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Resistance to b-lactam Antibiotics revisited Dr.T.V.Rao MD Dr.T.V.Rao MD 1

Resistance to b lactam antibiotics

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Resistance to b lactam antibiotics

Text of Resistance to b lactam antibiotics

  • Resistance to b-lactam Antibiotics revisited Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  • -lactam Antibiotics The -lactam ring is part of the core structure of several antibiotic families, the principal ones being the penicillins, cephalosporins, carba penems, and monobactams, which are, therefore, also called - lactam antibiotics. Nearly all of these antibiotics work by inhibiting bacterial cell wall biosynthesis. This has a lethal effect on bacteria
  • History of -lactams The first synthetic -lactam was prepared by Hermann Staudinger in 1907 by reaction of the Schiff base of aniline and benzaldehyde with diphenylketene in a cycloaddition: Upto 1970, most -lactam research was concerned with the penicillin and cephalosporin groups, but since then a wide variety of structures have been described.
  • Survival of the fittest Dr.T.V.Rao MD 4 Resistant bacteria survive, susceptible ones die Mutant emerges slowly Sensitive cells killed by antibiotic Mutants progeny overrun
  • Action of a b-lactamase N O COOH S HN O COOH S OH Active penicillin Inactive penicilloateH2O
  • Mechanism of b-Lactam Action Bactericidal b-lactams bind and inhibit penicillin binding proteins (PBPs) PBPs are responsible for assembly, maintenance, and regulation of peptidoglycan (cell wall) metabolism. Disruption of peptidoglycan synthesis
  • Spread of TEM plasmid b-lactamases 1963 Ampicillin; 1st broad spectrum penicillin 1965 TEM b-lactamases in E. coli 1969 TEM b -lactamase in P. aeruginosa 1974 TEM in H. influenzae & N. gonorrhoeae Now TEM in 30-60% E. coli & enterobacteria & in 5- 20% of H. influenzae & gonococci
  • PPID, 6th ed. 2005 ESBL Introduction B-lactamases conferring resistance to the penicillin's, first- second-, and third-generation cephalosporins and aztreonam Mechanism is via hydrolysis Inhibited by B-lactamase inhibitors such as clavulanic acid B-lactamases in group 2d and group 2be Group 2b: TEM-1, TEM-2, & SHV-1 Group 2d: OXA B-lactamase in group 1 AmpC*
  • Mechanisms of GNR Resistance to b-lactams Porin-mediated resistance Antibiotic does not reach target b-lactamase Majority of resistance to b- lactam antibiotics mediated through b-lactamases. Many different types of b- lactamases with different substrate (antibiotic) specificities.
  • How are b-lactamases transferred? Transfer of Plasmids. Extra chromosomal DNA Usually carry antibiotic resistance genes These genes can be encoded on transposons, which are also mobile. TEM-1 has been transferred between the Enterobacteriaceae and H. influenzae and the Neisseriaceae
  • b-lactam antibiotics Penicillins Ampicillin Piperacillin Beta-lactam/beta-lactamase inhibitors Ampicillin/sulbactam Amoxicillin/clavulanate Ticarcillin/clavulanate Piperacillin/Tazobactam
  • The -lactam family of antibiotics Ceftriaxone 3rdTicarcillin Ceftazidime 3rdMezlocillin Cefotaxime 3rdCarbenicillin ErtapenemCefmetazoleCefuroxime 2ndAmpicillin MeropenemCefotetanCefamandole 2ndMethicillin AztreonamImipenemCefoxitinCephalothin 1stBenzyl- penicillin MonobactamsCarbapenemsCephamycinsCephalosporinsPenicillins Cefepime 4th
  • b-lactam antibiotics First Generation cephalosporins Cefazolin Cephalothin Second Generation oral antibiotics Cefuroxime (many others) Second Generation cephamycins Cefoxitin
  • b-lactam antibiotics Third generation cephalosporins Cefotaxime Ceftriaxone Ceftazidime Fourth generation cephalosporins Cefepime Monobactams Aztreonam
  • b-lactam antibiotics Carbapenems Impenem Meropenem Ertapenem Doripenem
  • 17 Plasmid-mediated TEM and SHV b-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 b-Lactamases Look and you will find ESBL
  • Classification of lactamases Dr.T.V.Rao MD 18 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
  • Classification Dr.T.V.Rao MD 19 Ambler Classification Molecular class A D A Bush-Jacoby-Medeiros Classification Functional group 1 4 2 2b 2be Paterson and Bonomo, 2005
  • ESBLs Enzymes capable of hydrolyzing third-generation cephalosporins. Plasmid-mediated Derivatives (mutants) of original TEM-1 and SHV-1 b- lactamases. Susceptible in-vitro to clavulanate and cefoxitin.
