Course:PATH4172019W2/Case 4

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Case 4 : One Too Many Hamburgers

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.


Q1. The Body System Questions

(i) What are the signs (objective characteristics usually detected by a healthcare professional) and symptoms (characteristics experienced by the patient, which may be subjective).

(ii) Which body system is affected? In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful.

(iii) What treatment(s) will be prescribed to help Ronnie recover from this infection and how do they work

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

Q2. The Microbiology Laboratory Questions

(i) Including the stated bacteria, what are the most common bacterial pathogens associated with this type of infectious scenario.

(ii) What samples are taken for laboratory testing and how important is the Microbiology Laboratory in the diagnosis of this infectious disease?

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

(iv) What are the results expected from these tests that might allow the identification of the bacteria named in this case.

Q3. Bacterial Pathogenesis Questions

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

(i) Encounter: where does this 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

(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).

(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.

(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?

Q4. The Immune Response Questions

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

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

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

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

Reports: One Too Many Hamburgers

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.

Q1. The Body Systems Questions

Question (i)

(i) What are the signs (objective characteristics usually detected by a healthcare professional) and symptoms (characteristics experienced by the patient, which may be subjective).

Signs are objective characteristics detected by healthcare professionals whereas symptoms are subjective and are experienced and reported by the client (1). The distinction here is: who is the observer and whether they have medical expertise.

Strains of E.coli, such as 0157:H7, that cause illness in humans are enterohemorrhagic E.coli (EHEC) (2). It typically causes foodborne illness through the consumption of contaminated, undercooked liquids and foods such as raw milk and undercooked ground beef (2). EHEC is characterized by the production of verotoxin or Shiga toxins (Stx) (3). This makes Infection with E coli O157:H7 a type of verotoxigenic infection (VTEC) (4). Its pathogenesis involves toxins causing inhibition of human cell protein synthesis resulting in cell injury and cell death (4). The bacteria require a low infectious dose (only around 300 bacteria) which allows for person to person spread (4).

A sign is an effect of a health problem that can be observed objectively by a third party, for example Ronnie’s doctor, while a symptom is an effect experienced only by the person who has the health problem. The signs and symptoms that Ronnie is experiencing point towards a gastrointestinal infection, which are typically caused by bacteria like E. coli following consumption of uncooked meat (5). According to the CDC, infection with E.coli typically occurs 2-8 days post consumption, which aligns with Ronnie’s timeline (5). The bloody diarrhea and low-grade fever Ronnie is experiencing are hallmark symptoms of inflammatory diarrhea, notably following a specific infection by E.coli 0157:H7 (6).

Bloody diarrhea

Infection with the 0157:H7 strain is characterized specifically by bloody diarrhea or “hemorrhagic colitis” (6). Bloody diarrhea is a symptom of E.coli infection, as it is only experienced by the patient, as well as confirmation from investigations of analysis of the stool sample at the Microbiology Laboratory. The collected stool sample would also show no fecal leukocytes or fever upon laboratory analysis, another sign of inflammatory diarrhea (6).

Fever

Illness with E. coli 0157:H7 is almost always accompanied by fever (6). The fever itself is a sign of Enterohemorrhagic E.coli, as it can be objectively diagnosed using a thermometer, with an oral temperature of 37.8 C or higher or armpit temperature of 37.2 C or higher (7). However, the chills, sweats, malaise and fatigue that accompany a fever are symptoms, subjectively experienced only by Ronnie.

Abdominal cramps

Abdominal pain/cramps are a common symptom of E.coli infection experienced by the patient. Ronnie’s doctor performed a physical examination to test the nature of the abdominal pain by pressing lightly into his stomach, asking Ronnie to express where he felt pain. The periumbilical tenderness expressed by Ronnie is a common symptom of E.coli 0157:H7 infection (6).

Table 1. Typical Early Signs & Symptoms of an E. coli infection.

Early Symptoms: Earlier Signs:
  • Upset stomach
  • Nausea
  • Stomach cramps (abdominal pains)
  • Malaise
  • Loss of appetite
  • Mild dehydration
  • Persistent diarrhea (especially bloody stool)
  • Fever of about 100 F to 101 F (37.7 C to 38.3 C)

E. coli 0157:H7 is notorious because it can cause additional complications in children and the elderly.

Late Symptoms: The majority of people (especially normal adults) that are infected resolve the infection without antibiotics (self-limiting) in about five to seven days. However, some people (about 10% of people infected and especially children under the age of 5 and the elderly) develop more severe signs and symptoms, and these people usually require hospitalization and aggressive treatment (8). These patients develop the usual early symptoms listed above, but do not resolve the infection (8,9). They develop symptoms that last longer (at least a week) and, if not treated promptly, the infection may lead to disability or death.

Table 2: Later or late signs & symptoms of E. coli infections

Later Symptoms Later Signs
  • Hemorrhagic diarrhea * (large amounts of blood in the stools)
  • Anemia *
  • Pale skin color
  • Little or no urine output *
  • Severe abdominal pains
  • Easy bruising
  • Shortness of breath *
  • Generalized swelling
  • Jaundice *
  • Mental changes in the elderly (termed TTP* or thrombotic thrombocytopenic purpura)
  • Severe dehydration * especially for children (termed HUS or hemolytic uremic syndrome)
  • Nosebleeds
  • Kidney (renal) failure
  • Excessive + Spontaneous bleeding
  • Seizures
  • Organ failure
  • Death
Terms that are bolded with an asterisk (*) imply that these conditions may also be

regarded as signs (or vice versa) if monitored under a physician and using particular tests to confirm the

condition

These symptoms or complications fall into three main categories:

Hemorrhagic (bloody) diarrhea: Hemorrhagic (bloody) diarrhea is defined as an increased amount of blood in the diarrheal stool, which does not seem to decrease over time, and usually is accompanied by severe abdominal pain [10]. Although this may resolve within a week, some individuals can develop anemia and dehydration that can cause death.

Hemolytic-uremic syndrome (HUS): Hemolytic-uremic syndrome symptoms of pallor (due to anemia), fever, bruising or nosebleeds (due to destruction of blood platelets that are needed for blood to clot) [11,10], fatigue, shortness of breath, swelling of the body, especially hands and feet, jaundice, and reduced flow of urine may be seen. HUS symptoms usually develop at about 7-10 days after the initial diarrhea begins. HUS is the most common cause of kidney failure in children [12]; children under 10 years old are the most likely to develop HUS [10]. With this fact in mind, Ronnie is 10 years old and therefore is susceptible to developing HUS if his symptoms worsen. It is important to monitor his progress if he shows any signs of recovery in order not to succumb to developing HUS. HUS also produces toxins that damage the kidneys and destroys platelets that can lead to kidney failure, excessive bleeding, seizures, or death [12].

Thrombotic thrombocytopenic purpura (TTP): Thrombotic thrombocytopenic purpura is caused by the loss of platelets; however, the symptoms that occur are somewhat different and occur mainly in the elderly [13]. The symptoms are fever, weakness, easy, rapid or "spontaneous" bruising, kidney failure, and mental impairment that can rapidly progress to organ failures and death. Until the 1980's, TTP was considered a fatal disease, but since the 1980's, plasma exchange and infusion techniques have reduced the death rate in TTP patients to about 10% [13]. For most people (about 90%), the infection clears and a good outcome or and prognosis is good. However, if any of the previously mentioned complications occur, the prognosis may range from good to poor. The variable prognosis depends on the severity of the complication, the rapidity of diagnosis and treatment, the response of the individual to adequate treatment and the overall health of the individual [10]. Children and the elderly are at higher risk for adverse outcomes [13,14].

References:

  1. Niamh K. Signs and Symptoms: PATH 417A 99C Human Bacterial Infections - HMN BACT INFCTNS n.d.
  2. Gossman, William. “Escherichia Coli (E Coli 0157 H7).” StatPearls [Internet]., U.S. National Library of Medicine, 11 July 2019, www.ncbi.nlm.nih.gov/books/NBK507845/.
  3. Todar, Kenneth, and Madison. Pathogenic E. Coli, textbookofbacteriology.net/e.coli_4.html.
  4. Shilkofski, N., & Cheng, T. L. (2004). Escherichia coli O157:H7. Pediatrics in Review, 25(2), 75–76. doi:10.1542/pir.25-2-75
  5. Signs & Symptoms. (2014, June 20). Retrieved from https://www.cdc.gov/ecoli/2014/o157h7-05-14/signs-symptoms.html
  6. Evans DJ Jr., Evans DG. Escherichia Coli in Diarrheal Disease. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 25. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7710/
  7. Fever: First aid. (2019, September 11). Retrieved from https://www.mayoclinic.org/first-aid/first-aid-fever/basics/art-20056685
  8. Davis CP., MD, PhD, E. coli 0157:H7 Infection Early Symptoms, Treatment, & Prevention. Medicine Net. Retrieved from ://www.medicinenet.com/e_coli__0157h7/article.htm
  9. Clements, A., Young, J. C., Constantinou, N., & Frankel, G. (2012). Infection strategies of enteric pathogenic Escherichia coli. Gut microbes, 3(2), 71–87.
  10. Walterspiel JN, Ashkenazi S, Morrow AL, Cleary TG (1992). Effect of subinhibitory concentrations of antibiotics on extracellular Shiga-like toxin I. Infection. 20 (1): 25–29. doi:10.1007/BF01704889.
  11. Sweetser S. (2012). Evaluating the patient with diarrhea: a case-based approach. Mayo Clinic proceedings, 87(6), 596–602. https://doi.org/10.1016/j.mayocp.2012.02.015.
  12. Wedro B. Hemolytic Uremic Syndrome (HUS). Medicine Net. Retrieve from: https://www.medicinenet.com/hemolytic_uremic_syndrome/article.htm#what_causes_hemolytic_uremic_syndrome
  13. Maluykova I, Gutsal O, Laiko M, Kane A, Donowitz M, Kovbasnjuk O. Latrunculin B facilitates Shiga toxin 1 transcellular transcytosis across T84 intestinal epithelial cells. Biochim Biophys Acta. 2008;1782:370–7.
  14. Olesen B, Jensen C, Olsen K, Fussing V, Gerner-Smidt P, Scheutz F. VTEC O117:K1:H7. A new clonal group of E. coli associated with persistent diarrhoea in Danish travellers. Scand J Infect Dis 2005;37:288--94.

Question (ii)

(ii) Which body system is affected? In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful.
Fig 1. Digestive Tract
Overview

The body system that would be infected by this infection with E.coli O157:H7 would be the digestive system aka the gastrointestinal tract (Fig 1). This system includes the mouth, salivary glands (exocrine gland) esophagus, stomach, pancreas (endo/exocrine gland), small intestine, gallbladder (exocrine gland), and the large intestine (Fig 1). There are 4 general functions of the digestive system: 1) Assimilation of dietary food substances, where ingested food/nutrients are prepared to absorbable forms for cellular usage; 2) Fluid and electrolyte balance; 3) Excretion of waste products, notably iron, copper, bile and drugs; 4) Immune response via gut-associated lymphoid tissue, carried out by lymphocytes and macrophages[1.2 1]. Contents moving through the gastrointestinal tract are propelled through each compartment through a series of muscular contractions called peristaltic contractions. These are cued by the enteric nervous system which provides autonomic nervous control to the GI tract. The gastrointestinal tract is a hollow tube from mouth to anus that starts developing during gastrulation at around the 3-week mark of embryogenesis [1.2 2]. During development the endoderm gives rise to the epithelial lining of the gastrointestinal tract, liver, gallbladder, pancreas [1.2 3]. The mesoderm gives rise to the connective tissue including the smooth muscle and walls that line the gut tube [1.2 3]. The ectoderm separates into three components including the neural crest which differentiate into neurons that will innervate the GI tract (aka enteric nervous system, ENS) [1.2 3].  The tubes of the gastrointestinal tract contain an interior muscular layer surrounded by a mucous membrane (Fig 2.). The layers can be separated into the inner mucosa (epithelium, lamina propria, muscularis mucosae), followed by the submucosa, muscularis, and the outer serosa (Fig 2)[1.2 3].

Fig 2. Architecture of Gut Mucosal Wall

The mucosa is made up of the epithelium which lines the lumen of the GI tract, the lamina propria which are supportive tissue containing lymphocytes and plasma cells, and the muscularis mucosae which contain multiple layers of smooth muscle [1.2 3]. The submucosa is collagenous connective tissue that supports the mucosa and is highly vascularized (lymphatic and circulatory system) and innervated by the ENS [1.2 3]. The muscularis mucosae contain layers of longitudinal and circular smooth muscles responsible for peristaltic contractions [1.2 3]. The serosa is another outer layer of supportive tissue which is vascularized and innervated by nerves [1.2 3].

Physiological Functions of GI Tract
GI Tract

The following table describes, in general, the different functions of the various segments of the GI tract and its accessory organs[1.2 4].

Organs Secretions Functions
Mouth

Salivary Glands

Saliva: Salt, Water, Mucus, Amylase, lysozyme

The mouth functions to break food down through chewing, and to initiate the swallowing reflex. Saliva functions to moistening and lubricate food, as well as some digestion of carbohydrates
Pharynx and Esophagus Mucus Swallowing via peristalsis
Stomach gastric juice (HCl, Pepsinogen, mucus, intrinsic factor) Carbohydrate and protein digestion, mixing, storage, and emptying of chyme, neutralization of some pathogens
Exocrine Pancreas Water and bicarbonate; digestive enzymes (lipase, colipase, amylase) and proenzymes/zymogens Secretion of digestive enzymes to breakdown fats, proteins and carbohydrates, as well as bicarbonate to neutralize HCl in the small intestine
Liver Bile: Bile salts, bicarbonate, Bilirubin Bile secretion solubilizes water-insoluble fat, neutralizes HCl, and helps to eliminate organic waste
Gallbladder Stores and concentrates bile by absorbing water and electrolytes
Small intestine Mucus, salt and membrane-bound digestive enzymes (not secreted) Digest and absorb dietary nutrients, electrolytes, and water
Large intestine Mucus Absorbs remaining fluid and electrolytes and stores undigested material before defecation
Effect on Normal GI Function

E.coli O157:H7 (EHEC) do not invade mucosal cells, however they do produce a toxin known as the Shiga toxin [1.2 5]. These toxins play a significant role in the inflammatory response against EHEC strains and could possibly explain the ability of EHEC strains to cause hemolytic uremic syndrome [1.2 5]. Toxin production is further enhanced by iron deficiency [1.2 5]. The cytotoxic toxins induce cell death by inhibition of protein synthesis [1.2 6]. The toxin will therefore, directly damage mucosal cells and vascular endothelial cells of the gastrointestinal tract [1.2 7]. Furthermore the immune response conducted by the host will further potentiate the damage to the GI tract via local inflammation (gastroenteritis). The effects of infection by E.coli O157:H7 can thus be seen in the mouth, esophagus, stomach, small intestine, and large intestine.

In the mouth, diarrhea and subsequent dehydration caused by infection with E.coli O157:H7 would decrease secretion of saliva and can cause dry mouth [1.2 8]. This would impair the actions of saliva (lubrication, protection, digestion) and the ability to taste [1.2 8]. This phenotype can be accompanied by a loss of appetite [1.2 8].

In the stomach, E.coli O157:H7 are resistant to the acidic environment greatly contributing to their virulence [1.2 9]. The inflammatory response caused by E.coli O157:H7 (gastroenteritis) can result in symptoms of nausea and vomiting [1.2 10]. Communication between the brain-stem and the stomach triggers the nausea sensation and can induce the vomiting reflex by which the stomach will trigger gastroesophageal reflux [1.2 11].

EHEC Infection

In the small intestine, gastroenteritis will cause contents of the small intestine to pass through faster than normal [1.2 10]. This results in reduced digestion and absorption of nutrients, water, and vitamins. The dysregulation and spastic contractions caused by gastroenteritis contribute abdominal cramping experienced by patients [1.2 10]. Furthermore, as a result of reduced water absorption patients experience diarrhea, and dehydration [1.2 10]. In this compartment the Shiga toxin will also disrupt protein synthesis leading to cell death, the sloughing of the mucosa and an overall decrease in the surface area for nutrient absorption [1.2 8]. As infection and inflammation progresses, this ultimately results in bloody diarrhea [1.2 8]. This is achieved through adherence to intestinal epithelium and the injection of bacterial effector proteins into the host cells through a type III secretion system (T3SS) [1.2 12]. These effector proteins would then interfere with host cell signalling and functions such as tight junction integrity and alteration of ion channels [1.2 12]. The pathogen achieves this through Stx2 holotoxin, which will cause important brush border proteins such as villin to mistarget and cannot attach to the brush border [1.2 12].

In the large intestine, infection with E.coli O157:H7 can cause Haemorrhagic colitis which is responsible for causing many of the symptoms associated with the disease [1.2 13]. E.coli O157:H7 induced colitis disrupts the processes of the colon importantly water and electrolyte absorption (causing diarrhea and great dehydration) [1.2 13]. E.coli 0157:H7 decreases ion reabsorption via the secretion of effectors through T3SS [1.2 12]. This will increase the osmolality in the gut lumen and result in diarrhea . The increased amount of water in the intestinal lumen will also result in villus cell death and lead to reduced reabsorption [1.2 12]. Colitis by infection with E.coli O157:H7 also greatly contributes to the abdominal pain experienced by patients [1.2 13]. Antibiotics prescribed in the cases of these bacterial infections can also harm the normal gut flora impairing their ability to aid in the healthy function of the digestive system [1.2 13].

HUS

HUS Syndrome

Complications can lead to hemolytic uremic syndrome (HUS) [1.2 7]. HUS is caused by toxins entering the blood and killing red blood cells, which can lead to kidney failure due to the buildup of nitrogen wastes in the blood (uremia) [1.2 7]. The toxin can also enter glomerular endothelial cells, podocytes, and tubular epithelial cells in the kidney through the blood and cause kidney damage [1.2 6]. Kidneys are responsible for maintaining overall fluid balance as well as regulating and filtering minerals from the blood [1.2 14]. In severe cases, when E.Coli 0157:H7  colonizes the kidney, release of their toxin causes renal dysfunction, since the renal glomeruli have a particular vulnerability to microthrombi formation.[1.2 8] Microthrombi are small clots that form in the glomerulus, which inhibit the kidney from reabsorbing nutrients, ultimately resulting in a more hypotonic filtrate, and therefore less frequent urination[1.2 1]. HUS can develop approximately a week after symptoms first appear [1.2 15]. Signs and symptoms include: decreased frequency of urination, fatigue, and pale skin due to hemolytic anemia . Oftentimes, people who develop HUS need to be hospitalized as their kidney filters have been blocked and damaged [1.2 15]. Very few (5-10%) people actually develop HUS [1.2 7]. Children, the elderly, and people who are immunocompromised are usually at a higher risk for experiencing complications with E.coli infections [1.2 7].

