Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 527-532
doi:10.1111/j.1745-7270.2007.00304.x
Incidence of Extended-Spectrum
b-Lactamases and Characterization of
Integrons in Extended-Spectrum b-Lactamase-producing
Klebsiella pneumoniae Isolated in Shantou, China
Fen YAO1, Yuanshu
QIAN1*, Shuzhen CHEN2, Peifen WANG2, and
Yuanchun HUANG2
1 Department of
Pharmacology, Shantou University Medical College, Shantou 515041, China;
2 Department of Clinical
Laboratory, First Affiliated Hospital, Shantou University Medical College, Shantou
515041, China
Received: March 4,
2007
Accepted: April 18,
2007
This work was
supported by a grant from the Natural Science Foundation of Guangdong Province
(021222)
*Corresponding
author: Tel, 86-754-8900432; Fax, 86-754-855-7562; E-mail,
Abstract This study is concerned with the level of
antibiotic resistance of extended-spectrum b-lactamase
(ESBL)-producing Klebsiella pneumoniae, isolated in Shantou, China, and
its mechanism. Seventy-four non-repetitive clinical isolates of K. pneumoniae
producing ESBLs were isolated over a period of 2 years. Antibiotic
susceptibility, carried out by Epsilometer test, showed that most of the
isolates were multiresistant. Polymerase chain reaction showed that, among the
several types of b-lactamases, SHV was the most
prevalent, TEM was the second most prevalent, and CTX-M was the least
prevalent. Sixty-nine isolates were positive for integrase gene IntI1,
but no IntI2 or IntI3 genes were found. The variable region of
class 1 integrons were amplified and further identified by sequencing. Thirteen
different gene cassettes and 11 different cassette combinations were detected.
Dfr and aadA cassettes were predominant and cassette combinations dfrA12,
orfF and aadA2 were most frequently found. No gene cassettes
encoding ESBLs were found. Integrons were prevalent and played an important
role in multidrug resistance in ESBL-producing K. pneumoniae.
Keywords Klebsiella pneumoniae; extended-spectrum b-lactamase (ESBL); integron; gene
cassette
Klebsiella pneumoniae is an important
hospital or community-acquired pathogen that is naturally susceptible to
extended-spectrum cephalosporins (ESCs). However, strains resistant to these
antibiotics mediated by extended-spectrum b-lactamases (ESBLs) have
now spread worldwide. ESBLs contain several types of b-lactamases, including SHV,
TEM, CTX-M and OXA [1]. Dissemination of antibiotic resistance genes by
horizontal transfer has led to the rapid emergence of antibiotic resistance
among clinical isolates. In the 1980s, genetic elements termed integrons were
identified [2]. To date, at least eight classes of integrons, with different Int
genes, have been described [3]. Among the different integron families, class 1
integrons are found to be most prevalent in drug-resistant bacteria [4]. Class
1 integrons are mobile DNA elements with a specific structure consisting of two
conserved segments flanking a central region containing ?assettes that usually code
for resistance to specific antimicrobials [5]. The 5‘-conserved segment
contains the integrase gene (IntI1), a promoter region, and the IntI1-specific
integration site attI1. The 3‘-conserved segment usually contains
a combination of the three genes qacED1 (antiseptic resistance), sulI (resistance to sulfonamides),
and an open reading frame (orf5) of unknown function [6]. Between the
two conserved segments, the central variable region can contain from zero to
multiple cassettes [7]. The acquisition of resistance genes in bacteria is
often facilitated by integrons. The presence of integrons among clinical K.
pneumoniae isolates might account for multiple-antibiotic resistance. In this study, we determined the incidence of ESBL-coding genes and
characterized the different variable regions of the class 1 integrons in order
to identify the mechanism of resistance in clinical K. pneumoniae isolates.