  • Clin Microbiol Rev. 2005;18:657-686 J Clin Microbiol.2001;39:2206-2212. ESBL In Vitro Susceptibility NCCLs established breakpoints 1980s In vitro, MICs of ceph rise as inoculum of ESBL prod organisms rise inoculum effect NCCLs subcommittee convened working group recommending K. spp and E. coli screened for ESBL prod Suspected ESBL tested for phenotypic confirmation 1998 survey of 369 laboratories only 32% performed tests to detect ESBL production Most liberal interpretation of ceph susceptibility by CLSI w/ MIC=8ug/ml
  • Clin Microbiol Infect. 2008;14:169-174. ESBL In Vitro Susceptibility Currently accepted that cephalosporin breakpoints used in Europe (EUCAST) and US (CLSI) fail to detect most ESBL Published data suggests that clinical outcome with 3rd gen ceph related more to MICs and not presence of ESBL arguing against inoculum effect New breakpoints adopted by EUCAST March 2006 Existing breakpoints do not allow for detection of important resistance mechanisms Question if breakpoints correlate with clinical outcome Controversy re: contradicting 3rd gen ceph as S or R is ESBL pos CLSI Working Group on Enterobacteriacea have been proposed but not accepted as of Jan 2008 Suggested CLSI breakpoints for senstivity pre/post (ug/ml) Cefuroxime (8/8), Cefotaxime (8/1 ), Ceftriaxone (8/1), Ceftazidime (8/4), Cefepime (8/8)
  • E. coli susceptibility Report Ampicillin R Piperacillin R Cephalothin R Cefoxitin S Cefotaxime R Ceftazidime I Ceftriaxone R Aztreonam I Cefepime S Pip/Tazo I Imipenem S
  • Laboratory detection of ESBLs Resistance or intermediate to third-generation cephalosporins. Cefoxitin and cefotetan susceptible. ESBL disk diffusion test (clavulanate inhibition) E-test ESBL strip Confirmatory ESBL MIC test (Microscan) K. pneumoniae, K. oxytoca, E. coli, P. mirabilis
  • Double disc antagonism for inducible AmpC Cefoxitin Ceftazidime
  • ESBL Confirmatory Tests Double-disk synergy (DDS) test CAZ and CAZ/CA disks CTX and CTXCA 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
  • Combination Disk Method CLSI Approved Method
  • 28AmpC Disk Test Lawn culture: E. coli ATCC 25922 Test Organism on disk
  • E. coli ESBL susceptibility report Ampicillin R PiperacillinR Cephalothin R Cefoxitin S Cefotaxime R Ceftazidime IR Ceftriaxone R AztreonamIR Cefepime SR Pip/Tazo I Imipenem S
  • Enterobacter cloacae susceptibility report Ampicillin R PiperacillinR Cephalothin R Cefoxitin R Cefotaxime R Ceftazidime I Ceftriaxone R AztreonamI Cefepime S Pip/Tazo R Imipenem S
  • AmpC b-lactamases Chromosomally encoded-cell wall turnover Enterobacter sp., Citrobacter sp., Serratia sp., Morganella sp. Even E. coli. Third-generation cephalosporins are not good inducers of AmpC b-lactamase Third-generation cephalosporin resistant strains are derepressedmeaning that the AmpC b- lactamase is not inducible anymore. AmpC mutants are cephamycin resistant
  • Other concepts to know about AmpC b-lactamases They are transferred on plasmids as well. CMY, LAT, BIL, MOX, ACC, FOX, DHA Almost all ceftriaxone-resistant Salmonella isolated in the United States carry a plasmid-mediated AmpC b-lactamase called CMY-2. E. coli UTI isolates carry plasmid-mediated
  • Beta-lactamase inhibitors Dr.T.V.Rao MD Resemble -lactam antibiotic structure Bind to -lactamase and protect the antibiotic from destruction Most successful when they bind the -lactamase irreversibly Three important in medicine Clavulanic acid Sulbactam Tazobactam
  • 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 Dr.T.V.Rao MD 34 Know the species
  • Why Test for -lactamases ? Improve clinical outcome Inappropriate treatment leads to poor outcome Each 1 hour delay increases mortality by 7.6% in septic shock1 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 1Kumar, Crit Care Med, 2006
  • Clin Microbiol Rev. 2005;18:657-686. ESBL Epidemiology North America National Nosocomial Infections Surveillance (NNIS) Jan 1998-June 2002 6.1% of Klebsiella pneumoniae isolates resistant to 3rd gen ceph in 110 ICUs >10% of ICUs, resistance exceeds 25% Non-ICU inpt, 5.7% of Klebsiella pneumoniae isolates resistant Outpt, 1.8% of Klebsiella pneumoniae resistant Prevalence of ESBL underestimated due to MIC S/I Europe France in early 1990s, 25-35% of nococomial Klebsiella pneumoniae were ESBL producing N. France in 2000, 7.9% of nosocomial Klebsiella pneumoniae were ESBL producing Discordance between Western and Eastern Europe
