirect [Internet]. [cited 2022 Mar 17]. Available from: https://www.sciencedirect.com/science/article/pii/S1044532307001145?via%3Dihub

15. Riaz S, Steinsland H, Hanevik K. Human Mucosal IgA Immune Responses against Enterotoxigenic Escherichia coli. Pathogens. 2020 Aug 29;9(9):714.

16. Uematsu S, Fujimoto K, Jang MH, Yang B-G, Jung Y-J, Nishiyama M, et al. Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5. Nat Immunol. 2008 Jul;9(7):769–76.

17. Bunker JJ, Bendelac A. IgA Responses to Microbiota. Immunity. 2018 Aug 21;49(2):211–24.

18. Guglani L, Khader SA. Th17 cytokines in mucosal immunity and inflammation. Curr Opin HIV AIDS. 2010 Mar;5(2):120–7.

19. Gong J, Zhan H, Liang Y, He Q, Cui D. Role of Th22 Cells in Human Viral Diseases. Front Med [Internet]. 2021 [cited 2022 Mar 11];8. Available from: https://www.frontiersin.org/article/10.3389/fmed.2021.708140

ii) Host damage: what damage ensues to the host from the immune response?

Inflammation

Intestinal infection by EHEC results in the infiltration of immune cells to the site of infection and damage to the epithelial barrier (8). Moreover, recent clinical studies have shown elevated levels of proinflammatory cytokines, such as IL-8, TNF-a, and IL-1b, to be correlated to a greater likelihood of the individual developing HUS during an EHEC infection (8). As such, a majority of the host damage resulting from infection with EHEC can be attributed to the inflammation arising from activation of the innate immune response (8).

Figure 5. Response to gut epithelial barrier failure. Source: https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.13428

Upon infection with EHEC O157:H7, TLR-dependent signaling leads to the recruitment and activation of innate immune cells, specifically neutrophils and macrophages through chemokine release (8). In the case of intestinal mucosa, severe inflammation is followed by a loss of epithelial cells and a degradation of the extracellular matrix in the lamina propria, leading to ulcerations (2). Enzymes and mediators mainly secreted by monocytes, intestinal macrophages and granulocytes are responsible for this tissue damage (2). This continuous inflammation and tissue degradation may consequently lead to fibrosis and stricture formation (2). As well, a consequence is that luminal contents can also enter circulation, for instance, the damage on the wall results in more bacteria entering the tissues (3). Additionally, The bacterium has a myriad of effectors that inhibit NF-kB signaling and allow colonization of the bacteria in the intestinal tract, which can eventually spread to the kidneys and cause HUS (4).

Kidney Damage

Kidney damage can be induced via the inflammatory response by damaging small blood vessels in the kidney, leading to red blood cell breakdown and clot formation (5). This can be fatal due to the kidney’s blood filtration function (5). As well, there is dysregulation in the alternative complement pathway which leads to the endothelial cells being vulnerable to complement-mediated destruction causing microvascular thrombosis (6).

Additionally, microbicidal proteins, CC-chemokines and CXC-chemokines are released upon the degradation of platelets during HUS and cause proinflammatory and prothrombotic responses (13). This leads to increased inflammation in the kidney, causing further renal damage (13).This may also be due to leukocyte influx that causes increased chemoattractant levels such as IL-8 and granulocyte colony-stimulating factor (13).

Oxidative Stress

Figure 6. Cell response to oxidative stress. Source: https://www.scientificanimations.com/oxidative-stress-effects-risk-factors-managing-preventing/

In addition to causing host damage through the production of proinflammatory cytokines, neutrophils and macrophages also exacerbate host damage through the production of reactive oxygen species (ROS) (9). Oxidants are important contributors to mucosal, and submucosal tissue destruction (2). Oxygen metabolites, such as oxygen or hydroxide radicals, are produced in large amounts by infiltrating leucocytes in the inflamed mucosa (2). ROS are produced by complexes in the electron transport chain, dehydrogenases found in the mitochondrial matrix, as well as other enzymes such as NADPH oxidase (10,11). Activation of receptors on the surface of neutrophils and macrophages results in phagocytosis of pathogens, resulting in NADPH oxidase assembly at the phagosome membrane (10). NADPH oxidase reduces O2 to O2-, which then results in the production of other ROS (10). ROS release into the phagosome causes bacterial degradation (10). ROS can also be released into the extracellular environment in a process known as oxidative burst (11). Although ROS do play a role in the clearance of pathogenic bacteria by crossing the bacterial cell membrane and damaging nucleic acids and proteins, these molecules can also lead to cell and tissue injury and can even contribute to chronic inflammation (10). This occurs when ROS is overproduced by the mitochondria and NADPH oxidase without sufficient activation of antioxidant systems (10). This results in increased oxidative stress within the cell, which alters the cell’s redox status in favor of oxidation (10). Subsequent adverse events occur in the cell, such as lipid peroxidation of membrane phospholipids, oxidation of tyrosine, serine, and cysteine in proteins, DNA damage, and mitochondrial damage (10). As such, overproduction of ROS leads to cell death (10). ROS overproduction has also been implicated in neurodegenerative, cardiovascular, and metabolic (10).

