Multiplex PCR for Rapid Detection of Genes Encoding Class A Carbapenemases

Abstract

In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-β-lactamase nonproducers.

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ISSN 2234-3806 • eISSN 2234-3814
359
http://dx.doi.org/10.3343/alm.2012.32.5.359
www.annlabmed.org
Ann Lab Med 2012;32:359-361
http://dx.doi.org/10.3343/alm.2012.32.5.359
Brief Communication
Clinical Microbiology
Multiplex PCR for Rapid Detection of Genes Encoding
Class A Carbapenemases
Sang Sook Hong, M.D.
1
, Kyeongmi Kim, M.D.
1
, Ji Young Huh, M.D.
1
, Bochan Jung, M.D.
2
, Myung Seo Kang, M.D.
1
,
and Seong Geun Hong, M.D.
1
Department of Laboratory Medicine
1
, CHA Bundang Medical Center, CHA University, Seongnam; Department of Laboratory Medicine
2
, CHA Gumi Medical
Center, CHA University, Gumi, Korea
In recent years, there have been increasing reports of KPC-producing
Klebsiella pneu-
moniae
in Korea. The modied Hodge test can be used as a phenotypic screening test for
class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish
between carbapenemase types. The conrmation of type of CAC is important to ensure
optimal therapy and to
prevent transmission. This study applied a novel multiplex PCR as-
say to detect and differentiate CAC genes in a single reaction. Four primer pairs were de-
signed to amplify fragments encoding
4 CAC families (SME, IMI/NMC-A, KPC, and GES).
The multiplex PCR detected all genes tested for
4 CAC families that could be differentiated
by fragment size according to gene type. This multiplex PCR offers
a simple and useful
approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that
are metallo-
β
-lactamase nonproducers.
Key Words:
Carbapenemase, Multiplex PCR, KPC, GES
Received: January 26, 2012
Revision received: May 21, 2012
Accepted: July 9, 2012
Corresponding author: Seong Geun Hong
Department of Laboratory Medicine, CHA
Bundang Medical Center, CHA University,
59 Yatap-ro, Bundang-gu, Seongnam
463-712, Korea
Tel: +82-31-780-5463
Fax: +82-31-780-5476
E-mail: hlseo@cha.ac.kr
© The Korean Society for Laboratory Medicine.
This is an Open Access article distributed under
the terms of the Creative Commons Attribution
Non-Commercial License (http://creativecom-
mons.org/licenses/by-nc/3.0) which permits
unrestricted non-commercial use, distribution,
and reproduction in any medium, provided the
original work is properly cited.
Carbapenems are important antibiotics for the treatment of in-
fections caused by multidrug-resistant gram-negative bacilli [
1,
2]; however, carbapenem-resistance is increasing, causing in-
fections that are difcult to treat [
2-4]. Bacterial production of
carbapenemases is one of the most important mechanisms of
carbapenem resistance [
1]. There are 3 molecular classes of
carbapenemases: A (penicill
inases); B (metallo-
β
-lactamases,
MBLs); and D (oxacillinases). The class A carbapenemases
(CACs) include the SME, IMI/NMC-A, SFC, BIC, KPC, and some
type of GES family proteins. The genes for the SME, IMI/NMC-A
(except IMI-
2), SFC, and BIC enzymes are chromosomal, and
the genes for KPC and GES are carried on plasmids [
1]. KPC
producers have caused severe treatment problems in hospitals
around New
York and have also been reported in Europe, South
America, and China [
5-8]. In Korea, KPC-producing
Klebsiella
pneumoniae
have rarely been detected. However, several cases
were reported in
2010 and 2011 (Interscience Conference on
Antimicrobial Agents and Chemotherapy
2011 poster c2-652,
unpublished observation) [
9, 10].
The modied Hodge test (MHT) can be used as a phenotypic
conrmatory test f
or suspected carbapenemase production in
Enterobacteriaceae
[11]. However, it is reported that the MHT
shows approximately
25% false positive results among carbap-
enemase nonproducers, mainly AmpC hyperproducers and
strains harboring CTX-M [
12]. Moreover, it does not distinguish
between carbapenemase types or CAC types.
The major concern from the therapeutic and epidemiologic
perspective is with
transmissible and not chromosomal carbap-
enemases [
1, 2], and this information cannot be acquired by
the phenotypic methods. Conrmation of the CAC type is impor-
tant to ensure optimal therapy and to prevent transmission [
3].
In this study, we developed a multiplex PCR assay to detect and
differentiate multiple CAC genes in a single reaction.
Eleven CAC producers (1 SME-producing
Serratia marce-

