Documentation:PATH417archive2020W2/Case 1

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Case 1

The Raw Food Diet

25-year-old Johnny has been eating raw eggs as part of his new body building diet. One week into his new diet, he develops a mild fever, severe abdominal cramps and watery diarrhea. After 4 days of diarrhea, he goes to a walk-in clinic where the doctor finds that Johnny is volume depleted and has some abdominal tenderness. She gives him a container to collect a stool sample to send to the Microbiology Laboratory and suggests that he stop eating the raw eggs. Johnny’s stool sample grows Salmonella enteritidis.

1. The Body System

(i) Describe the signs (objective characteristics usually noted/detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient) reported. Also make note of any key history findings, what samples are taken (and why) and the meaning of any laboratory results reported. (There is no need to describe the laboratory testing as this is the basis of another question).

(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) might the doctor suggest or prescribe and how do these treatments work? If antibiotics are prescribed what is the antibacterial treatment of choice and how does the antibiotic work to rid the body of the organism?

(iv) Why did the doctor tell Johnny to stop eating raw eggs? Is this a reportable communicable disease?

2. The Microbiology Laboratory

(i) Other than the stated bacterial cause, 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 particular 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 allowing for the identification of the bacteria named in this case.

3. Bacterial Pathogenesis

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

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

ii. Entry: what facilitates the entry of the bacteria into the human host? What are the molecular, cellular and/or physiological factors at play in the initial entry/adherence step from the point of view of the organism 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?

4. The Immune Response

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 from this particular bacteria?

Q1. The Body System

Question (i) Describe the signs (objective characteristics usually noted/detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient) reported. Also make note of any key history findings, what samples are taken (and why) and the meaning of any laboratory results reported. (There is no need to describe the laboratory testing as this is the basis of another question).

The signs of infection are often nonspecific but include volume depletion of the body, presence of S.enteritidis in the stool and abdominal tenderness (1). Volume depletion is defined as a deficit in extracellular fluid (ECF) volume. In humans, this is due to a net reduction in the amount of sodium in the body (2), perhaps due to the fluid loss in diarrhea. Clinical manifestations of volume depletion such as decreased skin turgor (skin with decreased skin turgor stays elevated after being pulled up and released), dry mucous membranes, tachycardia and orthostatic hypotension (2). It can also include abnormal respiratory findings, such as rapid respiratory rate or chest crepitations suggestive of pneumonia, a lung infection, and hepatosplenomegaly, or enlargement of the liver (1). This may suggest invasive infection. The symptoms include vomiting, abdominal cramps, watery diarrhea, mild fever, abdominal cramps, and blood in the stool (3) In some immunosuppressed individuals, there is no diarrhea despite infection (4).

Johnny’s symptoms have been continuing for a few days, as evidenced by the mild fever, abdominal cramps and 4-day duration of diarrhea. The doctor also notes volume depletion and abdominal tenderness as signs for Johnny’s infection. Clinical diagnosis of volume depletion typically involves comparing patient weights before and after fluid loss (2). Assuming this individual is otherwise healthy (good physical condition, not immunocompromised), his symptoms should improve soon. His history of infection is unknown but Johnny has been eating raw eggs for the past week as part of his new diet regiment. This piece of history provides further evidence of S.enteritidis infection.  

A stool sample was collected to determine the presence of bacterial growth and determine the type of bacteria. Different complications can result depending on the type of bacteria. S. enteritidis results in nontyphoidal Salmonella. There are typhoidal salmonella bacteria such as Typhi, Paratyphi A and Paratyphi which can infect individuals and cause enteric fever such as typhoid fever and require additional treatment (4). Using the stool, one can determine the cause of infection and treatment as well.

Question (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.

The gastrointestinal tract, specifically the small and large intestine, is the main body system affected by a S.enteritidis infection (5). Salmonella are gram-negative bacteria belonging to the Enterobacerieae family; in this case study, the patient is infected with Salmonella enterica serovar enteritidis, a strain of Salmonella that is non-typhoidal and capable of infecting humans (6, 7). The most common outcome of an infection with non-typhoidal Salmonella results in self-limited acute gastroenteritis, a localized infection of the terminal ileum and colon that can produce symptoms such as fever, diarrhea, abdominal pain and nausea (8, 5). It is possible that the bacteria may enter the bloodstream, resulting in bacteremia, however, this is more common in immunocompromised individuals (8). Systemic disease is more likely in the human-restricted Salmonella serovars Typhi and Paratyphi A, and results in enteric typhoid fever, which is fatal without the treatment of antibiotics (7)

Salmonella enters the body via ingestion of contaminated food or water (9). The bacteria have adapted to survive within the harsh environment of the stomach in order to colonize the ileum and colon, and infect the intestinal epithelium (5). Salmonellae infect the intestinal epithelial cells by a specific process called bacteria mediated endocytosis; a process where cytoskeleton rearrangements occur on the apical side of the intestinal epithelium which allows for membrane ruffles to surround and internalize the bacteria (9).

The lower gastrointestinal tract contains the gut microbiota, a large population of resident bacteria (8). The gut microbiota has a mutually beneficial relationship with the human body as it helps to inhibit the colonization of pathogens (8). However, if the mucosal layer is breached by a pathogen, such as S.enteritidis, acute inflammation will occur as an immune response (8). The normal physiological function of Johnny’s GI tract is disturbed due to the invasion of the mucosa  by Salmonellae, which results in the release of proinflammatory cytokines, leading to an acute inflammatory response and intestinal damage (9) Pathogen-associated molecular patterns (PAMP) which are molecular markers specific to a pathogen that stimulate a host response, of Salmonella include the bacterial flagellum which activate pattern recognition receptors (PRR) on the intestinal epithelium (TLR4, LPS, TLR5) or in the cytosol (10). Stimulation of PRRs results in the secretion of proinflammatory cytokines, such as IL-1, 1L-6 and IL-8, which contribute to the inflammatory response (10). Symptoms such as abdominal pain, as seen in Johnny’s case, and fever can result from this.

Inflammation may also occur as a result of Salmonella virulence factors; Salmonellae encode a type III secretion system (T3SS) within their genome, specifically within the Salmonella pathogenicity island 1(SPI-1) (10). T3SS are macromolecular complexes that disrupt host cell functions by transporting virulence proteins from the bacterial cytoplasm and into the host cell (10). SPI-1 protein SopE and SopE2 activate certain signalling pathways to initiate inflammation and fluid secretion (10). Also, several SPI-1 proteins, such as SipA, SipC and SopB function to aid the entry of Salmonellae into the host cell by increasing cytoskeleton rearrangements and membrane ruffling.

Diarrhea is caused by excessive secretion and/or reduced absorption of fluid and electrolytes across the epithelium (11). Normally, the intestines are responsible for absorbing fluid. However, the intestinal inflammation as a result of Salmonella infection can result in diarrhea if the intestinal epithelia is disrupted, leading to an influx of water (10). As described above, SPI-1 proteins can also contribute to fluid secretion, resulting in diarrhea. Intestinal inflammation can produce other common inflammatory symptoms such as fever, chills, abdominal pain and leukocytosis (5).

Several strains of S.enteritidis have been found to produce enterotoxins and cytotoxins (12, 13); although their mechanisms of pathogenesis remain unclear, it is predicted that both contribute to diarrheal symptoms alongside intestinal inflammation (14). Certain S.enteritidis strains produce a thermolabile enterotoxin that is similar to cholera toxin, as both result in increased water secretion in intestinal loops, likely leading to diarrhea (13, 14). Cholera toxin, an enterotoxin, activates the host cell adenylate cyclase which results in increased intracellular cAMP levels (15). An accumulation of cAMP stimulates the chloride channel contained in intestinal epithelial cells, leading to a net increase in chloride secretion which causes the osmotic movement of water into the intestinal lumen, manifesting as the clinical symptom of diarrhea (15). Salmonella strains encoding the enterotoxin gene (stn) have been shown to activate the adenylate cyclase in rabbit intestinal epithelial cells which results in increased cAMP levels, similar to Cholera toxin (16). However, the exact mechanism of Salmonella enterotoxin and its contribution to diarrheal symptoms in humans is unknown (16).

S.enteritidis strains may also produce a cytotoxin that is similar to Shiga toxin as both have been shown to inhibit protein synthesis (13, 17). Shiga toxin disrupts the eukaryotic ribosome in order to inhibit protein synthesis; this damages the cells and results in their apoptosis (17). The damage within the intestine created by the toxin can lead to fluid secretion and diarrhea (18). However, as stated previously, research regarding the use of cytotoxins within Salmonella pathogenesis is ongoing.

After invading the intestinal epithelia, S.enteritidis can spread to mesenteric lymph nodes and to the blood which leads to bacteremia (19). Since the inflammatory response can lead to ulceration, the bacteria may be able to enter the bloodstream and spread systemically (9). Symptoms of bacteremia range from gastroenteritis, prolonged fever to death from septic shock (8). Clinical signs and symptoms of bacteremia may include fever, rapid heart rate, chills, low blood pressure, gastrointestinal symptoms, rapid breathing and disorientation (2). Bacteremia can cause extraintestinal focal infections and diseases such as osteomyelitis, endocarditis, mycotic aneurysms, pneumonia and others (19).

Question (iii) What treatment(s) might the doctor suggest or prescribe and how do these treatments work? If antibiotics are prescribed what is the antibacterial treatment of choice and how does the antibiotic work to rid the body of the organism?

Salmonella Enteritidis typically results in self-limiting gastroenteritis causing fever, abdominal pain and diarrhea that lasts for 4 to 7 days (14, 10). Gastroenteritis is primarily treated with the replacement of fluid and electrolytes lost as a result of diarrhea, and with the controlling of abdominal pain, nausea and vomiting when necessary (5, 10). Medication may also be recommended to limit diarrhea (21). Antibiotics are not suggested for the treatment of Salmonella gastroenteritis because they have not been proven to shorten illness and they may increase the prevalence of antibiotic resistant strains (5). However, antibiotics may be prescribed if the patient is immunocompromised or if the doctor suspects that the bacteria has entered the bloodstream, as that can lead to systemic infection which can cause septicemia or enteric fevers (9). There are several oral antibiotics available to treat Salmonella infections (20). Due to the high prevalence of antibiotic resistance in Salmonella enterica, antibiotics may be chosen based on if the specific strain isolated in the lab is susceptible to the specific antibiotic treatment (14).

Commonly prescribed oral antibiotics to treat Salmonella induced bacteremia are fluoroquinolone, azithromycin, or amoxicillin (20). Fluoroquinolone is a quinolone antibiotic, which acts as an inhibitor to block bacterial DNA replication by inhibiting topoisomerases, which will kill the bacteria. Azithromycin is part of the macrolide class and it binds to the bacterial ribosomes, inhibiting bacterial protein synthesis and resulting in bacterial cell death (22). Amoxicillin is a beta-lactam antibiotic which inhibits the final stages of cell wall synthesis, ultimately leading to cell lysis of the bacteria (23).

The patient in the case study is displaying signs and symptoms of uncomplicated Salmonella gastroenteritis such as a fever, diarrhea and abdominal pain. From the information provided, Johnny is a young and otherwise healthy individual that is not at risk for bacteremia. Therefore, the doctor might recommend that Johnny consumes a lot of fluid and electrolytes. Johnny should keep monitoring his symptoms to confirm that his infection does not progress into something more severe such as bacteremia.

Question (iv) Why did the doctor tell Johnny to stop eating raw eggs? Is this a reportable communicable disease?

Salmonella Enteritidis- Body Systems

Salmonella lives in the intestines of most food animals and as a result, both the inside and outside of raw eggs can be contaminated with the bacteria though direct and indirect methods (24). Direct contamination occurs during the formation of the egg in the reproductive tract of hens and indirect contamination occurs after the egg has been laid and Salmonella contaminating the outside of the egg penetrates through the shell (24). Inadequate cooking before consumption can result in infection (3). Therefore, Johnny’s doctor recommended that Johnny stop eating eggs to prevent infection.

The Centre for Disease Control (CDC) defines a ‘reportable’ or ‘notifiable’ disease as “one for which regular, frequent, and timely information regarding individual cases is considered necessary for the prevention and control of the disease” (25). Reporting of reportable diseases by healthcare professionals protects public health by aiding public health officials in measuring disease trends, assessing the effectiveness of control and prevention measures, recognise certain populations or areas at risk and prepare specific resources (25). The CDC defines a communicable disease as “an illness caused by infectious agent or its toxins that occurs through the direct or indirect transmission of the infectious agent or its products from an infected individual or via an animal, vector or the inanimate environment to a susceptible animal or host” (26). Salmonella is a reportable communicable disease according to the BC Center for Disease Control (27) and the CDC (25). Salmonella Enteritidis is considered a reportable communicable disease because it is spread to humans via the fecal-oral route from contaminated animal products. In severe cases it has the potential to be fatal, and the reporting of the disease may help to track the source of the infection to a specific farm or producer to prevent transmission and spread of disease (28).

References

1. Crump, J. A., Sjölund-Karlsson, M., Gordon, M. A., & Parry, C. M. (2015). Epidemiology, Clinical Presentation, Laboratory Diagnosis, Antimicrobial Resistance, and Antimicrobial Management of Invasive Salmonella Infections. Clinical Microbiology Reviews, 28(4), 901–937. https://doi.org/10.1128/CMR.00002-15
2. Volume Depletion - Endocrine and Metabolic Disorders [Internet]. Merck Manuals Professional Edition. [cited 2021 Jan 22]. Available from: https://www.merckmanuals.com/professional/endocrine-and-metabolic-disorders/fluid-metabolism/volume-depletion
3. Mayo Clinic. (2021). Salmonella - Symptoms and causes. Available at: https://www.mayoclinic.org/diseases-conditions/salmonella/symptoms-causes/syc-20355329
4. Brown M, Eykyn SJ. 2000. Non-typhoidal Salmonella bacteraemia without gastroenteritis: a marker of underlying immunosuppression: review of cases at St. Thomas' Hospital 1970-1999. J Infect 41:256–259.
5. Giannella RA. Medical microbiology 4^th edition. Texas: University of Texas Medical Branch at Galveston; 1996. 21, Salmonella.
6. Brunet P. Procedure for Isolation and Identification of Salmonella from Poultry Carcasses. Poult. Sci. 1986;65(2):405.
7. Schlute M, Hensel M. Models of intestinal infection by Salmonella enterica: introduction of a new neonate mouse model. F1000Res. 2016 Jun 24; 5.
8. Santos R, Raffatellu M, Bevins C, Adams L, Tükel Ç, Tsolis R et al. Life in the inflamed intestine, Salmonella style. Trends Microbiol. 2009;17(11):498-506.
9. Salmonella - Medical Microbiology - NCBI Bookshelf [Internet]. [cited 2021 Jan 22]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK8435/
10. Bennett JE, Dolin R, Blaser MJ. Mandell, Douglas, and Bennett's principles and practice of infectious diseases. 9th ed. Philadelphia, PA: Elsevier; 2020. Salmonella Species.
11. Thiagarajah JR, Donowitz M, Verkman AS. Secretory diarrhoea: mechanisms and emerging therapies. Nat Rev Gastroenterol Hepatol. 2015;12(8):446–57.
12. Ashkenazi S, Cleary TG, Murray BE, Wanger A, Pickering LK. Quantitative analysis and partial characterization of cytotoxin production by salmonella strains. Infect Immun. 1988; 56(12): 3089- 3094.
13. Rumeu MT, Suárez MA, Morales S, Rotger R. Enterotoxin and cytotoxin production by Salmonella enteritidis strains isolated from gastroenteritis outbreaks. J Appl Microbiol. 1997; 82(1): 19-31.
14. Kenneth Todar M. Salmonella and Salmonellosis [Internet]. Online Textbook of Bacteriology. [cited 23 January 2021]. Available from: http://textbookofbacteriology.net/
15. Vanden Broeck D, Horvath C, De Wolf MJS. Vibrio cholerae: cholera toxin. Int J Biochem Cell Biol. 2007 Jan 1; 39(10): 1771-1775.
16. Chopra AK, Huang JH, Xu X, Burden K, Niesel DW, Rosenbaum MW, Popov VL, Peterson JW. Role of Salmonella enterotoxin in overall virulence of the organism. Microb Pathog. 1999 Sep 1; 27(3): 155-171.
17. Melton-Celsa AR. Shiga toxin (stx) classification, structure and function. Microbiol Spectr. 2014; 2(4).
18. Mattock E, Blocker AJ. How do the virulence factors of Shigella work together to cause disease? Front Cell Infect Microbiol. 2017 Mar 2; 7(64).
19. Chen, P.-L., Chang, C.-M., Wu, C.-J., Ko, N.-Y., Lee, N.-Y., Lee, H.-C., Shih, H.-I., Lee, C.-C., Wang, R.-R., & Ko, W.-C. (2007). Extraintestinal focal infections in adults with nontyphoid Salmonella bacteraemia: Predisposing factors and clinical outcome. Journal of Internal Medicine, 261(1), 91–100. https://doi.org/10.1111/j.1365-2796.2006.01748.x
20. Cianflone NFC. Salmonellosis and the GI Tract: More than Just Peanut Butter. Curr Gastroenterol Rep. 2008 Aug;10(4):424–31.
21. Salmonella infection - Diagnosis and treatment - Mayo Clinic [Internet]. [cited 2021 Jan 22]. Available from: https://www.mayoclinic.org/diseases-conditions/salmonella/diagnosis-treatment/drc-20355335
22. Louie A, Drusano GL. Antibacterial Chemotherapy. In: Goldman-Cecil Medicine [Internet]. Twenty Sixth. Philadelphia, PA: Elsevier; 2020 [cited 2021 Jan 22]. p. 1850-1862.e2. Available from: https://www-clinicalkey-com.ezproxy.library.ubc.ca/#!/content/book/3-s2.0-B978032353266200271X?scrollTo=%23hl0001562
23. Amoxicillin- ClinicalKey [Internet]. [cited 2021 Jan 22]. Available from: https://www-clinicalkey-com.ezproxy.library.ubc.ca/#!/content/drug_monograph/6-s2.0-31
24. Howard Z.R., O’Bryan C.A., Crandall P.G., Ricke S.C. Salmonella Enteritidis in shell eggs: Current issues and prospects for control. Food Res. Int. 2012;45:755–764. doi: 10.1016/j.foodres.2011.04.030.
25. CDC, 2014. Summary of notifiable diseases – United States, 2012. Morbidity and Mortality Weekly Report 61, 1–23.
26. Menu of Suggested Provisions For State Tuberculosis Prevention and Control Laws [Internet]. Centers for Disease Control and Prevention; 2012 [cited 23 January 2021]. Available from: https://www.cdc.gov/tb/programs/laws/menu/definitions.htm
27. BC Center for Disease Control. (2021). Communicable Diseases. Retrieved from: http://www.bccdc.ca/health-professionals/data-reports/communicable-diseases
28. Centers for Disease Control and Prevention. (2013). Infection with Salmonella. Available at: https://www.cdc.gov/training/SIC_CaseStudy/Infection_Salmonella_ptversion.pdf

Q2. The Microbiology Laboratory

Question (i) Other than the stated bacterial cause, what are the most common bacterial pathogens associated with this type of infectious scenario.

