ESBL - Dynamics and Detection ESBL

ESBl extended spectrum beta lactamases dynamics and detection : 

ESBl extended spectrum beta lactamases dynamics and detection Dr.T.V.Rao Dr.T.V.Rao MD 1

Survival of the fittest: 

2 Dr.T.V.Rao MD Survival of the fittest Resistant bacteria survive, susceptible ones die Mutant emerges slowly Sensitive cells killed by antibiotic Mutant’s progeny overrun

PowerPoint Presentation: 

3 BETA LACTAM RING PENICILLIN BETA LACTAM RING CEPHALOSPORIN BETA LACTAMASES enzymes that inactivate the beta-lactam ring Dr.T.V.Rao MD

Action of a b-lactamase: 

Action of a b -lactamase N O COOH S HN O COOH S OH Active penicillin Inactive penicilloate H 2 O Dr.T.V.Rao MD 4

Basis of Betalactamse Activity: 

Basis of Betalactamse Activity Time-delayed Growth. Beta-lactamase (red) is produced by the central colony, promoting growth of nearby, non-resistant colonies as it deactivates ampicillin (blue). Diffusion of beta lactamase through agar leads to time-delayed growth of non-resistant colonies Dr.T.V.Rao MD 5

b-lactam antibiotics: 

b -lactam antibiotics Penicillin's Ampicillin Piperacillin Beta-lactam/beta-lactamase inhibitors Ampicillin/sulbactam Amoxicillin/clavulanate Ticarcillin/clavulanate Piperacillin/Tazobactam Dr.T.V.Rao MD 6

Penicillins: 

Penicillins 1 st gen – strep infection (G+) Ex. Penicillin, Cloxacillin Extended-spectrum – have broader spectrum against G- including E.Coli Ex. Amoxicillin With inhibitor would protect against some beta-lactamase producers Ex. Amoxicillin/Clav Broad spectrum – many Enterobacteriaceae Ex Piperacillin/Tazobactam Dr.T.V.Rao MD 7

PowerPoint Presentation: 

Some beta-lactamases only inactivate a small number of antibiotics e.g. penicillin Others have extended spectrum to all the penicillins and cephalosporins e.g. cefuroxime, ceftriaxone ( ESBLs ) In addition may also carry resistance to other antibiotics e.g. ciprofloxacin. 8 Dr.T.V.Rao MD

Definition of ESBL : 

Dr.T.V.Rao MD 9 Definition of ESBL : Class A by Ambler or Group 2be by Bush classifications Typically, enzymes are plasmid-mediated derived from older ß-lactamases of TEM and SHV In early 2000s, CTX-M derived ß-lactamases are included

ESBL Evolution: 

ESBL Evolution Mid 1980s Variants of TEM and SHV Breakdown 3 rd generation cephalosporins Mainly in hospital Klebsiella Spread world wide 10 Dr.T.V.Rao MD

What are extended-spectrum β-lactamases?: 

What are extended-spectrum β- lactamases? ESBLs are enzymes that mediate resistance to extended-spectrum (third generation) cephalosporins (e.g., ceftazidime, cefotaxime, and ceftriaxone) and monobactams (e.g., aztreonam) but do not affect cephamycins (e.g., cefoxitin and Cefotetan) or carbapenems (e.g., meropenem or imipenem). Dr.T.V.Rao MD 11

Ambler Classification of β-Lactamases: 

12 Ambler Classification of β -Lactamases Active site Nucleotide sequence Four evolutionarily distinct molecular classes A C D Serine-enzymes B Zinc-enzymes β -lactamases Dr.T.V.Rao MD

What is a beta-lactam?: 

What is a beta-lactam? Abx Penicillin Cephalosporin Monobactam Carbapenem Bacteriocidal Google Images Dr.T.V.Rao MD 13

Cephalosporins: 

Cephalosporins Willey, et al., 2008 1 st 2 nd 3 rd 4 th Dr.T.V.Rao MD 14

Cephalosporins-uses: 

Cephalosporins-uses 1 st gen: strep, staph, G- including E.coli Ex. Cefazolin 2 nd gen: greater spectrum against G- Ex. Cefoxitin 3 rd gen: even greater activity, combat narrow-spectrum beta-lactamase producers  ESBLs emerged Ex. Ceftazidime 4 th gen: effective against G- bacilli expressing Xm AmpC resistant to 3 rd gen Ex. Cefepime Dr.T.V.Rao MD 15

Others: 

