Name of Organism Disease/symptoms Gram Stain Colony Morphology

Salmonella sp. It causes a variety of human disease called salmonellosis. Disease is from mild self limiting gastroenteritis to more severe form with possibility of bacteraemia or typhoid fever, which can be life threatening. Severe disease associated with S.typhi and S.paratyphi A & S. paratyphi B whereas the other 2,300 or more strains are associated with gastroenteritis. S.typhi is unique because humans only carry it. This intracellular parasite can cause typhoid fever, which is characterised by fever, diarrhoea and inflammation of infected organs. Other symptoms include headache, abdominal cramping and vomiting. motile, facultatively anaerobic, gram-negative rod shaped bacterium On XLD, Salmonella sp. Produces red colonies usually with black centre Agglutination with polyvalent O, H & Vi antiserum followed by further O sub-antisera and H sub-antisera are carried out once the biochemical tests suggests? Salmonella sp.

S.paratyphi A, B & C and S.typhi positive agglutination is transferred to CAT III laboratory and API 20E and EUCAST sensitivities are performed. Isolate is sent to reference laboratory for confirmation of id and serotype Salmonella sp produces acid from glucose usually with production of gas and are oxidase, indole and urea negative. Most produces hydrogen sulphide except S. paratyphi A and Salmonella typhi, which is weak producer.

Presumptive positive identification of Salmonella sp is sent to MALDI for ID and manual sensitivities done Shigella sp. People infected with Shigella develop diarrhoea, fever and abdominal cramps. Severity of the disease ranges from mild to very severe diarrhoea. Diarrhoea is bloody 25-50 % of the time and most often contains mucus. The illness starts 12 hours to 6 days after exposure. Dehydration is common symptoms of shigella infection. Based on serological antigen the disease is named into 4 species.

Serotype A- S.dysenteriae

Serotype B- S.flexneri

Serotype C- S.boydii

Serotype D- S.sonnei non motile, facultative anaerobes, Gram negative rods. On XLD agar, shigella sp. Appears as red colonies (no black centre). The Shigella sp. can be subdivided according to their somatic(cell-wall) or O antigens. S.dysenteriae contains 12 serotype, S.flexneri contains 8 serotypes, S,boydii 18 serotypes and S.sonnei 1 serotype.

Shigella dysenteriae type 1 positive agglutination is transferred to cat III laboratory and API20E and EUCAST sensitivity are performed. Isolate is sent to the reference laboratory for confirmation of id and serotype. Shigella sp. is oxidase, indole and urea negative and does not decarboxylate lysine and all except S.dysenteriae type 1 are catalase positive. The majority of Shigella sp. Except S.flexneri 6 and S.boydii 13 and 14 ferment sugars without gas production. S.sonnei and S.dysenteriae type 1 are only species that are ONPG positive.

Presumptive positive identification of Shigella sp. is sent to MALDI for identification and manual sensitivities done Campylobacter sp. Campylobacter sp. is the commonest cause of enteritis in the UK. Initial symptoms may be severe with fever and abdominal pain suggesting appendicitis. Faeces frequently contain mucus with blood and leucocytes. Severe infection can cause reactive arthritis, bursitis, endocarditis and neonatal sepsis. Gram negative curved rods look like as spiral. Charcoal cefoperazone deoxycholate agar (CCDA) incubated microaerobically at 42ºC for 48 hours.

Culture may be incubated for a further 24 hours if required.

On selective agar, the colonies are grey/white or creamy gray and moist in appearance. They are motile, microaerophilic (optimum 5 -10% oxygen).

NA Oxidase Test

Campylobacter sp. is oxidase positive. If positive MALDI is done for identification.

For confirmation, colonies are subculture on Blood agar and incubated aerobically at 37ºC for 24 hours. No growth on BA after 24 hours confirms the campylobacter sp.

