Variability in gene cassette patterns of class 1 and 2 integrons associated with multi drug resistance patterns in Staphylococcus aureus clinical isolates in Tehran-Iran

Background To investigate antibiotic resistance, the occurrence and distribution of class 1 and 2 integrons in multidrug- resistant Staphylococcus aureus isolates from hospitals in Tehran, Iran. The isolates were examined for susceptibility to antimicrobial agents. The mecA gene, class 1 and 2 integrons were detected by PCR. Integrase positive strains were further analysed for the presence of resistance gene cassettes using specific primers and were sequenced. Results Among 139 S.aureus isolates, 109 (78.4 %) and 112 (80.5 %) strains were considered as multidrug resistant and mecA positive, respectively. Class 1 integrons and internal variable regions were found in 72.6 % (101/139) and 97 % (98/101) and class 2 integrons and variable regions also in 35.2 % (49/139) and 65.3 % (32/49) of S.aureus clinical isolates, respectively. Twelve distinct cassette arrays were found, containing genes encoding resistance to β-lactams, aminoglycosides, streptothricin, trimethoprim, chloramphenicol,a putative glucose dehydrogenase precursor and a protein with unknown function. Gene cassette arrays aadB, aadA2 and dhfrA1-sat2-aadA1 were common in S.aureus isolates. We detected a completely new gene cassettes which contained aadB, oxa2, aacA4, orfD-aacA4-catB8, aadB-catB3, orfD-aacA4 and aadB-aadA1-cmlA6 of class 1 and dhfrA1-sat2-aadA1, dhfrA11, dhfrA1-sat2 of class 2 integrons. Conclusions This is the first study to report carriage of class 1 and 2 integrons and associated gene cassettes among in S.aureus isolates from Iran.


Background
Staphylococcus aureus has long been recognized as a major human pathogen responsible for a wide range of infections, from mild skin infections to wound infections and bacteraemia [1]. Although the introduction of antibiotics has lowered the mortality rate from S. aureus infections, the bacteria have developed resistance mechanisms to all antimicrobial agents that have been produced [1].
Despite antibiotic therapy, staphylococcal infections occur regularly in hospitalized patients and have severe consequencess [2,3]. Due to an increasing number of infections caused by methicillin-resistant S. aureus (MRSA) strains, which are now most often multiresistant, therapy has become problematic [4]. Since this organism can spread easily by direct or indirect contact between patients and the environment, or among patients and medical personnel, S. aureus is an important cause of nosocomial infection, and major outbreaks are common [5,6].
Dissemination of antibiotic resistance genes by horizontal gene transfer has led to the rapid emergence of antibiotic resistance among bacteria, thus complicating the treatment of infections [7]. Acquired resistance evolves via horizontal transfer of antimicrobial resistance genes located on various types of mobile DNA elements.
A key system involved in spreading antibiotic multiresistance is the integron, an element that, although normally immobile itself, can be transferred through mobile genetic elements [8,9]. Integrons are genetic elements that incorporate exogenous open reading frames by recombination and convert them to functional genes [10]. All integrons characterized to date are composed of these major elements: a gene (intI) encoding an integrase; a primary recombination site (attI); and an outward orientated promoter (Pc) that directs transcription of the captured genes [11].
In this study, the presence of integrons and gene cassettes in S. aureus has been studied for the first time in Iran.
The objective of this research was to identified multiple drug resistance in clinical isolates of s. aureus by antibiotic sensitivity testing, detection of mecA gene for methicillin-resistant S. aureus strains, and distribution of class 1 and 2 integrons and associated gene cassettes among S.aureus isolates collected from clinical sources in Tehran by PCR assays and investigates associations between multidrug resistance and the existence of integrons.