Others Monobactams Monobactams very active against G- including E.coli Ex. Aztreonam Carbapenems Carbapenems have an extremely broad spectrum. Cross-reactivity with penicillins or cephalosporins Ex. Imipenam Dr.T.V.Rao MD 16

The Fight goes on ..: 

The Fight goes on .. Beta-lactam Beta-lactamase Beta-lactamase inhibitor ESBL Google Images Dr.T.V.Rao MD 17

Common ESBL producers: 

Klebsiella pneumoniae Escherichia coli Proteus mirabilis Enterobacter cloacae Non-typhoidal Salmonella (in some countries) Common ESBL producers Dr.T.V.Rao MD 18

The Fight Beta-Lactam: 

The Fight Beta-Lactam cell PG N O LYSIS Dr.T.V.Rao MD 19

The Fight Beta-lactamase: 

The Fight Beta-lactamase cell PG N O beta-lactamase Dr.T.V.Rao MD 20

The Fight Beta-lactamase: 

The Fight Beta-lactamase cell PG NH O OH Dr.T.V.Rao MD 21

The Fight Beta-lactamase inhibitor: 

The Fight Beta-lactamase inhibitor cell PG N O beta-lactamase Inhibitor Dr.T.V.Rao MD 22

The Fight Beta-lactamase inhibitor: 

The Fight Beta-lactamase inhibitor cell PG N O beta-lactamase Inhibitor LYSIS Dr.T.V.Rao MD 23

ESBLs: 

ESBLs Enterobacteriaceae Resistance to oxyimino-cephalosporins and Monobactams but not cephamycins and carbapenem Susceptible to beta-lactamase inhibitors Genes SHV TEM CTX-M OXA AmpC Oteo, et al., 2010 Dr.T.V.Rao MD 24

PowerPoint Presentation: 

Dr.T.V.Rao MD Plasmid-mediated TEM and SHV -lactamases Ampicillin 1965 TEM-1 E.coli S.paratyphi 1970s TEM-1 Reported in 28 Gm(-) sp 1983 ESBL in Europe 1988 ESBL in USA 2000 > 130 ESBLs Worldwide Extended-spectrum Cephalosporins 1963 Evolution of  -Lactamases Look and you will find ESBL 25

Classification of β lactamases: 

Classification of β lactamases Richards and Sykes (1971) substrate Ambler (1969) structure Bush, Jacoby, Medeiros (1995) Substrate; correlation with molecular structure 150 TEM; 88 SHV; 88 OXA, 53 CTX-M; 22 IMP; 12 VIM + smaller number of other enzymes (http://www.lahey.o Dr.T.V.Rao MD 26

Classification: 

Classification Ambler Classification Molecular class A – D A Bush-Jacoby-Medeiros Classification Functional group 1 – 4 2 2b 2be Paterson and Bonomo, 2005 Dr.T.V.Rao MD 27

Beta-lactamase inhibitors: 

28 Beta-lactamase inhibitors Resemble β-lactam antibiotic structure Bind to β-lactamase and protect the antibiotic from destruction Most successful when they bind the β-lactamase irreversibly Three important in medicine Clavulanic acid Sulbactam Tazobactam Dr.T.V.Rao MD

Resistance and genetics: 

Resistance and genetics AmpC Hi-level TEM ESBL CTX-M K1 Ceftazidime R R v S Cefotaxime R v R S Cefoxitin R S S S Aztreonam R v v R Synergy + clav No +++ +++ No Know the species Dr.T.V.Rao MD 29

PowerPoint Presentation: 

Why Test for β -lactamases ? Improve clinical outcome Inappropriate treatment leads to poor outcome Each 1 hour delay increases mortality by 7.6% in septic shock 1 Encourage antimicrobial stewardship Spare carbapenems.. Reduce C. difficile / antibiotic associated diarhoea Enhanced surveillance Identify emerging resistance problems Develop structures to prevent dissemination Infection Control ‘Search and Destroy’ analogous to MRSA ? Laboratory Detection is not always easy… OR Rapid 1 Kumar, Crit Care Med , 2006

Types of ESBLs: 

31 Types of ESBLs TEM SHV CTX-M OXA Mutations ESBL Phenotype Plasmid-mediated Dr.T.V.Rao MD

Choice of Indicator Cephalosporin: 

32 Choice of Indicator Cephalosporin TEM & SHV – obvious resistance to ceftazidime, variable to cefotaxime CTX-M – obvious resistance to cefotaxime, variable to ceftazidime All ESBLs – obvious resistance to cefpodoxime Cefuroxime, cephalexin and cephradine are unreliable indica tors Livermore D and Woodford N HPA Guidance 2004