In Johnny’s case, it has been determined that he has been infected with Salmonella enterica ssp. Enteritidis. This bacteria is a reasonable culprit, as it is well-associated with the presented symptoms and raw eggs being newly introduced in the patient’s diet. Salmonella enterica is one of two Salmonella species and is associated with causing gastroenteritis— a syndrome referred to as Salmonellosis (1). Gastroenteritis is characterized by diarrhea, abdominal cramps, and fever (1) which are all consistent with Johnny’s symptomatic presentation. The Salmonella enterica species is a gram negative, facultative-anaerobic bacilli with flagella, and can be further subdivided into serotypes by their surface lipopolysaccharide O antigens, heat-liable flagellar protein H antigens, and Vi antigens (note that Vi is only possessed by select serovars such as S. Typhi) (1). Salmonella Enteritidis (SE) is a serovar of the S. enterica species, and along with Salmonella Typhimurium, are the two most common serovars observed to cause infection in humans (2, 3). Salmonellosis is typically a food-borne illness and can be contracted by eating contaminated eggs, poultry, meat, unpasteurized milk or juice, cheese, along with some fruits and vegetables (4). Specifically, for Salmonella-induced gastroenteritis, symptoms are known to usually present within 6 to 48 hours, sometimes longer, and can last for about 2 to 7 days (1), which is consistent with Johnny's case.

Based on the Johnny's clinical presentation, incubation period, and recent exposure to raw eggs, a few pathogens may be associated with this given case. These include: Escherichia coli, Campylobacter jejuni, Cryptosporidiosis and Shigella (4), these bacteria are also commonly known to be responsible for food borne illness similar to Salmonella. Enteropathogenic E. coli (EPEC), Campylobacter jejuni (Campylobacteriosis), and Shigella infection can also occur from eating contaminated eggs, also making them candidates for our given scenario (4). Other potential infectious causes of Johnny’s symptoms to consider are presented in Table 1; the most likely organisms are discussed below.

Table 1 (Hatchette and Farina, CMAJ 2010)

Escherichia coli:

Escherichia coli inhabits the human gut either as a commensal or pathogenic bacteria. In particular, E. coli serotypes O157 and O104 are commonly responsible for outbreaks of human infection. There are five main strains of diarrhea-inducing E. coli: enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC) (5). Infection among these five strains share common symptoms as seen in Johnny (watery diarrhea, abdominal cramps, and fever), with certain strains having potential for more serious complications including intestinal perforation by EIEC and hemolytic uremic syndrome by STEC (5). Furthermore, depending on the virulence factor of the E. coli, it may cause either noninflammatory or inflammatory diarrhea (1). As an example, Shiga Toxin producing E. coli can result in severe stomach cramps, diarrhea (often bloody), vomiting, or fever, which is usually not very high (6). It is noted that people who get infected with this bacteria will start feeling sick 3 to 4 days after eating or drinking something that is contaminated. However, illness may begin anywhere from 1 to 10 days after exposure (7). In addition, similar to Salmonella, these bacteria are also Gram-negative bacillus (1). Note that only the E. coli that possess genetic elements encoding for virulence factors and invasion factors are ones that cause symptoms that align with Salmonella enteritidis and therefore, our case.  Transmission of pathogenic E. coli can occur through contamination by animal faeces, soil, or water at any stage in food production, and is therefore preventable by improved sanitation. Reported outbreaks by E. coli include a wide range of food items, including raw eggs (5). Laboratory testing for E. coli can include faecal sample culturing for bacterial detection, or in the case of STEC infection, Shiga toxin production can be assessed (8). To detect an E. coli outbreak, fingerprinting may be conducted across infected individuals (8).

Campylobacter jejuni

Symptoms resulting from an infection of the Campylobacter species include acute gastroenteritis which includes diarrhea, abdominal pain, fever, nausea and vomiting (1) which are similar to Johnny's scenario.  Comparable to the Salmonella species, the Campylobacter species are also Gram-negative bacteria. In addition, they are microaerophilic, non-fermenting motile rods that have a single polar flagellum (1). Like Salmonella, these bacteria have O and H protein antigens and also have porin protein and flagellin surface-exposed antigens (1). Furthermore, Campylobacter bacteria cause inflammatory diarrhea and fever through colonization of the small and large intestines (1), normally taking around 2-5 days to see symptoms with the exception of cases taking as little as 1 day or as long as 10 days (9). Consequently, Campylobacter bacterial infection typically comes from raw or undercooked poultry, but can also be transmitted through seafood, produce, meat and untreated water (10). The infection will typically resolve on its own with antibiotic treatment as a possible therapy. Complications may arise, especially in patients with underlying conditions or chemotherapy, these include irritable bowel syndrome and temporary paralysis (10). Outbreaks related to this bacteria  are especially common in low-income countries and are carried in the gastrointestinal tract of animals, and can therefore be transmitted through animal faeces (10)

Cryptosporidium

This organism is a parasite and when it infects a human it has a very similar symptom profile to Johnny’s, including (usually) watery diarrhea, stomach cramps, upset stomach, and slight fever (4). Commonly a person can contract this parasite through uncooked food or food contaminated by an ill food handler after cooking, or contaminated drinking water (4). Typically, this infection takes about seven days to show symptoms and lasts about one or two weeks (11). Furthermore, this case would be possible if the batch of eggs that Johnny was using was infected with Cryptosporidium; however, this option is unlikely because generally this disease is contracted through exposure with stool or drinking unprotected wells. (11)

Shigella

Shigella can be described as Gram-negative, nonmotile, facultatively anaerobic, non-spore-forming rods (1). Based on their pathogenicity, physiology, and serology, Shigella are differentiated from Escherichia coli (1). Shigella bacteria can cause infection of the intestinal lining predominantly by four disease-causing species: Shigella sonnei, flexneri, dysenteriae, and boydii (12, 13). Of these species, S. sonnei is most commonly responsible for disease cases in the USA and Canada, followed by S. flexneri (12). Upon infection with these bacteria, the host defense includes inflammation (1) which allows for these bacteria to have a similar symptom profile as our case which includes abdominal pain, tenesmus, watery diarrhea, and or dysentery (1). The clinical presentation of a Shigella infection can be both watery diarrheas associated with vomiting and mild to moderate dehydration and dysentery characterized by a small volume of bloody, mucoid stools and abdominal pain. Compared to the case given, the clinical presentation of shigellosis aligns with the symptoms that we see in Johnny. In addition, it is noted that raw foods are more likely contaminated with these bacteria (14) including raw produce but not limited to contaminated drinking water, uncooked foods and cooked foods that are not reheated after contact with an infected food handler (4). Complications of Shigella infection can include encephalopathy and febrile seizures in children in severe cases. The spread of Shigella bacteria is especially prominent in tightly crowded spaces with poor sanitation, such as refugee camps (12). Testing for Shigella can include assessing for increased heart rates, decreased blood pressure, dehydration, and abdominal tenderness. Additionally, stool samples can be analyzed to check for increased white blood cell concentrations, indicating infection. Although Shigella infections will tend to recover on their own, fluid replacement and antibiotic treatments are available for severe cases (12).

References

1. Giannella RA. Salmonella. In: Baron S, editor. Medical Microbiology [Internet]. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2021 Jan 22]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK8435/

2. Desin TS, Lam PK, Koch B, Mickael C, Berberov E, Wisner AL, Townsend HG, Potter AA, Köster W. Salmonella enterica serovar Enteritidis pathogenicity island 1 is not essential for but facilitates rapid systemic spread in chickens. Infection and immunity. 2009 Jul 1;77(7):2866-75.

3. Fabre L, Zhang J, Guigon G, Le Hello S, Guibert V, Accou-Demartin M, de Romans S, Lim C, Roux C, Passet V, Diancourt L. CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections. PloS one. 2012 May 18;7(5):e36995.

4. Nutrition C for FS and A. What You Need to Know about Foodborne Illnesses [Internet]. FDA. FDA; 2020 [cited 2021 Jan 22]. Available from: https://www.fda.gov/food/consumers/what-you-need-know-about-foodborne-illnesses

5. Yang SC, Lin CH, Aljuffali IA, Fang JY. Current pathogenic Escherichia coli foodborne outbreak cases and therapy development. Archives of microbiology. 2017 Aug;199(6):811-25.

6. Enterotoxigenic E. coli (ETEC) | E. coli | CDC [Internet]. 2019 [cited 2021 Jan 22]. Available from: https://www.cdc.gov/ecoli/etec.html

7. Symptoms | E. coli | CDC [Internet]. 2019 [cited 2021 Jan 22]. Available from: https://www.cdc.gov/ecoli/ecoli-symptoms.html

8. E.coli Infection [Internet]. Bccdc.ca. 2021 [cited 22 January 2021]. Available from: http://www.bccdc.ca/health-info/diseases-conditions/e-coli-infection#:~:text=Tests%20and%20diagnosis,coli%20has%20occurred.

9. Government of Ontario M of H and L-TC. Campylobacteriosis - Diseases and Conditions - Publications - Public Information - MOHLTC [Internet]. Government of Ontario, Ministry of Health and Long-Term Care; [cited 2021 Jan 22]. Available from: http://www.health.gov.on.ca/en/public/publications/disease/campylobacter.aspx

10. Campylobacter (Campylobacteriosis) | Campylobacter | CDC [Internet]. Cdc.gov. 2021 [cited 22 January 2021]. Available from: https://www.cdc.gov/campylobacter/index.html

11. General Information for the Public | Cryptosporidium | Parasites | CDC [Internet]. 2019 [cited 2021 Jan 22]. Available from: https://www.cdc.gov/parasites/crypto/general-info.html

12. Encyclopedia M. Shigellosis: MedlinePlus Medical Encyclopedia [Internet]. Medlineplus.gov. 2021 [cited 22 January 2021]. Available from: https://medlineplus.gov/ency/article/000295.htm

13. Boadi S, Wren MW, Morris-Jones S. Selective testing of β-galactosidase activity in the laboratory identification of Salmonella and Shigella species. Journal of clinical pathology. 2010 Dec 1;63(12):1101-4.

14. Sources of Infection & Risk Factors | Shigella – Shigellosis | CDC [Internet]. 2019 [cited 2021 Jan 22]. Available from: https://www.cdc.gov/shigella/infection-sources.html

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

Based on Johnny’s presentation of signs and symptoms, including multiple days of diarrhea, severe abdominal cramping, mild fever and watery diarrhea, a stool sample is most appropriate. As many different pathogens can cause infection with these symptoms, Salmonellosis infection cannot be diagnosed solely based on Johnny’s clinical presentation; laboratory testing is required to isolate the pathogenic bacteria and make a diagnosis (1). Feces is the sample of choice collected during the acute phase of a diarrheal disease when bacterial gastroenteritis is suspected (2). Analysis of the stool sample can help confirm whether the patient’s illness is due to a bacterial infection, or other issues such as viruses or poor nutrient absorption. Blood, urine, and bile samples may be collected if enteric fever is suspected; however Johnny’s symptoms are mild, and therefore these samples are not likely to be collected (1, 3, 4). Rectal swabs can also be used in cases of gastroenteritis, however it is less sensitive than stool samples for culture and primarily used for children and infants, and is therefore not applicable in this case (2, 5).

Bacterial pathogens that cause acute gastroenteritis, such as Salmonella enterica, ssp Enteritidis, often shed in the feces of an infected patient; therefore, the stool sample is the most commonly tested clinical material (6).  Since the clinical symptoms associated with Salmonella enterica, ssp Enteritidis are similar to those caused by several other bacterial pathogens, its diagnosis requires isolation from stool samples. Once the organism is isolated from the patient’s stool, further testing, such as biochemical and serotyping tests, are required to confirm the diagnosis (2).

Additionally, some environmental samples may be sent to the laboratory for analysis. Any remaining eggs or eggshells left at Johnny’s house are of particular interest to confirm the source of Salmonella infection. Other recent foods Johnny has consumed can provide insight into potential sources of Salmonella; outbreaks of Salmonella enterica have been linked to frozen breaded chicken (6 unique outbreaks in 2018), chocolate eclairs (85 case outbreak in 2019) and red onions (515 case outbreak in 2020), to name a few Canadian examples (7-9).

Stool samples can be collected at home, in medical clinics, or in hospitals. Typically, the patient is given stool collection kits, for each day that collection is needed. It is important that physicians collect a sufficient amount of samples for adequate testing (10). For instance, three stools should be submitted for culturing in order to maximize the clinical yield for testing (4). The use of multiple fecal samples rather than one can increase the sensitivity of detection from 10% to 30% (3). In addition, it is important to provide the laboratory with adequate patient information, including age, sex, medical history, underlying conditions, date of onset, and any tentative diagnoses​ ​to help streamline the selection of appropriate testing methods (10).

The fecal samples are collected in clean, dry containers that are tightly sealed to prevent contamination (2, 11). Furthermore, the stool should not be collected from the toilet bowl, or contact any toilet paper or urine. Stool collection kits may include “toilet hats” or stool-specific vials for easy collection (12, 13).

LifeLabs Stool Collection Vials (13).

If the stool is liquid or soft, a minimum of 5 mL of stool should be collected. If the stool is in a firm solid form, 0.5-2 grams are adequate (2). Once collected, fresh stool samples should be transported to the laboratory and processed within 2 hours. If stool samples cannot immediately be sent to the laboratory for testing within a 2-hour period (2), they should be refrigerated and can also be placed in a Cary-Blair transport medium, which helps conserve bacterial enteric pathogens. Keeping the stool samples at room temperature can allow for commensal bacteria to overgrow, creating difficulties isolating the pathogenic Salmonella bacteria in the laboratory. Refrigerating the samples will inhibit all bacterial growth in the stool (6). Upon initial collection of the fecal samples in the laboratory, the specimens are macroscopically examined, and any mucus or blood is noted (14). Further screening of the samples can involve multiple identification protocols. Common agars and biochemical tests used to identify the pathogenic micro-organism in cases of gastroenteritis include MacConkey agar (MAC), triple sugar iron (TSI) agar, desoxycholate citrate agar (DCA), indole test, urease test, xylose lysine desoxycholate agar (XLD) and selenite broth (15).

