|Year : 2014 | Volume
| Issue : 3 | Page : 256-260
Transfer patterns of integron-associated and antibiotic resistance genes in S. flexneri during different time intervals in Tianjin, China
J Wang1, F Liang2, X-Mei Wu1, W Qi1
1 Department of Infection Disease, Second Hospital of Tianjin Medical University, Tianjin, China
2 Department of Infection Disease , The Children's Hospital, Tianjin, China
|Date of Submission||02-May-2013|
|Date of Acceptance||28-Nov-2013|
|Date of Web Publication||10-Jul-2014|
Department of Infection Disease, Second Hospital of Tianjin Medical University, Tianjin
Source of Support: The Indian Council of Medical Research,
New Delhi, India,, Conflict of Interest: None
Background: Shigella is one of the common genera of pathogens responsible for bacterial diarrhoea in humans. According to World Health Organisation (WHO), 800,000-1,700,000 patients in China were infected with Shigella spp. in 2000, and Shigella flexneri is the most common serotype (86%). Objectives: We investigated the transfer patterns of integron-associated and antibiotic resistance genes in S. flexneri during different time intervals in the city of Tianjin in the People's Republic of China. Materials and Methods: The integrase-encoding and variable regions of the integrons of the bacterial strains were amplified by polymerase chain reaction (PCR), followed by gene sequencing. Fifty-six S. flexneri strains, 32 of which were stored in our laboratory and the other 24 were isolated from tertiary hospitals in Tianjin during different time intervals, were tested for their sensitivity to 12 antibiotics by using the Kirby-Bauer antibiotic testing method (K-B method). Results and Conclusion: Of the 32 strains of S. flexneri isolated from 1981 to 1983 and stored in our laboratory, class 1 integron was detected in 28 strains (87.50%), while 27 strains (84.37%) harboured an aminoglycoside resistance gene, aadA, in the variable region of their integrons. Class 1 integron was identified in 22 (91.67%) of the 24 S. flexneri strains isolated from 2009 to 2010, whereas the variable region and 3′-end amplification were not present in any of the strains. Class 2 integron was not found in the 1981-1983 group (group A) of strains; although 19 (79.17%) of the 24 strains in the 2009-2010 group (group B) possessed class 2 integron, and the variable region of the integron harboured dfrA1 + sat1 + aadA1 genes, which, respectively, mediate antibiotic resistance to trimethoprim, streptothricin and streptomycin. Seventeen strains of the total 56 possessed both class 1 and 2 integrons. Strains belonging to group A were highly resistant to tetracycline, chloramphenicol and a combination of trimethoprim-sulfamethoxazole; 65.63% of the strains were multi-resistant to three or more antibiotics. In group B, the strains showed high resistance to ampicillin, trimethoprim-sulfamethoxazole, piperacillin and tetracycline; 83.33% of the strains were multi-resistant to three or more antibiotics. Class 1 and 2 integrons exist extensively in S. flexneri, and the 3′-conserved segments of class 1 integron may have deletion or other types of mutations. Comparing the antibiotic and multi-drug resistance of group A with that of group B, it is apparent that the antibiotic resistance and the incidence of genes that confer multi-drug resistance have increased over the years in S. flexneri.
Keywords: Drug resistance, Integron, S. flexneri, Tianjin
|How to cite this article:|
Wang J, Liang F, Wu XM, Qi W. Transfer patterns of integron-associated and antibiotic resistance genes in S. flexneri during different time intervals in Tianjin, China. Indian J Med Microbiol 2014;32:256-60
|How to cite this URL:|
Wang J, Liang F, Wu XM, Qi W. Transfer patterns of integron-associated and antibiotic resistance genes in S. flexneri during different time intervals in Tianjin, China. Indian J Med Microbiol [serial online] 2014 [cited 2019 Dec 9];32:256-60. Available from: http://www.ijmm.org/text.asp?2014/32/3/256/136556
| ~ Introduction|| |
Shigella is one of the common genera of pathogens responsible for bacterial diarrhoea in humans. According to World Health Organisation (WHO),  800,000-1,700,000 patients in China were infected with Shigella spp. in 2000, and Shigella flexneri is the most common serotype (86%). Since the 1980s, Shigella infection has gradually increased despite the widespread application of antibiotics. In the past, studies on the mechanism of bacterial antibiotic resistance mainly focused on gene mutation. Subsequent research has found that bacteria acquire external antibiotic resistance genes through horizontal gene transfer, which accelerate the emergence of clinical antibiotic-resistant strains. The hereditary structures, which are closely related to horizontal transfer of bacterial antibiotic resistance genes, include plasmids, transposons, integrated phages and a recently discovered gene capture and dissemination system called integron. Previous studies ,, have recently reported that class 1 and 2 integrons are linked to drug resistance in Shigella. In this study, we analysed the antibiotic resistance of 56 S. flexneri strains carrying class 1 and class 2 integrons, which were isolated during different time intervals. We aimed to understand the horizontal gene transfer of class 1 and class 2 integrons and the changes in antibiotic resistance in the S. flexneri strains during different time intervals in Tianjin.