Hong SS, et al.
Multiplex PCR for class A carbapenemases
360
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http://dx.doi.org/10.3343/alm.2012.32.5.359
scens
, 2 IMI/NMC-A-producing
Enterobacter cloacae
, 2 KPC-
producing
Enterobacteriaceae
, and 6 GES-producing
Klebsiella
pneumoniae
); 7 MBL producers (3 VIM-producing
Pseudomo-
nas aeruginosa
, 2 IMP-producing
P. aeruginosa
, 1 IMP-produc-
ing
Acinetobacter baumannii
, and 1 SIM-producing
A. bauman-
nii
); and 5 non-carbapenemase-producing
Enterobacteriaceae
were studied (Table
1).
The bacterial cells
were lysed by heating at 95
°
C for 10 min,
and cellular debris was removed by centrifugation at
13,000 rpm
for
5 min. The supernatant was used as the source of amplica-
tion templates. PCR was performed with a nal volume of
20
μ
L
in
0.2 mL thin-walled tubes (Accupower
TM
HotStart PCR PreMix;
Bioneer, Daejeon, Korea).
We designed
4 primer pairs for 4 CAC families (SME, IMI/NMC-
A, KPC, and GES). The
genes encoding IMI and NMC-A type
CACs are similar to each other and could not be differentiated by
conventional PCR. Therefore, a single primer pair for the detection
of these
2 CAC families was designed. The SFC-1 and the BIC-1
enzymes have been found in environmental isolates, and the cor-
responding genes are chromosomally encoded [
13, 14]. For this
reason, we did not design pairs of primers fo
r these genes. The
primers used in this study were GES primers for
bla
GES1-9
and
bla
GES11-20
(GES-F: 5
’
-GCTTCATTCACGCACTATT-3
’
; GES-MR:
5
’
-CGATGCTAGAAACCGCTC-3
’
; product size: 323 bp), IMI/NMC-
A primers for
bla
IMI1-3
and
bla
NMC-A
(IMI(NMC)-F1: 5
’
-TGCGGTC-
GATTGGAGATAAA-
3
’
; IMI(NMC)-R1: 5
’
-CGATTCTTGAAGCTTCT-
GCG-
3
’
; product size: 399 bp), SME primers for
bla
SME1-3
(SME-F1:
5
’
-ACTTTGATGGGAGGATTGGC-3
’
; SME-R1: 5
’
-ACGAATTCGAG-
CATCACCAG-
3
’
; product size: 551 bp), and KPC primers for
bla
KPC2-13
(KPCF2: 5
’
-GTATCGCCGTCTAGTTCTGC-3
’
; KPCFR:
5
’
-GGTCGTGTTTCCCTTTAGCC-3
’
; product size 638 bp). The PCR
program consisted of an initial denaturation step at
94
°
C for 5 min,
followed by
25 cycles of DNA denaturation at 94
°
C for 30 sec,
primer annealing at
50
°
C for 30 sec, and primer extension at 72
°
C
for
1 min. After the last cycle, a nal extension step at 72
°
C for 7
min was added.
The GenBank nucleotide sequence accession numbers for
the sequences studied here were as follows: GES-
1 (AF156486);
GES-
2 (AF326355); GES-3 (AB113580); GES-4 (AB116260); GES-5
(AY
494717); GES-6 (AY494718); GES-7 (IBC-1, AF208529); GES-8
(IBC-
2, AF329699); GES-9 (AY920928); GES-11 (FJ854362); GES-
12 (FN554543); GES-13 (GU169702); GES-14 (GU207844); GES-
15 (GU208678); GES-16 (HM173356); GES-17 (HQ874631); GES-
18 (JQ028729); GES-19 (JN596280); GES-20 (JN596280); IMI-1
(U
50278); IMI-2 (DQ173429); IMI-3 (GU015024); NMC-A (Z21956);
SME-
1 (Z28968); SME-2 (AF275256); SME-3 (AY584237); KPC-2
(AY
034847); KPC-3 (AF395881); KPC-4 (AY700571); KPC-5
(EU
400222); KPC-6 (EU555534); KPC-7 (EU729727); KPC-8
(FJ
234412); KPC-9 (FJ624872); KPC-10 (GQ140348); KPC-11
(HM
066995); KPC-12 (HQ342889); and KPC-13 (HQ342890).
The CAC families could be differentiated into
4 groups, SME,
IMI/NMC-A, KPC, and GES, by the PCR product size (Fig.
1).
None of the non-CAC producers included in this study produced
PCR product bands. Not all genotypes of CAC were tested: only
SME-
1, IMI-1, NMC-A, KPC-2, KPC-3, and GES-5-type enzyme-
producing strain
s were included in this study. The primers for
the genotypic detection of SME, KPC and GES enzymes were
exactly complementary to the corresponding GenBank sequences,
but the primers for IMI/NMC-A were not complementary at
1
base each in the forward and reverse sequences of IMI-
3 and
NMC-A. Therefore, it is somewhat uncertain whether this multi-
plex PCR assay would be able to detect all of the geno
types of
CACs described above.
Table 1. Bacterial strains used for class A carbapenemase multiplex
PCR
Enzyme family
N of
strains
Strain
Enzyme
type*
Source
†
Class A carbapenemase
SME 1
Serratia marcescens
SME-1 CRAB
IMI 1
Enterobacter cloacae
IMI-1 CRAB
NMC-A 1
Enterobacter cloacae
NMC-A CRAB
KPC 1
Klebsiella pneumoniae
KPC-2 CRAB
1
Escherichia coli
KPC-3 CRAB
GES 6
Klebsiella pneumoniae
GES-5 like Our laboratory
Metallo-ß-lactamase
VIM 3
Pseudomonas aeruginosa
VIM-2 like Our laboratory
IMP 2
Pseudomonas aeruginosa
IMP-6 like Our laboratory
1
Acinetobacter baumannii
IMP-25 like Our laboratory
SIM 1
Acinetobacter baumannii
SIM-1 like Our laboratory
Non-carbapenemase
ESBL 1
Escherichia coli
TEM type Our laboratory
1
Escherichia coli
SHV type Our laboratory
1
Klebsiella pneumoniae
SHV type Our laboratory
1
Citrobacter freundii
TEM type Our laboratory
ESBL+AmpC
(EBC
‡
)
1
Klebsiella pneumoniae
SHV type Our laboratory
*ESBLs were detected by CLSI phenotypic conrmatory tests and type spe-
cic PCR;
†
CRAB, Center for Research in Anti-Infectives and Biotechnology,
Department of Medical Microbiology and Immunology, School of Medicine,
Creighton University, Omaha, Nebraska;
‡
EBC is a group of AmpC
β
-lactamase
originated from
E. cloacae
.
Abbreviation: ESBL, extended-spectrum
β
-lactamase.

