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. agglutination result 3d Susc. test Penicill. Vanc. Gluc. fermentation Task 4 (Hajna) Oxidase test Task 5a NEFERMtest 24 Task 5b (code) 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 (demonstrated by only one agar plate on the side table of the practical hall), describe their growth on chocolate agar instead. Then describe the growth of bacteria on Endo agar. Use “–“ no growth, “L–“ for growing, but lactose non-fermenters, “L+” for lactose fermenters. Pay attention: some strains may mimic lactose positivity although they are lactose negative – they produce pigments, so the colonies are dark, but the surroundings are pale. In doubts compare with Hajna medium (Task 4): completely yellow colour = lactose and glucose fermenter, complete red colour = lactose and glucose non-fermenter, half-yellow-half-red = glucose fermenter, lactose non-fermenter. As to MH agar: check only one strain, and only for eventual pigment presence. Use the plate from for Task 1 or from Task 6b, there is no special MH plate for Task 2 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 (performed for two strains) and connect the terms below with the features on your picture. Write your results to the basic table on the first page. 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) – write the result for all antigenic types to the table. 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. e) Confirmation of Pasteurella multocida determination using MALDI-TOF Look at the MALDI-TOF results of our Pasteurella strain. Decide: Determination of Pasteurella multocida by means of previous test is – is not (delete as appropriate) confirmed by MALDI-TOF test. Task 4: Hajna medium (Triple Sugar Iron Agar in A. A. Hajna’s modification) 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 “–”. Other results (lactose, sulphan) are not important for today’s topic, but the lactose result (complete yellow vs. red-and-yellow) might be used for comparison between lactose-fermenters and lactose-non-fermenters in Task 2. 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) *valid also for doxycyklin Interpre-tation Penicillin (P) S ≥ 12 / R < 12 Co-amoxicillin (AMC) S ≥ 15 / R < 15 *valid also for doxycyklin Cefuroxime (CXM) S ≥ 26 / R < 25 Nalidixic acid (NA) S ≥ 23 / R < 23 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 Co-amoxicillin (AMC) S ≥ 15 / R < 15 Cefotaxime (CTX) S ≥ 26 / R < 26 Ciprofloxacin (CIP) S ≥ 27 / R < 27 Tetracyclin (TE)* S ≥ 24 / R < 24 Co-trimoxazole (SXT) S ≥ 23 / R < 23 Penicillin (P) S ≥ 17 / R < 17 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 ≥ 26 / R < 26 gentamicin (CN) S ≥ 15 / R < 15 ceftazidime (CAZ) S ≥ 17 / R < 17 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 In the diagram, prepared by EUCAST# you can see intrinsic (primary) resistances of the most common G– non-fermenters. On the side table you can see susceptibility tests for Burkholderia and Stenotrophomonas. You do not need to measure zones – they have been already measured. # EUCAST = The European Committee on Antimicrobial Susceptibility Testing On the lid of your Petri dishes you can find: 1^st column: intrinsic resistance for the given strain (re-written from the EUCAST table above): R = intrinsic resistance, – = no intrinsic resistance. Only intrinsic resistances for antibiotics from our set (PS1) are written here. Note: The table above does not contain ofloxacin (OFX), but it is possible to consider strains primarily resistant to ciprofloxacin as resistant to ofloxacin, too. The table also does not contain namely gentamicin (CN), but its results can be derived from “Aminoglycosides”. 2^nd column: results from measuring the zones and comparing them with reference zones: R = resistant, S = susceptible Write on the next page, what is intrinsic resistance of B. cepacia and S. maltophilia according to EUCAST (copy from the first column). Then check, if all intrinsic resistances are expressed in our test according to following table: Intrinsic resistance (R)? Measured as Conclusion – susceptible (S) the result is OK, the strain may be reported as “susceptible” R resistant (R) the result is OK, the strain is to be reported as “resistant” – resistant (R) the result is OK, the strain is to be reported as “resistant” (it is a secondary resistance) R susceptible (S) the result is not in accordance, the strain should be reported as “resistant”, it should be understood as “false susceptibility” Finally, write if the strain is susceptible for any antibiotics (only such that may be reported as susceptible, not those measured susceptible, but they should be considered resistant as they have an intrinsic resistance!). Write: Strain ___ (B. cepacia) has, according to EUCAST, intrinsic resistance to antibiotics: ____________________ _________________________________________________________________________________________. Susceptibility assessed by diffusion disc test is in accordance with this intrinsic resistance (= no “false susceptibility”) is not in accordance with this intrinsic resistance for antibiotic(s): _________________________________* Strain may be reported as “susceptible“ to: ______________________________________________________ Strain ___ (S. maltophilia) has, according to EUCAST, intrinsic resistance to antibiotics: __________________ _________________________________________________________________________________________. Susceptibility assessed by diffusion disc test is in accordance with this intrinsic resistance (= no “false susceptibility”) is not in accordance with this intrinsic resistance for antibiotic(s): _________________________________* Strain may be reported as “susceptible“ to: ______________________________________________________ In case of more discrepancies it is usually recommended to check the susceptibility by quantitative tests, eventually to check, whether the genus and species determination of the strain was performed correctly. 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