Class 1 integron in staphylococci

Abstract

As a major concern in public health, methicillin-resistant staphylococci (MRS) still remains one of the most prevalent pathogens that cause nosocomial infections throughout the world and has been recently labeled as a “super bug” in antibiotic resistance. Thus, surveillance and investigation on antibiotic resistance mechanisms involved in clinical MRS strains may raise urgent necessity and utmost significance. As a novel antibiotic resistance mechanism, class 1 integron has been identified as a primary source of antimicrobial resistance genes in Gram-negative organisms. However, most available studies on integrons had been limited within Gram-negative microbes, little is known for clinical Gram-positive bacteria. Based on series studies of systematic integrons investigation in hundreds of staphylococci strains during 2001–2006, this review concentrated on the latest development of class 1 integron in MRS isolates, including summary of prevalence and occurrence of class 1 integron, analysis of correlation between integron and antibiotic resistance, further demonstration of the role integrons play as antibiotic determinants, as well as origin and evolution of integron-associated gene cassettes during this study period.

Staphylococci are a group of Gram-positive, facultative aerobic and usually unencapsulated organisms, which are responsible for various tissues infection and a multitude of diseases. These bacterium, are carried, mostly transiently, by approximately 20 and 30% of healthy adults on the skin and anterior nares, respectively. Over 30 different types of staphylococci are infectious for humans, and its related illness can range from mild to severe, from no treatment required to even potentially fatal. Most of these infections are caused by Staphylococcus aureus, which has been regarded as leading issues both in medicine and food safety, and can typically causes a wide variety of infections, including skin infections and sometimes pneumonia, endocarditis, osteomyelitis, gastroenteritis, scalded skin syndrome and toxic shock syndrome [103]. Coagulase-negative staphylococci (CoNS) are regarded as a frequent cause of nosocomial infection and bacteremia, especially in patients with indwelling medical devices [5]. CoNS have also become the most frequently isolated pathogens in intravascular catheter related infections (CRI), accounting for an estimated 28% of all nosocomial bloodstream infections [63]. Since the first discovery in 1961, methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens that cause nosocomial infections throughout the world. As this pathogen can spread easily by either direct or indirect contact between patients and environment, or via patients and medical personnel, it is considered to be an important risk factor for nosocomial infection, which continues to be a challenge for clinicians, hospital epidemiologists and administrators [11]. Methicillin resistance in staphylococci is caused by PBP2a protein encoded by the mecA gene. The mecA gene is located on a mobile genetic element, designated as 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 [39]. Methicillin-resistant coagulase-negative staphylococci (MRCNS), which are more frequent carriers of SCCmec than MRSA, have been postulated to be the reservoir for the transfer of methicillin resistance to S. aureus. One assumes that the ccr and mec genes were bought together in CoNS from an unknown source, where deletion in the mec regulatory genes occurred, before the genes were transferred into S. aureus [34]. As one example of the leading “Super Bugs”, methicillin-resistant staphylococci (MRS) strains show resistance to practically all β-lactam antibiotics and usually other multiple drugs due to the mecA and associated resistance genes carried by SCCmec, respectively [77]. China remains one of the worst areas for antibiotics abuse, with an estimate annual consumption of 140 g per person, which is ten times higher than that in the United Kingdom and the United States. General concerns for the threaten of unleashing waves of “Super Bugs” in China raised necessity for surveillance and investigation on antibiotic resistance mechanisms involved in clinical MRSA and MRCNS strains.

Introduction of integrons

Indiscriminate use of existing antibiotics leads to proliferation of antibiotic resistance and poses a dilemma for the future treatment of bacterial infection. Antibiotic resistance in microbes still remains one of the leading concerns in global public health, and several mechanisms involving mobile genetic elements such as plasmids and transposons, have been shown to contribute to the wide spread and distribution of antibiotic resistant genes among bacteria. In recent years, the role of integrons as a mobile genetic mechanism in horizontal transfer of antibiotic resistance has been well established [29, 31, 32, 84]. A complete functional integron platform comprises three elements (Fig. 1): the integrase gene (intI) encoding an integrase, a proximal primary recombination site attI and a promoter gene (Pc) which had been functionally demonstrated for all integrons [43]. IntI encodes a tyrosine-recombinase family integrase, which is characterized by the presence of invariant RHRY amino-acids in the conserved motifs (termed as box 1 and box 2), and mediates recombination between the attI site and a secondary target called an attC site (also known as 59 base elements or 59-be sites [59be]). The simple attI1 site consists of two inverted sequences that bind the integrase, and two additional integrase-binding sites termed strong (DR1) and weak (DR2) binding sites which are located 24–37 and 41–55 bp to the left of the cross-over point, respectively [22–24, 33, 78]. The attC sites comprise a family of diverse sequences which are not highly conserved and vary considerably in size from 57 to 141 bp [15, 16, 50, 69]. The attC region consists of four essential sites called 1R, 2R, 1L and 2L, with 1R and 2R as part of the RH consensus sequence, and 1L and 2L as part of the LH consensus sequence [23, 24]. The similarities of the attC sites are primarily restricted to their boundaries, which correspond to the inverse core site (ICS) as RYYYAAC and the core site (CS) as GTTRRRY [15, 85]. The attC sites are generally associated with a single ORF in a structure termed gene cassettes, which are not necessarily observed in integrons, but once integrated they become part of the integron [22]. These smallest known mobile genetic elements can exist in one of two forms, including the independent circular DNA molecule which is unable for stably maintain during cell division, and the linear form which is created by a highly orientation-specific insertion of the free circular element into the integron [43]. They contain a coding sequence, but are usually lack of promoters to constitute the mobile component of the system [31, 69, 72, 85, 87]. Mostly gene cassettes encode resistance against antibiotics cover a wide range of antibiotics, and up to date, more than 100 different antibiotic resistance gene cassettes have been characterized, with mostly unique attC sites. The position of a cassette in the integron, including both order and distance, is strictly related to the level of antibiotic resistance. Insertion of the gene cassette at the attI site, which is located downstream of a resident promoter internal to the intI gene, drives expression of the encoded proteins. In class 1 integron, gene cassettes are expressed from a common promoter located in the 5′-conserved segment (5′-CS) region, where two potential promoter sites Pc and P2 locate. Pc, also known as P_ANT, locates around 200 bp upstream of the integration site; and P2 is inactive for the replacement of the optimal 17 nucleotides between the −35 and −10 boxes to only 14 nucleotides [17]. Though not a part of site-specific recombination platform, Pc plays key role in the functioning of integron as it ensures the correct expression of gene cassette [43]. The 3′-conserved segment (3′-CS) of class 1 integrons possesses the genes qacEΔ1 and sul1, encoding resistance to quaternary ammonium salts and sulfonamide, respectively [70].

Fig. 1. Structure of class 1 integron.