Sampling and bacterial isolation
One hundred and thirty nine S. aureus isolates were collected from hospitals in Tehran, over a six month period, starting from the first of October 2011 till the end of March 2012 . The isolates were recovered from wounds (n = 31), blood (n = 31), urines (n = 28), nasal swabs (n = 21), catheters (n = 5), sternum samples (n = 5), sputa (n = 4), eyes (n = 2), CSF (n = 1), synovial fluid (n = 1), throat samples (n = 3) and abscesses (n = 7). The isolates were identified as S. aureus by standard biochemical tests for catalase, coagulase, clumping factor, DNase, and thermostable nuclease. All isolates were also evaluated for the presence of the sa442 gene by PCR [3,12]. The research was approved by the Ethics committee of the Pasteur Institute of Iran (no = 402) and acquire either verbal or written informed consent from the patients to take their samples for this study.

Antibiotic susceptibility test
The isolated S. aureus strains were tested for their in vitro antimicrobial susceptibility using the disk diffusion technique on Mueller-Hinton agar (Mast, UK). The results were recorded after 18 h of incubation at 37єC. . The MIC to vancomycin was determined by Etest (bioMeґrieux). Enterococcus faecalis ATCC 29212 and Enterococcus faecium BM4147 were used as controls for vancomycin susceptibility and vancomycin resistance, respectively. The breakpoints for antibiotic susceptibility were determined according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [13]. E. coli ATCC 25922 was used as a reference control strain. Isolates of S.aureus that show resistance to three or more than three classes of antibiotics, considered as multidrugresistant strains.
The extraction of S. aureus plasmid and genomic DNA The genomic DNA preparation used in this study was performed following Sambrook et al. [14]. The DNA was extracted with phenol-chloroform and precipitated by ice-cold ethanol. The DNA pellet was washed with 70 % ethanol and resuspended in 50 μl of TE buffer (10 mM Tris; 1 mM EDTA, pH8) and stored at -20єC until used and 1 μl of the suspension were used as the template DNA for PCR. Using the primers determined [3] mecA gene was detected by PCR. Size of the amplified fragment was 533 bp.
The Qiagen Plasmid Midi Kit was used for the S. aureus plasmid DNA extraction; the manufacturer's instructions were followed.

Detection of class 1 and 2 integrons
All isolates were screened for detection of class 1 and 2 integrons by the primers described by Moura et al. [15] designed for the intI1 and intI2 genes respectively. Primers intI1 F/R and intI2 F/R were used to amplify 280 bp and 232 bp fragments, respectively. The amplification program was performed by termocycler (Eppendorf Mastercycler®, MA) and started with initial denaturation of 4 min at 94°C and programmed wi th 35 cycles of each: 1 min at 94°C, 30 s at 55°C, 1 min at 72°C. The program finished with the final extension of 10 min at 72°C.

Mapping of class 1 and 2 integrons
The gene cassettes inserted in the variable regions of class 1 and 2 integrons (IVR) were amplified using the primer pairs introduced by Moura et al. [15].

Sequencing of amplified integron gene cassettes
For each PCR product that had a unique size when visualized on the gel, a number of samples of the post-PCR mixture was processed with the High Pure PCR Product Purification kit (Roche,USA) and used for direct sequencing. The purified amplicons were sequenced using the ABI Capillary System (Pasteur Institute, Tehran, Iran). Resulting sequences were assembled by using SeqMan program within the Lasergene suite version 7 (DNAstar Inc, Madison, WI, USA). Potential open reading frames (ORFs) were predicted by using the NCBI (National Center for Biotechnology Information) ORF Finder tool (http://www.ncbi.nlm.nih.gov/ gorf/gorf.html). BLAST (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) against GenBank database and The Integron Database INTEGRALL (http://integrall.bio.ua.pt/) were performed repeatedly for sequence comparison and annotation.

Statistical analysis
The antibiotic resistance data between integron-positive isolates and integron-negative isolates was compared and the χ2 test was used to calculate the P value in terms of resistant and susceptible numbers by SPSS version 18 software. A P value of <0.05 was considered statistically significant.

