Healthcare: Salmonella Enteritidis

An outbreak of food poisoning was traced to an event where food dressed in a fresh-egg based mayonnaise, had been eaten by all those who subsequently went to their general practitioner where gastroenteritis was diagnosed.

Faecal samples were sent to the laboratory for investigation for suspected Salmonella enteritidis infection.

1. Salmonella enteritidis gastroenteritis (not typhoid) is relatively common in the UK. Using HPA (Health Protection Agency) data (you must use these to plot your own single graph), comment on the incidence of S. enteritidis as a proportion of total Salmonella outbreaks over the last 10 years. 4 marks [up to 1 page for graph and text]

Salmonella enterica subspecies continues to cause a great public health concern in the UK. The infections arising from Salmonella Enteritidis has commonly been linked to eggs as well as egg products. This has been common in England and Wales where it’s known to cause diarrhea dehydration and even death. The infected eggs and eggs products are mostly imported. Salmonella has a rod shape. In wales for instance, Enteritidis phage type 4 has been on the rise since nineteen eighty six and peak infections in nineteen ninety seven. The rise in infection was a related to the global infection of the poultry, which infect the eggs. However, related infections, have declined in UK as a result of rising public awareness regarding food safety as well as compulsory vaccination of the poultry in UK done in nineteen ninety eight against the bacterium. These infections are especially during summer as a result of heat, which facilitate their reproduction where food safety is neglected and its present in buffets and barbecues where food consumption is delayed (PHWHPD, 2011).

The infections with Salmonella Enteritidis in Wales, UK for the last ten years is depicted below.

The salmonella was detected by analyzing fecal samples followed by phage typing. From the graph, infections arising from Salmonella Enteritidis are more common in Wales as compared to those caused by other salmonella species such as salmonella Typhimurium (PHWHPD, 2011).

1. Discuss some (at least 2) recent outbreaks of Salmonella infection associated with eggs. You MUST reference your account in the text, using the CU Harvard format. 5 marks (1 page)

Salmonella Enteritidis outbreaks are associated with undercooked or ven raw eggs notably in the U.S, South and North Carolina. A recent outbreak occurred in South Carolina on February to March of 2001 among inmates from 4 correctional facilities. The initial one happened in male correctional facility on sixth of February while the others occurred on March 2^nd in a second male correctional facility and two others for women. Out of two thousand, three hundred and seventeen inmates from the facilities, six hundred and eighty eight persons admitted to have gastrointestinal symptoms. From fecal sample tests from the inmates there were salmonella Enteritidis phage types 2, 13a as well as 23 (CDC, 2003). From a study carried out by the South Carolina Department of Health and Environmental Control on case patients and controls, it was notable that the inmates had been fed on eggs from a particular vendor and from his farm, the eggs tested positive for salmonella Enteritidis phage types, 28, 22, 23, 13a and 2. Type 2 was notable in the ailing inmates contacted from the facility meals. The flood borne diseases in such institutions is risky since the infection can spread quite fast (CDC, 2003).

Additionally, in North Carolina, in June of 2001 there was a notable Salmonella Enteritidis outbreak by the Statistical Outbreak Detection Algorithm at CDC where The North Carolina State Laboratory of Public Health identified fifty one cases in July and thirty one in august 2001. From the study it was clear that the outbreak was linked to phage type 13a that was traced to eggs retail outlets in the region. The egg could however not be implicated with any farm in the state (CDC 2003).

2. The laboratory instructions stated that to try to identify the Salmonella strain the sample would have to be processed according to the following scheme:

1. 1. Enrichment culture to enable sufficient Salmonella for subsequent isolation.
   2. Use of particular selective and indicator culture media to isolate Salmonella.
   3. Serotype the isolate, based on specific antigens according to the Kauffman and White classification scheme.

Describe each of these, including the biochemical, serological and microbiological detail to explain the specificity of each, with named examples of media and serotypes. 6 marks [⅓ page for each section]

The first procedure involves pre-enrichment using non-selective broth. The second stage involves enrichment in selective broth while the last one is to detect the strains using selective and differential agar media. The procedure is used in testing food fecal and organ samples. To isolated and detect salmonella there has to be media and culture conditions. Pre-enrichment involves use of Buffered Peptone Water (BPW). Alternatively, Lactose Broth can be used and incubation of samples at 37^oC for 18 hours follows (Riemann & Cliver, 2006). For selective enrichment, Rappaport- Vassiliadis Broth (RV), Tetrathionate Broth (TB) and Selenite Cystine Broth (SC) can be used at this stage while incubation of samples for 24 hours at 41.5^oC for RVS follows. The last stage involves plating and selective enrichment and uses Brilliant Green Agar (BGA), Hektoen Agar (HA), Bismuth Sulfite Agar (BSA) and Xylose-Lysine-Desoxycholate (XLD) agar which is incubated at 37^oC for 24 hours. Using several culture medium enhance sensitivity. The cells are cultured to achieve 10⁴– 10⁵ cells/ml and current methods used to detect them include PCR and ELISA (Riemann & Cliver, 2006).

