Topic P04: Diagnostics of enterobacteria and bacterial agents of gastrointestinal infections To study: Enterobacteriaceae, Vibrionaceae, Campylobacter, Helicobacter (from textbooks, WWW etc.) From spring term: Microscopy, culture, biochemical identification, antigen analysis Table for major results of Task 1 to Task 5 (to be filled step by step): Strain K L M N P Q R S Gram stain – Task 1 Culture (blod agar and Endo agar) Task 2 Size on BA Colour on BA Other on BA Size on Endo Colour on Endo Other on Endo Hajna medium Task 3a Oxidase test Task 3b PARTIAL CONCLUSION More media Task 4a XLD agar MAL agar CIN agar EnteroTest 16 (Task 4b) Antigen analysis (Tasks 5a and 5b) FINAL CONCLUSION Task 1: Microscopy of suspicious strains There are letter-labelled strains on the table. Gram-stain them and write your results to the table. Strain that is NOT G– rod should not be used in tasks 3 to 5 (but in Task 2 it should be described, for comparison) Task 2: Cultivation on blood agar and Endo agar Using standard procedure, describe colonies of all strains on blood agar and Endo agar. If the strain on the medium does not grow, write a zero to the corresponding cell of the table. Bacteria, that do not grow on any of the media and morphologically look like curved gram-negative rods, might be Campylobacter – see later. A G– rod, that does not grow on any of the media, but is not curved, will be studied in P05. For comparison describe also the strain, that appeared morphologically as a gram-positive coccus. Task 3: Group diagnostics of the most imporant gram-negative rods growing on Endo agar (differentiation of enterobacteriae, Vibrionaceae and G– non-fermenters) a) Reading of an examination on oblique agar according to Hajna Agar according to Hajna is a combined diagnostic medium. Nevertheless, in this task we will mostly search for biochemically non-active, neither glucose nor lactose splitting and sulphan non forming rods – the gram-negative non-fermenting bacteria („non-fermenters“). All strains, growing on Endo, were inoculated on Hajna medium. Have a look to the result. Where the medium remained fully red, it is a biochemically non-active strain – very likely, a gram-negative non-fermenter. This strain will not be used in Task 4 and Task 5. b) Oxidase test The teacher will do as a demonstration oxidase test for all Gram-negative, on Endo agar growing bacteria. Oxidase-positive are members of family Vibrionaceae and some gram-negative non-fermenters; the Enteobacteriaceae are (with exception of Plesiomonas) oxidase negative. Make partial conclusion after tasks 1 to 3. What bacteria are enterobacteria? Tasks 4 and 5 will be only performed with strains proven to be enterobacteria. Task 4: Genus and species determination of enterobacteria a) Culture of enterobacteria on more media You have already seen, how the colonies look like on BA and Endo agar. Add shortly your description of appearance of the colonies on CIN, XLD and MAL. b) Biochemical behaviour of enterobacteria Evaluate given results of ENTEROtest 16, beeng incubated a day berfore. Check, wethrer the results with other, already done tests; e.g. strains with sulphan formation lead to black colour of Hajna medium, Yersinia has tiny pink colonies, Salmonella pale transparent colonies with black centre on XLD and MAL medium… For the strain found to be Salmonella, write Salmonella sp. only. Count % of probability as a total of all % of probability of individual Salmonellas at the code; T index should be taken from the first Salmonella. ONPG 1H 1G 1F 1E 1D 1C 1B 1A 2H 2G 2F 2E 2D 2C 2B 2A Strain: 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 Code: Identification % of prob. T index ONPG 1H 1G 1F 1E 1D 1C 1B 1A 2H 2G 2F 2E 2D 2C 2B 2A Strain: 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 Code: Identification % of prob. T index ONPG 1H 1G 1F 1E 1D 1C 1B 1A 2H 2G 2F 2E 2D 2C 2B 2A Strain: 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 Code: Identification % of prob. T index ONPG 1H 1G 1F 1E 1D 1C 1B 1A 2H 2G 2F 2E 2D 2C 2B 2A Strain: 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 Code: Identification % of prob. T index Task No. 5 Antigen analysis to intra-species diagnostics of enterobacteriae We will perform the antigen analysis in strains