The Microbiology Laboratory is crucial in the diagnosis of this infectious disease, as it is needed to properly diagnose and classify the disease-causing pathogenic bacteria. Correctly identifying the bacterial pathogen is important to determine the correct therapeutic action and outline the proper treatment framework for the patient. Additionally, this information can be used for surveillance or epidemiological studies (2, 16). The laboratory diagnosis of Salmonella enterica, ssp Enteritidis in this scenario is important because this pathogen can be transmitted from person to person via the fecal-oral route (16). This information can then help the patient understand how to avoid future re-infection or transmission. In the case of Salmonella enterica, ssp Enteritidis, this may mean avoiding the ingestion of raw or undercooked poultry. Apart from patient care, the microbiology laboratory analysis of stool samples is an important aspect of public health, because isolating organisms like Salmonella enterica, ssp Enteritidis can help identify and track outbreaks of bacterial gastroenteritis within the community (2). As previously mentioned, the Microbiology Laboratory plays a significant role in identifying new food-borne outbreaks, using Whole Genome Sequencing to link and cluster related cases (7).

Additionally, proper laboratory testing and diagnosis will guide the appropriate treatment measures for this infection. Gastroenteritis-causing pathogenic bacterial infections are self-limiting and often resolve on its own without treatment intervention (2, 16, 17). A common treatment measure for Salmonella enterica, ssp Enteritidis infections is rehydration, which replaces fluid loss due to ongoing diarrhea (18). Furthermore, microbiological testing can confirm that Johnny’s illness is a case of non-typhoidal salmonellosis. This information is pertinent in the treatment regimen, so that antibiotic administration can be avoided. Antibiotics are not recommended for Salmonella enterica, ssp Enteritidis infections, because they may prolong the illness and fecal excretion of the organisms (2, 3, 16, 19). Furthermore, the number of antibiotic-resistant strains continues to rise, so the use of antibiotics should be limited to necessary cases, such as in the treatment of typhoid and enteric fevers (typhoidal salmonella) (16, 20).

References:

1. Giannella RA. Salmonella. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 21. Available from: https://www.ncbi.nlm.nih.gov/books/NBK8435/
2. Humphries RM, Linscott AJ. Laboratory diagnosis of bacterial gastroenteritis. Clinical microbiology reviews. 2015 Jan 1;28(1):3-1.
3. Crump JA, Sjölund-Karlsson M, Gordon MA, Parry CM. Epidemiology, clinical presentation, laboratory diagnosis, antimicrobial resistance, and antimicrobial management of invasive Salmonella infections. Clinical microbiology reviews. 2015 Oct 1;28(4):901-37.
4. Jenkins, C, Gillespie, SH. 2006. Salmonella spp. In S. H. Gillespie, & P. M. Hawkey (eds.), Principles And Practice of Clinical Bacteriology. John Wiley & Sons, Ltd, Chichester, UK. doi:10.1002/9780470017968.
5. Nakanishi K, Tsugawa T, Honma S, Nakata S, Tatsumi M, Yoto Y, Tsutsumi H. Detection of enteric viruses in rectal swabs from children with acute gastroenteritis attending the pediatric outpatient clinics in Sapporo, Japan. Journal of Clinical Virology. 2009 Sep 1;46(1):94-7.
6. Food Microbiology Testing. Traditional and Alternative Methods. 2019. https://www.rapidmicrobiology.com/test-method/salmonella-detection-and-identification-methods
7. [Internet]. Bccdc.ca. 2021 [cited 30 January 2021]. Available from: http://www.bccdc.ca/resource-gallery/Documents/Statistics%20and%20Research/Statistics%20and%20Reports/Epid/Enterics/2018%20BCISFP%20Annual%20Report.pdf
8. Canada P. Public Health Notice - Outbreak of Salmonella infections - Canada.ca [Internet]. Canada.ca. 2021 [cited 30 January 2021]. Available from: https://www.canada.ca/en/public-health/services/public-health-notices/2019/outbreak-salmonella.html
9. Canada P. Public Health Notice: Outbreak of Salmonella infections linked to red onions imported from the United States - Canada.ca [Internet]. Canada.ca. 2021 [cited 30 January 2021]. Available from: https://www.canada.ca/en/public-health/services/public-health-notices/2020/outbreak-salmonella-infections-under-investigation.html#a7
10. Washington, JA. 1996. Principles of Diagnosis. In S. Baron (ed.), Medical Microbiology. University of Texas Medical Branch at Galveston, Galveston, Texas.
11. HealthLinkBC. Medical Tests. Stool Analysis. https://www.healthlinkbc.ca/medical-tests/aa80714#tp16699d
12. Abrahamson M, Hooker E, Ajami NJ, Petrosino JF, Orwoll ES. Successful collection of stool samples for microbiome analyses from a large community-based population of elderly men. Contemporary clinical trials communications. 2017 Sep 1;7:158-62.
13. LifeLabs - Bacterial Stool Collection Vials [Internet]. Physician Report. 2011 [cited 29 January 2021]. Available from: https://lifelabs.azureedge.net/lifelabs-wp-cdn/wp-content/uploads/2018/08/2011_November_Lab_Update.pdf
14. Stool Culture | Lab Tests Online [Internet]. [cited 2021 Jan 22]. Available from: https://labtestsonline.org/tests/stool-culture
15. Boadi S, Wren MW, Morris-Jones S. Selective testing of β-galactosidase activity in the laboratory identification of Salmonella and Shigella species. Journal of clinical pathology. 2010 Dec 1;63(12):1101-4.
16. Gal-Mor, O. 2019. Persistent infection and long-term carriage of Typhoidal and Nontyphoidal salmonellae. Clinical Microbiology Rev 32:e00088-18. https://doi.org/10.1128/CMR .00088-18
17. U.S. National Library of Medicine. Medicine Plus- Medical Encyclopedia. Bacterial Gastroenteritis. https://medlineplus.gov/ency/article/000254.htm
18. Sattar SBA, Singh S. Bacterial Gastroenteritis. 2020. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing. Available from: https://www.ncbi.nlm.nih.gov/books/NBK513295/
19. Ventola CL. The antibiotic resistance crisis: part 1: causes and threats. P T. 2015;40(4):277-283.
20. Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Available from: https://www.ncbi.nlm.nih.gov/books/NBK8417/

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

Salmonella enterica is a gram negative, rod shaped, and facultatively anaerobic bacteria (1). They are generally non-lactose fermenting, citrate utilizing and acetyl methyl carbinol negative (1). These biochemical characteristics can be used to identify this species in cultures isolated from clinical samples, using microbiological cultures and biochemical tests (1). Identification of suspected Salmonella colonies is often based on Iysine decarboxylation, H2S production and the absence of lactose fermentation (2). The identification can then be confirmed and the serotype can be determined with serological tests, such as polymerase chain reaction, enzyme linked immunosorbent assays and mass spectrometry. The diagnosis of Salmonella spp. can be divided into four phases: initial isolation, screening identification, definitive identification, and serotyping.

Microbiological Cultures and Biochemical Tests

The diagnosis of bacterial gastroenteritis pathogens generally involves culturing a collected stool sample from the patient (3). Performing stool cultures can provide useful information for patients infected with Salmonella, Shigella, E. coli, and Campylobacter, which are some of the main pathogens that may be suspected given Johnny’s symptoms (3). The specimen will be cultured in a lab on various selective and non-selective agars and the results will be used to narrow down the suspected pathogen.

First, the clinical specimens are plated on a variety of media and incubated under aerobic and anaerobic conditions (4). Several non selective and selective agar plates are used (5). Non selective agars do not have special additives and are used to cultivate the growth of almost all types of bacteria in the lab, which can be useful in increasing the amount of bacteria in the sample for future agars and tests (4, 6). Some examples include tryptic soy agar and nutrient agar (6). The clinical specimen is also plated on enrichment broth (4). Enrichment broth contains specific essential growth factors that encourage the growth of certain microorganisms (6). They are also used to suppress the growth of normal fecal flora (6). Overall, these broths serve to increase the amount of desired microbes to an adequate level and minimize the presence of other organisms, which is critical to the isolation of the relevant pathogen from the sample (6). Selenite F and tetrathionate are two enrichment broths that are used to support the growth of Salmonella serovars (5). In fact, Selenite is also known as the Salmonella enrichment broth (7). Any growth in the enrichment broth is sub cultured onto various agars to continue to observe biochemical characteristics (4). Isolation is necessary to separate the relevant bacteria from a stool or blood sample that has a mixed population of microorganisms, such as microbiota and other contaminants. After isolation is complete, the colonies of the pathogen can be expanded in culture for other tests.

Selective and Differential Media

Selective media are selective for certain genus types due to the addition of inhibitory agents that inhibit the growth of other organisms, such as other microbes or contaminants (6). Common selective agents include selenite, bismuth, brilliant green, crystal violet, bile and specific antibiotics (6). Differential media distinguish between different microorganisms growing in the medium by observing their biochemical characteristics in the presence of specific nutrients and indicators (6). Selective media focuses on inhibiting growth of non-desired organisms, whereas differential media take advantage of the different biochemical properties of organisms to cause visible indicators of the growth of the desired organism (6). Together, this can visibly indicate unique characteristics of a microorganism.

MacConkey, Salmonella-Shigella, eosin-methylene blue, deoxycholate citrate, bismuth sulfite and brilliant green agars are some common selective and differential agars used in the isolation and culturing of Salmonella spp. (5). MacConkey agar and eosin methylene blue are commonly used to isolate enteric bacteria (4). MacConkey agar, Hektoen agar, eosin methylene blue and brilliant green agar are relevant media for Salmonella spp (8). The components and purpose of these agars are listed in Table 1. Solid culture media are used in order to produce isolated colonies that can be quantified and identified in the laboratory (4). Carbohydrates are also included in the culture medium with a pH indicator to utilize the different carbohydrate fermentation abilities of microorganisms to observe different growth patterns (4).

It is important to note that because the most common discrimination feature is Salmonella’s inability to ferment lactose, so the majority of relevant isolation media contain lactose and a pH indicator, as well as selective agents to inhibit the growth of other organisms (9). It is also common to have media that contain ferrous citrate to detect the H₂S production by Salmonella spp. (9). It is important to note that many other agars, not listed in the table, such as sorbitol MacConkey agar, which is selective for Escherichia coli, and Campylobacter selective agar would also be used (8). These negative results would rule out these other pathogens and help to converge to the identification of the pathogen in Johnny’s stool sample as one of the Salmonella genus.

Table 1: Name, composition, purpose and relevant results of common selective and differential media used in Salmonella spp. isolation and identification (6,8,9)

Name	Important Components	Purpose	Salmonella spp. Results
MacConkey agar	-lactose -pH indicator: neutral red -selective agent: crystal violet, bile salts	-commonly used to isolate gram negative enteric bacteria -crystal violet and bile salts inhibit growth of gram positive bacteria -neutral red differentiates bacteria based on lactose fermentation (turns red pH < 6.8)	-no lactose fermentation: colourless colonies
Eosin methylene blue agar	-lactose -selective agent and pH indicator: eosin Y, methylene blue	-commonly used to isolate gram negative enteric bacteria -coloured dyes inhibit growth of gram positive bacteria -differentiates bacteria based on lactose fermentation (fermentation encourages uptake of purple/blue dye into colonies	-no lactose fermentation: colourless colonies
Brilliant green agar	-lactose -pH indicator: phenol red -selective agent: brilliant green	-isolates gram negative bacteria -brilliant green selective agent inhibits growth of gram positive bacteria -phenol red changes from pink (pH ≥ 8) to orange (pH ≤ 6.8) upon fermentation of lactose	-no lactose fermentation: pink colonies
Hektoen agar	-lactose, sucrose, salicin -Andrade’s mix -peptone -pH indicator: bromothymol blue -H2S production indicator: ferric citrate -selective inhibitor: sodium deoxycholate, bile salts	-isolates gram negative bacteria -sodium deoxycholate and bile salts inhibit growth of gram positive bacteria -discriminate between Shigella and Salmonella based on H2S production	-no sugar fermentation: blue-green colonies -H2S production: black centered colonies
Deoxycholate citrate agar	-lactose -pH indicator: neutral red -selective agent: deoxycholate -H2S production indicator: ferric ammonium	-isolates enteric pathogens due to increased concentrations of both deoxycholate salts and citrate -inhibitory to most gut bacteria	-no lactose fermentation: pale colonies -H2S production: black centered colonies
Xylose lysine deoxycholate agar	-xylose, lactose, sucrose -lysine -thiosulfate	-isolates Shigella and Salmonella from clinical samples -discriminate between Shigella + Salmonella based on xylose fermentation and H2S production	-ferment xylose: yellow colonies  initially -decarboxylate lysine: red colonies later -H2S production: black centered colonies

Biochemical Tests

The growth characteristics and patterns under various conditions, such as the time, atmosphere required and substrates used in the media, are observed (4). Cell and colony morphology and biochemical characteristics are often used to determine the bacteria genus (4). The selection and amount of further tests for more identification varies depending on the category of the bacteria present (4). In this case of gram negative, facultatively anaerobic Salmonella bacteria, many tests are often required for definitive biochemical and physiologic characteristic determination (4). However, fortunately, systems have been developed to simplify the identification of Salmonella by permitting the rapid and simultaneous testing of 10-20 different biochemical parameters (5). These biochemical test results can often be used to make the presumptive identification of the Salmonella genus (5).

In the screening identification phase, a screening test is performed to exclude colonies of other organisms that are examined in the next biochemical tests (1). Common screening techniques include short sugar series, such as Kohn’s tubes and the Kliger iron medium, and commercial screening tests like APIZ (1). Kliger’s is the most popular composite media and contains sucrose, lactose, ferric ammonium citrate and an indicator (1). Organisms like Salmonella spp. that ferment glucose, but not lactose or sucrose, are inoculated (1). The medium is blackened by the H₂S production (1). In addition, the biochemical reactions of colonies are identified on triple sugar iron agar and lysine iron agar to make an initial identification of the pathogen (5). The triple lysine sugar agar is used to differentiate enteric pathogens based on their ability to reduce sulfur and ferment carbohydrates (5). Salmonella spp. would be expected to ferment glucose and form H_sS (4,5). These tests can be used alongside indole and urease tests, where Salmonella spp. do not produce indole or hydrolyse urea, to select for cultures that should be further examined for identification (1). The definitive identification phase involves using biochemical and serological tests to determine Salmonella spp. features (2). Biochemical tests are similar to those described in the initial isolation phase that use selective and differential media to observe growth and biochemical reactions (2).

Serological Tests

Serological tests can involve blood samples and identifying the flagella, H, O antigens or LPS types that are present (2). This involves using polyvalent and specific antisera raised by bacterial agglutination for antigenic analysis (2,5). Detection methods using polymerase chain reaction and enzyme linked immunosorbent assays have also been used (10,11).

Polymerase Chain Reaction

Polymerase Chain Reaction (Khan Academy) (20)

Nucleic acid based methods are generally more rapid, sensitive and specific than culture-based techniques, which reduces the need for enrichment procedures (12). These nucleic acid methods are usually based on polymerase chain reaction (PCR), which is a technique that uses short nucleic acid primers to identify and bind specific nucleic acid sequences in the sample that are unique and characteristic of the pathogen (12). In the presence of a heat stable enzyme and the cycling temperatures characteristic of PCR for denaturation, annealing and elongation steps, amplification of this target sequence will occur and reach high amounts that can be visualized (12). This indicates the presence of the specific bacteria (12). For Salmonella serotypes, many genes could be used, such as genes for fimbrial subunits, O and H antigens or virulence factors (12).

Multiplex polymerase chain reaction (PCR) is a molecular based technology that has been developed and used to characterize different Salmonella spp. (10). This technique is based on O serotyping and H typing and has been used to differentiate between S. Typhimurium, S. Enteritidis and S. dublin (10). These experiments use a variety of specific primers to detect serotype antigenic characteristics (10). It is completed with a variety of primers for a variety of DNA sequences, such as sdfF and sdfR to amplify Sd I, a DNA sequence unique to S. Enteritidis, and H-for to amplify fliC, common to S. typhimurium and S. dublin (10). Gel electrophoresis results are then analyzed to identify the serotype (10). In these tests, a band in the gel electrophoresis results representing the presence of Sdf I would be observed (10). There would be no other bands, which would indicate the absence of fli-C-g,p, fliC-i , fliC-z4, z23 (10). Overall, this pattern in the gel electrophoresis would be able identity the serotype as Salmonella Enteritidis (10).

Recently, a multiplex quantitative PCR was developed to detect S. Typhimurium and S. Enteritidis serotypes (13). The serovar specific genomic sequences targeted were determined using VISTA tools (13). Three primer and probe sets were used to detect the presence of invA, STM4200 and SEN1392 genes to differentiate between these two non typhoidal serotypes of Salmonella enterica serotypes, which are two of the most common causes of human salmonellosis cases (13). The InvA gene is considered a standard gene for the detection of Salmonella spp (13). STM4200 encodes a phage tail fiber protein specific to S. Typhimurium and SEN1392 encodes a phage protein specific to S. Enteritidis (13). In silico analysis of the primer and probe sets can then be used to determine the presence of these unique DNA sequences (13).

