|Year : 2013 | Volume
| Issue : 4 | Page : 385-389
Class 1 integrons contributes to antibiotic resistance among clinical isolates of Escherichia coli producing extended-spectrum beta-lactamases
T Chen, Y Feng, JL Yuan, Y Qi, YX Cao, Y Wu
Department of Clinic Laboratory, The Third Xiangya Hospital of Central South University, Changsha, Hunan, 410013, China
|Date of Submission||23-Nov-2012|
|Date of Acceptance||06-Jun-2013|
|Date of Web Publication||25-Sep-2013|
Department of Clinic Laboratory, The Third Xiangya Hospital of Central South University, Changsha, Hunan, 410013
Source of Support: This work was supported by the Hunan Nature
Science Foundation of Hunan, China (Key Program) (No. 08JJ3069)
and the Hunan Science & Technology Project of Hunan, China
(2010FJ3079), Conflict of Interest: None
Objectives: The objective of this study is to determine the prevalence of antibiotic resistance factors, including the production of extended-spectrum beta-lactamases (ESBLs) and the presence of class 1 integrons among Escherichia coli isolated from clinical specimens. Materials and Methods: Bacterial species identification was performed using a VITEK-2 system (VITEK2 GN-card; bioMérieux, France). Antimicrobial susceptibility testing was determined using the disk diffusion method according to the 2010 Clinical and Laboratory Standards Institute guidelines. Polymerase chain reaction (PCR) was used to detect integrons and amplify variable regions of the bla TEM, bla SHV and bla CTX-M genes. Gene cassettes were detected by deoxyribonucleic acid sequencing. Results: In this study, 58% (100/172) of clinical E. coli isolates were identified as ESBL producers. We found that 90% of the ESBL-producing E. coli isolates harbored the blaCTX-M gene, whereas only 59% and 32% possessed the blaTEM and blaSHV genes respectively. The presence of class 1 integrons was based on the detection of the integrase gene by PCR. A total of 69% of the ESBL-producing isolates were integron-positive. Resistance to 10 antibiotics, including quinolones, sulfonamides and β-lactam/enzyme inhibitors, was significantly higher in the class 1 integron-positive isolates (P < 0.05). The occurrence of class 1 integrons in blaTEM , blaSHV and blaCTX-M gene carriers was 72.9%, 84.4% and 68.9%, respectively. Class 1 integrons were detected in 61.5% of the isolates with only one ESBL genotype, but in 69.0% and 92.3% of the isolates with two or three different ESBL genotypes, respectively. Conclusions: Our findings indicate that clinical strains of bacteria with multiple ESBL genotypes may have greater opportunities to carry class 1 integrons.
Keywords: Antibiotic resistance, Class 1 integron, Escherichia coli, extended-spectrum beta-lactamase
|How to cite this article:|
Chen T, Feng Y, Yuan J L, Qi Y, Cao Y X, Wu Y. Class 1 integrons contributes to antibiotic resistance among clinical isolates of Escherichia coli producing extended-spectrum beta-lactamases. Indian J Med Microbiol 2013;31:385-9
|How to cite this URL:|
Chen T, Feng Y, Yuan J L, Qi Y, Cao Y X, Wu Y. Class 1 integrons contributes to antibiotic resistance among clinical isolates of Escherichia coli producing extended-spectrum beta-lactamases. Indian J Med Microbiol [serial online] 2013 [cited 2019 Jun 26];31:385-9. Available from: http://www.ijmm.org/text.asp?2013/31/4/385/118903
| ~ Introduction|| |
The spread of extended-spectrum beta-lactamases (ESBLs) in Enterobacteriaceae has become an ever-increasing problem. ,, In China, the CHINET national bacterial surveillance project found a 56.2% detectable rate of ESBL-producing Escherichia More Details coli in 2009 based on studies from 12 research laboratories in different hospitals.  Gene transfer between bacteria has been shown to play an emerging role in the acquisition of drug resistance and the horizontal dissemination of resistance genes is now known to be the main cause of resistance transmission. ,
Integrons, natural genetic elements that are capable of capturing and excising exogenous antibiotic resistance gene cassettes, play important roles in the horizontal dissemination of antibiotic resistance genes in bacteria. ,,, Meanwhile, integrons can be present on plasmids or as a part of a transposon and transfer along with them, facilitating the spread of antibiotic resistance genes among bacteria.  Class 1 integrons have been examined most extensively and are the most common type of integron found in clinical isolates. 
Additional investigations into class 1 integrons and ESBL bla genes are necessary, such as what are they and what is their relationship between them? How were they studied in other published papers? What are the unanswered questions of those papers and why should another study be performed? How important is this investigation?
