Searching for microbes Part VI. Testing of microbial susceptibility and their resistance factors Ondřej Zahradníček To practical of VLLM0421c Contacts to me: 777 031 969 zahradnicek@fnusa.cz ICQ 242-234-100 Content of this slideshow Tale • There was an antibiotic, let's call him ampicilline. And it decided to go out and to help people to fight with microbes. • It was a brave antibiotic, decided to destroy any microbe, that would be found around. Nevertheless… Ampicilline meets microbes… • The first microbe was called Proteus mirabilis and really it was destroyed by ampicilline. Ampicilline was pleasured by that, and continued it‘s trip. • But it met another microbe. Klebsiella pneumoniae, and it was disgusted. Ampicilline said: Oh, no, Klebsiella, I cannot destroy you, you are primarilly resistant to me, I can do nothing… I will continue my trip, and I will send you a more effective brother to destroy you… And the third meeting • Ampicilline 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 got secondary resistance, and so you can never catch me! Ampicilline was very sad… • But a microbiologist came and told him: don‘t worry, be happy, ampicillin, next time we‘ll into better. Each microbe, that could be either susceptible or resistant to you, should be tested, to see the situation. And if you will be no help? One of your bros will help us! • So ampicilline dried its tears, and run to help people again. What to learn from our tbut • Some microbes are primarily resistant to some antibiotics. It has no sesne to test their antibiotic suspeptibility – it is zero. • Other microbes may (not necessarilly) get secondarily resistant. Then – either we test microbial antibiotic in vitro susceptibility to the given antimicrobial agent – or we search for a certain factor, responsible for the microbial resistance Antimicrobial agents – overview Methods of „fight“ with microbes • Immunisation – 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 Types of antimicrobial agents • Agents acting to the whole body: – Antiparasital agents against parasites – Antimycotics against yeasts and molds – Antivirotics against viruses – Antituberculotics against mykobacteria – Antibiotics against bacteria (natural origin) – Antibacterial chemoterapeutics also against bacteria, but syntetic In recent period, the last two groups are often put into one group called „antibiotics“ • Locally acting agents: antiseptics 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. • Aplication into the wound means allready use of an antimicrobial agent (antiseptic) • It has its legal consequences: decontamination agents have more simple system of certification than antimicrobial agents (and other drugs) MIC, MBC, bacteriostatic and bactericidal antibiotics Action of certain influences on microbes I • At action of an influence like pH the axe has both upper and lower extremes Action of influences on microbes II • At decontamination we insist on killing 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 accute states or imunocompromised patients, where we try to ensure microbicidal action always 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 concetration 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.) Later, you will meet also terms MBIC and MBEC, that are connected with action of on antibiotic in biofilm Primarily bactericidal and primarily bacteriostatic antibiotics Primarily bactericidal are atb-s, where MIC and MBC are nearly equal Primarily bacteriostatic are atb-s, where values over MIC, but not over MBC are exploited (they are inhibitory in substance) In practice, never use bacteriostatic antibiotics in serious accute states, imunosupressed patients etc. Primarily bactericidal and primarily bacteriostatic atb Mechanisms of action and mechanisms of resistance 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 Microbial resistance to antimicrobial agents • Primary resistance: all strains of a given species are resistant. Example: betalactamic atb 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 ampicilline, although in recent period, resistant strains become very common) 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. Betalactamase inhibitors – 1 Betalactamase inhibitors – 2 Examples of antibiotics potentiated by betalactamase inhibitors Epidemio- logically importants resistances Epidemiologically important resistances – 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 (vancomycine, teicoplanin) remain effective. • VISA, VRSA – staphylococci partially or fully resistant to glycopeptids, too • VRE – vancomycin resistant enterococci. They sperad easily – many people have enterococci in their intestine Epidemiologically important resistances – 2 • ESBL (Extended Spectrum Beta Lactamase) producers. G- bacteria (Klebsiella, but also E. coli and other) may produce extended spectre betalactamase, where even inhibitor effect is not sufficient. Only carbapenems and sometimes certain non-betalactam atb-s are effective. • Simillar are ampC betalactamases. Besides carbapenems also 4th generation cephalosporins remain effective. • MLS resistance is a resistance to macrolids and lincosamids (and streptogramins) in streptocoi and enterococci. In S. aureus by good luck still rare. „Antibiotic politicy“, atb centres • Use of broad sprectre antibiotics performs a selection pressure – resistant strains of bacteria survive. • In countries, where atbs are used freely, there is usually high high ratio 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. Methods of susceptibility I: Diffusion disc test In vitro susceptibility observation methods • Assessment of susceptibility in vitro = in the laboratory • No guarantee of 100% treatment effect • Nevertheless, usefull in majority of findings of culurable bacteria l In common cases, qualitative tests (susceptible – resistant) l In indicated cases, quantitative tests (assessment of MIC). Usually risky strains at risky patients. 