|Year : 2015 | Volume
| Issue : 2 | Page : 231-236
High prevalence of class 1 integrons in clinical isolates of methicillin-resistant Staphylococcus aureus from India
NP Marathe1, SS Nagarkar2, AA Vaishampayan1, MH Rasane1, SA Samant3, V Dohe4, A Kagal4, YS Shouche1, N Deshpande2
1 Microbial Culture Collection, National Center for Cell Science, Ganeshkhind, Pune 411004, India
2 Department of Microbiology, Abasaheb Garware College, Karve Road, Pune 411004, India
3 Department of Microbiology, Mahatma Gandhi Memorial Medical College, Kamothe, Navi Mumbai, India
4 Department of Microbiology, Byramjee Jeejeebhoy Government Medical College and Sassoon General Hospital, Pune, India
|Date of Submission||18-Mar-2014|
|Date of Acceptance||21-Jun-2014|
|Date of Web Publication||10-Apr-2015|
N P Marathe
Microbial Culture Collection, National Center for Cell Science, Ganeshkhind, Pune 411004
Department of Microbiology, Abasaheb Garware College, Karve Road, Pune 411004
Source of Support: None, Conflict of Interest: None
Introduction: Class1 integrons are one of the prevalent mechanisms of antibiotic resistance gene transfer in Gram-negative organisms, but their prevalence and role in the spread of antibiotic resistance genes in methicillin-resistant Staphylococcus aureus (MRSA) is unexplored. The purpose of this study was to investigate the prevalence of class 1 integrons in clinical isolates of MRSA. Materials and Methods: Total 143 MRSA isolates obtained from two different cities in India (Pune and Mumbai) were characterized by biochemical tests, and the antibiotic sensitivity was performed using the Clinical and Laboratory Standards Institute (CLSI) guidelines. The presence of class 1 integrons, sul1/qacE0Δ1 region of class 1 integron and mecA gene among these isolates was determined by polymerase chain reaction (PCR). Results: All 143 isolates were mecA positive and coagulase-positive. Overall, 71% of the MRSA isolates carried class 1 integrons; 58% (45/77) of the isolates obtained from Mumbai and 85% (56/66) of the isolates from Pune carried class 1 integrons. In all, 39% of these isolates carried sul1/qacEΔ1 region, thus confirming the association of class 1 integrons with antibiotic resistance genes. Along with β-lactam antibiotics the MRSA isolates were resistant to several other antibiotics, with resistance to erythromycin, ciprofloxacin and trimethoprim-sulfamethoxazole being observed in 75%, 66% and 60% of the isolates, respectively. Conclusion: To the best of our knowledge, this is the first report of class 1 integrons in MRSA isolates from India. The study provides insights into the prevalence of a novel mechanism adapted by MRSA for the propagation of antibiotic resistance genes.
Keywords: Antibiotic resistance, class 1 integrons, India, methicillin-resistant Staphylococcus aureus
|How to cite this article:|
Marathe N P, Nagarkar S S, Vaishampayan A A, Rasane M H, Samant S A, Dohe V, Kagal A, Shouche Y S, Deshpande N. High prevalence of class 1 integrons in clinical isolates of methicillin-resistant Staphylococcus aureus from India. Indian J Med Microbiol 2015;33:231-6
|How to cite this URL:|
Marathe N P, Nagarkar S S, Vaishampayan A A, Rasane M H, Samant S A, Dohe V, Kagal A, Shouche Y S, Deshpande N. High prevalence of class 1 integrons in clinical isolates of methicillin-resistant Staphylococcus aureus from India. Indian J Med Microbiol [serial online] 2015 [cited 2019 Jun 17];33:231-6. Available from: http://www.ijmm.org/text.asp?2015/33/2/231/154905
N P Marathe, S S Nagarkar, A A Vaishampayan
These authors equally contributed to this work
| ~ Introduction|| |
Staphylococcus aureus is an adaptable human pathogen causing a variety of infections ranging from skin and soft tissue infections to fatal sepsis. It is a leading cause of nosocomial infections today.  The rate of MRSA infections and the associated economic losses are rising in Europe in the past few years.  MRSA infections are prevalent in India, with prevalence ranging from 24% to 68%. But the epidemiological studies on MRSA infections are lacking. 
Being multiple drug resistant, the treatment options for MRSA infections are limited. A mobile genetic element, called staphylococcal cassette chromosome mec (SCCmec), plays a vital role in developing multidrug resistance among MRSA. The SCCmec contains mecA gene responsible for the resistance to most β-lactam antibiotics and the ccr gene complex encoding site-specific recombinases responsible for the mobility of SCCmec.  The resistance genes associated with resistance to other classes of antibiotics are also carried by SCCmec, thus conferring MRSA resistance against multiple drugs. 
