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This document provides information about antimicrobial susceptibility testing (AST), including definitions of key terms, the importance and reasons for performing AST, methods for determining minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), classification of different classes of antibiotics, and manual techniques for AST including disk diffusion and ATB strips. Resistance mechanisms of common bacteria are also discussed.

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10. SUSCEPTIBILITY TESTING AND RESISTANCE MECHANISMS
CONTENT 1- What is AST ? 2- Why and when to perform AST ? 3 - Evolution of the resistances 4- Classification of antibiotics 5- The different manual techniques 6- Disk diffusion 7- Antibiotics action mode 8- Resistance mechanisms of bacteria 8 - 1 Enterobacteriaceae 8 - 2 Staphylococci 8 - 3 Streptococci 9- Choice of antibiotics panel composition 10- Interest of Expert System 11- Study of phenotypes
WHAT IS IT ? 1 - Antimicrobial Susceptibility Testing
Recover quickly THE PATIENT
Rapid diagnosis Effective treatment THE CLINICIAN
Reliable answers Least possible risk THE MICROBIOLOGIST
Microbiologist's answer The ideal result would be :  Therapy with antibiotic A will be successful  the bacteria is Susceptible to the antibiotic  Therapy with antibiotic B will lead to a failure  the bacteria is Resistant to the antibiotic
Microbiologist's answer Failure ? Success
Microbiologist's answer Sucess of therapy depends on :  Resistance of bacteria  Site of infection  Dosage  Patient
Microbiologist's answer Based on the :  Activity of the antibiotic for this strain  Pharmacokinetics of the drug  Normal dosage  Results of clinical studies
How to do it ?  Determine antibiotic activity in vitro  Interpret results for in vivo : potential risk of success and failure
2 - Why and when to perform a susceptibility testing?
WHY PERFORM A SUSCEPTIBILITY TEST ? In vitro susceptibility is one of the prerequisites for in vivo efficacy of antibiotic therapy To orient individual therapeutic decisions
WHY PERFORM A SUSCEPTIBILITY TEST ?  To have an epidemiological follow-up at different levels : wards, department, region or country  To adapt empiric antibiotic therapy  To check the clinical spectrum of antibiotics But also : helps for healthcare decisions, and prevention program
WHY PERFORM A SUSCEPTIBILITY TEST ? THERAPY EPIDEMIOLOGY DUAL INTEREST
WHEN TO PERFORM A SUSCEPTIBILITY TEST ? - Each time a bacteria considerated to be responsible for an infection is isolated from a bacteriological specimen - The technician selects the bacterium among normal flora (if multimicrobial specimen)  MICROBIOLOGIST APPROACH
WHEN TO PERFORM A SUSCEPTIBILITY TEST ? - SEVERE INFECTIONS : septicemia, meningitis, pyelonephrititis, bronchopneumopathy.... - CHRONIC INFECTIONS : urinary infections, otitis - INFECTIONS ON RISK PATIENTS : Old people, pregnant women, kidney and cardiac failure, immunosuppressed patients - THERAPEUTIC FAILURE - INFECTIONS ON RECENTLY HOSPITALIZED PATIENT : multiresistant bacteria  CLINICIAN APPROACH
Sometimes microbiologists cannot determine if AST is required, without obtaining the clinical information that only a clinician can provide. WHEN TO PERFORM A SUSCEPTIBILITY TEST ? Example: a commensal bacterium can cause an infection in an immunocompromised patient or in a specific body site.  importance of patients data information, and clinical symptoms...
WHEN TO PERFORM A SUSCEPTIBILITY TEST ? NEED FOR CLOSE WORKING RELATIONSHIP BETWEEN MICROBIOLOGISTS CLINICIANS
CAN WE PREDICT SUSCEPTIBILITY OR RESISTANCE OF A BACTERIUM TO AN ANTIBIOTIC ?
Direct impact on patient recovery The prescription of first-line antibiotics is poorly adapted Recovery ??? 10 % of therapeutic failure
ANTIBIOTICS Natural resistance Systematic sensitivity ANTIBACTERIAL ACTIVITY SPECTRUM Each antibiotic is characterized by a natural spectrum : of some bacterial species of some bacterial species
NATURAL RESISTANCE Permanent characteristic of all strains of the same bacterial species  stable  predictable  helps for confirmation of identification
EXAMPLES OF NATURAL RESISTANCE Antibiotics Amino penicillin penicillin G Cephalosporin 1st G Cephalosporin 2nd G Cephalosporin 3rd G Nalidixic acid E coli Salmonella Klebsiella Enterob Serratia Natural Resistance Amino penicillin + Betalactamase inhibitor Pseudo aeruginosa Entero cocci staph
NATURAL RESISTANCE  Wild Strain : = bacteria with just its natural resistance = No acquired resistance yet
 Characteristic specific to some strains  The genotype has been modified by gene mutation or gene acquisition. ACQUIRED RESISTANCE
ACQUIRED RESISTANCE Evolutive and unpredictable characteristic of the isolated bacterial strain  justifys the use of susceptibility testing Example : E. coli  wild strain : Amoxicillin (S)  Now : 30 - 60% Amoxicillin (R)
ACQUIRED RESISTANCE Given the evolution of acquired resistance:  the natural spectrum of activity is no longer sufficient to guide the choice of treatment. Example : Acute otitis caused by Pneumococci with decreasing susceptibility to Penicillin G : - 1985 : < 1% - 1998 : 10 - 50% (depends on the country)
CLINICAL SPECTRUM OF ACTIVITY Bacteriological data : natural spectrum + acquired resistance Pharmacokinetic data Clinical data  For each antibiotic, bacteria are divided in 3 classes : S, I, R Frequent updates : take into account the evolution of the resistances = + +
3 - Evolution of resistance
 Resistance must be monitored  Today, the over-use of antibiotics increases the frequency of resistant strains.  The antibiotic does not create resistance but selects resistant bacteria by eliminating the commensal and susceptible bacteria. EVOLUTION OF RESISTANCE
EVOLUTION OF RESISTANCE LARGE ANTIBIOTIC USE POORLY ADAPTED TREATMENT THERAPY FAILURE INCREASE IN RESISTANCE PRESSURE OF SELECTION
Situation of Methicillin Resistant S. aureus in Europe 33.6% 0% 1.5% 0.3% 1.8% 5.5% 21.6% 25.1% 30.3% 34.4% Marty, V. Jarlier- Surveillance des bactéries multirésistantes : Justification, rôle du laboratoire, indicateurs, données françaises récentes - Path Biol April 1998 ; 217-26 A few figures on resistance...
Evolution of bacterial resistances Community - acquired in Europe 1975 90 30 20 10 0 1985 1998 % of resistant strains Strepto A / Penicillin G H . influenzae / Ampicillin S. Pneumoniae/ Penicillin G S .pneumoniae / Erythromycin E . coli / Ampicillin S. aureus / Penicillin G 40 80 70 Questions and answers on ST : J . louis Tissier
Evolution of bacterial resistances Hospital - acquired in Europe 1975 40 30 20 10 0 1985 1998 % of resistant strains Enterococcus / Vancomycin K. pneumoniae / 3rd generation cephalo. P. aeruginosa / Fluoroquinolones E. cloacae / 3rd generation cephalo. S. aureus / Oxacillin E. coli / Ampicillin Questions and answers on ST : J louis Tissier
4 - Classification of antibiotics
DEFINITION OF AN ANTIBIOTIC  Natural substance produced by molds and bacteria  Inhibits or destroys other bacteria. All substances with an antibacterial activity (produced by synthesis or semi-synthesis)
 B- Lactams : - Penicillins / Inhibitors - Cephalosporins - Carbapenems - Monobactams  Aminoglycosides  Cyclins  Quinolones  MLSK  Polypeptides  Imidazoles  Rifamycins  Phenicols  Glycopeptides  Furanes  Sulfonamides  Miscellaneous SIMPLIFIED CLASSIFICATION OF ANTIBIOTICS 13 families
b LACTAMS Marker Gram (+) Gram (-) - PEN G Penicillin G Nat. R. - PEN M Oxacillin Nat. R. - PEN A Amox - Ampi - PEN C Ticarcillin - PEN-U Piperacillin Penicillins Systematic S of S. pyogenes
b LACTAMS Marker Gram (+) Gram (-) - 1st generation Cefalotin Nat R. of E. faecalis Cefaclor - 2nd generation Cefuroxime - Cephamycins Cefoxitin Cefotetan - 3rd generation Cefotaxime Ceftazidime - 4th generation Cefepime Cefpirome Cephalosporins
These enzymatic inhibitors have no true antibacterial activity, but a strong affinity for b-lactamases : Clavulanic acid Sulbactam Tazobactam Aim : blocks beta-lactamases Combination b-lactams - b-lactamase inhibitor : - Group 1 Amoxicillin + Clavulanic Ac. Ampicillin + sulbactam - Group 2 Ticarcillin + Clavulanic Ac. Piperacillin + tazobactam - Group 3 Sulbactam + 3rd generation cephalosporins Never alone ! INHIBITORS OF BETA-LACTAMASES
b LACTAMS Carbapenem  Imipenem  Meropenem Monobactam Marker Gram (+) Gram (-) Aztreonam Nat. R.
AMINOGLYCOSIDES Marker Gentamicin, Tobramycin, Nat. R. of Amikacin, Streptomycin, Streptococci Kanamycin, Netilmicin, and Nat. R. of Neomycin, Spectinomycin, Anaerobes - G(-) Bacilli : test GEN, TOB, AKN, NET - Enterococci and deficient Streptococci : test STR HC, GEN HC, KANA HC (Aminoglycosides high concentration) - Staphylococci : test GEN, TOB, KAN  NCCLS recommendations:
 Tetracycline  Doxycycline  Minocycline Extended spectrum  Tetracycline is the MARKER for all cyclines but for certains organisms doxycycline and minocycline are more susceptible than tetracycline CYCLINES
QUINOLONES Marker Gram (+) Gram (-) Quinolones Nalidixic acid Nat. R. Pipemidic acid Norfloxacin Pefloxacin Fluoroquinolones Ofloxacin Ciprofloxacin
M.L.S : MACROLIDES / LINCOSAMIDES / STREPTOGRAMINS Marker Gram (+) Gram (-) Macrolides Erythromycin Nat. R. except Haemophilus Lincosamides Natural resistance Clindamycin for Staph / Enterococci Streptogramins Quinupristin/ dalfopristin*  *Synercid
NITRO - IMIDAZOLES  Metronidazole Active on anaerobes, parasites RIFAMYCINS (ANSAMYCINS)  Rifampicin Used in anti-stapylococci eye lotions, gonococci, TB POLYPEPTIDES Marker Gram (+) Gram (-) Colistin Nat. R. P. aeruginosa Bacitracin = Syst. S.