| ~ Materials and Methods|| |
Our study included 56 heterologous S. flexneri strains, 32 of which were stored in our laboratory from 1981 to 1983 (group A). The other 24 strains were isolated from stools of patients (ages ranging from 2 to 70 years) who presented with bacillary dysentery at the gastrointestinal clinics of some tertiary hospitals in Tianjin from 2009 to 2010 (group B).
Twelve types of antibiotic disks produced by the Beijing Tiantan Institute of Biological Products Co., Ltd were used in our study. Each disk was impregnated with antibiotics such as trimethoprim-sulfamethoxazole (SMZ-TMP), norfloxacin, levofloxacin, ampicillin, piperacillin, ceftriaxone, cefotaxime, ceftazidime, cefepime, imipenem, tetracycline and chloramphenicol. Primer synthesis was performed by Sangon Biotech Co., Ltd (Shanghai, China), and gene sequencing was performed by BGI Sequencing (Beijing, China).
Antimicrobial susceptibility testing
Antimicrobial susceptibility tests were performed by the Kirby-Bauer antibiotic testing method (K-B method) and the bacteriostatic diameter of the zones of inhibition was used as a standard criterion. The CLSI 2010 criterion was used as a standard for the criteria and interpretation of our experiment's results. Escherichia More Details coli (ATCC25922) was adopted as a control strain for antibiotic susceptibility.
Detection and analysis of the integrons
Class 1 integron integrase, 3′-conserved segment and class 2 integron integrase were, respectively, amplified using the primers intI1L/intI1R,  qacE∆1-F/sul1-B  and intI 2L/intI 2R.  The variable regions of class 1 and class 2 integrons were amplified using the primers hep58/hep59  and hep74/hep51  [Table 1]. We designed downstream primers dfrA12R, dfrA17R, aadAR and aadA5R based on the GenBank genetic sequences, and amplified corresponding resistance gene cassettes with hep58 as an upstream primer. PCR-amplified integron sequences were analysed by using agarose gel electrophoresis on a 1% gel at 100 V for 30 min, while amplified sequences of the variable region were analysed by electrophoresis on a 1.5% agarose gel at 100 V for 45 min. A gel imaging system (Gel Doc 2000; Bio-Rad) was subsequently used for photographing the gels. Results of direct sequencing of PCR products were compared with standard strains of GenBank's databank using NCBI's BLAST.
Restriction endonuclease reaction
Ten micro litre PCR amplification products, 1.5 μl restriction enzyme buffer and 1 μl HinfI endonucleases were incubated together at 37°C for 2 h. After adding 8 μl loading buffer to the endonuclease products, we performed electrophoresis on a 2% agarose gel at 110 V for 90 min followed by photography of the gels by using a gel imaging system (Gel Doc 2000; Bio-Rad).
