Resistance to  -lactam antibiotics within the Enterobacteriaceae Paul D. Fey, Ph. D. University of Nebraska Medical Center

Objectives Understand the mechanisms of  -lactam resistance in GNRs Understand how  -lactamases are transferred. Know the characteristics of three classes of  -lactamases ESBLs AmpC KPC

Mechanism of  -Lactam Action Bactericidal  -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

Mechanisms of GNR Resistance to  - lactams Porin-mediated resistance Antibiotic does not reach target.  -lactamase Majority of resistance to  -lactam antibiotics mediated through  -lactamases. Many different types of  -lactamases with different substrate (antibiotic) specificities.

Common  -Lactamases in GNRs NEJM 352:

How are  -lactamases transferred? Transfer of Plasmids. Extrachromosomal 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

 -lactam antibiotics Penicillins Ampicillin Piperacillin Beta-lactam/beta-lactamase inhibitors Ampicillin/sulbactam Amoxicillin/clavulanate Ticarcillin/clavulanate Piperacillin/Tazobactam

 -lactam antibiotics First Generation cephalosporins Cefazolin Cephalothin Second Generation oral antibiotics Cefuroxime (many others) Second Generation cephamycins Cefoxitin Cefotetan

 -lactam antibiotics Third generation cephalosporins Cefotaxime Ceftriaxone Ceftazidime Fourth generation cephalosporins Cefepime Monobactams Aztreonam

 -lactam antibiotics Carbapenems Imipenem Meropenem Ertapenem Doripenem

ESBLs Enzymes capable of hydrolyzing third-generation cephalosporins. Plasmid-mediated Derivatives (mutants) of original TEM-1 and SHV-1  - lactamases. Susceptible in-vitro to clavulanate and cefoxitin.

E. coli susceptibility Report AmpicillinR PiperacillinR CephalothinR CefoxitinS CefotaximeR CeftazidimeI CeftriaxoneR AztreonamI CefepimeS Pip/TazoI ImipenemS

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

E. coli ESBL susceptibility report AmpicillinR PiperacillinR CephalothinR CefoxitinS CefotaximeR CeftazidimeI  R CeftriaxoneR AztreonamI  R CefepimeS  R Pip/TazoI ImipenemS

Enterobacter cloacae susceptibility report AmpicillinR PiperacillinR CephalothinR CefoxitinR CefotaximeR CeftazidimeI CeftriaxoneR AztreonamI CefepimeS Pip/TazoR ImipenemS

AmpC  -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  -lactamase Third-generation cephalosporin resistant strains are derepressed—meaning that the AmpC  - lactamase is not inducible anymore. AmpC mutants are cephamycin resistant

E. coli susceptibility report AmpicillinR PiperacillinR CephalothinR CefoxitinR CefotaximeR CeftazidimeI CeftriaxoneR AztreonamI CefepimeS Pip/TazoR ImipenemS

What do you do?

Other concepts to know about AmpC  -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  - lactamase called CMY-2. E. coli UTI isolates carry plasmid-mediated AmpC  - lactamases

Mechanisms of Carbapenem Resistance Carbapenemase hydrolyzing enzymes Porin loss “OprD” ESBL or AmpC + porin loss

Carbapenemases The most versatile family of  -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 ABD Functional Group 2f32d Aztreonam Hydrolysis +-- EDTA Inhibition -+- Clavulanate Inhibition +- 

Klebsiella pneumoniae AmpicillinR PiperacillinR CephalothinR CefoxitinS CefotaximeR CeftazidimeI CeftriaxoneR AztreonamI CefepimeS Pip/TazoR ImipenemI 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  -lactamases (IMI) Plasmid encoded Klebsiella pneumoniae carabapenemases (KPC) Guiana Extended-Spectrum (GES)

KPC Molecular class A and functional group 2f Inhibited by clavulanic acid but not by EDTA Confers resistance to ALL  -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

KPC Epidemiology KPC producers have been identified outside USA France Brazil Columbia China Not detected at the University of Nebraska Medical Center 45 ESBL-like isolates collected-6 had elevated carbapenem MICs-none contained KPC

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 JCM 45 (8): 2723

How to Detect a KPC Producer Commercial systems Inconsistent detection of KPC-producing isolates Tenover et al EID. 12: 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 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. meropenemertapenem imipenem pos neg neg neg pos pos Janet Hindler, What’s 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 Feb;52(2):570-3 Minocycline A strategy for susceptibility testing is needed

Conclusions ESBL detection—CLSI 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.