Resistance to b-lactam Antibiotics

revisited
Dr.T.V.Rao MD

Dr.T.V.Rao MD

-lactam Antibiotics
The -lactam ring is part of the core structure of several antibiotic families, the principal ones being the penicillins, cephalosporins, carbapenems, 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

Resistant bacteria survive, susceptible ones die

Mutant emerges slowly

Dr.T.V.Rao MD

Sensitive cells killed by antibiotic

Mutants progeny overrun

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Action of a b-lactamase
S

O
COOH

H2O

Inactive penicilloate
S

Active penicillin
HN
O OH COOH

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
1965 1969 1974

Ampicillin; 1st broad spectrum penicillin

TEM b-lactamases in E. coli TEM b -lactamase in P. aeruginosa TEM in H. influenzae & N. gonorrhoeae

Now TEM in 30-60% E. coli & enterobacteria & in 520% of H. influenzae & gonococci

ESBL Introduction
B-lactamases conferring resistance to the penicillin's, firstsecond-, 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*
PPID, 6th ed. 2005

Mechanisms of GNR Resistance to b-lactams

Porin-mediated resistance
Antibiotic does not reach target

b-lactamase
Majority of resistance to blactam antibiotics mediated through b-lactamases. Many different types of blactamases with different substrate (antibiotic) specificities.

PENICILLIN

BETA LACTAM RING

BETA LACTAMASES enzymes that inactivate the beta-lactam ring

CEPHALOSPORIN

BETA LACTAM RING

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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

Penicillins Benzylpenicillin Methicillin Ampicillin Carbenicillin Mezlocillin Ticarcillin Cephalosporins Cephamycins Carbapenems Monobactams Cephalothin 1st Cefamandole 2nd Cefoxitin Cefotetan Cefmetazole Imipenem Meropenem Ertapenem Aztreonam

Cefuroxime 2nd
Cefotaxime 3rd Ceftazidime 3rd Ceftriaxone 3rd 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

Evolution of b-Lactamases
Plasmid-mediated TEM and SHV b-lactamases

Ampicillin
1963 1965

Extended-spectrum Cephalosporins
1970s
1983

1988

2000

Look and you will find ESBL

TEM-1 TEM-1 E.coli Reported in S.paratyphi 28 Gm(-) sp

ESBL in Europe

ESBL in USA

> 130 ESBLs Worldwide

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Classification of 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

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Classification
Ambler Classification Molecular class A D
A

Bush-Jacoby-Medeiros Classification Functional group 1 4

2 2b 2be
Dr.T.V.Rao MD

Paterson and Bonomo, 2005

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

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 Rev. 2005;18:657-686 J Clin Microbiol.2001;39:2206-2212.

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)
Clin Microbiol Infect. 2008;14:169-174.

E. coli susceptibility Report

Ampicillin Piperacillin Cephalothin Cefoxitin S Cefotaxime Ceftazidime Ceftriaxone Aztreonam R R R
Cefepime Pip/Tazo Imipenem S I S

R I R I

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 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

Combination Disk Method

CLSI Approved Method

Lawn culture: E. coli ATCC 25922

Test Organism on disk

AmpC Disk Test

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E.

Ampicillin R Piperacillin R Cephalothin Cefoxitin S Cefotaxime Ceftazidime Ceftriaxone Aztreonam IR

coli ESBL susceptibility report

R R IR R Cefepime SR Pip/Tazo Imipenem

I S

Enterobacter cloacae susceptibility report

Ampicillin R Piperacillin R Cephalothin Cefoxitin R Cefotaxime Ceftazidime Ceftriaxone Aztreonam I R R I R Cefepime Pip/Tazo Imipenem S R 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 blactamase 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
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
Dr.T.V.Rao MD 33

Resistance and genetics

AmpC Hi-level R R R R No TEM ESBL R v S v +++ CTX-M v R S v +++ K1 S S S R No

Ceftazidime Cefotaxime Cefoxitin Aztreonam Synergy + clav

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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 1Kumar, Crit Care Med, 2006 Search and Destroy analogous to MRSA ? Laboratory Detection is not always easy OR Rapid

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 Clin Microbiol Rev. 2005;18:657-686. 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 Functional Group Aztreonam Hydrolysis EDTA Inhibition Clavulanate Inhibition

A 2f + +

B 3 + -

D 2d

Klebsiella pneumoniae
Ampicillin R Piperacillin R Cephalothin Cefoxitin S Cefotaxime Ceftazidime Ceftriaxone Aztreonam I R R I R

Cefepime Pip/Tazo Imipenem

S R 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 (KPC1) 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

 Modified Hodge test

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.

Definitive ID of a KPC Producer

100% sensitivity to detect KPC
pos pos

pos

3. Incubate overnight.
4. Look for growth of E. coli around test isolate streak - indicates carbapenem-hydrolyzing enzyme.

neg

neg

neg
meropenem ertapenem imipenem
Janet Hindler, Whats New in the 2008 CLSI Standards for (AST)?

Definitive ID of a KPC Producer

The method of choice to confirm KPC

PCR

 Tigecycline (100.0% effective) Colistin (88.1% effective) Minocycline A strategy for susceptibility testing is needed

Alternative Treatment for a KPC Producer

SENTRY report. AAC. 2008. Feb;52(2):570-3

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
Clin Microbiol Rev. 2005;18:657-686.

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

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

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