PHENICOLS  Chloramphenicol Extended spectrum : Typhoid fever, Gonococci, bronchopulmonary infections GLYCOPEPTIDES Marker Gram (+) Gram (-) - Vancomycin Nat. R. - Teicoplanin
FURANE  Nitrofurantoin Urinary infections : Gram (+) cocci, Enterobacteriaceae Natural resistance for P.mirabilis SULFONAMIDES (FOLATE PATHWAY INHIBITORS) Marker Gram (+) Gram (-) Cotrimoxazol P. aeruginosa = Nat. R. Sulfamides Digestive, genital and urinary infections
MISCELLANEOUS  Fusidic acid (French drug) Local anti-staphylococcal treatment  Fosfomycin - Cystitis for Gram + cocci and Gram- bacilli : «Monuril» - Systemic infection (multiresistant bacteria)  Natural resistance for Staphylococcus saprophyticus
5 - The different manual techniques  Reference technique  Routine techniques
MIC DETERMINATION Definition : Minimum Inhibitory Concentration of an antibiotic inhibiting the visible growth of a bacterium overnight in standardized conditions for rapid growth bacteria.  Bacteriostatic effect of an antibiotic on a bacterium
Mueller Hinton 1 ml 1 ml 0 0.5 1 2 4 8 16 µg/ml 107 CFU 35°C - 16 to 20 hrs 0 0.5 1 2 4 8 16 µg/ml 0 0.5 1 2 4 8 16 µg/ml MIC DETERMINATION liquid medium Reference technique for NCCLS* *National Committee for Clinical Laboratory Standarts MIC =
MIC DETERMINATION
MBC DETERMINATION Definition : Minimum Bactericidal Concentration  Bactericidal effect of an antibiotic on a bacterium
MBC - PROCEDURE 10 µl 0 µg/ml 4 µg/ml 8 µg/ml 16 µg/ml MBC = 0 0.5 1 2 MIC = 4 8 16 µg/ml 35°C - 16 to 20 hrs
Sub-cultured MBC - PROCEDURE
MIC - MBC MIC MBC Bactericidal MIC MBC Bacteriostatic 1 2 8 16 32 64 128 4 1 2 8 16 32 64 128 4
CATEGORIZATION OF STRAINS  2 concentrations set by the NCCLS :  c = low breakpoint  C = high breakpoint  According to the following criteria : - Bacteriological studies - Pharmacokinetics - Clinical experience Breakpoints :
c C S I R MIC < c c < MIC < C MIC > C CATEGORIZATION OF STRAINS
ROUTINE TECHNIQUES  Disk diffusion  ATB Strips  ViteK 2 Compact  E test
DISK DIFFUSION
DIFFUSION CHARACTERISTICS Diffusion Radial in depth surface base Agar (4mm) Diffusion of antibiotic
DIFFUSION READING Measure MIC Zone Diameter Sensitive Intermediate Resistant
DIFFUSION Methodology 1 hour < 15 mins Streak twice 5-15 mins 10 mins 30°C 37°C 37°C CO2 4°C 0.5 McF
DIFFUSION CONTROL  Control of each cartridge of antibiotics  Stability after opening the cartridge : 1 week (CLSI and CA-SFM recommendations)
DIFFUSION Summary  Not ready to use  Flexibility of antibiotics to be tested  Solid technique  Visual reading  Indirect technique
24 Hrs ATB STRIPS bioMérieux
 Thermoformed transparent plastic strip consisting of 32 cupules  Cupules containing dehydrated antibiotics  Inoculation medium : ATB medium (semi solid Mueller-Hinton)  2 empty cupules for individual antibiotic tests 24 Hrs ATB STRIPS
24 Hrs ATB STRIPS  READING OF A BACTERIAL GROWTH : turbidity  DETERMINATION OF S-I-R DIRECTLY  READING : VISUAL OR AUTOMATIC  For manual customers  Or customers equipped with mini API or ATB Expression system (automatic reading)
24 Hrs ATB STRIPS  Principle : Each antibiotic (A, B, C, D…) is tested at 2 different concentrations (Breakpoints: c, C) c C C B A 0 Growth control S (Susceptible) R (Resistant) I (Intermediate) = No bacterial growth = Bacterial growth
24 Hrs ATB STRIPS  Susceptible strain : MIC < c The antibiotic tested is active on the bacteria  Intermediate strain : c < MIC < C The bacteria is not affected by the treatment at usual doses, but may be affected by a high-dose intravenous or local treatment.  This is the risk range.  Resistant strain : MIC > C The bacteria resists to the strongest concentrations. The antibiotic will not be active in vivo.
24 Hrs ATB STRIPS 8 32 32 32 8 8 MIC < 8 (S) 8 < MIC  32 (I) MIC > 32 (R)  example : CFT 8 - 32 mg/l
VITEK 2 COMPACT BIOMERIEUX
MICROSCAN 24 Hrs CHARACTERISTICS  Broad range of MIC concentrations is tested for each antimicrobic which gives information on emerging and low level resistance. 20 antimicrobics in each card let the lab give a wide response. This is also a good basis for the deduction function of AES:3 - 6 concentrations for each antibiotics A rapid response can be used by clinicians when they are making the change from empiric to directed therapy.
SYSTEM CONFIGURATION CAPACITY= 15/30 /60 cards PC WINDOWS XP DENSICHEK inoculum Standardisatio CODE BARR
E-TEST
ME E 256 6 3 1.5 .75 .38 .19 .094 .047 .023 192 128 64 32 16 8 4 2 1.0 .50 .25 .125 .064 .032 .016 96 48 24 12 E-TEST : CHARACTERISTICS reading scale exponential and continuous antibiotic gradient antibiotic code
E.TEST READING
E-TEST PROCEDURE McF regarding ID 5 mins at 37 °C Streak twice < 15 mins saline water 30°C 37°C 37°C CO2
6 -DISK DIFFUSION
The way the microbiologist thinks :  the reference method  reliable  easy-to-use  cheap  flexible
LET’S LOOK AT EACH POINT ...
Disk diffusion is a commonly used method not the reference one REFERENCE METHOD ?
MIC is the REFERENCE Disk Diffusion is an INDIRECT METHOD REFERENCE METHOD ?
REFERENCE METHOD ? Do not use disk diffusion for Anaerobes Yeasts
"For generation of reproducible results, all technical details of the test procedure must be carefully standardized and controlled" NCCLS Dec. 93 - Vol.13 N°24 P.1 RELIABILITY ?
RELIABILITY ?  Depends on :  Choice of media  Agar thickness  Standardization of the inoculum  Disk stability
Mueller Hinton HTM GC Supplement MH + 5% Sheep Blood Chocolate 8 Chocolate + Supplement 8 Enterobacteriaceae Pseudomonas aeruginosa Staphylococci Enterococci Haemophilus Gonococci Pneumococci Others 8 NO 1- Choice of the medium NO NO
Diffusion of the antibiotic Agar (4 mm) 2- Agar thickness
Media too thin excessive diffusion Media too thick too little diffusion Media sloping uneven diffusion Disk not flat too little diffusion Plate too wet surface diffusion 2- Agar thickness
0.5 Mac Farland 3- Standardization of the inoculum
Inoculum too heavy Inoculum too light Inoculum uneven 3- Standardization of the inoculum
Time (months) 1 18 2 weeks 70% 150% % Antibiotic activity 4 - Disk stability
- Use disks within 5 days - Daily quality control for each disk 4 - Disk stability
 Adequate media  4 mm-thick media  Standardized inoculum  Daily quality control RELIABILITY ?
RELIABILITY ?  Correct reading ? Measure the diameter
RELIABILITY ? Human error and subjectivity make standardization difficult
Interpretation is SUBJECTIVE About 5% of discrepancies RELIABILITY ?
Difficult to perform EASY-TO-USE ?
CHEAP ?  Cost of disk diffusion = Reagent cost (media + disks) Labor cost QC cost + +
When you can report on so many antibiotics by testing few, why do you need more flexibility? FLEXIBILITY ?
Drug Tested Drug reported Penicillin G All Penicillin G Ampicillin All Penicillin A Cephalothin All Cephalosporins 1st Gen. Piperacillin Mezlocillin Cefuroxime Cefamandole, Cefomicin Cefotaxime Ceftriaxone, Ceftizoxime Oxacillin All beta-lactams Norfloxacin Ofloxacin, Lomefloxacin (Urinary Tract Infection) Tetracycline All Tetracyclines Use of markers FLEXIBILITY ?
Common Method Cheap reagent Reliable technique Easy-to-use Flexible but Depends on strictly following the procedure and the lab technologist Quality control difficult Costly method Time consuming Everybody uses a different technique but but but but CONCLUSION
7 - Antibiotics action mode
ANTIBIOTIC ACTION MODES  Aminoglycosides  Macrolides  Tetracyclines  Chloramphenicol R R E Chromosome  Quinolones  Nitro-imidazoles  b Lactams  Fosfomycin  Glycopeptides  Sulfonamides 1 2 3 4 = DNA gyrase E R = Ribosomes
External membrane Peptido glycane Cytoplasmic Membrane Cytoplasm PS PS P P PBP PBP PO PS = Periplasmic space P = Permeases PBP = Penicillin binding proteins PO = porines PO Polysaccharides Phospholipids (Except Neisseria and Haemophilus) Gram + Gram - BACTERIA CELL-WALL
External membrane Hydrophilic Antibiotic Lipophilic Antibiotic* Lipophilic Antibiotic* Hydrophilic Antibiotic Peptido glycane Cytoplasmic Membrane Cytoplasm PS PS P P PBP PBP PO * = fusidic acid, Macrolides, Rifampicin Intracytoplasmic targets Intracytoplasmic targets PO Polysaccharides Phospholipids (Except Neisseria and Haemophilus) Gram + Gram - ANTIBIOTICS ACTION MODES PS = Periplasmic space P = Permeases PBP = Penicillin binding proteins PO = porines
External membrane Antibiotic active on PBP Hydrophilic Antibiotic active on PBP Peptido glycane Cytoplasmic membrane Cytoplasm PS PS P P PBP PBP PO PO Polysaccharides Phospholipides (Except Neisseria and Haemophilus) Gram + Gram - = b lactamases ACTION MODE OF BETA-LACTAMS PS = Periplasmic space P = Permeases PBP = Penicillin binding proteins PO = porines
External membrane Lipophilic antibiotic * Lipophilic antibiotic * Peptido glycane Cytoplasmic membrane Cytoplasm PS PS P P PBP PBP PO PO Polysaccharides Phospholipides (Except Neisseria and Haemophilus) Gram + Gram - 50S 50S ACTION MODE OF MACROLIDES
Aminoglycosidases: AAC APH ANT External membrane Hydrophilic antibiotic Hydrophilic antibiotic Peptido glycane Cytoplasmic membrane Cytoplasm PS PS P P PBP PBP PO PO Polysaccharides Phospholipids (Except Neisseria and Haemophilus) Gram + Gram - 30S 30S ACTION METHOD OF AMINOGLYCOSIDES
External membrane Hydrophilic antibiotic Hydrophilic antibiotic Peptido glycane Cytoplasmic membrane Cytoplasm PS PS P P PBP PBP PO PO Polysaccharides Phospholipids (Except Neisseria and Haemophilus) Gram + Gram - DNA gyrase DNA gyrase ACTION METHOD OF QUINOLONES
8 - Resistance mechanisms of bacteria
RESISTANCE MECHANISMS MODIFICATION OF THE TARGET EFFLUX PHENOMENON ENZYME PRODUCTION IMPERMEABILITY resistance of Enterobacteriaceae to macrolides 1- b lactamases inactivating b lactams 2- Enzymes degrading aminoglycosides resistance of Enterobacteriaceae to tetracycline and quinolones 1- Penicillin binding proteins for b lactams (PBP) 2- Ribosomes for macrolides 3- DNA gyrase for quinolones R R E Plasmid Chromosome
RESISTANCE MECHANISMS P P PBP PBP PO PO (Except Neisseria and Haemophilus) Gram + Gram - ENZYMES ENZYMES ENZYMES ENZYMES Hydrophilic antibiotic Hydrophilic antibiotic EFFLUX EFFLUX Antibiotic active on PBP Hydrophilic antibiotic active on PBP Lipophilic antibiotic Decreasing size of porine Mutation of DNA gyrase Mutation of DNA gyrase Mutation of ribosome Mutation of ribosome
GENETIC SUPPORT OF RESISTANCE 1) The chromosome  stable  vertical transmission (to descendants)  no horizontal transmission (not transferable from one bacteria to another)
GENETIC SUPPORT OF RESISTANCE 2) The Plasmid  Unstable in the absence of the antibiotic which selected it  Transferable from one bacterium to another, sometimes between different species
GENETIC SUPPORT OF RESISTANCE CHROMOSOME PLASMID Natural resistance : - stable - no horizontal transmission Acquired resistance : - unstable - risk of horizontal transmission
CROSS AND ASSOCIATED RESISTANCE  Cross resistance The same resistance mechanism affects several antibiotics within a same family. Eg : a Gentamycin-resistant staphylococci is resistant to all aminoglycosides.  Associated resistance affects several antibiotics in different families Eg : Associated resistance of Enterobacteriaceae to b-lactams and aminoglycosides (ESBL and AAC 6').