Current Modern Methods : 

Slide 33 Current Modern Methods CLSI – Clinical Laboratory and Standards Institute ARMRL - Antibiotic Resistance Monitoring and Reference Laboratory, Health Protection Agency Centre for Infections, London EUCAST- European Society of Clinical Microbiology & Infectious Diseases Commercial methods – Etest, BD Phoenix, Vitek Neo tabs & others

Detection of ESBLs: 

Detection of ESBLs Seek ceph/clav synergy in ceph R isolates Double disc Combination disc Etest Dr.T.V.Rao MD 34

Challenges for the diagnostic lab: 

Challenges for the diagnostic lab Detection…. Hemophilus , Neisseria etc. Predicting b -lactamase types. Have GNB got ?: ESBL, AmpC Metallo types, VIM, IMP etc … S potting unusual patterns; knowing what to refer ???

Detection Strategy: step 1: 

Detection Strategy: step 1 Screen Enterobacteriaceae with : Cefpodoxime - best general ESBL substrate Cefotaxime & ceftazidime - good substrates for CTX-M & TEM/SHV, respectively Dr.T.V.Rao MD 36

Combination disk method Carter MW et al: J Clin Microbiol 2000; 38: 4228 - 4232: 

Combination disk method Carter MW et al: J Clin Microbiol 2000; 38: 4228 - 4232 Difference > 5 mm Dr.T.V.Rao MD 37

PowerPoint Presentation: 

Dr.T.V.Rao MD 38

PowerPoint Presentation: 

Dr.T.V.Rao MD 39

Klebsiella pneumonia producing a higher activity ESBL: 

Klebsiella pneumonia producing a higher activity ESBL The higher level of ESBL production is indicated by the inhibition of the β‑lactamase by clavulanic acid and the resulting elliptical inhibitory zone between cefotaxime (CTX 5) and Augmentin (AMC 60). Dr.T.V.Rao MD 40

PowerPoint Presentation: 

Double disc antagonism for inducible AmpC Cefoxitin Ceftazidime Dr.T.V.Rao MD 41

AmpC inducibility- when to look: 

AmpC inducibility- when to look Rarely!!!!! Risk is mutation, not inducibility per se Best to identify & predict risk from species Biggest risk Enterobacter & C freundii Avoid cephalosporins against them Identify means identify TO SPECIES LEVEL all Enterobacteriaceae (‘coliforms’) ex serious infections

ESBLs Detection Methods: Inhibition by Clavulanic Acid: 

43 ESBLs Detection Methods: Inhibition by Clavulanic Acid Co-amoxiclav disc surrounded by cefotaxime, ceftriaxone, ceftazidime and aztreonam discs (30 mcg each)

ESBL detection: 

ESBL detection Screen cefpodoxime ; cefotaxime & ceftazidime Synergy test with ceph/clav Combination discs are most cost effective synergy tests; Etests a good alternative.. or automate Dr.T.V.Rao MD 44

PowerPoint Presentation: 

ESBL Confirmatory Test Positive for ESBL Ceftaz/CA Cefotax/CA Ceftaz Cefotax 45 Dr.T.V.Rao MD 45

ESBL Confirmatory Test Negative for ESBL : 

46 ESBL Confirmatory Test Negative for ESBL Ceftaz/CA Cefotaxime/CA Ceftaz Cefotax 46 Dr.T.V.Rao MD

ESBL Confirmatory Test: 

ESBL Confirmatory Test Ceftaz/CA Ceftaz Etest 47 Dr.T.V.Rao MD 47

ESBLs: times a’ changing with CTX-M: 

ESBLs: times a’ changing with CTX-M Old advice- test ceftazidime; ESBL test if R New advice- test ceftazidime & cefotaxime; ESBL test if R to either Alternative- test cefpodoxime; ESBL test if R Still true- Only testing cefuroxime is inadequat e Dr.T.V.Rao MD 48

Comparing Disk diffusion with minimum inhibitory concentrations: 

Comparing Disk diffusion with minimum inhibitory concentrations Disk diffusion MICs cefpodoxime < 22 mm cefpodoxime > 2 µg/ml ceftazidime < 22 mm ceftazidime > 2 µg/ml aztreonam < 27 mm aztreonam > 2 µg/ml cefotaxime < 27 mm cefotaxime > 2 µg/ml ceftriaxone < 25 mm ceftriaxone > 2 µg/ml Dr.T.V.Rao MD 49

ESBL Confirmatory Tests: 

