Ondřej Zahradníček To practical of aVLLM0421c and aZLLM0421c Contact to me: zahradnicek@fnusa.cz

1. Searching for microbesPart VI.Testing of microbial susceptibility to antibacterial agents and assessment of microbial resistance factors Ondřej Zahradníček To practical of aVLLM0421c and aZLLM0421c Contact to me: zahradnicek@fnusa.cz

2. Introduction & tale Content of this slideshow Antimicrobial agents – overview MIC, MBC, bactericidal and bacteriostatic antibiotics Mechanisms of action of atb-s and mechanisms of resistance Epidemiologically important types of resistance Methods of susceptibility testing I: Diffusion disc test Methods of susceptibility testing II: E-test Methods of susceptibility testing III: Microdilution test Methods of assessment of resistance factors + phage therapy Bonus: Survey of antimicrobial drugs

3. Tale • There was an antibiotic, let's call him ampicillin. And he decided to go out and to help people to fight with microbes. • It was a brave antibiotic, decided to destroy any microbe, that might walk around. Nevertheless…

4. Ampicillin meets microbes… • The first microbe was called Proteus mirabilis and really, it was destroyed by ampicillin. And ampicillin was pleasured by that, and continued it‘s trip. • But he met another microbe. Klebsiella pneumoniae, and he was disgusted. Ampicillin said: Oh, no, Klebsiella, I cannot destroy you, you are primarily resistant to me, I can do nothing… I will continue my trip, and I will send you a more effective brother to destroy you…

5. And the third meeting • Ampicillin met a microbe called Escherichia coli. It is OK, it said to itself, I could destroy it. Nevertheless… Escherichia escaped, and giggled: Ha, ha, you thought you can destroy me! Well, some time ago, I was susceptible to you, but then I have developedsecondary resistance, and so you can never catch me!

6. Ampicillin was very sad… • But a microbiologist came and told him: don‘t worry, be happy, ampicillin, next time it‘ll be better. Each microbe, that could be either susceptible or resistant to you, should be tested, to see the situation. And if you will be found to be no help for treatment? After susceptibility testing we will see, who of your brothers could be sent to fight the microbe! • So ampicillin dried his tears, and run to help people again.

7. What to learn from our tale • Some microbes are primarily resistant to some antibiotics. It has no sense to test their antibiotic susceptibility – it is zero. • Other microbes may (not necessarily) get secondarily resistant. Then • either we test microbial antibiotic in vitro susceptibility to the given antimicrobial agent • or we search for a certain factor, produced by bacteria, that is responsible for the microbial resistance

8. Antimicrobial agents – overview

9. Methods of „fight“ with microbes • Immunization – exploits natural mechanisms of a macroorganism • Decontamination methods – crude physical and chemical influences, action outside the organism (see last practical) • Antimicrobial agents – fine, targeted action inside the organism with aim of maximal effect of the microbe and minimal influence on the host macroorganism • And also some more methods, for example use of bacteriophages (= „biological fight“)

10. Types of antimicrobial agents First antibiotic was penicillin, derived by A. Fleming • Agents acting to the whole body: • Antiparasital agents against parasites • Antimycotics against yeasts and molds • Antiviroticsagainst viruses • Antituberculotics against mycobacteria Antibioticsagainst all other microbes (today usually including also synthetic drugs, sooner put aside as antibacterial chemoterapeutics) • Locally acting agents:antiseptics

11. Decontamination, or an antimicrobial agent? • The borderline between decontamination methods and antimicrobial agents is not sharp. • Usually action on intact skin is still considered to be a decontamination. • Application into the wound means already use of an antimicrobial agent (antiseptic) • It has its legal consequences: decontamination agents are not considered „remedies“

12. MIC, MBC, bacteriostatic and bactericidalantibiotics

13. lower survival limit (bactericidal) lower growth limit (inhibitory) upper growth limit (inhibitory) upper survival limit (bactericidal) Action of influences on microbes I • At action of an influence like pH the axe has both upper and lower extremes upper growth limit (inhibitory) upper survival limit (bactericidal) • At action of antimicrobial agents (but also e. g. disinfectants)only right part of the axe has a logical sense

14. Action of influences on microbes II • At decontamination we insist that we have to kill the microbes (microbicidal effect) • At use of antimicrobial agents we can count with cooperation of patient‘s immunity, therefore even microbistatic (inhibitory) effect is usually sufficient • This is not valid for acute states or immunocompromised patients, where we try to ensure microbicidal action always As you would see soon, it is question of choice of the antibiotic rather than choice of its concentration