of bacteria, where it is performed routinelly. Antigen analysis is performed in enterobacteria mainly for one of two reasons: (a) to diferenciate antigen types with elevated virulence – especially in E. coli to differenciate EPEC, STEC etc. (b) of epidemiological reasons, sometimes in combination with (a) reasons – Salmonella, Shigella, Yersinia etc. a) Excluding of EPEC In strain identified as Escherichia coli, perform antigen analysis using slide agglutination with two polyvalent sera (one nonavalent, one trivalent). If both results will be negative, the strain does not belong into EPEC group. b) Assessing the serovar in Salmonella In strain identified as Salmonella enterica, perform antigen analysis using slide agglutination and discover the serovar. Let us suppose that in the pacient there was allready found a strain of serovar Enteritidis and now we only want to be sure, that it is the same strain once more. Perform a test with body antigen O: 9 and flagellar antigen H: g, m. Write the result to the table. Task No. 6: Susceptibility tests of enterobacteria to antibiotics On your table, you will find diffusion disc tests for strains found to be Enterobacteriaceae. Write abbreviations of antibiotics according to the card and measure susceptibility zones for all tested strains. Borderline zones are written on the cards; using them, interprete the strains as susceptible (S) rezistant (R) and dubious (D). Strain à Antibiotic (full name) Zone Æ (mm) Interpr. Zone Æ (mm) Interpr. Zone Æ (mm) Interpr. Zone Æ (mm) Interpr. Task No. 7 Diagnostics of Campylobacter Observe the cultivation appearance that did not grow neither on BA nor on Endo agar and which, according to the morphology, is supposed to be a Campylobacter (because of being curved), on a special medium. Remember four main conditions for cultivation of Campylobacter: (a) special medium with charcoal and addition of antibiotics and antimycotics to prevent growth of other microbes, (b) microaerofilic conditions, (c) temperature elevated to 42 °C, what correspons to body temperature of birds – natural hosts, and (d) prolongation of the cultivation to 48 hours. Describe the colonies write down the result of oxidase test (teacher will perform it as a demonstration). For Campylobacter a retarded positivity is typical, e. g. the strip becomes blue, but not immediatelly, but after a while. Description of colonies Result of oxidase test More notes Task No. 8: Urease test in diagnostics of Helicobacter In diagnostics of helicobacters we use the urease test, performed directly with a bioptic specimen of gastric mucosa. A pozitive result is red, negative yellow. Among two specimens (X and Y) find the positive one. Result: Positive urease test was found in specimen ____, negative in specimen ____ Task No. 9 Diagnostics of the family Vibrionaceae Vibrionaceae is a bacterial family simillar to Enterobacteriaceae, but oxidase-positive. We use special media to culture Vibrionaceae. Mutual differentiation is possible through biochemical tests like for enterobacteria. even Enterotest 16 could be used, but a special codebook would be required. Antigen analysis could be used, too. Draw here, how a Vibrio looks like microscopically, and add some more properties according to the slideshow. Microscopy: Most important solid medium for Vibrio: Most important liquid medium for Vibrio: The two most important serovars of V. cholerae The two most important biovars of V. cholerae O1 Check-up questions: 1. Do you know, what is the result of the catalase test in enterobacteria? 2. For practical reasons, one medium used in diagnostics of enterobacteria is missing: – selenite broth. What type of medium is it and what is its use? (See practical J03) 3. Do you know at least some antigenic types of EPEC? 4. What would be difference in Task 5b, when the patient would have no evidence of previous Salmonella? 5. What pathogen is diagnosed by Widal reaction? Is it a direct, or indirect method? Which type of reaction is it? 6. Is it recommended to use antibiotic treatment for intestinal infection? Why? 7. Do you know, what the Urea breath test is and what is its principle? 8. In which clinical material it is more likelly to find Salmonella Typhi in typhoid fever rather than in the stool?