Hong SS, et al.
Multiplex PCR for class A carbapenemases
361
http://dx.doi.org/10.3343/alm.2012.32.5.359
www.annlabmed.org
In summary, this multiplex PCR method appears to be a sim-
ple and useful approach for detecting and distinguishing CAC
genes in MBL-negative carbapenem-resistant strains. Therefore,
this method should be helpful for characterization of CACs and
prevention of the spread of pathogens producing these enzymes.
Authors’ Disclosures of Potential Conicts of
Interest
No potential conicts of interest relevant to this article were re-
ported.
Acknowledgement
We thank Dr. Kenneth S. Thomson (Center for Research in Anti-
Infectives and Biotechnology, Department of Medical Microbiol-
ogy and Immunology, School of Medicine, Creighton University,
Omaha, Nebraska) for providing class A carbapenemase-pro-
ducing isolates.
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700 bp
500 bp
400 bp
300 bp
200 bp
100 bp
KPC
SME
IMI/NMC-A
GES
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Fig. 1. Results of multiplex PCR for class A carbapenemase (CAC)-
producing strains. Multiplex PCR products were separated on a 2%
agarose gel. Lanes 1 and 14 show the 100-bp DNA ladder; lane 2,
the PCR product of the negative control (distilled water); lanes 3
and 4, KPC-type enzyme-producing strains; lane 5, SME-type; lanes
6 and 7, NMC-A and IMI-type, respectively; lanes 8-13, GES-type.
The amplied product from each PCR is indicated on the right, and
the size of the marker in base pairs is shown on the left.