Proteases

Influx of neutrophils to the site of infection leads to degranulation, resulting in the release of toxic substances such as proteases (12). Neutrophil-derived serine and matrix metalloproteases (MMPs) cleave molecules making up the extracellular matrix (i.e., elastin and collagen) and degrade receptors involved in the immune response (12). This feeds into a positive feedback loop that further promotes the influx of neutrophils to the site of infection, thus contributing to tissue injury given the proinflammatory capacity of neutrophils (12). In addition to this, matrix metalloproteinases, MMP-8 and MMP-9 in particular, result in the production of the tripeptide proline-glycine-proline, which acts as a chemokine, thus increasing the influx of neutrophils into the site of infection (12). This further contributes to cell and tissue damage (12).

Figure 7. Down-regulation of immune receptors by serine proteases from degranulated neutrophils. Source: https://ccforum.biomedcentral.com/articles/10.1186/s13054-016-1250-4

Complement

The complement system is a key innate immune defense against infection and an important driver of inflammation; however, these very properties can also cause harm (1). Inappropriate or uncontrolled activation of complement can cause local and/or systemic inflammation, tissue damage and disease (1). Thus, complement is tightly regulated by the fluid-phase regulatory proteins FH and FI and the membrane-bound regulators CD46, CD55, and CD59 (23).

Histamine

Histamine is released from mast cell degranulation under antigen stimulation and it causes vasodilation, increasing blood flow into the area and makes the vessel more hyperpermeable (7). These effects will increase the number of immune cells migrating to the area and make it easier for immune cells to squeeze through the endothelial lining to reach the area of infection (7). However, this leaky barrier can also allow bacterial pathogens such as EHEC to permeate, contributing to the spread of bacteria to the kidney which can develop into HUS (7).

Antimicrobial Peptide

The proteolytic and destructive properties of the cathepsins have a role in several chronic inflammatory diseases (2). Cathepsin family members are involved in the remodeling of ECM proteins under inflammatory conditions (2). The aspartic proteinase cathepsin D secreted by activated local macrophages or neutrophils has the potential to initiate a proteolytic cascade, and to degrade and remodel ECM (2).

References

Takano T, Elimam H, Cybulsky AV. Complement-mediated cellular injury. Seminars in Nephrology. 2013;33(6):586–601.
Rieder F, Brenmoehl J, Leeb S, Scholmerich J, Rogler G. Wound healing and fibrosis in intestinal disease. Gut. 2007;56(1):130–9.
Basset C, Holton J, O’Mahony R, Roitt I. Innate immunity and pathogen–host interaction. Vaccine. 2003;21.
Wan F, Weaver A, Gao X, Bern M, Hardwidge PR, Lenardo MJ. IKKβ phosphorylation regulates RPS3 nuclear translocation and NF-ΚB function during infection with escherichia coli strain O157:H7. Nature Immunology. 2011;12(4):335–43.
Hemolytic uremic syndrome (HUS) [Internet]. Mayo Clinic. Mayo Foundation for Medical Education and Research; 2021 [cited 2022Mar18]. Available from: https://www.mayoclinic.org/diseases-conditions/hemolytic-uremic-syndrome/symptoms-causes/syc-20352399
Tecklenborg J, Clayton D, Siebert S, Coley SM. The role of the immune system in kidney disease. Clinical and Experimental Immunology. 2018;192(2):142–50.
Ashina K, Tsubosaka Y, Nakamura T, Omori K, Kobayashi K, Hori M, et al. Histamine induces vascular hyperpermeability by increasing blood flow and endothelial barrier disruption in vivo. PLOS ONE. 2015;10(7).
Lebeis SL, Sherman MA, Kalman D. Protective and destructive innate immune responses to enteropathogenic escherichia coli and related A/E pathogens. Future Microbiology. 2008;3(3):315–28.
Kaplan MJ, Radic M. Neutrophil extracellular traps: Double-edged swords of innate immunity. The Journal of Immunology. 2012;189(6):2689–95.
Chelombitko MA. Role of reactive oxygen species in inflammation: A Minireview. Moscow University Biological Sciences Bulletin. 2018;73(4):199–202.
Ray PD, Huang B-W, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cellular Signalling. 2012;24(5):981–90.
Kruger P, Saffarzadeh M, Weber AN, Rieber N, Radsak M, von Bernuth H, et al. Neutrophils: Between host defense, Immune Modulation, and tissue injury. PLOS Pathogens. 2015;11(3).
Gear A, Camerini D. Platelet chemokines and chemokine receptors: Linking hemostasis, inflammation, and host defense. Microcirculation. 2003;10(3):335–50.

iii) Bacterial evasion: how does the bacteria attempt to evade these host response elements.