Experimental data were analysed using SPSS 17.0. Statistical analysis was performed using Fisher's exact test. A P < 0.05 was considered statistically significant.
| ~ Results|| |
Results of the drug sensitivity test
With regard to the Shigella sp. in group A, the strains showed highest resistance to tetracycline (93.75%), followed by the resistance rates to chloramphenicol (78.13%), and trimethoprim-sulfamethoxazole (31.25%). The drug resistance rates of the strains to ampicillin and other antibiotics were lower than 20%, discounting the drug resistance to third and fourth generation cephalosporin, fluoroquinolonese and imipenem. Twenty-one strains of the 32 were multi-resistant to three or more antibiotics, which accounted for 65.63%. In group B, the resistance of the strains to ampicillin was the highest (95.83%), followed by the resistance rates to tetracycline (91.67%), trimethoprim-sulfamethoxazole (91.67%), chloramphenicol (58.33%) and piperacillin (54.17%). The resistance rates of the strains to cefotaxime and ceftriaxone were higher than 30%. The strains' rate of resistance to ceftazidime, cefepime, levofloxacin and norfloxacin was lower, discounting the resistance to imipenem [Table 2]. Twenty of the 24 strains (88.33%) were multi-resistant to three or more antibiotics.
Detection of class 1 integron
With respect to the 32 strains in group A, class 1 integron was detected in 28 strains (87.50%), and 27 of those 28 integron-positive strains also possessed the variable region sequences [Table 3], [Figure 1]. The PCR-amplified products of the sequences were identical in size, and confirmed to be copies of a 1000 bp fragment through restriction enzyme analysis. The gene sequencing analysis showed that the sequence harboured an aminoglycoside drug resistance gene cassette aadA. Among the 24 strains of the group B, 22 strains (91.67%) contained class 1 integron, while both the variable region and the 3′-end amplification were absent [Table 3]. PCR amplification of the gene cassettes showed that five strains were positive for blaoxa-30 and aadA, while four strains were positive for dfrA12 [Table 4], [Figure 2], which had a coincidence rate of 99% according to homology analysis (BLAST) with the sequence obtained from the GenBank database. The amplification of DfrA17 and aadA5 was negative.
|Figure 1: PCR amplification of class 1 integrase M, DL2000 DNA Marker; 1, Negative control; 2-4, class 1 integrase amplification-positive strains|
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|Figure 2: PCR amplification of aadA, dfrA12 gene cassettes (a) M, DL2000 DNA Marker; 1, Negative control; 2, aadA-positive strains N8; (b) M, DL 2000 DNA Marker; 1-5, dfrA12 amplification-positive strains; 6, negative control|
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Results of class 2 integron detection
Class 2 integron amplification of 32 strains in group A was negative. Class 2 integron in group B was detected in 19 strains (79.17%) [Table 3], the variable region of which harboured dfrA1 + sat1 + aadA1 genes that mediate antibiotic resistance to trimethoprim, streptothricinm and streptomycin. Among those 19, 17 strains were positive for class 1 integron amplification. Nineteen strains with both class 1 and 2 integrons were analysed via HinfI restriction enzyme digestion. The band pattern of the sequences obtained via restriction enzyme digestion were identical with the sequence of the class 2 integron detected in the strains of our study [Figure 3]. The gene sequencing of a randomly selected sample with positive class 2-integron amplification showed that the class 2 integron sequence was about 2200 bp, which was identified to harbour the dhfl gene responsible for antibiotic resistance to trimethoprim, the satl gene encoding for streptothricin resistance, and the aadA gene conferring streptomycin resistance. These findings were verified by homologous comparison with the corresponding sequences of standard strains from the GenBank databank (No. AY140652) with a coincidence rate of 99%.
|Figure 3: Electrophoretogram of class 2 integron variable region M, DL 2000 DNA Marker; 1, strains A11; 2, strains A6; 3, strains A4; 4, strains N10|
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| ~ Discussion|| |
The common epidemic Shigella genotypes in China are mainly S. flexneri and S. sonnei. In 2007, results from national monitoring sites for bacillary dysentery showed  that S. flexneri and S. sonnei were the dominant causative pathogens of bacillary dysentery in China. Nearly 57.21% cases of bacterial diarrhoea were caused by S. flexneri and 42.41% cases of bacterial diarrhoea were caused by S. sonnei. The drug sensitivities of S. flexneri strains, which were isolated from 2009 to 2010, showed that the resistance of S. flexneri to antibiotics such as ampicillin, piperacillin, trimethoprim-sulfamethoxazole, and third and fourth generation cephalosporin and fluoroquinolones had evidently increased than the antibiotic resistance of the S. flexneri strains found in group A with significant differences, while the resistance rates of the strains to tetracycline and chloramphenicol remained essentially similar.