15. MIC and MBC MIC – minimal inhibitory concentration is a term, that is used in antibiotics for growth (multiplication) limit of a microbe MBC – minimal bactericidal concentration is a term used in antibiotics for survival limit of a microbe. (For simplicity, we talk about bacteria only. In viruses, we would use term „minimal virucidal concentration“ etc.) Notes: • In practice, MIC is quite routinely measured, especially in seriously ill patients. On the contrary, MBC is normally not measured in practice. Try to find the reason (with help of next slides) • Besides MIC and MBC, we have also MBEC – minimal biofilm eradication concentration. See Topic J07

16. Primarily bactericidal and primarily bacteriostatic antibiotics Primarily bactericidal are atb-s, where the primary effect is killing. MIC and MBC are nearly equal in these antibiotics Primarily bacteriostatic are atb-s, where values over MIC, but not over MBC are exploited (they are inhibitory in substance) In practice, we avoid using bacteriostatic antibiotics in serious acute states, imunosupressed patients etc. (This rule is not absolute – e. g. atb-s with very good effect to bone tissue are used for acute osteomyelitis treatment despite being bacteriostatic only)

17. Primarily bactericidal and primarily bacteriostatic antibiotics Primarily bactericidal antibiotic THERAPEUTICAL CONCENTRATIONS atb concentration toxicity for the macroorganism Primarily bacteriostatic antibiotic

18. Mechanisms of action and mechanisms of resistance

19. Mechanisms of antibiotic action • To the cell wall (bactericidal) • Betalactamic antibiotics • Glycopeptidic antibiotics (partially) • To cytoplasmic membrane – polypeptids (bactericidal) • To nucleic acids – quinolones (bactericidal) • To proteosynthesis:aminoglykosides (bactericidal); makrolids, tetracyclins, linkosamids, amphenicols (bacteriostatic) • To metabolism – sulfonamids, bacteriostatic • Primarily bacteriostatic antibiotics are not recommended for treatment in acute states and immunocompromised persons! BAKTERICIDAL BAKTERIO- STATIC

20. Why we do say more details Details concerning individual groups and their use will be discussed on the lectures or you would find them in a textbook Survey of antibiotics can be also found in the bonus material Some more information would be told in pharmacology education

21. Microbial resistance to antimicrobial agents • Primary resistance: all strains of a given species are resistant. Example: betalactamic atb-s do not act on mycoplasms, that do not have any cell wall at all. • Secondary resistance: non-susceptible mutants raise, and under selection pressure of an antibiotic they start to be in majority. (Escherichia may be susceptible to ampicillin, although in recent period, resistant strains become very common)

22. Mechanisms of resistance • Blocade of entrance of an atb into the cell • Active efflux of an atb from a cell • A false receptor is offered to an atb • Microbes split antibiotics enzymatically (e. g. betalactamase split betalactamic antibiotic) Knowledge of the resistance mechanism enable us to try to defence ourselves against such a resistances.

23. Betalactamase inhibitors – 1 Bacterium „BELA“ The Dog (a betalactamase) • When we act by a single antibiotic, it is inactivated by a bacterial betalactamase.

24. Betalactamase inhibitors – 2 • When a beta-lactamase has a more attractive substrate to be chosen, it is used instead, and the antibiotic can act. ACID

25. Examples of antibiotics strengthened by betalactamase inhibitors From FN USA intranet

26. Beta-lactamase inhibitors are not always sufficiently effective • Unfortunately, beta-lactamase inhibitors are effective only in some „not so strong“ types of beta-lactamases • There exist also extended spectre beta-lactamases (see later), where inhibitors are not sufficient (the treatment is not effective although we use them) • Nevertheless, some effect can be seen also in these beta-lactamases. This is used in diagnostics, where we observe difference in effect of certain antibiotic with : without inhibitor

27. Epidemio- logically important types of resistance

28. Epidemiologically important resistance patterns – 1 • MRSA – methicillin resistant staphylococci. Oxacilin or other beta lactams are not able to enter their cells. Many MRSA are also resistant to more antibiotics (macrolids, lincosamids). Glycopeptids (vancomycin, teicoplanin) remain effective. • VISA, VRSA – staphylococci partially or fully resistant to glycopeptids, too • VRE – vancomycin resistant enterococci. They spread easily – many people have enterococci in their intestine

29. MRSA in Europe 2016 http://ecdc.europa.eu/en/healthtopics/antimicrobial_resistance/database/Pages/map_reports.aspx

30. VRE 2016

31. Epidemiologically important resistances – 2 • ESBL (Extended Spectrum Beta Lactamase)producers. G- bacteria (Klebsiella, but also E. coli and other) may produce extended spectre beta-lactamase, where even inhibitor effect is not sufficient. Only carbapenems and sometimes certain non-betalactam atb-s are effective. • Similar are ampC betalactamases. Besides carbapenems also 4th generation cephalosporins remain effective. • MLSB resistance is a resistance to macrolids and lincosamids and streptogramin B in streptococci and staphylococci.

