|Year : 2016 | Volume
| Issue : 3 | Page : 303-307
Distribution of Class II integrons and their contribution to antibiotic resistance within Enterobacteriaceae family in India
P Singha1, DD Chanda2, AP Maurya1, D Paul1, A Chakravarty2, A Bhattacharjee1
1 Department of Microbiology, Assam University, Silchar, Assam, India
2 Department of Microbiology, Silchar Medical College and Hospital, Silchar, Assam, India
|Date of Submission||16-Dec-2015|
|Date of Acceptance||30-Jun-2016|
|Date of Web Publication||12-Aug-2016|
Department of Microbiology, Assam University, Silchar, Assam
Source of Support: None, Conflict of Interest: None
Background: Integrons are the main contributors to the development of multidrug resistance (MDR) among Gram-negative bacilli. There is a lack of knowledge about the molecular relation between gene cassettes and antibiotic resistance in India. Objective: In this study, we have investigated the occurrence of Class II integron and their cassette array among Enterobacteriaceae. Materials and Methods: A total of 268 MDR non-duplicate strains of Enterobacteriaceae were collected from Silchar Medical College and Hospital, Silchar, Assam, India, during June 2012 to May 2013. Polymerase chain reaction was performed for detection of the integrase genes and gene cassettes within the Class II integron which were further analysed by sequencing. Results: Class II integron was observed in 47 isolates. Four different gene cassette arrangements were detected: dfrA1-sat2-aadA1; dfrA1-sat2-aadA1-orfX-ybeA-ybfA-ybfB-ybgA; dfrA12-sat2-aadA1; and dfrA1-linF-aadA1. The most prevalent cassette combination was dfrA1-sat2-aadA1. This study has also identified a set of gene cassette associated with linF gene instead of sat2 gene. Conclusion: Further investigation is required to determine the current situation and important reservoir of Class II integron for the transmission of drug resistance among Enterobacteriaceae and their contribution to antimicrobial resistance in hospital environment .
Keywords: Class II integron, Enterobacteriaceae, gene cassette, integrase, multidrug resistance
|How to cite this article:|
Singha P, Chanda D D, Maurya A P, Paul D, Chakravarty A, Bhattacharjee A. Distribution of Class II integrons and their contribution to antibiotic resistance within Enterobacteriaceae family in India. Indian J Med Microbiol 2016;34:303-7
|How to cite this URL:|
Singha P, Chanda D D, Maurya A P, Paul D, Chakravarty A, Bhattacharjee A. Distribution of Class II integrons and their contribution to antibiotic resistance within Enterobacteriaceae family in India. Indian J Med Microbiol [serial online] 2016 [cited 2020 Feb 21];34:303-7. Available from: http://www.ijmm.org/text.asp?2016/34/3/303/188319
| ~ Introduction|| |
Integrons are genetic elements that contain the components of a site-specific recombination system that recognises and captures antibiotic resistance gene. , The multidrug resistance (MDR) integrons are considered to be the important contributors to the development of antimicrobial resistance among Gram-negative bacilli. ,, Class I and Class II integrons are both usually associated with bacterial resistance to antimicrobials in hospital environment. In contrast to Class I integron, there is less knowledge about the architecture of Class II integron and their association with MDR within this subcontinent. The backbone structure of integron comprises an integrase (intI), a recombination site (attI) and a promoter (Pc) and variable region with integrated gene cassette.  Class II integron is situated in a decreased diversity of non-replicative transposons Tn7 and Class II integrase genes represents an internal stop codon (TAA). ,, Most of the gene cassette arrays of Class II integron are conserved and show the lack of dynamics recombination due to integrase inactivation.  The variable region of Class II integron mostly carries the three antibiotic resistance gene, namely, dfr1, sat2 and aadA.  The resistance against trimethoprim is encoded in dfr gene within the Class II integron and high level of resistance to trimethoprim associated with the presence of dfrA1, dfrA5, dfrA7 and dfrA17.  This study was performed to describe the occurrence of Class II integron and contribution of their genetic architecture in accumulation of cassette array among Enterobacteriaceae in hospital environment and their contribution to antibiotic resistance.