Several classes of integrons have been identified and distinguished by differences and divergence in the intI sequences, and integron classes 1–3 are so-called multiresistant integron (RIs) which appear to be able to acquire same gene cassettes [30]. Class 4 integron is considered to be a distinct type of integron and termed super integron (SI), which was found on the small chromosome of Vibrio cholerae and known to be an integral component of many γ-proteobacterial genomes [3, 49, 74]. As approximately 9% of the sequenced bacterial genomes containing integrons, of these, class 1 integron platform is the most ubiquitous among clinical microbes and remains the focus of numerous studies [4, 43, 98]. As a direct result of the linkage of class 1 integrons with Tn402-like transposons, this integron had been reported to be associated with Tn3 transposon family (Tn21 or Tn1696) [43]. Class 1 integron has been reported in a large variety of clinical Gram-negative organisms, including Acinetobacter, Aeromonas, Alcaligenes, Burkholderia, Campylobacter, Citrobacter, Enterobacter, Escherichia, Klebsiella, Mycobacterium, Pseudomonas, Salmonella, Serratia, Shigella and Vibrio, as well as in a few Gram-positive bacteria [9, 12, 19, 21, 25, 27, 40, 42, 44, 46, 57, 59, 61, 62, 71, 89, 91, 94–96, 98–100]. Class 2 integron has an organization similar to that of class 1 but is associated with the Tn7 transposon family [31, 79]. The typical intI2 gene, with the amino-acid sequences less than 50% homologous to the IntI1 integrase, is not functional due to the replacement of the internal termination codon with a codon for glutamic acid (amino acid 179). IntI2 may be a pseudogene, however, the reason for the stop codon still remains unclear. The two possible explanations for the truncated intI2 may be the regulatory function, and the presence of another type of integrase, such as intI1. The latter hypothesis is supported by the frequently detection of class 1 integrons in isolates simultaneously with class 2 integrons and the small number of different gene cassettes observed in class 2 integrons comparing with class 1 integrons. This mutation has been attributed to the low diversity of integrated gene cassettes and most reported class 2 integron carry three specific gene cassettes, dfrA1, sat1 and aadA1, which confer resistance to trimethoprim, streptothricin and streptomycin/spectinomycin, respectively [35, 43]. Thus, class 2 integron has been regarded as a contributor to the antibiotic resistance issue, and commonly observed in some species of Gram-negative organisms such as Acinetobacter, Enterobacteriaceae, Salmonella and Psuedomonas [1, 51, 58, 67, 68, 92, 97]. IntI2 is capable of site specific excision and integration of gene cassettes precisely into attI2. However, this integrase is unable to recognize gene cassettes from class 1 integrons, despite the identical gene cassettes found in both class 2 and class 1 integrons. Recombination site attI2 and promoter Pc are found within transposons such as Tn7, and the 3′-CS contains five tns genes involved in the movements of the transposon, which mediates the mobility of class 2 integron via a preferential insertion into a unique site within bacterial chromosomes [35, 43]. Class 3 integron contains a comparable structure to that of class 2 integron, and up to date has only been described in Psuedomonas, Alcaligenes, Serratia marcescens and Klebsiella pneumoniae [2, 18, 58, 79]. Both IntI1 and IntI3 are part of the soil/freshwater Proteobacteria group, as class 2 integrases within the marine γ-Proteobacteria group. Functionally similar to IntI1, IntI3 is able to recognize different attC sites and integrate the cassettes into the attI3 site. Class 4 integron harbors hundreds of gene cassettes encoding adaptations that extend beyond antibiotic resistance and pathogenicity, and has been detected in isolates from the last century and indicates its existence predating the antibiotic era [73]. The two key features that define class 4 integron and distinguish it from other RIs includes: (1) A large number of cassettes that are incorporated, which in the case of V. cholerae, the cluster of VCR-associated ORFs represents at least 216 unidentified genes in an array of 179 cassettes and occupies about 3% of the genome; (2) The high homology between the attC sites of those gathered cassettes [72]. Class 4 integron has been identified and characterized among the Vibrionaceae, Shewanella, Xanthomonas, Pseudomonas, as well as other proteobacteria [13, 36, 60, 72, 74]. The remaining classes of integrons may also contain antibiotic resistance gene cassettes, but their worldwide prevalence remains low [31, 60].

The integron platforms are defective for self-transposition, however, the transposons and conjugative plasmids associated can serve as vehicles for the intra- and inter-species transmission of genetic material [72]. This site-specific recombination reaction can be mediated by either the Tn21 integrase or the integron integrase IntI1 when the integration sites conform to the consensus sequence GWTMW or GNT (Fig. 2), respectively [22]. IntI1 recognises three types of recombination sites including attI1, attC and secondary sites, and via this site-specific recombination event, class 1 integron is capable of capturing gene cassettes. Recombination event between attI1 site and attC is slightly more efficient than recombination between two attC sites, but those between two attI1 sites is far less efficient. For secondary sites, recombination with attC is more efficient than attI.

Fig. 2. Mechanism of class 1 integron-mediated excision and integration.

Development of class 1 integron

Since class 1 integron has been identified as a primary source of antimicrobial resistance genes and suspected to serve as reservoirs and exchanging platforms of resistance genes within microbial populations, its role in spread and dissemination of antibiotic resistance genes in a variety of Gram-negative bacteria had been well investigated and documented, with a broad distribution of 22–59% among Gram-negative organisms [43, 45, 47, 75]. Nevertheless, little is known about the prevalence of class 1 integron in Gram-positive bacteria. In 1998, a typical class 1 integron associated streptomycin/spectinomycin resistance determinant has been detected on a 29-kb plasmid pCG4 from Corynebacterium glutamicum, which showed even higher expression ability comparing to integron found in Escherichia coli, representing the first identification of class 1 integron in Gram-positive bacterial [59]. In 2002, an intI1-like gene together with a novel aminoglycoside adenyltransferase gene cassette aadA9 had been observed on a 27.8-kb R-plasmid pTET3 from C. glutamicum, which encodes streptomycin, spectinomycin, and tetracycline resistance [89]. Nandi et al. screened class 1 integron in Gram-positive organisms isolated from poultry litter, and found class 1 integron in several species of Gram-positive bacteria as Corynebacterium sp. (including C. ammoniagenes, C. casei and C. glutamicum), Aerococcus sp., Brevibacterium thiogenitalis and Staphylococcus sp. (including S. lentus, S. nepalensis and S. xylosus) [57]. Enterococci bacterium are another genus with frequent detection of integrons, with the first observation of class 1 integron-related gene, aadA, was found in E. faecalis strain W4470 [12]. In our novel report, class 1 integron was detected in 11 E. faecalis and two E. faecium strains, with two E. faecalis also positive for class 2 integron [98]. This is the first time to report class 2 integron in E. faecalis and class 1 integron in E. faecium, which is the first evidence of class 2 integron outside the Gram-negative organisms. Nevertheless, illustration and demonstration of the correlation between genetic integrons and clinical antibiotic resistance requires large scale number of clinical microbes and long-term track surveillance, which were limitations of the studies aforementioned. Based on series studies of systematic integrons investigation in hundreds of staphylococci strains during 2001–2006 [94–96, 99], this review concentrated on the latest development of class 1 integron in MRS isolates, including summary of prevalence and occurrence of class 1 integron, analysis of correlation between integron and antibiotic resistance, further demonstration of the role integrons play as antibiotic determinants, as well as origin and evolution of integron-associated gene cassettes during this study period.

