Difficult or Tricky Antibiotic

Resistance Phenotypes to
Recognize
Dr Koh Tse Hsien
Department of Pathology
Singapore General Hospital
 Contents
• Gram-positive
– Vancomycin Resistant Enterococci (VRE)
– Staphylococcus aureus with reduced susceptibility to
glycopeptides (hVISA, VISA, VRSA)
• Gram-negative
– Extended-spectrum beta-lactamases (ESBLs)
– ampC Cephalosporinases ( AmpC and pAmpC)
– KPC Carbapenemases (KPC)
– Metallo-beta-lactamases (MBL)
 Mechanisms of Resistance

Target modification

Antibiotic sequestration
 Vancomycin-resistant
Enterococcus
VanA VanB VanC

Vanc MIC 64->1000 4-1024 2-32

mg/L Resistant Resistant Intermediate
Teic MIC 16-512 ≤0.5 ≤0.5
Mg/L Resistant Susceptible Susceptible
E. Faecium E. Faecium E. Gallinarum
Species E. faecalis E. faecalis E. Casseiflavus
E. flavescens

Genetic Acquired Acquired Intrinsic

determinant
Transferable Yes Yes No
VRE
VanA Mechanism of Resistance
• VanA, VanR, VanS, VanH, VanX, VanZ located
on Tn1546 often on plasmid
• VanA produces new cell wall building block with
reduced affinity for vancomycin
• VanH is an enzyme which synthesizes new cell
wall building block precursor
• VanX and Y are enzymes that breaks down the
original cell wall building block
• VanR and VanS regulate transcription of
VanHAX cluster
• VanZ increases MIC of teicoplanin
VRE
 VRE Lab Detection
• Disk diffusion-incubate full 24 h, view with
transmitted light for hazy zones
• MIC methods
• Screening plates (vancomycin 6 mg/L)
• Multiplex PCR to detect vanA, vanB
Enterococcus gallinarum
vanC motile Enterococci
Vancomycin intermediate
Staphylococcus aureus
 Vancomycin-intermediate S.
aureus (VISA)
• First described in Japan in 1997
• Intermediate resistant (MIC 4-8 mg/L)
• Hetero-intermediate resistant (MIC <4 mg/L)
• Increased production of cell wall precursors and
PBP 2’
• Reduced cross linking in cell wall (less target for
vancomycin)
• Lower growth rates and thicker cell walls
(vancomycin sequestration)
• Prolonged exposure to vancomycin
 MRSA VISA

Micky LEONG (Ms) :: Laboratory Technologist, Electron Microscopy Unit YLLSOM

 Lab detection of VISA
• Not detected by routine disk or automated
methods
• Etest (consistently one twofold dilution
higher)
• Broth microdilution
hVISA
 Lab detection of hVISA
• Population analysis
• Macrodilution Etest (not true MIC)
• Etest GRD
• MHA5T screening plate (5 µg/ml
teicoplanin)
• hVISA may predict potential failure of
daptomycin therapy
 FIG. 8. Examples of Etest methodology used to detect hVISA

Howden, B. P. et al. 2010. Clin. Microbiol. Rev. 23(1):99-139

 Vancomycin-resistant S. aureus
(VRSA)
• In-vitro transfer of VanA in 1992
• Michigan (June 2002), Pennsylvania (Sept
2002)
• MIC 1024 mg/L, 32 mg/L
• VanA positive
• Plasmid-mediated
• Detected by disk diffusion but may not be
detected by automated methods
FIG. 1. Disk diffusion and Etest analysis of the PA-VRSA isolate on Mueller-Hinton agar

Tenover, F. C. et al. 2004. Antimicrob. Agents Chemother. 48(1):275-280

FIG. 1. Determination of MICs for VRSA-7 by Etest

Moubareck, C. et al. 2009. Antimicrob. Agents Chemother. 53(9):3657-3663

 Mechanisms of Resistance
Loss of membrane permeability

Enzymatic inactivation

inner membrane
periplasmic space

outer membrane

Efflux
 Classification of β-lactamases
• Ambler (sequence based)