8.1 - Main resistance mechanisms of Enterobacteriaceae
b-Lactamases Inducible Chromosomic Constitutive Plasmidic ENZYMATIC RESISTANCE Enzymes produced by the bacteria to inactivate the antibiotic
Constitutive : constant level of prodution independent of the presence of an inductor Synthesis of b-Lactamases Inducible : produced in small quantities but increases in the presence of the inductor Eg : Cefoxitin, Imipenem Presence of the antibiotic in the periplasmic space
b-Lactamases  Penicillinase  Cephalosporinase  ESBL : Extended spectrum B-Lactamase  IRT : TEM resistant to inhibitors
b-Lactamases Pase Case ESBL Chromosome Plasmid     IRT 
NATURAL RESISTANCE PHENOTYPES Group I : E. coli, Proteus mirabilis, Salmonella, Shigella  Wild strain : high level of susceptibility but very low level of cephalosporinase for E. coli, Shigella Group II : Klebsiella spp, Citrobacter koseri, Citrobacter amalonaticus, Escherichia hermanii,  Wild strain : natural penicillinase (Nat. Pase).  K. oxytoca : hyper production of natural Pase
NATURAL RESISTANCE PHENOTYPES Group III : Serratia spp, Citrobacter freundii, Enterobacter spp, Pantoea agglomerans Morganella morganii, Hafnia alvei, Proteus vulgaris, Providencia spp,  Wild strain : cephalosporinase (Nat. Case) low level enzyme production Group IV : Yersinia spp  Wild strain : Penicillinase and cephalosporinase ( Nat. Pase and Case)
RESISTANCE PHENOTYPES : WILD STRAINS AMO AMC TIC CFT S S S S  Group 1 :  No natural resistance Except low level case for E. coli and shigella AMO AMC TIC CFT R S R S  Group 2 :  Natural Pase AMO AMC TIC CFT R R* S R  Group 3 :  Natural Case (* :for most Group 3 bacteria) AMO AMC TIC CFT R R R R  Group 4 :  Case + pase
ENTEROBACTERIACEAE AMO AMC MEC TIC CFT CXT TET COL FUR Escherichia coli S S S S S S S S S I Shigella/Salmonella S S S S S S S S S Proteus mirabilis S S S S S S R R R Klebsiella pneumoniae/ II oxytoca R S S R S S S S S Citrobacter diversus (Lev. amalonatica) R S S R S S S S S Enterobacter cloacae/ aerogenes R R S S R R S S S Citrobacter freundii R R S S R R S S S Proteus vulgaris R S R S R S R R R III Serratia marcescens R R R S R In R R R Morganella morganii R R R S R In R R R Hafnia alvei R R R S R S S S S Providencia rettgeri R R R S R S R R R Providencia stuartii R R R S R S R R R IV Yersinia enterocolitica R R S R R r S S S Natural resistance r = low level resistance, in = Inducible
WILD STRAINS  Group 1 :  Group 2 : Natural Pase  Group 3 : Natural Case  Group 4 : Case + pase Low case for E. coli, Shigella
Wild strains and acquired resistances  Group 1 :  Group 2 :  Group 3 :  Group 4 : IRT ESBL ESBL ESBL Acquired PASE Acquired Pase Natural Pase Natural Case Acquired Pase Natural Case + Pase HL case (E. coli, Shigella) HL case ESBL
GROUP 1: P.mirabilis / Salmonella Observed phenotypes AMO AMC TIC PIC IMI 1GC 3GC Wild S S S S S S S Acquired Pase I-R S-I-R I-R I-R S S-I S ESBL R S-I I-R I-R S S-I-R S-I-R Acquired resistance phenotypes : b lactams
GROUP 1: E.coli / Shigella Observed phenotypes Acquired resistance phenotypes : b lactams AMO AMC TIC PIC TZP IMI 1GC CXT 3GC Wild S S S S S S S S S Low Case S-I S-I S S S S I-R S S HL Case R R S S S S R R I Acquired Pase I-R S-I I-R R S S S-I S S Pase resistant to inhibitors R R R R I-R S S-I S S ESBL R S-I R R S S S-I-R S S-I-R * Isolated R to Mecillinam possible for E. coli but impossible for the others ( inoculum too heavy)
GROUP 2 : Observed phenotypes Acquired resistance phenotypes : b lactams AMO AMC TIC PIC TZP 1GC 3GC Wild (Natural Pase) I-R S R S-I-R S S S Acquired Pase R S-I R I-R S I-S S ESBL R S I-R S-I-R S I-R S-I-R Hyper production of natural Pase (for K. oxytoca) R I-R R I-R S-I I-R S
GROUP 3 : Observed phenotypes Acquired resistance phenotypes : b lactams AMO AMC TIC PIC TZP IMI 1GC 3GC Wild: R (R) S S S S RS (Nat Case) Pase R (R) R R S-I S R S HL Case R R I-R I-R I-R S R I-R ESBL R R R R S-I-R S R S-I-R ( ) = possible S for Proteus vulgaris
b-Lactams and Enterobacteriaceae (SUMMARY) Antibiotics PASE PASE IRT ESBL CASE CASE NAT ACQUIRED NAT high level Penicillins : Amino P I/R R R R R R Carboxi P I/R R R R S R Ureido P S/I I-R R R S R Cephalo : C1G S S/I S/I/R R R R C2G S S S/I/R R S/I/R R C3G S S S S/I/R S R Inhibitors: S S/I R S/I/R R R Imipenem S S S S S S
Techniques for the detection of ESBL
WHAT ARE ESBL ?  Extended Spectrum Beta-Lactamase  mutation of a traditional Penicillinase - plasmid-origin - thus transferable  Described for Enterobacteriaceae 80 % of cases concern Klebsiella pneumoniae ALL BETA-LACTAMS ARE AFFECTED except Cephamycins and Carbapenems
Techniques for the detection of ESBL - CAZ at 1 mg/l on ATB G- 5 - Interpretative reading - ATB BLSE - Disk method (Champagne cork)  DETECTION OF ESBL  CONFIRMATION OF ESBL
Detection of ESBL using ATB G - 5  Objective : Distinguish acquired Penicillinase and ESBL when there is a doubt Depends on the MIC to Ceftazidim TEST CAZ 1 :
Detection of ESBL using ATB G - 5 Test CAZ 1 = Ceftazidim at 1 µg/ml enables the detection of low level resistance of ESBL (Low MIC) Breakpoints N° of strains 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 CAZ 1 1 µg/ml R S I MIC to Ceftazidim
DETECTION OF ESBL USING DIFFUSION: «Champagne cork» or «key hole effect»  Objective : Distinguish ESBL and HL Case when there is a doubt  Technique : Deposit 3 disks (Distance to be adapted ) - Inhibitor of B-Lactamase - 3rd gen. Cephalosporin - Aztreonam C3G AZT Inhibitor
DETECTION OF ESBL USING DIFFUSION: Amoxicillin + Clavulanic acid Ceftazidime Aztreonam ESBL HL CASE «Champagne cork» or «key hole effect» No action of inhibitor Action of inhibitor
DETECTION OF ESBL USING DIFFUSION: «Champagne cork» or «key hole effect»
Detection of ESBL using ATB BLSE  Objective : Distinguish ESBL and HL Case when there is a doubt  Technique : Comparaison of - MIC determination of 3rd gen. Cephalosporin - MIC determination of 3rd gen. Cephalosporin + Inhibitor of B-Lactamase If at least 4 dilutions difference : ESBL +
Detection of ESBL using ATB BLSE ref. 14 100 mg/l Example 1 ATM ATM+S Aztreonam Aztr.+Sulbactam 0.5 0.06 + + 2 ATM ATM+S Aztreonam Aztr.+Sulbactam 1 0.12 + + ESBL(+) : MIC range 3 ATM ATM+S Aztreonam Aztr.+Sulbactam 2 0.25 + + from 8 to 0.5 (4 4 ATM ATM+S Aztreonam Aztr.+Sulbactam 4 0.5 + - dilutions). Image + - 5 ATM ATM+S Aztreonam Aztr.+Sulbactam 8 1 - - 6 CAZ CAZ+S Ceftazidime Cefta+Sulbactam 0,5 0.06 + + 7 CAZ CAZ+S Ceftazidime Cefta+Sulbactam 1 0.12 + + 8 CAZ CAZ+S Ceftazidime Cefta+Sulbactam 2 0.25 + + ESBL(+) : MIC range 9 CAZ CAZ+S Ceftazidime Cefta+Sulbactam 4 0.5 + + from 32 to 1 (10 10 CAZ CAZ+S Ceftazidime Cefta+Sulbactam 8 1 + - dilutions). 11 CAZ CAZ+S Ceftazidime Cefta+Sulbactam 16 2 + - Image + - 12 CAZ CAZ+S Ceftazidime Cefta+Sulbactam 32 4 - - 13 CTT CTT Cefotetan 4 32 ESBL: S 14 IMI IMI Imipenem 4 8 ESBL: S 15 SCTX1 SCTX2 Cefotaxime + Sulbactam 0.06 0.12 Control of the recovery activity
ENTEROBACTERIACEAE GEN TOB NET AKN KAN APH 3' S S S S R AAC 3I R S S S S AAC 3II R R R S R AAC 6' S R R R R AAC 2' R R R S S ANT 2" R R S S S Impossible S S S R S Impossible S S R S S Impossible S R S S S NB : * AAC 6' : Often associated with an ESBL, except for S. marcescens * P. stuartii has an AAC 2' in the wild state Acquired resistance : Aminoglycosidases
8.2 - Main resistance mechanisms of Staphylococci
GRAM POSITIVE COCCI  GRAM POSITIVE COCCI  Natural resistance : Aztreonam Nalidixic acid (QUINOLONES 1) Polypeptides  STAPHYLOCOCCI  Natural susceptibility : Vancomycin  At least one case of Staphylococcus aureus has been detected VAN: I Natural resistance and susceptibility
STAPHYLOCOCCI  b lactams PEN G OXA S S Absence of pase Check with chromogenic test R S Acquired Pase R R Modification of PBP (MRSA) Acquired resistance
PENICILLINASE TEST  Cefinase  For rapid detection of B-Lactamase production  Chromogenic cephalosporin (= Nitrocefin) Ref : 55 622 colony (+) 5 mins to 1 hour Also for H. influenzae, N. gonorrhoae, Enterococci, and anaerobes
Cefinase (+)  change Pen G : S to R Cefinase (-)  do not change Pen G PENICILLINASE TEST  ATB Staph  Cupule Pen G at 0.125 g/l If Penicilline G is S, confirm by Cefinase  test
METICILLIN RESISTANCE : OXACILLIN TEST  NCCLS : broth or agar dilution  Mueller Hinton+ 2% Nacl  oxacillin 2 µg/ml  24 h at 35°C  MOLECULAR BIOLOGY  "Mec A"gene detection  Slidex MRSA detection  PBP 2a detection  ATB Staph  Cupule Oxa ( 2 mg/l )  ATB Na medium semi solid 5%Nacl  18- 24 H at 37°C Reference Method
8.3 - Main resistance mechanisms of Streptococci
STREPTOCOCCI  Natural resistance to Aminoglycosides  Usual susceptibility of Streptococcus pyogenes to PEN G  Enterococcus faecalis - faecium : Natural resistance to Cephalosporins (1st, 2nd, 3rd Gen.), Penicillin M (OXA) and Lincosamides E. faecium frequently Ampicillin : I or R Natural resistance and susceptibility
PNEUMOCOCCI  ATB STREP 5 PEP OXA (0.06-1) S S Wild I - R I - R Decreased susceptibility to PEN G by PBP mutation Test MIC before treatment ! Acquired resistance PEP = detection test of the susceptibility of Pneumococci to Penicillin
STREPTOCOCCI  Aminoglycosides : NATURAL RESISTANCE Detection of resistance ruling out the association with betalactams. KANHC GENHC R R No synergy blactams + Gentamycin and Kanamycin I I Synergy blactams + Gentamycin and Kanamycin I R Synergy blactams + Kanamycin No synergy blactams + Gentamycin Acquired resistance
9 - Choice of antibiotic panel compositions
HOW TO CHOOSE THE ANTIBIOTIC PANEL ?  Drugs with therapeutic interest  Markers enabling an equivalence with other drugs  Molecules tested for detection of natural and acquired resistances
 Notion of markers The molecule chosen in an antibiotic group must be the most regularly affected by the resistance mechanism  Notion of equivalence Also enables results to be extended to other molecules. Eg: cephalotin enables a result to be given for all first generation cephalosporins. HOW TO CHOOSE THE ANTIBIOTIC PANEL ?