Epidemiologic study of MRS isolates

During 2001–2006, a total of 262 MRS (209 MRSA and 53 MRCNS) strains isolated from various clinical samples were studied, including 20 MRSA and 13 MRCNS strains in 2001, 20 MRSA and 20 MRCNS strains in 2002, 15 MRSA and nine MRCNS strains in 2003, 34 MRSA and 11 MRCNS strains in 2004, 80 and 40 MRSA strains in 2005 and 2006, respectively. Two hundred and sixty-two MRS strains were isolated from First Affiliated Hospital of Jinan University (FAHJU), with 30 MRSA isolates sampled from Guangdong Provincial People's Hospital (GDPPH). Both of the medical settings are located in Guangzhou, China. During this period, the hospital staff conducted an active surveillance and data collection for staphylococci-colonized or -infected patients, with a tracking log used to identify epidemiologic relatedness between patients in order to select isolates for testing and thus document cluster. Each isolate was from an individual subject, and no repeat isolates were included, using the CDC definitions for nosocomial infections [7]. Only first patient isolates obtained from cultures performed more than 48 h after admission were included in the analysis. All MRS strains were identified to the species level using standard procedures: colony morphology, Gram staining, catalase test, the Vitek 2 automated system and the API-Staph commercial kit. Methicillin resistance was determined by susceptibility testing on oxacillin-screening agar, confirmed by latex agglutination for PBP2a and mecA detection by PCR as described previously [56, 96]. Detection of five exotoxin genes, encoding for staphylococcal enterotoxins SEA (sea), SEB (seb), SEC (sec), SED (sed), SEE (see); exfoliative toxin A, B (ETA; eta, ETB; etb); TSST-1 (tst); Panton-Valentine leukocidin (lukS and lukF) genes were performed as described previously [38, 52], and none of the tested strains were found to carry any of toxin genes. SCCmec types were assigned by PCR analysis of the cassette chromosome recombinase (ccr) and mec gene complexes (ccr-mec genes), using the primers of Ito et al. and Hisata et al. to identify the ccr and mec genes, followed by a further multiplex PCR confirmation [37, 38, 65]. The distribution of SCCmec type in 262 MRS strains showed that the classic nosocomial SCCmec type (I, II and III) dominated among the tested strains. For MRSA strains, three and 198 strains belonged to SCCmec type II and III, with eight strains untypable; for MRCNS strains, nine, 24 and 12 strains were classified as SCCmec type I, II and III, respectively, with eight strains untypable. None of the tested strain carried type IV or V. Genotyping were performed by distinctly individual RAPD assays, in brief, RAPD typing for 179 MRSA from FAHJU and 30 MRSA from GDPPH were performed as described by Van Belkum et al. and Obayashi et al., respectively [64, 90]; and 23 I-MRCNS isolates of three Staphylococcal species carrying a highly prevalent array of dfrA12-orfF-aadA2 gene cassettes were subjected to separate RAPD fingerprinting using the assay of Obayashi et al. as aforementioned [64]. For MRSA isolates, At least one strain representative of each RAPD type was selected randomly for further analyses by multilocus sequence typing (MLST), spa and coa typings [20, 82, 83]. MLST was performed by amplification of the internal region of seven housekeeping genes, including arc, aroE, glpF, gmk, pta, tpi and yqiL. The sequences of PCR products were compared with the existing sequences available in the MLST website (http://www.mlst.net) for S. aureus, and the allelic number was determined for each sequence. The sequence type (ST) was determined according to the pattern of the combination of the seven alleles, and the clonal complex (CC) was defined by the BURST (based upon related sequence types) program by accessing the MLST website. Typing of the polymorphic repeat region of protein A (spaA typing) and coagulase gene (coa typing) were performed by analyzing the spaA and coa repeats with the FINDPATTERNS program from Genetics Computer Group Wisconsin Package 9.1 and comparing the spaA repeat sequences as well as their distribution through http://www.ridom.de/spaserver. Thirty MRSA strains from GDPPH were classified into six distinct genotypes by RAPD-PCR, with 16 integron-positive and 14 integron-negative strains belonging to two and four genotypes, respectively. RAPD-PCR with primers AP1, AP7 and E2 classified 179 MRSA strains from FAHJU into eight, six and 12 distinct groups, respectively, with a total of 16 RAPD types detected. All MRSA strains fell into ST239-MRSA-III group (clonal complex, CC239), with the same coa type HIJKL. SpaA type of most MRSA isolates was WGKAOMQ (t037), with 12 I-MRSA and five non I-MRSA strains while belonged to WGKAQQ (t030). For 23 I-MRCNS isolates harbouring dfrA12-orfF-aadA2 array, all tested strains were phylogenetically unrelated and exhibited distinct RAPD patterns with low Dice coefficients.

Investigation of class 1 integron

Integron characterization was performed by PCR amplification for the integrase gene, variable region and 3′-conserved region. In brief, 262 MRS strains were screened by multiple PCR amplification for three classes of integrase genes. One hundred and twenty-two strains yielded a 565-bp PCR product, suggesting the existence of class 1 integrase (intI1), with none of class 2 and 3 integrase gene obtained (Table 1). IntI1-positive strains were further characterized for the variable region and 3′-conserved region. Most reported class 1 integron has classic 3′-CS including a ΔqacE and a sulI gene and ORF5 [54, 66], and in this series of studies, one hundred and fifteen strains yielded an 800-bp amplicons in the PCR amplification of 3′-CS of qacEΔ1-sul1, with a rate of 94.3% (115/122). Variable region was determined by PCR with primers in-F and in-B, and PCR products were characterized by restriction fragment length polymorphism (RFLP) and at least three strains representative of each cassette type was selected randomly for further confirmation by sequencing. The PCR products of variable region were cut out from the agarose gel, purified by the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany) and 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–Bertani agar containing ampicillin (100 μg/ml). Recombinant plasmid DNA was purified by standard method and subjected for DNA sequencing for further analyses. The nucleotide sequences of gene cassette were determined by BigDye Terminator Cycle Sequencing FS Ready Reaction Kit on ABI PRISM 310 Genetic Analyzer (Perkin-Elmer Japan Applied Biosystems, Tokyo, Japan). Nucleotide sequence homology searches were performed against all sequences in the 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). A total of four different arrays of gene cassette arrays were found in tested staphylococci strains, with fragments varied in length between 975 and 2360-bp. A 975-bp amplicon was obtained from 53 strains, and the sequence demonstrated an aadA2 gene cassette encoding resistance to aminoglycoside. Sixty-one strains gave a 1913-bp PCR product, the sequence of which demonstrated a dfrA12-orfF-aadA2 array of gene cassettes. The dfrA12 and aadA2 conferred resistance to trimethoprim and aminoglycoside respectively, and the second ORF encodes an unknown function protein. A 1664-bp product was found from three strains and the sequence confirmed the presence of gene cassettes dfrA17 and aadA5, which were resistant to trimethoprim and aminoglycoside, respectively. Five strains yielded an amplicon of 2360-bp, harboring aacA4 and cmlA1 genes which conferred resistant to aminoglycoside and chloramphenicol, respectively. To further determine the location of class 1 integron, intI1 and gene cassettes of 28 MRSA and 30 MRCNS were investigated by Southern blot hybridization. According to the result of Southern hybridization, no signal had been observed on plasmid DNA, whereas the PCR-generated intI and cassette probes hybridized with genomic DNA, demonstrating the class 1 integrons were located on chromosomes, not plasmid.

Table 1. Phenotypic and genotypic characteristics of I-MRS strains.