Materials and Methods
Clinical isolates
From February 2001 to June 2003, 74 non-repetitive (one per patient)
clinical isolates of K. pneumoniae producing ESBLs were isolated from
hospitalized patients in the First Affiliated Hospital, Shantou University
Medical College (Shantou, China). Twenty-three strains were isolated from the
Department of Neurosurgery, 14 from the Neonatology Center, 11 from the Surgery
Intensive Care Ward, 7 from the Department of Pediatrics, 5 from the Department
of Neurology and 14 from other wards. Sputum was the most frequent type of
sample (68 strains), followed by exudates (three strains), blood (one strain),
urine (one strain), and stool (one strain). Production of ESBLs was determined
by an agar dilution method and the double-disk synergy test by
ceftazidime/cefotaxime with and without clavulanate on Mueller-Hinton agar. The
results were interpreted according to Clinical and Laboratory Standards (CLSI)
antimicrobial susceptibility testing standards (2006) [8].
Antimicrobial susceptibility
determination
Minimal inhibitory concentrations to antimicrobial agents including cefotaxime,
ceftazidime, ceftriaxone, cefepime, imipenem, gentamicin, amikacin,
ciprofloxacin and tetracycline were determined. Epsilometer test (E-test) was
carried out according to the manufacturer’s recommendations with E-test strips
(AB BIODISK, Solna, Sweden). Escherichia coli ATCC 35218 was used as the
quality control strain.
Polymerase chain reaction
(PCR), cloning, sequencing and protein analysis
Template DNA was prepared as follows: a cell pellet from 1.5 ml of
overnight culture was resuspended in 500 ml of TE (10 mM Tris, 1 mM
EDTA, pH 8.0) after centrifugation and boiling for 10 min. After
centrifugation, the supernatant was used for PCR. The primers and conditions
for PCR are listed in Table 1 [9–15]. Strains containing the
IntI1 gene were subsequently subjected to PCR for amplification of the
class 1 integron gene cassettes with primers RB317 and RB320 as described [13].
Amplicons of the same size obtained with primers RB317 and RB320 were digested
with EcoRI, HindIII and BspI. PCR product with different
restriction profiles was purified with a UNIQ-10 column PCR product
purification kit (Sangon, Shanghai, China) and cloned into pUCm-T vector by T4 ligase (Sangon). After incubation at 16 ?C for 1 h, ligation
mixtures were used to transform into E. coli JM109. Transformants
containing inserts were screened by blue/white colony on a Mueller-Hinton agar
plate containing ampicillin (100 mg/ml), IPTG plus X-gal, then identified by PCR
analysis. Recombinant plasmid DNA extracted from transformants was sequenced by
Invitrogen (Shanghai, China). DNA sequences were translated into protein
sequences using Web-based analysis tools (http://www.expasy.ch/tools/dna.html)
then compared with the protein sequence of the GenBank database using the BLAST
network service (http://www.ncbi.nlm.nih.gov/blast).
Results
Antimicrobial susceptibility
determination
Most of the isolates were highly resistant (minimal inhibitory
concentration>128 mg/ml) to gentamicin and amikacin. More than half of the isolates
showed resistance or decreased
susceptibility (intermediate resistance) to ESCs except cefepime.
Although most of the isolates were multiresistant (resistant to more than two
classes of antibiotics), they all remained susceptible to imipenem (Table 2).
Prevalence of ESBL-coding IntI1,
IntI2 and IntI3 genes
Most of the isolates contained either blaSHV, blaTEM,
or both. The blaSHV was amplified from 63 isolates, blaTEM
was amplified from 39 isolates, blaCTX-M was amplified from 21
isolates, blaOXA-1 was amplified from six isolates, and blaOXA-2
was amplified from only one isolate. The combinations of genotypes of ESBLs are
listed in Table 3. The IntI1 gene was detected in 69 of the 74
isolates included in this study. IntI2 and IntI3 genes were not
detected.
Characterization of cassette
arrays
Twelve isolates containing the IntI1 gene failed to produce
an amplicon by RB317 and RB320. Thirteen different gene cassettes and 11 groups
of variable segment were detected within the integrons (Fig. 3).