10 - Interest of the Expert system
A CONTINUOUS QUALITY CONTROL The Expert checks that each observed result is possible = interpretative reading
EXPERT  INTERPRETATIVE READING  avoids therapeutic failure  Rapid evolution of acquired resistance mechanisms  Weak expression of resistance «in VITRO»
EXPERT Interpretative reading permits:  To find out resistance mechanisms  To deduce non tested drugs  To realise epidemiological study
HOW THE EXPERT WORKS  Needs ID  Checks natural resistances  Observes if acquired resistances  Deduces mecanisms of resistance  Sometimes – correct : S  R, or I  R – comment
HOW THE EXPERT WORKS Raw results IN VITRO IN VIVO Interpreted results for : EXPERT
THEN Incoherence Phenotype Identification etc. Ampicillin Ticarcillin Imipenem R R R If AMP = R If TIC = R If IMI = R and ID .... = Fact base = Knowledge base RAW RESULTS RULES Comments HOW THE EXPERT WORKS
If Klebsiella pneumoniae R R THEN S Natural resistance of K. pneumoniae (Penicillinase) S S AMOXICILLIN TICARCILLIN IMIPENEM AMOX. CLAVU CEFOTAXIM Test CAZ 1 S EXAMPLE
If R R THEN S ESBL I I R EXAMPLE Klebsiella pneumoniae AMOXICILLIN TICARCILLIN IMIPENEM AMOX. CLAVU CEFOTAXIM Test CAZ 1
If R R THEN R Impossible phenotype S S R EXAMPLE Klebsiella pneumoniae AMOXICILLIN TICARCILLIN IMIPENEM AMOX. CLAVU CEFOTAXIM Test CAZ 1
DEDUCE DRUGS  Testing MARKERS is essential  to deduce non tested drugs  So, the choice of drugs to be tested must respond to rules and not (only) to the request of physician
EPIDEMIOLOGICAL INTEREST  Follow up evolution of resistance  Epidemiological surveillance tools for local resistances statistical analysis of resistance per species, type of specimen  in order to adapt the initial choice of antibiotic therapy  Intra-hospital epidemics caused by multiresistant bacteria  justify an appropriate infection control measures
 Non transferable mechanisms  stable, chromosomic  Transferable mechanisms – Transmission of the resistance (plasmid) – loose of the resistance possible EPIDEMIOLOGICAL INTEREST Not transferable transferable
EPIDEMIOLOGY ISOLATION OF PATIENT NO ISOLATION OF PATIENT Appropriate hygiene mesures + OR Transferable resistance Ex : BLSE Multiresistant bacteria Ex : MRSA Not transferable resistance Ex : Natural case
THE EXPERT Check Validate Correct Expert analysis
THE EXPERT SYSTEMS  Deduce resistance mechanisms  Deduce results for antibiotics not tested (equivalence)  Make therapeutic corrections  Base for Epidemiological studies
11 - Study of phenotypes
Amoxicillin R Amo / Clav Ac. S Ticarcillin S Ticar+ clav Ac. S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin S Cefoxitin R Cefotaxime R Ceftazidime R Ceftazidime - 1 R Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole S raw value raw value 1 - Enterobacter cloacae
Amoxicillin R Amo / Clav Ac. S Ticarcillin R Ticar + clav. Ac S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin S Cefoxitin S Cefotaxime S Ceftazidime S Ceftazidime - 1 S Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. R Pefloxacin S Ciprofloxacin S Cotrimoxazole S raw value raw value 2 - Escherichia coli
Amoxicillin R Amo / Clav Ac. R Ticarcillin S Ticar +clav Ac S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin R Cefoxitin S C efotaxime S Ceftazidime S Ceftazidime - 1 S Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole S raw value raw value 3 - Pantoea agglomerans
Amoxicillin R Amo / Clav Ac. S Ticarcillin S Ticar +clav Ac S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin I Cefoxitin S C efotaxime S Ceftazidime S Ceftazidime - 1 S Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole S raw value raw value 4 - Proteus vulgaris
Amoxicillin R Amo / Clav Ac. S Ticarcillin S Ticar +clav Ac S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin S Cefoxitin S C efotaxime S Ceftazidime S Ceftazidime - 1 S Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole S raw value raw value 5 - Yersinia enterocolitica
Amoxicillin R Amo / Clav Ac. R Ticarcillin R Ticar +clav Ac R Piperacillin R Pip / Tazobactam S Imipenem S Cefalotin I Cefoxitin S C efotaxime S Ceftazidime S Ceftazidime - 1 S Cefepime S Cefpirome S Tobramycin S Gentamicin R Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole R raw value raw value 6 - Klebsiella pneumoniae
Amoxicillin R Amo / Clav Ac. R Ticarcillin R Ticar +clav Ac R Piperacillin R Pip / Tazobactam I Imipenem S Cefalotin R Cefoxitin R C efotaxime R Ceftazidime R Ceftazidime - 1 R Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole R raw value raw value 7 - Enterobacter cloacae
Amoxicillin R Amo / Clav Ac. R Ticarcillin S Ticar+ clav Ac. S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin R Cefoxitin S Cefotaxime S Ceftazidime S Ceftazidime - 1 S Cefepime S Cefpirome S Tobramycin S Gentamicin R Amikacin S Netilmicin S Nalidixic Ac. R Pefloxacin I Ciprofloxacin S Cotrimoxazole S raw value raw value 8 - Morganella morganii
Amoxicillin R Amo / Clav Ac. R Ticarcillin R Ticar+ clav Ac. S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin R Cefoxitin R Cefotaxime I Ceftazidime I Ceftazidime - 1 R Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole S raw value raw value 9 - Serratia marcescens
Amoxicillin S Amo / Clav Ac. S Ticarcillin S Ticar+ clav Ac. S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin I Cefoxitin S Cefotaxime S Ceftazidime S Ceftazidime - 1 S Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole S raw value raw value 10 - E.coli
Amoxicillin R Amo / Clav Ac. I Ticarcillin R Ticar +clav Ac S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin R Cefoxitin S C efotaxime S Ceftazidime S Ceftazidime - 1 R Cefepime S Cefpirome S Tobramycin R Gentamicin S Amikacin R Netilmicin S Nalidixic Ac. R Pefloxacin S Ciprofloxacin S Cotrimoxazole R raw value raw value 11- Proteus mirabilis
Penicillin G S Oxacillin S AMPI/Sulbact S Cefalotin S Gentamicin S Netilmicin S Erythromycin S Clindamycin S Nitrofurantoïn S Pefloxacin / Q2G S Ciprofloxacin S Rifampicin S Vancomycin S Teicoplanin S Cotrimoxazole S Tetracyclin S raw value raw value 12 - Staphylococcus aureus
Penicillin G R Oxacillin S AMPI/Sulbact S Cefalotin S Gentamicin S Netilmicin S Erythromycin S Clindamycin S Nitrofurantoïn S Pefloxacin / Q2G S Ciprofloxacin S Rifampicin S Vancomycin S Teicoplanin S Cotrimoxazole S Tetracyclin S raw value raw value 13 - Staphylococcus aureus
Penicillin G R Oxacillin R AMPI/Sulbact S Cefalotin S Gentamicin R Netilmicin S Erythromycin R Clindamycin S Nitrofurantoïn S Pefloxacin / Q2G R Ciprofloxacin S Rifampicin S Vancomycin S Teicoplanin S Cotrimoxazole S Tetracyclin S raw value raw value 14 - Staphylococcus epidermidis
Penicillin G R Oxacillin S AMPI/Sulbact S Cefalotin S Gentamicin S Netilmicin S Erythromycin S Clindamycin S Nitrofurantoïn S Pefloxacin / Q2G S Fosfomycine S Rifampicin S Vancomycin S Teicoplanin S Cotrimoxazole S Tetracyclin S raw value raw value 15- Staphylococcus saprophyticus
Penicillin Pneumo R Penicillin Strepto R Ampicillin Strepto S Penicillin entero R Ampicillin entero S Cefalotin S Cefuroxime R Piperacillin S Oxacillin R Ciprofloxacin S Tetracyclin R Erythromycin R Clindamycin R Cotrimoxazol S Nitrofurantoïn S Rifampicin S Vancomycin S Teicoplanin S Kanamycin HC R Gentamicin HC I raw value raw value 16 - Enterococcus faecalis
Penicillin Pneumo S Penicillin Strepto S Ampicillin Strepto S Penicillin entero S Ampicillin entero S Cefalotin S Cefuroxime S Piperacillin S Oxacillin S Ciprofloxacin S Tetracyclin S Erythromycin S Clindamycin S Cotrimoxazol S Nitrofurantoïn S Rifampicin S Vancomycin S Teicoplanin S Kanamycin HC I Gentamicin HC I raw value raw value 17 - Streptococcus agalactiae
Penicillin Pneumo R Penicillin Strepto R Ampicillin Strepto R Penicillin entero R Ampicillin entero I Cefalotin R Cefuroxime R Piperacillin S Oxacillin R Ciprofloxacin S Tetracyclin R Erythromycin R Clindamycin R Cotrimoxazol S Nitrofurantoïn S Rifampicin S Vancomycin S Teicoplanin S Kanamycin HC R Gentamicin HC I raw value raw value 18 - Enterococcus faecium
Penicillin Pneumo S Penicillin Strepto S Ampicillin Strepto S Penicillin entero S Ampicillin entero S Cefalotin S Cefuroxime S Piperacillin S Oxacillin S Ciprofloxacin S Tetracyclin S Erythromycin S Clindamycin S Cotrimoxazol S Nitrofurantoïn S Rifampicin S Vancomycin S Teicoplanin S Kanamycin HC I Gentamicin HC I raw value raw value 19 - Streptococcus pyogenes
Penicillin Pneumo R Penicillin Strepto S Ampicillin Strepto S Penicillin entero S Ampicillin entero S Cefalotin S Cefuroxime S Piperacillin S Oxacillin R Ciprofloxacin S Tetracyclin S Erythromycin R Clindamycin S Cotrimoxazol R Nitrofurantoïn S Rifampicin S Vancomycin S Teicoplanin S Kanamycin HC I Gentamicin HC I raw value raw value 20 - Streptococcus pneumoniae

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AST.ppt

  • 2. CONTENT 1- What is AST ? 2- Why and when to perform AST ? 3 - Evolution of the resistances 4- Classification of antibiotics 5- The different manual techniques 6- Disk diffusion 7- Antibiotics action mode 8- Resistance mechanisms of bacteria 8 - 1 Enterobacteriaceae 8 - 2 Staphylococci 8 - 3 Streptococci 9- Choice of antibiotics panel composition 10- Interest of Expert System 11- Study of phenotypes
  • 3. WHAT IS IT ? 1 - Antimicrobial Susceptibility Testing
  • 6. Reliable answers Least possible risk THE MICROBIOLOGIST
  • 7. Microbiologist's answer The ideal result would be :  Therapy with antibiotic A will be successful  the bacteria is Susceptible to the antibiotic  Therapy with antibiotic B will lead to a failure  the bacteria is Resistant to the antibiotic
  • 9. Microbiologist's answer Sucess of therapy depends on :  Resistance of bacteria  Site of infection  Dosage  Patient
  • 10. Microbiologist's answer Based on the :  Activity of the antibiotic for this strain  Pharmacokinetics of the drug  Normal dosage  Results of clinical studies
  • 11. How to do it ?  Determine antibiotic activity in vitro  Interpret results for in vivo : potential risk of success and failure
  • 12. 2 - Why and when to perform a susceptibility testing?