Strain	Species	Date	Infection site	Dept	Antibiotic resistance	SCCmec		Integron
						ccr	mec	3′-CS	Gene cassettes
010808	S. aureus	2001	O	O	AcChCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
010912	S. aureus	2001	O	S	AcChCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
011000	S. aureus	2001	P	D	AcChCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
011001	S. aureus	2001	B	G	AcChCiClELOTs	3	A	+	dfrA12-orfF-aadA2
011016	S. aureus	2001	U	S	AcCiEGLOTs	3	A	+	dfrA12-orfF-aadA2
011024	S. aureus	2001	S	I	AcChCiClEGLOTc	3	A	−	dfrA12-orfF-aadA2
011025	S. aureus	2001	S	S	AcCiEGLOTs	3	A	+	dfrA12-orfF-aadA2
011045	S. aureus	2001	S	I	AcChCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
011052	S. aureus	2001	S	I	AcChCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
011055	S. aureus	2001	P	O	AcChCiClELOTs	3	A	+	dfrA12-orfF-aadA2
011058	S. aureus	2001	P	N	AcChCiClELOTs	3	A	+	dfrA12-orfF-aadA2
011083	S. aureus	2001	P	N	AcChCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
011098	S. aureus	2001	S	I	AcCiEGLOTs	3	A	+	dfrA12-orfF-aadA2
021138	S. aureus	2002	S	I	AcChCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
021153	S. aureus	2002	O	S	AcClOTcTs	3	A	+	dfrA12-orfF-aadA2
021206	S. aureus	2002	S	I	AcClOTcTs	3	A	+	dfrA12-orfF-aadA2
021207	S. aureus	2002	S	I	AcClOTcTs	3	A	+	dfrA12-orfF-aadA2
021238	S. aureus	2002	S	I	AcChCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
021261	S. aureus	2002	U	N	AcChCiClELOTs	3	A	+	dfrA12-orfF-aadA2
021266	S. aureus	2002	U	S	AcCiEGLOTs	3	A	+	dfrA12-orfF-aadA2
021267	S. aureus	2002	S	I	AcChCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
021268	S. aureus	2002	P	S	AcCiEGLOTs	3	A	+	dfrA12-orfF-aadA2
021296	S. aureus	2002	B	O	AcCiEGLOTs	3	A	−	dfrA12-orfF-aadA2
021542	S. aureus	2002	P	S	AcChCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
031788	S. aureus	2003	S	I	AcChCiClELOTs	3	A	+	dfrA12-orfF-aadA2
032142	S. aureus	2003	S	I	ChCiClELOTs	3	A	+	dfrA12-orfF-aadA2
032267	S. aureus	2003	O	S	AcChCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
032371	S. aureus	2003	S	I	AcClOTcTs	3	A	+	dfrA12-orfF-aadA2
032415	S. aureus	2003	S	S	AcChCiClGLO	3	A	+	aacA4-cmlA1
032423	S. aureus	2003	S	I	AcChClEGLOTc	3	A	+	dfrA17-aadA5
032439	S. aureus	2003	S	I	AcChCiClEGLOTs	3	A	+	aacA4-cmlA1
042457	S. aureus	2004	S	I	AcChCiClGLO	3	A	+	aadA2
042497	S. aureus	2004	U	I	AcChCiClEGLOTcTs	3	A	+	aadA2
042547	S. aureus	2004	S	I	AcCiClEGLOTcTs	3	A	+	aadA2
042564	S. aureus	2004	S	I	AcCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
042637	S. aureus	2004	S	I	AcChCiClGLOTcTs	3	A	+	aadA2
042649	S. aureus	2004	S	I	AcChCiClGLOTcTs	3	A	+	aadA2
042772	S. aureus	2004	B	I	AcCiClEGLOTcTs	3	A	+	aadA2
042848	S. aureus	2004	S	D	AcChCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
042885	S. aureus	2004	O	I	AcChCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
042887	S. aureus	2004	U	G	AcChCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
042898	S. aureus	2004	P	OG	AcCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
042923	S. aureus	2004	O	I	AcCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
042954	S. aureus	2004	P	P	AcCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
042966	S. aureus	2004	O	I	AcCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
043000	S. aureus	2004	S	I	AcCiClEGLOTcTs	3	A	+	dfrA12-orfF-aadA2
032147	S. aureus	2005	O	I	AcChCiClEGOTcTs	3	A	+	aadA2
032148	S. aureus	2005	O	I	AcChCiClEGOTcTs	3	A	+	aadA2
050511	S. aureus	2005	O	I	AcChCiClEGOTcTs	3	A	+	aadA2
050512	S. aureus	2005	R	I	AcChCiClEGOTcTs	3	A	+	aadA2
050513	S. aureus	2005	R	I	AcChCiClEGOTcTs	3	A	+	aadA2
050518	S. aureus	2005	R	I	AcChCiClEGOTcTs	3	A	+	aadA2
050557	S. aureus	2005	R	I	AcChCiClEGOTcTs	3	A	+	aadA2
050558	S. aureus	2005	R	I	AcChCiClEGOTcTs	3	A	+	aadA2
050559	S. aureus	2005	R	I	AcChCiClEGOTcTs	3	A	+	aadA2
050560	S. aureus	2005	R	I	AcChCiClEGOTcTs	3	A	+	aadA2
050561	S. aureus	2005	R	I	AcChCiClEGOTcTs	3	A	+	aadA2
050562	S. aureus	2005	R	I	AcChCiClEGOTcTs	3	A	+	aadA2
050581	S. aureus	2005	O	I	AcChCiClEGOTcTs	3	A	+	aadA2
050582	S. aureus	2005	R	I	AcChCiClEGOTcTs	3	A	+	aadA2
050583	S. aureus	2005	R	I	AcChCiClEGOTcTs	3	A	+	aadA2
050585	S. aureus	2005	R	I	AcChCiClEGOTcTs	3	A	+	aadA2
053001	S. aureus	2005	O	I	AcCiClEGLOTcTs	3	A	+	aadA2
053059	S. aureus	2005	R	I	AcCiClEGLOTc	3	A	+	aadA2
053147	S. aureus	2005	B	I	AcCiClEGLOTcTs	3	A	+	aadA2
053182	S. aureus	2005	R	I	AcCiClEGLOTcTs	3	A	+	aadA2
053224	S. aureus	2005	R	I	ChCiClGLTcTs	1	N	+	aadA2
053332	S. aureus	2005	B	I	AcChCiClGLOTcTs	3	A	−	aadA2
053333	S. aureus	2005	O	I	AcChCiClGLOTc	3	A	+	aadA2
053401	S. aureus	2005	O	I	AcCiClEGLOTcTs	3	A	+	aadA2
053423	S. aureus	2005	U	I	AcChCiClGLOTcTs	3	A	+	aadA2
053443	S. aureus	2005	B	I	AcChCiClGLOTc	3	A	+	aadA2
053474	S. aureus	2005	B	I	AcCiClEGLOTcTs	3	A	+	aadA2
053564	S. aureus	2005	O	I	AcCiClEGLOTcTs	3	A	+	aadA2
053610	S. aureus	2005	O	I	AcCiClEGLOTcTs	3	A	+	aadA2
053658	S. aureus	2005	R	I	AcCiClEGLOTcTs	3	A	+	aadA2
053685	S. aureus	2005	R	I	AcCiClEGLOTcTs	3	A	+	aadA2
053845	S. aureus	2005	R	I	AcCiClEGLOTc	3	A	+	aadA2
053899	S. aureus	2005	O	I	AcCiClEGLOTcTs	3	A	+	aadA2
064043	S. aureus	2006	R	I	AcCiClEGLOTcTs	3	N	+	aadA2
064050	S. aureus	2006	R	I	AcChCiClGLOTcTs	3	A	+	aadA2
064064	S. aureus	2006	R	I	AcCiClEGLOTcTs	N	–	+	aadA2
064100	S. aureus	2006	R	I	AcCiClEGLOTcTs	3	A	+	aadA2
064163	S. aureus	2006	R	I	AcChCiClGLOTc	3	A	+	aadA2
064221	S. aureus	2006	R	I	AcChCiClGLOTcTs	3	A	+	aadA2
064249	S. aureus	2006	R	I	AcChCiClGLOTcTs	3	A	+	aadA2
064278	S. aureus	2006	O	I	AcChCiClGLOTcTs	3	A	+	aadA2
064375	S. aureus	2006	R	I	AcChCiClGLOTcTs	3	A	+	aadA2
065212	S. aureus	2006	R	I	AcChCiClGLOTcTs	3	A	+	aadA2
065217	S. aureus	2006	R	I	AcChCiClGLOTc	3	A	+	aadA2
065260	S. aureus	2006	R	I	AcChCiClGLOTcTs	3	A	+	aadA2
012216	S. epidermidis	2001	R	I	AcChCiELTcTs	2	A	+	dfrA12-orfF-aadA2
012219	S. epidermidis	2001	R	I	AcCiClGLTs	3	A	+	dfrA12-orfF-aadA2
012228	S. epidermidis	2001	U	I	AcCiClLTs	2	A	+	dfrA12-orfF-aadA2
022212	S. epidermidis	2002	B	S	AcChEGLTcTs	3	A	+	dfrA12-orfF-aadA2
022218	S. epidermidis	2002	R	I	AcCiClELTcTs	2	A	+	dfrA12-orfF-aadA2
022225	S. epidermidis	2002	B	I	AcChCiTs	1	B	+	dfrA12-orfF-aadA2
022230	S. epidermidis	2002	R	I	AcClEGLTc	2	A	−	aadA2
022237	S. epidermidis	2002	B	I	AcClEGLTcTs	2	A	+	dfrA12-orfF-aadA2
022244	S. epidermidis	2002	R	O	AcChCiClELTcTs	3	A	+	dfrA12-orfF-aadA2
022256	S. epidermidis	2002	R	I	AcClEGLTs	N	–	+	dfrA12-orfF-aadA2
022258	S. epidermidis	2002	U	S	AcChEGLTcTs	N	–	+	dfrA12-orfF-aadA2
032237	S. epidermidis	2003	R	I	AcChCiClEGTc	2	A	−	aacA4-cmlA1
032211	S. epidermidis	2003	R	S	AcChEGLTcTs	N	–	+	dfrA12-orfF-aadA2
032224	S. epidermidis	2003	R	I	ChCiTs	1	B	+	dfrA12-orfF-aadA2
042219	S. epidermidis	2004	U	S	AcChCiClEGL	2	A	−	aacA4-cmlA1
042237	S. epidermidis	2004	U	S	AcCiTcTs	3	A	+	dfrA17-aadA5
012303	S. hominis	2001	R	I	AcEGTcTs	1	B	−	dfrA12-orfF-aadA2
012305	S. hominis	2001	R	O	AcChELTs	1	B	+	dfrA12-orfF-aadA2
012306	S. hominis	2001	U	S	EGTcTs	1	B	+	dfrA12-orfF-aadA2
022303	S. hominis	2002	B	I	AcChClEGLTcTs	2	A	+	dfrA12-orfF-aadA2
032309	S. hominis	2003	R	I	AcClETcTs	2	A	+	dfrA12-orfF-aadA2
032315	S. hominis	2003	R	S	AcChCiLTs	3	A	+	dfrA17-aadA5
042306	S. hominis	2004	U	I	AcChCiClEGLTcTs	2	A	+	aacA4-cmlA1
042315	S. hominis	2004	U	S	AcCiETs	2	A	+	dfrA12-orfF-aadA2
022405	S. haemolyticus	2002	R	I	AcCiClETs	2	A	+	dfrA12-orfF-aadA2
022407	S. haemolyticus	2002	B	S	AcChEGLTs	2	A	+	dfrA12-orfF-aadA2
022411	S. haemolyticus	2002	R	S	AcEGLTs	2	A	+	dfrA12-orfF-aadA2
022413	S. haemolyticus	2002	B	I	AcCiTcTs	N	–	+	dfrA12-orfF-aadA2
042403	S. haemolyticus	2004	U	I	AcCiTcTs	N	–	+	dfrA12-orfF-aadA2
012501	S. warneri	2001	B	I	AcCiClEGLTs	2	A	+	dfrA12-orfF-aadA2