Enzyme-linked Immunosorbant Assay (ELISA) adapted from BioTek (21)

Enzyme Linked Immunosorbent Assays

Visualizing the specific interaction between characteristic antigens and antibodies is another way to detect Salmonella and differentiate between its various serotypes (12). These interactions are commonly used for agglutination assays for diagnosis confirmation and serotyping. The majority of these techniques employ the most common immunoassay, enzyme linked immunosorbent assays (ELISA), antibodies that target specific and unique antigens to certain pathogens are immobilized and plated on a surface (12). Then, the pathogen will be exposed to this plate and will bind to the corresponding antibodies (12). The presence of these cells will be detected using other antibodies that are conjugated to an enzyme that converts its substrate to coloured product that can be detected by the eye or spectrophotometry (12).

Indirect ELISA involves using a detection antigen coated onto wells of a microtiter plate then, detecting specifically bound antibodies with an antibody-enzyme conjugate (11). Competitive ELISA involves coating antigens, specifically monoclonal antibodies, to wells, then following up with a pure or crude antigen preparation (11). Test samples and conjugated monoclonal antibodies are then applied (11). ELISAs can be based on a variety of models to detect the presence of different pathogens and specific bacterial serotypes. For instance, ELISAs based on lipopolysaccharide and Vi antigens have been used to detect and differentiate between S. Typhimurium and S. Typhi serotype (11). ELISAs can also be developed to detect the production of certain immune molecules, such as specific IgM and IgG antibodies, that are expected in the presence of certain bacteria (11). For S. Enteritidis, indirect ELISAs with antigens, such as LPS, flagella, specific SEF14 fimbriae preparations have been used for detection (11,14). Additionally, direct ELISAs with monoclonal antibodies for flagella, LPS and SEF14 of S. Enteritidis have been used (11).

In a recent study, recombinant flagellins were purified to be used as antigens in an ELISA to detect Salmonella enterica serotypes, S. Enteritidis and S. Typhimurium, in poultry with a higher specificity than culture tests and previous ELISA tests using lipopolysaccharide as an antigen (15). This purified recombinant flagellin contained H1 flagellar antigens g, m, i, r, z₁₀ and H2 flagellar antigens 1, 2, e, n, x (15). Different combinations of antigens unique to S. Enterica serotypes, S. Enteritidis, S. Hadar, S. Heidelberg and S. Typhimurium were created (15). An ELISA based on the unique antigens of H1 g, m were used to detect the S. Enteritidis serotype (15).

Mass Spectrometry

Matrix-Assisted Desorption/Ionization-Time of Flight (Singhal, et al. 2015) (16)

Increasingly, classic biochemical assays are being replaced by proteomic techniques such as Mass Spectrometry (MS). Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry represents one of the most commonly employed MS technologies (16). Briefly, this consists of blasting apart the proteins of a sample into smaller peptide fragments with a laser, and measuring the ionized fragments with a detector (16). The basis of this technique relies on the fact that proteins will scatter into these specific fragments in a predictable and reproducible fashion, based on fragment mass and charge (16). A visual representation of this is presented on the right (16).

MALDI-TOF MS is used to analyze all proteins contained in a sample and may be used to identify pathogen-specific molecules, sometimes referred to as 'direct bacterial profiling' (17, 18, 19). This represents a powerful advancement in organism identification, as this can provide serovar specific identifiers, without the need to screen and selectively identify Salmonella Enteritidis using the classical biochemical methods described above (17). Although this remains a costly approach, MS methods may present a technique that will replace classic microbiological and biochemical methods for diagnosis of Salmonella infection and serotype identification in the near future (16).

Overall, serological tests will determine the serological type of the Salmonella species (5). The tests can be used for diagnosis confirmation and to provide insights on the epidemiology of the infection and potential treatment (5). Moreover, it is important to note that this is a rapidly developing field and researchers have been advocating to use these more rapid and sensitive serological tests prior to bacterial culture for rapid detection of infection.

References

1. Jenkins C, Gillespie SH. 2006. Salmonella spp. In S. H. Gillespie, & P. M. Hawkey (eds.), Principles And Practice of Clinical Bacteriology. John Wiley & Sons, Ltd, Chichester, UK. doi:10.1002/9780470017968.
2. Busse M. 1995. Media for Salmonella. Int J of Food Microbiol. 26:117-131.doi:10.1016/0168-1605(93)E0030-U
3. Humphries RM, Linscott AJ. Laboratory diagnosis of bacterial gastroenteritis. Clinical microbiology reviews. 2015 Jan 1;28(1):3-1.
4. Washington JA. 1996. Principles of Diagnosis. In S. Baron (ed.), Medical Microbiology. University of Texas Medical Branch at Galveston, Galveston, Texas.
5. Giannella R. 1996. Salmonella. In S. Baron (ed.), Medical Microbiology. University of Texas Medical Branch at Galveston, Galveston, Texas.
6. Chauhan A, Jindal T. 2020. Microbiological culture media: Types, role and composition. In: Springer, C (ed.), Springer International Publishing, Cham, Switzerland
7. Sawadogo S, Diarra B, BIsseye C, Compaore TR, Djigma FW, Ouermi D, Ouattara AS, Simpore J. 2017. Molecular diagnosis of Shigella, Salmonella and Campylobacter by multiplex Real-time PCR in stool culture samples in Ouagadougou (Burkina Faso). Sudan J of Med Sci (SJMS), 12: 163-173. https://doi.org/10.18502/sjms.v12i3.93
8. de la Maza LM, Pezzlo MT, Bittencourt CE, Peterson EM. 2020 Escherichia, shigella, and salmonella. In L.M de la Maza, M.T. Pezzlo, C.E. Bittencourt, E.M. Peterson (eds.), Colour Atlas of Medical Bacteriology. John Wiley & Sons, Inc, Hoboken, New Jersey.
9. Popoff MY, Le Minor LE. 2015. Salmonella. In M. E. Trujillo, S. Dedysh, P. DeVos, B. Hedlund, P. Kämpfer, F. A. Rainey, W. B. Whitman (eds.), Bergey’s Manual of Systematics of Archaea and Bacteria. John Wiley & Sons, Ltd, Chichester, UK. doi:10.1002/9781118960608.gbm01166
10. Kariuki S, Kiiru J. (2020). Detection and characterization of Salmonella enterica serotypes by simple PCR technologies. In J. Walker. Methods in Molecular Biology. Springer US, New York, New York. 2182:161-177.
11. Barrow PA. 1994. Serological diagnosis of Salmonella serotype enteritidis infections in poultry by ELISA and other tests: Salmonella Enteritidis. Int J of Food Microbiol. 21: 55-68. https://doi.org/10.1016/0168-1605(94)90200-3
12. Dykes GA. 2016. Salmonella: Detection. In B. Caballero, P.M. Finglas, F. Toldra (eds). Encyclopedia of Food and Health.  Elsevier Ltd, Kidlington, Oxford.
13. Heymans R, Vila A, van Heerwaarden CAM, Jansen CCC, Castelijn GAA, van der Voort M, Biesta-Peters EG. 2018. Rapid detection and differentiation of salmonella species, salmonella typhimurium and salmonella enteritidis by multiplex quantitative PCR. PloS One, 13: 1-15. doi:10.1371/journal.pone.0206316
14. Mirhosseini SA, Fooladi AAI, Amani J, Sedighian H. 2017. Production of recombinant flagellin to develop ELISA-based detection of Salmonella Enteritidis. Brazilian J of Microbiol. 48:774-78. https://doi.org/10.1016/j.bjm.2016.04.033
15. Minicozzi J, Sanchez S, Lee M, Holt P, Hofacre C, Maurer J. 2013. Development of recombinant flagellar antigens for serological detection of salmonella enterica serotypes enteritidis, hadar, heidelberg, and typhimurium in poultry. Agriculture (Basel), 3: 381-397. doi:10.3390/agriculture3030381
16. Singhal N, Kumar M, Kanaujia PK, Virdi, JS. 2015. MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis. Frontiers in microbiology, 6:791. https://doi.org/10.3389/fmicb.2015.00791
17. Alispahic M, Hummel K, Jandreski-Cvetkovic, Nobauer K, Razzazi-Fazeli E, Hess M, Hess C. 2010. Species-specific identification and differentiation of Arcobacter, Helicobacter and Campylobacter by full-spectral matrix-associated laser desorption/ ionization time of flight mass spectrometry analysis. J Med Microbiol. 59:295–301.
18. Carbonnelle E, Bertti JL, Cottyn S, Quesne G, Berche P, Nassif X, Ferroni A. 2007. Rapid identification of staphylococci isolated in clinical microbiology laboratories by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 45:2156–2161.
19. Dieckmann R, Malorny B. 2011. Rapid Screening of Epidemiologically Important Salmonella enterica subsp. enterica Serovars by Whole-Cell Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry. Appl Enviro Microbiol. 77: 4136-4146; doi: 10.1128/AEM.02418-10
20. Khan Academy. Article is a modified derivative of "The polymerase chain reaction," by CK-12 Foundation (CC BY-NC 3.0). 2021 [cited 29 January 2021]. Available from: https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/biotechnology/a/polymerase-chain-reaction-pcr
21. BioTek - ELISA and related immunoassays [cited 29 January 2021]. Available from: https://www.biotek.com/applications/elisa-and-related-immunoassays.html

Question (iv): What are the results expected from these tests allowing for the identification of the bacteria named in this case.

Classical Microbiological Culture:

Salmonella​ enteritidis are Gram-negative, rod shaped, fermentative, and facultatively anaerobic bacteria [1]. Identification of suspected ​S. enteritidis colonies is often based on Iysine decarboxylation, H₂S production, and the absence of lactose fermentation [2]. Certain results are expected from these tests to identify the bacterial pathogen in Johnny’s case as Salmonella enterica ssp. Enteritidis. The fecal specimen containing the S. Enteritidis will produce certain characteristics when inoculated on the following agar media:

Figure 1. Visual representation of MacConkey Selective Agar. Non-fermenting Salmonella will appear colourless, as shown on the right.

Figure 2. Visual Representation of Xylose Lysine Deoxycholate (XLD) agar

MacConkey agar isolates gram negative and enteric bacteria and differentiates them based on lactose fermentation [3].  It contains lactose, neutral red and selective inhibitors, like crystal violet and bile salts [3]. Crystal violet and bile salts prevent the growth of Gram-positive bacteria by degrading the thick peptidoglycan layer. Neutral red is a pH indicator that turns red in pH below 6.8. The fermentation of lactose will cause a drop in pH through the production of metabolic wastes and secondary metabolites. Therefore, in the presence of lactose fermenters, the MacConkey agar would become pink. Since S. enteritidis is a lactose non-fermenter, the pH remains unchanged, resulting in colourless colonies, as shown in Figure 1. XLD agar contains xylose, lactose, and sucrose with lysine. If S. enteritidis is present in the stool specimen and inoculated onto an XLD agar, the agar will turn red and produce black precipitates, as shown in Figure 2. This is because Salmonella is a lactose non-fermenter that produces H₂S, so it will produce red colonies with a black centre [2].  In the presence of lactose fermenters, the XLD agar colour would become yellow [4].

Figure 3. Salmonella sp. cultured onto an HE Agar

Hektoen agar contains lactose, sucrose, salicin and a pH indicator of bromothymol blue and Andrade’s mix [3]. If S. enteritidis is present in the stool specimen and inoculated onto an HE agar, the agar will produce colonies that are blue or green in colour, with black precipitates, as shown in Figure 3. This is because Salmonella is a lactose non-fermenter that produces H₂S. In the presence of lactose fermenters, the HE agar colour would become yellow-orange [4]. Brilliant green agar contains lactose, phenol red, and the selective agent brilliant green [3]. If Salmonella enteritidis is present in the stool sample, the Brilliant green agar will show a presentation of colonies that are red, pink, or white with halo. This is because it is unable to ferment lactose [2]. This agar inhibits the growth of Salmonella Typhi and Paratyphi [4]. Deoxycholate citrate agar contains lactose, neutral red, and the selective agent deoxycholate. It also contains ferric ammonium citrate, which is an H₂S production indicator [3]. In this agar, Salmonella​ will produce pale colonies with a black center because it will not ferment lactose but will reduce the ferric ammonium citrate and produce H₂S [2,3]. However, it is important to note that because this pattern can be mimicked by other organisms, XLD agar is another medium that should be used in parallel [2]. ​

​Biochemical Assays:

Figure 4. SE in a TSI agar test.

Once the results have been obtained from a combination of the agars above, secondary biochemical screening is traditionally carried out using TSI agar, LIA agar, and urease tests [4,5]. The fecal specimen containing S. enteritidis will produce certain characteristics when tested using these biochemical approaches:

​-If S. enteritidis is present in the stool sample, the TSI agar test tube will have a yellow colour in the butt and a red colour in the slant, with black precipitates [6]. This is because glucose, not lactose, is being fermented in the butt and acid is being oxidized in the slant. Furthermore, the black colour in the tube will be due to the production of H₂S [6]. This reaction is depicted on the left in Figure 4.

​-If S. enteritidis is present in the stool sample, the LIA test tube will produce an alkaline purple colour in both the butt and the slant, with black precipitates [7]. This is because lysine decarboxylate will be produced which will turn the butt purple, and the lysine will not be deaminated so the slant colour will remain purple.  Furthermore, the black colour in the tube will be due to the production of H₂S [7].

​-In the urease test, if ammonia is formed, then the indicator colour of the media will change from light orange to magenta. S. enteritidis does not hydrolyze urea to produce urease [8]. Therefore, if S. enteritidis is present in the stool sample, the urease test will be negative and the indicator colour of the media will remain light orange [8].

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​PCR Based Methods:

Figure 5. PCR product distribution for positive identification of Salmonella Enteritidis. (Kariuki and Kiiru, 2020) (9)

​As described, DNA amplification methods may be used to achieve serovar-level identification of Salmonella enterica ssp. Enteritidis. Simple polymerase chain reaction (PCR) methods can performed on the bacterium isolated from the stool, or from the stool sample directly.

​PCR products are then visualized on an agarose gel, using gel electrophoresis. In this example shown on the right, the target amplicon specific for Salmonella Enteritidis appears at 300 base pairs, and is specific for the Sdf 1 gene, Salmonella difference fragment-1 (9). Please also note the absence of bands at 850, 600, and 430 base pairs (approximate values). The 16S ribosomal RNA subunit is used as a positive control for Salmonella amplification; this is expected in any Salmonella enterica subspecies.

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​A more advanced approach, can utilize specific PCR-probes or sequencing techniques to differentiate among different Salmonella serovars. Single nucleotide polymorphisms (SNPs) in select genes can also provide a positive identification for Salmonella Enteritidis. Presented in Figure 6 below, S. Enteritidis may be identified from S. Typhimurium by targeting 3 genes: fliD, sopE2, and spaO in a single multi-plexed PCR (10). These single base changes may be detected allowing for rapid serovar-level identification without needing to run the PCR products on a gel. SNP-based techniques are becoming increasingly common for the rapid identification of specific Salmonella serovars for outbreak investigations in Canada (10).

Figure 6. Rapid detection and serovar identification of Salmonella enterica serovars based on relevant SNP (Furukawa et al, 2017) (10)

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​Mass Spectrometry (MALDI-TOF) Identification and Serotyping

Figure 7. Decision tree classification for identification of selected Salmonella enterica subsp. enterica serovars using MALDI-TOF MS. (Dieckmann and Malorny, 2011) (11)

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​Mass spectrometry represents an advanced and quickly accelerating method for pathogen identification and classification (10). Presented on the left, a representation of the many different Salmonella enterica serovars, and their specific and respective mass peaks. In bold, are the peaks expected for the given serotype. In our case, a peak at 6,036 Da confirms the identification of Salmonella enterica ssp. Enteritidis.