Results
A total of 139 S. aureus isolates were collected from clinical specimens of hospitals in Tehran, over a 6 month period, starting from the first of October 2011 till the end of March 2012.

Antibiotic susceptibility test
The antimicrobial resistance patterns of isolates are shown in Tables

Detection of class 1 and 2 Integrons
One hundred and thirty-nine of S.aureus, were examined for the presence of known markers for integrons by PCR amplification. The 101 (72.6 %) isolates were positive for class 1 integron with the amplicon size of 280 bp and class 2 integron was found in 49 (35.2 %) isolates with the amplicon size of 232 bp. Information about integron positive isolates among different sources of S.aureus isolates are shown in Table 3. In the present study, among of 112 strains of MRSA, 95 (84.8 %) isolates carrying class 1 integron as well as 46 (41 %) isolates carrying class 2 integron.
The results indicate that resistance to several different antibiotics is associated with the presence of the integron. Tables 1 and 2 shows the antibiotic susceptibilities of integron-positive and integron-negative isolates to each of the antibiotics. Both class 1 and 2 integrases genes were present together in 44 (31.6 %) strains of S.aureus.
The integron cassette region could not be amplified by PCR in 17 (34.6 %) of the class 2 integron-containing isolates (Table 5). Schematic representation of the various cassette arrays found in class 1 and class 2 integrons are shown in Fig. 2.