| ~ Materials and Methods|| |
The study was carried out with 268 consecutive, non-duplicated clinical isolates, collected from patients admitted to various wards or attended clinics of Silchar Medical College and Hospital, Silchar, Assam, India, between June 2012 and May 2013. The clinical isolates were identified by the standard biochemical tests.  The susceptibility was performed against ampicillin (10 μg), co-trimoxazole (1.25/23.75 μg), ciprofloxacin (5 μg), gentamicin (10 μg) and cefepime (30 μg) (Hi-Media, Mumbai, Maharashtra, India), by Kirby-Bauer disc diffusion method and results were interpreted as per the Clinical and Laboratory Standards Institute criteria.  Escherichia coli ATCC 25922 was taken as negative control.
Template DNA preparation
DNA templates were prepared by boiling centrifugation method. Overnight culture was suspended in 200 μl of sterile distilled water and heated at 90°C for 10 min. The boiled cell suspension was centrifuged and the subsequent supernatant was used as template for polymerase chain reaction (PCR).
Screening of Class II integron
PCR was performed for the presence of Class II integrase gene  [Table 1] using bacterial DNA as template. The PCR conditions were as follows: 94°C for 3 min, followed by 32 cycles at 94°C for 20 s, 54°C for 20 s, 72°C for 1 min and final extension at 72°C for 5 min.
Amplification of Class II integron
Class II integrase-positive isolates were further subjected to hep PCR  [Table 1] for amplification of whole Class II integrin.  The PCR conditions were 95°C for 2 min, followed by 35 cycles at 95°C for 20 s, 56°C for 40 s, 72°C for 2 min and final extension at 72°C for 5 min.
Cloning of Class II integron
To determine the genetic array of gene cassettes within the variable region of Class II integron, amplified products were cloned using pGEM-T vector (Promega, Madison, WI, USA) and transformed into E. coli JM107. Plasmid was purified from the transformants and sequenced. Sequences were compared using BLAST search (http://www.ncbi.nlm.nih.gov/BLAST).
Antimicrobial susceptibility of bacterial strains and clones
Based on the gene cassette array, transformants and wild-type isolates were further subjected to antibiotic susceptibility against gentamicin (10 μg), netilmicin (30 μg), amikacin (30 μg), sulphafurazole (300 μg), trimethoprim (5 μg), kanamycin (30 μg) and ciprofloxacin (5 μg) (Hi-Media, Mumbai, Maharashtra, India).
| ~ Results|| |
Among the isolated organisms (n = 268), E. coli was the most predominant type (n = 174) followed by Klebsiella pneumoniae (n = 71) and Proteus mirabilis (n = 23) [Table 1]. A majority of isolates were obtained from urine, followed by pus, stool and other specimens [Table 2].
Susceptibility profiling was performed against five different types of antibiotics and results had shown that high levels of resistance was found against co-trimoxazole (91%), ampicillin (87%), gentamicin (81%) ciprofloxacin (60%) and cefepime (58%) [Table 3].
Thirty-two isolates were harbouring both Class I and Class II integron and 15 isolates were carrying single Class II integron [Table 4]. To study the genetic context of the variable regions of all the Class II integron-positive isolates, four samples failed to fetch any amplified product [Figure 1]. The variable size of Class II integrons investigated has size ranging from 2 to 2.5 kb. Sequencing results revealed four different types of cassettes array. Amplicon with sizes of 2-2.3 kb was found in 11 stains [Figure 2] and was found harbouring arrangement I with dfrA1, sat2 and aadA1 cassette array. In one strain, the amplified product of 2.3 kb showed arrangement II, array of ybeA gene, which carried a truncated attI2 site with three additional genes (ybfA, ybfB and ybgA) in the downstream of the integron II. In arrangement III, dfrA12 was followed by sat2 and aadA1 and this particular arrangement was observed in one isolate [Figure 3]. The arrangement IV was unusual and was only observed in two isolates of P. mirabilis where linF was flanked by dfrA1 and aadA1 in upstream and downstream region, respectively, with an amplified product size 2.5 kb [Figure 3].