Recently, class 1 integrons have been detected in MRSA. , Class 1 integrons are a natural gene capture and expression system in bacteria known to be associated with antibiotic resistance genes. They capture exogenous open reading frames (ORFs) by site-specific recombination and ensure their correct expression under the control of a strong promoter. The captured ORFs are called the gene cassettes.  Class1 integrons are a prevalent mechanism of antibiotic resistance gene transfer in Gram-negative pathogens. But its role and prevalence in Gram-positive pathogens, especially in MRSA, is largely unclear. 
With this background, the aim of the current study was to investigate the prevalence of class 1 integrons in clinical MRSA isolates, obtained from two different cities in India and collected over a period of 1 year.
| ~ Materials and Methods|| |
Collection of MRSA isolates
MRSA isolates were obtained from the following hospitals from two different cities in Maharashtra, India. 1) B. J. Govt. Medical College, Pune 2) Padmashree Dr. D. Y. Patil Medical College, Hospital and Research Centre, Pimpri, Pune 3) Sahyadri hospital, Karve road, Pune 4) MGM Medical College, Navi Mumbai. These MRSA isolates were collected by the hospital as a part of the standard practice and made available for the study. The MRSA strains were isolated from different sources such as pus, urine, blood and sputum samples, pleural fluids and tissue bits of the patients, as well as from indwelling devices including catheter tips, endotracheal tubes and cannula tips, presented in [Table 1].
|Table 1: The presence of class 1 integrons and sul1/qacEΔ1 region of the class 1 integrons in the MRSA isolates under study and their isolation sources |
Click here to view
Characterization of the isolates
The S. aureus strains were identified by Gram staining, colony morphology on Blood agar, mannitol fermentation and coagulase test. The coagulase test was carried by both tube and slide method as described earlier.  The methicillin resistance among the isolates was detected by the disc diffusion method on Mueller-Hinton (MH) Agar media, using cefoxitin and oxacillin (HiMedia, Mumbai, India) discs as per CLSI guidelines. 
Further confirmation of methicillin resistance was done by polymerase chain reaction (PCR) detection of mecA gene, responsible for methicillin resistance, as described in the next section.
DNA extraction and PCR
Genomic DNA was extracted from freshly grown cultures using PureLink® Pro 96 Genomic DNA Purification Kit (Invitrogen, USA). Polymerase chain reaction amplification of 16S rRNA gene was carried out, as an internal control for DNA quality, by procedure described earlier.  The PCR for mecA gene was carried out as described earlier.  Class 1 integrons were detected by amplification of integrase gene (intI1) of class 1 integrons as described by Hardwick et al. 2007.  The gene cassettes were amplified using primers targeting the conserved 5' and 3' region of class 1 integron; this enables amplification of variable gene cassettes inserted between the conserved regions of integron.  Bacillus cereus strain ER5, reported to carry class 1 integron was used as a positive control for intI1 gene and gene cassette PCR, whereas Escherichia More Details coli DH10B was used as a negative control. 
All the PCRs were carried out in a total volume of 25 µl. The reaction constituted 1X standard Taq Buffer, 200 nM dNTPs, 0.4 ΅M of each primers [Table 2], 0.625 U Taq Polymerase (New England Biolabs, USA) and 20 ng of template DNA. All PCRs were performed for 35 cycles using a GeneAmp® PCR System 9700 Thermal Cycler (Applied BiosystemsInc, USA). The list of the PCR primer used, annealing temperature and expected product size is represented in [Table 2].
| ~ Results and Discussion|| |
Characterization of the MRSA isolates
Total 143 MRSA isolates were obtained from the hospitals; 77 isolates were from Mumbai and 66 isolates from Pune. Pus samples constituted a major source of these isolates (63%), followed by blood samples (12%) and indwelling devices (10%). Sputum and urine samples were the source of the remaining isolates, presented in [Table 1].
The mecA gene was detected in all the 143 isolates, thus confirming the methicillin resistance of the isolates. Along with resistance to β-lactam antibiotics, the MRSA isolates were resistant to many other antibiotics. Highest resistance was observed against erythromycin (75% isolates), ciprofloxacin (66% isolates) followed by trimethoprim-sulfamethoxazole (60% isolates), gentamicin (47% isolates) and tetracycline (42% isolates).