References
  1. 1.0 1.1 Kenny Kwok. Gastrointestinal Physiology [unpublished lecture notes]. CAPS301: Human Physiology, University of British Columbia; lectures given 2019 Jan-Feb
  2. Hill, M.A. 2020. Embryology Gastrointestinal Tract Development. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Gastrointestinal_Tract_Development
  3. 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 Bhatia A, Bordoni B. Embryology, Gastrointestinal. 2018. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; Available from: https://www.ncbi.nlm.nih.gov/books/NBK537172/
  4. Kenny Kwok. Gastrointestinal Physiology [unpublished lecture notes]. CAPS301: Human Physiology, University of British Columbia; lectures given 2019 Jan-Feb.
  5. 5.0 5.1 5.2 Todar, Kenneth, and Madison. Pathogenic E. Coli, textbookofbacteriology.net/e.coli_4.html.
  6. 6.0 6.1 "Enterohaemorrhagic Escherichia Coli (EHEC)." World Health Organization. Web. 20 Mar. 2016.
  7. 7.0 7.1 7.2 7.3 7.4 “Lab-Test: Shiga Toxin-Producing Escherichia Coli.” Merck Manuals Professional Edition, www.merckmanuals.com/professional/multimedia/lab-tests/v42968541.
  8. 8.0 8.1 8.2 8.3 8.4 8.5 Gossman W, Wasey A, Salen P. 2019. Escherichia Coli (E Coli 0157 H7) In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; Available from: https://www.ncbi.nlm.nih.gov/books/NBK507845/
  9. Lim JY, Yoon J, Hovde CJ. 2010. A brief overview of Escherichia coli O157:H7 and its plasmid O157. J Microbiol Biotechnol; 20(1):5–14.
  10. 10.0 10.1 10.2 10.3 Sattar SBA, Singh S. Bacterial Gastroenteritis. 2019. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; Available from: https://www.ncbi.nlm.nih.gov/books/NBK513295/
  11. Chaudhry SR, Liman MNP, Peterson DC. Anatomy, Abdomen and Pelvis, Stomach. 2019. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; Available from: https://www.ncbi.nlm.nih.gov/books/NBK482334/
  12. 12.0 12.1 12.2 12.3 12.4 Tran, Seav-Ly, et al. “Shiga Toxin Production and Translocation during Microaerobic Human Colonic Infection with Shiga Toxin-ProducingE. Coli O157:H7 and O104:H4.” Cellular Microbiology, vol. 16, no. 8, 2014, pp. 1255–1266., doi:10.1111/cmi.12281.
  13. 13.0 13.1 13.2 13.3 Azzouz LL, Sharma S. 2019. Physiology, Large Intestine. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; Available from: https://www.ncbi.nlm.nih.gov/books/NBK507857/
  14. "The Digestive System Diagram, Organs, Function, and More." WebMD. WebMD. Web. 20 Mar. 2016
  15. 15.0 15.1 “Symptoms.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 20 Nov. 2017, www.cdc.gov/ecoli/ecoli-symptoms.html.

Question (iii)

(iii) What treatment(s) will be prescribed to help Ronnie recover from this infection and how do they work?

Treatment of a E.Coli 0157:H7 gastrointestinal infection is mainly centered around supportive care and maintenance of hydration, since patients like Ronnie lose lots of water with the excessive diarrhea.[Q1-3 1] Within 10 days, most patients recover from an enterohemorrhagic E.coli infection without pharmacological treatment.[Q1-3 1] Moreover, antibiotics (ex. trimethoprim, quinolones, or furazolidone) have not been shown to be efficacious in treating 0157:H7 infection, and actually worsen outcomes in some cases by increasing the likelihood of HUS.[Q1-3 1] This is due to the fact that when antibiotics lyse E.coli’s cell well, it releases more of their enterotoxin into systemic circulation, ultimately leading to acute renal failure.[Q1-3 1] Additionally, usage of antiperistaltic agents typically prescribed for diarrhea (such as loperamide or dicyclomine) that slow intestinal motility are advised against, since they delay the clearance of E. coli via decreasing gastrointestinal motility, allowing the bacteria to generate more damage and increase the likelihood of further complications such as HUS.[Q1-3 2]

Specifically, rehydration therapy is used in the form of blood volume expansion administered intravenously. This involves balancing electrolytes and preventing oligo-anuria (pre-HUS symptoms) allowing the kidneys to be perfused sufficiently. Nutritional support via IV is essential as nutrients are lost via diarrhea. Diuretic therapy could also aid with replenishing fluids via keeping the fluids in the body (preventing excretion) but this therapy is only used should acute renal failure occurs, such as in the case of HUS. Patients with severe HUS may also benefit from a hemodialysis to treat the volume, electrolyte, acidosis and uremia issues that develop from acute renal failure.[Q1-3 1] Since HUS also affects the blood, blood transfusions (providing packed red blood cells for severe anemia) could be beneficial but be sure to not administer platelets as this could worsen clinical course of the infection.

Additionally, there has been limited evidence suggesting the efficacy of the monoclonal antibody eculizumab in expediting recovery from 0157:H7 infection. Eculizumab inhibits the complement cascade, thus interfering with recruitment of inflammatory cells, which decreases the damage done to renal vasculature.[Q1-3 1] Typically, this treatment is reserved as a rescue agent for very extreme cases, as it’s extremely expensive, and lacks clinical consensus.[Q1-3 1] Antibody therapy with antibodies to the Shiga toxin have been shown to be well-tolerated in initial pediatric clinical studies, and efficacious in reducing the likelihood of HUS.[Q1-3 2]

Due to the historic lack of treatment options for E.coli 0157:H7 infection, novel approaches have recently been investigated. One of which is carbosilane dendrimers, which have been shown to bind the enterotoxin with high affinity, and inhibit its entry into host cells.[Q1-3 3] IV injection of carbosilane dendrimers decreased brain damage, and prevented the lethal effect of the toxin when tested in mice.[Q1-3 3]

The use of natural products to treat and prevent E.coli infection have also been widely assessed, such as natural probiotic agents.[Q1-3 3] Common in yogurt, cultures of Lactobacillus casei have been shown to have a protective effect against the toxin and are effective in delaying the growth of pathogenic E.coli.[Q1-3 3] Other natural remedies like components of green tea and cranberries have also been shown to have similar protective effects.[Q1-3 3]

Preliminary data support that certain antibiotic agents may in fact be used for treatment of E.coli 0157:H7, mainly rifampin and gentamicin.[Q1-3 3] Rifampin is an RNA polymerase inhibitor, thus it inhibits DNA-dependent RNA synthesis.[Q1-3 3] Gentamicin is a ribosome inhibitor, that stops bacterial protein synthesis by binding to 30S ribosomes, and blocking translation.[Q1-3 3] These agents have been previously shown – with other bacterial infections – to inhibit toxin expression.[Q1-3 3] Injection of E.coli 0157:H7 in mice followed by treatment with rifampin coupled with gentamicin resulted in a 50% survival rate.[Q1-3 3] While this method has yet to be tested on humans, the work done on mice shows promising results for future treatment implementation.

For children like Ronnie that are infected, treatment should also include the counselling for parents and children on the importance of avoiding re-entry to any childcare center until the diarrhea is resolved (which is measured by two stool cultures being negative for E. coli O157:H7) as to not still infect other children.[Q1-3 4] This counselling should also touch on the expectations throughout treatment (and even more so as prevention) on correct hand hygiene and sanitation of oneself, one’s food (as well as correct cooking of meats) and one’s environment according to guidelines. Washing one’s hands limits transmission to others and prevents transmission from the environment to oneself. Washing of food such as fruits and vegetables and ensuring no cross contamination from raw meat or unpasteurized dairy products with the fruit and vegetables is crucial as well as sanitation following exposure to farm or petting zoo environments is important.[Q1-3 4]

References:

  1. 1.0 1.1 1.2 1.3 1.4 1.5 1.6 Gossman W, Wasey A, Salen P. Escherichia Coli (E Coli 0157 H7) [Updated 2019 Jul 11]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK507845/
  2. 2.0 2.1 Rahal, E. A., Kazzi, N., Nassar, F. J., & Matar, G. M. (2012). Escherichia coli O157:H7—Clinical aspects and novel treatment approaches. Frontiers in Cellular and Infection Microbiology, 2. doi:10.3389/fcimb.2012.00138
  3. 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 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. Published 2012 Nov 15. doi:10.3389/fcimb.2012.00138
  4. 4.0 4.1 Shilkofski, N., & Cheng, T. L. (2004). Escherichia coli O157:H7. Pediatrics in Review, 25(2), 75–76. Doi:10.1542/pir.25-2-75


Question (iv)

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

Because it was determined that Ronnie had contracted a stain of the Shiga-like producing E. coli O157:H7, this should be reported to both the provincial department of health and the federal government.[Q1-4 1] As mentioned by the BCCDC, all communicable diseases, as all food-borne illnesses are, must be reported in British Columbia as a result of the Public Health Act. [Q1-4 2] Even though ­E. coli infections have a much lower incidence rate than other food-borne pathogens like Salmonella, they have a much higher hospitalization rate.[Q1-4 3] Tracking reportable and notifiable diseases are important to both the provincial ministry of health and the federal government because it could have federal implications for trade, provincial implications for animal health, and public health implications for the general populace.[Q1-4 4] E. coli 0157:H7 infection has been a major public health concern for decades in most areas of the world, including North America.[Q1-4 3] For example, two BC E. coli outbreaks in 2017 were associated with contaminated flour, as well as several cases associated with international travel, with 176 cases in total in BC.[Q1-4 2] Reporting the occurrence of these E. coli infections was crucial to identifying their source, and limiting the distribution of the contaminated source to larger groups of the population. While most infected individuals recover without medical intervention, children and elderly are at an increased risk of developing more severe clinical complications such as HUS[Q1-4 3], emphasizing the importance of reporting E. coli 0157:H7 infection as early as possible.

If an outbreak is imminent, the government must quickly act to determine the source of infection and take appropriate measures for the safety of everyone in the community, province, and country. For instance, in this situation, the government may use this information to determine whether the source of Ronnie’s hamburgers was contaminated, and if a recall is required. Additionally, sharing this finding with the government will help ensure that research will be done on this new strain and efforts will be made to limit the amount of new cases. Increasing the public’s knowledge about this situation will help avoid the use of unnecessary antibiotics and antidiarrheals as they could make the disease worse.

References:

  1. Canada PHA of. E. coli (Escherichia coli) infection. Aem 2019. https://www.canada.ca/en/public-health/services/diseases/e-coli.html (accessed March 27, 2020).
  2. 2.0 2.1 BCCDC. Communicable Diseases n.d. http://www.bccdc.ca/health-professionals/data-reports/communicable-diseases (accessed March 27, 2020).
  3. 3.0 3.1 3.2 Nguyen, Y., & Sperandio, V. (2012). Enterohemorrhagic E. coli (EHEC) Pathogenesis. Front. Cell. Inf. Microbio. Frontiers in Cellular and Infection Microbiology/ Perez-Perez, G., & Blaser, M. (1996). Campylobacter and Helicobacter. Medical Microbiology. 4th Edition.
  4. Agriculture M of. Reportable and Notifiable Diseases - Province of British Columbia n.d. https://www2.gov.bc.ca/gov/content/industry/agriculture-seafood/animals-and-crops/animal-health/reportable-notifiable-diseases (accessed March 27, 2020).

Q2. The Microbiology Laboratory

Question (i)

(i) Including the named bacteria, what are the most common bacterial pathogens associated with this type of infectious scenario
E. coli O157:H7:
E. coli O157 H7 image.

Theodor Escherich first described E. coli in 1885 as Bacterium coli commune, which he isolated from the feces of newborns (1). Later it was called Escherichia coli and for many years it was thought to be only a commensal organism that inhibited the large intestine. However, in 1935 a strain of E. coli was found that showed to cause diarrhea among infants.

Depending on the virulence factors they possess, virulent Escherichia coli strains cause either noninflammatory diarrhea (watery diarrhea) or inflammatory diarrhea (dysentery with stools usually containing blood, mucus, and leukocytes) (2). it seems like from the bloody diarrhea that Ronnie has the inflammatory diarrhea.

They are Gram negative bacilli bacteria and belong to the family Enterobacteriaceae. The only difference between the virulent and nonvirulent strains is that virulent possessing genetic elements for virulence factors. Strains producing enterotoxins are enterotoxigenic E coli (ETEC) (2).

Transmission is by the fecal-oral route. Pili (fimbriae) allow the bacteria to colonize the ileal mucosa. Cytotonic enterotoxins (encoded on plasmid or bacteriophage DNA) induce watery diarrhea (2).

Shigella:
Several media have been designed to selectively grow enteric bacteria and allow differentiation of Salmonella and Shigella from E. coli. The primary plating media shown here are eosin methylene blue (EMB) agar, MacConkey agar, ENDO agar, Hektoen enteric (HE) agar and Salmonella-Shigella (SS) agar (1)

Is a genus of gamma proteobacteria in the family Enterobacteriaceae. Shigellae are Gram-negative, nonmotile, non-spore forming, rod-shaped bacteria, very closely related to Escherichia coli (1).

Shigellosis is an infectious disease caused by various species of Shigella. People infected with Shigella develop diarrhea, fever and stomach cramps starting a day or two after they are exposed to the bacterium – the diarrhea is often bloody, which are the symptoms that Ronnie had.

Infection is initiated by ingestion of shigellae, usually via fecal-oral contamination. An early symptom is diarrhea, which is possibly elicited by enterotoxins and/or cytotoxin. It may occur as the organisms pass through the small intestine (2). The hallmarks of shigellosis are bacterial invasion of the colonic epithelium and inflammatory colitis (2).

Salmonella enterica (serotype typhimurium):
Salmonella typhi, the agent of typhoid.

The two species of Salmonella are Salmonella enterica and Salmonella bongori (1). S. enterica is the type species and is further divided into six subspecies, that include over 2,600 serotypes (2). We will focus on Salmonella enterica, serotype typhimurium.

Is a Gram-negative facultative rod-shaped bacterium in the same proteobacterial family as Escherichia coli, the family Enterobacteriaceae, trivially known as "enteric" bacteria (1).

Salmonellae live in the intestinal tracts of warm and cold blooded animals. Some species are ubiquitous. Other species are specifically adapted to a particular host.

In humans, Salmonella are the cause of two diseases called salmonellosis: enteric fever (typhoid), resulting from bacterial invasion of the bloodstream (1), and acute gastroenteritis (most common form), resulting from a foodborne infection/intoxication, which could have possibly been the cause of Ronnie’s infection.

Some species of Salmonella are ubiquitous (e.g. Typhimurium) whereas others are specifically adapted to a particular host (6). In human adults, ubiquitous Salmonella organisms are most responsible for foodborne toxic infections (1).

Salmonella species are Gram-negative, flagellated facultatively anaerobic bacilli characterized by O, H, and Vi antigens. There are over 1800 known serovars which current classification considers to be separate species.

Pathogenic salmonellae ingested in food can survive the passage through the gastric acid barrier and invade the mucosa of the small and large intestine and produce toxins. Upon invasion of epithelial cells, it can stimulate the release of proinflammatory cytokines which induce an inflammatory reaction. The acute inflammatory response causes diarrhea and may lead to ulceration and destruction of the mucosa. Which could have been the case with Ronnie.

Campylobacter jejuni:
Campylobacter Jejuni on gram stain.

Campylobacter jejuni is a gram-negative, spirally curved microaerophilic bacterium that is recognized as a significant cause of human enteritis (inflammation of the intestine, especially the small intestine) and is associated with diarrheic illness in several animal species, including dogs, cats, cows, goats, pigs, mink, ferrets, and sheep (4)

Transmission occurs by ingestion of organisms through direct contact with feces or contaminated food and water. This can be a possible cause infection because Ronnie had beef burgers at the BBQ and then developed bloody diarrhea.

C. jejuni causes inflammatory enteritis and acute dysentery with severe abdominal pain and fever as common symotoms (4). Ronnie presents with abdominal cramps, bloody diarrhea and a low grade fever, which could possibly hint at this infection.

Yersinia enterocolitica:
Yersinia enterocolitica as a bacillus shaped bacterium.

Yersinia enterocolitica is a gram-negative bacillus shaped bacterium that causes a zoonotic disease called yersiniosis (2).

The infection is manifested as acute diarrhea, mesenteric adenitis, terminal ileitis, and pseudoappendicitis. In rare cases, it can even cause sepsis (2). The pathogen passes into the stomach, traverses the gut wall, and localizes in lymphoid tissue and mesenteric lymph nodes (2). In some counties Yersinia has more cases of infection than Salmonella or Shigella.

Route of entry is consumption of contaminated food as well as a blood transfusion, so Ronnie might have acquired these bacteria through eating those contaminated burgers. The infection is transmitted predominantly through fecal-oral route.

The one key feature of yersinia is that the individual will continue to shed the organism in feces for nearly 3 months after symptoms have subsided- thus detection of yersinia in stools is critical (2), and hence why a stool test one which was ordered by the doctor.

3 of the 11 species of Yersinia are most significant for humans:

  • Yersinia pestis
  • Yersinia enterocolitica (our specie of interest)
  • Yersinia pseudotuberculosis

Species of Yersinia have a 70 kilodalton virulent plasmid known as pYV. The bacteria also produce ureases that metabolized urea and forms ammonia to protect itself from the harsh acidic environment of the stomach (2). Another virulence factors is the ability of the bacteria to produce Ail (attachment invasion focus) and YadA, that confers resistance to complement-mediated opsonization and prevents phagocytosis (2). The bacteria also contains Yops (Yersinia outer membrane proteins) that arrests phagocytosis by block secretion of mediators including TNF-alpha and IL-8 (2). Certain strains produce yersiniabactin that is an iron-binding agent that can effectively bind iron in a depleted state. This further allows the bacteria to thrive and grow.

Reference:

1.     Todar K, Madison. Online Textbook of Bacteriology. Online Textbook of Bacteriology. http://textbookofbacteriology.net/. Accessed March 28, 2020.

2.     Evans DJ, Jr. Escherichia Coli in Diarrheal Disease. Medical Microbiology. 4th edition. https://www.ncbi.nlm.nih.gov/books/NBK7710/. Published January 1, 1996. Accessed March 28, 2020.

3.     Campylobacter - Classification, Jejuni, Infection and Gram Stain. MicroscopeMaster. https://www.microscopemaster.com/campylobacter.html. Accessed March 28, 2020.

4.     Campylobacter Jejuni. Campylobacter Jejuni - an overview | ScienceDirect Topics. https://www.sciencedirect.com/topics/medicine-and-dentistry/campylobacter-jejuni. Accessed March 28, 2020.

5.     Yersinia enterocolitica (Yersiniosis). Centers for Disease Control and Prevention. https://www.cdc.gov/yersinia/index.html. Published October 24, 2016. Accessed April 3, 2020.

6.     Ashurst JV, Truong J, Woodbury B. 2019. Salmonella Typhi. In StatPearls (ed), Salmonella Typhi [Internet]. StatPearls Publishing, Treasure Island, FL.

Question (ii)

(ii) What samples are taken for laboratory testing and how important is the Microbiology Laboratory in the diagnosis of this infectious disease?

Ronnie’s symptoms (abdominal cramps, bloody diarrhea, low-grade fever, periumbilical tenderness) and provided history (possible consumption of undercooked meat) suggest that he has a form of gastroenteritis. A detailed history and physical examination are the main tools used to diagnose gastroenteritis and assess hydration status (11). The diagnosis is generally suggested by the clinical information and then confirmed by stool culture (1). Initial evaluation generally includes detailed history taking and a physical exam (specifically food and medical history), assessment of duration, frequency, volume status, and other abnormal signs/symptoms of the patient.

  • Stool and blood samples are collected from Ronnie for laboratory testing. Many cases of acute bacterial gastroenteritis may not require any additional testing to determine a specific etiology, especially when it will not affect the physician’s prescribed treatment plan. However, in cases of severe volume depletion, a serum electrolyte panel should be completed to check for electrolyte disturbances (1).