3. Briefly explain the importance of ‘phage typing in the identification of Salmonella species. 2marks [½ page]

Salmonella strains have prophages, which the toxins release spontaneously. Hence, bacteriophages enable phage typing which is not only fast but also allow cheap classification of common serovars. This is crucial for primary analysis prior to expensive molecular procedures e.g. sequencing. Prophages vary elementally in a chromosome and module exchange in prophage genome hence give great discerning potential (Porwollik, 2011). Phage typing enables identification of salmonella constituents in a two letter prefix. For instance Salmonella Enteritidis has phage types PT e.g. PT4. Phage typing is essential to monitor and document global trends and patterns related to salmonella epidemiology. As a result, even with advancing molecular techniques phage typing remains impotent for phenotypic and genotypic typing (Riemann & Cliver, 2006).

4. Give some examples of how ELISAs may be used in the identification of Salmonella. Ensure that you specify whether each is a direct or indirect ELISA. 2 marks [½ page]

ELISA is a procedure with high specificity of the antibody used to salmonella. Salmonella antibodies are stuck to a solid substrate that is applied in attracting salmonella antigen contained in a enrichment broth added to the solid substrate such as microtitre plate wells. When positive results are achieved from carling out ELISA, a confirmation is done by streaking the initial enrichment broth on selective agar. ELISA enables fast sample screening whose outcome is negatives in twenty four hours as well as early identification of a probable positive result. Micro-plates are used in ELISA, dipsticks or strip wells that are coated with antibodies to bind salmonella antigens of a sample (Bell & Kyriakides, 2002). The resulting complex is analyzed calorimetrically or using fluorometric reactions with conjugate, enzyme substrate and chromogen. ELISA has sensitivity of 10⁴ -10⁶ salmonellae/ml of sample culture. The procedure relies on monoclonal antibodies and used to test flocks. Indirect ELISA use single enzyme conjugate and allows for detection of wider range of strains as well as serologically liked viruses. This is done by using non-pre-coated antigen-first which is highly sensitive as compared to direct ELISA which is better used to detect infected samples. Direct ELISA apply labeled prime antibody that binds directly with antigen where direct detection is possible (Lund et al., 2000).

5. If problems were encountered attempting to identify Salmonella in a human isolates and therefore there was a need to consult the UK experts, which reference unit would you contact? Where is it located and what services does it offer? 2 marks [Answer in a few sentences]

The UK experts aids with Identification of Salmonella spp. phage typing, serotyping, Serodiagnosis, molecular techniques in identifying salmonella and Sources and/or occurrence of Salmonella spp. The contact is; Salmonella Reference Unit, HPA Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, Tel: 0208 3276136. Unit Head: Elizabeth De Pinna (HPA, 2011).

6. How would Salmonella enteritidis infection be routinely treated? How may treatment vary with particularly vulnerable patients? 2 marks [½ page].

Patients infected with salmonella Enteritidis have gastrointestinal symptoms of dirrhoea, abdominal pains and fever where hospitalization might be necessary. Self-care is esential during treatment by taking medication to manage diarrhea. Medications with loperamide are effective to hold stool and therefore, less dehydration. Taking plenty of water is crucial since dehydration might result. If the infection persists, it is essential to accuurately diagnose the infection and antibiotics may be given when it’s positive. These may include ciprofloxacin as well as Ampicillin. The main aim of routinely treating salmonella enteritidis is to curb dehydration by taking a lot of fluids. Such may be electrolyte drips during severe diarrhea, administered intravenously particulary in small children whose fever can be managed by Ibuprofen, paracetamol or even acetaminophen. In case of dirrhea, firm foods are essential such as bananas and toast to firm the stools while avoiding milk and eggs. Vulnerable patients such as children should be breastfed continously and have electrolyte fluids administered intravenoiuisly. They therefore, require hospitalizatoion and avoidance of anti-emetics. Immunocompromised and elderly individuals have to manage the symptoms and seek treatment and hospitalization (Saeed et al., 1999).