Table 4 showed an overview of the ESBLs
and various cassettes arrays detected in isolates of different resistance
phenotypes.
discussion
The introduction of ESCs has facilitated effective treatment of
severe infections caused by gram-negative bacteria. However, resistance to
these agents increased in recent years and this correlated with the increasing
use of ESCs [16]. According to the susceptibility test, imipenem and the
fourth-generation cephalosporin, cefepime, showed better in vitro
activity than third-generation cephalosporin, such as cefotaxime, ceftazidime
and ceftriaxone to ESBL-producing K. pneumoniae.
Resistance to ESCs is
primarily mediated by b-lactamases especially ESBLs and AmpC b-lactamases. To date,
although a variety of ESBLs have been described, SHV, TEM and CTX-M enzymes are
the three main types of EBSLs among members of the family Enterobacteriaceae
[17]. In our study, SHV b-lactamase was most prevalent, TEM b-lactamase was the second
most prevalent, and CTX-M b-lactamase was less than both. This prevalence of ESBLs appeared to
be different from those seen in other areas of China [18,19]. In fact,
ESBL-encoding genes in our study were not sequenced. Because primers for SHV
and TEM b-lactamases can amplify non-ESBLs SHV-1 and TEM-1 b-lactamases,
respectively, some SHV-positive and TEM-positive isolates might produce SHV-1
and TEM-1 b-lactamases [9,10].
The dissemination of antibiotic resistance genes among bacterial
strains is an increasing problem in bacterial infections. Integron had become
an important horizontal gene transfer system of resistance genes in clinical
isolates. Incidence of class 1 integron was high in ESBL-producing K.
pneumoniae. Twelve isolates containing the IntI1 gene failed
to produce an amplicon using primers RB317 and RB320. This was probably due to
the lack of a 3‘ conserved segment or the variable region was too long
to be amplified in these isolates. This phenomenon had been reported previously
[14].Integron-positive isolates were more likely to be multiresistant
than integron-negative isolates [20]. Multiresistant integrons are considered
to be important contributors to the development of antibiotic resistance among
Gram-negative bacteria [21,22]. In our study, high prevalence of class 1
integron contributed to the multiresistance in most isolates. PCR sequencing
analysis of the cassette arrays revealed a predominance of dfr and
aadA cassettes that confer resistance to trimethoprim and aminoglycosides.
The high incidence of aadA and aacA gene cassettes, confering
resistance to aminoglycosides, was an important reason for the high prevalence
of resistance to gentamicin and amikacin. The cassette combinations dfrA12,
orfF and aadA2 were most frequently found in this study and also
very prevalent in other areas. The reason for the wide distribution of some
integrons with a specific cassette combination is so far unknown [23,24]. To date, genes resistant to nearly every major class of antibiotics
including ESBL-coding genes such as blaCTX-M, blaGES, blaOXA
and blaVEB integrated into integron had been reported, but blaSHV
and blaTEM had not been found within integron [25–29]. In our
study, although all the isolates exhibited ESBLs activity, no cassette encoding
ESBLs was found, indicating that ESBL genes were not spread by integron. In our
previous study, 37 isolates in this study had been typed by pulsed-field gel
electrophoresis. Data showed that most of the isolates belong to a different
genotype. Isolates in the same pulsed-field gel electrophoresis type had
different resistance profiles, and most of them contained different types of
ESBL-coding genes and different gene cassettes [30]. It seemed that clonal
spread was not important for the dissemination of ESBLs and integron. As many
ESBLs and integrons are on conjugative plasmids, horizontal spread by
conjugation might be a major mechanism for their dissemination.These data indicated that integrons were very prevalent and played
an important role in multidrug resistance in ESBL-producing K. pneumoniae.
The production of ESBLs and integrons will continue to threaten the usefulness
of antibiotics as therapeutic agents.
Acknowledgement
We thank Shengping HU (Shantou University
Medical College, Shantou, China) for providing technical assistance.
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