E. coli O157:H7 has developed many mechanisms to evade the host’s immune response. The complement system, in particular, is a crucial aspect of the innate immune response given its involvement in the recognition and subsequent destruction of pathogenic microbes (1). The complement system is composed of three different pathways: the alternative pathway, the classical pathway, and the lectin pathway (1). Following infection with E. coli O157:H7, all three pathways converge, resulting in the formation of a membrane attack complex (MAC) on the surface of the pathogenic microbe (1). Over time, pathogenic E. coli strains have developed a number of strategies to prevent complement-mediated bacteriolysis (1).

(i) Capsular Polysaccharides:

Capsular polysaccharides are important virulence factors that are usually involved in adhesion and resistance to the host immune response (1). In the case of E. coli, the K1 capsular polysaccharide promotes downregulation of the alternative complement pathway (1). It is able to accomplish this largely due to its composition. The K1 capsular polysaccharide in E. coli is made up of sialic acid (1). Complement factor H, a plasma glycoprotein that inhibits the alternative complement pathway, recognizes and binds to sialic acid (2). This results in an increase in factor H in the vicinity of pathogenic E. coli, which leads to downregulation of the alternative complement pathway (1). As a result, E. coli is protected from the formation of a MAC complex on its surface, and subsequent bacterial cell lysis (1).

(ii) Degradation of complement proteins by secreted proteases:

Complement evasion strategies of Escherichia coli

Enterohemorrhagic E. coli uses the serine protease activity of two autotransporters to inactivate proteins that are essential to the complement cascade (1). These autotransporters are called extracellular serine protease P (EspP) and protein involved in colonization (Pic) (1). EspP, a 105-kDa protease, is able to cleave pepsin A and human coagulation factor V to promote hemorrhagic colitis; however, in terms of the complement cascade, its main role is cleavage of C3/C3b AND C5 (1). Pic, on the other hand, is a 116-kDa protease that cleaves C3/C3b, C4/C4b, and C2 (1). By cleaving proteins essential to the complement system, EspP and Pic downregulate complement activation, thus decreasing complement-mediated bacteriolysis (1). In addition to this, E. coli O157:H7, specifically, secretes StcE, which is a metalloprotease that binds to C1-INH (C1 esterase inhibitor) and sequesters it onto the surface of its target cells (1). By doing so, StcE is able to downregulate complement activation (1).

(iii) Recruitment of negative complement regulatory proteins:

C4BP (C4 Binding Protein) is a soluble regulator that inhibits the classical and lectin pathways (1). Factor H, on the other hand, inhibits the alternative pathway (1). E. coli recruits these molecules. To further elaborate, OmpA (outer membrane protein A), which is found embedded into the outer membrane of E. coli, binds to the complement control protein domain 3 of the C4BP a-chain, resulting in decreased deposition of C3b onto the surface of E. coli, and consequently decreased MAC-mediated bacteriolysis (1). In addition to OmpA, new lipoprotein I (NlpI), which is embedded into the outer membrane of E. coli, also recruits negative complement regulatory proteins to the surface of E. coli (1). NlpI specifically recruits C4BP, thus preventing complement activation through the classical pathway (1). Lastly, OmpW (outer membrane protein W) binds factor H, thus downregulating complement activation through the alternative pathway (1).

Type III Secretion System (T3SS)

Enterohemorrhagic Escherichia coli interacts with the colonic epithelium through the type III secretion system (T3SS).

T3SS is a contact-dependent translocation of the bacterial protein into a host cell and is known to be a crucial virulence factor for EHEC(3). Depending on the environment, the T3SS can coordinate to the stage of infection for its activity, for instance changes in temperature, pH, oxygen tension, contact with host cells can affect the regulation of transcription of the T3SS genes(3). T3SS effectors in infected cells can subvert the immune response to ensure their survival by blocking the delivery of hydrolytic enzymes to bacteria-containing vacuoles, autophagosomal degradation, transcription and secretion of pro-inflammatory cytokines. Moreover, a crucial aspect of it is the ability to inhibit NF-kB and MAPK activation early in infection (3). Especially in macrophages, T3SS effectors can block inflammasome activation, phagolysosome degradation and phagocytosis. This type of mechanism can also protect the epithelial cells injected with TS33 from undergoing apoptosis to ensure the effectors are still being transcribed for evading the immune response for the bacteria’s survival (3).

Translocated Intrimin Receptor Protein (Tir) –T3SS

TAK1 is involved in many signalling pathways, that once activated it will cause the activation of cellular signalling cascades to lead to the translocation of transcription factors NF-kB and AP-1 into the nucleus to produce more pro-inflammatory cytokines (4). Tir is an EHEC effector protein that is injected into the plasma membrane of the host cells via the Type III Secretion System (T3SS) to act as a receptor for bacterial surface adhesion intimin (4). The Tir can recruit SHP-1 to inhibit the immune response by using SHP2 to inhibit TAK1 activity to downregulate the signal transduction pathways which will in turn downregulate cytokine production (4).