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​-Salmonella enterica ssp. Enterica-specific mass peak at m/z 14,395, corresponding to the ribosomal protein L17

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​-Salmonella enterica ssp. Enteritidis serovar-specific mass peak at 6036

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​References:

1. ​Jenkins, C, Gillespie, SH. 2006. Salmonella spp. In S. H. Gillespie, & P. M. Hawkey (eds.), Principles And Practice of Clinical Bacteriology. John Wiley & Sons, Ltd, Chichester, UK. doi:10.1002/9780470017968.
2. ​Busse, M. 1995. Media for Salmonella. Int J of Food Microbiol. 26:117-131. doi:10.1016/0168-1605(93)E0030-U
3. ​Popoff, MY, Le Minor, LE. 2015. Salmonella. In M. E. Trujillo, S. Dedysh, P. DeVos, B. Hedlund, P. Kämpfer, F. A. Rainey & W. B. Whitman (eds.), Bergey’s Manual of Systematics of Archaea and Bacteria. John Wiley & Sons, Ltd, Chichester, UK. doi:10.1002/9781118960608.gbm01166
4. ​Humphries RM, Linscott AJ. 2015. Practical Guidance for Clinical Microbiology Laboratories: Diagnosis of Bacterial Gastroenteritis. Clinical Microbiology Review. 28:3-31. doi:10.1128/CMR.00073-14
5. ​Gal-Mor, O. 2019. Persistent infection and long-term carriage of Typhoidal and Nontyphoidal salmonellae. Clinical Microbiology Rev 32:e00088-18. https://doi.org/10.1128/CMR .00088-18
6. ​Aryal S. The Triple Sugar Iron (TSI) Test - Procedure, Uses and Interpretation [Internet]. Microbiology Info.com. 2021 [cited 29 January 2021]. Available from: https://microbiologyinfo.com/triple-sugar-iron-tsi-test/
7. ​Aryal S. Lysine Iron Agar (LIA) Slants Test - Procedure, Uses and Interpretation [Internet]. Microbiology Info.com. 2021 [cited 29 January 2021]. Available from: https://microbiologyinfo.com/lysine-iron-agar-slants-test/
8. ​Aryal S. Urease Test- Principle, Media, Procedure and Result [Internet]. Microbiology Info.com. 2021 [cited 29 January 2021]. Available from: https://microbiologyinfo.com/urease-test-principle-media-procedure-and-result/
9. ​ Kariuki S, Kiiru J. (2020). Detection and characterization of Salmonella enterica serotypes by simple PCR technologies. In J. Walker. Methods in Molecular Biology. Springer US, New York, New York. 2182:161-177.
10. ​ Furukawa M, Goji N, Janzen TW, Thomas MC, Ogunremi D, Blais B, Misawa N, Amoako KK. Rapid detection and serovar identification of common Salmonella enterica serovars in Canada using a new pyrosequencing assay. Canadian Journal of Microbiology. 64(1): 75-86. https://doi.org/10.1139/cjm-2017-0496
11. ​ Dieckmann R, Malorny B. 2011. Rapid Screening of Epidemiologically Important Salmonella enterica subsp. enterica Serovars by Whole-Cell Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry. Appl Enviro Microbiol. 77: 4136-4146; doi: 10.1128/AEM.02418-10

Q3. Bacterial Pathogenesis Questions

Question (i)

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

Geographic Location:

Salmonella enterica, serovar enteriditis (S. enteritidis) is a major cause of food-borne illness in humans, and is primarily associated with the consumption of contaminated poultry and eggs (Desin et al., 2009). S. enteritidis is the most common salmonella serotype world-wide but is especially prevalent in Europe (85% of all salmonella cases), Asia (38% of all salmonella cases), Latin America and the Caribbean (31% of all salmonella cases). There is additionally predominance in both North America as well as Africa, thus S. enteritidis is a pathogen of concern to animal and human health globally (Galanis et al., 2006)

S. enteritidis is a zoonotic pathogen with a broad host range and an enormous animal reservoir including livestock integrated into the human food chain such as chickens, turkeys, pigs, cows and their associated animal products such as eggs or dairy (Giannella, 1996). Salmonella bacteria are ubiquitous due to their ability to withstand pH, extreme temperatures, and dry climates, which can be attributable to a number of cellular adaptations. Sigma factors found in S. enteritidis are specialized proteins that initiate transcription of cellular components in response to environmental stressors. RpoH sigma factor encodes proteins in response to extreme heat, and these heat shock proteins are heat stable and thus allow for cell survival during extreme heat (Bang et al., 2005). Conversely, cold shock proteins protect salmonella in extreme cold by ensuring stability and promoting protein synthesis and cellular growth during cold temperatures (Kim et al., 2001). Lastly, osmoprotectants such as L-proline and trehalose are molecules that limit cell water loss in Salmonella bacteria to ensure cell survival in dry conditions. An osmoprotectant transporter uptakes L-proline and other solutes to ensure water retention within the bacterial cell (Dunlap & Csonka, 1985). The sigma factor RpoS is involved in trehalose synthesis, which is integral in forming a protective layer to prevent lipid membranes from drying out and inhibiting protein denaturation due to water loss (Nickerson & Curtiss, 1997).

During infection of chicken eggs, hens are most often infected with S. enteritidis by vertical transmission, and thus contaminate eggs through transovarian infection (Andino & Hanning, 2015). S. enteritidis bacteria infect the ovaries and upper oviduct of the chicken, allowing the bacteria to localize to the egg before the shell is formed, and infect the egg yolk and albumen (Todar, 2012). Eggs contain antimicrobial factors like lysozyme and defensins, however Salmonella enteritidis has developed several molecular mechanisms to optimize survival in eggs. Lysozyme inhibitors are present in many serovars of Salmonella, and PliC has been identified in S. enteritidis as a lysozyme inhibitor contributing to bacterial survival. It is speculated that PliC interferes with bacteria-host interactions by modulating the production of peptidoglycan fragments, which are important for the host immune response. This modulation of peptidoglycan fragments thus interferes with bacterial-host communication, allowing S. enteritidis to survive (Wagner & Hensel, 2011). S. enteritidis proliferate most effectively in the albumen of eggs, and must pass through the vitelline membrane to access the egg yolk. It is hypothesized that structural components, such as curli fimbrae allow S. enteritidis bacteria to attach to the vitelline membrane, and thus invade the yolk (Gantois et al., 2009).  TolC, an outer membrane channel protein is also important for the survival of S. enteritidis in eggs . TolC is part of multidrug efflux pumps, which prevent damaging host molecules from entering the cell, and promoter controlling expression of this protein is upregulated by egg whites, indicating it is important for the survival of Salmonella enteritidis in eggs (Raspoet et al., 2019)

Figure 2: Proposed infection route of S. enteritidis in poultry. A broad range of environments are colonized and infected throughout the infection cycle (Guard-Petter, 2001).

S. enteritidis can be transmitted through excretory materials, from those infected or recovered, and spread to different parts of the environment such as water, soil and plants, in addition to animal reservoirs such as insects and rodents (Todar, 2012).  S. enteritidis cannot multiply in these conditions, however, it can survive years in soil and a few weeks in water given the right pH, temperature and humidity conditions (Todar, 2012). The resiliency and broad host range of S. enteritidis causes risk for many livestock populations. The ability of S. enteritidis to be transmitted through vectors such as rodents and insects represents a large animal reservoir that can easily and rapidly infect animal processing environments. The conditions of these processing environments often require animals to live in close proximity and have poor sanitation systems thus increasing the likelihood of bacterial spread. Additionally, it has been indicated that S. enteritidis can form biofilms outside of hosts, and this multicellular behaviour could contribute to prolonged environmental survival of bacteria (Waldner et al., 2012). S. enteritidis poses a major threat to public health as the pathogen can survive in meats and animal products that are not thoroughly cooked, thus causing foodborne illness. In rare occasions, S. enteritidis has been noted to transmit from human to human (Giannella, 1996).

Host Location:

Once inside a host, S. enteritidis colonizes in gastrointestinal tracts of human and animal hosts. Within humans, S. enteritidis colonizes the distal ileum to the proximal colon. Two specific structures have been identified as particularly habitable by S. enteritidis, the ileum within the small intestine, and cecum within the large intestine (Giannella, 1996).

Stomach acid is the one of the first anti-bacterial barriers that S. enteritidis will encounter upon ingestion.  It has been suggested that S. enteritidis bacteria may undergo an acid tolerance response, wherein they can temporarily survive in acidic environments, such as the stomach. This response is generated through the synthesis of acid shock proteins (ASPs). ASPs are induced when the bacteria is exposed to acidic environments, and are responsible for regulating many cellular processes in an effort to prevent and repair damage to the bacterial cell due to acidic environments (Foster, 1991). ASPs are able to repair damage by modulating processes such as molecular chaperoning, energy metabolism, transcription and translation, fimbriae synthesis, and regulation of cellular membrane. An example of an acid-inducible protein is RpoS, which forms a protective layer to inhibit protein denaturation.  An example of regulation of the cellular membrane is the ability of S. enteritidis bacteria to alter the membrane composition to favour cyclic fatty acids over unsaturated fatty acids. S. enteritidis bacterium containing higher concentrations of cyclic fatty acids in their membranes are less susceptible to acid damage, which may contribute to survival (Álvarez-Ordóñez et al., 2011).

Figure 1: Representation of Salmonella bacteria evading mucus entrapment to invade the intestinal epithelium (Furter et al., 2019).

There are multiple bacterial characteristics that leave S. enteritidis suited to the gastrointestinal tract, and specifically allow the invasion of Salmonella into enterocytes. Salmonella is considered bile tolerant, and thus can survive against the antimicrobial properties present in bile salts. It is proposed that the LPS present in Salmonella provide an envelope barrier and protect the bacterial cell from damage and degradation. Efflux pump systems promptly remove any bile that has entered the bacterial cell to reduce cellular damage (Hernández et al., 2012). Mucus also poses as a barrier for bacterial entry into enterocytes along the intestines. S. enteritidis is able to exploit host physiology to evade entrapment in mucus. Mucus only covers the bottom of intestinal crypts, therefore the epithelium between crypts is unshielded. S. enteritidis swims near the surface of the mucus layer to prevent entrapment, until it is able to locate the epithelium between crypts that is mucus deficient.  Flagella-driven motility allows S. enteritidis to use chemotaxis to evade mucus entrapment (Furter et al., 2019).

There are several characteristics contributing to the survival of S. enteritidis involving attachment to host cells. As aforementioned, flagellum contributes to bacterial motility and is integral to chemotaxis. In the context of the gastrointestinal tract, flagellum contributes to both attachment and colonization within mucosal tissues. (Wagner & Hensel, 2011). Adhesin proteins are critical in allowing salmonella to adhere to epithelial cells, and the largest adhesin within the Salmonella proteome is SiiE, a 595 kDa protein composed of 53 repetitive immunoglobulin domains. S. enteritidis is able to apically invade intestinal epithelial cells through interaction between SiiE and the host transmembrane mucin MUC1, which is constitutively expressed on nearly all surface epithelial cells (Li et al., 2019). This contact between SiiE and MUC1 allows for downstream processes such as actin remodeling and bacterial internalization (Barlag & Hensel, 2015). These characteristics allow S. enteritidis to thrive in both animal and human reservoirs.

How our patient came into contact with this bacteria:

In the context of our patient, shell eggs are a major source of S. enteritidis foodborne illness in humans. As reference, 75% of S. enteritidis outbreak cases from 1985 to 2003 were able to confirm that eggs were the primary ingredient or food vehicle of contamination (Andino & Hanning, 2015). Johnny’s frequent consumption of raw eggs as a part of his body-building diet certainly could be the point of transmission of S. Enteritidis.

References

Álvarez-Ordóñez, A., Begley, M., Prieto, M., Messens, W., López, M., Bernardo, A., & Hill, C. (2011). Salmonella spp. Survival strategies within the host gastrointestinal tract. Microbiology, 157(12), 3268–3281. https://doi.org/10.1099/mic.0.050351-0

Andino, A., & Hanning, I. (2015). Salmonella enterica: Survival, Colonization, and Virulence Differences among Serovars. The Scientific World Journal, 2015. https://doi.org/10.1155/2015/520179

Bang, I.-S., Frye, J. G., McClelland, M., Velayudhan, J., & Fang, F. C. (2005). Alternative sigma factor interactions in Salmonella: ΣE and σH promote antioxidant defences by enhancing σS levels. Molecular Microbiology, 56(3), 811–823. https://doi.org/10.1111/j.1365-2958.2005.04580.x

Barlag, B., & Hensel, M. (2015). The giant adhesin SiiE of Salmonella enterica. Molecules (Basel, Switzerland), 20(1), 1134–1150. https://doi.org/10.3390/molecules20011134

Desin, T. S., Lam, P.-K. S., Koch, B., Mickael, C., Berberov, E., Wisner, A. L. S., Townsend, H. G. G., Potter, A. A., & Köster, W. (2009). Salmonella enterica Serovar Enteritidis Pathogenicity Island 1 Is Not Essential for but Facilitates Rapid Systemic Spread in Chickens. Infection and Immunity, 77(7), 2866–2875. https://doi.org/10.1128/IAI.00039-09

Dunlap, V. J., & Csonka, L. N. (1985). Osmotic regulation of L-proline transport in Salmonella typhimurium. Journal of Bacteriology, 163(1), 296–304.

Foster, J. W. (1991). Salmonella acid shock proteins are required for the adaptive acid tolerance response. Journal of Bacteriology, 173(21), 6896–6902.

Furter, M., Sellin, M. E., Hansson, G. C., & Hardt, W.-D. (2019). Mucus Architecture and Near-Surface Swimming Affect Distinct Salmonella Typhimurium Infection Patterns along the Murine Intestinal Tract. Cell Reports, 27(9), 2665-2678.e3. https://doi.org/10.1016/j.celrep.2019.04.106

Galanis, E., Wong, D. M. A. L. F., Patrick, M. E., Binsztein, N., Cieslik, A., Chalermchaikit, T., Aidara-Kane, A., Ellis, A., Angulo, F. J., & Wegener, H. C. (n.d.). Web-based Surveillance and Global Salmonella Distribution, 2000–2002—Volume 12, Number 3—March 2006—Emerging Infectious Diseases journal—CDC. https://doi.org/10.3201/eid1203.050854

Gantois, I., Ducatelle, R., Pasmans, F., Haesebrouck, F., & Immerseel, F. V. (2009). The Salmonella Enteritidis Lipopolysaccharide Biosynthesis Gene rfbH is Required for Survival in Egg Albumen. Zoonoses and Public Health, 56(3), 145–149. https://doi.org/10.1111/j.1863-2378.2008.01195.x

Giannella, R. A. (1996). Salmonella. In S. Baron (Ed.), Medical Microbiology (4th ed.). University of Texas Medical Branch at Galveston. http://www.ncbi.nlm.nih.gov/books/NBK8435/

Guard-Petter, J. (2001). The chicken, the egg and Salmonella enteritidis. Environmental Microbiology, 3(7), 421–430. https://doi.org/10.1046/j.1462-2920.2001.00213.x

Hernández, S. B., Cota, I., Ducret, A., Aussel, L., & Casadesús, J. (2012). Adaptation and Preadaptation of Salmonella enterica to Bile. PLoS Genetics, 8(1). https://doi.org/10.1371/journal.pgen.1002459

Kim, B. H., Bang, I. S., Lee, S. Y., Hong, S. K., Bang, S. H., Lee, I. S., & Park, Y. K. (2001). Expression of cspH, Encoding the Cold Shock Protein in Salmonella enterica Serovar Typhimurium UK-1. Journal of Bacteriology, 183(19), 5580–5588. https://doi.org/10.1128/JB.183.19.5580-5588.2001

Li, X., Bleumink-Pluym, N. M. C., Luijkx, Y. M. C. A., Wubbolts, R. W., Putten, J. P. M. van, & Strijbis, K. (2019). MUC1 is a receptor for the Salmonella SiiE adhesin that enables apical invasion into enterocytes. PLOS Pathogens, 15(2), e1007566. https://doi.org/10.1371/journal.ppat.1007566

Nickerson, C. A., & Curtiss, R. (1997). Role of sigma factor RpoS in initial stages of Salmonella typhimurium infection. Infection and Immunity, 65(5), 1814–1823.

Raspoet, R., Eeckhaut, V., Vermeulen, K., De Smet, L., Wen, Y., Nishino, K., Haesebrouck, F., Ducatelle, R., Devreese, B., & Van Immerseel, F. (2019). The Salmonella Enteritidis TolC outer membrane channel is essential for egg white survival. Poultry Science, 98(5), 2281–2289. https://doi.org/10.3382/ps/pey584

Todar, K.  (2012). Salmonella and Salmonellosis. Textbook of Bacteriology. Retrieved January 28, 2021, from http://textbookofbacteriology.net/salmonella_4.html

Wagner, C., & Hensel, M. (2011). Adhesive Mechanisms of Salmonella enterica. In D. Linke & A. Goldman (Eds.), Bacterial Adhesion: Chemistry, Biology and Physics (pp. 17–34). Springer Netherlands. https://doi.org/10.1007/978-94-007-0940-9_2

Waldner, L. L., MacKenzie, K. D., Köster, W., & White, A. P. (2012). From Exit to Entry: Long-term Survival and Transmission of Salmonella. Pathogens, 1(2), 128–155. https://doi.org/10.3390/pathogens1020128

Question (ii)

Entry: what facilitates the entry of the bacteria into the human host? What are the molecular, cellular and/or physiological factors at play in the initial entry/adherence step from the point of view of the organism and the host.