  • 13. WHY PERFORM A SUSCEPTIBILITY TEST ? In vitro susceptibility is one of the prerequisites for in vivo efficacy of antibiotic therapy To orient individual therapeutic decisions
  • 14. WHY PERFORM A SUSCEPTIBILITY TEST ?  To have an epidemiological follow-up at different levels : wards, department, region or country  To adapt empiric antibiotic therapy  To check the clinical spectrum of antibiotics But also : helps for healthcare decisions, and prevention program
  • 15. WHY PERFORM A SUSCEPTIBILITY TEST ? THERAPY EPIDEMIOLOGY DUAL INTEREST
  • 16. WHEN TO PERFORM A SUSCEPTIBILITY TEST ? - Each time a bacteria considerated to be responsible for an infection is isolated from a bacteriological specimen - The technician selects the bacterium among normal flora (if multimicrobial specimen)  MICROBIOLOGIST APPROACH
  • 17. WHEN TO PERFORM A SUSCEPTIBILITY TEST ? - SEVERE INFECTIONS : septicemia, meningitis, pyelonephrititis, bronchopneumopathy.... - CHRONIC INFECTIONS : urinary infections, otitis - INFECTIONS ON RISK PATIENTS : Old people, pregnant women, kidney and cardiac failure, immunosuppressed patients - THERAPEUTIC FAILURE - INFECTIONS ON RECENTLY HOSPITALIZED PATIENT : multiresistant bacteria  CLINICIAN APPROACH
  • 18. Sometimes microbiologists cannot determine if AST is required, without obtaining the clinical information that only a clinician can provide. WHEN TO PERFORM A SUSCEPTIBILITY TEST ? Example: a commensal bacterium can cause an infection in an immunocompromised patient or in a specific body site.  importance of patients data information, and clinical symptoms...
  • 19. WHEN TO PERFORM A SUSCEPTIBILITY TEST ? NEED FOR CLOSE WORKING RELATIONSHIP BETWEEN MICROBIOLOGISTS CLINICIANS
  • 20. CAN WE PREDICT SUSCEPTIBILITY OR RESISTANCE OF A BACTERIUM TO AN ANTIBIOTIC ?
  • 21. Direct impact on patient recovery The prescription of first-line antibiotics is poorly adapted Recovery ??? 10 % of therapeutic failure
  • 22. ANTIBIOTICS Natural resistance Systematic sensitivity ANTIBACTERIAL ACTIVITY SPECTRUM Each antibiotic is characterized by a natural spectrum : of some bacterial species of some bacterial species
  • 23. NATURAL RESISTANCE Permanent characteristic of all strains of the same bacterial species  stable  predictable  helps for confirmation of identification
  • 24. EXAMPLES OF NATURAL RESISTANCE Antibiotics Amino penicillin penicillin G Cephalosporin 1st G Cephalosporin 2nd G Cephalosporin 3rd G Nalidixic acid E coli Salmonella Klebsiella Enterob Serratia Natural Resistance Amino penicillin + Betalactamase inhibitor Pseudo aeruginosa Entero cocci staph
  • 25. NATURAL RESISTANCE  Wild Strain : = bacteria with just its natural resistance = No acquired resistance yet
  • 26.  Characteristic specific to some strains  The genotype has been modified by gene mutation or gene acquisition. ACQUIRED RESISTANCE
  • 27. ACQUIRED RESISTANCE Evolutive and unpredictable characteristic of the isolated bacterial strain  justifys the use of susceptibility testing Example : E. coli  wild strain : Amoxicillin (S)  Now : 30 - 60% Amoxicillin (R)
  • 28. ACQUIRED RESISTANCE Given the evolution of acquired resistance:  the natural spectrum of activity is no longer sufficient to guide the choice of treatment. Example : Acute otitis caused by Pneumococci with decreasing susceptibility to Penicillin G : - 1985 : < 1% - 1998 : 10 - 50% (depends on the country)
  • 29. CLINICAL SPECTRUM OF ACTIVITY Bacteriological data : natural spectrum + acquired resistance Pharmacokinetic data Clinical data  For each antibiotic, bacteria are divided in 3 classes : S, I, R Frequent updates : take into account the evolution of the resistances = + +
  • 30. 3 - Evolution of resistance
  • 31.  Resistance must be monitored  Today, the over-use of antibiotics increases the frequency of resistant strains.  The antibiotic does not create resistance but selects resistant bacteria by eliminating the commensal and susceptible bacteria. EVOLUTION OF RESISTANCE
  • 32. EVOLUTION OF RESISTANCE LARGE ANTIBIOTIC USE POORLY ADAPTED TREATMENT THERAPY FAILURE INCREASE IN RESISTANCE PRESSURE OF SELECTION
  • 33. Situation of Methicillin Resistant S. aureus in Europe 33.6% 0% 1.5% 0.3% 1.8% 5.5% 21.6% 25.1% 30.3% 34.4% Marty, V. Jarlier- Surveillance des bactéries multirésistantes : Justification, rôle du laboratoire, indicateurs, données françaises récentes - Path Biol April 1998 ; 217-26 A few figures on resistance...
  • 34. Evolution of bacterial resistances Community - acquired in Europe 1975 90 30 20 10 0 1985 1998 % of resistant strains Strepto A / Penicillin G H . influenzae / Ampicillin S. Pneumoniae/ Penicillin G S .pneumoniae / Erythromycin E . coli / Ampicillin S. aureus / Penicillin G 40 80 70 Questions and answers on ST : J . louis Tissier
  • 35. Evolution of bacterial resistances Hospital - acquired in Europe 1975 40 30 20 10 0 1985 1998 % of resistant strains Enterococcus / Vancomycin K. pneumoniae / 3rd generation cephalo. P. aeruginosa / Fluoroquinolones E. cloacae / 3rd generation cephalo. S. aureus / Oxacillin E. coli / Ampicillin Questions and answers on ST : J louis Tissier
  • 36. 4 - Classification of antibiotics
  • 37. DEFINITION OF AN ANTIBIOTIC  Natural substance produced by molds and bacteria  Inhibits or destroys other bacteria. All substances with an antibacterial activity (produced by synthesis or semi-synthesis)
  • 38.  B- Lactams : - Penicillins / Inhibitors - Cephalosporins - Carbapenems - Monobactams  Aminoglycosides  Cyclins  Quinolones  MLSK  Polypeptides  Imidazoles  Rifamycins  Phenicols  Glycopeptides  Furanes  Sulfonamides  Miscellaneous SIMPLIFIED CLASSIFICATION OF ANTIBIOTICS 13 families
  • 39. b LACTAMS Marker Gram (+) Gram (-) - PEN G Penicillin G Nat. R. - PEN M Oxacillin Nat. R. - PEN A Amox - Ampi - PEN C Ticarcillin - PEN-U Piperacillin Penicillins Systematic S of S. pyogenes
  • 40. b LACTAMS Marker Gram (+) Gram (-) - 1st generation Cefalotin Nat R. of E. faecalis Cefaclor - 2nd generation Cefuroxime - Cephamycins Cefoxitin Cefotetan - 3rd generation Cefotaxime Ceftazidime - 4th generation Cefepime Cefpirome Cephalosporins
  • 41. These enzymatic inhibitors have no true antibacterial activity, but a strong affinity for b-lactamases : Clavulanic acid Sulbactam Tazobactam Aim : blocks beta-lactamases Combination b-lactams - b-lactamase inhibitor : - Group 1 Amoxicillin + Clavulanic Ac. Ampicillin + sulbactam - Group 2 Ticarcillin + Clavulanic Ac. Piperacillin + tazobactam - Group 3 Sulbactam + 3rd generation cephalosporins Never alone ! INHIBITORS OF BETA-LACTAMASES
  • 42. b LACTAMS Carbapenem  Imipenem  Meropenem Monobactam Marker Gram (+) Gram (-) Aztreonam Nat. R.
  • 43. AMINOGLYCOSIDES Marker Gentamicin, Tobramycin, Nat. R. of Amikacin, Streptomycin, Streptococci Kanamycin, Netilmicin, and Nat. R. of Neomycin, Spectinomycin, Anaerobes - G(-) Bacilli : test GEN, TOB, AKN, NET - Enterococci and deficient Streptococci : test STR HC, GEN HC, KANA HC (Aminoglycosides high concentration) - Staphylococci : test GEN, TOB, KAN  NCCLS recommendations:
  • 44.  Tetracycline  Doxycycline  Minocycline Extended spectrum  Tetracycline is the MARKER for all cyclines but for certains organisms doxycycline and minocycline are more susceptible than tetracycline CYCLINES
  • 45. QUINOLONES Marker Gram (+) Gram (-) Quinolones Nalidixic acid Nat. R. Pipemidic acid Norfloxacin Pefloxacin Fluoroquinolones Ofloxacin Ciprofloxacin
  • 46. M.L.S : MACROLIDES / LINCOSAMIDES / STREPTOGRAMINS Marker Gram (+) Gram (-) Macrolides Erythromycin Nat. R. except Haemophilus Lincosamides Natural resistance Clindamycin for Staph / Enterococci Streptogramins Quinupristin/ dalfopristin*  *Synercid
  • 47. NITRO - IMIDAZOLES  Metronidazole Active on anaerobes, parasites RIFAMYCINS (ANSAMYCINS)  Rifampicin Used in anti-stapylococci eye lotions, gonococci, TB POLYPEPTIDES Marker Gram (+) Gram (-) Colistin Nat. R. P. aeruginosa Bacitracin = Syst. S.