Tir can also suppress autophagy with cAMP independent PKA activation. Autophagy is a critical self-defence mechanism during infection, and it is the process of degrading intracellular proteins and organelles (5). Infection with this strain of E. coli caused a robust activation of PKA that is associated with a decrease in cAMP level as shown by this study (5). Therefore, this PKA pathway is used by these pathogens to promote their survival by negatively regulating the ERK signalling and ER stress that promotes the autophagy response(5). This is beneficial for the bacterium as if autophagy of the host cell is activated it impairs the adhesion of this strain of E. coli to intestinal epithelial cells

Non-locus of enterocyte effacement (LEE) encoded Proteins -T3SS

These non-LEE encoded effectors have been studied to show that these pathogens inject multiple of these effectors into the host cell that will target and repress host immune factors. Particularly they interfere with NF-kb and MAPK activation. The proteins include: NIeB, NIeC, NIeE and NIeH (6).

NF-kB are transcription factors that control the gene expression of IL-8 importantly during inflammation and are activated by pro-inflammatory cytokines such as TNF- α and IL-1β (6). Each of these proteins will target a different component of the pathway as NF-Kb refers to the collection of dimeric transcription factors for pro-inflammatory cytokine production (7).

NIeE is cysteine methyltransferase and one of the first effectors to inhibit NF-kB signalling NIeE has been found to block the TNF-induced IkB degradation and translocation of activated NF-kB into the nucleus of epithelial cells(6). It facilitates this inhibition using S-adenosyl-L-methionine-dependent methyltransferase activity that changes a cysteine residue on of the signal adaptor proteins TAB2 or TAB3 that is needed for NF-kB activation(6). This inhibition of NF-kB translocation and activation causes low levels of IL-8 production in the epithelial cells.

NIeC and NIeD are metalloprotease effectors and inhibitors of two signalling networks. NIeC and NIeD targets the NF-kB Rel Proteins for degradation which cleaves MAPK enzymes, JNK and p38. These two proteins will amplify the NIeE inhibition of IL-8 production(6).

NIeB is a glycosyltransferase that inhibits IkB degradation and NF-Kb activation only in response to TNF-- α (6). It does this by exhibiting GIcNAc transferase activity which allows GAPDH to be modified and prevent IkB degradation and p65 translocation that may potentially dampen inflammatory responses. There is still a lot unknown about this effector (6).

NIeH contributes to the suppression of NF-kB activation by allowing it to evade the immune response by directing the host immune enzyme IKK-beta and it allows the bacterium to survive longer and spread to other individuals(8).

Shiga Toxin (stx)

Mechanism of action of Shiga toxin (Stx). (1) Stx is internalized by endocytosis (2) Subsequently, Stx undergoes retrograde transport to the trans-Golgi network (TGN) (3) and then to the endoplasmic reticulum (ER) (4) In the ER, Stx encounters its target, the ribosome, inactivating it. As a consquence, Stx inhibits protein synthesis, causing cell death by apoptosis.

This type of toxin is the predominant cause of mortality within EHEC infection, as it is a potent cytotoxin that is produced by enteric pathogens. The production of stx is noted to be the link between EHEC and the development of HUS (9). Stx is an AB5 toxin, meaning it has a catalytic subunit A bound non-covalently to a pentamer B subunit. Stx has 5 identical B subunits that bind the toxin to the glycolipid globotriaosylceramide (Gb3) on the cell surface and the A subunit will cleave the ribosomal subunit to inhibit protein synthesis (9). Stx first enters circulation by being absorbed by the epithelium to gain access into the tissues with a goal to travel to the kidneys. Damage to the tissue caused by the bacterium can enhance this absorption of stx. The main goal of the toxin is to inhibit protein synthesis and induce apoptosis. It does this by binding to the target eukaryotic cell and being endocytosed (9). Stx will bypass the endocytic pathway and enter the trans golgi network and head to the endoplasmic reticulum where it will meet its target, the ribosome. Stx A subunit’s N-glycosidase activity cleaves the adenosine residue from 28S ribosomal RNA of the 60S ribosomal unit (9). If the ribosome cannot function then the proteins cannot be translated, which will lead to cell death.

There are different classes of Stx: Stx1 and Stx2, which consist of different variants that may differ in their biological activity and what receptors they may bind to. Regardless both classes share the same enzymatic activity and Stx2 is known to cause more severe disease than Stx1 (10).

Studies have shown that it also inhibits the expression of chemokine mRNAs CCL20 and IL-8 in human enterocytes by suppressing the PI3K/Akt/NF-kB signalling pathway meaning that Stx can be an indirect suppressor of gene transcription (11). In addition, another study showed that EHEC particularly can subvert the IFN- γ pathway in different human epithelial cell lines by inhibiting stat-1 tyrosine phosphorylation (12).

This toxin also damages the intestinal wall which is what causes the bloody diarrhea Ronnie is experiencing as a part of this infection because it causes the cells in the tissue to undergo apoptosis, damaging the lining that separates the outside from the inside of the body (12).