Resistance profile, antibiotics used in the current study included: amoxicillin/clavulanic acid (Ac), chloramphenicol (Ch), ciprofloxacin (Ci), clindamycin (Cl), erythromycin (E), gentamicin (G), levofloxacin (L), oxacillin (O), tetracycline (Tc), trimethoprim-sulfamethoxazole (Ts)

Infection site: B bloodstream, R respiratory tract, S skin and soft tissue, U urinary tract, O others

Dept: D dept. infectious disease, G general ward, I internal medicine, N neurology, O orthopedics, OG obstetrics and gynecology, P pediatrics, S surgery

+, carrying a 3′-CS of qacEΔ1-sul1; −, not carrying 3′-CS; N not typeable

Class 1 integron was commonly found in the tested staphylococci isolates (46.6%, 122/262), and the proportion had been decreasing during this study time span. During 2001–2004, the detection rate of class 1 integron for MRSA and MRCNS was 51.7% (46/89) and 56.6% (30/53), respectively. Nevertheless, only 38.3% (46/120) of MRSA isolates carried class 1 integron. Concerning cassette types, dfrA12-orfF-aadA2 and aadA2 remained prevalent, taking up 50.0% (61/122) and 43.4% (53/122) among all I-MRS strains (Fig. 3). For I-MRSA, major types as dfrA12-orfF-aadA2 and aadA2 consisted of 40.2% (37/92) and 56.5% (52/92), respectively. However, for I-MRCNS, dfrA12-orfF-aadA2 dominated during the study period, with only one aadA2 case observed. The most frequently detected resistance genes in class 1 integron were aadA and dfrA family, with the rate 95.9% (117/122) and 52.5% (64/122), respectively, which was similar to previous studies [28, 97] (Figs. 3, 4). It was noticed that most of the known gene cassettes encoded resistance to the oldest groups of antibiotics which have been used for more than 20 years, in spite of an increasing number of new gene cassettes defining resistance against newer groups of antibiotics. It should also be noted that a large proportion of gene cassettes encoding resistance against streptomycin and spectinomycin, despite the fact that use of these antibiotics (at least in a clinical setting) has long ago been discontinued. Nevertheless, since it has been hypothesized that the presence of an integron may lead to a more extensive exchange of resistance determinants than gene cassettes alone, the consistent detection of class 1 integron in MRS isolates strongly suggest class 1 integron may serve as reservoirs of antimicrobial resistance and contribute to increased rates of treatment-resistant staphylococci infections in both the hospital and community setting.

Fig. 3. Proportion of array types of gene cassettes in I-MRS strains.

Fig. 4. Occurrence and prevalence of integrons and array types during 2001–2006.

Antibiotic resistance determinant

In order to investigate the role of class 1 integron played as antibiotic resistance determinant in MRS strains, antimicrobial susceptibility testing was performed by standard disk diffusion method, and minimum inhibitory concentration (MIC) were determined for amoxicillin/clavulanic acid, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, levofloxacin, oxacillin, teicoplanin, tetracycline, trimethoprim-sulfamethoxazole and vancomycin according to Clinical Laboratory and Standards Institute (CLSI) methods [14]. Furthermore, associations between integron-bearing and non integron-bearing MRS were analyzed by the χ² test or analysis of variance. A P value of <0.05 was considered statistically significant. All analyses were performed with SPSS 12.0G for Windows. Antimicrobial susceptibility testing showed the multidrug resistance (defined as resistance to six or more antibiotics) rates of integron-positive and -negative strains were 85.2% (104/122) and 79.3% (111/140), with the highest number of amoxicillin/clavulanic acid resistance (93.5%, n = 245), followed by ciprofloxacin (84.0%, n = 220) and clindamycin (81.0%, n = 214). The percentage of resistance to chloramphenicol, erythromycin, gentamicin, levofloxacin, oxacillin, tetracycline and trimethoprim-sulfamethoxazole ranged from 51.5 to 76.7% (Table 2). None of the tested isolates showed resistance to vancomycin and teicoplanin. The MIC of oxacillin ranged from 16 to 512 μg/ml. The antibiotic resistance data between I-MRS and non I-MRS isolates was compared and the χ² test was used to calculate the P value in terms of resistant and susceptible numbers. Class 1 integron was significantly associated with resistance to certain antibiotics in both types of strains, including erythromycin, gentamicin, tetracycline and trimethoprim-sulfamethoxazole. Regular treatment of MRS infection is usually with penicillinase-resistant β-lactams, however, since antibiotic resistance becomes common, vancomycin or other newer antibiotics may be required. Some strains had been reported partially or totally resistant to all but the newest antibiotics, including linezolid, quinupristin/dalfopristin, daptomycin, telavancin, dalbavancin, and tigecycline. In the current investigation, erythromycin, gentamicin, tetracycline and trimethoprim-sulfamethoxazole had been found correlated strongly with the presence of class 1 integron. Therapeutic options for deep-seated infection due to MRS strains are limited. In treating complicated MRS infections, gentamicin has been often used by many clinicians as combination with vancomycin in the expectation of a more rapid bacteriologic response based on a synergistic interaction between the two antibiotics [53]. Erythromycin has been commonly used for penicillin-allergic patients. Trimethoprim/sulfamethoxazole is often the first choice to cure community-acquired cutaneous infections likely to be due to MRS strains, and its combination use with rifampin has been useful in treating MRSA in carriers, though the organism recurs in up to 50% and frequently becomes resistant.

