Topic J05: Determination of susceptibility of bacteria to antimicrobial drugs, assessments of resistance factors For study: textbooks, www, keywords e. g. Diffusion disc test ; E-test ; dilution micromethod ; minimal inhibition concentration Bacterial susceptibility to antimicrobial drugs may be tested qualitativelly (diffusion disc test) or quantitativelly (assessment of minimal inhibitory/bactericidal concentration). Important is detection of eventual bacterial rezistance af a given strain. Task 1: Preparing of a diffusion disc test Take two colonies of Escherichia coli from Endo agar using a loop and suspend in saline with glucose. With a cotton swab, suspend densely to the Mueller-Hinton agar. Then rotate the agar plate to 90 and inoculate once more. After drying, place clock-wise the following set of discs Name of antibiotic Name of antibiotic 1. 4. 2. 5. 3. 6. Look also at a dispensor that serves to quick placing of disc sets in case of often used discs. The plate should be incubated overnight at 37 C. The antibiotics diffuse to surroundings of the discs. When the bacterium is susceptible, a zone of growth inhibition is formed. Task 2: Qualitative test of antibiotic susceptibility (diffusion disc test), comparison of susceptibility of Gram-positive and Gram-negative bacteria In diffusion disc test we compare eventually found susceptibility zones with reference zones. When the zone is larger than reference limit, we say that the strain is susceptible to given antibiotic. Sometimes the zones are so large that it is not possible to measure them. Obviously in such cases, too, we describe such result as susceptible. When the zone is smaller than the reference limit, or we cannot find any zone at all, we describe such result as resistant. Sometimes we have also borderline cases (the zone is just the same as limit zone). Such results are usually described as D (dubious, borderline, query, unsure, plus-minus ). The zone is larger than reference limit. We can evaluate such result as S (stain is susceptible to given antibiotic) The susceptibility zone is present, but is is smaller than the limit. so the result of the strain is reported as R (the strain is resistant to given antibiotic) The zone is so large that it cannot be measured, but it does not matter, it is obvious that the strain is susceptible ( S ) There is no visible zone here, so it is obvious that the strain is resistant, R We use various sets according to tested bacteria. Sets for gram-negative bacteria contain mostly antibiotics effective against gram-negative bacteria, sets for staphylococci or streptococci contain rather antibiotics with supposed effectivity against such bacterial genera. Read, to what antibiotics two given bacterial strains are susceptible. At inhibition zone diameter larger than reference limit sign S (susceptible), in smaller zone or no zone at all write R (resistant). Name General Medicine Date. 3. 2014 Page 1/5
Set for Gram-positive bacteria (write names, NOT abbreviations) Staphylococcus aureus (a G+ coccus) Escherichia coli (a G- rod) Set for Gram-negative bacteria (write names, NOT abbreviations) Staphylococcus aureus (a G+ coccus) Escherichia coli (a G- rod) Task 3: Assessment of susceptibility of urinary tract infections For the urinary tract infections (UTI), we use specific antibiotics that concentrate in urinary bladder. Some of them concentrate sufficiently only in urinary bladder; such antibiotics can be only used for cystitis, as in case of pyelonephritis a sufficient concentration in tissues is also required. In practice, we use test for enterobacteria from urine, test for staphylococci from urine etc., but also a universal urine set that is exploited e. g. for children s policlinic. With help of the slideshow and your teacher, write at least five antibiotics suitable for treatment of UTI infections. Underline antibiotics suitable only for cystitis treatment. Task 4: Assessment of MIC using the dilution micromethod The dilution micromethod (microdilution test) is a quantitative way of testing antibiotic susceptibitlity of bacteria. Each well contains a substrate and some concentration of an antibiotic the antibiotic is diluted by a geometric row. The growth is seen as turbidity (eventually a dot made of sedimented bacterial cells), inhibition of growth is indicated by a clear well. The lowest concentration of an antibiotic that is inhibitory for bacterial growth is the minimal inhibitory concentration (MIC). Encircle all clear wells (wells that are not turbid and with no visible dot inside). Double encircle the lowest clear well (= minimal inhibition concentration) For each antibiotic, compare the MIC with the breakpoint (marked as >X<) and judge, whether the strain is susceptible (S) or resistant (R) to given antibiotic If there are no clear wells for a certain antibiotic, do not encircle anything and write R. The MIC is higher than the highest value mentioned in the table If there are no turbid wells for a certain antibiotic, encircle all of them and double encircle the lowest one, although the MIC value might be even lower. Obviously, such strain is S. Write full names of all given antibiotics, write to which group they belong (penicillins, cephalosporins) and what are their side effect for treatment (according to guides on your tables) Name General Medicine Date. 3. 2014 Page 2/5