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 infekctions, MIC is not measured) • In abscessi, processi in bones and mostly in meningitis: breakpoints are derived from serum concentrations, but in various parts of body the concentration 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 Diffusion disc test – 1 • To MH agar (or another one) a bacterium is inoculated from a saline suspension using a cotton swab • After that, antibiotic discs are added – round bits of papers with antibiotic • Atb diffunds from disc through the agar • In a standard Petri dish we use mostly six discs, sometimes the seventh to the middle How to prepare a diffusion disc test • Prepare a bacterial suspension in physiological saline with glucose • Suspension shoud be distributed regularly onto the MH agar surface • After drying of the suspension, place carefully and uniformly the atb discs onto the agar surface 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). Results of the diffusion disc test Sets of 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 relativelly „universal“ antibiotics (but because of polyresitstant strains, no „universal“ antibiotics are really universal) Diffusion disc test in practice: zones are measured and compared with reference zohes Sometimes the zones are too large Methods of susceptibility II: E-test E-tests • Principially simillar to diffusion disc test • Instead of a disc, a strip is used • The strip has raising atb concentration from one end to another ( grace to a special technology – that is why they are expensive) • The zone is not round, but egg-shaped • The test is quantitative • The strip has a scale – sipmle reeding(see image on the next screen) E-tests – reading We can read the MIC value directly on the strip – in place, where the margins cross the strip Somewhere, special large dishes are used Methods of susceptibility III: Microdi-lution test Microdillution test • Atb-s are in a row of wells in a plastic microtitration plate, concetration decreases • The lowest concetration, that inhibits the growth, is the MIC value • For interpretation, we need breakpoint values for each antibiotic. MIC < breakpoint => the strain is susceptible. MIC > breakpoint => resistance • One plate is usually used for one strain,e. g. 12 antibiotics, each in 8 concentrations Practical reading a microdilution test • Find and write MIC values for twelfve tested antibiotics • Turbid well = it grows • Clear well = no growth • No growth = it is inhibited • The lowest inhibitory concentration = minimal inhibitory concentration • MIC £ breakpoint à strain susceptible • MIC > breakpoint à strain resistant Microdilution test – an example Microdilution test – reading • In case of collumns 1, 3, 4 & 5, MIC value is too high and cannot be measured using this test. Example of reading • E: MIC >32, breakpoint =16, conclusion: resistant • F: MIC = 32, breakpoint = 16, conclusion: resistant • G: MIC = 8, breakpoint = 32 conclusion: susceptible • H: MIC £ 1, breakpoint = 8, conclusion: susceptible Methods of assessment of resistance factors Assessment of resistance factors • Sometimes, instead of susceptibility testing, we should rather assess the presence of individual resistance factors by special methods, e. g. betalactamases • The reason for this may be following: – susceptibility testing does not give sure results (bad diffusion in diffusion test, antibiotic does not work directly, but as a metabolite…) – we want to know, whether the resistance belongs to a specific type (ESBL, ampC) Nitrocephine test for presence of common betalactamase Two ways of nitrocephin test use Testing for production of broad specter betalactamase type ESBL Detection of broad specter betalactamases (ESBL) in two ways • Both tests are based on comparison of an effect of the same antibiotic (cefotaxime, ceftazidime) without / with clavulanic acid • In synergism test, diffusion of clavulanic acid from co-amoxicilin disc (amoxicilin + clavulanic acid) is used • In the second test we have directly discs of the same antibiotic (1) alone and (2) in combination with clavulanic acid Double synergy test On a picture, the result is positive – the zone is enlarged to the left side (presence of co-amoxicillin disc) An example of a positive result CLSI test Testing for constitutive ampC betalactamase production The end Sir Alexander Fleming • Sir Alexander Fleming was born at Lochfield near Darvel in Ayrshire, Scotland on August 6th, 1881.