The systems of the acid tolerance response of Salmonella enteritidis including a) the pH homeostatic systems, b) the acid shock proteins, and c) the changes in membrane fluidity. (Álvarez-Ordóñez et al., 2011)

S. enteritidis enters humans via the oral route, usually by eating contaminated food that is already infected by the bacteria (Pegues & Miller, 2020). Different doses of bacteria have been reported to cause disease, with some studies reporting only 200 bacteria were needed to cause gastroenteritis and others requiring up to 10⁶ (Pegues & Miller, 2020). It has also been shown that the ingestion of food may neutralize gastric acids and temporarily increase the stomach pH, and thus an ID of ≤100 cells may be sufficient to induce an infection (Álvarez-Ordóñez et al., 2011). Solid food sources, and foods with high fat and protein content have been shown to protect S. enteritidis against stomach acidity. Upon ingestion, S. enteritidis encounters numerous host barriers prior to colonization in the intestinal tract (Giannella, 1996). Some of these barriers include the acidic pH of the stomach, bile salts, osmotic shock, low oxygen conditions of the intestine and bacterial metabolites (Álvarez-Ordóñez et al., 2011). Despite these host defense mechanisms, S. enteritidis has established numerous adaptations to thrive in these conditions. Salmonella enteritidis have an acid tolerance response that allows them to survive the low pH of the stomach (Ye et al., 2019). One important component of this response is the molecular systems in Salmonella that help maintain their internal pH of 7.6-7.8, like efflux pumps that remove hydrogen ions from the cell and the lysine and arginine decarboxylase systems (Álvarez-Ordóñez et al., 2011). When the bacteria are exposed to very low pH environments in which lysine is present, the lysine decarboxylase system is activated, starting with the cadBA operon becoming activated by the transcription factor, CadC (Álvarez-Ordóñez et al., 2011). This results in the transcription of CadA, a lysine decarboxylase enzyme which converts intracellular lysine to cadaverine and consumes one proton in the process, increasing the internal pH of the bacteria (Álvarez-Ordóñez et al., 2011). Another enzyme, CadB, exchanges the cadaverine within the cell for an extracellular lysine, allowing the process to repeat (Álvarez-Ordóñez et al., 2011). The arginine decarboxylase system is very similar, with arginine being converted to agmatine by the arginine decarboxylase, AdiA, and the enzyme AdiC exchanging the agmatine for external arginine. S. enteritidis also produce acid shock proteins that prevent and repair damage caused by stomach acid (Álvarez-Ordóñez et al., 2011). They are typically involved in transcription, translation, metabolism, regulation of cellular envelopes, molecular chaperoning, and virulence. One example of an important acid shock protein is Fur, which is involved in the regulation of iron metabolism in S. Enteritidis (Álvarez-Ordóñez et al., 2011). Fur regulates the expression of other acid shock proteins by controlling the iron concentration in the bacteria, resulting in protection from acid stress. S. Enteritidis can also modify its membrane composition in response to acidic conditions to decrease the fluidity of the membrane by decreasing the ratio of unsaturated to saturated fatty acids and increasing the amount of cyclic fatty acids in the membrane (Álvarez-Ordóñez et al., 2011). Some studies suggest this protects the bacteria from oxidative damage, as cyclic fatty acids are less reactive to oxidation than unsaturated fatty acids (Álvarez-Ordóñez et al., 2011).

The acid tolerance response allows the bacteria to progress to the intestines, where it faces another environment with different host barriers. Bile, which is produced by the gallbladder and is present in the intestines, acts as a detergent and can break down the cell membrane of bacterial cells, as well as cause damage to the DNA and proteins (Álvarez-Ordóñez et al., 2011). Salmonella are naturally resistant to bile exposure, and genes that have been linked to bile tolerance encode efflux pumps and proteins involved in cell membrane maintenance and DNA repair. Features of the outer membrane including lipopolysaccharide (LPS) and enterobacterial common antigen also contribute to Salmonella’s resistance to damage from bile (Álvarez-Ordóñez et al., 2011). The intestinal lumen also has a high salt concentration relative to other tissues, and is approximately 0.3M NaCl (Zhou et al., 2011). S. enteritidis bacteria prevent osmotic shock by increasing potassium uptake, followed by an increase in cytoplasmic concentration.  As S. enteritidis passes through the gastrointestinal tract, oxygen availability also decreases, and thus bacterial cells must be able to regulate metabolic processes anaerobically. S. enteritidis encodes a cytoplasmic oxygen sensor that can upregulate transcription of genes required for anaerobic metabolism in low oxygen environments. This mechanism allows S. enteritidis to adapt and continue metabolic processes in accordance with environmental cues (Álvarez-Ordóñez et al., 2011).

S. enteritidis requires adhesion to cell surfaces within the host in order to facilitate infection. Peristalsis, or muscular movement as part of digestion, is a barrier to bacterial adhesion. S. enteritidis uses flagella for directed movement such as chemotaxis, and additionally, is able to utilize flagella for stabilization to withstand peristalsis. It has been noted that flagella are also crucial in initiating attachment to mucosal tissues (Wagner & Hensel, 2011). FliC, the major subunit of flagella, appears to be the most critical for attachment, specifically interacting with cholesterol (Wagner & Hensel, 2011). There are several other cellular mechanisms that S. enteritidis utilizes for the adhesion process. Three translocon proteins that are part of the type-III secretion system (T3SS) on the Salmonella pathogenicity island 1 (SPI-1), SipB, SipC, and SipD, have been identified as mediating the intimate attachment of S. Enteritidis bacteria and epithelial cells (Barlag & Hensel, 2015). The T3SS is later utilized by the bacteria to translocate bacterial effector proteins important for invasion and pathogenesis into the host cell (Wagner & Hensel, 2011).

Both fimbrial and non-fimbrial adhesins assembled by multiple pathways are major adhesive structures used by S. enteritidis bacteria for host cell attachment. Fimbriae take on a number of physiological morphology including short appendages, long polar follicles and thin aggregative structures (Wagner & Hensel, 2011). Each distinct structure is assembled by a different cellular pathway and mediates a specific attachment relationship between bacteria and host epithelium. Most fimbriae bind a specific structure on the surface of the host cell, like lipid structures, membrane proteins, or sugar domains (Wagner & Hensel, 2011). FimH, the subunit at the tip of the fimbriae interacts with the host cells, and FimA is the major structural subunit that forms the shaft of the fimbriae (Wagner & Hensel, 2011). Salmonella FimH is specific for mannose residues, and this interaction between mannose on the host cell and FimH helps the bacteria adhere to the host cell (Wagner & Hensel, 2011). Non-Fimbrial adhesins such as SiiE (a giant adhesin) and autotransporter adhesins work together intricately to secure adherence. The vast array of adhesive mechanisms used by S. Enteritidis could suggest that the pathogen is highly adapted to multiple host organisms (Wagner & Hensel, 2011).

The host gastrointestinal microflora may also compete with S. enteritidis bacteria for epithelium adhesion receptors, and such microflora may contain bacteriocins and short chain fatty acids with antimicrobial properties. Animal models have indicated that the inflammatory state triggered by S. enteritidis pathogen may permit bacteria to outcompete the host microflora and thus colonize in the intestine (Hallstrom & McCormick, 2011).

The host innate immune response will likely become activated when the presence of bacteria is detected by the host toll-like receptors on immune cells and the intestinal epithelium (Pegues & Miller, 2020). Pro-inflammatory cytokines will be released by these cells, triggering neutrophils to localize to the site of infection (Pegues & Miller, 2020).

References:

Álvarez-Ordóñez, A., Begley, M., Prieto, M., Messens, W., López, M., Bernardo, A., & Hill, C. (2011). Salmonella spp. Survival strategies within the host gastrointestinal tract. Microbiology, 157(12), 3268–3281. https://doi.org/10.1099/mic.0.050351-0

Barlag, B., & Hensel, M. (2015). The giant adhesin SiiE of Salmonella enterica. Molecules (Basel, Switzerland), 20(1), 1134–1150. https://doi.org/10.3390/molecules20011134

Giannella, R. A. (1996). Salmonella. In S. Baron (Ed.), Medical Microbiology (4th ed.). University of Texas Medical Branch at Galveston. http://www.ncbi.nlm.nih.gov/books/NBK8435/

Pegues, D. A., & Miller, S. I. (2020). Salmonella Species. In Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases (9th ed., pp. 2725-2736.e3). Elsevier. http://www.clinicalkey.com/#!/content/book/3-s2.0-B978032348255400223X?scrollTo=%23hl0000728

Wagner, C., & Hensel, M. (2011). Adhesive Mechanisms of Salmonella enterica. In D. Linke & A. Goldman (Eds.), Bacterial Adhesion: Chemistry, Biology and Physics (pp. 17–34). Springer Netherlands. https://doi.org/10.1007/978-94-007-0940-9_2

Ye, B., He, S., Zhou, X., Cui, Y., Zhou, M., & Shi, X. (2019). Response to Acid Adaptation in Salmonella enterica Serovar Enteritidis. Journal of Food Science, 84(3), 599–605. https://doi.org/10.1111/1750-3841.14465

Zhou, K., George, S. M., Métris, A., Li, P. L., & Baranyi, J. (2011). Lag Phase of Salmonella enterica under Osmotic Stress Conditions. Applied and Environmental Microbiology, 77(5), 1758–1762. https://doi.org/10.1128/AEM.02629-10

Question (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.

S. enteritidis is a gram negative, facultative pathogen meaning this bacterium can survive in conditions with or without oxygen (Pegues & Miller, 2020). It also considered an intracellular pathogen therefore, it must enters cells in order to multiply and spread (Pegues & Miller, 2020). Once S. enteritidis has arrived at the ileum and colon, it adheres to the apical surface of intestinal epithelial cells through fimbriae structures (Pegues & Miller, 2020). Adhesins, located at the end of fimbriae, bind to extracellular matrix components of the host cells, such as glycoproteins (Rehman et al., 2019).

The roles of SPI-1 T3SS effector proteins in membrane ruffling and phagocytosis, resulting in bacterial-mediated endocytosis of S. enteritidis (Manon et al., 2012).

This binding allows for the entry of S. enteritidis which occurs through bacterial-mediated endocytosis. This process is controlled by a Type III Secretion System (T3SS) encoded in the Salmonella Pathogenicity Island 1 (SPI-1) found in the genome of S. enteritidis (Lou et al., 2019). SPI-1 transcribes six effector proteins (SipA, SipC, SopB/SigD, SopD, SopE and SopE2) with multiple functions including actin manipulation (Perrett & Jepson, 2009). T3SS acts like a “molecular syringe” by injecting multiple proteins or virulence factors, including the six effector proteins encoded by SPI-1, into the plasma membrane and cytoplasm of the host cell (Ly & Casanova, 2007; Manon et al., 2012). Bacterial-mediated endocytosis of S. enteritiditis occurs through two steps: 1) cytoskeletal rearrangement known as membrane ruffling and 2) phagocytosis. First, the proteins SipC and SipA are injected into the plasma membrane and create a protein complex in the membrane acting as a translocation pore to allow for the transfer of more proteins into the host (Ly & Casanova, 2007). In addition, SipC stimulates actin filament bundling and SipA stabilizes actin filaments (Pegues & Miller, 2020). SopE and SopE2 proteins aid with membrane ruffling as well through interacting with Rho GTPases, responsible for stimulating actin polymerization (Perrett & Jepson, 2009). This actin reconstruction facilitates S. enteritidis entry, since the rearranged actin cellular network is able to extend, surround, and engulf the bacterial cell. The proteins SopB/SigD aid in the final steps of phagocytosis through activating inositol phosphatase in order for phagosome closure and engulfment (Ly & Casanova, 2007). Lastly, SopD works together with SopB/SigD to achieve membrane fission and the formation of a Salmonella Containing Vacuole (SCV) within the host cell (Manon et al., 2012). Along with the T3SS SPI-1 dependent pathway just mentioned, the Salmonella enteritidis serotype has been studied to enter host intestinal cells through other methods. One of these methods include through the Rck invasin (Rosselin et al., 2012). Rck is an outer membrane protein encoded in the plasmid of Salmonella enteritidis. The main function of Rck is to enable adherence and entry of Salmonella enteritidis to the host cell through a receptor medicated entry process, which results in downstream actin reorganization, and ultimately leads to Salmonella enteritidis entry (Rosselin et al., 2012).

Once entry is complete, the bacteria multiply within the SCV and escape the host epithelial cell through the basal side to the blood stream. S. enteritidis can circulate and spread to other areas of the body through the infection of macrophages present in the spleen, liver and lymph nodes (Mastroeni & Grant, 2011). This systemic spread is not common and in most cases the bacteria localize strictly to the intestinal epithelium, causing the manifestation of gastroenteritis which is characterized by diarrhea, fever and abdominal pain (Giannella, 1996). This type of systemic spread is most often seen in people who are immunocompromised, including infants, the elderly, and those with underlying infections such as HIV (Schulte & Hensel, 2016). The bacteria infect macrophages via phagocytosis and survive long term in SCVs (Mastroeni & Grant, 2011; Pegues & Miller, 2020). S. enteritidis is able to evade host immune mechanisms by hiding in SCVs in macrophages, however, these SCVs undergo normal phagosome maturation which involves an increasing vacuole acidity. The bacteria are able to adapt to the rising acidity through the help of proteins encoded in another T3SS located in the Salmonella Pathogenicity Island 2 (SPI-2) (Pham & McSorley, 2015). SPI-2 is necessary for intracellular and systemic infection (Ohl & Miller, 2001). The proteins of SPI-2 interfere with vesicle trafficking in the macrophage and inhibits the accumulation of NADPH-oxidase in SCVs (Chakravortty et al., 2002). Moreover, these proteins help to remodel the bacterial cell surface and provide nutrients to the SCV required for survival (Pegues & Miller, 2020).

References

Chakravortty, D., Hansen-Wester, I., & Hensel, M. (2002). Salmonella Pathogenicity Island 2 Mediates Protection of Intracellular Salmonella from Reactive Nitrogen Intermediates. The Journal of Experimental Medicine, 195(9), 1155–1166. https://doi.org/10.1084/jem.20011547

Giannella, R. A. (1996). Salmonella. In S. Baron (Ed.), Medical Microbiology (4th ed.). University of Texas Medical Branch at Galveston. http://www.ncbi.nlm.nih.gov/books/NBK8435/

Lou, L., Zhang, P., Piao, R., & Wang, Y. (2019). Salmonella Pathogenicity Island 1 (SPI-1) and Its Complex Regulatory Network. Frontiers in Cellular and Infection Microbiology, 9. https://doi.org/10.3389/fcimb.2019.00270

Ly, K. T., & Casanova, J. E. (2007). Mechanisms of Salmonella entry into host cells. Cellular Microbiology, 9(9), 2103–2111. https://doi.org/10.1111/j.1462-5822.2007.00992.x

Manon, R., Nadia, A., Fatemeh, N., Isabelle, V.-P., Philippe, V., & Agnes, W. (2012). The Different Strategies Used by Salmonella to Invade Host Cells. In B. Annous (Ed.), Salmonella—Distribution, Adaptation, Control Measures and Molecular Technologies. InTech. https://doi.org/10.5772/29979

Mastroeni, P., & Grant, A. J. (2011). Spread of Salmonella enterica in the body during systemic infection: Unravelling host and pathogen determinants. Expert Reviews in Molecular Medicine, 13. https://doi.org/10.1017/S1462399411001840

Ohl, M. E., & Miller, S. I. (2001). Salmonella: A Model for Bacterial Pathogenesis. Annual Review of Medicine, 52(1), 259–274. https://doi.org/10.1146/annurev.med.52.1.259

Pegues, D. A., & Miller, S. I. (2020). Salmonella Species. In Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases (9th ed., pp. 2725-2736.e3). Elsevier. https://www.clinicalkey.com/#!/content/book/3-s2.0-B978032348255400223X?indexOverride=GLOBAL

Perrett, C. A., & Jepson, M. A. (2009). Regulation of Salmonella-induced membrane ruffling by SipA differs in strains lacking other effectors. Cellular Microbiology, 11(3), 475–487. https://doi.org/10.1111/j.1462-5822.2008.01268.x

Pham, O. H., & McSorley, S. J. (2015). Protective host immune responses to Salmonella infection. Future Microbiology, 10, 101–110. https://doi.org/10.2217/fmb.14.98

Rehman, T., Yin, L., Latif, M. B., Chen, J., Wang, K., Geng, Y., Huang, X., Abaidullah, M., Guo, H., & Ouyang, P. (2019). Adhesive mechanism of different Salmonella fimbrial adhesins. Microbial Pathogenesis, 137, 103748. https://doi.org/10.1016/j.micpath.2019.103748

Rosselin, M., Abed, N., Namdari, F., Virlogeux-Payant, I., Velge, P., Wiedemann, A. (2012). The Different Strategies Used by Salmonella to Invade Host Cells. 339-364. https://cdn.intechopen.com/pdfs/37807/InTechThe_different_strategies_used_by_salmonella_to_invade_host_cells.pdf

Schulte, M., & Hensel, M. (2016). Models of intestinal infection by Salmonella enterica: introduction of a new neonate mouse model. F1000Research, 5, F1000 Faculty Rev-1498. https://doi.org/10.12688/f1000research.8468.1

Question (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?

