First observation of excision and integration in Class 1 integron in staphylococci

1 Department of Urology, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080, China. 2 Guangzhou Women and Children’s Medical Center, Guangzhou 510623, China. 3 School of Food Science and Nutrition, Faculty of Mathematics and Physical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom. 4 Food Safety Key Laboratory of Guangdong Province, College of Food Science, South China Agricultural University, 510642 Guangzhou, China. 5 Key Laboratory for Green Chemical Process of Ministry of Education, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073, China. 6 Guangdong Pharmaceutical University, Guangzhou 510006, China. 7 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China. 8 Department of Microbial Pathogenesis, Dental School, University of Maryland, Baltimore, MD 21201, USA


INTRODUCTION
In recent years, the role of integrons and gene cassettes in the dissemination of resistance genes has been well established, which is responsible for the facile spread of resistance genes and the rapid evolution of resistance to a wide range of unrelated antibiotics among diverse bacteria (Tauch et al., 2002;Yang et al., 2004;Yu et al., 2003).An integron comprises three elements including the integrase gene (intI) encoding an integrase, a recom-*Corresponding authors.E-mail: xhzhao2006@gmail.com,chenjunxing@hotmail.com; Tel: +86-13419663487; Fax: +86-13419663487; Tel: +86-20-87333300; Fax: +86-20-87333300.†These authors contributed equally to this work.bination site, attI and a promoter gene.Several classes of integrons have been reported on the basis of the intI gene and classes 1 to 3 are so-called multiresistant integron.Of these, the class 1 integron platform is the most ubiquitous among resistant clinical isolates of gram-negative bacteria and is found to be associated with the Tn21 transposon family, while the class 2 integrons are associated with the Tn7 transposon family (Arakawa et al., 1995;Barlow et al., 2004;Correia et al., 2003;Hall and Stokes, 1993;Nandi et al., 2004;Nesvera et al., 1998;Nield et al., 2001;Sun et al., 2002).Class 1 integron can capture the gene cassettes, which also contain attC site for recombination, via a site-specific recombination event between attI and attC.Its 3' conserved segment (3'CS) possesses qacE≥1 and sul1 genes, encoding resistance to quaternary ammonium salts and sulfonamide, respectively (Recchia and Hall, 1997).Although, the role of class 1 integron in the spread of antibiotic resistance genes in gram-negative bacteria is clear, little is known about the prevalence of class 1 integron in gram-positive bacteria, especially in Staphylococcus aureus (Clark et al., 1999;Nandi et al., 2004).Increasng antibiotic resistance in gram-positive bacteria has become a great concern.
In the past decades, methicillin-resistant S. aureus (MRSA) has become one of the most prevalent pathogens that cause nosocomial infections throughout the world.Since it can spread easily by direct or indirect contact between patients and environment or through patients or medical personnel, MRSA is an important risk factor for nosocomial infection and MRSA mediated nosocomial outbreaks are common in hospitals.As consequence, MRSA mediated nosocomial infections continue to be a challenge for clinicians, hospital epidemiologists and administrators since such infections may lead to a serious problem for therapy of patients and infection control.MRSA shows resistance to practically all β-lactam antibiotics, which is caused by mecA gene.The mecA gene is carried by a mobile genetic element, designated by staphylococcal cassette chromosome mec (SCCmec), which contains the mec gene complex (the mecA gene and its regulators) and the ccr gene complex encoding site-specific recombinases responsible for the mobility of SCCmec (Ito et al., 2001;Kuroda et al., 2001;Xu et al., 2007Xu et al., , 2008aXu et al., ,b, 2010cXu et al., , 2011a and b) and b).
In recent years, class 1 integron had been found commonly existed in MRSA and methicillin-resistant coagulase negative staphylococci (MRCNS) strains (Xu et al., 2007(Xu et al., , 2008a(Xu et al., and b, 2009(Xu et al., , 2010a and b) and b).In this study, we investigated class 1 integron-mediated recombination in staphylococci, including class 1 integron mediated excision of different gene cassettes with 2 att sites and 3 arrays of cassettes and further integration of them by site-specific recombination.

Electroporation of cassettes carried plasmids into RN4220
To establish different transformants for the excision and integration assay, the aforementioned plasmids had been transformed into RN4220 by electroporation, which had been performed as described previously (Hauschild et al., 2003).In brief, 100 of the competent cells and 1 or 2 µg of each plasmid were subjected to electroporation, 200 µl aliquots were then streaked on plated supplemented with proper antibiotics according to relevant phenotypes.