Discussion
In the current study, prevalence of intI 1 and intI 2 were examined among multidrug resistant S.aureus strains, isolated from different specimens. The prevalence of antimicrobial-resistant bacterial pathogens has become a major public health concern [16,17]. Horizontal gene transfer has already been proved a significant mechanism for disseminating antimicrobial resistance in bacterial populations. Antimicrobial drug resistance can also be facilitated by integrons in case of many other bacteria [18][19][20][21]. Since the integrons were detected first in Gram-negative bacteria, many studies have been conducted on Gram-negative bacteria. Although the role of class 1integrons is well known in the spread of antibiotic resistance genes in gram-negative bacteria, much less is known about gram-positive bacteria and very few studies have reported the presence of class 1 integrons in grampositive bacteria so increasing antibiotic resistance mediated by integrons in gram-positive bacteria has become a great concern in the medical field [22].
Integrons are genetic elements able to recognize and capture mobile gene cassettes carrying antibiotic resistance genes leading to MDR distribution and subsequently limitation of treatment options for infections [23].
To our knowledge, this is the first study to report carriage of class 1 and 2 integrons and associated gene cassettes in S.aureus isolates from Iran. Our findings demonstrate that    [24,25]. Among different classes of integrons, class 1 integrons have been found more frequently in different species of bacteria, especially Gram-negative bacteria [11,26,27]. The classification of different integrons is mainly based on differences in the gene structure of integrases [11].
In this survey, presence of integrons in strains of S.aureus as compared to similar studies conducted by Xu et al. and Ren et al. has been increased [24,25]. The rate of detection of integron class 1 was more than integron class 2. One hundred and one (72.6 %) isolates were positive for class 1 integron and 49 (35.2 %) isolates were positive for class 2 integron.
The nine different cassettes arrangements wich class 1 integrons and another three arrangements within class 2 integrons were identified.
Twelve different gene cassettes were detected. Cassette genes encoding resistance to aminoglycosides were found to be predominant in the class 1 integron. The aadB and aadA types genes that encode resistance to aminoglycosides (aadB gene cassette conferring resistance to gentamicin, tobramycin, and kanamycin; aadA gene cassette conferring resistance to streptomycin and spectinomycin) were most commonly found [11].
The B-lactamase cassettes (oxa2 or blaoxa2) that confer resistance to oxacillin and ampicillin [28], were found in three isolates with amplicon size 1200 bp. The aacA4 cassette (aminoglycoside acetyl transferase) that confer resistance to gentamicin, amikacin and tobramycin, was found with amplicon size 800 bp [11,28].
The diversity of gene cassettes inserted in IVRs of class 2 integrons is much lower than class 1 integrons. This reduction in diversity is probably owing to a nonsense mutation in codon 179 (ochre 179) in the integrase gene of class 2 integrons, thereby yielding a truncated, non-functional protein, the resultant integrase is therefore unable to excise existing cassettes or insert new ones [10].
Phenotypic and genotypic characterizations of integrase-positive S.aureus are shown in Table 6. Ninety percent (91 out of 101) of class 1 and 8.16 % (4 out of 49) of class 2 integrons harboured at least one gene cassette. Eleven arrays were detected containing one to three cassettes, out of which eight distinct arrays were present in integrons of class 1 and three arrays in class 2 integrons. Common gene cassettes arrays were identified in S.aureus strains. In comparison with other sequences deposited in databases, the following gene cassettes were identified: gentamicin, tobramycin and kanamycin resistance genes (aadB), gentamicin, amikacin and tobramycin resistance genes (aacA4), streptomycin and spectinomycin resistance genes (aadA1, aadA2), trimethprim resistance genes (dfrA1, dfrA11), β-lactam resistance genes (blaoxa2), chloramphenicol resistance genes (catB3, catB8 and cmlA6), streptothricin resistance genes (sat2), an orfD encoding a protein with unknown function, a putative Fig. 2 Schematic representation of the various cassette arrays found in class 1 and class 2 integrons. The arrows display the open reading frames of the different genes. All aadA and aadB genes are presented as white arrows, dhfrA, aacA4, orfD, oxa2, catB, cmlA6, sat2 genes are presented as black, yellow, light grey, grey, blue and light blue arrows respectively. The grey light boxes indicate the 3′ and 5′ CSs of class1 or class 2 integrons glucose dehydrogenase. Moreover, the arrays aadB and aadA2 in class 1 integrons were found during the entire process, indicating the most common array. Previous studies have reported aadA2, aacA4-cmlA1, dfrA17-aadA5 and dfrA12-orfF-aadA2 genes as frequently found gene cassettes both in clinical and environmental strains [30,31].
Common gene cassette arrangements in class 2 integrons were found in S.aureus strains (dfrA1-sat2-aadA1). It should be noted, the cassette array dfrA1-sat2-aadA1 was detected most frequently in class 2 integrons [15,[30][31][32]. Furthermore, integron-positive isolates were resistant to different classes of antimicrobial agents but related resistance gene cassettes were not found to be harbored on the integron, implying the nonintegron sources of resistance to these antibiotics [33]. In this study, integron cassette region could not be amplified by PCR in 3 (2.9 %) and 17 (34.6 %) of the class 1 and 2 integroncontaining isolates respectively. In the cases where the cassette array could not be amplified, there is no cassettes or in the absent, significant alter-ation in the 3 CS primer binding site or a very large size of the Integron cassette are the most likely explanations for the negative PCRs [3].
The total prevalence of integrases carried in plasmids 36 % (50/139) was similar to that previously reported in S.aureus [34]. Nevertheless, we are aware that the prevalence obtained may be biased due to the fact that plasmid and chromosomal DNA can display the same electrophoretic mobility. The dissemination of the class 1 integrons, in many instances, has been attributed to the spread of an integron-containing transposon, Tn21 [35]. The distribution of Tn7 in clinical isolates correlates with the frequency of trimethoprim resistance [36]. Trimethoprim resistance is due to the dihydrofolate reductase enzyme encoded by the dhfr gene in Tn7 [29]. Class 2 integron variable regions were amplified using primers that bind to attI2 and to orfX, situated downstream of the cassette region within transposon Tn7 [37]. Therefore, the presence of such an integron in S.aureus may have had its origin in a transposition event.  I  blaoxa2  C  CRO,LEV,TN,E