S. enteritidis is considered an intracellular pathogen and causes some direct damage to the host since it aids in the development of intestinal inflammation. There has been some speculation that translocated proteins from the SPI-1 type III secretory system, such as SopA and SopD, contribute to this inflammation and intestinal secretion (Pegues and Miller, 2020). SopB from SPI-1 has also been linked to intestinal inflammation and secretion through hijacking inositol phosphate signaling (Ohl and Miller, 2001). Through this pathway, SopB is responsible for promoting fluid secretion into the lumen by increasing Cl​^{­​ -} efflux (Santos et al., 2003). This is beneficial to S. enteritidis because this increase in fluid secretion leads to increase of diarrhea, which helps promote S. enteritidis spreading into the environment and therefore to eventually invade another host (Santos et al., 2003). S. enteritidis also produce both exotoxins and endotoxins which can cause damage to host cells. It has been hypothesized that the release of cytotoxins (a type of exotoxin) shortly after mucosal invasion by S. enteritidis may contribute to the inflammatory response and cause ulceration - however, evidence supporting the mode of action of the Salmonella cytotoxin is rather limitted (van Asten & van Dijk, 2005). In particular, the S. enteritidis serotype has been shown to produce a heat labile, trypsin-sensitive cytotoxin which is able to inhibit protein synthesis in the host cell, thus killing mammalian cells (van Asten & van Dijk, 2005; Todar, n.d.). Also, S. enteritidis can also release endotoxins, specifically lipopolysaccharide (LPS). Bacterial LPS, found within the cell envelopes of S. enteritidis, can potentially cause fever, activate serum complement, and ultimately can be responsible for the symptoms of septic shock (Giannella, 1996). The constituents of LPS include Lipid A, core oligosaccharide and O-antigen polysaccharide, which are prominent elicitors of the immune response and inflammation. Through interaction with host receptors, LPS sparks the innate immune functions such as phagocytic cells and cytokine production (Ohl and Miller, 2001).

While some damage can be attributed to the direct action of S. enteritidis, the majority of the damage caused by S. enteritidis is caused by the host response. The presence of S. enteritidis in epithelial cells induces the production and release of pro-inflammatory cytokines, such as IL-1, IL-6, IL-8, TNF-2, IFN-U, MCP-1, and GM-CSF (Giannella, 1996). The release of these cytokines triggers an acute inflammatory response, causing symptoms such as fever, chills, abdominal pain, and diarrhea. For instance, Toll-like receptor 4 (TLR4) is activated by the presence of LPS, inducing the production of proinflammatory cytokines (Pham and McSorley, 2015). Similarly, TLR5 is activated by the presence of flagellin, a globulin protein that makes up the filament of bacterial flagella. The presence of these cytokines induces inflammation and disrupts the intestinal epithelium, contributing to the loss of fluid through diarrhea. The fever and abdominal pain that Johnny experienced was also likely a result of the rapid inflammatory response triggered by the release of pro-inflammatory cytokines.

In addition to the SopB virulence factor which promotes Cl​^{­​ -} secretion, the release of LPS in response to bacterial cell death can be responsible for activating mucosal adenylate cyclase, which would subsequently increase intracellular cAMP production. The increase in cAMP production inhibits the influx of Na​⁺​ and causes an efflux of Cl​^{­​ -} from the intestinal environment. This change in ion concentration causes cellular water loss (Hoare et al., 2006). The efflux of Cl​^{­​ -} from the intestinal environment causes water to also be drawn across the intestinal epithelial barrier through osmosis. Water then leaves the body, causing diarrhea, and thus contributes as to why Johnny was volume depleted.

References

Hoare, A., Bittner, M., Carter, J., Alvarez, S., Zaldívar, M., Bravo, D., Valvano, M. A., & Contreras, I. (2006). The Outer Core Lipopolysaccharide of Salmonella enterica Serovar Typhi Is Required for Bacterial Entry into Epithelial Cells. Infection and Immunity, 74(3), 1555–1564. https://doi.org/10.1128/IAI.74.3.1555-1564.2006

Ohl, M.E., Miller, S.I., 2001. Salmonella: A Model for Bacterial Pathogenesis. Annual Review of Medicine 52, 259–274. https://doi.org/10.1146/annurev.med.52.1.259

Pegues DA, Miller SI. Salmonella Species. In: Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases [Internet]. 9th ed. Philadelphia, PA: Elsevier; 2020 [cited 2021 Jan 22]. p. 2725-2736.e3. Available from: http://www.clinicalkey.com/#!/content/book/3-s2.0-B978032348255400223X?scrollTo=%23hl0000728

Pham, O.H., McSorley, S.J., 2015. Protective host immune responses to Salmonella infection. Future Microbiol 10, 101–110. https://doi.org/10.2217/fmb.14.98

Prasad, H., Shenoy, A. R., & Visweswariah, S. S. (2019). Cyclic nucleotides, gut physiology and inflammation. The FEBS Journal, 287(10), 1970-1981. https://doi.org/10.1111/febs.15198)

Santos, R.L., Tsolis, R.M., Bäumler, A.J., Adams, L.G. (2003, January). Pathogenesis of Salmonella-induced enteritis. Brazilian Journal of Medical and Biological Research, 36(1) 3-12. https://doi.org/10.1590/S0100-879X2003000100002

Todar, K. (n.d.). Salmonella and Salmonellosis. Online Textbook of Bacteriology. Available from: http://textbookofbacteriology.net/

van Asten, A. J. A. M., van Dijk, J. E. (2005). Distribution of “classic” virulence factors among Salmonella spp., FEMS Immunology & Medical Microbiology, 44(3), 251–259. https://doi.org/10.1016/j.femsim.2005.02.002

Q4. The Immune Response

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

Salmonella enteritidis is a Gram-negative, food-borne pathogen that has been linked to the consumption of contaminated eggs leading to salmonellosis (1). As a Gram-negative bacterium, Salmonella enteritidis has a thin peptidoglycan layer and an outer lipid membrane which contains lipopolysaccharides (LPS), proteins and phospholipids. Unlike Gram-positive bacteria, the presence of LPS may function as an endotoxin which is an essential virulence factor of Salmonella enteritidis(2). This specific form of Salmonella is a non-typhoidal serotype of Salmonella enterica that causes gastroenteritis which leads to the common symptoms of fever, diarrhea and abdominal cramps in humans (1). Gastroenteritis is a self-limiting form of food poisoning that remains confined in the intestines causing mild inflammation of the gastrointestinal tract (1).  Since Salmonella enteritidis has become a leading cause of food-borne illness related to the consumption of contaminated eggs, information regarding the innate and adaptive immune responses to this infection are still becoming researched and understood.

Innate Immune Response

Infection by Salmonella enteritidis is initiated when contaminated eggs are ingested orally therefore surpassing the physical barriers of the skin. As this food-borne pathogen moves from the mouth towards the highly acidic environment of the stomach, some bacteria may survive or become eliminated (3). The bacteria that survive the low pH environment of the stomach will then travel towards the small intestine where they will compete against the normal gut microbiota in order to exploit host nutrients and resources necessary for their pathogenesis (3). For example, glucose has been found to be an essential host nutrient that Salmonella relies on in order to acquire energy through the process of glycolysis (4). Therefore, individuals who produce less acidic or low amounts of stomach acid are more susceptible to this bacterial infection as they lack this feature of innate immunity.

Neutrophil killing of bacteria by release of NETs. Adapted from Encyclopedia of Cell Biology, L-A. H. Allen, 2016.

Microfold (M) cells are specialized epithelial cells found in mucosa-associated lymphoid tissues of the gastrointestinal tract in addition to other tissue sites (5). Intestinal M cells function as specialized antigen sampling cells that transport material across follicle-associated epithelium to trigger the activation of innate immune responses, however this mechanism is often exploited by pathogenic bacteria (5). M cells are specifically targeted by Salmonella enteritidis in order to gain entry to the mucosal epithelia of the small intestine in the host (5). Once entering, Salmonella is exposed to several destructive elements essential to the host innate immune response which include the mucous produced by goblet cells, antimicrobial peptides produced by Paneth cells, enzymes, bile containing secretory IgA and intestinal microbiota (6,7). In the small intestine, the recognition of bacterial LPS by Toll-like receptor 4 (TLR-4) expressed on epithelial and lamina propria mononuclear cells will initiate the release of cytokines and chemokines including Interleukin 1 (IL-1), IL-6, IL-8, and TNF-2 (2,8). These cytokines and chemokines signal the recruitment of other innate immune cells such as neutrophils and macrophages (9). Specifically, neutrophils can kill pathogens through release of reactive oxygen species (ROS) and other antimicrobial proteins/peptides (10). Neutrophil extracellular traps (NETs) containing chromosomal DNA, histones and proteases can also be released to kill bacteria (10). In addition, the detection of LPS will trigger macrophages to produce nitric oxide (NO), a toxic defense molecule against Salmonella enteritidis (11). Both of these cell types also participate in the inflammatory response by phagocytosing pathogens intracellularly to clear infection at the level of the innate immune response.

Adaptive Immune Response

Another type of cell that engulfs Salmonella during infection are dendritic cells which form the transition from innate to adaptive immunity. Dendritic cells (DCs) ingest S. enteritidis in the gut and once the TLRs and other PRRs in the DC detect Salmonella PAMPs, they will undergo a process of maturation while they travel to local lymph nodes such as the mesenteric lymph nodes (9,12,13). Once they have arrived at the lymph node, the DC will have digested the bacterial antigens and have expressed them on Major Histocompatibility Complex Class I and II (MHC class I and II) proteins on their cell surfaces in order to activate T cells in the lymph node. CD4 T cells bind to the MHC II on DCs and a signalling cascade occurs to activate the T cell into a CD4 helper T cell.

Activated CD4 helper T cells then migrate to the site of infection via a cytokine and chemokine gradient where they will secrete molecules that facilitate a TH1-type immune response which is best suited for defence against extracellular organisms (14). TH1 responses are primarily humoural immunity and are characterized by the release of pro-inflammatory molecules such as IFN-gamma, TNF-alpha, IL-6, and IL-12 to facilitate a dramatic increase in phagocytic activity by macrophages causing accelerated clearance of the bacteria.

In addition to amplifying innate cell responses, CD4 helper T cells activate B cells which produce and secrete antibodies specific against S. enteritidis. To promote a more effective antibody response against salmonella, CD4 helper T cells aid in the class switching process of antibody production (12). There are two main types of antibody in Salmonella infection: Immunoglobulin G (IgG) and Immunoglobulin A (IgA). IgA in its secreted form (sIGa) is produced by B cells where they will bind to the surface proteins of Salmonella and reduce the adherence of the bacteria to the intestinal mucosa, reducing levels of infection and breach in the epithelium (12). IgG in its pentameric form is important in clearing bacteria from the patient. IgG to Salmonella and facilitates their removal via Fc-receptor-mediated phagocytosis, known as opsonization, or through the activation of complement through the classical pathway (13).

Complement is a vital part of humoural immunity and is a series of protein cascades that ultimately lead to death of Salmonella either by opsonization or by cell lysis via the MAC complex. The MAC complex is formed when complement proteins C5b, C6, C7, C8, and C9 bind to the bacterial cell surface and create a pore. This is done multiple times on each bacteria, resulting in eventual lysis of the bacteria.

Another class of T cells that participate in this immune response are CD8 cytotoxic T cells that are part of cell-mediated immunity. These T cells are highly efficient killers that target infected cells and destroy them by releasing perforin and granzyme which cleaves to disrupt the cell wall can cause cell lysis. CD8 T cells are activated by CD4 helper T cells and travel to the site of infection via a chemokine gradient. CD8 T cells bind to the MHC class I molecule expressed by all human cells and detect foreign antigen. If a foreign antigen is detected on MHC I, the CD8 T cell releases its cytolytic payload to kill the cell. While CD8 T cells play large roles in viral infections, they play more of a secondary role during a S. enteritidis infection.

At the end of the infection, a subset of B cells will become memory B cells which are important in long-term protection against S. enteritidis. Upon reinfection, memory B cells can secrete Salmonella-specific antibodies without the help of helper T cells which will dramatically increase the speed and efficiency of the immune response (15).

References

1. Desin TS, Lam P-KS, Mickael C, Wisner ALS, et al. Salmonella enterica serovar enteritidis pathogenicity island 1 is not essential for but facilitates rapid systemic spread in chickens. Infect Immun. 2009;
2. Baron S. Medical Microbiology. 4th ed. University of Texas Medical Branch; 1996.
3. Jones BD. Host responses to pathogenic Salmonella infection. Genes Dev. 1997;
4. Bowden SD, Rowley G, Hinton JCD, Thompson A. Glucose and glycolysis are required for the successful infection of macrophages and mice by Salmonella enterica serovar typhimurium. Infect Immun. 2009;
5. Jepson MA, Clark MA. The role of M cells in Salmonella infection. Microbes Infect. 2001;1183–90.
6. Griffin AJ, McSorley SJ. Development of protective immunity to Salmonella, a mucosal pathogen with a systemic agenda. Mucosal Immunol. 2011;4(4):371–82.
7. Khan CMA. The dynamic interactions between Salmonella and the Microbiota, within the challenging niche of the gastrointestinal tract. Int Sch Res Not. 2014;
8. Dheer R, Santaolalla R, Davies JM, Lang JK, Phillips MC, Pastorini C, et al. Intestinal epithelial Toll-like receptor 4 signaling affects epithelial function and colonic Microbiota and promotes a risk for transmissible colitis. Infect Immun. 2016;84(3):798–810.
9. Hurley D, McCusker MP, Fanning S, Martins M. Salmonella-host-interactions-modulation of the host immune system. Front Immunol. 2014;
10. Su Y, Gao J, Kaur P, Wang Z. Neutrophils and macrophages as targets for development of nanotherapeutics in inflammatory diseases. Pharmaceutics. 2020;12(12):1222.
11. Sharma JN, Al-Omran A, Parvathy SS. Role of nitric oxide in inflammatory disease. Inflammopharmacology. 2007;15(6):252–9.
12. Cummings L, Deathreage BL, Cookson B. Adaptive Immune Responses During Salmonella Infection. Ecosal Plus. 2009;
13. Goldman A. Immunology Overview. Med Microbiol. 1996;
14. Tubo NJ, Jenkins MK. CD4+ T Cells: “Guardians of the Phagosome.” Clin Microbiol Rev.
15. Parham P. The Immune System.

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

The severe water efflux into the intestinal lumen is the driving factor for diarrhea that patients may experience. Adapted from: Urdaneta et al, 2017.

Oxidative Damage

The reactive-oxygen-species (ROS) such as superoxide, hydrogen peroxide and hydroxyl radicals produced by neutrophils (1) and reactive nitrogen species produced by macrophages (2), while helpful in killing Salmonella, can also cause inflammation and disruption of tissue epithelial integrity through ROS-induced oxidative DNA and RNA damage (3). DNA damage can lead to cell-mediated apoptosis and cell death (3). S. enteritidis can also use byproducts of ROS as nutrients, promoting the infection (4). ROS oxidize thiosulfate to tetrathionate and Salmonella is able to anaerobically respire tetrathionate, which can allow it to utilize non-fermentable carbon sources such as ethanolamine, which is generated from the membranes of dead enterocytes (4). This results in promotion of Salmonella reproduction and further intestinal damage and possible ulceration. Whereas Salmonella is resistant to moderate levels of ROS, these responses transform the gut into an inhospitable environment for many types of anaerobic microbiota, leading to imbalances in the normal flora and further potential infections (4).

Similar to the effects of ROS, inflammatory cytokines such as IL-1, IL-6, IL-8 and TNF-2 produced by epithelial cells also result in intestinal damage and symptoms of inflammation such as fever and abdominal pain (5). This response is largely attributable to the presence of neutrophils and macrophages which are secreted to the lamina propria of the intestinal lumen through chemo attractants at the beginning of an infection (5).

Secretion of Fluid

Invasion of the intestinal mucosa and inflammatory reaction also activates the release of prostaglandins by neutrophils (5). These prostaglandins, which are a type of hormone, cause increased levels of mucosal adenyl cyclase which results in increased cyclic adenosine monophosphate (cAMP) (5). This signal transduction pathway increases secretion of fluid and electrolytes, causing diarrhea and can negatively impact nutrient and water absorption (5). This is what causes the severe diarrhea that patients with Salmonella may experience, resulting in dehydration and electrolyte imbalance. The sudden rush of water displaces commensal microbiota and so does the suddenly accelerated passage of any residual chyme that may be in the intestinal tract, leaving the host potentially vulenarable to future infections (4). In addition, cytokines such as TNF-α, IL-1, and IFN-γ released during infection from stimulated macrophages and T lymphocytes can lead to fever (6).