Routine bloodwork is conducted to determine the patient’s overall health status, renal function, electrolytes, and other relevant factors. It can also be used to rule out the presence of bacteremia and/or other health conditions (14). It is important to note that a complete blood count (CBC) cannot differentiate between bacterial etiologies, but it can help suggest severe disease or possible complications (1). For example, high white blood count signifies invasive bacteria or pseudomembranous colitis, while low platelet counts suggest development of hemolytic-uremic syndrome (1). Blood culture should be done for patients with high fever or other severe constitutional symptoms.

  • For blood sample collection, only 3 mL of blood is collected since Ronnie is a 10-year-old child (13). It should be collected in tubes without anti-coagulants and then centrifuged before  the serum is sent for analysis. If a centrifuge is not available, the blood sample should be stored in a refrigerator until a clot forms so the serum can be removed and emptied into a sterile tube using a Pasteur pipette. Tubes of both spun and unspun serum should be refrigerated until transportation(13).

Stool testing for bacterial pathogens is required for the presence of severe illness (e.g. signs of dehydration/hypovolemia, severe abdominal pain, need for hospitalization), high-risk host factors (e.g. pregnant women, 70+ years of age, immunocompromised, other co-morbidities), or other signs/symptoms of inflammatory diarrhea (e.g. mucus or blood in diarrhea, high-grade fever) (1)(8)(11). Routine stool culture can identify common bacterial pathogens such as Salmonella, Campylobacter, Shigella, and pathogenic Escherichia coli. Suspicion of other bacterial pathogens (e.g. Vibrio, Yersinia, Aeromonas, Listeria) requires specific microbiology and culture analysis (11). In cases with bloody diarrhea, additional testing for Shiga toxin and leukocytes in stool for EHEC (enterohemorrhagic E. coli) should be requested in addition to routine stool culture (8). In cases of persistent diarrhea, the physician should send stool samples for ova and parasite testing. In the case of ETEC (enterotoxigenic E. coli), diarrheal stool produces a virtually pure culture of E coli (8). Since the disease is self-limiting, virulence testing of isolates and serotyping is impractical except in an outbreak situation. Confirmation is achieved by serotyping, serologic identification of a specific colonization factor antigen (CFA) on isolates, demonstration of heat-labile enterotoxin (LT) or heat-stabile enterotoxin (ST) production, and identification of genes encoding these virulence factors. Isolates recovered from stool cultures can also be used to identify and track outbreaks (8).

  • For stool specimen collection, feces is collected in the acute phase of diarrheal disease is the choice specimen when bacterial gastroenteritis is suspected (14). This is because responsible bacteria are constantly being shed through fecal matter. If stool is liquid/soft, approximately 5 mL should be collected for culture. If it is formed, approximately 2 g should be collected for culture. Feces should be collected in a clean, dry container with a tight lid and should not be contaminated with urine, barium salts, or toilet paper (14). While about 94% of cases determine the etiologic agent from the first submitted specimen, collection of additional fecal specimens may be required to rule out a bacterial cause of infection, especially in cases where patient symptoms persist (15). Transport media for fecal specimens includes Stuart’s, Aimes, or Cary-Blair medium (16). Fresh stool specimens should be transported to the laboratory and processed within 2 hours of collection (17), which is particularly important to the survival of Shigella and Campylobacter (18). If specimens cannot be processed within 2 hours, they should be placed in Cary-Blair transport medium (19). Refrigeration of specimens in Cary-Blair medium at 4°C prior to processing will best preserve bacterial entomopathogens, with the exception of Shigella (19).
Importance of Microbiology Laboratory to Diagnose Infectious Disease:

Most pathogenic infections produce a variety of non-specific clinical syndromes in humans that often overlap with other pathogens. As a result, laboratory testing is crucial to identify the specific etiology of the disease (i.e. the specific pathogen) (12). A single disease could also result from an infection with one of many individual pathogens, or a combination of multiple pathogens (12). Identification of the specific pathogen(s) that cause the patient’s symptoms are vital for efficient and effective treatment selection.

In Ronnie’s case, his symptoms of abdominal cramps, bloody diarrhea, low-grade fever, periumbilical tenderness, and the fact that he may have consumed undercooked meat could be attributed to a variety of pathogens as mentioned in part 1 (Campylobacter, Shigella, Salmonella, Yersinia, Listeria, or pathogenic Escherichia coli). Without laboratory testing to determine the specific disease etiology, it is challenging for physicians to properly prescribe specific and effective antibiotics rather than broad-spectrum antibacterial agents. Prescription of overly broad-spectrum antibiotics as an alternative to specific diagnosis is a large concern worldwide today. Broad-spectrum antibiotics are one of the many causes that have allowed bacteria to adapt and change to buildup antimicrobial resistance. Acquired antibiotic resistance jeopardizes the ability to treat many common infections and can also promote the spread of bacterial “superbugs.”

In the specific case of E. coli O157:H7, bacterial culturing can confirm the diagnosis and also identity specific toxins as produced by E. coli O157:H7 (12. Furthermore, characterization of bacterial enteropathogens from stool cultures in clinical laboratories is one of the primary methods by which public health authorities identify and track outbreaks of bacterial gastroenteritis (14). Evidently, the microbiology laboratory is vital in the diagnosis of the correct pathogen(s) causing the patient’s symptoms, thus ruling out other potential pathogens that could cause similar clinical manifestations. Specific pathogen identification aids the physician in efficiently prescribing a specific and effective antimicrobial treatment before allowing the disease to progress to more severe stages. This will also minimize the use of unnecessary broad-spectrum antibiotics.

References:

1) Sattar SBA, Singh S. Bacterial Gastroenteritis. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019

8) Evans DJ, Evans DG. Chapter 25: Escherichia Coli in Diarrheal Disease. In: Baron S, editor. Medical Microbiology. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996.

11) Elsevier Point of Care. Clinical Overview Gastroenteritis in Children. ClinicalKey. 2019May29.

12) Washington JA. Chapter 10: Principles of Diagnosis. In: Baron S, editor. Medical Microbiology. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996.

13) Centers for Disease Control and Prevention. Recommendations for Collection of Laboratory Specimens Associated with Outbreaks of Gastroenteritis [Internet]. Centers for Disease Control and Prevention; 1990 [cited 2020Mar27]. Available from: https://www.cdc.gov/mmwr/preview/mmwrhtml/00001829.htm

14) Humphries RM, Linscott AJ. Laboratory Diagnosis of Bacterial Gastroenteritis. Clinical Microbiology Reviews. 2015;28(1):3–31.

15) Gilligan PH, Nolte FS. Laboratory diagnosis of bacterial diarrhea. American Society for Microbiology; 1992.

16) York MK, Rodrigues-Wong P, Church DL. Fecal culture for aerobic pathogens of gastroenteritis. Clinical Microbiology Procedures Handbook, 2d ed. Washington, DC: American Society for Microbiology. 2004:3.

17) Baron EJ, Thomson RB. Specimen collection, transport, and processing: bacteriology. In: Manual of Clinical Microbiology, 10th Edition 2011 Jan 1 (pp. 228-271). American Society of Microbiology.

18) Wells JG, Morris GK. Evaluation of transport methods for isolating Shigella spp. Journal of clinical microbiology. 1981 Apr 1;13(4):789-90.

19) Nataro JP, Bopp CA, Fields PI, Kaper JB, Strockbine NA. Escherichia, shigella, and salmonella. In: Manual of Clinical Microbiology, 10th Edition 2011 Jan 1 (pp. 603-626). American Society of Microbiology.

Question (iii)

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

Fecal specimens should first be observed for blood and mucus, as these contain the highest number of enteric pathogens and should be used for culturing. Different media and mediums can be used to differentiate the pathogens [2.3 1]. Routine fecal culture setup should be designed to optimize the recovery of Salmonella, Shigella, Campylobacter, and STEC. Therefore, fecal specimens received should be planted into 4 media.

  1. MacConkey (MAC) agar for general recovery of gram-negative, rod-shaped bacteria [2.3 1] [2.3 2]
  2. Selective medium designed to recover Salmonella and Shigella [2.3 1]
  3. Medium designed to recover Campylobacter
  4. Medium designed for recovery of STEC O157 and enrichment broth to test for Shiga toxins such as MacConkey agar with sorbitol (SMAC) or cefixime-tellurite SMAC (CT-SMAC)

Selection of testing should be based on a combination of host and epidemiologic risk factors and ideally in coordination with public health authorities. Laboratory tests can be split into culture and non-culture tests.:

Medium Tests
  1. MacConkey (MAC) Agar Medium  
  2. Xylose-lysine-deoxycholate (XLD) agar
  3. Hektoen enteric (HE) agar  
  4. Salmonella Shigella Agar  
  5. Brilliant Green (BG) agar/Bismuth sulfite (BS) agar
  6. Campy Blood Agar  
  7. MacConkey agar with sorbitol (SMAC)
  8. MacConkey agar with sorbitol, cefixime and tellurite (CT- SMAC)
  9. CIN agar  
  10. Eosin-Methylene Blue Agar
General Tests
  1. Gram Stain
  2. Oxidase Test
  3. Enzyme- Linked Immunosorbent Assay (ELISA)
  4. Antibiotic Sensitivity Test (AST)
  5. Triple Sugar Iron (TSI) test
  6. Lysine Iron Test (LIA)
  7. Polymerase Chain Reaction (PCR)
Medium Tests
MacConkey (MAC) Agar

MacConkey Agar is used to isolate and differentiate Gram-negative enteric bacilli in microbiology laboratories [2.3 3]. It is a selective and differential medium that isolates and differentiates bacterial pathogens based on their ability to ferment lactose [2.3 3]. It contains a pancreatic digest of gelatin and peptones which provides essential nutrients, vitamins, and nitrogenous factors that bacteria require for growth. Agar provides structure (solidifies medium), and lactose monohydrate acts as a fermentable source.

The presence of bile salts and crystal violet inhibit the growth of Gram-positive organisms [2.3 3]. Neutral red is a pH indicator that turns red at < pH6.8 and is colourless at > pH 6.8 [2.3 3]. Organisms that ferment lactose (producing acidic environment) will appear pink due to neutral red turning red. Enough acid production will also cause the precipitation of bile salts as a bile precipitate or halo around lactose fermenting bacteria. Non-fermenters will produce colourless colonies.

Xylose-lysine-deoxycholate (XLD) agar

XLD agar is a selective growth medium for the isolation and identification of Gram-negative enteric pathogens [2.3 4]. It is most efficient for the detection of Shigella and Salmonella species [2.3 4]. It is able to differentiate lactose fermenters (appears yellow) from non-fermenters (appears red) [2.3 1]. It can also detect H2S production (black precipitate) [2.3 1]. The selective agent is sodium deoxycholate, which inhibits the growth of Gram-positive organisms; the carbohydrate source is xylose [2.3 4]. The addition of lysine helps with the second differentiation of Salmonella. Lysine decarboxylation reverts pH of the medium from acidic to more neutral. Under this condition, sodium thiosulfate is added as a sulfur source and ferric ammonium is added as an indicator to allow H2S-producing organisms to appear black.

Hektoen enteric (HE) agar

HE agar is a selective and differential medium for the isolation and differentiation of Gram-negative enteric pathogens [2.3 5]. It is particularly useful for Shigella species. It uses large amounts of peptide to offset the inhibitory effect of bile salts. Bile salts allow for the selective nature of HE agar by inhibiting Gram-positive organisms, and can also be toxic for some Gram-negative strains. Salicin, sucrose and lactose are the fermentable carbohydrates present and provide optimal differentiation of enteric pathogens. Bromothymol blue and acid fuchsin are added as acid-base indicators and ferric ammonium citrate is also added as an indicator. Sodium thiosulfate provide sulfur and enable the detection of H2S, which is visible as black-centered colonies.

Salmonella-Shigella (SS) agar

SS agar is used as a selective and differential medium for the isolation of Salmonella and some Shigella species [2.3 6]. It also aids in the differentiation of lactose and non-lactose-fermenters. Bile salts, sodium citrate and brilliant green inclusion inhibit gram-positive and coliform organisms. The basis of differentiation depends on the fermentation of lactose and the absorption of neutral red as the bile salts precipitate in the acidic condition. Neutral turns red (appear pink) in acidic pH, indicating fermentation. Sodium thiosulfate is added as a sulfur source, and ferric citrate is added as an indicator for H2S production. Gram-negative and lactose-fermenting bacteria will form red colonies and those that are gram-negative and do not ferment lactose will form clear colonies. The colonies with black centers will indicate that they can produce H2S.

Brilliant Green (BG)/ Bismuth sulfite (BS) agar

Brilliant Green (BG)

BG agar is used as a primary plating medium for the selective enrichment and isolation of Salmonella species other than S. typhi and S. paratyphi [2.3 6]. Phenol red is used as the pH indicator and brilliant green is used as an inhibitory agent that inhibits Gram-positive organisms and Gram-negative bacilli. Organisms that ferment lactose and/or sucrose exhibit yellow to yellow-green colonies surrounded by a yellow-green zone.

Bismuth Sulfite (BS) agar

BS agar is a highly selective and differential medium [2.3 7]. It is used for the isolation of Salmonella species, especially S. typhi from dairy products. This is more useful and suitable than the BG agar if we are looking for S. typhi specifically. BS agar utilizes inhibitors to inhibit most commensal Gram-positive and Gram-negative species and is differential on the basis of H2S production; dextrose is an energy source. Bismuth sulfite indicators inhibit gram-positive and growth of coliform bacteria.

Campy Blood Agar    

This agar medium is specifically for the isolation and cultivation of Campylobacter jejuni [2.3 8]. It is made up of brucella Agar, where there are options for blood based and blood free agar (where blood free is used more in developing countries). Casein and meat peptones provide nutrients for Campylobacter growth, sodium provides electrolytes and maintains osmotic balance, dextrose provides an energy source, while yeast extract provides essential vitamin B.

Antimicrobics such as cephalothin, amphotericin B, trimethoprim, vancomycin, and polymyxin B inhibit the growth of other flora such as Enterobacteriaceae, Staphylococcus, and yeast, to suppress the growth of other fecal flora, allowing for growth of C. jejuni, C. coli, and C. laridis from feces. Colonies should be yellowish/greyish and non-hemolytic after growth. With this agar, as with many others, further testing is recommended to narrow down the exact strain that is the cause of the infection.

Sorbitol-MacConkey (SMAC) agar

SMAC is used as a selective and differential medium for detection of E. coli O157:H7 [2.3 9]. It has the same composition as MacConkey agar, except that lactose is replaced with sorbitol, which E.coli O157:H7 is unable to ferment. This medium has proved to be inexpensive, rapid, simple yet reliable.

MacConkey agar with Sorbitol, Cefixime, and Tellurite (CT-SMAC)

CT-SMAC is used for the selective and differential isolation of E. coli O157:H7 [2.3 10]. It inhibits growth of most non-verocytotoxigenic E. coli strains and other non-sorbitol fermenting species. Many organisms that are generally mistaken for E. coli O157:H7 on SMAC are inhibited on CT-SMAC.

Cefsulodin-Irgasan-novobiocin (CIN) agar

Several enteric pathogens require highly selective media and will not be optimally recovered by the selection of the media described above [2.3 1]. CIN agar is used to recover both Aeromonas and Yersinia enterocolitica. It is highly selective against the growth of E. coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella enterica, Shigella sonnei and Streptococcus. Y. enterocolitica ferments mannitol in the medium, producing an acidic pH which gives the colonies their red colour and the “bull’s eye” appearance [2.3 11]. Sodium deoxycholate, cefsulodin, irgasan and novobiocin are added as selective agents.

Eosin-Methylene-Blue (EMB) agar

EMB agar is used as selective and differential media for the isolation of Gram-negative bacilli, including coliform organisms and enteric pathogens [2.3 12]. Eosin dye and methylene blue are used to inhibit the growth of Gram-positive bacteria. The dyes also act as differential indicators for fermentation products; lactose and sucrose provide a carbohydrate source. Similarly to MAC, gelatin is the nitrogen source and lactose is the carbohydrate source, where the bacteria must be streaked in order to ensure isolation. Acid production from fermentation results in the eosin-methylene blue dye complex being taken up by bacterial cells to produce a brown to blue-black colony appearance. However, this medium does not differentiate between carbohydrate utilization.

General Tests
Gram Stain

Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents, by separating gram positive and gram negative groups by red or violet groups [2.3 13]. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process.

  1. Cells are first stained with crystal violet dye, followed by an iodine solution of iodine and potassium iodide, which is added to form a complex between the crystal violet and iodine. This iodine is insoluble in water.
  2. A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria. However with Gram negative bacteria, the OM is degraded and the thinner PG layer is unable to retain the crystal complex and the color is lost.
  3. A counterstain, such as the weakly water soluble safranin, is added to the sample. As it is lighter than crystal violet, it will not disrupt the purple coloration but will instead stain the gram negative strains red.

Gram-staining on stool specimens is not routinely performed as part of stool culture testing and should only be performed upon special request. Gram-staining is generally not useful when performed on stool with the key exception of campylobacters, for which characteristic seagull-shaped campylobacters can be visualized in stool when carbol-fuchsin is used as a counterstain.

Oxidase Test

The oxidase test is used to identify bacteria that produce cytochrome c oxidase, a bacterial ETC enzyme [2.3 14]. This enzyme catalyzes transport of electrons between electron donors within the bacteria of interest and redox dye (usually tetramethyl-p-phenylene-diamine), causing the dye to be reduced from colourless to a deep purple colour. If cytochrome c oxidase is present, it will oxidize the reagent, which is tetramethyl-p-phenylenediamine, into indophenol, creating a purple end product. When the enzyme is not present, the reagent remains reduced and is colorless. All bacteria that are oxidase positive are aerobic, and can use oxygen as a terminal electron acceptor in respiration. However, they may not be strict aerobes and a negative result may indicate that they are anaerobic, aerobic, or facultative, but simply lack the cytochrome c oxidase. Oxidase test is most helpful in screening colonies suspected of being one of the Enterobacteriaceae (all negative) and in identifying colonies suspected of belonging to other genera such as Aeromonas, Pseudomonas, Neisseria, Campylobacter, and Pasteurella (positive). Those that do not have cytochrome c leave the reagent colourless, meaning that they are oxidase-negative.  A positive test is indicated when a blue/purple colour appears within 30 seconds; any colour appearing after this time should be disregarded [2.3 15].

Catalase Test

Catalase is an enzyme produced by microorganisms that live in oxygenated environments to neutralize toxic oxygen metabolites [2.3 16]. This test is to determine the presence of catalase within bacteria as this enzyme breaks down hydrogen peroxide into water and oxygen to form oxygen bubbles (which can be visualized and indicated positive test. When a small amount of catalase-producing organism is introduced into H2O2 , bubbles form as a result of the enzyme’s activity producing oxygen [2.3 17]. Formation of rare bubbles after 20 to 30 seconds is considered a negative test result.

Possible results [2.3 16]

  • Positive: immediate bubble formation
  • Negative: no bubbles forming
Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones [2.3 18]. An antigen is immobilized on a solid surface and then complexed with an antibody linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product, detecting the highly specific antibody-antigen interactions.

For example, antibodies specific to a certain bacteria, will be attached to a microtiter well, and then inserted into the sample. If the bacteria is present, the antibodies will bind to antigens, and a secondary fluorescence coupled antibody can be used to visually determine any changes. In this case, we can use an antibody for the Shiga toxin to determine if there is either E. coli O157:H7 or Shigella dysenteriae.