• A serine (TEM, SHV, CTX-M, ESBLs,

KPC)
• B zinc (IMP, VIM, NDM-1)
• C serine (chromosomal ampC, DHA,
CMY)
• D serine (OXA)
 What is an ESBL?
• A beta-lactamase that hydrolyzes
extended -spectrum cephalosporins
(ESCs) and is inhibited by clavulanate
 Susceptibility characteristics
• Resistant to penicillins, most
cephalosporins and monobactams
• Susceptible to
– β-lactamase inhibitors (clavulanate,
tazobactam)
– Cephamycins (cefoxitin, cefotetan)
– Carbapenems (meropenem, imipenem)
• Resistance may develop due to hyper-
production and OMP loss
 ESBLs
• TEM-mutants of TEM-1,-2
• SHV-mutants of SHV-1
• CTX-M
• Others
 TEM and SHV
• Early 1960s-TEM-1 from E. coli isolated
from a patient named Temoniera
– Plasmid and transposon mediated
– Enterobacteriaceae, P. aeruginosa, H.
influenzae, N. gonorrhoeae
• SHV-1 (sulphdryl variable) common β-
lactamase chromosomally encoded in
most K. pneumoniae and plasmid-
mediated in E. coli
 TEM ESBLs
• Most commonly encountered β-lactamase
in GNB
• 90% of amp resistance in E. coli due to
TEM-1
• AA substitutions in TEM-1 result in
changes in substrate specificity
• Found in E. coli, K. pneumoniae, Proteus
spp. Enterobacter spp. etc...
 SHV ESBLs
• SHV-1 and ampicillin resistance in K.
pneumoniae
• Fewer ESBL derivatives compared with
TEM
• K. pneumoniae, C. diversus, E. coli, P.
aeruginosa
E. Coli ESBL TEM
E. Coli ESBL TEM
Klebsiella ESBL SHV
Klebsiella SHV ESBL
 Newer ESBLs
• CTX-M
• OXA
• PER-1
• VEB-1
• TLA-1
 CTX-M

• Not closely related to TEM and SHV

• Hydrolyze cefotaxime better than
ceftazidime
• Inhibited better by tazobactam than
clavulanate
• High homology to chromosomal
enzymes of Kluyvera spp.
Klebsiella CTX-M ESBL
Klebsiella CTX-M ESBL
 Distribution of ESBLs (NARSS 2007)

E. coli n % Klebsiella spp n %

Total with ESBL Total with ESBL
phenotype 36 phenotype 60
CTXM enzyme 33 92% CTXM enzyme 45 75%
CTXM Grp 1 28 78% CTXM Grp 1 45 75%
CTXM Grp 9 4 11% 0%
CTXM Grp 1 & Grp 4 1 3% 0%
TEM ESBL 3 8% TEM ESBL 1 2%
SHV ESBL 0 0% SHV ESBL 44 73%
OXA ESBL 0 0% OXA ESBL 2 3%
No gene detected 3 8% No gene detected 5 8%
 Class C beta-lactamases
• Gene normally found in most Gram negatives except
Klebsiella and Salmonella
• Cephalosporinases
• Not inhibited by clavulanate, tazobactam
• Resistant to cefoxitin
• Cefepime not generally affected
• Chromosomal
• Generally expressed at low level but are inducible
 ESCHAPPM

• Enterobacter cloacae/aerogenes
• Serratia marcescens
• Citrobacter freundii
• Hafnia alvei
• Aeromonas hydrophila/caviae
• Providencia stuartii/rettgeri
• Morganella morganii
 Induction vs Stable
Derepression
• Induction
– transient switching on of β-lactamase
synthesis in response to an inducer (cefoxitin,
imipenem)
• Stable Derepression
– permanent hyperproduction of the β-
lactamase independent of an inducer
Chromosomal AmpC in
Enterobacter spp.
 Ceftriaxone

Derepressed mutants

Cefoxitin
Derepressed Original
mutant strain
 >32 mg/L-
-<8 mg/L susceptible
-1.5 mg/L

Derepressed Original
mutant strain
DHA in Klebsiella pneumoniae
CMY in E. coli
 CLSI M100-S20 (Jan 2010)
Susceptible Susceptible
Zone Diameter (mm) Minimal Inhibitory Concentration
(mg/L)
CLSI CLSI EUCAST CLSI CLSI EUCAST
2009 2010 2010 2009 2010 2010