Excision and integration reaction
In vivo excision and integration tests were performed by double transformants (pSU2056 with one of the cassette containing PLQ clones) and triple transformants (R388, pSU2056 with one of the cassette containing PLQ clones), respectively.Growth condition and induction of IntI1 in transformants were performed as described previously (Leon and Roy, 2003;Martinez and de la Cruz, 1990).In brief, transformants were grown overnight at 37°C in blood medium supplemented with selective antibiotics (ampicillin 50 g/ml, chloramphenicol 50 g/ml) and 2% of the overnight culture was then inoculated in 15 ml of new medium (without antibiotic).Isopropyl-β-D-thiogalactopyranoside (IPTG) was added (with final concentration of 0.4 mM for overnight induction) when the optical density at 600 nm reached 0.6.Plasmid isolation was performed as described previously (Schwarz et al., 1989).Excision events were determined by polymerase chain reaction (PCR) amplification and further confirmed by excision-integration tests, with different primer pairs as indicated (Table 1).All experimental procedure had been performed as described previously with slight changes (Leon and Roy, 2003).In brief, for excision assays, sixty-four colonies had been randomly selected and further subjected to PCR amplification for each transformant.For integration assays, ten colonies recovered had been selected to sequencing.In detail, PCR products were purified from agarose gels and the DNA fragments were ligated with the pGEM-T easy vector (Promega, Madison, WI, USA).The ligation mixture was transformed into E. coli DH5α strain and the recombinants were selected on Luria agar containing ampicillin (100 µg/ml).Recombinant plasmid DNA was purified by standard method and subjected to sequencing and further analyses.The nucleotide sequences of the insert DNA was determined using the BigDye terminator cycle sequencing FS ready reaction kit on an ABI PRISM 310 genetic analyzer (Perkin-Elmer Japan Applied Biosystems, Tokyo, Japan).The nucleotide sequences were analyzed and compared using GenBank database by using the BLAST algorithm, which is available through the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov).Definition of excision and integration efficiency was according to previous report (Leon and Roy, 2003).

Excision and integration reaction
According to the results, excision efficacy ranged from 7.8 to 62.5%, which was thoroughly lower than previous reports when the assays induced in bacteria other than S. aureus (Leon and Roy, 2003).Two major factors affecting the excision included att site and gene cassettes.As the results showed, greater efficacy had been observed when attC site on the left-hand neighbour comparing with attI, based on 7.8% (5/64) for RN4220-PLQ425 with 31.2% (20/64) for RN4220-PLQ438, 25% (16/64) for RN4220-PLQ440 with 53.1% (34/64) for RN4220-PLQ444.In addition, transformants containing aadA1 and aacA1a-orfG+orfH obtained higher efficacy than pse-1 and bla imp , as excision efficacy was 15.6% (10/64) for RN4220-PLQ431 and 31.25% for RN4220-PLQ438, comparing with 53.1% for RN4220-PLQ444 and 62.5% (40/64) for RN4220-PLQ443.These results were similar to those published previously (Hansson et al., 1997;Leon and Roy, 2003) and had also been further confirmed by subsequent integrations of excised cassettes into relevant attI site.Amplicons obtained were around expected 200 bp and sequencing amplicons confirm that, the excised cassettes were integrated specifically into the G/TTRRRY consensus site of the attI1 or attC site.

DISCUSSION
Since the first description of class 1 integron in 1989 (Stokes and Hall, 1989) integrons have been identified as a primary source of resistance genes and were suspected to serve as reservoirs of antimicrobial resistance genes within microbial populations (Xu et al., 2007(Xu et al., , 2009)).However, most of these studies were done in gram-negative bacteria with only a few exceptions.While in recent years, integrons had been identified as a novel and unexpected antibiotic determinant in gram-positive organism, especially in MRSA and MRCNS (Xu et al., 2007(Xu et al., , 2008a(Xu et al., and b, 2009(Xu et al., , 2010).The Integron system had been well-known as a mobile genetic element, playing key role in antibiotic resistance in various bacteria (Rowe-Magnus and Mazel, 2001).In this study, we tested and investigated the class 1 integron-mediated excision and integration in S. aureus RN4220 and positive result had been observed for both assays.Up to date, no report is available on the dissemination of class 1 integron in any species of staphylococci strain and we are convinced that, the observation of the class 1 integron-mediated excision and integration in S. aureus may definitely raise the attention of integron as the novel antibiotic mechanism in gram-positive bacteria, especially in staphylococci, which had been neglected throughout the time.

Table 1 .
Primes used for the different mobile gene cassettes constructs.

Table 2 .
List of transformants and its characteristics.