Role of integrons in antimicrobial resistance : A review

1 First Affiliated Hospital of Jinan University, Guangzhou 510620, China. 2 Guangzhou Women and Children’s Medical Center, Guangzhou 510623, China. 3 College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China. 4 Key Laboratory for Green Chemical Process of Ministry of Education, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073, China. 5 Department of Food Science and Biotechnology and Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon, Gangwon 200-701, South Korea. 6 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China.


INTRODUCTION
Antibiotics are substances that kill or inhibit the growth of microorganisms.Regarded as one of the greatest contributions to medicine and humanity in the 20th century, antibiotics had been used to treat a wide range of infectious diseases caused by bacteria, for both animals and humans.Microorganisms mediated infectious diseases still remain one of the greatest concerns in public health (Wang et al., 2011(Wang et al., , 2012;;Zhao et al., 2010aZhao et al., , b, c, 2011;;Xu et al., 2011dXu et al., , 2012a, b), b).However, the indiscriminate use of existing antibiotics leads to the emergency of antibiotic resistance and poses a dilemma for the future treatment of bacterial infection, with an increasing number of clinical failures in bacterial mediated

INTEGRON
In recent years, the role of integrons as a mobile genetic mechanism in horizontal transfer of antibiotic resistance has been well established (Stokes and Hall, 1989;Hall, 1993Hall, , 1994Hall, , 1995Hall, , 1999)).A complete functional integron platform comprises three elements: 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 (Labbate et al., 2009).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 bp and 41-55 bp to the left of the cross-over point, respectively (Fluit andSchmitz, 1999, 2004;Francia, 1999;Hall, 1999;Segal , 1999).The attC sites comprise a family of diverse sequences which are not highly conserved and vary considerably in size from 57 to 141 bp (Collis et al., 1993;Collis and Hall, 1995;Mazel et al., 2000;Recchia and Hall, 1995).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 (Fluit and Schmitz, 2004;Francia, 1999).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 (Collis and Hall, 1995;Stokes et al., 1997).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 (Fluit and Schmitz, 1999).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 (Labbate et al., 2009).They contain a coding sequence, but are usually lack of promoters to constitute the mobile component of the system (Hall, 1993;Recchia and Hall, 1995;Rowe-Magnus and Mazel, 2001;Stokes et al., 1997;Sundstrom, 1998).Mostly gene cassettes encoding antimicrobial resistance 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 -35 and -10 boxes to only 14 nucleotides (Collis et al., 1998).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 (Labbate et al., 2009).The 3'-conserved segment (3'CS) of class 1 integrons possesses the genes qacEΔ1 and sul1, encoding resistance to quaternary ammonium salts and sulfonamides, respectively (Recchia, 1997).
Integrons have been considered to be a mobile genetic element, and its mobility 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 (Boucher Y, 2007;Mazel, 2006).Most of the reported class 1 integrons was 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.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 (Mazel, 2006;Nemergut, 2008).It is conceivable that any ORF can be structured as a gene cassette and crucial 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 (Rowe-Magnus and Mazel, 2001).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 (Labbate et al., 2009).
Several classes of integrons have been identified and distinguished by differences and divergence in the intI sequences, and integron classes 1 to 3 are so-called multi-resistant integron (RIs), which appear to be able to acquire same gene cassettes (Hall RM and CM, 1998).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 (Barker et al., 1994, Mazel et al., 1998, Rowe-Magnus et al., 2001).Nevertheless, up to date, most of the available investigations and studies on integron, had concentrated on class 1 integron with Gram-negative microorganisms.In the last decade, class 4 integron (also known as super integron) was considered to be a major concern on antimicrobial resistance and a significant interest in bacterial genome evolution, but most reports had been limited within the species of Vibrio.Thus, this review is aimed at summarizing the occurrence and prevalence of integrons in bacterial isolates from both clinical setting and environment, with the focus on class 1 integron within Gram-positive bacteria, class 2 and 3 integron.

