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Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 527-532 |
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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,
[email protected]
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 揷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 multi�resistant (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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