Table 2. Correlation of antibiotic resistance between I-MRS and non I-MRS strains.

Antibiotics	All MRS strains		I-MRS strains		Non I-MRS strains		P value	OR (95% CI)
	R	S	R	S	R	S
Amoxicillin/clavulanic acid	245 (93.5%)	17 (6.5%)	117 (44.7%)	5 (1.9%)	128 (48.9%)	12 (4.6%)	0.053	0.107–1.061
Chloramphenicol	135 (51.5%)	127 (48.5%)	67 (25.6%)	55 (20.9%)	68 (26.0%)	72 (27.6%)	0.249	0.461–1.222
Ciprofloxacin	220 (84.0%)	42 (16.0%)	102 (38.9%)	20 (7.7%)	118 (45.0%)	22 (8.4%)	0.893	0.493–1.854
Clindamycin	214 (81.7%)	48 (18.3%)	97 (37.0%)	25 (9.6%)	117 (44.7%)	23 (8.8%)	0.557	0.654–2.257
Erythromycin	118 (64.1%)	94 (35.9%)	88 (33.6%)	34 (12.9%)	85 (30.5%)	60 (23.0%)	0.007	0.292–0.828
Gentamicin	170 (64.9%)	92 (35.1%)	89 (34.0%)	33 (12.5%)	81 (30.9%)	59 (22.6%)	0.006	0.287–0.820
Levofloxacin	194 (74.0%)	68 (26.0%)	89 (34.0%)	33 (12.5%)	105 (40.1%)	35 (13.4%)	0.866	0.603–1.824
Oxacillin	201 (76.7%)	61 (23.3%)	90 (34.4%)	32 (12.1%)	111 (42.4%)	29 (11.2%)	0.464	0.718–2.262
Tetracycline	171 (65.3%)	91 (34.7%)	90 (34.4%)	32 (12.1%)	81 (30.9%)	59 (22.6%)	0.004	0.274–0.788
Trimethoprim-sulfamethoxazole	164 (62.6%)	98 (37.4%)	107 (40.8%)	15 (5.6%)	57 (21.8%)	83 (31.8%)	0.000	0.046–0.170

Mobility and evolution

As a mobile genetic element, the mobility of integrons is defined as being associated with mobile DNA elements (transposons or plasmids) and antibiotic-resistance genes in addition to having a small array size and substantial heterogeneity in the sequence of attC sites [6, 48]. It has been generally accepted most existent class 1 integrons were located on plasmids as facilitation of conjugative-mediated transfer, which was also supported by the frequently detected plasmid-integrons in non-staphylococci in this study. In previous studies, Southern hybridization analysis was used to determine the location and the copy numbers of integron in 58 staphylococci isolates (data of 28 MRSA unpublished) [95]. Strikingly, all strains were detected to carry one copy of class 1 integron on chromosomes, not plasmid. Integron is not self-movable, but it contains gene cassettes that can be mobilized to other integrons or to secondary sites in the bacterial genome. As a natural capture system and assembly platform, the class 1 integron system allows bacteria to incorporate gene cassettes and convert them to functional genes by ensuring their correct expression, which has been regarded as key player in the dissemination and spread of resistance genes, responsible for the facile spread of resistance genes and the rapid evolution of resistance to a wide range of unrelated antibiotics among diverse bacteria [48, 58]. It is conceivable that any ORF can be structured as a gene cassette and vital to decipher the mechanism governing cassette genesis. Through the recombination platform (IntI1 and attI), integron has the potentially limitless capacity to exchange and stockpile functional gene cassettes which permits rapid adaptation to selective pressure and may ultimately endow increased fitness and advantage to the host. Altogether, hundreds of gene cassettes, all types of other mobile DNA elements such as conjugative plasmids, transposons, insertion sequences, even entire chromosome, would probably be the vast reservoirs of integron, lending support to the longstanding concept of a single massive genetic pool that is available and shared among bacteria [72]. The common observation of integrons in microorganisms from general environment and its enormous sequence diversity detected from such microbes, as well as various products unrelated to antibiotic resistance encoded by integron-associated cassette genes, strongly suggests that integrons are ancient genetic element in structure of genomes and have played a general role in evolution and adaptation for a considerable period of time [43]. For the staphylococci strains, on the other side, SCCmec is a genomic island (G island) inserted at the 3′ end of orfX and located near the replication origin, which is defined as a basic mobile genetic element demarcated by a pair of direct repeats and inverted repeats, having a set of site-specific recombinase genes (ccrA and ccrB) required for its movement and carrying the mecA gene complex [41]. SCCmec may have evolved from a primordial mobile element, SCC, into which the mec complex was inserted, with the staphylococci chromosome. But there is no reason to limit the putative SCC to being only the conveyer of methicillin resistance (mediated by mecA gene) alone, while it might be serving as a vehicle for exchange of useful genes for the better survival for staphylococci in various environments, which means that SCCmec is a general genetic information exchange system of staphylococci with ccrA and ccrB involved in the recombination events (integration and excision) [38]. For both Integron and SCCmec serve as the reservoir of all kinds of genes and possess the function to exchange genes between species, so whether the simultaneous existence of Integron system and SCCmec would speed up the gene exchange and genome evolution in staphylococci require further investigation. As a novel antibiotic resistance determinant, general concerns for integrons in staphylococci may raise necessity for surveillance and investigation of its occurrence, mechanism and evolution.