  • 48. PHENICOLS  Chloramphenicol Extended spectrum : Typhoid fever, Gonococci, bronchopulmonary infections GLYCOPEPTIDES Marker Gram (+) Gram (-) - Vancomycin Nat. R. - Teicoplanin
  • 49. FURANE  Nitrofurantoin Urinary infections : Gram (+) cocci, Enterobacteriaceae Natural resistance for P.mirabilis SULFONAMIDES (FOLATE PATHWAY INHIBITORS) Marker Gram (+) Gram (-) Cotrimoxazol P. aeruginosa = Nat. R. Sulfamides Digestive, genital and urinary infections
  • 50. MISCELLANEOUS  Fusidic acid (French drug) Local anti-staphylococcal treatment  Fosfomycin - Cystitis for Gram + cocci and Gram- bacilli : «Monuril» - Systemic infection (multiresistant bacteria)  Natural resistance for Staphylococcus saprophyticus
  • 51. 5 - The different manual techniques  Reference technique  Routine techniques
  • 52. MIC DETERMINATION Definition : Minimum Inhibitory Concentration of an antibiotic inhibiting the visible growth of a bacterium overnight in standardized conditions for rapid growth bacteria.  Bacteriostatic effect of an antibiotic on a bacterium
  • 53. Mueller Hinton 1 ml 1 ml 0 0.5 1 2 4 8 16 µg/ml 107 CFU 35°C - 16 to 20 hrs 0 0.5 1 2 4 8 16 µg/ml 0 0.5 1 2 4 8 16 µg/ml MIC DETERMINATION liquid medium Reference technique for NCCLS* *National Committee for Clinical Laboratory Standarts MIC =
  • 55. MBC DETERMINATION Definition : Minimum Bactericidal Concentration  Bactericidal effect of an antibiotic on a bacterium
  • 56. MBC - PROCEDURE 10 µl 0 µg/ml 4 µg/ml 8 µg/ml 16 µg/ml MBC = 0 0.5 1 2 MIC = 4 8 16 µg/ml 35°C - 16 to 20 hrs
  • 58. MIC - MBC MIC MBC Bactericidal MIC MBC Bacteriostatic 1 2 8 16 32 64 128 4 1 2 8 16 32 64 128 4
  • 59. CATEGORIZATION OF STRAINS  2 concentrations set by the NCCLS :  c = low breakpoint  C = high breakpoint  According to the following criteria : - Bacteriological studies - Pharmacokinetics - Clinical experience Breakpoints :
  • 60. c C S I R MIC < c c < MIC < C MIC > C CATEGORIZATION OF STRAINS
  • 61. ROUTINE TECHNIQUES  Disk diffusion  ATB Strips  ViteK 2 Compact  E test
  • 65. DIFFUSION Methodology 1 hour < 15 mins Streak twice 5-15 mins 10 mins 30°C 37°C 37°C CO2 4°C 0.5 McF
  • 66. DIFFUSION CONTROL  Control of each cartridge of antibiotics  Stability after opening the cartridge : 1 week (CLSI and CA-SFM recommendations)
  • 67. DIFFUSION Summary  Not ready to use  Flexibility of antibiotics to be tested  Solid technique  Visual reading  Indirect technique
  • 68. 24 Hrs ATB STRIPS bioMérieux
  • 69.  Thermoformed transparent plastic strip consisting of 32 cupules  Cupules containing dehydrated antibiotics  Inoculation medium : ATB medium (semi solid Mueller-Hinton)  2 empty cupules for individual antibiotic tests 24 Hrs ATB STRIPS
  • 70. 24 Hrs ATB STRIPS  READING OF A BACTERIAL GROWTH : turbidity  DETERMINATION OF S-I-R DIRECTLY  READING : VISUAL OR AUTOMATIC  For manual customers  Or customers equipped with mini API or ATB Expression system (automatic reading)
  • 71. 24 Hrs ATB STRIPS  Principle : Each antibiotic (A, B, C, D…) is tested at 2 different concentrations (Breakpoints: c, C) c C C B A 0 Growth control S (Susceptible) R (Resistant) I (Intermediate) = No bacterial growth = Bacterial growth
  • 72. 24 Hrs ATB STRIPS  Susceptible strain : MIC < c The antibiotic tested is active on the bacteria  Intermediate strain : c < MIC < C The bacteria is not affected by the treatment at usual doses, but may be affected by a high-dose intravenous or local treatment.  This is the risk range.  Resistant strain : MIC > C The bacteria resists to the strongest concentrations. The antibiotic will not be active in vivo.
  • 73. 24 Hrs ATB STRIPS 8 32 32 32 8 8 MIC < 8 (S) 8 < MIC  32 (I) MIC > 32 (R)  example : CFT 8 - 32 mg/l
  • 75. MICROSCAN 24 Hrs CHARACTERISTICS  Broad range of MIC concentrations is tested for each antimicrobic which gives information on emerging and low level resistance. 20 antimicrobics in each card let the lab give a wide response. This is also a good basis for the deduction function of AES:3 - 6 concentrations for each antibiotics A rapid response can be used by clinicians when they are making the change from empiric to directed therapy.
  • 76. SYSTEM CONFIGURATION CAPACITY= 15/30 /60 cards PC WINDOWS XP DENSICHEK inoculum Standardisatio CODE BARR
  • 80. E-TEST PROCEDURE McF regarding ID 5 mins at 37 °C Streak twice < 15 mins saline water 30°C 37°C 37°C CO2
  • 82. The way the microbiologist thinks :  the reference method  reliable  easy-to-use  cheap  flexible
  • 83. LET’S LOOK AT EACH POINT ...
  • 84. Disk diffusion is a commonly used method not the reference one REFERENCE METHOD ?
  • 85. MIC is the REFERENCE Disk Diffusion is an INDIRECT METHOD REFERENCE METHOD ?
  • 86. REFERENCE METHOD ? Do not use disk diffusion for Anaerobes Yeasts
  • 87. "For generation of reproducible results, all technical details of the test procedure must be carefully standardized and controlled" NCCLS Dec. 93 - Vol.13 N°24 P.1 RELIABILITY ?
  • 88. RELIABILITY ?  Depends on :  Choice of media  Agar thickness  Standardization of the inoculum  Disk stability
  • 89. Mueller Hinton HTM GC Supplement MH + 5% Sheep Blood Chocolate 8 Chocolate + Supplement 8 Enterobacteriaceae Pseudomonas aeruginosa Staphylococci Enterococci Haemophilus Gonococci Pneumococci Others 8 NO 1- Choice of the medium NO NO
  • 90. Diffusion of the antibiotic Agar (4 mm) 2- Agar thickness
  • 91. Media too thin excessive diffusion Media too thick too little diffusion Media sloping uneven diffusion Disk not flat too little diffusion Plate too wet surface diffusion 2- Agar thickness
  • 92. 0.5 Mac Farland 3- Standardization of the inoculum
  • 93. Inoculum too heavy Inoculum too light Inoculum uneven 3- Standardization of the inoculum
  • 94. Time (months) 1 18 2 weeks 70% 150% % Antibiotic activity 4 - Disk stability
  • 95. - Use disks within 5 days - Daily quality control for each disk 4 - Disk stability
  • 96.  Adequate media  4 mm-thick media  Standardized inoculum  Daily quality control RELIABILITY ?
  • 97. RELIABILITY ?  Correct reading ? Measure the diameter
  • 98. RELIABILITY ? Human error and subjectivity make standardization difficult
  • 99. Interpretation is SUBJECTIVE About 5% of discrepancies RELIABILITY ?
  • 101. CHEAP ?  Cost of disk diffusion = Reagent cost (media + disks) Labor cost QC cost + +
  • 102. When you can report on so many antibiotics by testing few, why do you need more flexibility? FLEXIBILITY ?
  • 103. Drug Tested Drug reported Penicillin G All Penicillin G Ampicillin All Penicillin A Cephalothin All Cephalosporins 1st Gen. Piperacillin Mezlocillin Cefuroxime Cefamandole, Cefomicin Cefotaxime Ceftriaxone, Ceftizoxime Oxacillin All beta-lactams Norfloxacin Ofloxacin, Lomefloxacin (Urinary Tract Infection) Tetracycline All Tetracyclines Use of markers FLEXIBILITY ?
  • 104. Common Method Cheap reagent Reliable technique Easy-to-use Flexible but Depends on strictly following the procedure and the lab technologist Quality control difficult Costly method Time consuming Everybody uses a different technique but but but but CONCLUSION
  • 105. 7 - Antibiotics action mode
  • 106. ANTIBIOTIC ACTION MODES  Aminoglycosides  Macrolides  Tetracyclines  Chloramphenicol R R E Chromosome  Quinolones  Nitro-imidazoles  b Lactams  Fosfomycin  Glycopeptides  Sulfonamides 1 2 3 4 = DNA gyrase E R = Ribosomes
  • 107. External membrane Peptido glycane Cytoplasmic Membrane Cytoplasm PS PS P P PBP PBP PO PS = Periplasmic space P = Permeases PBP = Penicillin binding proteins PO = porines PO Polysaccharides Phospholipids (Except Neisseria and Haemophilus) Gram + Gram - BACTERIA CELL-WALL
  • 108. External membrane Hydrophilic Antibiotic Lipophilic Antibiotic* Lipophilic Antibiotic* Hydrophilic Antibiotic Peptido glycane Cytoplasmic Membrane Cytoplasm PS PS P P PBP PBP PO * = fusidic acid, Macrolides, Rifampicin Intracytoplasmic targets Intracytoplasmic targets PO Polysaccharides Phospholipids (Except Neisseria and Haemophilus) Gram + Gram - ANTIBIOTICS ACTION MODES PS = Periplasmic space P = Permeases PBP = Penicillin binding proteins PO = porines
  • 109. External membrane Antibiotic active on PBP Hydrophilic Antibiotic active on PBP Peptido glycane Cytoplasmic membrane Cytoplasm PS PS P P PBP PBP PO PO Polysaccharides Phospholipides (Except Neisseria and Haemophilus) Gram + Gram - = b lactamases ACTION MODE OF BETA-LACTAMS PS = Periplasmic space P = Permeases PBP = Penicillin binding proteins PO = porines
  • 110. External membrane Lipophilic antibiotic * Lipophilic antibiotic * Peptido glycane Cytoplasmic membrane Cytoplasm PS PS P P PBP PBP PO PO Polysaccharides Phospholipides (Except Neisseria and Haemophilus) Gram + Gram - 50S 50S ACTION MODE OF MACROLIDES
  • 113. 8 - Resistance mechanisms of bacteria
  • 114. RESISTANCE MECHANISMS MODIFICATION OF THE TARGET EFFLUX PHENOMENON ENZYME PRODUCTION IMPERMEABILITY resistance of Enterobacteriaceae to macrolides 1- b lactamases inactivating b lactams 2- Enzymes degrading aminoglycosides resistance of Enterobacteriaceae to tetracycline and quinolones 1- Penicillin binding proteins for b lactams (PBP) 2- Ribosomes for macrolides 3- DNA gyrase for quinolones R R E Plasmid Chromosome
  • 115. RESISTANCE MECHANISMS P P PBP PBP PO PO (Except Neisseria and Haemophilus) Gram + Gram - ENZYMES ENZYMES ENZYMES ENZYMES Hydrophilic antibiotic Hydrophilic antibiotic EFFLUX EFFLUX Antibiotic active on PBP Hydrophilic antibiotic active on PBP Lipophilic antibiotic Decreasing size of porine Mutation of DNA gyrase Mutation of DNA gyrase Mutation of ribosome Mutation of ribosome
  • 116. GENETIC SUPPORT OF RESISTANCE 1) The chromosome  stable  vertical transmission (to descendants)  no horizontal transmission (not transferable from one bacteria to another)
  • 117. GENETIC SUPPORT OF RESISTANCE 2) The Plasmid  Unstable in the absence of the antibiotic which selected it  Transferable from one bacterium to another, sometimes between different species
  • 118. GENETIC SUPPORT OF RESISTANCE CHROMOSOME PLASMID Natural resistance : - stable - no horizontal transmission Acquired resistance : - unstable - risk of horizontal transmission
  • 119. CROSS AND ASSOCIATED RESISTANCE  Cross resistance The same resistance mechanism affects several antibiotics within a same family. Eg : a Gentamycin-resistant staphylococci is resistant to all aminoglycosides.  Associated resistance affects several antibiotics in different families Eg : Associated resistance of Enterobacteriaceae to b-lactams and aminoglycosides (ESBL and AAC 6').