Extracellular Damage from CD8 T cells

Because Salmonella is an intracellular pathogen, CD8 cytotoxic T cells will target and destroy infected host cells (6). Although CD8 T cells are targeted to infected cells, some neighbouring tissues can still be harmed in the process (6). This was thought to be typically limited due to the highly controlled mechanism through which CD8 T cells are able to deliver these substances to infected cells. However, research has shown that leakage of granzymes into the extracellular space is possible during this mechanism (7).

References

1. Parham, Peter. The Immune System, 4th Edition.  New York, NW: Garland Science; 2014.
2. A. Mehta, S. Singh, N. K. Ganguly: Role of Reactive Oxygen Species in Salmonella typhimurium-induced Enterocyte Damage. Scandinavian Journal of Gastroenterology, 1998: 33(4), pp. 406–414.
3. Hu, J.-H., Nie, J.-J., Gao, Z.-X., Weng, Q.-H., Wang, Z.-H., Li, C.-B., Pian, Y.-Y., Zhang, R., Jiang, Z.-L., Xia, M.-M., & Cai, J.-P.. Oxidative DNA and RNA damage and their prognostic values during Salmonella enteritidis-induced intestinal infection in rats. Free Radical Research, 2018: 52(9), 961–969.
4. Behnsen J, Perez-Lopez A, Nuccio S, Raffatellu M.  Exploiting host immunity: the Salmonella paradigm. Trends in Immunology, 2015: 36(2): 112-120.
5. Giannella, R. 1996. Salmonella. In S. Baron (ed.), Medical Microbiology. Galveston: University of Texas Medical Branch at Galveston; 1996.
6. Goldman, A, Prabhakar, B. Immunology Overview. In S. Baron (ed.), Medical Microbiology. Galveston: University of Texas Medical Branch at Galveston; 1996.
7. Martin, S.  Cytotoxic and non‐cytotoxic roles of the CTL/NK protease granzyme B. Immunological Reviews. 2010: 235(1).

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

The physical barriers of the innate immune response and how Salmonella can overcome these barriers. Adapted from Bernal-Bayard, 2017

Salmonella have virulence genes to evade host defences that are often clustered in Salmonella pathogenicity islands (SPIs) (1). SPIs encode type III secretion systems 1 and 2 that can inject bacterial proteins called effectors directly into host cells (1). These effectors interfere with host cellular functions and subvert immunity to promote pathogen proliferation (2).

Innate Immunity Evasion

Salmonella can overcome the epithelial barrier through translocation across M cells of Peyer’s patches or by secretion of T3SS1 effector proteins that induce actin rearrangement to be internalized into the epithelial cell. After crossing the epithelial barrier, Salmonella are phagocytoses by macrophages and secrete effector proteins, T3SS1 and 2 that prevent fusion with the lysosome and help to develop a Salmonella-containing vacuole to survive and proliferate within. Adapted from Hurley, 2014.

Upon encountering the low pH of the stomach, Salmonella can enact an acid tolerance response (ATR) which uses acid shock proteins that can shield bacterial proteins from damage and repair damaged proteins (3). The ATR also utilizes proton-sodium and proton-potassium antiporters which can help to remove excess protons from bacterial cells in order to regulate intracellular pH (3).

Next up, Salmonella must face the intestine innate defences, which includes bile, antimicrobial peptides, the microbiota and mucus, before it is able to invade host cells and become an intracellular pathogen (1).

Bile is a bactericidal agent that disrupts bacterial cell membranes and can alter protein conformation (1). Salmonella has a bacterial cell envelope that can act as a barrier to bile salts. If bile enters the bacterial cell, efflux pumps can mediate export of bile salts out of the bacterial cytoplasm (1). The AcrAB-TolC efflux system is most common in enteric bacteria, and the genes encoding these proteins are synthesized in response to bile (1). Furthermore, one study showed that if Salmonella have been previously exposed to sublethal doses, they can adapt to grow in high bile concentrations through changes in their gene expression (1). Exposure to sublethal concentrations of bile salts effectively increased the minimal inhibitory concentration of bile salts (4). In particular, upregulation of the RpoS-dependent (RpoS is a sigma factor that regulates bacterial transcription and can be activated in response to environmental conditions) stress response is crucial to this adaptation (1,4).

In addition, Salmonella can sense and resist sublethal concentrations of antimicrobial peptides in the intestine (1). PhoQ is an inner-membrane histidine kinase that can sense antimicrobial peptides and phosphorylates the response regulator, PhoP (1). PhoP activation prevents dephosphorylation of PmrA, which is the protein that controls gene expression for LPS modifications that are used to resist antimicrobial peptides (1). PmrA can regulate the O-antigen length of LPS, and modify the phosphate groups and lipid A regions of LPS in order to reduce the negative charge of the bacterial outer membrane and reduce electrostatic interactions with cationic antimicrobial peptides (1).

The microbiota prevents infection by serving as a physical barrier to pathogen attachment and by competing for nutrients (1).There is some evidence that the host inflammatory immune response may lead to conditions that allow Salmonella to compete with microbiota in the gut by offering different nutrients or adhesion receptors types that can be used by the bacteria (1). One example is that the production or reactive oxygen species by neutrophils during inflammation can react with thiosulfate to form tetrathionate, which can be used as a terminal electron acceptor for anaerobic respiration by Salmonella, thereby conferring a growth advantage to the pathogen (1).

To penetrate the mucosal epithelium, S. enteritidis uses glycosyl hydrolases to degrade glycan layers which make up the mucus barrier (1). NanH and MalS have been identified as new virulence factors that are specific glycan-degrading enzymes in Salmonella (1). Salmonella can even use host glycan as a source of nutrients (1).The mucosal epithelia within the gastrointestinal tract consists of tight junctions between neighbouring epithelial cells (1). Salmonella can actively breach this mucosal barrier by injecting effector protein, T3SS-1, into host cell which induces actin rearrangement and disconnection of epithelial cell junctions, thereby facilitating the internalization of Salmonella into epithelial cells (1). Salmonella can also breach the epithelial barrier and tight junctions through passive transport mediated by dendritic cells, which can extend pseudopods between epithelial cells (2). This passive translocation of Salmonella occurs at microfold cells of Peyer’s patches which are located in the small intestine (2).

After crossing the intestinal epithelial barrier, Salmonella are engulfed by phagocytic cells (2). Because Salmonella is a facultative intracellular pathogen, it can replicate and persist within non-phagocytic epithelial cells as well as phagocytic dendritic cells and macrophages of the host innate immune system (1). Salmonella can establish an intracellular niche in a modified phagosome, called a Salmonella­-containing vacuole (SCV), where the pathogen can survive and proliferate in the absence of host antimicrobial defences (1,2). Salmonella can develop this SCV through the activity of effector proteins, T3SS 1 and 2, which can manipulate the endocytic pathway to avoid fusion and degradation by a lysosome (1). For instance, T3SS2 can mediate active suppression of autophagic signalling in macrophages and prevent the induction of a protective cytokine response (1). Furthermore, Salmonella can increase the expression of epithelial cell effectors, such as miR-128, which is responsible for regulating the level of expression of Macrophage-Colony stimulating factor which helps to recruit macrophages to the site pf infection and induce their phagocytic activity (5). The increased expression of miR-128 also decreases the production of reactive oxygen species by macrophages, which reduces the macrophages ability to clear the bacterial infection (5).

Adaptive Immunity Evasion

Salmonella can subvert the antigen presenting cell function to inhibit T cell activation, and evade the host immune response. As shown in Part F, Salmonella can regulate antigen expression, modify its surface and suppress APC activation, such that APCs become sites of bacterial survival and replication instead of presenters and activators for the host immune response. Adapted from Cummings 2009

Antigen presenting cells (APCs, like macrophages and dendritic cells, use major histocompatibility complexes (MHCs) to activate antigen-specific T-cells (6). S. enteritidis can interfere with APC function in that the pathogen can survive and replicate within APCs and interfere with presentation of antigens to T cells. In this way, the activation of the adaptive immune response is inhibited (6). Antigen FliC is a flagellar subunit protein that is expressed on the Salmonella surface (6). Once Salmonella transitions from an extracellular to an intracellular environment, it will reduce its antigen bioavailability by restricting FliC expression to an intracellular bacterial compartment and decrease FliC expression below the threshold needed for T-cell activation (6).

Salmonella can also suppress humoral response during primary infection, and thus the memory response for future infections (7). Salmonella is known to suppress B lymphopoiesis, which means that the differentiation of lymphoid precursors to B cells is limited (7). Salmonella protein, SteD, can deplete MHC class II on CD4 T cells to inhibit the T cell activation that is required to help activate and differentiate B cells (7). Furthermore, in a study with mice, it has been shown that Salmonella can reduce and suppress IgG-secreting plasma cells by secreting an adhesin protein called SiiE (7). SiiE-deficient Salmonella demonstrated enhanced production of IgG against Salmonella and a memory response in mice (7)

Salmonella is equipped to subvert multiple actors and pathways within the innate and adaptive immune responses in order to cause infection.

References

1. Bernal-Bayard, J, Ramos-Morales, F. Molecular mechanisms used by Salmonella to evade the immune system. Curr Issues Mol Biol. 2018. 25:133-169. doi: 10.21775/cimb.025.133.
2. Hurley D, McCusker MP, Fanning S, Martins M. Salmonella–host interactions – Modulation of the host innate immune system. Front Immunol [Internet]. 2014 [cited 2021 Jan 24];5. Available from: https://www.frontiersin.org/articles/10.3389/fimmu.2014.00481/full
3. Palmer AD, Slauch JM. Mechanisms of Salmonella pathogenesis in animal models. Human and Ecological Risk Assessment: International Journal. 2017 Nov 17; 23(8):1877–92.
4. Hernandez, S, Cota, I, Ducret, A, Aussel, L, Casadesus, J.  Adaptation and preadaptation of Salmonella enterica to bile. PLOS Genetics. 2012. 8(1). doi: https://doi.org/10.1371/journal.pgen.1002459.
5. Zhang, T, Yu, J, Zhang, Y, Li, L, Chen, Y, Li, D, Liu, F, Zhang, C, Gu, H, Zen, K. Salmonella enterica serovar enteritidis modulates intestinal epithelial miR-128 levels to decrease macrophage recruitment via macrophage colony-stimulating factor. J Infect Dis. 2014. 209(12).
6. Cummings LA, Deatherage BL, Cookson BT. Adaptive immune responses during Salmonella infection. EcoSal Plus [Internet]. 2009 Sep 17 [cited 2021 Jan 24];3(2). Available from: http://www.asmscience.org/content/journal/ecosalplus/10.1128/ecosalplus.8.8.11
7. Takaya A, Yamamoto T, Tokoyoda K. Humoral Immunity vs. Salmonella. Front Immunol. 2020 Jan 21;NA-NA.

iv. Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections from this particular bacteria?

Prognosis

In otherwise healthy individuals, Salmonella infections, including those caused by Salmonella enteritidis most often only result in localized, acute gastroenteritis and can normally be controlled by the innate host defenses within the small intestine (1,2). Thus, bacterial infections through the fecal-oral route by S. enteritidis are usually eliminated by the host immune system and treatment, in severe cases, can include fluid therapy through oral or intravenous routes to replenish the patient’s electrolytes and prevent severe dehydration (1,3). Patients suffering from S. enteritidis infections that present with self-limited, acute and localized gastroenteritis, such as Johnny, can usually recover from symptoms (such as fever, nausea, vomiting, etc.) within 2-7 days post infection (1). However, in susceptible populations, such as in the young, elderly and immunocompromised, bacterial infection by S. enteritidis can progress into a disseminated systemic infection, resulting in septicemia and potentially death (2).

Antibiotic Treatment

Antibiotics are not used for uncomplicated S. enteritidis gastroenteritis because they do not shorten the illness and can increase development of antibiotic resistant strains (3). Studies have shown that in these severe cases where antibiotic treatment is used, not only can these antibiotics clinically fail to rapidly resolve symptoms and completely clear infections (although the Salmonella bacteria remain susceptible to the drug in vitro) but can also result in the development of chronic infections, the evolution of antimicrobial resistant groups, prolonged periods of transmission and the potential for dangerous relapses (4). Antibiotic treatment in general has been demonstrated to result in large alterations to host microbiota compositions and greater preinfection perturbations to this microbial environment, as a result of such treatments, is associated with increased susceptibility of Salmonella colonization in the host intestine, larger postinfection changes in microbiota composition and the prevalence of more severe intestinal pathology with infection in mice (5).

Future Implications after Primary Infection

Even in healthy patients, asymptomatic carriage of the bacteria can occur for extended periods of time (from months to years after the primary infection) due to the continuous shedding of the bacteria in their stool after infection and have a potential for relapse of infection in the future (6). This shedding will continue until the normal gut microbiota has restored itself (6). If depletion of normal gut microbiota continues, the individual becomes more susceptible to Salmonella colonization (7). This is because as the normal, commensal intestinal microbiota is altered and depleted, there is a greater availability of nutrients and reduced competition for colonization, which allows pathogenic bacterial populations such as Salmonella enteritidis to expand and increases probability of infection (5). Reinfection by Salmonella enteritidis through the fecal-oral route occurs at a very low rate (<1%) (4). Conversely, it has been shown that secondary infections and the development of new foci of infection by Salmonella are largely clonal (4).

If someone who experienced a primary acute infection by S. enteritidis were to develop a secondary infection in the future, memory T cells are more able to control this secondary infection through the rapid and more robust release of cytokines and macrophage activation/recruitment to the intestine where the initial infection also occurred (1). Because S. enteritidis is a facultative intracellular pathogen, it replicates within host cells and is thus protected from recognition by circulating antibodies; thus, both cellular and humoral components will be required for immunity against subsequent infections (1). Protective immunity against secondary Salmonella infections is provided by both CD4 T cells and B cells (1,8). B cells can present antigens to Salmonella-specific TH1 cells which mediate bacterial clearance through production of specific inflammatory cytokines and activation of macrophages (8). Studies show that deficiency in either CD4 T cells or B cells in mice showed enhanced susceptibility to secondary Salmonella infection; this shows that CD4 T cells and B cells are critical for protective immunity for future infections (8). The protective role of antibodies is controversial due to limited conclusive evidence currently (8). Thus, there may be some acquired immunity in subsequent infections that will allow the body to clear the infection faster next time. However, Salmonella can suppress IgG plasma cell memory, decimating the memory humoral response (9). Furthermore, specific immunity mechanisms for S. enteritidis still have yet to be elucidated (3). Some types of Salmonella such as S. typhi have been shown to create specific anti-Salmonella serum IgA but it is unclear if S. enteritidis also has this property (3).

References

1. Cummings LA, Deatherage BL, Cookson BT. Adaptive Immune Responses during Salmonella Infection. EcoSal Plus [Internet]. 2009 Sep 17 [cited 2021 Jan 24];3(2). Available from: http://www.asmscience.org/content/journal/ecosalplus/10.1128/ecosalplus.8.8.11
2. Palmer AD, Slauch JM. Mechanisms of Salmonella pathogenesis in animal models. Human and Ecological Risk Assessment: An International Journal. 2017 Nov 17;23(8):1877–92.
3. Giannella RA. Salmonella. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 21. Available from: https://www.ncbi.nlm.nih.gov/books/NBK8435/
4. Rossi O, Vlazaki M, Kanvatirth P, Restif O, Mastroeni P. Within-host spatiotemporal dynamic of systemic salmonellosis: Ways to track infection, reaction to vaccination and antimicrobial treatment. Journal of Microbiological Methods. 2020 Sep 1;176:106008.
5. Sekirov I, Tam NM, Jogova M, Robertson ML, Li Y, Lupp C, et al. Antibiotic-Induced Perturbations of the Intestinal Microbiota Alter Host Susceptibility to Enteric Infection. Infect Immun. 2008 Oct;76(10):4726–36.
6. Hurley D, McCusker MP, Fanning S, Martins M. Salmonella–Host Interactions – Modulation of the Host Innate Immune System. Front Immunol [Internet]. 2014 [cited 2021 Jan 24];5. Available from: https://www.frontiersin.org/articles/10.3389/fimmu.2014.00481/full
7. Janeway CA, Travers P, Walport M, Shlomchik MJ. Immunobiology. 5th ed. Boca Raton, FL: CRC Press; 2001
8. Nanton MR, Way SS, Shlomchik MJ, McSorley SJ. Cutting Edge: B Cells Are Essential for Protective Immunity against Salmonella Independent of Antibody Secretion. The Journal of Immunology. 2012 Dec 15;189(12):5503–7.
9. Takaya A, Yamamoto T, Tokoyoda K. Humoral Immunity vs. Salmonella. Frontiers in Immunology. 2020 Jan 21;NA-NA.