ESBL Confirmatory Tests Double-disk synergy (DDS) test CAZ and CAZ/CA disks CTX and CTX\CA disks Confirmatory testing requires using both CAZ and CTX alone and with CA 5 mm enhancement of the inhibition zone of antibiotic/CA combination vs antibiotic tested alone = ESBL Dr.T.V.Rao MD 50

Synergy tests with 4-gen cephalosporins: 

Synergy tests with 4-gen cephalosporins Cefepime/clav (Mast & AB Biodisk) Cefpirome clav (Oxoid) Devt. driven by spread of clonal E. aerogenes with TEM-24 in Belgium & France Sensitivity for weak ESBLs remains to be proven Cefpirome & cefepime products need comparison Dr.T.V.Rao MD 51

Pitfalls in ESBL detection: 

Pitfalls in ESBL detection Methods optimised for E. coli & Klebsiella More difficult with Enterobacter clavulanate induces AmpC; hides ESBL Best advice is to do synergy test (NOT SCREEN) with 4 th gen ceph Dr.T.V.Rao MD 52

Risk Factors for ESBL Infection: 

53 Risk Factors for ESBL Infection Length of hospital stay Severity of illness Time in the ICU Intubation and mechanical ventilation Urinary or arterial catheterization Previous exposure to antibiotics Dr.T.V.Rao MD

Bacteria not to test for ESBL’s: 

Bacteria not to test for ESBL’s Acinetobacter Often S to clavulanate alone S. maltophilia +vet result by inhibition of L-2 chromosomal b -lactamase, ubiquitous in the species Dr.T.V.Rao MD 54

ESBL Reporting Rule: 

ESBL Reporting Rule The rule (CLSI =NCCLS) M100-S15) … “ Strains of Klebsiella spp. E. coli, and Proteus mirabilis that produce ESBLs may be clinically resistant to therapy with penicillin's, cephalosporins, or aztreonam, despite apparent in vitro susceptibility to some of these agents. ” The message … Report “ confirmed ” ESBL-producing strains as R to all penicillin's, cephalosporins, and aztreonam 55 Dr.T.V.Rao MD 55

Will CLSI confirmatory test detect ALL ESBL-producing GNB?: 

Will CLSI confirmatory test detect ALL ESBL -producing GNB? No - some isolates have ESBLs plus other resistance mechanisms that mask ESBL detection in the confirmatory test, e.g., > 1 ESBL ESBL + AmpC ESBL + porin mutation ESBLs occur in species other than E. coli, Klebsiella spp., and Proteus mirabilis which CLSI does not currently address 56 Dr.T.V.Rao MD 56

ESBL Detection: Automated Systems (AS): 

144 putative of ESBL producers ESBL detection: AS: Microscan, Vitek2, Phoenix Phenotypic tests: Etest, DDS Molecular tests: PCR, IsoElectric Focusing (IEF) Molecular identification: the reference method ESBL Detection: Automated Systems (AS) Dr.T.V.Rao MD 57

The resistance becoming complex: 

The resistance becoming complex Beta-lactamases are getting more complex Full I/D needs complex molecular methods Much can be inferred from simple tests. Needs I/D Testing wide panels of antibiotics; synergy tests Knowledge of what’s unusual Dr.T.V.Rao MD 58

Antibiotic Policy changes practised : 

Antibiotic Policy changes practised Nitrofurantion substituted for quinolones in UTIs Imipenem substituted for quinolones in serious sepsis Ertapenem introduced for ESBL sepsis Gentamicin substituted for cephalosporins in surgical prophylaxis Return to amoxycillin in respiratory tract infections 59 Dr.T.V.Rao MD

Microbiology Laboratories and ESBL’s: 

60 Microbiology Laboratories and ESBL’s Unfortunately, many clinical laboratories lack of understanding regarding ESBLs and Ampc ß-lactamase and their detection .This has been documented in a study in Connecticut USA, where it was found that 21% of laboratories failed to detect extended –spectrum cephalosporins and Aztreonam in ESBLs and Ampc . The true prevalence of ESBLs is not known and is probably underestimated because of difficulties encounter in their detection. However, it is clear that ESBLs –producing organisms are distributed worldwide and their prevalence is increasing. Dr.T.V.Rao MD

Hand washing still can reduce the ESBL spread: 

Hand washing still can reduce the ESBL spread Dr.T.V.Rao MD 61

PowerPoint Presentation: 

The programme created by Dr.T.V.Rao MD for basic understanding by Medical Microbiologists in the Developing World Email doctortvrao@gmail.com Dr.T.V.Rao MD 62