  • 120. 8.1 - Main resistance mechanisms of Enterobacteriaceae
  • 122. Constitutive : constant level of prodution independent of the presence of an inductor Synthesis of b-Lactamases Inducible : produced in small quantities but increases in the presence of the inductor Eg : Cefoxitin, Imipenem Presence of the antibiotic in the periplasmic space
  • 123. b-Lactamases  Penicillinase  Cephalosporinase  ESBL : Extended spectrum B-Lactamase  IRT : TEM resistant to inhibitors
  • 125. NATURAL RESISTANCE PHENOTYPES Group I : E. coli, Proteus mirabilis, Salmonella, Shigella  Wild strain : high level of susceptibility but very low level of cephalosporinase for E. coli, Shigella Group II : Klebsiella spp, Citrobacter koseri, Citrobacter amalonaticus, Escherichia hermanii,  Wild strain : natural penicillinase (Nat. Pase).  K. oxytoca : hyper production of natural Pase
  • 126. NATURAL RESISTANCE PHENOTYPES Group III : Serratia spp, Citrobacter freundii, Enterobacter spp, Pantoea agglomerans Morganella morganii, Hafnia alvei, Proteus vulgaris, Providencia spp,  Wild strain : cephalosporinase (Nat. Case) low level enzyme production Group IV : Yersinia spp  Wild strain : Penicillinase and cephalosporinase ( Nat. Pase and Case)
  • 127. RESISTANCE PHENOTYPES : WILD STRAINS AMO AMC TIC CFT S S S S  Group 1 :  No natural resistance Except low level case for E. coli and shigella AMO AMC TIC CFT R S R S  Group 2 :  Natural Pase AMO AMC TIC CFT R R* S R  Group 3 :  Natural Case (* :for most Group 3 bacteria) AMO AMC TIC CFT R R R R  Group 4 :  Case + pase
  • 128. ENTEROBACTERIACEAE AMO AMC MEC TIC CFT CXT TET COL FUR Escherichia coli S S S S S S S S S I Shigella/Salmonella S S S S S S S S S Proteus mirabilis S S S S S S R R R Klebsiella pneumoniae/ II oxytoca R S S R S S S S S Citrobacter diversus (Lev. amalonatica) R S S R S S S S S Enterobacter cloacae/ aerogenes R R S S R R S S S Citrobacter freundii R R S S R R S S S Proteus vulgaris R S R S R S R R R III Serratia marcescens R R R S R In R R R Morganella morganii R R R S R In R R R Hafnia alvei R R R S R S S S S Providencia rettgeri R R R S R S R R R Providencia stuartii R R R S R S R R R IV Yersinia enterocolitica R R S R R r S S S Natural resistance r = low level resistance, in = Inducible
  • 129. WILD STRAINS  Group 1 :  Group 2 : Natural Pase  Group 3 : Natural Case  Group 4 : Case + pase Low case for E. coli, Shigella
  • 130. Wild strains and acquired resistances  Group 1 :  Group 2 :  Group 3 :  Group 4 : IRT ESBL ESBL ESBL Acquired PASE Acquired Pase Natural Pase Natural Case Acquired Pase Natural Case + Pase HL case (E. coli, Shigella) HL case ESBL
  • 131. GROUP 1: P.mirabilis / Salmonella Observed phenotypes AMO AMC TIC PIC IMI 1GC 3GC Wild S S S S S S S Acquired Pase I-R S-I-R I-R I-R S S-I S ESBL R S-I I-R I-R S S-I-R S-I-R Acquired resistance phenotypes : b lactams
  • 132. GROUP 1: E.coli / Shigella Observed phenotypes Acquired resistance phenotypes : b lactams AMO AMC TIC PIC TZP IMI 1GC CXT 3GC Wild S S S S S S S S S Low Case S-I S-I S S S S I-R S S HL Case R R S S S S R R I Acquired Pase I-R S-I I-R R S S S-I S S Pase resistant to inhibitors R R R R I-R S S-I S S ESBL R S-I R R S S S-I-R S S-I-R * Isolated R to Mecillinam possible for E. coli but impossible for the others ( inoculum too heavy)
  • 133. GROUP 2 : Observed phenotypes Acquired resistance phenotypes : b lactams AMO AMC TIC PIC TZP 1GC 3GC Wild (Natural Pase) I-R S R S-I-R S S S Acquired Pase R S-I R I-R S I-S S ESBL R S I-R S-I-R S I-R S-I-R Hyper production of natural Pase (for K. oxytoca) R I-R R I-R S-I I-R S
  • 134. GROUP 3 : Observed phenotypes Acquired resistance phenotypes : b lactams AMO AMC TIC PIC TZP IMI 1GC 3GC Wild: R (R) S S S S RS (Nat Case) Pase R (R) R R S-I S R S HL Case R R I-R I-R I-R S R I-R ESBL R R R R S-I-R S R S-I-R ( ) = possible S for Proteus vulgaris
  • 135. b-Lactams and Enterobacteriaceae (SUMMARY) Antibiotics PASE PASE IRT ESBL CASE CASE NAT ACQUIRED NAT high level Penicillins : Amino P I/R R R R R R Carboxi P I/R R R R S R Ureido P S/I I-R R R S R Cephalo : C1G S S/I S/I/R R R R C2G S S S/I/R R S/I/R R C3G S S S S/I/R S R Inhibitors: S S/I R S/I/R R R Imipenem S S S S S S
  • 136. Techniques for the detection of ESBL
  • 137. WHAT ARE ESBL ?  Extended Spectrum Beta-Lactamase  mutation of a traditional Penicillinase - plasmid-origin - thus transferable  Described for Enterobacteriaceae 80 % of cases concern Klebsiella pneumoniae ALL BETA-LACTAMS ARE AFFECTED except Cephamycins and Carbapenems
  • 138. Techniques for the detection of ESBL - CAZ at 1 mg/l on ATB G- 5 - Interpretative reading - ATB BLSE - Disk method (Champagne cork)  DETECTION OF ESBL  CONFIRMATION OF ESBL
  • 139. Detection of ESBL using ATB G - 5  Objective : Distinguish acquired Penicillinase and ESBL when there is a doubt Depends on the MIC to Ceftazidim TEST CAZ 1 :
  • 140. Detection of ESBL using ATB G - 5 Test CAZ 1 = Ceftazidim at 1 µg/ml enables the detection of low level resistance of ESBL (Low MIC) Breakpoints N° of strains 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 CAZ 1 1 µg/ml R S I MIC to Ceftazidim
  • 141. DETECTION OF ESBL USING DIFFUSION: «Champagne cork» or «key hole effect»  Objective : Distinguish ESBL and HL Case when there is a doubt  Technique : Deposit 3 disks (Distance to be adapted ) - Inhibitor of B-Lactamase - 3rd gen. Cephalosporin - Aztreonam C3G AZT Inhibitor
  • 142. DETECTION OF ESBL USING DIFFUSION: Amoxicillin + Clavulanic acid Ceftazidime Aztreonam ESBL HL CASE «Champagne cork» or «key hole effect» No action of inhibitor Action of inhibitor
  • 143. DETECTION OF ESBL USING DIFFUSION: «Champagne cork» or «key hole effect»
  • 144. Detection of ESBL using ATB BLSE  Objective : Distinguish ESBL and HL Case when there is a doubt  Technique : Comparaison of - MIC determination of 3rd gen. Cephalosporin - MIC determination of 3rd gen. Cephalosporin + Inhibitor of B-Lactamase If at least 4 dilutions difference : ESBL +
  • 145. Detection of ESBL using ATB BLSE ref. 14 100 mg/l Example 1 ATM ATM+S Aztreonam Aztr.+Sulbactam 0.5 0.06 + + 2 ATM ATM+S Aztreonam Aztr.+Sulbactam 1 0.12 + + ESBL(+) : MIC range 3 ATM ATM+S Aztreonam Aztr.+Sulbactam 2 0.25 + + from 8 to 0.5 (4 4 ATM ATM+S Aztreonam Aztr.+Sulbactam 4 0.5 + - dilutions). Image + - 5 ATM ATM+S Aztreonam Aztr.+Sulbactam 8 1 - - 6 CAZ CAZ+S Ceftazidime Cefta+Sulbactam 0,5 0.06 + + 7 CAZ CAZ+S Ceftazidime Cefta+Sulbactam 1 0.12 + + 8 CAZ CAZ+S Ceftazidime Cefta+Sulbactam 2 0.25 + + ESBL(+) : MIC range 9 CAZ CAZ+S Ceftazidime Cefta+Sulbactam 4 0.5 + + from 32 to 1 (10 10 CAZ CAZ+S Ceftazidime Cefta+Sulbactam 8 1 + - dilutions). 11 CAZ CAZ+S Ceftazidime Cefta+Sulbactam 16 2 + - Image + - 12 CAZ CAZ+S Ceftazidime Cefta+Sulbactam 32 4 - - 13 CTT CTT Cefotetan 4 32 ESBL: S 14 IMI IMI Imipenem 4 8 ESBL: S 15 SCTX1 SCTX2 Cefotaxime + Sulbactam 0.06 0.12 Control of the recovery activity
  • 146. ENTEROBACTERIACEAE GEN TOB NET AKN KAN APH 3' S S S S R AAC 3I R S S S S AAC 3II R R R S R AAC 6' S R R R R AAC 2' R R R S S ANT 2" R R S S S Impossible S S S R S Impossible S S R S S Impossible S R S S S NB : * AAC 6' : Often associated with an ESBL, except for S. marcescens * P. stuartii has an AAC 2' in the wild state Acquired resistance : Aminoglycosidases
  • 147. 8.2 - Main resistance mechanisms of Staphylococci
  • 148. GRAM POSITIVE COCCI  GRAM POSITIVE COCCI  Natural resistance : Aztreonam Nalidixic acid (QUINOLONES 1) Polypeptides  STAPHYLOCOCCI  Natural susceptibility : Vancomycin  At least one case of Staphylococcus aureus has been detected VAN: I Natural resistance and susceptibility
  • 149. STAPHYLOCOCCI  b lactams PEN G OXA S S Absence of pase Check with chromogenic test R S Acquired Pase R R Modification of PBP (MRSA) Acquired resistance
  • 150. PENICILLINASE TEST  Cefinase  For rapid detection of B-Lactamase production  Chromogenic cephalosporin (= Nitrocefin) Ref : 55 622 colony (+) 5 mins to 1 hour Also for H. influenzae, N. gonorrhoae, Enterococci, and anaerobes
  • 151. Cefinase (+)  change Pen G : S to R Cefinase (-)  do not change Pen G PENICILLINASE TEST  ATB Staph  Cupule Pen G at 0.125 g/l If Penicilline G is S, confirm by Cefinase  test
  • 152. METICILLIN RESISTANCE : OXACILLIN TEST  NCCLS : broth or agar dilution  Mueller Hinton+ 2% Nacl  oxacillin 2 µg/ml  24 h at 35°C  MOLECULAR BIOLOGY  "Mec A"gene detection  Slidex MRSA detection  PBP 2a detection  ATB Staph  Cupule Oxa ( 2 mg/l )  ATB Na medium semi solid 5%Nacl  18- 24 H at 37°C Reference Method
  • 153. 8.3 - Main resistance mechanisms of Streptococci
  • 154. STREPTOCOCCI  Natural resistance to Aminoglycosides  Usual susceptibility of Streptococcus pyogenes to PEN G  Enterococcus faecalis - faecium : Natural resistance to Cephalosporins (1st, 2nd, 3rd Gen.), Penicillin M (OXA) and Lincosamides E. faecium frequently Ampicillin : I or R Natural resistance and susceptibility
  • 155. PNEUMOCOCCI  ATB STREP 5 PEP OXA (0.06-1) S S Wild I - R I - R Decreased susceptibility to PEN G by PBP mutation Test MIC before treatment ! Acquired resistance PEP = detection test of the susceptibility of Pneumococci to Penicillin
  • 156. STREPTOCOCCI  Aminoglycosides : NATURAL RESISTANCE Detection of resistance ruling out the association with betalactams. KANHC GENHC R R No synergy blactams + Gentamycin and Kanamycin I I Synergy blactams + Gentamycin and Kanamycin I R Synergy blactams + Kanamycin No synergy blactams + Gentamycin Acquired resistance
  • 157. 9 - Choice of antibiotic panel compositions
  • 158. HOW TO CHOOSE THE ANTIBIOTIC PANEL ?  Drugs with therapeutic interest  Markers enabling an equivalence with other drugs  Molecules tested for detection of natural and acquired resistances
  • 159.  Notion of markers The molecule chosen in an antibiotic group must be the most regularly affected by the resistance mechanism  Notion of equivalence Also enables results to be extended to other molecules. Eg: cephalotin enables a result to be given for all first generation cephalosporins. HOW TO CHOOSE THE ANTIBIOTIC PANEL ?