Antibiotic Sensitivity Test (AST)

This is a test for antibiotic resistance in pathogens and determines the best course of antibiotic treatment for a specific pathogen [2.3 19]. There are a number of methods, but disc diffusion is the most common. A bacterial inoculum is applied to the surface of a large Mueller-Hinton agar plate and up to 12 commercially prepared, fixed concentration, paper antibiotic disks are placed on the agar surface. Plates are incubated for 16 to 24 hours at 35˚C prior to determination of results. The zones of growth inhibition around each of the antibiotic disks are measured to the nearest mm. The larger the diameter, the more susceptible the organism is to the drug.

Triple Sugar Iron (TSI) Test

Involves 3 sugars, lactose, sucrose, and glucose to determine the fermentation preferences of the bacteria [2.3 20]. Glucose is added to the medium since most enteric pathogens ferment it [2.3 21]. Lactose and sucrose are added in ten times the amount of glucose, as most enteric pathogens do not ferment them. This addition causes non-pathogenic organisms that ferment these sugars to produce acid in the slant. Pathogenic organisms produce an initial acid slant but become more alkaline as growth continues. Sodium thiosulfate is added as a source of sulfur; ferrous ammonium sulfate serves as the indicator. Gas production may also occur and is seen as cracks and bubbles in the medium. The tubes are poorly oxygenated at the bottom and highly oxygenated on the top. If the sugar is oxygenated, the colour is yellow and red if the sugar is not oxygenated.

Possible Results [2.3 20] (K= alkaline, A= Acidic, NC = No Change)

  • Alkaline slant/ no change in butt (K/NC)
  • Alkaline slant/ alkaline butt (K/K)
  • Alkaline slant/ acidic butt (K/A)
  • Acidic slant/ acidic butt (A/A)
Lysine Iron Test (LIA)

The LIA test is used in differentiating members of the Enterobacteriaceae, especially Salmonella species, by H2S production and the decarboxylation or deamination of lysine [2.3 22]. The indicator is bromocresol purple; purple indicates an alkaline reaction whereas yellow indicates an acidic reaction. Sodium thiosulfate is added as the source of sulfur; ferric ammonium citrate is the indicator. This indicator turns the butt black in the presence of free H2S gas. Dextrose is the carbohydrate source and is added in a concentration of 0.1%. Bacterial pathogens that can ferment dextrose will produce acid (yellow butt and purple slant), and sometimes gas. Lysine is added to show the decarboxylation reaction, which causes an alkaline environment, seen as a purple butt. If lysine is deaminated in the presence of oxygen, red would be seen on the slant.

Possible results [2.3 23]

  • K/K = Slant purple, Butt purple = positive lysine decarboxylation (butt), negative deamination (slant)
  • A/K = Slant yellow, Butt purple = negative lysine decarboxylation (butt), negative deamination (slant)
  • R/A = Slant red, Butt yellow = negative decarboxylation (butt), positive lysine deamination (slant)
  • A/A = Slant yellow, Butt yellow = negative lysine decarboxylation (butt), positive lysine deamination (slant)
  • H2S = Black precipitate = H2S gas forming
Polymerase Chain Reaction (PCR)

PCR primers can be used to determine if any bacterial rDNA matches a certain predetermined sequence, in order to identify the bacterial strain [2.3 24]. A large number of DNA copies of the sample are made, which allows for amplification of the material and for detection of specific target genes. Increasing the temperature allows for the DNA to separate, and for primers to bind to the DNA strands. The DNA enzymes can then copy the sequence, before the reapplication of heat then separates these new strands to make four new single strands when cooled. If we already know the sequence of a specific section, such as the Shiga toxin, we can determine the presence of its component parts such as Stx, Stx1 and Stx2, encoding for Shiga toxin (Stx), Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) respectively. Depending on the type of toxin present, we can determine which components are present and use that to determine which specific type of toxin is being created.

References
  1. 1.0 1.1 1.2 1.3 1.4 1.5 Humphries, Romney M.; Linscott, Andrea J. (2015-01). "Laboratory diagnosis of bacterial gastroenteritis". Clinical Microbiology Reviews. 28 (1): 3–31. doi:10.1128/CMR.00073-14. ISSN 1098-6618. PMC 4284301. PMID 25567220. Check date values in: |date= (help)CS1 maint: PMC format (link)
  2. Chui, L.; Christianson, S.; Alexander, D. C.; Arseneau, V.; Bekal, S.; Berenger, B.; Chen, Y.; Davidson, R.; Farrell, D. J.; German, G. J.; Gilbert, L.; Hoang, Lmn; Johnson, R. P.; MacKeen, A.; Maki, A.; Nadon, C.; Nickerson, E.; Peralta, A.; Arneson, Sm Radons; Yu, Y.; Ziebell, K. (2018-11-01). "CPHLN recommendations for the laboratory detection of Shiga toxin-producing Escherichia coli (O157 and non-O157)". Canada Communicable Disease Report = Releve Des Maladies Transmissibles Au Canada. 44 (11): 304–307. doi:10.14745/ccdr.v44i11a06. ISSN 1188-4169. PMC 6449107. PMID 30996693.CS1 maint: PMC format (link)
  3. 3.0 3.1 3.2 3.3 Beaver, Linda; Rutter, Mary (2007). "Microbugz - MacConkey Agar". Retrieved 2020-04-04.
  4. 4.0 4.1 4.2 Hardy Diagnostics (1996). "XLD Agar". Hardy Diagnostics. Retrieved 2020-04-04.
  5. Hardy Diagnostics (1996). "Hektoen Enteric (HE) Agar - for stool cultures - for the isolation of enteric pathogens - Salmonella and Shigella". Hardy Diagnostics. Retrieved 2020-04-04.
  6. 6.0 6.1 Hardy Diagnostics (1996). "Brilliant Green Agar for the identification of Salmonella - Food Microbiology". Hardy Diagnostics. Retrieved 2020-04-04.
  7. Hardy Diagnostics (1996). "CRITERION Bismuth Sulfite Agar for Isolation of Salmonella". Hardy Diagnostics. Retrieved 2020-04-04.
  8. "Campylobacter Agar - CAMPY". Anaerobe Systems. Retrieved 2020-04-04.
  9. Hardy Diagnostics (1996). "MacConkey Agar with Sorbitol - (SMAC) for the detection of E. coli O157". Hardy Diagnostics. Retrieved 2020-04-04.
  10. Hardy Diagnostics (1996). "MacConkey Agar with Sorbitol, Cefixime and Tellurite (CT-SMAC)". Hardy Diagnostics. Retrieved 2020-04-04.
  11. Hardy Diagnostics (1996). "CIN Agar - for Yersinia and Aeromonas". Hardy Diagnostics. Retrieved 2020-04-04.
  12. Hardy Diagnostics (1996). "CRITERION EMB Agar, Levine - for gram negative bacilli". Hardy Diagnostics. Retrieved 2020-04-04.
  13. Bruckner, Monica Z. "Gram Staining". Microscopy. Retrieved 2020-04-04.
  14. Tankeshwar, Acharya (2012-12-29). "Oxidase test: Principle Procedure and oxidase positive organisms". Learn Microbiology Online. Retrieved 2020-04-04.
  15. Hardy Diagnostics (1996). "OxiStrips™ Oxidase Strips and OxiSticks™ Oxidase Swabs - rapid microbiology test". Hardy Diagnostics. Retrieved 2020-04-04.
  16. 16.0 16.1 Tankeshwar, Acharya (2013-10-07). "Catalase test: Principle, Procedure, Results and Applications". Learn Microbiology Online. Retrieved 2020-04-04.
  17. Hardy Diagnostics (1996). "Catalase Reagent - hydrogen peroxide for rapid bacterial catalase detection". Hardy Diagnostics. Retrieved 2020-04-04.
  18. "Overview of ELISA | Thermo Fisher Scientific - CA". Retrieved 2020-04-04.
  19. Reller, L. Barth; Weinstein, Melvin; Jorgensen, James H.; Ferraro, Mary Jane (2009-12-01). "Antimicrobial Susceptibility Testing: A Review of General Principles and Contemporary Practices". Clinical Infectious Diseases. 49 (11): 1749–1755. doi:10.1086/647952. ISSN 1058-4838. Retrieved 2020-04-04.
  20. 20.0 20.1 Tankeshwar, Acharya (2013-07-16). "Triple Sugar Iron Agar (TSI): Principle, Procedure and Interpretation". Learn Microbiology Online. Retrieved 2020-04-04.
  21. Hardy Diagnostics (1996). "Triple Sugar Iron (TSI) Agar". Hardy Diagnostics. Retrieved 2020-04-04.
  22. "Lysine Iron Agar (LIA) - for the identification of enteric bacteria)". Retrieved 2020-04-04.
  23. "Lysine Iron Agar (LIA) Slants Test - Procedure, Uses and Interpretation". Microbiology Info.com. Retrieved 2020-04-04.
  24. Barghouthi, Sameer A. (2011-10). "A Universal Method for the Identification of Bacteria Based on General PCR Primers". Indian Journal of Microbiology. 51 (4): 430–444. doi:10.1007/s12088-011-0122-5. ISSN 0046-8991. PMC 3209952. PMID 23024404. Retrieved 2020-04-04. Check date values in: |date= (help)CS1 maint: PMC format (link)

Question (iv)

(iv) What are the results expected from these tests that might allow the identification of the bacteria named in this case.
Stool Culture Results in Different Media
Medium Organism Results Image
MacKoney (MAC) Agar

[40]

E. coli O157:H7 Grow and colonies appear pink.

However, MAC agar alone cannot differentiate

between the different serotypes of E. coli, therefore,

another culture with SMAC is needed for isolation.

Figure 1. Culture of E. coli on MacConkey (MAC) agar [41]
Salmonella enterica

(serotype typhimurium)

Grow and colonies appear colourless
Campylobacter jejuni Grow and colonies appear colourless
Listeria monocytogenes Inhibited (Gram-positive)
Shigella sonnei Grow and colonies appear colourless
Yersinia enterocolitica Grow and colonies appear colourless
Xylose-lysine-deoxycholate

(XLD) Agar [42]

E. coli O157:H7 Grow and colonies appear yellow
Figure 2. Salmonella enterica colonies growing on XLD agar incubated aerobically for 24 hours at 35˚C [42].
Figure 3. Shigella colonies growing on XLD agar incubated aerobically for 24 hours at 35˚C [42].
Salmonella enterica

(serotype typhimurium)

Grow and colonies appear red with black centers
Campylobacter jejuni Grow and colonies appear yellow
Listeria monocytogenes Inhibited (Gram-positive)
Shigella sonnei Grow well and colonies appear red to pink
Yersinia enterocolitica Grow and colonies appear yellow
Hektoen enteric

(HE) agar [43]

E. coli O157:H7 Partial inhibition.

May have slight growth of yellow to salmon-coloured colonies

Figure 4. Salmonella enterica colonies growing on HE agar incubated aerobically for 24 hours at 35˚C [43].
Figure 5. Shigella colonies growing on HE agar incubated aerobically for 24 hours at 35˚C [43].
Salmonella enterica

(serotype typhimurium)

Grow and colonies appear green-bluish green.

Those that produce H2S appear as blue-green

colonies with black centre

Campylobacter jejuni Unknown
Listeria monocytogenes Inhibited (Gram-positive)
Shigella sonnei Grow and colonies appear green-bluish green
Yersinia enterocolitica Unknown
Salmonella-Shigella

(SS) agar [44]

E. coli O157:H7 Partial to complete inhibition.

Pink to rose red colonies with precipitate

Figure 6. E. coli colonies growing on SS agar incubated aerobically for 24 hours at 35˚C [44].
Figure 7. Salmonella enterica colonies growing on SS agar incubated aerobically for 24 hours at 35˚C [44].
Figure 8. Shigella colonies growing on SS agar incubated aerobically for 24 hours at 35˚C [44].
Salmonella enterica

(serotype typhimurium)

Grow and colonies appear transparent or

translucent. Those that produce H2S appear

with or without black centers

Campylobacter jejuni General Inhibition
Listeria monocytogenes Inhibited (Gram-positive)
Shigella sonnei Grow and colonies appear transparent or translucent
Yersinia enterocolitica General Inhibition
Brilliant Green

(BG) agar [45]

E. coli O157:H7 Partial to complete inhibition.

Small yellow to yellow-green colonies

Figure 9. Salmonella enterica colonies growing on BG agar incubated aerobically for 24 hours at 35˚C [45].
Salmonella Grow and colonies appear red to pink

(white colonies surrounded by a red centre) S. typhimurium growth is inhibited

Campylobacter jejuni Inhibited
Listeria monocytogenes Inhibited (Gram-positive)
Shigella sonnei Inhibited
Yersinia enterocolitica Inhibited
Bismuth Sulfite

(BS) agar [46]

E. coli O157:H7 Partial to complete inhibition.

Colonies appear to be brown-green

Figure 10. Salmonella typhi, E. coli and Proteus sp. colonies are cultured on BS agar and incubated for 48 hours [47].
Salmonella enterica

(serotype typhimurium)

Grow and colonies appear black or greenish-gray.

May have sheen. Not zones or halo effect

Campylobacter jejuni Inhibited
Listeria monocytogenes Inhibited (Gram-positive)
Shigella sonnei Inhibited
Yersinia enterocolitica Inhibited
Campylobacter Selective Media

[48]

E. coli O157:H7 Partial to complete inhibition
Figure 11. Campylobacter jejuni colonies growing on CAMPY agar [48].
Salmonella enterica

(serotype typhimurium)

Inhibited
Campylobacter jejuni Grow and colonies appear grey-white
Listeria monocytogenes General inhibition
Shigella sonnei Inhibited
Yersinia enterocolitica Partial to complete inhibition
Sorbitol-MacConkey

(SMAC) agar [49]

E. coli O157:H7 Grow and colonies appear clear.

No fermentation of sorbitol. (Other E. coli strains’ colonies appear pink)

Figure 12. E. coli O157:H7 colonies growing on SMAC agar incubated aerobically for 24 hours at 35˚C [49].
Figure 13. Other E. coli serotype colonies growing on SMAC agar incubated aerobically for 24 hours at 35˚C [49].
Salmonella enterica

(serotype typhimurium)

Inhibited
Campylobacter jejuni Inhibited
Listeria monocytogenes Inhibited
Shigella sonnei Inhibited
Yersinia enterocolitica Inhibited
MacConkey agar

with Sorbitol, Cefixime, and Tellurite

(CT-SMAC) [50]

E. coli O157:H7 Grow and colonies appear clear.

No fermentation of sorbitol. (Other E. coli colonies strains appear

pink, ferment sorbitol or are partially to

completely inhibited)

Figure 14. E. coli O157:H7 colonies growing on CT-SMAC agar incubated aerobically for 24 hours at 35˚C [50].
Salmonella enterica

(serotype typhimurium)

Inhibited
Campylobacter jejuni Partial to complete inhibition
Listeria monocytogenes Inhibited
Shigella sonnei Inhibited
Yersinia enterocolitica Partial to complete inhibition
Cefsulodin-Irgasan-novobiocin

(CIN) agar [51]

E. coli O157:H7 Partial to complete inhibition
Figure 15. Yersinia enterocolitica colonies growing on CIN agar incubated aerobically for 24 hours at 35˚C [51].
Figure 16. Aeromonas colonies growing on CIN agar incubated aerobically for 24 hours at 35˚C [51].
Salmonella enterica

(serotype typhimurium)

Partial to complete inhibition
Campylobacter jejuni Partial to complete inhibition
Listeria monocytogenes Partial to complete inhibition
Shigella sonnei Partial to complete inhibition
Yersinia enterocolitica Grow and colonies appear with

a red center and transparent border

Eosin-Methylene-Blue

(EMB) agar [52]

E. coli O157:H7 Grow and colonies are blue-black

centered with green metallic sheen

Figure 17. E. coli colonies growing on EMB agar incubated aerobically for 24 hours at 35˚C [52].
Figure 18. Salmonella enteric colonies growing on CIN agar incubated aerobically for 24 hours at 35˚C [52].
Salmonella enterica

(serotype typhimurium)

Grow and colonies appear colourless to amber
Campylobacter jejuni Unknown
Listeria monocytogenes Inhibited
Shigella sonnei Grow and colonies appear colourless to amber
Yersinia enterocolitica Grow and colonies appear blue-black

Table 1. Results of stool culture for E. coli O157:7, Salmonella enterica (serotype typhimurium), Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, Shigella sonnei and Yersinia enterocolitica on various culture media.

Stool Sample Workup Results
Test Organism Results Image
Gram Staining E. coli O157:H7 Gram-negative bacilli; stains pink N/A
Salmonella enterica

(serotype typhimurium)

Gram-negative bacilli; stains pink
Campylobacter jejuni Gram-negative helical-shaped; stains pink
Listeria monocytogenes Gram-positive bacilli; stains purple
Shigella sonnei Gram-negative bacilli; stains pink
Yersinia enterocolitica Gram-negative bacilli; stains pink
Oxidase Test

[34]

E. coli O157:H7 Negative
Figure 19. Pseudomonas aeruginosa applied to an OxiStrip. Development of a blue/purple colour indicates a positive oxidase test result [54]
Figure 20. Pseudomonas aeruginosa applied to an OxiSwab. Development of a blue/purple colour indicates a positive oxidase test result [54]
Figure 21. E. coli applied to an OxiStrip. No development of a blue/purple colour indicates a negative oxidase test result [54].
Salmonella enterica

(serotype typhimurium)

Negative
Campylobacter jejuni Positive
Listeria monocytogenes Negative
Shigella sonnei Negative
Yersinia enterocolitica Negative
Catalase Test

[55]

E. coli O157:H7 Positive
Figure 22. Positive catalase test (left) and negative catalase test (right) [55].
Salmonella enterica

(serotype typhimurium)

Positive
Campylobacter jejuni Positive
Listeria monocytogenes Positive
Shigella sonnei Positive
Yersinia enterocolitica Positive
Enzyme-Linked

Immunosorbent Assay

(ELISA) [56]

Antigen: shiga toxin

E. coli O157:H7 Colour change = positive N/A
Salmonella enterica

(serotype typhimurium)

No colour change = negative
Campylobacter jejuni No colour change = negative
Listeria monocytogenes No colour change = negative
Shigella sonnei Colour change = positive
Yersinia enterocolitica No colour change = negative
Antibiotic Sensitivity Test

(AST)

E. coli O157:H7 Resistant (smaller zones of inhibition)

to erythromycin, amoxicillin and tetracycline.

Sensitive (larger zones of inhibition) to

nitrofurantoin, norfloxacin, gentamicin and

ciprofloxacin. Regular monitoring of antimicrobial susceptibility is recommended [58].

Figure 23. Disk diffusion test for antibiotic sensitivity [57].
Salmonella enterica

(serotype typhimurium)

Resistant (smaller zones of inhibition)

to amoxicillin, ampicillin, tetracycline

and co-trimoxazole.

Sensitive (larger zones of inhibition)

to ciprofloxacin and ceftriaxone [59].

Campylobacter jejuni Resistant (smaller zones of inhibition)

to fluoroquinolones and ciprofloxacin. Sensitive (larger zones of inhibition) to erythromycin [60]

Listeria monocytogenes Resistant (smaller zones of inhibition)

to fluoroquinolones and cephalosporins. Sensitive (larger zones of inhibition) to erythromycin , clarithromycin,

streptomycin, gentamicin, vancomycin,

imipenem, trimethoprim and chloramphenicol

[61].

