Topic P04: Diagnostics of Enterobacteriaceae and bacterial agents of gastrointestinal infections To study: Enterobacteriaceae, Vibrionaceae, Campylobacter, Helicobacter (from textbooks, www etc.) From spring term: Microscopy, culture, biochemical identification, antigenic analysis Summary sheet 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 Colonies (blood 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 XLD agar Task 4a ENTEROtest 16 Task 4b Antigenic 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 in the summary sheet. Attention, strain N, more difficult for staining, is already stained, i. e. you do not have to stain it. A strain that is NOT a 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 summary sheet. 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. Do not use Petri dishes labelled “Úkol č. 2” for other tasks than Task 2. Task 3: Group diagnostics of the most important Gram-negative rods growing on Endo agar (differentiation of Enterobacteriaceae, Vibrionaceae and G– non-fermenters) a) Reading of an examination on oblique triple sugar iron 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 the strains growing on Endo were inoculated on Hajna medium. Examine 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 demonstrate an 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 the exception of Plesiomonas) oxidase-negative. Make partial conclusion after tasks 1 to 3. Which bacteria are Enterobacteriaceae? Tasks 4 and 5 will be only performed with strains proven to be Enterobacteriaceae. Task 4: Genus and species determination of Enterobacteriaceae a) Culture of Enterobacteriaceae on other media You have already seen what the colonies look like on BA and Endo agar. Describe in the summary sheet shortly the appearance of the colonies on XLD medium. Also look at MAL medium on the side table – it is very likely to the appearance of XLD medium (there exist more similar media, for example DC agar). Look also at CIN medium – Yersinia has tiny pink colonies, other bacteria are different or do not grow at all. Results on MAL and CIN are not written in the summary sheet. b) Biochemical properties of Enterobacteriaceae Evaluate the results of the ENTEROtest 16 incubated a day before. Check, whether the results correspond with other already performed tests; e.g. strains with sulphan formation lead to black colour of Hajna medium, Salmonella pale transparent colonies with black centre on XLD and MAL medium. For the strain found to be Salmonella, write just Salmonella sp. (or Salmonella enterica) as a result. For this strain, count the percent of probability as a total of all % of probability of all three individual salmonellae found in the book corresponding to your code; T index should be taken from the first Salmonella taxon in the codebook. All the names of bacteria should be copied to the summary sheet preceding Task 1. Tube First row Second row 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 5: Antigenic analysis to intra-species diagnostics of Enterobacteriaceae We will perform the antigenic analysis in strains of bacteria, where it is performed routinely. Antigenic analysis in Enterobacteriaceae is performed mainly for one of two reasons: (a) To differentiate antigenic types with elevated virulence – especially in E. coli to differentiate e.g. EPEC, ETEC and EIEC. (b) For epidemiological reasons, sometimes in combination with (a) reasons – Salmonella, Shigella, Yersinia etc. a) Excluding EPEC In the strain identified as Escherichia coli, perform the antigenic analysis using slide agglutination with two polyvalent sera (one nonavalent, one trivalent). If both results are negative, the strain does not belong in the EPEC group (as a final result, write “E. coli, non-EPEC”). b) Assessing the serovar in Salmonella In the strain identified as Salmonella enterica, perform the antigenic analysis using the slide agglutination and determine the serovar. Let us suppose that in the patient there has already been found a strain of the serovar Enteritidis and now we only want to make sure, that this is the same strain again. Perform a test with body antigen O: 9 and flagellar antigens H: g, m. Write the result in the summary sheet. Task 6: Antibiotic susceptibility tests of Enterobacteriaceae On your table, you will find diffusion disc tests for the strains found to be Enterobacteriaceae. There is no test for Salmonella – such isolates are usually stool origin and so antibiotic treatment is not indicated. The antibiotic set is one of suitable sets for urinary tract infections (UTI). Interpret the strains as susceptible (S), resistant (R) and intermediate (I) to given antibiotics. The interpretation is done according to actual EUCAST# recommendations, so for some antibiotics we have only “susceptible” or “resistant”, for some other we also have intermediate (I). Strain Antibiotic Suceptible if Inter-mediate if Resistant if Zone Æ (mm) Interpre- tation Zone Æ (mm) Interpre- tation Zone Æ (mm) Interpre- tation Ampicillin AMP ≥ 14 mm < 14 mm Cephalotin KF ≥ 18 mm 14–17 mm < 14 mm Co-trimoxa-zole SXT ≥ 16 mm 13–15 mm < 13 mm Nitrofuran-toin F ≥ 11 mm < 11 mm TetracyclineTE* ≥ 15 mm 12–14 mm < 12 mm Cefuroxime CXM ≥ 18 mm < 18 mm Norfloxacin NOR ≥ 22 mm 19–21 mm < 19 mm *also valid for doxycycline Task 7: Diagnostics of Campylobacter Observe the cultivation appearance of the strain that grew 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 the four main conditions needed for the cultivation of Campylobacter: (a) Special medium with charcoal and addition of antibiotics and antimycotics to prevent growth of other microbes. (b) Microaerophilic conditions. (c) Temperature elevated to 42 °C, which corresponds to the body temperature of birds – natural hosts. (d) The extension of the cultivation to 48 hours. Describe the colonies, write down the result of the oxidase test (the teacher will perform it as a demonstration). For Campylobacter, a retarded positive result is typical, e.g. the strip becomes blue not immediately, but only after a while. Description of colonies Result of oxidase test More notes Task 8: Urease test in the diagnostics of Helicobacter In the diagnostics of helicobacters we use the urease test, performed directly on a biopsy specimen of gastric mucosa (not with a cultured strain – an exception!). The specimen is put in a medium containing urea and an indicator. The positive result is red, the negative one yellow. Out of the two specimens (X and Y) find the positive one. Result: Positive urease test was found in specimen ____, negative in specimen ____. Task 9: Diagnostics of the Vibrionaceae family Vibrionaceae is a bacterial family similar to Enterobacteriaceae, but oxidase-positive. We use special media to culture Vibrionaceae. Mutual differentiation is possible using the same biochemical tests as with Enterobacteriaceae. Even Enterotest 16 can be used, but a special codebook would be required. Antigenic analysis can be used as well. Draw here what a Vibrio looks like microscopically, and add some more properties according to the slideshow. Microscopy: The most important solid medium for Vibrio: The most important liquid medium for Vibrio: The two most important serovars of V. cholerae: The two most important biovars of V. cholerae O1: #EUCAST = The European Committee on Antimicrobial Susceptibility Testing