The Beta-Lactamase Family:
Classification, Detection, and Interpretive
Criteria
COL Helen Viscount, PhD, D(ABMM)
LTC Steven Mahlen, PhD, D(ABMM)
Transplant patient
 Extremely resistant Klebsiella
pneumoniae recovered
 Sensitive only to colistin and
gentamicin
 Patient put in isolation
 Isolate transmitted to 10 other
patients
 Outcomes:
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
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
4/5 with bacteremia died
1 other died
2 with renal failure
Only 4/11 discharged without
renal failure
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Ampicillin: R
Pip/tazo: R
Ceftazidime: R
Ceftriaxone: R
Cefepime: R
Imipenem: R
Meropenem: R
Aztreonam: R
Amikacin: R
Tobramycin: R
Trimeth/sulfa: R
Fluoroquinolones: R
Gentamicin: S
Colistin: S
Nursing home resident
 83 years old
 Pneumonia
 Admitted to ICU
 Started on ceftriaxone and
levofloxacin
 Blood cultures +
 K. pneumoniae
 Based on sensi’s:
 No more levo
 Kept on ceftriaxone
 Patient got worse
 Had to be ventilated
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Ampicillin: R
Pip/tazo: S
Cefazolin: R
Ceftazidime: I
Ceftriaxone: S
Cefepime: S
Imipenem: S
Aztreonam: S
Tobramycin: S
Trimeth/sulfa: R
Levofloxacin: I
Ciprofloxacin: I
Gentamicin: S
Objectives
 At the end of this workshop
the attendee should be able to
distinguish ESBL positive
from carbapenemaseproducing bacteria
 At the end of this workshop
the attendee should be able to
describe a method to screen
for ESBLs
 At the end of this workshop
the attendee should be able to
interpret the results of the
modified Hodge Test
Beta-lactam antibiotics
 Penicillins
 Ampicillin
 Amoxicillin
 Piperacillin
 Cephalosporins (generations)
 1st gen: cephalothin
 2nd gen (cephamycins): cefoxitin, cefotetan
 3rd gen: ceftazidime, cefotaxime, ceftriaxone
 4th gen: cefepime
Beta-lactam antibiotics
 Monobactam: aztreonam
 Carbapenems:
 Imipenem
 Meropenem
 Ertapenem
 Inhibitors
 Sulbactam (ampicillin/sulbactam: Unasyn)
 Tazobactam (piperacillin/tazobactam: Zosyn)
 Clavulanate (amoxicillin/clavulanate: Augmentin)
Mechanisms of Resistance
 Altered target (Gram negative/positive)
 Altered permeability (Gram negative)
 Production of inactivating enzymes (Gram negative/positive)
Gram-negative cell
Gram-positive cell
Outer membrane
Peptidoglycan
Peptidoglycan
Penicillin
Binding proteins
(PBPs)
Inner (cytoplasmic) membrane
Alteration of Target
 Resistance to -lactams via altered penicillin-binding
proteins (PBPs)
 MRSA
 Vancomycin resistance in enterococci
 Fluoroquinolone resistance
Altered Permeability
 Passive diffusion of Gram-negative cell wall
 Mutate outer membrane proteins
 Active efflux
Active Efflux
Production of Inactivating Enzymes
 Chloramphenicol acetyltransferase
 Aminoglycoside-modifying enzymes
 -Lactamases
-Lactamases
 Well over 340 different enzymes
 Extended spectrum -lactamases (ESBLs)
 AmpC -lactamases
 Chromosomal
 Plasmid-mediated
 Carbapenemases
-Lactamases
 First -lactamase identified: AmpC beta-lactamase
 1940, Escherichia coli
 1940, penicillinase, Staphylococcus aureus
 First plasmid-mediated -lactamase: TEM-1
 1965, Escherichia coli, Greece
-Lactamase Activity
H
H
S
R-CONH
C
C
C
N
-lactam
CH3
CH3
O
COOH
Enzyme-Ser-OH
-Lactamase Activity
H
H
S
R-CONH
O
HOH
C
C
C
N
O
H
Ser
Enzyme
CH3
CH3
COOH
L
L L
L