Inhibitory immunoreceptor tyrosine-based activation motif (ITAM) hijacking

By hijacking inhibitory immunoreceptor tyrosine-based activation motif (ITAM) signalling, EHEC are able to escape macrophage receptor recognition with collagenous structure (MARCO) dependent killing(13). With low affinity, the non-opsonized EHEC bind to FcγRIII (CD16) and induces weak phosphorylation of the Fc receptor common y chain (FcRy)(13). This leads to SHP-1 recruitments, in which SHP-1 dephosphorylates PI3k and halts MARCO-dependent phagocytosis of EHEC(13). Mice studies show that mice that are deficient in FcrRIII or FcRy have greater survival rates in sepsis model that is associated with their ability to clear E. coli from their body. This indicates that through FcγRIII-FcRγ signaling complex manipulation, E. coli can overcome the inflammatory response(14).

STAT-1 signal inhibition

EHEC secretes a currently unknown effector that inhibits STAT-1 signalling as an immunoevasive strategy(15). This inhibit cytokine production through altering receptor localisation in epithelial cell line and IFN-gamma. In-vitro studies have shown that many EHEC strains encode metalloprotease that cleaves the C1 esterase inhibitor, which is a classical complement cascade regulator(15).

StcE protease

EHEC secretes different effector proteins that cause lesions in the intestinal and endothelium lining as well as the leukocyte and complement protein infiltration to the gut lumen(16). Leukocyte surface glycoprotein CD45 and CD43 that consist of mucin type glycosylation are targeted by secreted protease of C1-esterase inhibitor (StcE) to provide EHEC a competitive advantage to other pathogens and allows EHEC to modulate the host immune system(16). For instance, sialoglycoprotein loss due to StcE proteolytic activity prevent immune cells from moving to the infection site and initiating an effective inflammatory response. Neutrophils showed decreased chemotactic mobility when treated with StcE(17). StcE also targets the completed system by inhibiting proteolytic activation of complement cascades. They do this by recruiting human C1-esterase inhibitor (C1-INH), an essential serine protease regulator in the complement pathway, to the host cell surface(17). StcE then localizes the complement regulator to the cell membrane through interaction with mucin-like N-terminus of C1-INH to inactivate downstream complement cascade components. Research show that this downstream component inhibition allows E. coli to be more resistant to complement-mediated cell lysis when in the presence of C1-INH treated with StcE(17).

EspB is a LEE (locus of enterocyte effacement)-encoded protein that is required for type III secretion, and is an effector protein that is translocated into host cells(18). In a EspB- dependent manner, EHEC can use Stx to suppress cytokine and bacteria induced NF-kB activation that is in response to Stx presence, which indicates that EspB may have a indirect or direct role in inflammation suppression(18). EHEC and Stx are shown to inhibit gamma interferon-mediated epithelial cell activation, which mediates the host immune response and promotes EHEC colonization(18).

The human digestive tract has a protective mucosal lining that contains mucin glycoproteins which acts as a major challenge for bacteria to come into contact with the surface. The lining separates the host epithelial cells from microorganisms in the lumen and have a dynamic outer layer and adherent inner layer that is constantly shed and renewed(19). Microorganisms avoid agglutination by salivary mucins and avoid removal by mucociliary action. They also penetrate the thick mucus layer by masking the epithelial cells before colonization occurs(19). Mucins are proteins that are densely glycosylated and have Pro/Thr/Ser (PTS)-rich domains. The “bottle-brush” like structure of the mucins, due to the high-density oligosaccharides projecting from the peptide backbone causes electrostatic repulsion and steric exclusion from negatively charged O-linked sugars(19). This provides protease resistance to mucin peptide backbone and prevents access to the host epithelium layer. EHEC can penetrate the mucosal lining and intimately adhere to the host cells. EHEC secretes StcE via the type II general secretory pathway during infection, which helps with the EHEC penetration of the mucosal lining(17). StcE plays a role in EHEC colonization by proteolytically unmasking host epithelial cell surface and to allow EHEC to intimately adhere to the epithelial cells(20). The zinc metalloprotease mediated mucinase activity of StcE towards specific mucin-type glycoproteins is associated with EHEC virulence. StcE degrades and lowers the mucus layer viscosity by mucin 7 and glycoprotein 340 cleavage, which is found in saliva and other host tissues(19). Mucin 7 and glycoprotein 340 are also part of the innate immune system as they act as receptor decoys for microbial adhesins to facilitate bacteria-protein aggregate clearance, in which destroying these molecules with StcE allow EHEC to overcome the mucosal barrier defense against bacterial adhesion to the epithelium(19).

Acid Resistance

Model of ClC chloride channel involvement in acid resistance.

Acid resistance (AR) is the ability of bacteria to protect themselves from extremely low pH (<pH 3.0) (21). The low pH in the stomach (pH 1.5 to 3.0) is one of the first host defenses against foodborne pathogens like EHEC (21). To colonize and cause a disease, enteric pathogens must overcome environmental challenges that include acid stress in the stomach as well as short-chain fatty-acid stress in the intestine (21, 22). Acid resistance is associated with a lowering of the infectious dose of enteric pathogens (21). The low infectious dose is one of the best known characteristics of EHEC O157:H7 (as low as 10 organisms), making this bacterium highly infectious and illustrating the importance of acid resistance in the pathogenesis of EHEC (21, 22).