7. Using research literature, discuss some developments and applications of molecular analysis in the identification of Salmonella species. 5 marks [½-1 page]

From the journal of clinical microbiology “Molecular Analysis of and Identification of Antibiotic Resistance Genes in Clinical Isolates of Salmonella typhi from India” researchers studied twenty one salmonella typhi strains. The strains susceptibility to various antimicrobial agents was evaluated where 11 S. typhi strains were resistant to chloramphenicol, amoxicillin and trimethoprim and 4 were resistant to the named agents other than amoxicillin while 6 were entirely sensitive. All S. typhi isolates showed susceptibility to gentamicin, cephalosporin agents, imipenem as well amoxicillin plus clavulanic acid. Resistance to antibiotics in the isolates was encoded by a particular plasmid and the antibiotic-resistance genes were recognized. For chloramphenicol, it was chloramphenicol acetyltransferase type I while that for trimethoprim was identified as ihydrofolate reductase type VII and that for ampicillin was TEM-1 β-lactamase. Pulsed-field gel electrophoresis on XbaI- produced restriction fragments were 18-DNA for the resistant isolates while 6-profiles were identified from the sensitive isolates. Hence one strain with plasmid having multidrug resistance has cropped up and adapts to the alternative antibiotics used to treat S. typhi in India (Shanahan et al., 1998).

8. A bacterial isolate from the food sample, assumed to be a Salmonella species, was maintained on nutrient agar. A single colony was used to inoculate nutrient broth and the culture incubated at 37^OC for 18h. A drop of this culture was then viewed under the light microscope. After spending some hours watching bacteria dividing under the microscope, the laboratory notes read as follows:

Observation started at 2.15 p.m. with a single cell (A) at the center of the field of view. ‘A’ divided after 10 min to form cells B and C, and 10 min later C divided to form D and E. At 2.40 p.m. cell B divided giving two daughters, F and G, and 5 min later both E and D divided at the same moment, D giving rise to H and I whilst the offspring of E were J and K. At 2.55 p.m. G produced cells L and M, then G’s sister divided with a generation time only 2 min longer than G, forming two more cells, N and O. N divided 40 min later giving P and Q, and at 4 p.m. L divided giving rise to R and S. No further cell divisions were noted in the next 2 hours and all cells were assumed to be dead.

1. 1. Arrange and present the data above in a logical diagrammatic form so that the salient features (comparisons, generation times and correlations between offspring) are discernible at a glance. (Think about your labelling!). Comment on and suggest explanations for these results. 4 marks [Diagram and text up to 1½ pages]

During incubation in a selective enrichment broth, there is a notably competitive species thriving in selective agents dominating the pre-enrichment broth. A bacteriostatic effect follows where there is a drop of competitors that are incapable of growth. Then bactericidal effect occurs where competitors die. There is growth of less vigorous target strain which compose a lesser part of the entire population. A poorly growing target strain decline in number and eventually Jameson Effect occurs. This is the inhibition of other strains when the dominant one achieves a stationary phase in the enrichment broth (Lund et al., 2000).

Jameson evaluated the interaction of various enterobacteriaceae species in a selective both and notably they grew initially having same lag and generation times as if they were single strains in the media. However, as the culture moved towards the stationary phase the, dominant species causes a decrease in cell division of the other species contained in the media. This does not depend on the stage where the fewer number of species reached in the growth cycle. According to Jameson, this is referred to as primary stage of enrichment. Following this, an extended incubation was noted as a secondary stage where the numerous species succeeded in growing to become the larger proportion of the entire population. However the secondary stage of cell division was notably slower as compared to the one in the initial stage. Varied strains of salmonella as they grow collectedly depict the Jameson effect. As a result, salmonella cultures in selective enrichment media incubated could portray positive as well as negative result with time or salmonellae could even be isolated..this is however dependent upon the initial quantities of salmonella and competitive strains contained in the culture (Lund et al., 2000).

2. 2. Was the assumption that all cells were dead justified? Explain your reasoning. 1 mark [2-3 sentences]

No. the assumption was incorrect since the cells are not dead instead, Jameson effect has occurred and there is inhibition of other strains when the dominant ones achieves a stationary phase in the enrichment broth and no further cell division is notable.

3. 3. Knowing the presumed identity of the bacterium comment on the reality of the generation time/s obtained from these observations. 1 mark [1-2 sentences]

Cultures with salmonella on a selective media following incubation of 24,48,72 & 96 hours could be seen as negative or positive depending on the time while salmonella could be isolated at some time. However, the bacterium being presumed to be salmonella species exhibit a different generation time of just several minutes. Therefore the generation times of the salmonella species above are unrealistic.