Prevalence of class 1 integrons
High prevalence of class 1 integrons in MRSA isolates, i.e. 71% (101/143 isolates), was observed in the present study [Table 1]. The prevalence of class 1 integrons in MRSA isolates from Mumbai was 58% (45/77) and from Pune was 85% (56/66). To the best of our knowledge, this is the highest reported prevalence of class 1 integrons in MRSA. Previous studies from China reported the prevalence of class 1 integrons in MRSA to be 42.5% and ranged from 30% to 56% in different years. ,
Class 1 integrons are a natural gene capture, expression and transfer system in bacteria.  Several genes coding resistance against β-lactams, aminoglycosides, sulfonamides, macrolides and tetracyclines are frequently encountered in gene cassettes carried by class 1 integrons. , Moreover, class 1 integrons are usually associated with other mobile elements such as plasmids and transposons. Hence, class 1 integrons are considered as a marker of horizontal gene transfer potential of an organism. , Bacteria isolated from antibiotic-contaminated environments like waste water treatment plants and hospital effluents have been reported to have a high prevalence of class 1 integrons. , Thus, antibiotic selection pressure might be selecting out integron-carrying bacteria, which can acquire and express resistance genes through integron gene cassettes.
The rise of antibiotic resistance in India is largely contributed to the irrational use of antibiotics, which has created selection pressure in the environment consequently leading to the rise in antibiotic resistance.  Lately, India has been the focal point for the rise in antibiotic resistance in the clinics, especially with the rise of NDM-1 containing superbug.  This suggests that the pathogens are acquiring new mechanisms for gaining antibiotic resistance in India. The high prevalence of class 1 integrons in MRSA isolates from India reported in our study supports this view. Integrons have been extensively studied in Gram-negative pathogens like Pseudomonas aeruginosa, E. coli and Klebsiella pneumonia, but their role in dissemination of antibiotic resistance in Gram-positive bacteria is less clear.  Our study reveals that class 1 integrons are highly prevalent in MRSA isolates from India. It is possible that the high prevalence of class 1 integrons in MRSA contributes to the acquisition of antibiotic resistance genes by these organisms, via mobile gene cassettes.
The prevalence of sul1/qacEΔ1 (3' region of class 1 integron) region-bearing integrons was low among the isolates, with only 39% of the integron-bearing isolates being positive for the PCR, [Table 1]. The qacEΔ1 confers resistance to ethidium bromide and quaternary ammonium compounds while sul1 confers sulfonamide resistance.  Nevertheless, the presence of these genes signifies the association of class 1 integron in MRSA with antibiotic resistance genes. Studies have shown that bacteria do carry class 1 integrons that lack sul1/qacEΔ1 region at 3' end and have variable genes at 3'end.  This suggests that the MRSA isolates in this study might have class 1 integron-carrying genes other than sul1/qacEΔ1, at the 3' end.
The difference observed in the prevalence of class1 integrons in MRSA isolates from the two cities is very striking. The epidemiological survey conducted by Indian Network for Surveillance of Antimicrobial Resistance (INSAR) group, India suggests that Pune has higher prevalence of MRSA infections compared with Mumbai.  This is in accordance with the higher prevalence of class 1 integrons in MRSA isolates from Pune as compared with Mumbai. A recent study showed the presence of (NDM-1)-positive pathogens in a tertiary care centre in Pune, India.  The high prevalence of class 1 integrons in MRSA in Pune and the detection of NDM-1 positive pathogens calls for controlling measures and proper monitoring of spread of antibiotic resistance genes in the clinical settings. Proper management of antibiotic use and monitoring antibiotic resistance among pathogens is important to control the spread of antibiotic resistance observed in clinics, especially in India.
The present study thus provides important information for the epidemiology of class 1 integrons in MRSA in India. Methicillin-resistant Staphylococcus aureus isolates are gaining different mechanisms like class 1 integrons, which would aid in gaining antibiotic resistance genes.
| ~ Acknowledgements|| |
This work was supported by funding from Indian Council for Medical Research (ICMR), New Delhi, India (project number AMR/12/2011-ECD 1). Nachiket Marathe is grateful to Council of Scientific and Industrial Research (CSIR), New Delhi, India for the fellowship. We acknowledge Dr. Maithili Kavthekar, Microbiology and Pathology Department, Sahyadrispecialty Hospitals, Karve road, Pune and Dr. Mishra, Microbiology Department, Padmashree Dr. D. Y. Patil Medical College, Hospital and Research Centre, Pimpri, Pune for providing MRSA strains.
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[Table 1], [Table 2]