The integron system, first proposed by Stokes et al.,  in 1989, consists of three components: A 5′-conserved segment (5′-CS), a 3′-conserved segment (3′-CS) and a variable region between them. The 5′-CS is the elementary structure of an integron and includes the integrase-encoding intI, a recombination site attI and a variable region promoter (Pant). The variable region harbours different numbers of gene cassettes with different functions, but it is not an elementary structure of the integron, since some integrons have no gene cassettes between the 5′-CS and 3′-CS. An integron can recognise and capture (or delete) mobile gene cassettes through the action of integrase, and can be expressed under the action of a promoter located upstream of the integron in order to confer antibiotic resistance, and even multi-drug resistance, to the bacteria. Currently, several studies have been investigating the integron system, which has been found in various antibiotic-resistant bacteria, including the members of Shigella. Among them, class 1 and class 2 integrons are closely related to multi-drug resistance of Shigella spp., but the detection rate may vary between different regions and species. It has been reported that the detection rate of class 2 integron has been far higher than that of class 1 integron in Shigella spp. ,,
Our study showed that class 1 integron-positive rate of Shigella sp. in Tianjin was 87.50% in group A and 91.67% in group B. There was no significant statistical difference between the class 1 integron-positive rates of the two groups. The amplification of the variable region of class 1 integron showed that group A had 28 strains that contained the integron along with the 3′-CS, of which 27 were positive for the variable region containing the aminoglycoside-resistance gene cassette aadA, which was determined through gene sequencing. Group B had 22 integron-positive strains that did not have the variable region or the 3′-CS. To exclude the omission caused by the 3′-CS mutation, we designed downstream primers dfrA12R, dfrA17, aadA and aadA5 based on the drug-resistance gene cassettes dfrA12, dfrA17, aadA and aadA5, which are usually carried by the class 1 integron. With hep58 as an upstream primer, corresponding drug-resistance gene cassettes were amplified to confirm whether the variable region was present, which showed that four strains were positive for dfrA12 amplification, while five strains were positive for aadA amplification and the blaoxa-30 gene located upstream. We speculate that a 3′-CS deletion mutation or mutations in the class 1 integron might exist in the strains belonging to group B. Class 2 integron was absent in the Shigella strains of group A, while 79% of the strains in group B possessed the class 2 integron. Mammina et al.,  speculated that S. sonnei have been carrying the class 2 integron since the 1980s, a theory they put forth by comparing the incidence of class 2 integron in S. sonnei strains isolated during different time intervals from 1971 to 2003 in Southern Italy, with which our conclusion was similar. Class 2 integron-positive strains possessed the variable region, which was confirmed through restriction endonuclease analysis to be the same fragment, and gene sequencing analysis showed class 2 integron carried the trimethoprim resistance gene dfrA1, streptothricin resistance gene sat1, and streptomycin resistance gene aadA1.
Our results have shown that class 1 integron-positive rate of S. flexneri between 2009 and 2010 had slightly increased than that between 1981 and 1983 in Tianjin, but the difference was not statistically significant. Both, variable region of class 1 integron and the 3′-CS of Shigella sp. were absent in the strains of group B. We think that the 3′-CS might have deletion or other mutations through the amplification of common antibiotic-resistance gene cassettes. Class 2 integron-amplification positive rate of the Shigella strains in group B was 79.17%, of which 17 strains had both class 1 and class 2 integrons. Considering the drug sensitivity results, we believe that the increased resistance of the Shigella strains to gentamicin, trimethoprim-sulfamethoxazole and cephalosporin might be linked to mutations in the structure of the class 1 integron, the ability to carry more drug-resistance gene cassettes as well as possessing the class 2 integron. However, this needs to be confirmed through further research on larger quantities of samples.
In conclusion, with the wide use of broad-spectrum antibiotics, the rate of resistance to commonly used antibiotics and the incidence of multiple drug-resistant strains of Shigella have increased than earlier. Class 1 and class 2 integrons are widespread in the members of Shigella, and 3′-CS deletion or mutations in class 1 integron may exist in some strains.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]