Concentrations (mg/l) Antibiotic AMP AMS CZL CRX CXT GEN COT COL OXO OFL TET AZT S/R? 32 64 64 64 64 32 128 32 64 16 32 GC 16 32 32 32 32 16 >64< 16 32 8 16 64 >8< >16< 16 16 16 >8< 32 >8< >16< >4< 8 32 4 8 >8< >8< >8< 4 16 4 8 2 >4< >16< 2 4 4 4 4 2 8 2 4 1 2 8 1 2 2 2 2 1 4 1 2 0.5 1 4 0.5 1 1 1 1 0.5 2 0.5 1 0.25 0.5 2 0.25 0.5 0.5 0.5 0.5 0.25 1 0.25 0.5 0.125 0.25 1 GC = growth control (should be always turbid, no antibiotic present) Explanation of antibiotic abbreviations Abbr. Full name Group Unwanted side effects (if any) AMP AMS CZL CRX CXT GEN COT COL OXO OFL TET AZT Task 5: Evaluation of the MIC using the E-test Although principally similar to the diffusion disc test, E-test is a quantitative test. The concentration values are written directly on the strip. The site where the margin of the zone crosses the strip shows us the MIC value. Read an E-test for a given strain. Draw and evaluate the result. Name General Medicine Date. 3. 2014 Page 3/5
Tested strain Tested antibiotic / antimycotic MIC value Breakpoint: Conclusion (strain is susceptible/resistant to given antibiotic) Task 6: Test for common beta-lactamase production using nitrocephin strip method Draw the result of beta-lactamase test from the slideshow. Positive beta-lactamase Negative beta-lactamase Task 7: Detection of the extended-spectrum beta-lactamases (ESBL) using CLSI test A very important problem of today s medicine are so called ESBL producers (ESBL = extended-spectrum beta lactamases). The test is based on following of a difference in effect of antibiotic alone (CTX = cefotaxime or CAZ = ceftazidime) and antibiotic with inhibitor of betalactamase (CCTX or CTX CLA and CCAZ or CAZ CLA). An ESBL-producer is detected so, that the antibiotic with inhibitor has a zone at least 5 mm larger than the zone of the same antibiotic without inhibitor. To avoid mistakes, we do the test twice with two different antibiotics (CTX = cefotaxime, CAZ = ceftazidime). Draw and evaluate result of tests showing production of ESBL in two given bacterial strains according to your teacher and slideshow. Mind the letters written on discs! For negativity, fill upper and lower arm of a + by black ink or black pencil, making so a of it. Task 8: Detection of the extended-spectrum beta-lactamases (ESBL) and ampc betalactemases using ABCD test In recent days, ESBL beta-lactamases of classic type are not the only important betalactamases. Worldwide problem is related with so called metallobetalactamases and karbapenemases, that are resistant to even more beta-lactam antibiotic. There is also an important problem of so called ampc betalactamases. They are simillar to ESBL betalactamases, but often produced by other strains of enterobacteria. More and more laboratories try to detect classic ESBL beta-lactamases and ampc beta-lactamases simultaneously, for example with use of ABCD test. Using the picture, try to asess, whether you strain is ESBL producer, ampc producer, non-producer or producer of both types of beta-lactamases. Attention: Your test is original from the examination laboratory and your result may be different from your neighbour s result! Name General Medicine Date. 3. 2014 Page 4/5
A = CPD (cefpodoxime), B = CPD + ESBL inhibitor, C = CPD + ampc inhibitor, D = CPD + both inhibitors ESBL positive: B and D larger than A and C (difference 5 mm and more) ampc positive: C and D larger than A and B (difference 5 mm and more) ESBL and ampc pozitive: D larger than the others, the others have no differences Conclusion: My strain is ESBL positive negative and ampc positive negative (delete as appropriate) Task 9: Screening media for MRSA and VRE strains Besides betalactamases, another important problem of antibiotic resistance are methicilin resistant Staphylococcus aureus strains (MRSA) and vancomycin resistant enterococcus (VRE) strains. One of ways of detecting them is the use of screening media. Usually we use chromogenic media* containing antibiotics. In case of MRSA, it is not only important to differenciate between methicilin-resistant and methicilin-susceptible strains, but also between Staphylococcus aureus (an important pathogen, more in P01 topic) and so called coagulase-negative staphylococci (less pathogenous, also described in P01 topic). On the medium you can observe several combinations of the species and antibiotic susceptibility. Attention: The media are quite expensive, therefore the tast in only available in one exemplar on the side table. *Chromogenic media contain a substrate specific for a given species of bacterium or micromycete. The substrate is bound to a dye that becomes colourless. In situation that the substrate is utilized by the searched strain, the colourless substance turns back to its original colour, and so we can see coloured colonies. 9a) MRSA Strain Staphylococcus aureus, methicilin resistant (= MRSA) Staphylococcus aureus, methicilin susceptible A coagulase-negative staph, methicilin resistant A coagulase-negative staph, methicilin susceptible Does/does not grow, colour of colonies 9b) VRE Strain Vancomycin-resistant enterococcus Does/does not grow, colour of colonies Vancomycin-susceptible enterococcus Task 10: Bacteriophage therapy As bacteria become resistant to antibiotics, experiments with bateriophage therapy start to be actual again. It is either possible to use a complete phage, or a phage lysate (containing effective bacteriophage proteins hamful for the bacterium). Eventualy it may contain both lysate and living phages (e. g. STAPHAL in Czech conditions). The results can be checked in vivo, but experimentally also in vitro. STAPHAL is registered e. g. in Slovakia. Task: Observe a demonstration of bacteriophage preperation effect on a strain of Staphylococcus sp. Compare a strain susceptible to the given phage preparation (A) and a resistant strain (B). Draw schematically and describe according to teacher s comment. A B Name General Medicine Date. 3. 2014 Page 5/5