Shigella sonnei Resistant (smaller zones of inhibition)

to amoxicillin, ampicillin chloramphenicol,

tetracycline, and were multidrug resistant. Sensitive (larger zones of inhibition) to ciprofloxacin and ceftriaxone [59]

Yersinia enterocolitica Resistant (smaller zones of inhibition)

to amoxiclav. Sensitive (larger zones of inhibition) to azithromycin, gentamicin and levofloxacin.

Triple Sugar Iron

(TSI) Test [63]

E. coli O157:H7 Growth.

Yellow slant, yellow butt, gas positive, no H2S

Figure 25. E. coli growing on TSI agar incubated aerobically for 24 hours at 35˚C [63].
Figure 24. Salmonella enterica growing on TSI agar incubated aerobically for 24 hours at 35˚C [63].
Salmonella enterica

(serotype typhimurium)

Growth.

Red slant, yellow butt, gas positive, black butt (H2S produced)

Campylobacter jejuni N/A
Listeria monocytogenes N/A
Shigella sonnei Growth.

Red slant, yellow butt, no gas, no H2S produced

Yersinia enterocolitica Growth.

Glucose and lactose/sucrose fermented OR Only glucose fermented; peptone utilized

Lysine Iron Agar

(LIA) Test [64]

E. coli O157:H7 Growth.

Yellow slant, purple butt OR Yellow slant and butt

Figure 25. Salmonella typhimurium growing on Lysine Iron Agar (LIA) incubated aerobically for 24 hours at 35˚C [64].
Figure 26. Shigella growing on Lysine Iron Agar (LIA) incubated aerobically for 24 hours at 35˚C [64].
Salmonella enterica

(serotype typhimurium)

Growth.

Purple slant and butt, blackening with H2S positive

Campylobacter jejuni N/A
Listeria monocytogenes N/A
Shigella sonnei Growth.

Purple slant, yellow butt, H2S negative

Yersinia enterocolitica Growth.

Yellow slant, purple butt OR Yellow slant and butt

Polymerase Chain Reaction

(PCR) [66]

E. coli O157:H7 Positive N/A
Salmonella enterica

(serotype typhimurium)

Negative
Campylobacter jejuni Negative
Listeria monocytogenes Negative
Shigella sonnei Positive
Yersinia enterocolitica Negative

Table 2. Results of different biochemical tests of E. coli O157:7, Salmonella enterica (serotype typhimurium), Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, Shigella sonnei and Yersinia enterocolitica performed as a part of stool culture workup [34].

References

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[41] Smith KP. 2019. The Origin of MacConkey Agar, American Society for Microbiology.

[42] Hardy Diagnostics. 1996. XLD Agar, Hardy Diagnostics.

[43] Hardy Diagnostics. 1996. Hektoen Enteric (HE) Agar, Hardy Diagnostics.

[44] Hardy Diagnostics. 1996. SS Agar, Hardy Diagnostics.

[45] Hardy Diagnostics. 1996. Brilliant Green Agar, Hardy Diagnostics.

[46] Hardy Diagnostics. 1996. Criterion Bismuth Sulfite Agar, Hardy Diagnostics.

[47] CDC. 1964. Public Health Image Library (PHIL), U.S. Department of Health & Human Services.

[48] Hardy Diagnostics. 1996. Campylobacter Selective Agar (CAMPY), Hardy Diagnostics.

[49] Hardy Diagnostics. 1996. MacConkey Agar with Sorbitol (SMAC), Hardy Diagnostics.

[50] Hardy Diagnostics. 1996. MacConkey Agar with sorbitol, cefixime, and tellurite (CT-SMAC), Hardy Diagnostics.

[51] Hardy Diagnostics. 1996. CIN Agar, Hardy Diagnostics.

[52] Hardy Diagnostics. 1996. Criterion EMB (Eosin Methylene Blue) Agar, Levine, Hardy Diagnostics.

[53] Coico R. 2005. Gram staining. Curr Protoc Microbiol. https://doi.org/10.1002/9780471729259.mca03cs00

[54] Hardy Diagnostics. 1996. Oxistrips Oxidase Strips and Oxisticks Oxidase Swabs, Hardy Diagnostics.

[55] Hardy Diagnostics. 1996. Catalase Reagent, Hardy Diagnostics.

[56] ThermoFisher Scientific. n.d. Overview of ELISA, ThermoFisher Scientific.

[57] James HJ, Ferraro MJ. 2009. Antimicrobial Susceptibility Testing: A Review of General Principles and Contemporary Practices. Clin Infect Dis. https://doi.org/10.1086/647952

[58] Kibret M, Abera B. 2011. Antimicrobial Susceptibility Patterns of E. coli from Clinical Sources in Northeast Ethiopia. Afr Health Sci. https://doi.org/10.4314/ahs.v11i3.70069

[59] Assefa A, Girma M. 2019. Prevalence and antimicrobial susceptibility patterns of Salmonella and Shigella isolates among children aged below five years with diarrhea attending Robe General Hospital and Goba Referral Hospital, South East Ethiopia. Trop Dis. https://doi.org/10.1186/s40794-019-0096-6

[60] Luangtongkum T, Jeon B, Han J, Plummer P, Logue CM, Zhang Q. 2009. Antibiotic resistance in Campylobacter: emergence, transmission and persistence. Future Microbiol. https://doi.org/10.2217/17460913.4.2.189

[61] Arslan S, Özdemir F. 2020. Prevalence and antimicrobial resistance of Listeria species, and virulence genes, serotyping, PCR-RFLP and PFGE typing of Listeria monocytogenes isolated from retail ready-to-eat-foods. FEMS Microbiol Lett. https://doi.org/10.1093/femsle/fnaa006

[62] Nwankwo EO, Nasiru MS. 2011. Antibiotic sensitivity pattern of Staphylococcus aureus from clinical isolates in a tertiary health institution in Kano, Northwestern Nigera. Pan Afr Med J. https://doi.org/10.4214/pamj.v8i1.71050

[63] Hardy Diagnostics. 1996. Triple Sugar Iron (TSI) Agar, Hardy Diagnostics.

[64] Hardy Diagnostics. 1996. Lysine Irone Agar (LIA), Hardy Diagnostics.

[65] Valones MA, Guimarães RL, Brandão LA, deSouza PR, de Albuquerque Tavares CarvalhoA, Crovela S. 2009. Principles and application of polymerase chain reaction in medical diagnostic fields: a review. Braz J Microbiol. https://doi.org/10.1590/S1517-83822009000100001

[66] Aryal S. 2018. Polymerase Chain Reaction (PCR) – Principle, Procedure, Types, Applications and Animation, Microbiology Info.

[67] Austin JW, Pagotto FJ. 2003. Detection of Foodborne Pathogens and their Toxin. In Trugo L, Fingals PM (ed), Encyclopedia of Food Sciences and Nutrition, 2nd edition. Elsevier Science Ltd, Baltimore, MD.

[68] Creative Proteomics. n.d. MALDI-TOF Mass Spectrometry, Creative Proteomics.

Q3. Bacterial Pathogenesis Questions

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

Question (i)

(i) Encounter: where does this 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

E. coli O157:H7 is defined as an Enterohaemorrhagic E. Coli (EHEC), a subcategory of the Shiga toxin-producing E.coli (STEC). EHEC primarily cause hemorrhagic colitis (HC) or bloody diarrhea in humans (1). The O157:H7 strain is a particularly important food and waterborne pathogen that results in these life-threatening conditions (2). It may also progress to potentially fatal hemolytic uremic syndrome (HUS) (1). Typically, it resides in the lower intestine of warm-blooded organisms (3). This includes the lower intestine of cattle (which is most common), humans, and other ruminants such as sheep, goats and deer (3). In humans, EHEC will enter the body through ingestion. It resides in the colon where it can bind to intestinal mucosa and release its toxins (2,4). It is important to note that commensal E. coli strains are normal habitants of the human intestinal tract, where the bowel system is usually colonized within the first 40 hours after birth. Although a part of the normal flora, virulence genes can be acquired from the ecosystem and can lead to the bacteria’s increased pathogenicity (5).

Geographical Distribution

While this bacteria is found world-wide, incidence rates of E. coli O157:H7 infection vary between countries. This is demonstrated by the following statistics: in 2004 the number of laboratory confirmed cases in the European Union and Norway was 1.3 cases per 100 000 population while in the same year the incidence in the USA was 0.9 cases per 100 000 people (6).

Since discovering this bacteria, there have been large food-borne outbreaks in North American, Europe and Japan. As of 2014, E. coli O157:H7 has been found to be the etiological agent for 73,000 illnesses and 61 deaths annually in the US (2,5). The outbreak surveillance data from the US Centre for Disease Control reports that E. coli O157:H7 infections are decreasing after its peak in 1999. However, large outbreaks and sporadic cases continue to occur (7). To make matters worse, treating E. coli with antimicrobials has been shown to worsen disease progression. Antibiotics are thought to result in bacterial lysis which induces the potent release of Shiga toxins (8). Without an effective treatment, this generates a public health dilemma (5). Despite the current reports, comprehensive data on EHEC infections is lacking. In particular, data on EHEC from developing countries where populations are often exposed to contaminated environments is especially limited (6). In the summer months, elevated temperatures may be responsible for favouring bacterial proliferation and survival (4).

Survival Mechanisms

The bacteria can survive a large range of environments due to its altered genotype conveying hardy survival characteristics that exceeds those found in commensal E. coli (2). This enables the bacteria to survive harsh conditions frequently encountered within the human food chain (2). Remarkably so, E. coli O157:H7 has been found to survive for months in manure and water-trough sediments (3). Furthermore, E. coli O157:H7 are facultative anaerobes. In anaerobic conditions, the bacteria can grow by means for fermentation or utilize NO3, NO2 or fumarates as final electron acceptors for cellular respiration. Their ability to grow in the presence of absence of oxygen adapts the bacterium to the intestinal (anaerobic) and extra-intestinal (aerobic/anaerobic) habitats (1). E. coli are also able to metabolically transform glucose into all of the macromolecular components that make up the cell, contributing to the bacteria’s versatility in different environments (1).

On top of all this, one of the bacteria’s most notable abilities is its ability to respond to environmental signals. This includes changes in chemicals, pH, temperature and osmolarity (1). Below are specific examples employed by E. coli O157:H7.

  • In the presence of absence of certain chemicals, the bacteria can move towards it or away (1).
  • If a specific cell or surface receptor is recognized, the bacteria can stop swimming to grow fimbriae for attachment to the host (1).
  • The bacteria has Acid Resistance (AR) or the ability to protect itself from extremely low pH environments (<pH 3.0). E. coli O157:H7 has 3 AR systems in which the molecular control and requirements vary. In addition, the production of exopolysaccharide (EPS) is associated with heat and acid tolerance. For these mechanisms, E. coli O157:H7 can survive stomach acid, which grants the bacteria a chance to colonize in the intestines and cause infection (7). The low infectious dose of E. coli O157:H7 (10-100 cells) has also been found to be associated with Acid Resistance. This confers for a highly infectious bacteria (1,7).
  • Changes in temperature and osmolarity can lead to changes in the bacteria’s outer membrane porin diameter to accommodate the influx of nutrients or to exclude toxic substances (1).

With these complex regulatory mechanisms that adjust the bacteria to specific environments, another benefit for the bacteria is that molecular resources and nutrients are not wasted when they are not needed (1). Despite all this, E. coli O157:H7 is heat sensitive, which is why it is important to cook food thoroughly (3). This Shiga toxin-producing E.coli (STEC) can grow in temperatures ranging from 7-50 degrees Celsius, where its optimum temperature is 37 degrees Celsius (3). It is destroyed by thorough cooking of food when all parts of the product reach a temperature of 70 degrees Celsius or higher (3). 

Transmission

Cattle is the primary reservoir for E. coli O157:H7, which was found by tracing outbreaks of Shiga Toxin enterohemorrhagic diarrhea to domesticated animals (2). For this, E. coli O157:H7 infection can be caused by zoonotic transmission after the consumption of undercooked meat or inadequately pasteurized dairy products (2). Other causes include exposure to contaminated water from drinking sources, swimming pools and lakes, inadequately washed leafy greens and fruits, unpasteurized drinks (such as apple juice) and direct contact with animals in petting farms (2). The contamination of fruits and vegetables occurs through fecal contamination in agricultural irrigation water or runoff (2). In fact, E. coli has been observed to enter the lettuce plant through the root system and migrate to the edible portion of the plant (5). E. coli O157:H7 can also be passed from person to person through the fecal-oral route or through fecal shedding (which account for 11% of infections) (2).

How did Ronnie get infected?

When Ronnie ate 2 hamburgers that were not thoroughly cooked, this was likely how the E. coli O157:H7 entered his alimentary tract and caused infection. Evidently, the meat was contaminated with E. coli O157:H7, and because it was not thoroughly cooked, the bacteria were able to continue living in the meat and subsequently infect Ronnie who ate it. As explained earlier, this is quite probable as human exposure to contaminated meat is a common mode of transmission for E. coli. Furthermore, diarrhea often develops 3 days after exposure to contaminated food which was why the Doctor asked Ronnie what he had eaten in the past week (2)

References

1.         Pathogenic E. coli [Internet]. [cited 2020 Mar 25]. Available from: http://textbookofbacteriology.net/e.coli.html

2.         Gossman W, Wasey A, Salen P. Escherichia Coli (E Coli 0157 H7). In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 [cited 2020 Mar 24]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK507845/

3.         E. coli [Internet]. [cited 2020 Mar 24]. Available from: https://www.who.int/news-room/fact-sheets/detail/e-coli

4.         Fatima R, Aziz M. Enterohemorrhagic Escherichia Coli (EHEC). In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 [cited 2020 Mar 26]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK519509/

5.         Md Tamrin S. A Review on Escherichia coli O157:H7-The Super Pathogen. Health Environ J. 2014 Oct 1;5:118–34.

6.         Havelaar AH, Cawthorne A, Angulo F, Bellinger D, Corrigan T, Cravioto A, et al. WHO Initiative to Estimate the Global Burden of Foodborne Diseases. The Lancet. 2013 Jun;381:S59.

7.         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.   

Question (ii)

(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).

Commensal E. coli strains are normal habitants of the human intestinal tract, where the bowel system is usually colonized within the first 40 hours after birth (1). On the other hand EHEC, such as E. coli O157:H7, will enter the body through ingestion of contaminated food or water(2). Colonization and invasion of the digestive or mucosal surfaces requires bypassing physical barriers, such as the mucosa and a thin layer of smooth muscles(3). Such strategies can include overcoming the mucus layer via production of proteases the target host mucins (that normally function to prevent entry of pathogens). The bacteria also has plasmid-encoded invasion factors which permits invasion of the mucosa, while encoding cytotoxic enterotoxins that induces tissue damage in the areas they colonize (3). Cytotoxic enterotoxins encoded by bacteriophage DNA or plasmids can induce the secretion of water and electrolytes, which are enabling resources for bacterial colonization. This results in watery diarrhea for the host.

The infectious process of E. coli O157:H7 is initiated by the ingestion of a small number of bacteria. In response to acid stress that E.coli O157:H7 encounters during its passage in the stomach and gastrointestinal tract, E.coli O157:H7 activates its sigma factor RpoS which upregulates the expression of acid shock proteins that allow E.coli to survive in acidity as low as pH 2(4). The Rpos sigma factor can also regulate the expression of proteins that increase resistance to heat, salt and desiccation when the bacterium is exposed to stress. Furthermore, the exopolysaccharide produced by the bacteria helps with acid resistance(3).

The bacterial fimbrial attachment onto the mucosa of the distal ileum and the colon followed by translocation of the bacterial Tir protein to the host cell membrane is how bacterial attachment starts(5).  Multiple fimbrae (thread like- structures) protruding from the bacterial surface contribute to adherence of this organism to host cells(5)(6). Type 1 fimbrae – the most common adhesin produced in E.coli allows the pathogen to bind to glycoproteins that contain mannose on the surface of host cells(6).

E. coli O157:H7 encodes the Pathogenic Island Locus of Enterocyte Effacement (LEE). A pathogenic Island is a group of genes on a chromosome that encode virulence factors. Pathogenic islands can be aquired via horizontal gene transfer and allow bacteria to evolve. The pathogenic island LEE contains genes for the type III secretion system (T3SS),  intimin and its translocated receptor Tir (2). It also contains genes for several secreted proteins (eg. Esp) which are important in modification of host cell signal transduction during the formation of  attaching and effacing (A/E) lesions(2).

Attachment of EHEC to intestinal epithelial cells is necessary for colonization and is mediated by localized actin assembly beneath the attached bacteria. The formation of these actin “pedestals” is dependent on effectors translocated via a T3SS (7). Tir, an effector required for pedestal formation, localizes in the host cell plasma membrane after being translocated by T3SS and promotes attachment of bacteria to mammalian cells by binding to the EHEC outer surface protein Intimin. Attached bacteria stimulate host cell actin polymerization accumulation, resulting in a raised attachment actin pedestal formation (A/E) lesion(2). The A/E lesion is characterized by effacement of microvilli and bacterial adherence to the epithelial cell membrane(2). The presumed functions of these pedestals are prevention of dislodgement of the bacterium during the host diarrheal response and inhibition of bacterial phagocytosis(5).

           The formation of a biofilm, consisting of one or many types of bacteria, can adhere to any surface. This forms a protected micro-environment that allows for growth and replication of bacteria in difficult environments(3). Biofilms may also serve as a platform for the aggregation and creation of antimicrobial bacteria, which overtime become more resistant to antibiotics and host defenses if the biofilms are maintained(3).

References

1.         Pathogenic E. coli [Internet]. [cited 2020 Mar 26]. Available from: http://www.textbookofbacteriology.net/e.coli.html

2.         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.

3.         Ribet D, Cossart P. How bacterial pathogens colonize their hosts and invade deeper tissues. Microbes Infect. 2015 Mar 1;17(3):173–83.

4.         Dong T, Schellhorn HE. Global effect of RpoS on gene expression in pathogenic Escherichia coli O157:H7 strain EDL933. BMC Genomics. 2009 Aug 3;10(1):349.

5.         Rahal EA, Kazzi N, Nassar FJ, Matar GM. Escherichia coli O157:H7—Clinical aspects and novel treatment approaches. Front Cell Infect Microbiol [Internet]. 2012 [cited 2020 Mar 27];2. Available from: https://www.frontiersin.org/articles/10.3389/fcimb.2012.00138/full

6.         Md Tamrin S. A Review on Escherichia coli O157:H7-The Super Pathogen. Health Environ J. 2014 Oct 1;5:118–34.

7.         Battle SE, Brady MJ, Vanaja SK, Leong JM, Hecht GA. Actin pedestal formation by enterohemorrhagic Escherichia coli enhances bacterial host cell attachment and concomitant type III translocation. Infect Immun. 2014 Sep;82(9):3713–22.

Question (iii)

(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.

E. coli O157:H7 has developed various mechanisms to inhibit internalization by phagocytic cells. (1) EHEC inhibits internalization of both un-opsonized and opsonized bacteria through the actions of its effector proteins. The first effector protein, EspF, targets PI3K signaling. In doing this, it inhibits its activation rather than recruitment to the site of attachment, thus preventing phagocytosis. Furthermore, EspB binds various members of the myosin superfamily. This prevents the interaction of Myosin -1c and actin, and in turn inhibits cis-phagocytosis. EspH, the effector that inactivates Rho GTPase signaling, has demonstrated inhibition of both cis- and trans-phagocytosis. Finally, effector protein EspJ, has been proven capable of preventing phagocytosis of both IgG and complement opsonized particles. Through these effector proteins, E. coli O157:H7 is able to prevent internalization by phagocytosis.