E. coli becomes acid resistant upon entry into stationary phase (22). Stationary phase triggers at three AR mechanisms (21, 22. 23):

Acid-induced oxidative system
1. Requires alternate sigma factor RpoS, and cyclic AMP (21, 23).
Acid-induced arginine-dependent system
1. Requires the addition of arginine during acidic conditions, the structural gene for arginine decarboxylase (adiA), and the regulator of adiA, cysB (21, 23).
Glutamate-dependent system
1. The third AR system requires glutamate for protection at low pH conditions, one of two genes encoding glutamate decarboxylase (gadA or gadB), and the gene encoding the putative glutamate:γ-aminobutyric acid antiporter (gadC). Only one of the two glutamate decarboxylases is needed for protection at pH 2.5. However, survival at pH 2 requires both glutamate decarboxylase isozymes (21, 23).

Acid-induced oxidative system provides the least level of protection while the glutamate-dependent system provides the highest level of protection (22). The mechanisms of the acid-induced oxidative system are still unknown (22, 23). The two decarboxylase systems are believed to consume protons during the decarboxylation of glutamate or arginine (22, 23). The end products, γ-aminobutyric acid (GABA), are transported out of the cell (23). This transport process is catalyzed by specific antiporter systems, GadC for glutamate and an unknown antiporter for arginine (23). The result is that protons leaking into the cell during acid stress are consumed and excreted from the cell, thereby preventing the internal pH from decreasing to lethal levels (23).

Other than these three AR systems, several proteins involved in AR of E. coli O157:H7 have been identified (21). These proteins include chaperone HdeA, RNA polymerase-associated protein SspA, and DNA-binding protein Dps (21). Moreover, it was shown that alterations in the cell wall membrane or colonic acid production are associated with successful AR (21). Thus, E. coli O157:H7 utilizes different AR systems based on the type of acidic environment naturally encountered (21).

References

1. Abreu AG, Barbosa AS. How Escherichia coli Circumvent Complement-Mediated Killing. Front Immunol [Internet]. 2017 [cited 2022 Mar 12];8. Available from: https://www.frontiersin.org/article/10.3389/fimmu.2017.00452

2. Ferreira VP, Pangburn MK, Cortés C. Complement control protein factor H: the good, the bad, and the inadequate. Mol Immunol. 2010 Aug;47(13):2187–97.

3. Puhar A, Sansonetti PJ. Type III secretion system. Curr Biol. 2014 Sep 8;24(17):R784–91.

4. Zhou R, Chen Z, Hao D, Wang Y, Zhang Y, Yi X, et al. Enterohemorrhagic Escherichia coli Tir inhibits TAK1 activation and mediates immune evasion. Emerg Microbes Infect. 2019 Jan 1;8(1):734–48.

5. Xue Y, Du M, Sheng H, Hovde CJ, Zhu M-J. Escherichia coli O157:H7 suppresses host autophagy and promotes epithelial adhesion via Tir-mediated and cAMP-independent activation of protein kinase A. Cell Death Discov. 2017 Oct 2;3(1):1–11.

6. Pearson JS, Hartland EL. The Inflammatory Response during Enterohemorrhagic Escherichia coli Infection. Microbiol Spectr. 2014 Aug 15;2(4):2.4.11.

7. Vande Walle K, Vanrompay D, Cox E. Bovine innate and adaptive immune responses against Escherichia coli O157:H7 and vaccination strategies to reduce faecal shedding in ruminants. Vet Immunol Immunopathol. 2013 Mar 15;152(1–2):109–20.

8. How pathogenic E. coli bacterium causes illness [Internet]. ScienceDaily. [cited 2022 Mar 11]. Available from: https://www.sciencedaily.com/releases/2011/03/110314111222.htm

9. Kaper JB, Nataro JP, Mobley HLT. Pathogenic Escherichia coli. Nat Rev Microbiol. 2004 Feb;2(2):123–40.

10. Pacheco AR, Sperandio V. Shiga toxin in enterohemorrhagic E.coli: regulation and novel anti-virulence strategies. Front Cell Infect Microbiol. 2012 Jun 7;2:81.

11. Gobert AP, Vareille M, Glasser A-L, Hindré T, Sablet T de, Martin C. Shiga Toxin Produced by Enterohemorrhagic Escherichia coli Inhibits PI3K/NF-κB Signaling Pathway in Globotriaosylceramide-3-Negative Human Intestinal Epithelial Cells. J Immunol. 2007 Jun 15;178(12):8168–74.

12. Ho NK, Ossa JC, Silphaduang U, Johnson R, Johnson-Henry KC, Sherman PM. Enterohemorrhagic Escherichia coli O157:H7 Shiga Toxins Inhibit Gamma Interferon-Mediated Cellular Activation. Infect Immun. 2012 Jul;80(7):2307–15.

13. Avondt KV, Sorge NM van, Meyaard L. Bacterial Immune Evasion through Manipulation of Host Inhibitory Immune Signaling. PLOS Pathog. 2015 Mar 5;11(3):e1004644.