In this study, we examined the presence and distribution of class 1 integrons in ESBL-producing clinical isolates to determine their correlation with drug resistance. Furthermore, we studied the association between integrons and the bla genes that encode β-lactamases and thus explored how it may influence antibiotic resistance.
| ~ Materials and Methods|| |
Bacterial strains and antimicrobial susceptibility testing
Between June and December 2009, E. coli was isolated from sputum, blood, urine, secretion and other samples from 172 in-patients (duplicate samples from the same patient were excluded). Identification of bacterial species was performed using a VITEK-2 system (VITEK2 GN-card; bioMérieux, France). Antimicrobial susceptibilities to 12 antimicrobial agents, including carbapenems, aminoglycosides, quinolones, sulfonamides and β-lactam/enzyme inhibitors, were tested by the disk diffusion method according to Clinical Laboratory Standards Institute guidelines (CLSI, 2010).  Isolates were screened for ESBL production using the clavulanate double-disk synergy method (CLSI, 2010)  with combination A (cefotaxime and a cefotaxime/clavulanic acid) or combination B (ceftazidime and a ceftazidime/clavulanic acid).
E. coli ATCC25922 and Pseudomonas aeruginosa ATCC27853 were used as drug sensitivity controls while Klebsiella pneumoniae ATCC70060 was used as an ESBL production positive control. Vibrio cholerae O1 strain SK10 was used as a positive control for class I integrons. The E. coli C600 strain used as a negative control was provided by Prof. Shi Lei in South China University of Technology. E. coli strains producing CTX-M, TEM and SHV were provided by Prof. Ni Yuxing of the Department of Microbiology, Ruijin Hospital, Shanghai Jiao Tong University.
Identification of integron regions and ESBL genes
Genomic deoxyribonucleic acid (DNA) of E. coli from clinical isolates was extracted using an EasyPure™ Genomic DNA Kit (Beijing TransGen Biotech Co., Ltd., China) and used as a template for the polymerase chain reaction (PCR) experiments. Detection of integrase and amplification of the integron variable region (VR) and of ESBL genes, including bla TEM, bla SHV and bla CTX-M, were performed with the primers listed in [Table 1]. The PCR reaction was carried out in 50-μL reaction mixtures using 2 × Pfu PCR SuperMix (Beijing TransGen Biotech Co., Ltd., China). The PCR products of the VR were purified using the EasyPure™ PCR Purification Kit (Beijing TransGen Biotech Co., Ltd., China). The nucleotide sequences were determined by Shanghai Invitrogen Biotechnology Co., Ltd., China.
|Table 1: Sequences of the primers used to detect the intI1 and ESBL genes|
Click here to view
Data were analysed using the Chi-squared test (χ2 test) to determine the significant differences in resistance. Differences were considered significant at P < 0.05.
| ~ Results|| |
ESBL screening and conformational tests
A total of 172 consecutive non-duplicate E. coli isolates were investigated in this study. The combination disk diffusion method identified 58% (100/172) of these as ESBL producers.
Prevalence of class 1 integrons and drug resistance in ESBL-producing strains
The PCR amplification results showed that of the 100 ESBL-producing isolates, 69 were positive for the class 1 integrons as indicated by the presence of the integrase gene (intI1). [Table 2] provides a comparison of the resistance patterns for class 1 integron-positive and integron-negative ESBL-producing isolates. The results revealed that resistance rates to meropenem and amikacin were <16% in both groups with no differences between them (P > 0.05). However, for the other 10 antibiotics studied [Table 2], the integron-positive ESBL-producing isolates had significantly higher resistance than the integron-negative ESBL-producing strains (P < 0.05).
|Table 2: Drug resistance in class 1 integron‑positive and integron‑negative ESBL‑producing strains|
Click here to view
Genetic assay of the VR of class 1 integrons
We examined the VR of the resistance gene cassettes in 69 integron-positive strains and found that 58 strains (84.1%) had a class 1 integron VR with an amplification size of 200-2400 bp. A total of 37 of these strains (63.8%) carried one kind of integron VR while 21 strains (36.2%) carried two kind of integron VR. A 1600-bp VR was detected in 41.4% (24/58) of these strains while the co-occurrence of both 2400 bp and 1600 bp VR was observed in 19.1% (11/58). The distribution of VR sizes is shown in [Table 3]. The DNA fragments of the VR were sequenced and the results were compared with the GenBank database using BLASTN alignment analysis. All the gene sequences found in the class 1 integrons and their arrangements are shown in [Table 3].
|Table 3: Types and arrangements of the VR gene cassettes within class 1 integrons|
Click here to view
Correlation between β-lactamase genes and class 1 integrons
PCR amplification of all the 100 ESBL-producing bacterial strains was performed to assay for the bla TEM, bla SHV and bla CTX-M genes, which encode the class A β-lactamases that are commonly responsible for antibiotic resistance. A total of 97 of these ESBL-producing strains were found to have at least one of the β-lactamase gene types; none of these genes were detected in three E. coli strains. The bla TEM gene was detected in 59 strains (59%), the bla SHV gene was detected in 32 strains (32%) and the bla CTX-M gene was detected in 90 strains (90%). A total of 26 strains (26%) possessed only one type of β-lactamase gene while 71 strains (71%) had more than one kind of ESBL gene [Table 4].
