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
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 plasmid-mediated AmpC b-lactamase called CMY-2. E.
coli UTI isolates carry plasmid-mediated
Beta-lactamase inhibitors Dr.T.V.Rao MD 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
Resistance and genetics AmpC Hi-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 34 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 Search and Destroy analogous to
MRSA ? Laboratory Detection is not always easy OR Rapid 1Kumar,
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 Classification Molecular Class A B D Functional
Group 2f 3 2d Aztreonam Hydrolysis + - - EDTA Inhibition - + -
Clavulanate Inhibition + -
Klebsiella pneumoniae Ampicillin R PiperacillinR Cephalothin R
Cefoxitin S Cefotaxime R Ceftazidime I Ceftriaxone R AztreonamI
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, Whats 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 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 55
Dr.T.V.Rao MD 56 The Programme created by Dr.T.V.Rao MD for
Medical Microbiologists in the Developing World Email
[email protected]