14. Pinheiro da Silva F, Aloulou M, Skurnik D, Benhamou M, Andremont A, Velasco IT, et al. CD16 promotes Escherichia coli sepsis through an FcR gamma inhibitory pathway that prevents phagocytosis and facilitates inflammation. Nat Med. 2007 Nov;13(11):1368–74.

15. Lebeis SL, Sherman MA, Kalman D. Protective and destructive innate immune responses to enteropathogenic Escherichia coli and related A/E pathogens. Future Microbiol. 2008 Jun 1;3(3):315–29.

16. Szabady RL, Lokuta MA, Walters KB, Huttenlocher A, Welch RA. Modulation of neutrophil function by a secreted mucinase of Escherichia coli O157:H7. PLoS Pathog. 2009 Feb;5(2):e1000320.

17. Pearson JS, Hartland EL. The Inflammatory Response during Enterohemorrhagic Escherichia coli Infection. Microbiol Spectr. 2014 Aug 15;2(4):2.4.11.

18. Paton JC, Paton AW. Pathogenesis and Diagnosis of Shiga Toxin-Producing Escherichia coli Infections. Clin Microbiol Rev. 1998 Jul;11(3):450–79.

19. Yu ACY, Worrall LJ, Strynadka NCJ. Structural Insight into the Bacterial Mucinase StcE Essential to Adhesion and Immune Evasion during Enterohemorrhagic E. coli Infection. Structure. 2012 Apr 4;20(4):707–17.

20. Pillai L, Sha J, Erova TE, Fadl AA, Khajanchi BK, Chopra AK. Molecular and Functional Characterization of a ToxR-Regulated Lipoprotein from a Clinical Isolate of Aeromonas hydrophila. Infect Immun. 2006 Jul;74(7):3742–55.

21. Lim JY, Yoon JW, Hovde CJ. A brief overview of escherichia coli O157:H7 and its plasmid O157. Journal of Microbiology and Biotechnology. 2010;20(1):5–14.

22. Castanie-Cornet M-P, Penfound TA, Smith D, Elliott JF, Foster JW. Control of acid resistance in escherichia coli. Journal of Bacteriology. 1999;181(11):3525–35.

23. Richard HT, Foster JW. Acid resistance in escherichia coli. Advances in Applied Microbiology. 2003;:167–86.

iv) Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections with the candidate infectious agent?

Outcome:

Most E. coli strains harmlessly colonize the gastrointestinal tract of humans and animals as a normal flora (1). However, serotype EHEC O157:H7 is a pathogenic E. coli that can produce Stx and cause disease, thus it is not normally found in the body (1). Gastrointestinal symptoms due to infection with E. coli O157:H7 usually resolve within a week without treatment (2).

However, children such as Ronnie, or even older adults they have a higher risk of developing HUS which is a life-threatening form of kidney failure (3). 5–10% of patients under the age of 10 develop HUS approximately one week after onset of hemorrhagic colitis (4). HUS most commonly occurs in children between 1 and 5 years of age but it can also occur in hospitalized patients over the age of 60 years (2). The release of Stx plays a central role in the development of HUS (4).

After the onset of the acute phase of HUS, characterized by the triad of hemolytic anemia, thrombocytopenia, and acute renal injury, the patient's clinical disease may follow one of several patterns (2). More than 95% of cases recover from the acute phase of the disease, thus the mortality rate is 5% (4). Grave sequelae, such as end-stage renal disease or permanent neurologic damage, occur in about 5% of subjects who survive the acute phase of HUS (2). A condition known as thrombotic thrombocytopenic purpura (TTP) strikes mostly the adult population and is rarer than HUS (2). In TTP less marked renal damage is noted and fewer cases have a diarrheal prodrome (2).

Progression of infection resulting from E. coli O157:H7

A study done by Rosales et al., 2012, found that most patients recover completely and have no symptoms even after a five-year follow up as well there is no impaired renal function. However, some patients that developed HUS during the course of infection may still present hypertension, impaired renal function, and neurological symptoms in the follow-up after five years (5). However, if appropriate treatment is done in time for HUS patients, then usually this leads to a full recovery (6).

There is currently no treatment available for EHEC infections (7). Studies show that conventional antibiotics exacerbate Stx-mediated cytotoxicity in the host, and that administration of antibiotics to EHEC patients increased the risk of developing HUS (8). Children taking antibiotic therapy for hemorrhagic colitis associated with EHEC have a greater chance of developing HUS (9). Stx production is also enhanced by antibiotics by increasing the expression and replication of stx genes encoded within a chromosomally integrated lambdoid prophage genome (10). The main form of HUS management is intensive supportive therapy to main homeostasis, such a peritoneal dialysis or hemodialysis, hypertension treatment and fluid balance (11).