Origins and dissemination

In a long-term integron investigation of MRSA, 16 genotypes were identified from 179 MRSA strains, and I-MRSA and non I-MRSA representative of 14 specific clones, with two genotypes shared. It was noteworthy that some I-MRSA strains containing the same organization of gene cassettes were found in various RAPD-PCR genotypes (aadA2 and dfrA12-orfF-aadA2 shared three and five genotypes, respectively), suggesting a horizontal transfer of integrons, which had been suspected in other studies [75, 80, 88]. In contrast, certain strains grouped in the same genotype were found to carry different arrays of gene cassette, indicating the acquisition and exchange of different cassettes by site-specific recombination of IntI1. In a preliminary investigation on an outbreak of 30 MRSA isolates, six genotypes were obtained, with I-MRSA representative of two specific clones. Despite the 3-month outbreak, large proportion of I-MRSA strains were isolated during a short time span of 12 days (May 19–30), with non I-MRSA strains prevalent for more than 2 months. Remarkably, strains with the same genotype were obtained from clinical specimens on consecutive days or patients in adjacent beds, as well as both from environment and patients, strongly suggesting pathogen contamination and transmission may occur by direct or indirect contact between patients, medical personnel and environment. Many potential reservoirs of S. aureus in the hospital environment, including medical equipment such as parenteral solution and rinse solution and ward stuff such as doorknob and console are probably important risk factors and serve as vehicle for pathogen spreading. Strikingly, high diversity in genetic background was observed in the I-MRCNS investigation. Twenty-three CoNS strains with array of dfrA12-orfF-aadA2 exhibited distinct RAPD patterns and were divided into different genetic groups, demonstrating that they were phylogenetically unrelated. The results strongly suggested that the wide spread and distribution of class 1 integron in diverse clones of CoNS strains may be mainly due to the horizontal transfer of integrons, not by specific clone. Remarkably, the arrays of gene cassettes detected in staphylococci, had been previously reported in clinical isolates of various negative bacteria [59, 89, 101, 102], with identical sequences. For example, cassettes dfrA12-orfF-aadA2 within class 1 integron was also present in the Gram-negative enteroinvasive E. coli O164 strain RIMD05091045 isolated from a patient in Japan [1]. When the cassette arrays dfrA12-orfF-aadA2, aadA2, dfrA17-aadA5 and aacA4-cmlA1 were compared to those deposited in GenBank, homology of nucleotides ranged from 95 to 100%, including array dfrA12-orfF-aadA2 in E. coli (AB154407, DQ157 751), Salmonella enterica (DQ238105), K. pneumoniae (AF180731), Acinetobacter baumannii (DQ141318) and P. aeruginosa (AB191047), array dfrA17-aadA5 in E. coli (AB189264, AB194702, DQ663488, DQ322597, AY82 8551, AY748452) and K. pneumoniae (AF220757, AY994 155), array aacA4-cmlA1 in E. coli (AB212941). The integron sequences identified in staphylococci were at least 99.5% homologous to those from isolates sampled in the same hospital setting (Table 3). It is noteworthy that in staphylococci strains, prevalent array cassettes such as dfrA12-orfF-aadA2 and aadA2 also dominated in other species. The observance of identical integrons in genomes of phylogenetically distant bacteria and the similarity of prevalent arrays in diverse clinical organisms strongly suggest intergeneric horizontal transfer of genetic cassettes in the hospital setting. Several classes of integrons had been reported capable of spreading among Gram-negative bacteria, with one example being the transfer of class 1 integron via plasmid from E. faecalis strain W4770 to a recipient strain of E. faecalis [12]. However, issues concerning horizontal transfer and dissemination of integrons between Gram-positive and Gram-negative organisms, as well as the origins of class 1 integron in Gram-positive bacteria, still remained unclear and required further investigation.

Table 3. Comparison of integron sequences between MRS and other species isolated from FAHJU.

S. aureus	Other organisms	Homology	GenBank no.	Reference
Class 1 integron
dfrA12-orfF-aadA2	S. epidermidis	100% homology	AB297447	95
	S. hominis	100% homology	AB297448	95
	S. haemolyticus	99.9% homology with 860 T–C	AB297449	95
	S. warneri	100% homology	AB297450	95
	E. faecalis	99.8% homology with 398 A–G, 458 A–G, 984 T–G and 1603 G–A	Not deposited	98
	E. faecium	99.8% homology with 398 A–G, 458 A–G, 984 T–G and 1603 G–A	Not deposited	98
dfrA17-aadA5	S. epidermidis	99.9% homology with 1289 T–C	AB291061	95
	S. hominis	100% homology	AB291062	95
	E. faecalis	99.9% homology with 1289 T–C	Not deposited	98
	E. coli	99.9% homology with 1289 T–C	AB189264	86
aadA2	S. epidermidis	100% homology	AB481131	95
	E. faecalis	99.5% homology with 217 C–T, 322 C–A, 586 G–A, 717 G–T and 857 A–G	Not deposited	98
dfrA17-aadA5	S. epidermidis	99.9% homology with 1291 T–C	AB291061	95
	S. hominis	99.9% homology with 1291 T–C	AB291062	95

Development of I-MRS

Contemporary integron investigation had been conducted on a large scale of clinical organisms isolated from FAHJU, involving 254 Gram-negative and 163 Gram-positive strains, with a total detection rate of 73.4% (248/338) (Table 4). Class 1 integrons were found in 72.8% (185/254) of the Gram-negative strains, of which 91.3% (21/23) were in Acinetobacter spp., 86.7% (13/15) in Enterobacter cloacae, 81.2% (56/69) in E. coli [86], 87.5% (28/32) in K. pneumoniae, 47.4% (49/95) in P. aeruginosa [97] and 90% (18/20) in other organisms. For Gram-positive isolates, class 1 integrons were detected in 86.7% (13/15) of enterococci and 83.3% (5/6) of streptococci strains [86], and for MRS the detection rate was 46.6% (122/262), with 42.5% (76/179) for I-MRSA and 56.6% (30/53) for MRCNS. Remarkably, a significant decrease in class 1 integron positive rate was found during this studying time period, with 68.3% (28/41) in 1998, 100% (14/14) in 2000, 88.9% (40/45) in 2001, 78.4% (29/37) in 2002, 55.3% (26/47) in 2003, 80.9% (106/131) in 2004 and 21.7% (5/23) in 2005. This tendency had also been observed in I-MRS, with 60.6% (20/33) in 2001, 60.0% (24/40) in 2002, 50.0% (12/24) in 2003, 45.5% (20/44) in 2004, 42.5% (34/80) in 2005 and 30.0% (12/40) in 2006. It was noteworthy that class 2 integron was commonly found in 9.8% (33/338) of tested strains, including E. coli, P. aeruginosa and E. faecalis, which represented the first evidence of class 2 integron in Gram-positive bacteria and indicated the potential risk of spread of class 2 integron from Gram-negative to -positive bacteria, as staphylococci, though none of class 2 integron were found in staphylococci strains. Since integron is best known for its role in contributing to clinical antibiotic resistance and a large variety of different gene cassettes containing genes that confer resistance to antibiotics had been found, gene cassettes-associated multiple insertion events can lead to the accumulation of many cassettes within an integron, thus contributing to multidrug resistance [43]. Multiple copies of class 1 integron had been observed in 9.8% (33/338) isolates and 22 of them showed different arrays of gene cassettes, however, this had not been observed in staphylococci isolates as aforementioned. The prevalent gene cassette arrays were dfrA12-orfF-aadA2 and aadA2, taking up 50.0% (61/122) and 42.6% (52/122) of the integron-positive strains. Nevertheless, a significant tendency of changing of gene cassettes was observed, cassette array dfrA12-orfF-aadA2 dominated during 2001–2004, with 100% in 2001 (20/20) and 2002 (23/23), 58.3% (7/12) in 2003, 55.0% (11/20) in 2004, and disappeared afterwards. Nevertheless, gene cassette aadA2 appeared in 2004 (6/20, 30.0%) and became prevalent during 2005–2006 (46/46, 100%), however, it was noticed that one MRCNS strain isolated in 2002 also carried aadA2. Gene cassettes carried by isolates from 2003 and 2004 revealed the most diversity, with four different arrays of gene cassettes. A striking good concordance was observed between staphylococci and non-staphylococci isolates. As far as those cassette types identified in staphylococci were concerned, dfrA12-orfF-aadA2 first emerged in 2000 (42.9%, 6/14) and had been frequently obtained in 2001 (80.0%, 32/40) and 2002 (69.0%, 20/29), however, its proportion decreased in 2003 (65.4%, 17/26) and 2004 (57.5%, 61/106), with none of this cassette observed afterwards. Another prevalent cassette aadA2 was first acquired in 2002 (6.9%, 2/29), and had been slightly increased in 2003 (19.2%, 5/26) and 2004 (14.2%, 15/106). Cassette array dfrA17-aadA5 dominated in 1998 (46.4%, 13/28) and 2000 (64.3%, 9/14), but the detection rate decreased from then on, with 15.0% (6/40) in 2001, 6.9% (2/29) in 2002, 11.5% (3/26) in 2003 and 21.7% (23/104) in 2004. Detection rate of another infrequently detected array aacA4-cmlA1 ranged from 1 to 21% during 2001–2004. For non-staphylococci isolates, a total of six cassette genes had been found, including aadA (217/248), dfrA (200/248), aacA (11/248), cmlA (10/248), sul3 (5/248), bla (2/248) and pse (2/248). Family aadA and dfrA remained the most frequently detected, which was similar in staphylococci strains, with 95.9% (117/122) and 52.5% (64/122), respectively. In the recent decade, as regulations on the appropriate use of antibiotics have been effectively enforced in developed countries, community-associated methicillin-resistant staphylococci (CA-MRS) emerged and infections caused by CA-MRS have been observed with increasing frequency in a variety of countries and geographic regions [8, 10, 26, 93, 102]. CA-MRS, characterized by several distinctive properties such as: more susceptible antimicrobial phenotype, the presence of different exotoxin gene profiles and a much smaller SCCmec (type IV or V), is becoming more and more prevalent and has the tendency to replace traditional hospital-associated methicillin-resistant staphylococci (HA-MRS) worldwide [55, 76]. While in South China, according to our studies, classic nosocomia SCCmec still dominated, with no strain of type IV or V SCCmec acquired. As generally accepted, indiscriminate use of existing antibiotics resulted in antibiotic selective pressure and proliferation of antibiotic resistance, which was the rudimentary and intrinsic cause of the emergence and development of mobile genetic resistance mechanism as integron and gene cassettes, and was reflected by the domination of nosocomial SCCmec and prevalence of integron in the series studies. In South China, as trained practitioners were unavailable in many areas, regulations on the clinical use of antibiotics had been poorly enforced or absent and the surveillance system was not as effective, there may be more opportunity for the inappropriate use of antibiotics, resulting in heavier antibiotic selective pressure. Since the present existence and distribution of integron is due to multiple losses and gene transfer events and the ability of excision and integration may be selectively advantageous with the selective impact of integron on genomes [58] it is reasonable to presume in this region, MRS strains developed into a different direction, which inclined to carry genetic elements that would endow more fitness and advantage, resulting in their rapid adoption to selective pressure and survival.