9. Produce a table, in the format shown below, to compare and contrast the bacterial characteristics and diseases produced by Salmonella typhimurium or Salmonella enteritidis and Salmonella typhi. (You need to expand the boxes as required)

10. Subsequent investigations revealed that the suspect mayonnaise had been used to prepare a prawn cocktail starter. Knowing this, what other microbes should the laboratory attempt to isolate from the suspect food? Include at least one example each of a virus and another bacterium. Briefly explain the tests that you would suggest, including detail of the characteristic properties that are targeted. 3 marks [½ page]

Bacteria to test on would include Staphylococcus aureus. This is implicated with food poisoning particularly in UK although the symptoms are a bit mild and disappears within a short time. The bacterium is liked to toxic bacteriophages which causes nausea and vomiting. It results to food poisoning in foods which they are handled more to prepare them such as prawn cocktails starter. To detect it, there should be selective absorption from the sample to ion exchange resins. Additionally chemical and physical tests have to be carried out to selectively isolate the enterotoxin in a solution (FDA, 2009).

Rotavirus is pathogenic and related to severe diarrhea in children. It is transmitted from the stool and ingested. It leads to gastroenteritis as contaminated food could infect individuals. It is detected by through conducting monoclonal antibody enzyme immunoassay, latex agglutination assay, polyacrylamide gel electrophoresis as well as electron microscopy and reverse transcription polymerase chain reaction. Rotavirus could contaminate the prawn starter during preparation and transmitted through fecal-oral route, could be from contaminated water or persons. The virus however, dos not multiply in food but can be detected (Gómara et al., 2000).

11. A friend of one of the patients suggested that they should eat live yoghurt. Briefly discuss the microbiological validity of this approach. 2 marks [few sentences]

Salmonella species infect the gastrointestinal tract in humans to cause what is identified as food poisoning. Therefore, ingesting live yoghurt with probiotics bacteria facilitate digestion and absorption of nutrients. This contains active cultures which wipe out food poisoning arising from salmonella Enteritidis (Stein et al., 2000).

List of References

Bell, C. and Kyriakides, A. (2002) Salmonella: A Practical Approach to the Organism and Its Control in Foods. Malden, MA, Blackwell Science Ltd.

Centers for Disease Control and Prevention (CDC) (2003) Outbreaks of Salmonella Serotype Enteritidis Infection Associated with Eating Shell Eggs — United States, 1999–2001. Morbidity and Mortality Weekly Report 51(51);1149-1152. Web.

Engelkirk, P. G. and Duben-Engelkirk, J. (2010) Burton’s Microbiology for the Health Sciences, North American Edition. Baltimore, MD Lippincott Williams & Wilkins.

Gómara, M.I., Green, J., and Gray, J. (2000) Methods of Rotavirus Detection, Sero- and Genotyping, Sequencing, and Phylogenetic Analysis. Methods in Molecular Medicine Volume 34, 189-216, Web.

Hawley, L. (2006) High-Yield Microbiology and Infectious Diseases. Baltimore, MD, Lippincott Williams & Wilkins.

Health Protection Agency (2011) Salmonella Reference Unit. Web.

Kiyono, H. (1996) Mucosal Vaccines. San Diego, CA, Academic Press.

Lund, B. M., Baird-Parker, T.C., and Gould, G.W. (2000) The Microbiological Safety and Quality of Food, Volume 2. Gaithersburg, Maryland, Aspen Publishers Inc.

Porwollik, S. (2011) Salmonella: From Genome to Function. Norfolk, UK, Caister academic Press.

Public Health Wales Health Protection Division (PHWHPD) (2011) Salmonella. Public Heath Wales. Web.

Riemann, H. P. and Cliver, D. O. (2006) Foodborne Infections and Intoxications. San Diego, CA, Academic Press.

Saeed, A. M., Gast, R.K. and Potter, M. E. (1999) Salmonella Enterica Serovar Enteritidis in Humans And Animals: Epidemiology, Pathogenesis, and Control. Ames, Lowa, Lowa State University Press.

Shanahan, M. A., Jesudason, M.V., Thomson, C. J. and Amyes, G. B. (1998)^. Molecular Analysis of and Identification of Antibiotic Resistance Genes in Clinical Isolates of Salmonella typhi from India. Journal of Clinical Microbiology p. 1595-1600, Vol. 36, No. 60095-1137/98/$04.00+0. Web.

Stein, M., Zei, P., Hwang, G., and Breaden, R. (2000) Cracking the Boards: USMLE Step 1. New York, The Princeton Review.

Torrence, M.E. and Isaacson, R.E. (2003) Microbial Food Safety in Animal Agriculture: Current Topics. Ames, Lowa, Wiley-Blackwell.

U.S. Food and Drug administration (FDA). BBB – Staphylococcus aureus Bad Bug Book: Foodborne Pathogenic Microorganisms and Natural Toxins Handbook Staphylococcus aureus April 2009. US Department of Health and Human Services. Web.