Upon translocation across the mucosal lining via M cells, E. coli is taken up by the resident macrophages. Through this, it is able to survive phagocytosis through use of its various virulence factors. (20) E. coli is able to occupy an intracellular state as it multiplies inside the macrophage until cell death is induced. This results in the subsequent release of various bacterial virulence factors including endotoxins and Shiga toxins. Shiga toxins are produced and secreted by E. coli O157:H7, and can cause significant damage to host cells. The potent virulent effects of Shiga toxin, or Stx, will be discussed in further detail in the following questions.  The secretion of Shiga toxins is significant, as it allows for these toxins to spread to various tissues through the bloodstream where they can then bind to Shiga toxin receptors, or Gb3. Gb3, a glycosphingolipid that is composed of a lipid or a ceramine component and a trisaccharide. (4) The cellular function of Gb3 remains unknown, however those with excess Gb3 tend to exhibit kidney disease. Gb3 is present on several human epithelial and endothelial cells including vascular endothelial, renal tubular, glomerular epithelial, and intestinal epithelial cells, thus resulting in cell death at these sites. This is significant, as this spread of the pathogen through various parts of the body is what results in the secondary infection sites.

Although bacteremia by infection with E. coli O157:H7 is rare, the Shiga toxins that are produced and secreted by the bacteria can enter the blood and result in systemic damage. (6) Shiga toxins bind to Gb3, a glycosphingolipid that is comprised of a lipid or a ceramine component and a trisaccharide. (4) The cellular function of Gb3 remains unknown, however individuals with excess Gb3 tend to exhibit kidney disease. Although during infections with E. coli O157:H7 the bacteria most often remain at the site of infection, it is a possibility that the bacteria may spread to secondary sites of infection. The bacteria hone in on these secondary sites of infection due to their expression of Gb3, as this is what Shiga toxin binds to. The host damage that occurs as a result of Shiga toxin will be further described in the following question.

EHEC has demonstrated the ability to form distinct bacterial communities known as macrocolonies. These macrocolonies encompass multiple host cells. (10) It has been made evident that Tir, EspFU, and the host Arp2/3 complex are all critical for the expansion of macrocolonies. The process of colonization involves EspFU (EHEC effector protein)-mediated actin assembly. EHEC is capable of reorganizing actin, however the pathogen must remain extracellular and signal across the plasma membrane in order to create attaching and effacing lesions. These lesions are characterized by their intimate attachment of the bacteria to the host cell membrane, microvilli loss, and the subsequent assembly of filamentous actin “pedestals” underneath the bacteria. (10) To generate these actin pedestals, EHEC must use the T3SS to inject effector proteins into the host cells. The intimin receptor, Tir, is translocated and inserted into the plasma membrane. It then binds to the adhesin intimin that is expressed on the bacterial surface, forming a close attachment to the host cell. In EHEC infection, Tir recruits the host proteins IRTKS and IRSp53. These host proteins interact with EspFU, the EHEC effector protein, and a multivalent and highly potent activator of N-WASP. N-WASP is an adaptor/scaffold protein that regulates actin polymerization by stimulating the actin-nucleating activity of the Arp2/3 complex. N-WASP is involved in several processes including mitosis and cytokinesis through its role of regulating actin polymerization. Along with CDC42, it is involved in the extension and maintenance of forming thin, actin-rich surface projections known as filopodia. (19) This results in the formation of actin pedestals. Their formation is vital to the infection, as they allow the bacteria to glide along the tops of cultured cells. The actin pedestals enable bacterial movement, thus allowing for the bacteria to further infect other host cells.

References:

1. Clements A, Young JC, Constantinou N, Frankel G. Infection strategies of enteric pathogenic Escherichia coli. Gut microbes. 2012 Mar 1;3(2):71-87.

4. Melton-Celsa AR. Shiga toxin (Stx) classification, structure, and function. Microbiology spectrum. 2014;2(2).

6. Sheng H, Wang J, Lim JY, Davitt C, Minnich SA, Hovde CJ. Internalization of Escherichia coli O157: H7 by bovine rectal epithelial cells. Frontiers in microbiology. 2011 Feb 22;2:32.

10. Velle KB, Campellone KG. Extracellular motility and cell-to-cell transmission of enterohemorrhagic E. coli is driven by EspFU-mediated actin assembly. PLoS pathogens. 2017 Aug 3;13(8):e1006501.

19. (n.d.). Retrieved from https://www.phosphosite.org/proteinAction?id=3960&showAllSites=true)

20. Paton, J. C., & Paton, A. W. (1998). Pathogenesis and Diagnosis of Shiga Toxin-Producing Escherichia coli Infections. Clinical Microbiology Reviews, 11(3), 450–479. doi: 10.1128/cmr.11.3.450

Question (iv)

(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?

Direct bacterial damage is mediated mostly by Shiga-like toxins (Stx) in EHEC infection. In the colon, the pathogen of concern causes haemorrhagic colitis (HC). Stx starts off an inflammation of the gut lining at the recto-anal junction (RAJ), which yields hemorrhage and edema in the lamina propria. Localized necrosis of the gut tissue and neutrophil infiltration are characteristic of HC, and result in the bloody diarrhea, as the one presented in Ronnie’s case.

Shiga toxin acts on the lining of the blood vessels, also known as the vascular endothelium. (2) The toxin’s B subunits bind to Gb3, a component of the host cell membrane. This binding allows the complex to enter the cell. Following this, the A subunit interacts with the ribosomes to inactivate them. The Shiga toxin A subunit is an N-glycosidase which modifies the RNA component of the ribosome to inactivate it. This brings a halt to protein synthesis and results in cell death. The death of these cells causes the lining of the vascular endothelium to break down, thus resulting in hemorrhage. As Shiga toxins are released into the gut lumen, they facilitate the destruction of the vascular endothelium in the GI tract. Thus, some of the first symptoms we see from this infection are often bloody diarrhea as a result of vascular endothelium hemorrhage, and stomach cramping. Pathogenic E. coli strains can produce a toxin that promotes the apoptosis of host cells in the intestine and cells in the endothelial lining of blood vessels. This lysis of endothelial cells leads to bloody diarrhea as the blood vessels are leaking. The exotoxin causes many of the other signs and symptoms, such as abdominal cramps and periumbilical tenderness. The lysis of the host cells destroys the microvilli structure of the intestines. Since the structure of the gut is not maintained, many molecules can pass through this barrier and cause tenderness in the abdominal area. The exotoxin also induces the recruitment of neutrophils to the site of infection (4). Neutrophils will cause the release of chemokines and this leads to gut inflammation. The release of chemokines and the activation of the immune system and of the complement system are manifested in the low-grade fever. Most people recover from this infection within 5-7 days.

If the bacteria are successful at invading the gut and gain access to the bloodstream, the produced bacterial Stx can be translocated to the kidneys. The renal endothelium express a large number of Gb3, a glycolipid receptor for Stx (3). The renal endothelium then becomes destroyed (induced apoptosis by Stx) and the blood may leak into the urinary space. An EHEC-infected person may detect hematuria (blood in urine), a characteristic sign of hemolytic-uremic syndrome (HUS). In severe cases, HUS may progress to acute renal failure (ARF). Fortunately, Ronnie does not display signs or symptoms of HUS. Additionally, CNS endothelium expresses Gb3, implying the possibility of secondary infection and bacterial damage in the CNS. If the CNS is affect, it results in neonatal meningitis, which affects 1/ 2,000-4000 infants. ​E. coli strains spread to this infection site by invading the blood stream of infants through either the nasopharynx or the GI tract, and are then carried to the meninges (2). The major determinant of virulence among strains of ​E. coli that cause neonatal meningitis is the K-1 antigen. The K-1 antigen inhibits phagocytosis, complement, and responses from the host’s immunological mechanisms. It is believed that siderophores and endotoxins are involved as well. It has been demonstrated through epidemiological studies that pregnancy is associated with higher rates of K-1 colonization. Thus, the K-1 strains are what become involved in the subsequent cases of meningitis in newborns. It appears that the infant GI tract is the portal of entry into the bloodstream. Fortunately, catastrophic sequelae such as neonatal meningitis are rare, even though colonization is rather common.

Shiga toxin is also known to have prominent effects on the lungs. In HUS, pulmonary involvement is uncommon, however potentially life-threatening. Sudden unexpected pulmonary edema and pulmonary hemorrhage have both been described at the ultimate cause of death of patients suffering from HUS. The presence of Gb3 and CD77 expressing cells in lung tissue was confirmed (3). This is significant as Stx binds to Gb3, suggesting that not only vascular endothelial cells contain Gb3 expressing cells, but that portions of the pulmonary epithelium do as well. The study indicates that certain sections of the lung epithelium express receptors for Stx, and can be directly damaged during STEC infection. Therefore, both vascular endothelium and lung epithelium are major targets to Shiga toxins. It is evident that the damage mediated by Shiga toxin contributes to the pathogenesis of pulmonary involvement that is associated with Stx-induced HUS.

The infectious colonies of bacteria can be removed completely by the host immune systems. With consistent hydration and rest, patients can fully recover their health within a week since the onset of infection. Like Salmonella, B lymphocytes proliferates and differentiate into antibody-secreting plasma cells and memory B cells. Therefore, upon re-infection, the presence of antibodies and memory B cells specifically against E.coli O157:H7 will act quicker than the initial encounter to the pathogen. Other serovars of E.coli may not induce the same immune response if they don’t share particular antigenic sequence recognized by antibodies and memory B cells.

Recommended treatment: There are difficulties with providing protection against human gastrointestinal infections because potential effectors (i.e. secretory IgA or lymphocytes trafficking to the gut) are difficult to obtain and transfer to recipients who have never had the initial infections. In addition, vaccines protective against different types of E. coli have yet to be developed (1).

Preventative measures one can take to reduce the likelihood of exposure to both pathogens and reducing the rate of infections includes:

  • Establishing control measures at all stages of the food chain, including agricultural production, to processing, manufacturing and preparation of foods in both commercial establishments and at home (same as E. coli)
  • Careful monitoring of contact between infants/young children and pet animals that may be carrying.
  • Proper hand sanitation and washing of fruits/vegetables and before preparing food.
  • Separating raw and cooked food, ensuring meats and other foods are cooked thoroughly (i.e. centre of the food reaches at least 70°C), and still hot when served.
  • Avoiding raw milk and other meat products.
  • Changing animal slaughtering practices to reduce cross contamination of animal carcasses, which protects processed foods from contamination.

References

  1. Escherichia Coli O157:H7 Infection.” Canadian Journal of Gastroenterology 27.5 (2013): 281-5. Wijburg, O. L. C., & Strugnell, R. A. (2006). Mucosal immune responses to escherichia coli and salmonella infections. EcoSal Plus, 2(1) doi:10.1128/ecosalplus.8.8.12 World Health Organization (WHO). (2016, October). E. coli. Retrieved March 22, 2017, from http://www.who.int/mediacentre/factsheets/fs125/en/
  2. Pathogenic E. coli [Internet]. Available from: http://textbookofbacteriology.net/tuberculosis.html
  3. Uchida H, Kiyokawa N, Taguchi T, Horie H, Fujimoto J, Takeda T. Shiga toxins induce apoptosis in pulmonary epithelium-derived cells. Journal of Infectious Diseases. 1999 Dec 1;180(6):1902-11
  4. Welinder-Olsson, C., & Kaijser, B. (2005). Enterohemorrhagic Escherichia coli (EHEC). Scandinavian Journal of Infectious Diseases, 37(6-7), 405–46. doi: 10.1080/00365540510038523

Q4. The Immune Response Questions

Question (i)

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

Our immune system is broken down into the (InformedHealth.org, 2006) innate immune system and the (Janeway, 2005) adaptive immune system (InformedHealth.org, 2006). The innate immune system is general and non-specific for pathogens, whereas the adaptive immune system is specialized and specific. The innate immune system consists of both cellular and humoral elements, as well as anatomical (chemical, physical and biological) barriers which all work together to protect against and destroy microorganisms. The innate immune system, although generic, is able to discriminate effectively between host cells and pathogens and is thus able to provide initial defenses and also contribute to the activation of the adaptive immune system. The adaptive immune system is capable of making distinctions between pathogens, and is thus more specific than the innate immune system. Similar to the innate immune system, the adaptive immune system also comprises both cellular and humoral elements.

The major differences between the innate and adaptive immune systems are summarized in Table 1 below:

Innate Adaptive
Cellular Elements
  • Natural killer cells
  • Phagocytes:
    • Macrophages
    • Neutrophils
    • Dendritic cells
  • B Cells
    • Plasma cells
  • T cells
    • T helper cells
    • Cytotoxic T cells
Humoral Elements
  • Complement proteins
  • Antimicrobial peptides
  • Antibodies
Other Elements
  • Anatomical barriers
    • Chemical
    • Physical
    • Biological
/
Self & Non-Self Discrimination Present: there is a reaction against foreign organisms and molecules Present: there is a reaction against foreign organisms and molecules
Lag Phase Absent: immediate response Present: response takes a couple days to get started
Diversity Limited (therefore decreased specificity) Elaborate: results in a variety of antigen receptors
Memory Absent: same response to a pathogen regardless of exposure Present: amplified and quicker response upon exposure after the first infection
Specificity Limited: same response for majority of encountered organisms and molecules Elaborate: response is targeted to the invasive agent

Table 1: Major differences between innate and adaptive immunity

Figure 1: Three phases of immune response to a pathogen

Innate Immune Response

The first line of innate immune defense against E. coli O157:H7 involves antimicrobials found in saliva and this contributes to the humoral aspect of innate immunity (Nguyen and Sporandio, 2012). For example, lysozyme is a key antimicrobial of the saliva that functions to hydrolyze beta-linkages in bacterial peptidoglycan.

Gastrointestinal (GI) Tract:

One of the first lines of defense against this bacterium is the low pH of gastric secretions, which can range from pH 1.5 to 5.5 (Waterman and Small, 1998). When the pH of gastric juice, which consists of pepsin and HCl, is less than 3.0, bacteria can be killed within 15 minutes (Tennant et al., 2008). Moreover, the epithelial cells of the GI tract function as a physical barrier to pathogens and also work with immune and stromal cells (Takiishi et al., 2017). The epithelium consists of a single layer of intestinal epithelial cells (IECs), including enterocytes, goblet cells, enteroendocrine cells, and Paneth cells (Takiishi et al., 2017). Paneth cells and enterocytes produce antimicrobial peptides like phospholipases, lysozyme C, and alpha-defensins (Takiishi et al., 2017). Goblet cells secrete mucins to lubricate and protect the epithelial intestinal surface and also serve as antigen presenting cells (APCs) when delivering antigens to dendritic cells (Takiishi et al., 2017). The constant regulated turnover of IECs prevents pathogen attachment to the intestinal wall and colonization of the gut (Takiishi et al., 2017). Tight junction complexes (occludins, claudins, junction adhesion molecules, and zonula occludens) seal spaces between neighbouring IECs and block entry of pathogens (Takiishi et al., 2017).

Figure 2: Mucosa, villi, crypts and cells of the small intestine

The mucous membrane of the gastrointestinal tract also serves as an innate defense system. Goblet cells that are located in the large and small intestines release mucopolysaccharides and IgA antibodies which serve to lubricate and protect the epithelium from harm (Winter and Man, 2010). Additionally, paneth cells found in the small intestine secrete α-defensins as well as lysozymes and phospholipase A2 (PLA2s) which serve to inhibit microbial growth. Binding of bacterial antigens to the Toll-like receptors found on Paneth cells leads to release of these enzymes. PLA2s are enzymes that cleave fatty acids from the second carbon of glycerol and this modification can induce changes in membrane composition and activates the inflammatory cascade. Furthermore, α-defensins form pores to lyse the bacteria and this is facilitated by the binding of the defensins to the bacterial membrane (Defensin, 2005).

The GI tract can contain 70% of the body’s lymphocyte population; under the epithelium, the lamina propria (LP) harbors the gut-associated lymphoid tissue (GALT) and dendritic cells (DCs) (Takiishi et al., 2017). GALT includes Peyer’s patches (PP), intraepithelial lymphocytes (IELs), and LP-lymphocytes (Takiishi et al., 2017). PP are more common in the ileum and contain microfold (M) cells that transport antigens to the mucosal lymphoid tissue to initiate immune responses (Takiishi et al., 2017).

Innate Immune Cells and Processes:

Another part of the first line of defense against an E. coli 0157:H7 infection is the activation of the innate immune system (Ho et al., 2013). Pathogen-associated molecular patterns (PAMPs) expressed by the microbes are detected using pattern recognition receptors (PRRs), to elicit an antimicrobial immune response (Ho et al., 2013). The main host PRRs are the Toll-like receptors (TLRs) and Nod-like receptors (NLRs) (Ho et al., 2013). TLRs are membrane-bound receptors that recognize gram-negative bacterial flagellin (TLR-5) and lipopolysaccharide (TLR-4) (Ho et al., 2013). TLR-2 (which detects molecule with diacyl and triacylglycerol moieties, proteins, and polysaccharides) is expressed on intestinal epithelium (Naik et al., 2001; Oliveira-Nascimento, Massari and Wetzler, 2012). TLR-5 may also play a role through detection of flagellin (Trinchieri and Sher, 2007). NLRs detect peptidoglycan (Nod-2) in the host cell cytoplasm (Ho et al., 2013). Activation of NLRs and TLRs activates the immune system leading to activation of immune cells and release of immune mediators that function in clearing the bacterial infection (Ho et al., 2013).

The interferon-gamma (IFNγ), Jak 1, 2, Stat1 signal transduction pathway is a signaling cascade activated upon infection with E. coli 0157:H7 (Ho et al., 2013). Natural killer T cells, macrophages and active T cells secrete proinflammatory cytokines such as IFNγ into the extracellular environment (Ho et al., 2013). IFNγ production causes an antimicrobial state in host cells by tyrosine phosphorylation of the activator and signal transducer of transcription 1 (Stat-1) molecule (Ho et al., 2012). This leads to its dimerization, translocation to the nucleus, binding to the gamma-activating sequence (GAS), and upregulation of as many as 2000 IFNγ-stimulated proinflammatory genes in host cells (Ho et al., 2012). These genes encode monocyte chemoattractant protein 1 (MCP-1), inducible nitric oxide synthase (iNOS), and lymphocyte adhesion protein ICAM-1 (Ho et al., 2012). These stimulated genes mount the host defense against the E. coli bacteria (Université de Montréal, 2004). IFNγ production also increases expression of major histocompatibility complex (MHC) class II (Université de Montréal, 2004).

M Cells (Lebeis et al., 2008)

These cells, found in the intestinal lymphoid follicles, are capable of transcytosis. This process facilitates the access of luminal antigens to lymphocytes so that an antibody response can be generated to the bacterium.

Dendritic Cells (Lebeis et al., 2008)

These cells play a major role in providing antigens to activate the third phase of the immune response (adaptive immunity). They are located below the M cells in the intestines, and can sample antigens within the lymphoid follicles (antigens come in via transcytosis from M cells). However, there are also some dendritic cells that are dispersed throughout the intestinal tract and have access to luminal antigens as well as migrate to inflamed areas. Dendritic cell-associated cytokines such as IL-10 influence the host immune response, and dendritic cells bridge the innate and adaptive immune response (Ho et al., 2013).