Class 1 integron
Approximately 9% of the sequenced bacterial genomes contain integrons, and class 1 integron platform is the most ubiquitous among clinical microbes and remains the focus of numerous studies (Barlow RS, 2004, Labbate et al., 2009, Xu et al., 2010, 2011c).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) (Labbate et al., 2009).Class 1 integron is defective for self-transposition, however, the transposons and conjugative plasmids associated can serve as vehicles for the intra-and interspecies transmission of genetic material (Rowe-Magnus and Mazel, 2001).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 (Table 1), respectively (Fluit and Schmitz, 1999).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 (Hall, 1993(Hall, , 1994)).For secondary sites, recombination with attC is more efficient than attI (Hall, 1993(Hall, , 1994)).
Class 1 integron has been commonly 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, and occasionally observed Yu et al. 1303 in Campylobacter jejuni, Providencia stuartii, Serratia marcescens, Stenotrophomonas maltophilia, with a broad distribution of 22-59% (Clark et al., 1999;Nandi et al., 2004;Nesvera J, 1998;Tauch et al., 2002;Arakawa et al., 1995;Lee et al., 2002;Chang et al., 2004;Li et al., 2002;Isabelle Plante, 2003;Ruiz et al., 2004).Regarded as a primary source of antimicrobial resistance genes and reservoirs and exchanging platforms of resistance genes within microbial populations, the role of class 1 integron in the dissemination of antibiotic resistance genes had been well investigated and documented in a large number of gram-negative bacteria (Labbate et al., 2009;Maguire et al., 2001;Martinez-Freijo et al., 1998;Sallen et al., 1995).Nevertheless, investigations on Gram-positive bacteria still remained limited.The first evidence of class 1 integron in Gram-positive bacteria was the detection of a 29-kb plasmid pCG4 associated streptomycin/ spectinomycin resistance determinant from Corynebacterium glutamicum in 1998, which was found to be part of a typical class 1 integron (Nesvera, 1998).The sequence of this integron was almost identical to the InC in P. aeruginosa, and this plasmid exhibited even higher expression ability compare to integron found in E. coli (Nesvera, 1998).
In 2002, an intI1-like gene truncated by the insertion sequence IS6100 had been reported on a 27.8-kb R-plasmid pTET3 from C. glutamicum LP-6, which encodes streptomycin, spectinomycin, and tetracycline resistance (Tauch et al., 2002).Within pTET3, a novel aminoglycoside adenyltransferase gene cassette aadA9 was found, and its protein AAD9 showed 61% identity and 71% similarity to AAD6 of integron In51 from P. aeruginosa.(Nandi et al., 2004).Integronss have been frequently reported in Enterococci bacterium, with the first identification of class 1 integron-related gene, aadA, in E. faecalis strain W4470 (Clark et al., 1999).Lately, class 1 integron was reported in 11 E. faecalis and 2 E. faecium strains, with 3 different types of cassette arrays (dfrA12-orfF-aadA2, dfrA17-aadA5 and aadA2) obtained (Xu et al., 2010), representing the first evidence of class 1 integron in E. faecium.Nevertheless, these aforementioned reports had been limited by the lack of large scale number of clinical microbes and long-term track surveillance.

Name
Genes Accession no.