32. Klebsiella pneumoniae 2016 The data are for total resistance to 3rd generation cephalosporins, but majority of cases are probably ESBL beta-lactamases.

33. Polyresistant Klebsiella – combined resistance to cephalosporins and other antibiotics

34. Polyresistant Pseudomonas

35. „Antibiotic politics“, atb centres • Use of broad spectre antibiotics performs a selection pressure – resistant strains of bacteria survive. • In countries, where atb-s are used freely, there is usually high of atb resistance • In Czechia there exist „free atb-s“, that can be prescribed freely, and „special atb-s“; their use should be approved by antibiotic centre. • Atb centre is usually part of microbiological labs in big hospitals. They do advisor work, too.

36. Methods of susceptibility I: Diffusion disc test

37. In vitro susceptibility observation methods • Assessment of susceptibility in vitro = in the laboratory • No guarantee of 100% treatment effect • Nevertheless, useful in majority of findings of cultivable bacteria • In common cases, qualitative tests (susceptible – resistant) • In indicated cases, quantitative tests (assessment of MIC). Usually risky strains at risky patients.

38. When „in vitro“ does not correspond with „in vivo“ • At urinary infections we should use a breakpoint derived from urinary concentrations, not serum concentrations. (In majority of UTI infections, MIC is not measured) • In abscesses, processes in bones and mostly in meningitis: breakpoints are derived from serum concentrations, but in various parts of body the concentration they may be much lower • It is necessary to count that microbes may exist in a biofilm form– so, MBIC and MBEC values (biofilm inhibitory/eradication concentrations) should be assessed rather than MIC values

39. Diffusion disc test – 1 • To MH agar (or another one) a bacterium is inoculated from a saline suspensionusing a cotton swab • After that, antibiotic discs are added – round bits of papers with antibiotic • Atb diffuses from disc through the agar • In a standard Petri dish we use mostly six discs, sometimes the seventh to the middle

40. How to prepare a diffusion disc test • Prepare a bacterial suspension in physiological saline with glucose • Suspension should be distributed regularly onto the MH agar surface • After drying of the suspension, place carefully and uniformly the atb discs onto the agar surface

41. Diffusion disc test – 2 • Concentration of atb(and its inhibitory properties)decrease with distance from the disc • When a microbe growth to a disc, or its inhibition zone is very small, it is resistant (not susceptible, not sensitive) • When a zone large enough is present around the disc (more than a limit for the given antibiotic), it is susceptible (sensitive). • The reference zones are usually available at the disc producer, or at EUCAST: http://www.eucast.org

42. Results of the diffusion disc test CITLIVÝ REZISTENTNÍ 1 Bacteria are affraid of antibiotics. Large zone (sometimes so large that it is impossible to measure it) 2 Bacteria are not affraid of antibiotics, they are resistant.A small zone around the disc, or no zone.

43. Sets of antibiotic discs • Usually we use sets of atb discs. Some sets are suitable for G+ or G– bacteria, but it is also possible to construct a set of relatively „universal“ antibiotics (but because of polyresitstant strains, no „universal“ antibiotics are really universal)

44. Diffusion disk test in practice:Zones are measured and compared with given reference limits No zone at all: Microbe is resistant. Photo: Institute for microbiology Zone diametre is larger that the limit: Microbe is susceptible Zone exists, but its diametre is smaller than the reference limit: Microbe is resistant.

45. How to measure a zone • In practical sessions, we use a simple ruler; such ruler cannot be officially calibrated • In practice, we mostly use calibrated callipers, either manual or electronic ones Photo: O. Z.

46. Automated measuring systems • It is also possible to use automated systems, like ADAGIO system. Nevertheless, the reading should be confirmed by a living person, so surprisingly the whole procedure is usually rather more time consummating than time saving. Photo: O. Z.

47. Adagio system interface I

48. Adagio system interface II

49. Diffusion disc test performed with a pigmented microbe (Pseudomonas aeruginosa) Photo: Institute for microbiology

50. Sometimes the zones are too large When zones are so large that it is impossible to measure the diameter, it is possible to measure the radius and to multiply by two. In green, you have theoretical margins of zones – as you can see, majority of them are confluent or they go through the margin of the dish Photo O. Z.