  • Mechanisms of Carbapenem Resistance Carbapenemase hydrolyzing enzymes Porin loss OprD ESBL or AmpC + porin loss
  • Carbapenemases The most versatile family of b-lactamases Two major groups based on the hydrolytic mechanism at the active site Serine at the active site: class A and D Zinc at the active site: class B All carbapenemases hydrolyze penicillins, extended spectrum cephalosporins, and carbapenems
  • Carbapenemase Classification Molecular Class A B D Functional Group 2f 3 2d Aztreonam Hydrolysis + - - EDTA Inhibition - + - Clavulanate Inhibition + -
  • Klebsiella pneumoniae Ampicillin R PiperacillinR Cephalothin R Cefoxitin S Cefotaxime R Ceftazidime I Ceftriaxone R AztreonamI Cefepime S Pip/Tazo R Imipenem I Might need to screen for carbapenemase
  • Carbapenemases Class A First identified 1982 in UK Four major families Chromosomally encoded Serratia marcescens enzyme (SME) Not metalloenzyme carbapenemases (NMC) Imipenem-hydrolyzing b-lactamases (IMI) Plasmid encoded Klebsiella pneumoniae carabapenemases (KPC) Guiana Extended-Spectrum (GES)
  • Etest for metallo-b-lactamase Imipenem Imipenem + EDTA
  • Etest for metallo-b-lactamase Imipenem Imipenem + EDTA
  • KPC Molecular class A and functional group 2f Inhibited by clavulanic acid but not by EDTA Confers resistance to ALL b-LACTAM antibiotics Plasmid-encoded Associated with other resistant genes (aminoglycosides, fluoroquinolones) Transferable
  • KPC Epidemiology Predominantly in K. pneumoniae (KP) Reported in Enterobacter spp., Salmonella spp., E. coli, P. aeruginosa, and Citrobacter spp. First identified in KP clinical isolate from North Carolina in 1996 (KPC- 1) KPC-2, -3, and -4 have been reported. Mostly identified on the East cost
  • When to Suspect a KPC Producer Enterobacteriaceae Resistance to extended spectrum cephalosporins (cefotaxime, ceftazidime, and ceftriaxone) Variable susceptibility to cephamycins (cefoxitin, cefotetan) Carbapenem MICs 2 g/ml
  • How to Detect a KPC Producer Antimicrobial susceptibility tests (ASTs) MIC Carbapenem MIC 2 g/ml Disk diffusion Carbapenem: I or R Among carbapenems, ertapenem: Most sensitive less specific Anderson et al. 2007. JCM 45 (8): 2723
  • How to Detect a KPC Producer Commercial systems Inconsistent detection of KPC-producing isolates Tenover et al. 2006. EID. 12:1209-1213 Breakpoints do not match CLSI recommendations
  • Definitive ID of a KPC Producer Modified Hodge test 100% sensitivity to detect KPC 1. Swab E. coli ATCC 25922 onto plate to create lawn Place imipenem disk in center. 2. Streak test isolates from edge of disk to end of plate. 3. Incubate overnight. 4. Look for growth of E. coli around test isolate streak - indicates carbapenem-hydrolyzing enzyme. meropenem ertapenem imipenem pos neg neg neg pos pos Janet Hindler, Whats New in the 2008 CLSI Standards for (AST)?
  • Definitive ID of a KPC Producer PCR The method of choice to confirm KPC
  • Alternative Treatment for a KPC Producer Tigecycline (100.0% effective) Colistin (88.1% effective) SENTRY report. AAC. 2008. Feb;52(2):570-3 Minocycline A strategy for susceptibility testing is needed
  • Clin Microbiol Rev. 2005;18:657-686. ESBL Antibiotic Choice Cefepime should not be used as first-line against ESBL-producing organisms MICs rise with inoculum effect size High dose 2 gm iv 12 +/- amikacin B-lactam/B-lactamase inhibitor MICs rise with inoculum size Reduced activity in presence of porin loss and b-lactamase production Quinolones option for complicated UTI due to ESBL organism In vitro synergy with fq + b-lactam (cefotax) Carbapenems first line for serious ESBL organisms Meropenem preferred over Imipenem for nosocomial meningitis No evidence of combination superior to alone
  • Conclusions ESBL detectionCLSI guidelines present Need to have guidelines to detect ESBLs present in other species besides E. coli, K. pneumoniae, K. oxytoca, and P. mirabilis. AmpC detection-No guidelines available KPC detection-Not widespread, need to have lower concentrations of carbapenems on panels.
  • The message 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 whats unusual
  • Hand washing still can reduce the ESBL spread in the Hospitals Dr.T.V.Rao MD 55
  • Dr.T.V.Rao MD 56 The Programme created by Dr.T.V.Rao MD for Medical Microbiologists in the Developing World Email [email protected]