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.
Dr.T.V.Rao MD 10 BETA LACTAM RING PENICILLIN BETA LACTAM RING CEPHALOSPORIN BETA LACTAMASES enzymes that inactivate the beta-lactam ring
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 p