Neutrophils (Lebeis et al., 2008)

These cells are usually the first that are recruited to the site of infection and produce chemokines similar to the ones released by epithelial cells, as well as lysozyme and cytokines. IL-8 is a strong chemoattractant for neutrophils, which is released by the epithelium after it recognizes the pathogen (Lim et al., 2007).

Macrophages (Lebeis et al., 2008)

Macrophages play a role in the repair of the intestinal epithelium via MyD88.  E. coli invasion involves compromising the epithelial barrier to allow the bacteria to have access to areas such as the subepithelium. To combat this, the host requires TLR4 (toll-like receptor 4) and MyD88. MyD88 signaling on macrophages allows for the host to replenish the epithelium damage produced by the bacteria.This process involves stem cells that lie at the base of intestinal crypts which then differentiate into enterocytes that then divide and migrate along the crypt to the site of damage. In addition to this, they function to phagocytose bacteria and process antigens for presentation to T cells. Human macrophages bind E. coli by recognizing bacterial lipopolysaccharide (LPS) and binding them with LPS receptors (Wright and Jong, 1986). Three receptors in particular were shown to bind LPS: CR3, lymphocyte function-associated antigen (LFA-1) and p150,95 (Wright and Jong, 1986).

Mast Cells (Lebeis et al., 2008)

The intestines contain some mast cells and they are capable of secreting antimicrobial peptides and inflammatory mediators (such as TNF-alpha). While these cells are often associated more with a role in allergic responses, it has also been shown that they are usually necessary to prevent bacteremia. They are located in the perivascular space of most organs and likely prevent dissemination by releasing antimicrobial factors that can directly kill the bacterium. Histamine release by mast cells and prostaglandins production due to release of interleukins and tumor necrosis factors (TNF-α) result in an inflammatory state in the GI tract, which causes swelling, fever, and pain.

The complement system is also a part of the innate immune system and causes biochemical cascades which aim to eliminate the pathogen (Janeway et al., 2001). There are three pathways of complement activation and the alternate pathway is the one most prevalent in innate immune responses. These pathways and their cellular components are summarized in Figure 3. There are three ways in which the complement system protects against infection. First, it generates large numbers of activated complement proteins that bind covalently to pathogens, opsonizing them for engulfment by phagocytes bearing receptors for complement. Second, the small fragments of some complement proteins act as chemoattractants to recruit more phagocytes to the site of complement activation, and also to activate these phagocytes. Third, the complement components damage certain bacteria by creating pores in the bacterial membrane.

Figure 3: Three Pathways of Complement

Bridge to Adaptive Immune Response

Additionally, microfold (M) cells are distinct cells in the epithelial cell layer that phagocytose bacteria through rearrangements of their actin cytoskeleton on the apical membrane and then exocytose the bacteria on the basolateral membrane in a process called transcytosis (Jung et al., 2010). This process allows bacterial antigen to be received by antigen-presenting cells, such as dendritic cells. Dendritic cells (DCs) abundantly express TLRs, so when they are activated by antigens that have undergone exocytosis from M cells, they similarly secrete cytokines and chemokines but they also migrate to secondary lymphoid tissue of the gut-associated lymphoid tissue (GALT). Most notably, DCs migrate to mesenteric lymph nodes and Peyer’s patches to present antigen to and activate naïve T cells (Kobayashi et al., 2019). Peyer’s patches are lymphoid follicles found in the mucosa of the small intestine that contain many germinal centers where B cells proliferate and undergo somatic hypermutation. The activation of naïve T cells by DCs is essential for T cell-dependent IgA class switch recombination in Peyer’s patches, thus allowing for the development of IgA-secreting plasma cells and a subsequent mucosal IgA response (Kobayashi et al., 2019).

Adaptive Immune Response

Cellular Immunity

Since E. coli O157:H7 is an extracellular pathogen, cell mediated adaptive immunity doesn’t play as great of a role in containing and eliminating the infection as does humoral immunity. Phagocytosis of E. coli by macrophages allows the leukocyte to present antigens specific to the bacterium on its cell surface via MHC II (Todar, 2008-2012). The TCR-CD3 complex on helper T cells (Th) bind strongly to the MHC-peptide complex on the macrophages. Activation of CD4+ helper T- cells via co-stimulation of MHC II and CD80/86 leads to release of interleukin 2 (IL-2) to locally stimulate the development of other T-cells. Helper-T cells are able to release cytokines to activate and recruit macrophages, as well as stimulate B-cell activation and subsequent antibody production. As well, a portion of helper T-cells become memory T-cells which are able to respond quickly when re-exposed to the same antigen in the future.

Triggered by IL-2, IL-12, and IFN-gamma, helper T cells differentiate into Th1 cells, which are good at clearing intracellular bacteria. Activated Th1 cells secrete more IL-2 and IFN-γ to activate macrophages for more active killing. IFN-γ can also result in the production of nitrogen oxides to directly kill intracellular E. coli. IFN-γ also increases the production of IL-12 from dendritic cells and macrophages, creating a positive feedback loop of amplified response. Th1 also promotes the production of IgG by plasma cells. IgG will opsonize E. coli when they are extracellular to minimize their ability to infect other cells (T helper cell, 2015).

IFN-gamma up-regulation occurs when cytokines released during infection bind to cell surface receptors (Shea-Donohue et al., 2010).  This  leads to receptor oligomerization and activation of tyrosine kinases from the janus kinase (JAK) family JAKs. From here, phosphorylate signal transducers and activators of transcription (STATs) dimerize and translocate to the nucleus, where they can increase transcription of selected genes . For example, IL-12 generated by dendritic cells can bind to STAT4, which upregulates T-bet, a T-box transcription factor, and differentiation into Th1 cells, which release IFN-γ

Humoral Immunity

In the humoral immune response, B cells produce antibodies that destroy pathogens and prevent the spread of intracellular infections (Janeway et al., 2001). CD4 T cells (carry the co-receptor protein CD4) recognize peptides from intravesicular sources bound to MHC class II molecules and differentiate into CD4 TH2 and CD4 TH1 effector cells that activate macrophages and B-cell responses to antigen (Janeway et al., 2001). The helper T cells with bound antigen induce B cells to proliferate and differentiate into plasma cells that secrete specific antibodies (Janeway et al., 2001).

Immunoglobulin molecules (antibodies) are made of two heavy chains and two light chains joined by disulfide bonds (Janeway et al., 2001).  IgG is the most common type of antibody found in the body, and functions in neutralizing toxins (Schroeder and Cavacini, 2010). The IgA antibody at the mucosa is known as secretory IgA (sIgA) and is a dimer associated with a secretory component (polypeptide chain) and a J-chain (Schroeder and Cavacini, 2010). IgA, through direct neutralization or by preventing binding to the mucosal surface, protects mucosal surfaces from bacteria and toxins (Schroeder and Cavacini, 2010). TH1 and TH2 cells can stimulate B cells to proliferate and differentiate into antibody-secreting effector cells as mentioned above, or into memory B cells (Alberts et al., 2002).

Memory B cells are long-lived plasma cells that provide long-term humoral immunity (Elsevier, 2015). After generation, memory B cells enter a resting state and remain in lymphoid organs for long periods of time when immunizing antigen is not encountered (Elsevier, 2015). When they do get exposed to antigen again, the memory B cells provide a quicker and greater antibody response than before (Elsevier, 2015).

Mucosal immunity in the gastrointestinal (GI) tract is a primary defence against GI pathogens. Adherence of E. coli O157:H7 to intestinal epithelial cells is essential to induce secretory immunoglobulin A (IgA) production (Nagano et al., 2014). There are four key E. coli virulence factors that induce antibody production:

  • Tir: translocated intimin receptor - essential to the adherence of E. coli to the gut epithelium and inserts into the host cell membrane
  • Intimin: bacterial outer membrane protein that binds to Tir
  • EspA: secreted protein which forms filamentous structures on E. coli surface
  • EspB: inserted into the host membrane and cytoplasm

Main antibodies of this infection:

IgG:

These antibodies activate complement, and the tail region of an IgG molecule binds to Fc receptors on neutrophils and macrophages (Alberts et al., 2002). These phagocytic cells bind pathogens that have been coated with IgG antibodies, ingest them, and destroy them (Alberts et al., 2002). IgG binds pathogens and immobilizes them, resulting in their agglutination (Starks, 2019). IgG coats pathogens (opsonization) for recognition and phagocytosis by immune cells, to eliminate the pathogen (Starks, 2019). IgG also activates the classical pathway of the complement system, binds and neutralizes toxins, and is involved in type II and type III hypersensitivity reactions (Starks, 2019). IgG (neutralizing) antibodies are detected against the A and B subunits of Stx2 (Guirro et al., 2014).

IgA:

These antibodies are found in tears, milk, saliva, and intestinal and respiratory secretions (Alberts et al., 2002). In secretions, IgA is found as an eight-chain dimer (Schroeder et al., 2010). Polymeric IgA can bind mannan binding lectin (MBL) and activate the lectin pathway (Woof and Kerr, 2005). IgA can inhibit bacterial adhesion to epithelial cells and can acts in neutralization of bacterial toxins (Queen’s University Belfast). sIgA facilitates the adaptive humoral response at mucosal surfaces such as respiratory, gastrointestinal, and urogenital tracts (LeBlanc et al., 2004). During this infection, sIgA levels increase to prevent adherence and colonization of the bacteria (Li et al., 2000). E. coli 0157:H7 binds to M cells in Peyer’s patches, inducing an increase in sIgA levels (Li et al., 2000).

Patients infected with this E. coli serotype develop an antibody response to O157 lipopolysaccharide (LPS) (Tankeshwar, 2013). It has been shown that IgG antibodies to E. coli LPS tend to be most dominant in healthy individuals, whereas patients with acute E. coli 0157 disease exhibit elevated levels of IgM antibodies (Currie et al., 2001). Furthermore, there is significant IgG and IgA response to the R3 oligosaccharide found in bacterial LPS, suggesting that this component may play a large role in our immunological memory to E. coli. The proteins that are secreted by the type III secretion system are strongly immunogenic, producing antibody responses (Tankeshwar, 2013). These proteins (virulence factors) include EspA, Tir, EspB, and the 280 carboxyl-terminal amino acids of intimin, with the anti-Tir immune response being the strongest (Tankeshwar, 2013). Intimin is an integral outer membrane protein, EspB and Tir are delivered to the host cell, and EspA bridges the bacterial surface to the host cell surface by forming filamentous surfaces on the bacterial surface (Tankeshwar, 2013). Once in the host cell, Tir acts as a receptor for intimin (Tankeshwar, 2013). The goal of the immune system in blocking these proteins is to prevent formation of actin pedestals (structures formed from host cell actin), inhibit adherence, and thus block disease (Tankeshwar, 2013).

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Question (ii)

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

There is various damage that is derived from bacterial action. Specifically, this section will focus on damage from the host immune response. Inflammation (characterized by redness, swelling, heat, pain, as well as disrupted function) has a protective function in the context of bacterial infection and can explain some of Ronnie’s signs and symptoms. However, inflammation can also cause damage to host tissue (Nathan, 2002). E.coli O157:H7 is classified as an extracellular pathogen. Therefore, the majority of the host tissue damage arises from the innate immune responses rather than cell-mediated immunity. Adaptive humoral immunity does not typically cause damage to the host since the antibodies produced by the B lymphocytes are specific for the antigen (and not for host cells) (Todar, 2012). Additionally, as there are always antibodies present in the body system even when there are no bacterial infections, it shows that their presence does not have damaging effects on the body. However, innate immune responses do not have the same targeted actions or specificity as adaptive immunity, and have the potential to cause a lot of host damage, particularly when they are hyperactivated.

Inflammatory Response (Cytokines)

LPS on the E. coli surface binds to the TLR4 receptor complex to activate NF-kB and lead to production of proinflammatory cytokines (Lebeis, Sherman, and Kalman, 2008). TLR4-dependent signaling induces inflammation and aggravates disease (Lebeis, Sherman, and Kalman, 2008). PRRs expressed on the innate immune cells such as macrophages can recognize host soluble molecules that are released after bacterial infection known as DAMPs (damage-associated molecular patterns), which can induce strong inflammation (Hato and Dagher, 2015). Ig antibodies, interleukins, lysozymes, and prostaglandins can all lead to an inflammatory response.

Additionally, a bacterial virulence factor called Shiga toxin is thought to disrupt protein synthesis in intestinal epithelial cells, thus resulting in cell death. Although this damage is induced by the bacterial pathogen, this toxin initiates a proinflammatory cascade in which the host inflammatory response contributes to further tissue damage (Gossman et al., 2020).  Shiga toxins induce an increase in chemokine synthesis from intestinal epithelial cells, which augment host mucosal inflammatory response. Such responses release interleukins, such as IL-8 and IL1, in addition to Tumor Necrosis Factor (TNF) (Rahal et al., 2012). This process of activating human endothelium leads to an increase in toxin receptor synthesis and increased sensitivity of the cell. This could lead to increased cell death after exposure to the toxin (Rahal et al, 2012).

Cytokines modulate the immune response; however, excessive amounts of proinflammatory cytokines (such as IL-1, IL-6, TNF-α, IFN-γ and more) can cause detrimental effects (Scarpioni, Ricardi and Albertazzi, 2016). Phagocytosis activates PRRs and inflammatory signaling cascades, and the resulting excessive inflammation can cause collateral tissue damage, hemodynamic (dynamic of blood flow) changes, organ failure, and possibly death (Czaja, 2014; Hato and Dagher, 2015). Proinflammatory cytokines also have a role in causing fever, tissue destruction, and in some cases even shock and death (Dinarello, 2000). Small amounts of nitric oxide (NO) play a role in maintaining vascular tone and vasodilation, but large amounts of NO can be stimulated through inflammatory cytokines, which can damage tissue (Edwards, n.d.). Inflammatory mediators released by activated T cells and neutrophils can stimulate Cl- secretion, thereby affecting intestinal fluid transport homeostasis (Gelbmann et al., 1995). Eventually, intestinal mucosa cells will be sloughed off, resulting in hemorrhagic diarrhea. Furthermore, this inflammatory response can also occur at vascular endothelial cells in the kidneys resulting in hemolytic uremic syndrome (HUS), or in a more severe manifestation resulting in multiple organ failure (Gossman et al., 2020)

Neutrophil Infiltration
Figure 1. Neutrophil effector mechanism (Kruger et al., 2017).

Activation of TLRs and other pathogen recognition receptors in epithelial cells results in production of cytokines through NF-kB activation and recruitment of neutrophils to the infection site (Lebeis, Sherman, and Kalman, 2008). Neutrophils significantly contribute to damage as they are often the first immune cell recruited to the site of infection and they release a lot of factors that are effective at killing pathogens (Figure 1), but can also damage host tissues (Wilgus, Roy, and McDaniel, 2013).

While a strong neutrophil response is beneficial because it fights off pathogens early on, it can also have a lot of harmful effects. The activity of many enzymes and chemokines secreted by neutrophils can be very damaging, especially because these factors have no specificity for pathogens or the host. Furthermore, E. coli is extracellular, so a lot of these factors would be released extracellularly, and this is even worse because they have a big chance to come in contact with host cells and tissues. Majority of the neutrophil damage comes from the proteases it releases, and this can be problematic for wounds as it can prevent them from healing. Proteases function to break molecules down, and high-levels of neutrophil-derived proteases are associated with chronic, non-healing wounds. In the case of E. coli, this could make it harder to heal the damaged parts of the intestinal epithelium (as it is injured by both the bacteria and the host), especially if their extracellular concentration becomes high.

Neutrophils also release extracellular traps (NETs) to eliminate pathogens (Dąbrowska et al., 2016). Although these factors aid in eliminating pathogens, they can also damage healthy host tissue (Nathan, 2006). Some examples of the adverse effects from neutrophil infiltration are inflammatory bowel diseases (IBD), arthritis, some cardiovascular conditions, inflammatory pulmonary and renal diseases, and viral/bacterial infection-associated damage.

Reactive Oxygen Species and Nitric Oxide

Cytotoxic substances (ex. ROS, NOS, proteases) are produced within the immune cells when the pathogen is phagocytosed, such as from macrophages. These molecules can sometimes leak out of these cells and into the surrounding environment and cause damage. Expression of inducible nitric oxide synthase (iNOS) is upregulated in this signal pathway leading to the production of large amounts of nitric oxide, a reactive nitrogen species (Ho et al., 2012). iNOS is expressed in macrophages, microglia, astrocytes, and other cell types (Sierra et al., 2014). This nitric oxide can then react with superoxide, a reactive oxygen species, leading to damage to host cells. In high amounts, this enzyme can also cause septic shock, possibly leading to multiple organ failure and death. TNF-a promotes the release of reactive oxygen radicals which not only harm the pathogen but also the host epithelium. Due to the damage of the epithelium in the large intestine in its ability to absorb fluids, the host may experience watery and/or bloody diarrhea

Chronic inflammation

While acute inflammation is a limited beneficial response during infection, chronic inflammation is a persistent phenomenon that can lead to tissue damage (Gabay, 2006). If inflammation becomes chronic, there can be large amounts of tissue destruction and fibrosis (scarring) can occur (Edwards, n.d.). Neutrophil infiltration plays a central role in inflammation and is a major cause of tissue damage. Neutrophils are recruited to the site of infection by damage-associated molecular patterns and CXCL8. They will kill pathogens through phagocytosis, degranulation, reactive oxygen species, and neutrophil extracellular traps. Chronic inflammation can occur if there is excess neutrophil infiltration and activation at the site of infection, resulting in local tissue damage via the release of proteases, reactive oxygen species, and matrix metalloproteases (de Oliveira et al., 2017). As an example, matrix metalloproteases released by neutrophils can cleave extracellular matrix proteins, such as elastin and collagen, into small fragments thus further stimulating the immune response to recruit more neutrophils to the site of tissue damage and continuing the cycle of damage to the host. Although there is damage done to the host, it is important to remember that this feed-forward loop likely benefits the bacterium as well. Chronic tissue damage and inappropriate immune cell activation can lead to intestinal inflammation seen in autoimmune diseases such as inflammatory bowel disease and coeliac disease (Gareau and Barrett, 2013).

Pain

Histamine produced by mast cells, platelets, and basophils can stimulate nociceptors responsible for causing pain. Histamine also acts as a vasodilator, causing an increase in vascular permeability by increased edema in the area of infection. This can lead to inflamed and swollen tissue. Bradykinin is also a major mediator involved in the pain response (Edwards, n.d.).

Fever

The LPS (lipopolysaccharide) of E. coli is an exogenous pyrogen (a substance that produces fever when introduced into the blood) that can cause leukocytes to release endogenous pyrogens such as IL-6, IL-1, TNF (tumor necrosis factor) and IFN-γ (interferon-γ) (“Inflammation and Fever”, n.d.). These molecules then cause other cells to release prostaglandin E2 (PGE2), which resets the hypothalamus to initiate fever (“Inflammation and Fever”, n.d.). A fever stimulates leukocytes to kill pathogens, enhancing the innate immune response (“Inflammation and Fever”, n.d.).

References

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Gastroenterology, 20(10), p.2515.