L





L
L




 
L
-lactamase
production


L 



Types of Beta-Lactamases
 ESBLs
 AmpCs
 Carbapenemases
ESBLs
ESBLs
 Extended-spectrum beta-lactamases (ESBLs) are mutant
enzymes with a broader range of activity than their parent
molecules
 They:
 Hydrolyze 3rd and 4th gen cephalosporins and aztreonam
 Do not affect cephamycins (2nd gen ceph) or carbapenems
 Remain susceptible to beta-lactamase inhibitors
ESBLs
 The most common plasmid-mediated ß-lactamases in
Enterobacteriaceae are TEM-1, TEM-2, and SHV-1
 TEM: Escherichia coli
 Named after first patient with a urinary tract infection that was not
treatable with ampicillin
 Her name: Temorina
 SHV: Klebsiella pneumoniae
 “Sulfhydryl variant”; amino acids in the enzyme that cross-link with other
molecules
 “Classical” ESBLs are derived from TEM and SHV enzymes
 “Non-classical” ESBLs are derived from enzymes other than
TEM or SHV
Classical ESBLs
 Primarily found in E. coli and Klebsiella spp.
 Differ from their parent TEM or SHV enzymes by only 1-4
amino acids
 >100 TEM- or SHV-derived beta-lactamases have been
described – most are ESBLs
Non-classical ESBLs
 Many described, but less common than classical ESBLs
 CTX-M
 Found in multiple genera of Enterobacteriaceae
 Preferentially hydrolyze cefotaxime
 U.S., Europe, South America, Japan, Canada
 OXA
 Mainly in P. aeruginosa
 Primarily hydrolyze ceftazidime
 France, Turkey
ESBL Epidemiology
 ESBLs first appeared in Europe in the mid-1980s
 Worldwide, but prevalence varies widely geographically and
between institutions
 U.S. national average for ESBLs in Enterobacteriaceae ~3%
ESBL Epidemiology
 ESBL producers especially prevalent in ICUs and long
term care facilities
 Becoming more widespread in the community also
 Have been associated with outbreaks
 Typically arise in ICU
 Plasmid transfer between GNRs
 Organism transfer between patients
 Control of outbreaks
 Infection control practice – isolation
 Restriction of 3rd and 4th generation cephalosporins
 Antimicrobial cycling
Clinical Significance
 Despite appearing susceptible to one or more penicillins,
cephalosporins, or aztreonam in vitro, the use of these agents
to treat infections due to ESBL-producers has been associated
with poor clinical outcome
Clinical Significance
 ESBL genes are often carried on plasmids that also
encode resistance to multiple classes of antimicrobials
 Aminoglycosides, Fluoroquinolones
 Trimethoprim/Sulfamethoxazole
 Treatment experience is largely based on classical ESBL
producers
 Carbapenems
 ß-lactam/inhibitor combinations
Typical ESBL Susceptibility Profile
 Amp: R
 Amp: R
 Piperacillin: R
 Piperacillin: R
 Pip/tazo: S
 Pip/tazo: S
 Cefazolin: R
 Cefazolin: R
 Cefoxitin: S
 Cefoxitin: S
 Ceftazidime: S
 Ceftazidime: R
 Ceftriaxone: R
 Ceftriaxone: R
 Cefepime: R
 Cefepime: R
 Aztreonam: S
 Aztreonam: R
 Imipenem/meropenem: S
 Imipenem/meropenem: S
AmpCs
AmpC: General
 Chromosomal
 Escherichia coli
 Citrobacter freundii
 Enterobacter aerogenes, E. cloacae
 Serratia marcescens
 Morganella morganii
 Hafnia alvei
 Providencia rettgeri, P. stuartii
 Pseudomonas aeruginosa
 Aeromonas sp.
AmpC: General
 Are not inhibited by -lactamase inhibitors