|Table 4: Characterization and screening of integron in clinical isolates |
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While testing the susceptibility patterns of clones of all respective arrangements, it was observed that the clones of the different arrangements showed variable resistance pattern. It was observed in arrangement I and II that the clones were resistant towards trimethoprim and kanamycin whereas found to be susceptible towards netilmicin, gentamicin and amikacin. In arrangement III and IV, resistance was observed towards trimethoprim and amikacin, whereas against rest of the tested aminoglycoside drugs, the clones were susceptible.
| ~ Discussion|| |
The variation of the gene cassettes in Class II integron may reflect the horizontal gene transfer among Enterobacteriaceae. In this study, four different types of Class II integron gene cassette arrays were identified. The phenotypic resistance to a specific drug was observed in all isolates carrying the corresponding gene cassette. The most common set of cassette array was aadA1-dfrA1-dfrA12-sat2 which has a size of 2.3 kb.
The presence of dfr cassettes is likely to be due to the selection pressure and high use of trimethoprim/sulfamethoxazole in this region which was also evidenced elsewhere. , The prevalence of aminoglycoside resistant genes associated with Class II integron has changed over the course of time where majority of isolates were carrying aadA1 gene. The aadA gene confers the resistance to streptomycin and spectinomycin and sat2 confers the resistance to streptothricin which are occasionally used therapeutically , with no reports of their use against Gram-negative bacilli from this country. Thus, even when antibiotics cease to be used therapeutically, genes encoding resistance to these antibiotics are not necessarily lost and maintained within the integron. 
Arrangement II showed an array where it was associated with three additional (ybfA-ybfB-ybgA) functional open reading frame with unknown function along with ybeA gene and other gene cassette array linked with intI2 (dfrA1-sat2-aadA1-orfX-ybeA-ybfA-ybfB-ybgA). This array of gene is not observed in any other sample in the present investigation. As claimed in the previous study, the arrangement might have horizontally transferred from a non-clinical isolates and maintained vertically in hospital environment in subsequent generation.  The IV arrangement with 2.5 kb amplicon size carries a unique set of cassette array, and based on the sequencing result, we could predict that this integron type might be formed by a complex recombination event (dfrA1, linF and aadA1). Except for IV arrangement, all the other types of cassette array have been described previously in members of Enterobacteriaceae. The LinF gene is of Gram positive bacterial origin which is responsible for conferring low level resistance to lincomycin and clindamycin. Thus presence of this gene cassette in our study isolates underscores potential transfer of this gene from Gram positive origin to a Gram negative host. 
Repeated exposure to antimicrobials in hospital environment is the key to maintain cassette array of resistant genes which reposition themselves according to the selective presence by a specific antibiotics, thus enable a set of resistance genes to be maintained under a common promoter. 
| ~ Conclusion|| |
This study highlighted the extensive variety of drug resistance phenotypes as well as clones and clonal lineages among bacterial isolates in hospital settings in Silchar Medical College, Silchar, Assam, India. This diversity of structure was despite the fact that many of these Class II integrons had identical cassette arrays. Accordingly, continued surveillance of the epidemiology as well as further investigation of those Class II integrons and their gene cassette is required to determine the impact and molecular mechanisms of acquisition and adaptation of MDR genes within members of family Enterobacteriaceae.
The authors would like to acknowledge HOD, Department of Microbiology, Assam University, and Assam University Biotech Hub for providing infrastructural facility.
Financial support and sponsorship
The Council of Scientific and Industrial Research (Scheme number 37/1632/14/EMR-II) and the Science and Engineering Research Board SR/FT/LS-72/2012 New Delhi, India, had provided the financial support.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]