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Biology of Neutrophil Extracellular Traps. Scandinavian Journal of Immunology, 84(6), pp.317-322.

de Oliveira, S., Rosowski, E. E., & Huttenlocher, A. (2016). Neutrophil migration in infection and wound repair: going forward in reverse. Nature Reviews Immunology, 16(6), 378.

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Question (iii)

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

Acid resistance in the stomach

As mentioned in Q1, E. coli O157:H7 has developed several techniques to survive beyond the acidic environment of the stomach. Firstly, the bacterium has a low infectious dose of about 50 CFU, meaning that only a few organisms are required to be ingested before initiating colonization followed by infection (Lim et al., 2010). As a result, even if only small amounts of pathogen survive beyond the low pH of the gastric acid, it may still be sufficient to cause infection.

Secondly, E. coli O157:H7 utilizes four different mechanisms to induce acid resistance in the stomach. In the first mechanism, alternative sigma factor RpoS is thought to regulate stress response systems necessary for the survival of the organism during conditions of hunger, such as glucose-repression (Lim et al., 2010). In the second mechanism, arginine decarboxylase (AdiA) and its transcriptional regulator cysB are used to catalyze the conversion of arginine into agmatine and carbon dioxide (Bearson et al., 2009). In this process, a proton is removed from the cytoplasm and transported out of the cell via the arginine/agmatine antiporter AdiC, thus increasing the pH inside of the bacterial cell (Lim et al., 2010). Since the pH of the cytoplasm does not decrease to that of the acidic environment, the bacterium’s intracellular processes will not be interrupted due to decreased enzymatic activity or protein denaturation due to unfolding. Similarly, in the third mechanism, glutamate decarboxylase (GadA or GadB) is used to catalyze the conversion of glutamate into γ-aminobutyric acid (GABA) and carbon dioxide via the γ-aminobutyric acid antiporter GadC (Bearson et al., 2009). These products are then exchanged for new amino acids through cognate antiporters, GadC and AdiC. Hence, in two of the four listed mechanisms for acid resistance, E. coli O157:H7 uses a virtual proton pump to avoid large decreases in intracellular pH due to the acidic environment in the stomach.

Finally, E. coli O157:H7 has been shown to express a small chaperone protein called HdeA that prevents acid-induced protein aggregation in the periplasm (Tapley et al., 2009). Normally, gastric acid induces bacterial protein unfolding and results in irreversible aggregation of periplasmic proteins because the unfolded proteins clump together. Once this occurs, it is very difficult for bacteria to dissolve these protein clumps leading to decreased pathogen activity and eventual death. As a result, HdeA functions by binding to unfolded proteins in the periplasmic space and preventing irreversible aggregation. To do so, HdeA is a stress-sensing protein that is activated by stress conditions (low pH) through the conversion of an inactive dimer to a chaperone-active monomer. When this pH-induced dissociation occurs, the hydrophobic amino acid residues are exposed, thus allowing the HdeA monomers to bind other unfolded proteins in the periplasm (Tapley et al., 2009). This binding prevents the unfolded proteins from sticking together and forming clumps, resulting in bacterial survival in the acidic conditions of the stomach.

Adhesion to Intestinal Epithelial Cells

Through the use of a virulence factor called LEE pathogenicity island, E. coli O157:H7 can overcome the mucus barrier of the intestinal mucosa to bind and attach to intestinal epithelial cells. LEE pathogenicity island is a 42 kb gene sequence that encodes several different bacterial proteins that allow this process to occur. Firstly, E. coli secreted proteins EspA, EspB, and EspD are structural proteins for a type 3 secretion system (T3SS) that acts as a molecular syringe in order to inject many other effector proteins into the cytoplasm of the host epithelial cell (Ho et al., 2013). Among these injected effector proteins, translocated intimin receptor (Tir) anchors itself on the surface of the epithelial cell plasma membrane and binds to intimin located on the surface of the bacterial cell, thus creating an attachment between the bacterial cell and the host epithelial cell. Furthermore, some of the effector proteins injected by T3SS act to induce actin polymerization at the binding site resulting in the formation of actin pedestals, actin-rich structures that are found beneath the site of bacterial adhesion, though their function is still unknown (MBInfo, 2018). Finally, the LEE pathogenicity island also encodes lymphostatin/EFA-1, a toxin that inhibits mitogen-activated expression and transcription of IL-4, IL-2 and IFN-γ, thus inducing an immunosuppressive effect (Klapproth et al., 2010). This process results in the formation of attaching and effacing (A/E) lesions on the mucosal epithelium at the recto-anal junction (RAJ). A/E lesions are characterized by “destruction of microvilli, intimate attachment of the bacteria to the cell, and accumulation of polymerized actin beneath the site of bacterial attachment to form a pedestal-like structure cupping individual bacterium” (Nguyen and Sperandio, 2012).

Recently, a transcriptional regulator called SdiA was shown to regulate the transcription of the LEE genes through the sensing of acyl-homoserine lactones (AHLs) produced by other bacteria (Nguyen and Sperandio, 2012). This process of detecting chemical signals produced by other bacteria is called quorum-sensing. By using a cow model, the researchers determined that the production of AHLs by commensal bacteria in the rumen of the cattle was detected by SdiA, which in turn inhibited the transcription of LEE genes to ensure that bacterial colonization did not commence in the acidic environment of the rumen (Nguyen and Sperandio, 2012). Then, when the bacteria have moved beyond the rumen, there are no AHLs detected by SdiA so it no longer represses transcription of LEE genes, thus permitting colonization to occur. As a result, it was demonstrated that the LEE genes are regulated by quorum-sensing using AdiA.

Suppression of IFN-γ Signal Transduction Pathway

E. coli O157:H7 has also been shown to suppress the IFN-γ signal transduction pathway discussed in Q1 by inhibiting IFN-γ-mediated phosphorylation of Stat-1 (Ho et al., 2012). By preventing this phosphorylation, Stat-1 does not dimerize and thus cannot translocate into the host cell nucleus to upregulate the expression of proinflammatory genes. This would dampen the immune response by suppressing innate immunity and would result in a failure for the body to activate the antibacterial state. This is speculated to be attributed to E. coli-derived Shiga toxin, which some studies claim is responsible for the suppression of IFN-γ-mediated phosphorylation of Stat-1 (Ho et al., 2012). Alternatively, E. coli O157:H7 secretes a virulence protein called NleH1 that has been shown to inhibit the phosphorylation of ribosomal protein S3 (RPS3), an NF-κB subunit required for the transcription of many critical chemokines and cytokines (such as IFN-γ) in the innate immune response (Wan et al., 2011). By inhibiting the phosphorylation of RPS3, NleH1 inhibits the protein’s ability to translocate into the nucleus and initiate gene transcription. Specifically, NleH1 prevents phosphorylation of a serine residue in the 209th position of the RPS3 sequence (RPS3 Ser209), a reaction that is normally catalyzed by a kinase called IKKβ (Wan et al., 2011). Thus, NleH1 also suppresses the host innate immune response by targeting the IFN-γ signal transduction pathway. Furthermore, other effector proteins, such as NleE and NleC, have also been shown to suppress NF-κB activation using similar mechanisms. It is suggested that NleE interferes with the activation of the IKK enzyme that normally phosphorylates RPS3 while NleC directly cleaves the p65 subunit of the NF-κB subunit (Yen et al., 2010).

Plasmid pO157 and its associated proteins

The pO157 plasmid of E. coli 0157:H7 encodes several key proteins involved in bacterial evasion, such as catalase-peroxidase (KatP) and metalloprotease StcE. Firstly, KatP catalase-peroxidase is a pO157 plasmid protein that detoxifies oxidants released by neutrophils and macrophages in their oxidative burst, thus preventing damage to the bacterial cells (Brunder et al., 1996). KatP is located in the bacterial periplasm and helps E. coli 0157:H7 to colonize the intestine by reducing the oxidative stress, as well as through the use of by-product oxygen in intestinal low oxygen conditions. Additionally, the bacterial chromosomal gene katG encodes two hydroperoxidases called HP1 and HP2 that perform similar functions. Specifically, HP1 is both a catalase and peroxidase that uses hydrogen peroxide as an electron acceptor, while HP2 only demonstrates catalase activity (Brunder et al., 1996). Secondly, plasmid pO157 encodes the StcE metalloprotease, which dampens the classical pathway of the complement cascade by cleaving a key regulator of this cascade called serpin C1 esterase inhibitor (C1-INH) (Lathem et al., 2004). Surprisingly, this cleavage has been shown to enhance the ability of C1-INH to inhibit the classical pathway of the complement cascade, thus protecting the bacterial cells from classical complement-mediated lysis. Furthermore, StcE has also been demonstrated to cleave glycosylated proteins found in saliva that are responsible for bacterial aggregation in the oral cavity, thus degrading the protective layer of mucins and glycoproteins on the host cell (Grys et al., 2005).

Suppression of Autophagy

Recent studies have demonstrated that the bacterial intimin receptor that is translocated into the host intestinal cell by T3SS is also capable of suppressing autophagy via activation of protein kinase A (Xue et al., 2017). Autophagy is the process of cellular degradation of intracellular proteins and organelles via the formation of an autophagosome that fuses with a lysosome. This process has been shown to participate in the removal of bacterial pathogens from host cells, thus it acts as a self-defence mechanism to microbial infection. Intimin receptor, also known as Tir, was shown to activate protein kinase A in a cAMP-independent manner, which then suppressed the ERK and PI3K/Akt signaling pathways that normally induces autophagy (Xue et al., 2017). Furthermore, autophagy was shown to prevent bacterial adhesion to epithelial cells, thus this bacterial evasion strategy allows E. coli O157:H7 to successfully adhere to host epithelial cells and avoid autophagy. This suppression pathway is demonstrated in Figure 1 below.

Figure 1: Suppression of autophagy via intimin receptor inhibition of protein kinase A

References:

Bearson, Bradley L., et al. “Escherichia Coli O157 : H7 Glutamate- and Arginine-Dependent Acid-Resistance Systems Protect against Oxidative Stress during Extreme Acid Challenge.” Microbiology, vol. 155, no. 3, Jan. 2009, pp. 805–812., doi:10.1099/mic.0.022905-0.

Brunder, W., et al. “KatP, a Novel Catalase-Peroxidase Encoded by the Large Plasmid of Enterohaemorrhagic Escherichia Coli O157:H7.” Microbiology, vol. 142, no. 11, 1 Nov. 1996, pp. 3305–3315., doi:10.1099/13500872-142-11-3305.

De Oliveira, Sofia, et al. “Neutrophil Migration in Infection and Wound Repair: Going Forward in Reverse.” Nature Reviews Immunology, vol. 16, no. 6, 2017, pp. 378–391., doi:10.1038/nri.2016.49.

Gossman, William, et al. “Escherichia Coli (E Coli 0157 H7).” Treasure Island (FL), StatPearls Publishing, 2020.

Grys, T. E., et al. “The StcE Protease Contributes to Intimate Adherence of Enterohemorrhagic Escherichia Coli O157:H7 to Host Cells.” Infection and Immunity, vol. 73, no. 3, 2005, pp. 1295–1303., doi:10.1128/iai.73.3.1295-1303.2005.

Ho, Nathan K., et al. “Enterohemorrhagic Escherichia Coli O157:H7 Shiga Toxins Inhibit Gamma Interferon-Mediated Cellular Activation.” Infection and Immunity, vol. 80, no. 7, 2012, pp. 2307–2315., doi:10.1128/iai.00255-12.

Ho, Nathan K, et al. “Pathogenicity, Host Responses and Implications for Management of Enterohemorrhagic Escherichia Coli O157:H7 Infection.” Canadian Journal of Gastroenterology, vol. 27, no. 5, May 2013, pp. 281–285.

Hunt, John M. “Shiga Toxin–Producing Escherichia Coli (STEC).” Clinics in Laboratory Medicine, vol. 30, no. 1, 2010, pp. 21–45., doi:10.1016/j.cll.2009.11.001.

Jung, Camille, et al. “Peyers Patches: The Immune Sensors of the Intestine.” International Journal of Inflammation, vol. 2010, 2010, pp. 1–12., doi:10.4061/2010/823710.

Kobayashi, Nobuhide, et al. “The Roles of Peyers Patches and Microfold Cells in the Gut Immune System: Relevance to Autoimmune Diseases.” Frontiers in Immunology, vol. 10, Sept. 2019, doi:10.3389/fimmu.2019.02345.

Klapproth, Jan-Michael A. “The Role of Lymphostatin/EHEC Factor for Adherence-1 in the Pathogenesis of Gram Negative Infection.” Toxins, vol. 2, no. 5, 5 May 2010, pp. 954–962., doi:10.3390/toxins2050954.

Kruger, Philipp, et al. “Neutrophils: Between Host Defence, Immune Modulation, and Tissue Injury.” PLOS Pathogens, vol. 11, no. 3, 2015, doi:10.1371/journal.ppat.1004651.

Lathem, Wyndham W., et al. “Potentiation of C1 Esterase Inhibitor by StcE, a Metalloprotease Secreted by Escherichia Coli O157:H7.” Journal of Experimental Medicine, vol. 199, no. 8, 2004, pp. 1077–1087., doi:10.1084/jem.20030255.

Lim, Ji Youn, et al. “A Brief Overview of Escherichia Coli O157:H7 and Its Plasmid O157.” Journal of Microbiology and Biotechnology, vol. 20, no. 1, 2010, pp. 5–14., doi:10.4014/jmb.0908.08007.

Ludwig, Kerstin, et al. “Escherichia Coli O157 Fails to Induce a Long‐Lasting Lipopolysaccharide‐Specific, Measurable Humoral Immune Response in Children with Hemolytic‐Uremic Syndrome.” The Journal of Infectious Diseases, vol. 186, no. 4, 2002, pp. 566–569., doi:10.1086/341781.

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Martorelli, L., et al. “Impact of Infection Dose and Previous Serum Antibodies against the Locus of Enterocyte Effacement Proteins OnEscherichia coliO157:H7 Shedding in Calves Following Experimental Infection.” BioMed Research International, vol. 2015, 2015, pp. 1–8., doi:10.1155/2015/290679.

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Question (iv)

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

Current treatment options for EHEC infection and HUS consist of fluid resuscitation, peritoneal dialysis and plasma exchange (Ho et al., 2013) During early stages of treatment, careful attention to “intravascular volume status” and liberal use of intravenous fluids”. May reduce the rates of developing acute renal failure in infected subjects. (Ho et al., 2013) Antibiotics are not recommended because this practice leads to an increased burden of antibiotic-resistant pathogenic E coli and of more life-threatening enteropathogens. (Todar, 2016) Additionally, antibiotics may lead to more toxin release from the pathogen and result in “increased systemic exposure to the adverse effects of the potent nephrotoxin.” It is found that antibiotic usage appears to increase the frequency of HUS among infected children. (Ho et al., 2013

There are also alternative strategies aiming to sequester and limit Shiga toxin (Stx) associated pathology being proposed. For example, Stx ligand mimics should sequester Stx from binding to host cells and thereby limit pathology. However, the treatment failed to diminish disease severity in infected children. Other methods being proposed include neutralizing Stx specific monoclonal antibodies to animals that are challenged with lethal doses of the toxin. (Ho et al., 2013

Prevention strategies are extremely important in controlling the sporadic outbreaks of EHEC O157:H7 infection since it is a significant cause of death all around the world.

Household prevention methods (WHO, E coli)

  • Good food hygiene practise (5 keys to safer food)
    • Keep clean.
    • Separate raw and cooked.
    • Cook thoroughly.
    • Keep food at safe temperatures.
    • Use safe water and raw materials.
  • Regular hand washing, before food preparation or consumption and after toilet contact

Industry prevention methods (WHO, E coli)

  • Various mitigation strategies for ground beef (for example, screening the animals pre-slaughter to reduce the introduction of large numbers of pathogens in the slaughtering environment)
  • Education in hygienic handling of foods for workers at farms, abattoirs and those involved in the food production
  • Bactericidal treatment, such as heading or irradiation
  • Protect drinking-water source, from animal waste

Fresh products prevention methods (WHO, E coli)

  • Good food hygiene practices (5 keys to growing safer fruits and vegetables)
    • Practice good personal hygiene.
    • Protect fields from animal faecal contamination.
    • Use treated faecal waste.
    • Evaluate and manage risks from irrigation water.
    • Keep harvest and storage equipment clean and dry.
      Immune response and secretion of antibodies (Pokhrel, 2015)

Usually for these sorts of bacterial infections, within five to seven days after the onset of an E. coli O157:H7 infection patients can make a full recovery with the help of proper hygiene, consistent hydration and adequate rest. However, complications may occur during prolonged or severe infections including: sepsis, dehydration, anemia and ulceration (Dipiro et al., 2008).

Memory T cells

E. coli O157:H7 infection in humans has been shown to induce a memory T cell response, suggesting that there would be immunity to future infections from the same pathogen. Type a mucosal T cells can develop into long-lived effector memory T cells that reside in the lamina propria of the GALT if they encounter a dendritic cell that has taken up bacterial antigen in the T cell areas of the Peyer’s patches or mesenteric lymph nodes (van Wijk and Cheroutre, 2010). Upon secondary infection by the same pathogen, these effector memory T cells can rapidly differentiate into CD8+ cytotoxic T cells that can kill pathogen-infected host cells through the action of perforin and granzymes which induce cellular apoptosis via the caspase cascade. Thus, these effector memory T cells can mount rapid and highly efficient immune responses upon secondary infection by the same pathogen.

Memory B cells

Memory B cells are long-lived cells that are defined as class-switched, somatically-mutated cells that are very responsive to specific antigens (Yates, et al., 2013). In germinal centers (GCs), which are specialized structures in secondary lymphoid organs, class-switched Ig memory B cells are produced (Yates, et al., 2013. When the memory B cells get exposed to their associated antigen, they proliferate and differentiate into antibody secreting cells, resulting in serum Ig that is antigen-specific and helps in clearing the bacteria from the host (Yates, et al., 2013. These IgM memory B cells are capable of class-switching to produce IgG, during secondary exposure to the bacterial antigen (Yates, et al., 2013. Therefore, the memory B cells lead to a secondary immune response that involves a larger and faster production of antibodies, generating future immunity (Yates, et al., 2013. Additionally, in a different study, a strong humoral response to the intimin receptor (anti-Tir antibodies), a bacterial virulence factor, was detected in the serum up to 60 days post-infection (Li et al., 2000). However, a long-term follow-up did not occur for the patients in this study, thus it is not known whether these anti-Tir antibodies persisted beyond 60 days post-infection or not.

If the bacteria re-enter the host, these antibodies and B lymphocytes will generate an immune response quickly depending on the time frame and the health conditions of the patient. The bacterial infection can elicit IgM memory B cells that maintain long-term immunity (Kieckens et al., 2016). For young children and older adults, they have a higher risk of developing a life-threatening form of kidney failure called hemolytic uremic syndrome (Mayo Clinic, 2019; Davis and Marks, 2019). Hemolytic uremic syndrome complicates up to 10% of E. coli O157:H7 infections, with higher risks in younger children. With hemolytic uremic syndrome, the risk of mortality approaches 5% (Gossman, Wasey and Salen, 2019). In this specific case, Ronnie should be able to recover fully, although may experience diarrhea and uncomfortable bowel movement even after the infection goes away for a few days (Todar, 2016). Due to the adaptive immune response to this infection, including the presence of memory T and B cells, the patient is able to be immune to many future infections with E. coli if reinfected with the same strain.

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