Ceftriaxone ≥21 ≥23 ≥23 ≤8 ≤1 ≤1

Ceftazidime ≤8 ≤4 ≤4

Aztreonam ≥22 ≥21 ≥27 ≤8 ≤4 ≤1

Cefepime ≥18 ≥18 ≥24 ≤8 ≤8 ≤1

 KPC
• Class A Carbapenemase
• Inhibited by clavulanic acid
• Mainly K. pneumoniae, but also Serratia spp.
Enterobacter spp. Citrobacter spp. E. coli and S.
enterica, P. aeruginosa, Acinetobacter spp.
• USA (NE), Israel, China (Zhejiang), South
America, Greece
• Carbapenem MICs may be in the susceptible
range (as low as 2 µg/ml)
Microbiological detection of KPC-2-producing K pneumoniae and
Escherichia coli. Clavulanic acid only weakly inhibits the
resistance of K pneumoniae to ertapenem and imipenem.
A=ertapenem. B=clavulanic acid. C=imipenem

Nordmann P et al. The real threat of Klebsiella pneumoniae carbapenemase

producing bacteria. The Lancet Infectious Diseases. April 2009: 228-236.
 Susceptibility testing using Etest methodologyMicrobiological detection of KPC-2-producing K
pneumoniae and E coli. The presence of scattered colonies makes it difficult to read the defined
endpoint. The numbers on the strips represent the MIC (µg/mL) for the antibiotic in question.
IP=imipenem. MP=meropenem. ETP=ertapenem.

Nordmann P et al. The real threat of Klebsiella pneumoniae carbapenemase

producing bacteria. The Lancet Infectious Diseases. April 2009: 228-236.
Hodge Test
Inhibited by boronic acid
 Class B β-lactamases
• Hydrolyze carbapenems
• Zinc dependent
• Spare aztreonam
• Inhibited by metal ion chelators
• Chromosomal vs acquired
 Chromosomal Class B β-lactamases

• Stenotrophomonas maltophilia (L1)

• Elizabethkingia meningoseptica (BlaB, GOB-1)
• Aeromonas hydrophilia (CphA, A2h), A. sobria
(ImiS, AsbM1), A. salmonicida (ASA-1)
• Legionella gormannii
• Bacillus cereus (II )
• Bacteroides fragilis (CfiA)
• Burkholderia cepacia (PCM-1)
 Acquired Class B β-lactamases

• IMP
• VIM
• SPM (Brazil, P. aeruginosa)
• SIM-1 (Korea, Acinetobacter spp.)
• GIM-1 (Germany, P. aeruginosa)
• DIM-1 (Netherlands, P. stutzeri)
• AIM-1 (Australia, P. aeurginosa)
• KHM-1 (Japan, Citrobacter freundii)
• NDM-1 (Indian subcontinent, K. pneumoniae, E.
coli)
 DDST
M
E
T
A
• double-disk synergy test (DDST)
L
L
O
-
B
E
T
A
L IPM
A MPA
C
T
A
M
A
S
E

Picão RC, et al. Metallo-beta-lactamase detection: comparative evaluation of double-disk synergy versus combined disk tests for
IMP-, GIM-, SIM-, SPM-, or VIM-producing isolates. J Clin Microbiol. 2008 Jun;46(6):2028-37.
 MBL & issues
M
E
T
A
• MBL inhibitors may themselves have
L
L bactericidal effects
O
-
B • Multiple combinations of substrates and
E
T MBL-inhibitors possible
A
L
A • MBL Etest strip unable to detect MBL-
C
T producing strains where imipenem MIC <4
A
M
A
µg/ml
S
E
DM23092 IMP+
CHROMagar KPC chromID ESBL
CLSI M100-S20 (2010) June update
Susceptible Susceptible
Zone Diameter (mm) Minimal Inhibitory Concentration
(mg/L)
CLSI CLSI EUCAST CLSI CLSI EUCAST
Jan Jun Jan Jun

Ertapenem ≥19 ≥23 ≥25 ≤2 ≤0.25 ≤0.5

Imipenem ≥16 ≥23 ≥21 ≤4 ≤1 ≤2

Meropenem ≥16 ≥23 ≥22 ≤4 ≤1 ≤2