Dr.T.V.Rao MD 1

Resistance to b-lactam Antibiotics

revisited Dr.T.V.Rao MD

β-lactam Antibiotics • The β-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 β-lactam antibiotics. Nearly all of these antibiotics work by inhibiting bacterial cell wall biosynthesis. This has a lethal effect on bacteria

History of β-lactams• The first synthetic β-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 β-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 MD4

• Resistant bacteria survive, susceptible ones die

Mutant emergesslowly

Sensitive cellskilled by antibiotic

Mutant’s progenyoverrun

Action of a b-lactamase

NO

COOH

S

HNO

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-

lactamsPorin-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 β-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• Cefotetan

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-1E.coliS.paratyphi

1970s

TEM-1Reported in 28 Gm(-) sp

1983

ESBL in Europe

1988

ESBL in USA

2000

> 130 ESBLsWorldwide

Extended-spectrumCephalosporins

1963

Evolution of b-Lactamases

Look and you will find ESBL

Dr.T.V.Rao MD 18

Classification of β 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

Dr.T.V.Rao MD 19

Classification•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• PiperacillinR• Cephalothin R• Cefoxitin S• Cefotaxime R• Ceftazidime I• Ceftriaxone R• AztreonamI

• 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 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

28AmpC Disk Test

Lawn culture: E. coli ATCC 25922

Test Organism on disk

E. coli ESBL susceptibility report

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

• CefepimeSR

• Pip/Tazo I• Imipenem S

Enterobacter 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

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 derepressed—meaning 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 AmpC b-lactamases

Dr.T.V.Rao MD

Beta-lactamase inhibitors

33

• Resemble β-lactam antibiotic structure• Bind to β-lactamase and protect the antibiotic from

destruction• Most successful when they bind the β-lactamase

irreversibly• Three important in medicine

• Clavulanic acid• Sulbactam• Tazobactam

Resistance and geneticsAmpCHi-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 34Know the species

Why Test for β-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 Rapid1Kumar, 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 ClassificationMolecular

ClassA B D

Functional Group

2f 3 2d

Aztreonam Hydrolysis

+ - -

EDTA Inhibition

- + -

ClavulanateInhibition

+ -

Klebsiella 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

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, 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. 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 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.

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 testsKnowledge of what’s unusual

Dr.T.V.Rao MD 55

Hand washing still can reduce the ESBL spread in the Hospitals

Dr.T.V.Rao MD 56

• The Programme created by Dr.T.V.Rao MD for Medical Microbiologists in the Developing

World •Email

•doctortvrao@gmail.com