Topic P05: Diagnostics of Pasteurellaceae and G– non-fermenters To study: Haemophilus, Pasteurella, Pseudomonas and G– non-fermenters (from textbooks, www etc.) From spring term: Microscopy, culture, biochemical identification, antigenic 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 Task 2 Cul-ture Growth on BA (Y/N) Growth characte- ristics on BA (ChA*) Endo agar (–/L–/L+^#) MH agar (colour) Task 3a Satelite phenomenon (+/–) Task 3b Factor test (X, V, X + V) Task 3c H. influen. capsular type 3d Susc. test Penicill. Vanc. Gluc. fermentation Task 4 (Hajna) Oxidase test Task 5a NEFERMtest 24 Task 5b FINAL CONCLUSION *Use ChA (chocolate agar) for bacteria not growing on BA (blood agar) ^#does not grow/does grow, Lactose- non-fermenter/does grow, Lactose fermenter Task 1: Microscopy of suspicious strains There are letter-labelled strains on the table. Gram-stain them and write your results in the table. The 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 agar media First write down which bacteria do grow on blood agar and which do not. Then, using the standard procedure, describe the colonies of all the strains on blood agar. In strains that do not grow on blood agar*, describe their growth on chocolate agar instead. Then describe the growth of bacteria on Endo agar (only “–” for not growing bacteria, “+” for growing ones; lactose fermentation cannot be seen, as the strains do not have isolated colonies). On MH agar check only one strain and only for eventual pigment presence (the plate serves also for Task 6b). *demonstrated by only one agar plate on the side table of the practical hall Task 3: Identification of Pasteurellaceae and their more precise determination a) Satellite phenomenon of haemophili Haemophili are typical for the so-called satellite phenomenon, which means that they are able to grow on blood agar only in the presence of a microbe able to release growth factors for the haemophili. Usually Staphylococcus aureus is used for this purpose. Draw the satellite phenomenon and connect the terms below with the features on your picture Staphylococcus aureus Colonies of haemophili b) Identification of the haemophili on the basis of the growth factors requirements Determine the given strains according to their requirements of the growth factors. Draw the growth factor tests for both strains. c) The detection of H. influenzae capsule antigens Describe the result of agglutination of H. influenzae capsule antigens by means of latex agglutination (from the slide-show). d) The detection of Pasteurella multocida using typical antibiotic susceptibility pattern P. multocida is characterized by its susceptibility to penicillin, which is very rare among G– rods. On the other hand, it is resistant to much stronger (but for G+ bacteria only) antibiotic – vancomycin. Fill in the table. Task 4: Hajna medium Observe the results of culture of four strains on Hajna medium. Mark the strains able to ferment glucose (yellow colour) as “+”, the strains unable to ferment it (red colour) as “–”. Task 5: Determination of G– glucose non-fermenters a) Oxidase test A demonstration of the oxidase test for the three strains determined as G– non-fermenters. Write down the results to the table (Pseudomonas should be always positive, Burkholderia is mostly positive but not necessarily; on the other hand, Stenotrophomonas tends to be negative). The oxidase positive bacterium with typical odour and pigmentation (mostly green, less often blue or maroon) is almost certainly Pseudomonas aeruginosa. In this bacterium, it is not necessary to perform further biochemical testing, described in Task 5a. In the other two strains, this biochemical testing is necessary. b) Detailed biochemical testing Evaluate the given results of NEFERMtest 24, incubated two days prior (unlike the other biochemical tests, where it is one day) at 30 °C (again a difference, other tests require 37 °C). The way of code counting is different, too, as there are three rows in the test. The upper row is always “1” when positive, the medium row is “2” and the lowest one “4”. The first number is for the oxidase test: write “1” when positive and “0” when negative. The results of “B” and “A” columns are NOT used for code counting. So, you obtain a 7-position code: The first number is “0” or “1” and the remaining six positions are for the results of the tests in columns H to C. Strain: OX H G F E D C B A Code: 1 Identification: 2 % of probability: 4 Typicity index: Code Strain: OX H G F E D C B A Code: 1 Identification: 2 % of probability: 4 Typicity index: Code Notes: Task 6: Antibiotics susceptibility tests of pathogenic bacteria Among your bacteria, there are five pathogens: two of the Pasteurellaceae family, three G– non-fermenters (but of them, you are supposed to measure zones for Pseudomonas only). Write the abbreviations of the antibiotics according to the card and measure the susceptibility zones for all the tested strains. Borderline zones are written on the cards; using them, interpret the strains as susceptible (S), resistant (R) and intermediate (I). 6a) Test for Haemophilus (Haemophilus influenzae was found to be strain ___) Antibiotic Zone Æ (mm) Interpre-tation Ampicillin (AMP) S ≥ 16 / R < 16 Co-amoxicillin (AMC) S ≥ 16 / R < 16 Cefuroxime (CXM) S ≥ 25 / R < 25 Chloramfenicole (C) S ≥ 28 / R < 28 Tetracyclin (TE)* S ≥ 25 / R < 22 Co-trimoxazole (SXT) S ≥ 23 / R < 20 6b) Test for Pasteurella (Pasteurella multocida was found to be strain ___) Antibiotic Zone Æ (mm) Interpre-tation Ampicillin (AMP) S ≥ 17 / R < 17 Co-amoxicillin (AMC) S ≥ 15 / R < 15 Cefotaxime (CTX) S ≥ 26 / R < 26 Ciprofloxacine (CIP) S ≥ 27 / R < 27 Tetracyclin (TE)* S ≥ 24 / R < 23 Co-trimoxazole (SXT) S ≥ 23 / R < 23 6c) Test for Pseudomonas (Pseudomonas aeruginosa was found to be strain ___) Antibiotic Zone Æ (mm) Interpre-tation Antibiotic Zone Æ (mm) Interpre-tation Piperacillin/tazobactam (TZP) S ≥ 18 / R < 18 ciprofloxacin (CIP) S ≥ 25 / R < 22 gentamicin (CN) S ≥ 15 / R < 15 ceftazidime (CAZ) S ≥ 16 / R < 16 ofloxacin (OFL) S ≥ 16 / R < 13 colistin (CT) S ≥ 11 / R < 11 Note. Tazobactam acts as betalactamase inhibitor, but it also has its own antimicrobial effect. 6d) Check-up for primary resistances for Burhkohleria and Stenotrophomonas strains This part is not performed by dental students. Task 7: Relations of bacteria to oxygen – comparison of Enterobacteriaceae, G– non-fermenters and anaerobes Look at the broth cultivated under aerobic and anaerobic conditions (layer of paraffin oil on the surface of VL-broth), evaluate bacterial growth and its character. Strain Growth in common broth Growth in VL-broth Conclusion