Table 4. Contemporary integron investigation in FAHJU during 2001–2004.

Strains	Class 1 integron-positve rate	Year	Prevalent gene cassettes	No.
Acinetobacter spp.	91.3% (21/23)	2001	dfrA12-orfF-aadA2	4
			dfrA17-aadA5	1
		2002	dfrA12-orfF-aadA2	2
	1/2	2003	dfrA12-orfF-aadA2	1
	11/12	2004	dfrA12-orfF-aadA2	11
			aacA4-catB3-dfrA1-noncoding	1
			dfrA12-orfF-aadA2; aadA2	1
Alcaligenes spp.	100% (2/2)	2001	dfrA12-orfF-aadA2	1
		2004	dfrA12-orfF-aadA2	1
Burkholderia pseudomallei	75.0% (3/4)	2002	dfrA12-orfF-aadA2	1
	2/3	2004	dfrA12-orfF-aadA2	2
Citrobacter freundii	100% (2/2)	2004	dfrA12-orfF-aadA2	1
			dfrA17-aadA5	1
Enterobacter cloacae	86.7% (13/15)	2001	dfrA12-orfF-aadA2	3
		2002	aadA2	1
	1/3	2003	dfrA17-aadA5	1
		2004	aadA2	1
			dfrA12-orfF-aadA2; dfrA17-aadA5	4
			Others	3
Enterococcus faecalis	83.3% (10/12)	2001	dfrA12-orfF-aadA2	3
		2002	dfrA12-orfF-aadA2	2
	1/3	2003	dfrA12-orfF-aadA2	1
		2004	dfrA12-orfF-aadA2	2
			dfrA17-aadA5	1
			dfrA12-orfF-aadA2; aadA2	1
Enterococcus faecium	100% (1/1)	2004	dfrA12-orfF-aadA2	1
E. coli	88.3% (98/111)	1998	aadA1-dfrA12	10
			dfrA17-aadA5	13
			Others	5
		2000	dfrA17-aadA5	7
			dfrA12-orfF-aadA2	4
			dfrA12-orfF-aadA2; dfrA17-aadA5	2
			Others
		2001	dfrA12-orfF-aadA2	7
			dfrA17-aadA5	3
		2002	dfrA12-orfF-aadA2	5
			dfrA17-aadA5	2
		2003	dfrA12-orfF-aadA2	3
			dfrA17-aadA5	1
			aacA4-cmlA1	1
		2004	dfrA12-orfF-aadA2	17
			dfrA17-aadA5	11
			dfrA12-orfF-aadA2; dfrA17-aadA5	3
			aacA4-cmlA1	1
			Others	2
Flavobacterium	100% (1/1)	2004	dfrA12-orfF-aadA2	1
Hemophilus influenza	100% (1/1)	2003	dfrA12-orfF-aadA2	1
Klebsiella pneumoniae	87.5% (28/32)	2001	dfrA12-orfF-aadA2	3
		2002	dfrA12-orfF-aadA2	2
	3/6	2003	dfrA12-orfF-aadA2	2
			dfrA17-aadA5	1
	20/21	2004	dfrA12-orfF-aadA2	6
			dfrA17-aadA5	1
			drfA1-orfX	3
			aadA2	2
			dfrA17-aadA5, aadA2	1
			Others	7
Pseudomonas aeruginosa	45.8% (54/118)	2001	dfrA12-orfF-aadA2	6
			aacA4-cmlA1	2
			dfrA17-aadA5	2
		2002	dfrA12-orfF-aadA2	6
			aacA4-cmlA1	6
		2003	dfrA12-orfF-aadA2	8
			aadA2	5
		2004	aadA2	7
			dfrA12-orfF-aadA2	3
			dfrA12-orfF-aadA2; aadA2	2
			Others	2
		2005	Sul3	3
			blaVIM4- pse1	2
Proteus spp.	80.0% (4/5)	2001	dfrA12-orfF-aadA2	2
	2/3	2004	dfrA17-aadA5	2
Salmonella typhi	100% (2/2)	2001	dfrA12-orfF-aadA2	1
		2004	dfrA12-orfF-aadA2	1
Streptococcus spp.	83.3% (5/6)	2001	dfrA12-orfF-aadA2	2
		2002	dfrA12-orfF-aadA2	1
			dfrA12-orfF-aadA2; aadA2	1
	1/2	2003	dfrA12-orfF-aadA2	1
Xanthomonas maltophilia	100% (3/3)	2004	dfrA12-orfF-aadA2	2
			dfrA17-aadA5	1

Data of E. faecalis, E. faecium, E. coli and P. aeruginosa were from previous studies [81, 86, 97, 98]

Concluding remarks

As a common genetic element existed in 9% of bacteria and representatives from a broad range of phyla and environments, integron plays core role in antibiotic resistance among clinical organisms and contributes to the evolution and adaption of bacteria. However, integrons in Gram-positive bacteria has barely been touched upon, which may be an unnoticed and neglected antibiotic resistance determinant. Many question have been arisen and posed regarding the number and types of cassettes, rate of integration and excision, diversity of cassette genes pool and its distinct access, the functional role and genetic regulation of cassettes, as well as the extent to which it facilitates adaption and evolution to environmental selection pressure. The review offers important information for the epidemiology of class 1 integron in staphylococci strains, which will aid in the investigation of integrons in Gram-positive organisms.