 Normally are repressed, so produced at low levels
 Chromosomal: inducible
 In all except E. coli
 In the presence of certain -lactam antibiotics
 Normally, produced at low levels
 Plasmid-mediated also
The AmpC of E. coli
 Chromosomal, but not
 Amp: S
inducible
 Normally expressed at low
levels
 Regulated by a growth
rate-dependent attenuation
mechanism
 Can become highly
expressed with mutations
 Amox/clav: S
 Piperacillin: S
 Pip/tazo: S
 Cefoxitin: S
 Ceftazidime: S
 Ceftriaxone: S
 Cefepime: S
 Aztreonam: S
 Imipenem/meropenem: S
AmpC Induction and Derepression
 Is induction clinically relevant?
 True danger—mutation in induction pathway
 “Derepressed mutant”
 150-1000 fold more enzyme produced than normal
Chromosomal AmpC profile
 Normal
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Amp: R
Amox/clav: R
Piperacillin: S
Pip/tazo: S
Cefoxitin: R
Ceftazidime: S
Ceftriaxone: S
Cefepime: S
Aztreonam: S
Imipenem/meropenem: S
 Derepressed profile
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Amp: R
Amox/clav: R
Piperacillin: R
Pip/tazo: R
Cefoxitin: R
Ceftazidime: R
Ceftriaxone: R
Cefepime: S
Aztreonam: R
Imipenem/meropenem: S
Plasmid-Mediated AmpCs (pAmpC)
 First true proof of AmpC on plasmid: 1988
 MIR-1, found in Klebsiella pneumoniae
 90% identical to E. cloacae ampC
 Some are also inducible (DHA-1)
 Most frequently found in K. pneumoniae
 Also commonly found in:
 K. oxytoca
 Salmonella sp.
 P. mirabilis
 E. coli, E. aerogenes also
pAmpCs: Distribution
 World-wide distribution
 Africa, Asia, Europe, Middle East, North America, South
America, Central America
 CMY-2 is most prevalent globally
 Algeria, France, Germany, Greece, India, Pakistan, Taiwan,
Turkey, UK, US
ESBLs vs AmpCs
ESBLs
AmpCs
Inhibitors (pip/tazo,
amp/sulbactam, amox/clav)
S
R
Cefoxitin, cefotetan
S
R
Ceftazidime,
ceftriaxone
R
R
S/R
S
Cefepime
Carbapenemases
Carbapenemases
 Carbapenem resistance:
 High level production of chromosomal AmpC with decreased
outer membrane permeability (porins)
 E. cloacae, E. aerogenes
 C. freundii
 E. coli
 S. marcescens
 K. pneumoniae (porins)
Carbapenemases
 Carbapenem resistance:
 Changes in affinity of PBPs for carbapenems
 Carbapenemases
 Frequently, bugs that produce a carbapenemase produce
other -lactamases
Carbapenemases
 KPC (plasmid, K. pneumoniae)
 “Klebsiella pneumoniae carbapenemase”
 IMI-1 (plasmid, E. cloacae)
 Nmc-A (plasmid, E. cloacae)
 Sme-1 (plasmid S. marcescens)
 IMP-1 (plasmid, S. marcescens, P. aeruginosa)
 L-1 (chromosomal, Stenotrophomonas maltophilia)
Carbapenemases: Profile
 R to carbapenems, penicillins, cephalosporins
 S or R to aztreonam, depending on enzyme
 So the key:
 Look for intermediate or R to imipenem or meropenem!
KPC
 Infection control emergency!!!
 May test sensitive to carbapenems though!
 Extensive multidrug resistance (XDR)
 Very rapid spread
 Empiric therapy: colistin + tigecycline
 KPC 1-8
Further reading
 Yang, 2007. Ann. Pharmocother. 41:1427-1435
 Jacoby, 2009. Clin. Microbiol. Rev. 22:161-182
 Black et al, 2005. J. Clin. Microbiol. 43:3110-3113
 Livermore et al, 2001. J. Antimicrob. Chemother. 48 Suppl
1: 87-102
 Pfaller and Segreti, 2006. Clin. Infect. Dis. 42: S153-163.