|Table 4: Occurrence of class 1 integrons in ESBL‑producing isolates with different ESBL genotypes|
Click here to view
An analysis of the relationship between class 1 integrons and the β-lactamase genes in the ESBL producers revealed that detection rates for class 1 integrons in bla TEM, bla SHV and bla CTX-M gene carriers were 72.9% (43/59), 84.4% (27/32) and 68.9% (62/90), respectively. The occurrence of class 1 integrons in strains carrying the bla SHV gene was significantly higher than that in strains carrying the other two β-lactamase genes (P < 0.05).
| ~ Discussion|| |
Class 1 integrons are the most common antibiotic resistant genes found in the clinical insolates of Gram-negative bacteria. ,, Until date, more than 130 gene cassettes have been identified and the encoded products would confer bacterial resistance to almost all antibiotics.  Phongpaichit et al.  reported that 74.7% of ESBL-producing E. coli were integron-positive isolates. Integrons has been identified as a primary source of resistance genes and claimed that they were reservoirs of antimicrobial resistance genes with microbial populations. , Our study found that 69% of clinical ESBL-producing isolates carry class 1 integrons, indicating that these integrons are widely present in ESBL-producing strains of bacteria and may influence the level of antibiotic resistance. Indeed, the accumulation of resistance genes by integrons may be an important factor in the development of multi-drug-resistant E. coli strains.
Between the integron-positive and integron-negative strains of ESBL-producing bacteria, the resistance rates to meropenem and amikacin were low with no significant differences. Because meropenem, a carbapenem antibiotic, can enter through the bacterial cell wall and is stable to β-lactamase, especially ESBL and the amikacin resistance gene, aacA7, was not detected in this study. As such, these antibiotics may be considered the drugs of choice for the treatment of ESBL-producing strain-induced infections. However, although carbapenems have higher activity, their widespread use will inevitably induce the development of drug-resistant strains.  As a result of the membrane permeability of these agents, carbapenem-resistant strains of ESBL-producing E. coli are already evolving,  posing a significant threat to the clinical treatment of ESBL-induced bacterial infections. Interestingly, significant differences in the resistance rates between integron-positive and integron-negative groups were observed for the other 10 antibiotics studied.
In our study, the occurrence of the resistance gene cassette in the class 1 integrons VR was 84.1% and six types of amplified fragment were detected in these instances. The sequencing results show that the 1600 bp resistance gene cassettes, which were the most common, were trimethoprim and aminoglycoside resistance genes. It may be that when bacteria are under antibiotic selective pressure, resistance gene cassettes are more easily captured and accumulated by integrons.
ESBL genes are primarily plasmid mediated, but studies have reported that the bla CTX-M-2 gene is found in the VR of class 1 integrons.  The incidence of CTX-M among plasmid-mediated ESBL strains is continuously increasing and bla CTX-M has already become the most common ESBL gene.  In this study, the occurrence of CTX-M was 90%, much higher than that of TEM (59%) and SHV (32%). Further analysis of the correlation among these three ESBL genes and class 1 integrons showed that the occurrence of class 1 integrons in bla TEM, bla SHV and bla CTX-M gene-positive strains was 72.9%, 84.4% and 68.95%, respectively. This indicates that class 1 integrons were more commonly associated with the bla SHV gene than with the other two genes, suggesting that in ESBL-producing strains, bla SHV carriers were more closely related to class 1 integrons carriers, probably due to genetic linkage between them. Although both ESBL- and integron-encoding genes are located in plasmids, , the ESBL genes in this study had not been captured and integrated by integrase. This suggests that further research is needed to determine whether these two genes are located within the same plasmid.
Analysis of the correlation between integrons and bla TEM, bla SHV or bla CTX-M genes indicated that 71% of the ESBL producers had two or more ESBL genotypes. Among strains with only one ESBL genotype, class 1 integrons were detected in 61.5% while among those with two or three different ESBL genotypes, the class 1 integron detection rates were 69.0% and 92.3%, respectively. This finding suggests that clinical strains of bacteria with multiple ESBL genotypes have a greater opportunity to carry class 1 integrons. Therefore, bacteria carrying both integrons and ESBL genes have much stronger multi-resistance activity.
| ~ Acknowledgement|| |
This work was supported by the Nature Science Foundation of Hunan, China (Key Program) (No. 08JJ3069) and the Science and Technology Project of Hunan, China (2010FJ3079).
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[Table 1], [Table 2], [Table 3], [Table 4]
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