  • 160. 10 - Interest of the Expert system
  • 161. A CONTINUOUS QUALITY CONTROL The Expert checks that each observed result is possible = interpretative reading
  • 162. EXPERT  INTERPRETATIVE READING  avoids therapeutic failure  Rapid evolution of acquired resistance mechanisms  Weak expression of resistance «in VITRO»
  • 163. EXPERT Interpretative reading permits:  To find out resistance mechanisms  To deduce non tested drugs  To realise epidemiological study
  • 164. HOW THE EXPERT WORKS  Needs ID  Checks natural resistances  Observes if acquired resistances  Deduces mecanisms of resistance  Sometimes – correct : S  R, or I  R – comment
  • 165. HOW THE EXPERT WORKS Raw results IN VITRO IN VIVO Interpreted results for : EXPERT
  • 166. THEN Incoherence Phenotype Identification etc. Ampicillin Ticarcillin Imipenem R R R If AMP = R If TIC = R If IMI = R and ID .... = Fact base = Knowledge base RAW RESULTS RULES Comments HOW THE EXPERT WORKS
  • 167. If Klebsiella pneumoniae R R THEN S Natural resistance of K. pneumoniae (Penicillinase) S S AMOXICILLIN TICARCILLIN IMIPENEM AMOX. CLAVU CEFOTAXIM Test CAZ 1 S EXAMPLE
  • 170. DEDUCE DRUGS  Testing MARKERS is essential  to deduce non tested drugs  So, the choice of drugs to be tested must respond to rules and not (only) to the request of physician
  • 171. EPIDEMIOLOGICAL INTEREST  Follow up evolution of resistance  Epidemiological surveillance tools for local resistances statistical analysis of resistance per species, type of specimen  in order to adapt the initial choice of antibiotic therapy  Intra-hospital epidemics caused by multiresistant bacteria  justify an appropriate infection control measures
  • 172.  Non transferable mechanisms  stable, chromosomic  Transferable mechanisms – Transmission of the resistance (plasmid) – loose of the resistance possible EPIDEMIOLOGICAL INTEREST Not transferable transferable
  • 173. EPIDEMIOLOGY ISOLATION OF PATIENT NO ISOLATION OF PATIENT Appropriate hygiene mesures + OR Transferable resistance Ex : BLSE Multiresistant bacteria Ex : MRSA Not transferable resistance Ex : Natural case
  • 175. THE EXPERT SYSTEMS  Deduce resistance mechanisms  Deduce results for antibiotics not tested (equivalence)  Make therapeutic corrections  Base for Epidemiological studies
  • 176. 11 - Study of phenotypes
  • 177. Amoxicillin R Amo / Clav Ac. S Ticarcillin S Ticar+ clav Ac. S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin S Cefoxitin R Cefotaxime R Ceftazidime R Ceftazidime - 1 R Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole S raw value raw value 1 - Enterobacter cloacae
  • 178. Amoxicillin R Amo / Clav Ac. S Ticarcillin R Ticar + clav. Ac S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin S Cefoxitin S Cefotaxime S Ceftazidime S Ceftazidime - 1 S Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. R Pefloxacin S Ciprofloxacin S Cotrimoxazole S raw value raw value 2 - Escherichia coli
  • 179. Amoxicillin R Amo / Clav Ac. R Ticarcillin S Ticar +clav Ac S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin R Cefoxitin S C efotaxime S Ceftazidime S Ceftazidime - 1 S Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole S raw value raw value 3 - Pantoea agglomerans
  • 180. Amoxicillin R Amo / Clav Ac. S Ticarcillin S Ticar +clav Ac S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin I Cefoxitin S C efotaxime S Ceftazidime S Ceftazidime - 1 S Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole S raw value raw value 4 - Proteus vulgaris
  • 181. Amoxicillin R Amo / Clav Ac. S Ticarcillin S Ticar +clav Ac S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin S Cefoxitin S C efotaxime S Ceftazidime S Ceftazidime - 1 S Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole S raw value raw value 5 - Yersinia enterocolitica
  • 182. Amoxicillin R Amo / Clav Ac. R Ticarcillin R Ticar +clav Ac R Piperacillin R Pip / Tazobactam S Imipenem S Cefalotin I Cefoxitin S C efotaxime S Ceftazidime S Ceftazidime - 1 S Cefepime S Cefpirome S Tobramycin S Gentamicin R Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole R raw value raw value 6 - Klebsiella pneumoniae
  • 183. Amoxicillin R Amo / Clav Ac. R Ticarcillin R Ticar +clav Ac R Piperacillin R Pip / Tazobactam I Imipenem S Cefalotin R Cefoxitin R C efotaxime R Ceftazidime R Ceftazidime - 1 R Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole R raw value raw value 7 - Enterobacter cloacae
  • 184. Amoxicillin R Amo / Clav Ac. R Ticarcillin S Ticar+ clav Ac. S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin R Cefoxitin S Cefotaxime S Ceftazidime S Ceftazidime - 1 S Cefepime S Cefpirome S Tobramycin S Gentamicin R Amikacin S Netilmicin S Nalidixic Ac. R Pefloxacin I Ciprofloxacin S Cotrimoxazole S raw value raw value 8 - Morganella morganii
  • 185. Amoxicillin R Amo / Clav Ac. R Ticarcillin R Ticar+ clav Ac. S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin R Cefoxitin R Cefotaxime I Ceftazidime I Ceftazidime - 1 R Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole S raw value raw value 9 - Serratia marcescens
  • 186. Amoxicillin S Amo / Clav Ac. S Ticarcillin S Ticar+ clav Ac. S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin I Cefoxitin S Cefotaxime S Ceftazidime S Ceftazidime - 1 S Cefepime S Cefpirome S Tobramycin S Gentamicin S Amikacin S Netilmicin S Nalidixic Ac. S Pefloxacin S Ciprofloxacin S Cotrimoxazole S raw value raw value 10 - E.coli
  • 187. Amoxicillin R Amo / Clav Ac. I Ticarcillin R Ticar +clav Ac S Piperacillin S Pip / Tazobactam S Imipenem S Cefalotin R Cefoxitin S C efotaxime S Ceftazidime S Ceftazidime - 1 R Cefepime S Cefpirome S Tobramycin R Gentamicin S Amikacin R Netilmicin S Nalidixic Ac. R Pefloxacin S Ciprofloxacin S Cotrimoxazole R raw value raw value 11- Proteus mirabilis
  • 188. Penicillin G S Oxacillin S AMPI/Sulbact S Cefalotin S Gentamicin S Netilmicin S Erythromycin S Clindamycin S Nitrofurantoïn S Pefloxacin / Q2G S Ciprofloxacin S Rifampicin S Vancomycin S Teicoplanin S Cotrimoxazole S Tetracyclin S raw value raw value 12 - Staphylococcus aureus
  • 189. Penicillin G R Oxacillin S AMPI/Sulbact S Cefalotin S Gentamicin S Netilmicin S Erythromycin S Clindamycin S Nitrofurantoïn S Pefloxacin / Q2G S Ciprofloxacin S Rifampicin S Vancomycin S Teicoplanin S Cotrimoxazole S Tetracyclin S raw value raw value 13 - Staphylococcus aureus
  • 190. Penicillin G R Oxacillin R AMPI/Sulbact S Cefalotin S Gentamicin R Netilmicin S Erythromycin R Clindamycin S Nitrofurantoïn S Pefloxacin / Q2G R Ciprofloxacin S Rifampicin S Vancomycin S Teicoplanin S Cotrimoxazole S Tetracyclin S raw value raw value 14 - Staphylococcus epidermidis
  • 191. Penicillin G R Oxacillin S AMPI/Sulbact S Cefalotin S Gentamicin S Netilmicin S Erythromycin S Clindamycin S Nitrofurantoïn S Pefloxacin / Q2G S Fosfomycine S Rifampicin S Vancomycin S Teicoplanin S Cotrimoxazole S Tetracyclin S raw value raw value 15- Staphylococcus saprophyticus
  • 192. Penicillin Pneumo R Penicillin Strepto R Ampicillin Strepto S Penicillin entero R Ampicillin entero S Cefalotin S Cefuroxime R Piperacillin S Oxacillin R Ciprofloxacin S Tetracyclin R Erythromycin R Clindamycin R Cotrimoxazol S Nitrofurantoïn S Rifampicin S Vancomycin S Teicoplanin S Kanamycin HC R Gentamicin HC I raw value raw value 16 - Enterococcus faecalis
  • 193. Penicillin Pneumo S Penicillin Strepto S Ampicillin Strepto S Penicillin entero S Ampicillin entero S Cefalotin S Cefuroxime S Piperacillin S Oxacillin S Ciprofloxacin S Tetracyclin S Erythromycin S Clindamycin S Cotrimoxazol S Nitrofurantoïn S Rifampicin S Vancomycin S Teicoplanin S Kanamycin HC I Gentamicin HC I raw value raw value 17 - Streptococcus agalactiae
  • 194. Penicillin Pneumo R Penicillin Strepto R Ampicillin Strepto R Penicillin entero R Ampicillin entero I Cefalotin R Cefuroxime R Piperacillin S Oxacillin R Ciprofloxacin S Tetracyclin R Erythromycin R Clindamycin R Cotrimoxazol S Nitrofurantoïn S Rifampicin S Vancomycin S Teicoplanin S Kanamycin HC R Gentamicin HC I raw value raw value 18 - Enterococcus faecium
  • 195. Penicillin Pneumo S Penicillin Strepto S Ampicillin Strepto S Penicillin entero S Ampicillin entero S Cefalotin S Cefuroxime S Piperacillin S Oxacillin S Ciprofloxacin S Tetracyclin S Erythromycin S Clindamycin S Cotrimoxazol S Nitrofurantoïn S Rifampicin S Vancomycin S Teicoplanin S Kanamycin HC I Gentamicin HC I raw value raw value 19 - Streptococcus pyogenes
  • 196. Penicillin Pneumo R Penicillin Strepto S Ampicillin Strepto S Penicillin entero S Ampicillin entero S Cefalotin S Cefuroxime S Piperacillin S Oxacillin R Ciprofloxacin S Tetracyclin S Erythromycin R Clindamycin S Cotrimoxazol R Nitrofurantoïn S Rifampicin S Vancomycin S Teicoplanin S Kanamycin HC I Gentamicin HC I raw value raw value 20 - Streptococcus pneumoniae