Class 2 integron
Class 2 integron has an organization similar to that of class 1 but is associated with the Tn7 transposon family, including Tn7 and its derivatives (Tn1825, Tn1826 and Tn4132), and its 3' segment contains five tns genes (tnsA, tnsB, tnsC, tnsD and tnsE) involved in transposon movements (Hall RM, 1993, Senda et al., 1996).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 frequent 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 3 specific and conserved gene cassettes, dfrA1, sat1 and aadA1, which confer resistance to trimethoprim, streptothricin and streptomycin/ spectinomycin, respectively (Hansson et al., 2002, Labbate et al., 2009).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 (Ahmed et al., 2005;McIver et al., 2002;Nemergut DR, 2008;Ramirez et al., 2005a, b;Xu et al., 2009).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 (Hansson et al., 2002;Labbate et al., 2009).
However, its low incidence had been reported from other studies.In Spain, 3 out of 100 (3.0%) E.coli strains revealed the presence of class 2 integron (Vinue et al., 2008).Su et al. reported 4 out of 111 (3.6%) E. coli strains harboring class 2 integron with a classic cassette array dfrA1-sat1-aadA1.In another study conducted on 322 E. coli strains previously isolated from irrigation water and associated sediments at the El Paso, Presidio, and Weslaco regions of the river, only one isolate was detected to contain class 2 integron (Matthew, 2003).In an investigation on antibiotic resistance mechanism in 183 coliforms isolated from ten rivers in northern region of Turkey, 2.7% (5/183) of the coliforms harbored class 1 and class 2 integrons, respectively (Ozgumus et al., 2009).As K. pneumoniae was concerned, the occurrence was low.Li et al. had reported one class 2 integron positive strain from 98 clinical Klebsiella pneumoniae strains (Li, 2007).In P. aeruginosa, clinical surveillance on class 2 integron remained limited.Lately, 23 out of 118 (19.5%) clinical strains were detected positive for class 2 integron, with identical dfrA1-sat1-aadA1 cassette arrays (Xu et al., 2009).As commonly reported, for the internal stop codon in IntI2 and the greater efficiency and looser specificity of IntI1, class 1 integron has shown a far greater occurrence and prevalence than the Tn7-based class 2 integron.The evolutionary success of an integron was determined by two important factors: the resistance cassettes it carries and the host range of the plasmid on which it occurs (Hansson et al., 2002).It is reasonable to presume the transferring of cassettes among different integrons (Hansson et al., 2002).As a Shigella strain was concerned, high incidence of class 2 integron had been reported (Ahmed et al., 2006;Dubois et al., 2007;Pan, 2006).In China, class 2 was found in 100% of S. flexneri (58/58) strains isolated from stool samples of sporadic diarrheic patients from different hospitals between 2005 and 2006, and identical dfrA1-sat1-aadA1 cassette arrays were detected (Zhu, 2011).
In a previous study conducted on 726 isolates, 130 of which harbored class 2 integron and only Acinetobacter baumannii and Enterobacter cloacae strains at a high frequency (Ramírez MS, 2010).In Serratia marcescens, 9 out of 30 (30.0%) were positive for class 2 integron, with identical dfrA1-sat1-aadA1 cassette arrays (Crowley, 2008).Also, class 2 integron had been occasionally reported in Pasteurella aerogenes (One porcine Pasteurella aerogenes strain 343) (Schwarz et al, 2010).The only available report on Gram-positive organisms was the observation of class 2 integron in E. faecalis, with the identical cassette array dfrA1-sat1-aadA1, class 1 integron in E. faecium, representing the first detection of class 2 integron outside of the gram-negative organisms and also the first identification of clinical E. faecalis carrying class 1 and 2 integrons simultaneously (Xu et al., 2010).
Comparing with class 1 integrons, class 2 integrons exhibited low diversity.The presence of a stop codon at amino acid 179 in the class 2 integrase (intI2) leads to the production of a shorter and inactive polypeptide, which was unable to catalyse the recombination reaction (Barlow and Gobius, 2006).As consequence, small number of different gene cassettes observed in class 2 integrons and most reported class 2 integron carry 3 resistance genes, namely, dihydrofolate reductase (dfrA1), streptothricin acetyltransferase (sat1), and aminoglycoside adenyltransferase (aadA1), conferring resistance to trimethoprim, streptothricin and streptomycin/ spectinomycin, respectively.Nevertheless, since the last decade, investigations on the variability of class 2 integrons had identified several novel rearrangements as well as antibiotic resistance genes which were previously unassociated with class 2 integron.An erythromycin esterase gene (ereA) in a class 2 integron had been lately identified, which contained its own promoter and was propagated by a class 2 integron with an insertion sequence element (IS1) upstream of the intI2 gene (Biskri, 2003).
In 2005, Ramirez et al described a novel reaarangement of a class 2 integron (Tn7::In2-8) with new cassettes in the variable region, from 3 isolates of.Acinetobacter baumannii (Ramirez et al., 2005a).The structure of this class 2 integron contained 6 antibiotic resistance genes within the variable region, with 3 additional genes sat2, aadB and catB2 inserted upstream of the 3 conventional antibiotic resistance genes of Tn7 class 2 integrons.Other novel cassette arrays of class 2 integron (Tn7:In2-1 in B. cenocepacia strain and an unusual array sat-sat1-aadA1 in S. enteritidis) had also been observed and identified (Ramirez et al., 2005b) However, the mechanism and benefit of such novel cassette arrays still remained unclear.

Class 3 integron
Class 3 integron contains a comparable structure to that of class 2 integron, and 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 has been demonstrated to catalyze excision of integrated cassettes and integration of circularized cassettes into the attI3 site, but the recombination between a 59-be and secondary sites occurred at frequencies significantly lower than observed with IntI1 (Collis et al., 2002).In addition, IntI3 is able to recognize different attC sites, and integrate the cassettes into the attI3 site which was localized to a short region adjacent to intI3 (Collis et al., 2002).

Class 4 integron: Super integron
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 pre-dating the antibiotic era (Rowe-Magnus et al., 1999).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 (Rowe-Magnus and Mazel, 2001).

CONCLUSION
As a common genetic element existed in 9% of bacteria and representatives from a broad range of phyla and environments, integrons play the key role in antibiotic resistance among clinical organisms and contributes to the evolution and adaption of bacteria.Of the available reports conducted on integrons, most of them have been restricted to class 1 integrons in Gram-negative bacteria.Nevertheless, class 1 integrons in Gram-positive bacteria, as well as class 2 and 3 integrons have barely been touched upon, which may potentially be unnoticed and neglected antibiotic resistance determinants.Many questions have been arisen and posed regarding the species of involved microorganisms, distribution and spread of integrons and cassettes arrays, occurrence and prevalence of integrons in certain species of bacteria.This Yu et al. 1307 review aimed at summarizing the occurrence and prevalence of integrons in bacterial isolates from both clinical setting and environment, with the focus on class 1 integron within Gram-positive bacteria, class 2 and 3 integron.
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 such 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)

Table 1 .
Summary of different structures of class 2 integrons.
. Xu et al identified 3 out of 16 multidrug-resistant strains of known or opportunistic gram-negative bacterial pathogens to be positive of class 2 integron from the Salmon River in south-central British Columbia from 2005 to 2009