Approximately 30% of EHEC infection survivors suffer from permanent disabilities. These include hypertension, neurological deficits, and chronic renal insufficiency (12). Furthermore, chronic renal abnormalities, such as decreased glomerular filtration rate, decreased urinary concentrating ability and proteinuria have manifestation after many years of supposed completed recovery (13). Patients can also have insulin-dependent diabetes mellitus after clearance of virus. There are also rare post infection gastrointestinal complications associated with EHEC (13). These include colonic stricture and biliary lithiasis secondary to pigment stones, likely precipitated during hemolysis in HUS. With or without HUS, patients may also have extended periods of crampy abdominal pain post EHEC infection (13). These abdominal pains may be causes residual disaccharidase deficiencies or colonic stricture (13).

Immunity:

Considering this pathogen stimulates a strong adaptive humoral response, there will be memory T and B cells that may recognize this pathogen if there were to be reinfection. In a study with a similar pathogenic E. coli, it was found that those who have previously experienced infection with the bacterium, had developed immunity that offered some protection in the case of reinfection with the bacterium, especially if it was for the same strain (14). This can also imply that infection with this strain O157:H7 can also provide some protection against other pathogenic strains of E. coli. It has been identified that the secreted form IgA plays an important role in reinfection as it can inhibit the attachment to the intestinal wall by binding to the bacterium (14).

In patients who had an EHEC infection developed a significant antibody response to O liposaccharide (O LPS), Tir and EspA, EspB and intimin which are essential EHEC virulence factors necessary for bacterial interactions with the host cells and disease (15). It was especially found that there were higher antibody titres of Tir visible in all patients. There was no variability in antibody responses from those who had infections that developed into HUS compared to those who did not (15).

References:

1. Lim JY, Yoon JW, Hovde CJ. A Brief Overview of Escherichia coli O157:H7 and Its Plasmid O157. J Microbiol Biotechnol. 2010 Jan;20(1):5–14.

2. Rahal EA, Kazzi N, Nassar FJ, Matar GM. Escherichia coli O157:H7-Clinical aspects and novel treatment approaches. Front Cell Infect Microbiol. 2012;2:138.

3. E. coli - Symptoms and causes [Internet]. Mayo Clinic. 2020 [cited 2022 Mar 18]. Available from: https://www.mayoclinic.org/diseases-conditions/e-coli/symptoms-causes/syc-20372058

4. Ameer MA, Wasey A, Salen P. Escherichia Coli (E Coli 0157 H7). In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Mar 18]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK507845/

5. Rosales A, Hofer J, Zimmerhackl L-B, Jungraithmayr TC, Riedl M, Giner T, et al. Need for long-term follow-up in enterohemorrhagic Escherichia coli-associated hemolytic uremic syndrome due to late-emerging sequelae. Clin Infect Dis Off Publ Infect Dis Soc Am. 2012 May;54(10):1413–21.

6. Hemolytic uremic syndrome (HUS) - Symptoms and causes [Internet]. Mayo Clinic. 2021 [cited 2022 Mar 18]. Available from: https://www.mayoclinic.org/diseases-conditions/hemolytic-uremic-syndrome/symptoms-causes/syc-20352399

7. Goldwater PN, Bettelheim KA. Treatment of enterohemorrhagic Escherichia coli (EHEC) infection and hemolytic uremic syndrome (HUS). BMC Med. 2012 Feb 2;10:12.

8. Slutsker L, Ries AA, Maloney K, Wells JG, Greene KD, Griffin PM. A nationwide case-control study of Escherichia coli O157:H7 infection in the United States. J Infect Dis. 1998 Apr;177(4):962–6.

9. Wong CS, Jelacic S, Habeeb RL, Watkins SL, Tarr PI. The risk of the hemolytic-uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections. N Engl J Med. 2000 Jun 29;342(26):1930–6.

10. Karch H, Schmidt H, Janetzki-Mittmann C, Scheef J, Kröger M. Shiga toxins even when different are encoded at identical positions in the genomes of related temperate bacteriophages. Mol Gen Genet MGG. 1999 Dec;262(4–5):600–7.

11. Paton JC, Paton AW. Pathogenesis and Diagnosis of Shiga Toxin-Producing Escherichia coli Infections. Clin Microbiol Rev. 1998 Jul;11(3):450–79.

12. Karmali MA. Infection by verocytotoxin-producing Escherichia coli. Clin Microbiol Rev. 1989 Jan;2(1):15–38.

13. Tarr PI. Escherichia coli O157:H7: clinical, diagnostic, and epidemiological aspects of human infection. Clin Infect Dis Off Publ Infect Dis Soc Am. 1995 Jan;20(1):1–8; quiz 9–10.

14. Riaz S, Steinsland H, Hanevik K. Human Mucosal IgA Immune Responses against Enterotoxigenic Escherichia coli. Pathog Basel Switz. 2020 Aug 29;9(9):E714.

15. Li Y, Frey E, Mackenzie AM, Finlay BB. Human response to Escherichia coli O157:H7 infection: antibodies to secreted virulence factors. Infect Immun. 2000 Sep;68(9):5090–5.

Image Reference:

16. Mead PS, Griffin PM. Escherichia coli O157:H7. The Lancet. 1998 Oct 10;352(9135):1207–12.