Title: Resistance to b-lactam antibiotics within the Enterobacteriaceae
1- Resistance to b-lactam antibiotics within the
Enterobacteriaceae - Paul D. Fey, Ph. D.
- University of Nebraska Medical Center
-
2Objectives
- Understand the mechanisms of ?-lactam resistance
in GNRs - Understand how ?-lactamases are transferred.
- Know the characteristics of three classes of
?-lactamases - ESBLs
- AmpC
- KPC
3Mechanism 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
4Mechanisms of GNR Resistance to ?-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.
5Common ?-Lactamases in GNRs
NEJM 3524. 2005
6How are b-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 -
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12b-lactam antibiotics
- Penicillins
- Ampicillin
- Piperacillin
- Beta-lactam/beta-lactamase inhibitors
- Ampicillin/sulbactam
- Amoxicillin/clavulanate
- Ticarcillin/clavulanate
- Piperacillin/Tazobactam
13b-lactam antibiotics
- First Generation cephalosporins
- Cefazolin
- Cephalothin
- Second Generation oral antibiotics
- Cefuroxime (many others)
- Second Generation cephamycins
- Cefoxitin
- Cefotetan
14b-lactam antibiotics
- Third generation cephalosporins
- Cefotaxime
- Ceftriaxone
- Ceftazidime
- Fourth generation cephalosporins
- Cefepime
- Monobactams
- Aztreonam
15b-lactam antibiotics
- Carbapenems
- Imipenem
- Meropenem
- Ertapenem
- Doripenem
16ESBLs
- 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.
17E. 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
18Laboratory 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
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20E. coli ESBL susceptibility report
- Ampicillin R
- Piperacillin R
- Cephalothin R
- Cefoxitin S
- Cefotaxime R
- Ceftazidime I?R
- Ceftriaxone R
- Aztreonam I?R
- Cefepime S?R
- Pip/Tazo I
- Imipenem S
21Enterobacter cloacae susceptibility report
- Ampicillin R
- Piperacillin R
- Cephalothin R
- Cefoxitin R
- Cefotaxime R
- Ceftazidime I
- Ceftriaxone R
- Aztreonam I
- Cefepime S
- Pip/Tazo R
- Imipenem S
22AmpC 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
23E. coli susceptibility report
- Ampicillin R
- Piperacillin R
- Cephalothin R
- Cefoxitin R
- Cefotaxime R
- Ceftazidime I
- Ceftriaxone R
- Aztreonam I
- Cefepime S
- Pip/Tazo R
- Imipenem S
24 25Other 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 AmpC
b-lactamases
26Mechanisms of Carbapenem Resistance
- Carbapenemase hydrolyzing enzymes
- Porin loss OprD
- ESBL or AmpC porin loss
27Carbapenemases
- 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
28Carbapenemase Classification
Molecular Class A B D
Functional Group 2f 3 2d
Aztreonam Hydrolysis - -
EDTA Inhibition - -
Clavulanate Inhibition - ?
29Klebsiella pneumoniae
- Ampicillin R
- Piperacillin R
- Cephalothin R
- Cefoxitin S
- Cefotaxime R
- Ceftazidime I
- Ceftriaxone R
- Aztreonam I
- Cefepime S
- Pip/Tazo R
- Imipenem I
- Might need to screen for carbapenemase
30Carbapenemases 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)
31KPC
- 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
32KPC 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
33KPC 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
34When 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
35How 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
36How to Detect a KPC Producer
- Commercial systems
- Inconsistent detection of KPC-producing isolates
- Tenover et al. 2006. EID. 121209-1213
- Breakpoints do not match CLSI recommendations
37Definitive ID of a KPC Producer
- Modified Hodge test
- 100 sensitivity to detect KPC
- Swab E. coli ATCC 25922 onto plate to create lawn
Place imipenem disk in center. - Streak test isolates from edge of disk to end of
plate. - Incubate overnight.
- Look for growth of E. coli around test isolate
streak - indicates carbapenem-hydrolyzing enzyme.
pos
pos
pos
neg
neg
neg
meropenem
ertapenem
imipenem
Janet Hindler, Whats New in the 2008 CLSI
Standards for (AST)?
38Definitive ID of a KPC Producer
- PCR
- The method of choice to confirm KPC
39Alternative Treatment for a KPC Producer
- Tigecycline (100.0 effective)
- Colistin (88.1 effective)
- SENTRY report. AAC. 2008. Feb52(2)570-3
- Minocycline
- A strategy for susceptibility testing is needed
40Conclusions
- 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.