Vibrio species Disease is associated with ingestion of contaminated water or seafood. The diarrhoea causing sp. most frequently isolated are vibrio parahaemolytics. A symptom ranges from mild to bloody diarhhoea (often accompanied by abdominal cramps and vomiting) to explosive diarrhoea. The main symptoms of cholera are passage of profuse watery diarrhoea with mucus but no blood, giving a ‘rice water’ appearance. Fluid loss and dehydration is the main cause of death. The incubation period is a few hours to a few days. V.cholerae 01 and 0139 are producers of cholera toxin (CT). V. parahaemolyticus associated with eating contaminated seafood particularly shellfish. Gram negative curved rods characteristically comma shaped. This characteristic appearance is not always observed when the organism is Gram stained from solid media. Vibrio sp. are facultative anaerobes, motile by a single polar flagellum. On blood agar colonies are 2-3 mm diameter. Some strains may be haemolytic. After 18-24 hours incubation colonies on TCBS are at least 2mm in diameter and yellow in case of sucrose fermenters (V.cholerae) and green non-sucrose fermenters (V.parahaemolyticus) Serology and sensitivity to pteridine O129

Most vibrio sp. are sensitive with 150µg but species differ with 10 µg discs. V. cholerae O1 depends on the detection of the O1 antigens on the surface of the bacterium. Presumptive identification of V.cholerae is sent to MALDI for ID and sensitivities are done using EUCAST. Isolate is sent to the reference laboratory. Oxidase test and API 20E

Vibrio species are oxidase positive. It is necessary to subculture colonies to blood agar for oxidase test as false oxidase test may result on media containing carbohydrate.

Ecoli O157 (VETEC O157) E.coli O157 is one of the verocytotoxin producer organisms. The toxin is similar to the shiga toxin of shigella dysenteriae and is associated with haemorrhagic colitis, haemolytic uramic syndrome and thrombocytopenic purpura (TTP) which causes a kidney failure, haemolytic anaemia and thrombocytopaenia. Infection vary in severity from mild to blood diarrhoea and may occur in any age group, however more common in children. Gram negative rods. On Sorbitol Macconkeys agar (SMAC) containing cefixime and tellurite, the colonies are colourless and 2-3 mm in diameter. VETC O157 differs from other members of the genus Escherichia in that it usually does not ferment sorbitol. Plates are usually incubated in air at 35-37ºC for 16-24 hrs.

Maldi is used for ID on both SMAC plates, once a positive ID of E. coli. The specimen and all the plates are transferred to CAT III.

API20E and sensitivity tests are performed in CAT III and isolate is sent to reference laboratory. E.coli 0157 strains are oxidase negative, indole positive, urea and citrate negative.

Yersinia sp.

Infection is usually acquired by the oral route from contaminated food, milk or water. Pigs are a frequently identified source of infection. Infection occurs more often in the young (<6y) and the elderly. After ingestion the organism proliferates in the lymphoid tissue of the small intestine where it may cause hyperaemia, neutrophil infiltration and ulceration. The incubation period is between 4 and 7 days. Occasionally, haematogenous spread occurs, producing septicaemia with the formation of abscesses in organs such as the liver and spleen.

Yersiniosis may therefore present with variety of clinical conditions such as acute diarrhoea, mesenteric lymphadenitis, terminal ileitis, “pseudo-appendicitis”, septicaemia, metastatic infection and immunological sequelae (e.g. reactive arthritis). Gram negative rods On CIN agar, colonies of suspected Yersinia sp. Shows typical red bull eyes pink colonies at 30ºC in aerobic condition.

NA Yersina sp. are urea positive and oxidase negative.

Presumptive positive identification of Yersinia is sent to MALDI for ID and manual sensitivities are done. Isolate is sent to the reference laboratory.

Other organisms of potential importance in enteric pathogens

Aeromonas species

Aeromonas sp. have been implicated as causative organisms of watery, no-blood diarrhoea. Young children and patients who are elderly may be more susceptible to infection. Although the organisms have been linked to food and water borne outbreaks, their significance is still uncertain.

Pseudomonas sp. should be excluded by confirmation of growth of pseudomonas agar/MALDI.

Aeromonas and plesiomonas show pink colonies, oxidase positive on XLD. Aeromonas can also grow on TCBS forming green bluish colonies.

Possible identification of Aeromonas and Plesiomonas sp. are send to MALDI for confirmation and sensitivities are done in PHOENIX. In addition, an isosensitest agar is inoculated with the addition of an 0129 disc(150µg)

Aeromonas = Resistant

Plesiomnas = Sensitive

Aeromonas sp. and Plesiomonas sp. can produces false negative oxidase result, if tested form Macconkey’s and therefore it is necessary to do oxidase from BA subculture.

Plesiomonas shigelloides

Plesiomonas shigelloided has been isolated from in patients with diarrhoea and abdominal cramps. It has been linked to food and water-borne outbreaks of gastrointestinal infection.

Bacterial Pathogens

Explain the composition and method of action on each type of medium used on the faeces bench

Media used on faeces bench Composition Method of action XLD (Xylulose lysine dextrose medium) Lysine, xylose, lactose, sucrose,sodium desoxycholate, sodium thiosulphate,ferric ammonium citrate,phenol red It utilizes sodium desoxycholate as the selective agent and inhibits the Gram positive organisms. Xylose fermentation differentiate Shigella sp. from other enteric pathogens. Because of this decarboxylation the pH changes and the colony appears pink by neutral red indicator. If the organism produces hydrogen sulphide then the colony appears pink with a black centre.

Selenite medium

Tryptone

Lactose

Disodium phosphate

L-cystine This is enrichment medium for Salmonella sp. Which inhibits the growth of coliforms and other faecal streptococci. Lactose is added as a fermentable carbohydrate to prevent a rise in pH value during incubation.

Sorbitol – Macconkey’s

Medium

Peptone, sorbitol, sodium chloride, Neutral red, Potassium tellurite and Cefixime. The medium is mainly designed for the non-sorbitol fermenting organism E.coli O157. This medium contains the main supplements cefixime and potassium tellurite, which inhibit the growth of other non-sorbitol fermenter organisms with no inhibitory effect on E. coli O157. The medium is only used for a screening purpose as some of the non-sorbitol fermenters may grow on this medium.

TCBS medium

Bacteriological peptone, sodium thiosulphate, sodium citrate, ox bile, sucrose, sodium chloride, ferric citrate, bromothymol blue, thymol blue The bile salt inhibits the growth of gram-positive organism whereas sodium thiosulphate serves as a sulphur source which, in combination with ferric citrate, detects the hydrogen sulphide production.

Alkaline peptone water This is an enrichment medium containing a high conc. of salt as Vibrio sp. requires a high salt conc. A loopful of the medium was cultured on to another TCBS agar and incubated at 37˚C for 24 hours in aerobic condition.

CCDA selective media Bacteriological charcoal, Casein hydrolysate, sodium desoxycholate, ferrous sulphate, sodium pyruvate, amphotericin B and cefaperazone This is the blood free selective medium plus it mainly contains two main antibiotics- amphotericin and cefaperazone which inhibits the growth of fungi and other gram negative organisms. The charcoal, ferrous sulphate and sodium pyruvate in the medium enhances the growth of Campylobacter sp.

If the organisms are suspected, they appear as creamy wet colonies which are oxidase positive.

Yersina selective agar Mannitole

Neutral red, crystal violet, Sodium desoxycholate, Cefsulodin, Irgasan, novobiocin. Selective medium inhibiting Gram negative and Gram positive organism by means of crystal violet, sodium desoxycholate and antimicrobial agents. Fermentation of manitole in presence of neutral red results in a characteristic bull-eye colony, colourless with red centres.

3a Bacterial Pathogens

C) Explain how and why the laboratory processes faecal specimens with regards to clinical details and patients demographics

Faecal specimens are cultured onto a range of selective media in order to isolate enteric pathogens.

Firstly, the appearance of sample is recorded on the form and on Winpath as: formed, semi-formed, liquid, blood stained specimen or mucoid specimen.

Specimens are then cultured according to following the criteria on the SOP.

The culture protocol depends on where the patient is (Community, Ward, Hospital), the age of the patient and the clinical and travel history. Briefly, for GP and outpatients, full culture with XLD, CAMP and Selenite is performed. Inpatient are screened with selenite and CAMP only, unless is a new admission or form from A&E, AAU, T8, T11, T12 and obstetrics patients, where full culture is performed. Neonate specimens are cultured onto MAC +gent, SAB & PSM agar. If the patient has travelled in tropical countries or fish consumptions indicated and specimen appears liquid, TCBS & APW is added to the culture. Also if a clinical detail suggests that patient have abdominal pain or acute appendicitis, CIN agar for investigation of Yersinia sp. is added for Yersinia screening.

Culture plates are incubated under appropriate conditions to screen out non-pathogens, leaving the enteric pathogens to be identified. Antibiotic susceptibility tests are performed and a preliminary report is issued. Isolates are further identified as necessary by referring them to the HPA reference laboratory, where on return of the result, an additional report is issued.