New structures simultaneously harboring class 1 integron and ISCR1-linked resistance genes in multidrug-resistant Gram-negative bacteria

Background The connection structure of class 1 integron and insertion sequence common region 1 (ISCR1) is called “complex class 1 integrons” or “complex sul1-type integrons”, which is also known to be associated with many resistance genes. This structure is a powerful gene-capturing tool kit that can mobilize antibiotic resistance genes. In order to look for and study the structure among clinical multidrug-resistant (MDR) Gram-negative isolates, 63 isolates simultaneously harbored class 1 integron and ISCR1-linked resistance genes were isolated from 2309 clinical non-redundant MDR Gram-negative isolates in Nanfang Hospital in 2008–2013. The connecting regions between the class 1 integrons and ISCR1 were examined using PCR and DNA sequencing to determine the structures in these isolates. Result The two elements (the variable regions of the class 1 integron structures and the ISCR1-linked resistance genes) are connected in series among 63 isolates according to long-extension PCR and DNA sequencing. According to the kinds and permutations of resistance genes in the structure, 12 distinct types were identified, including 8 types that have never been described in any species. Several types of these structures are similar with the structures of other reports, but not entirely same. Conclusion This study is the first to determine the structure simultaneously harboring class 1 integron and ISCR1-linked resistance genes by detecting the region connecting class 1 integrons and ISCR1 in a large number of MDR bacteria. These structures carrying various resistance genes were closely associated with multidrug resistance bacteria in Southern China.


Background
The increasing use of antimicrobial agents to treat Gramnegative bacterial infections has led to an increase in antibiotic resistance. Consequently, formerly routine therapies for many infectious diseases caused by multidrug-resistant (MDR) Gram-negative bacteria are now compromised. MDR bacteria evolve relatively quickly because the main driving force is lateral gene transfer, which is facilitated by a wide range of mobile genetic elements. The majority of these elements are integrons and transposons (including unit transposons and insertion sequences) [1]. Insertion sequences with common regions (ISCRs) are a type of insertion sequence.
In two previous studies, a total of 1329 and 1447 multidrug Gram-negative bacteria isolated in 2008-2009, was investigated for an ISCR1 [2] and a class 1 integron [3] respectively. In this study, 2309 clinical non-redundant MDR Gram-negative isolates were isolated between 2008 and 2013 at Nanfang Hospital, a 2200-bed tertiary-level teaching hospital in Guangzhou, China. Here, strains which carry a physical linkage between class 1 integrons and ISCR1 were focused. The ISCR1 and class 1 integrons were characterized using PCR and DNA sequencing as the methods described previously [2,3]. Of these strains, 63 isolates simultaneously harbored class 1 integrons and ISCR1-linked resistance genes were selected. The results were shown on the Table 1. Based on the results, the region connecting the ISCR1 and the 3′-CS of the integron and the overall structures were investigated.
This structure which is usually called "complex class 1 integrons" or "complex sul1-type integrons", [4] is the large genetic element in which different class 1 integrons is associated with ISCR1. These elements are known to be associated with many resistance genes, encoding resistance to chloramphenicol, trimethoprim, quinolone, and β-lactam, [5] and have two notable structures. Besides, this structure is a powerful gene-capturing tool kit that can mobilize antibiotic resistance genes. The most of their structure comprises a typical class 1 integron with a 5′ conserved segment (5′-CS), a 3′-CS, and an intervening variable region (VR1), followed by a copy of ISCR1 and then by an ISCR1-linked resistance gene region (VR2), which accommodates a variety of resistance genes. This region is, in turn, followed by a repetition of the 3′-CS [6,7].

Methods
According to the previous study, sixty three isolates simultaneously harbored class 1 integrons and ISCR1-linked resistance genes were selected for further analysis of the connecting the ISCR1 and the 3′-CS of the integron.
According the hypothesis connecting structure (Fig. 1) between the ISCR1 and the 3′-CS of the integron, primers A and B were designed to amplify the 2045-bp fragment at the junction between ISCR1 and the 3′-CS of the class 1 integrons and used to preliminarily confirm that ISCR1 was inserted downstream from the sul1 gene in the class 1 integrons. Primers I and X, which are specific for the VR1 and VR2 resistance genes investigated in this study, were used to identify the connecting regions: downstream of class 1 integrons and upstream of ISCR1. The amplification products of primers I and X were used as the templates in subsequent PCR analyses. Five set primers ( Fig. 1) were used to verify this region with the ISCR1 and the class 1 integrons connected in series.
Primers of the connection region of integron and ISCR1 are listed in Table 2 and were synthesized by Beijing Genomics Institute (Shenzhen, China). PCR amplifications Table 1 Characterisation of complex class 1 integron in multidrug-resistant Gram-negative bacteria and resistance profiles of sequenced strains

Species
No. of isolates Class 1 integron ISCR Type of complex class 1 integron
In this study, the structures (the variable regions of the class 1 integron structures and the ISCR1-linked resistance genes) are connected in series among 63 isolates. We found 12 distinct structures connecting the ISCR1 and the class 1 integron, with a different genecassette variable regions, composed of the 5′-CS and the 3′-CS but displaying another unique variable region located between ISCR1 and the second copy of the 3′-CS (Fig. 2). The PCR products amplified from the regions connecting the first 3′-CS and ISCR1 are shown in Fig. 3.

Discussion
To the best of our knowledge, this is the first study to investigate the structure connecting the ISCR1 and the 3′-CS of the integron in different species in clinical isolates of MDR Gram-negative bacteria on a large scale. It was found that 63 isolates simultaneously carried class 1 integrons and ISCR1. The variable regions of the class 1 integron structures to the ISCR1-linked resistance genes were linked successfully using long-extension PCR, suggesting that these two structures are connected in series.
Of the 12 type structures, 8 types (A, C, F, H, I, J, K and L type) were first found in any species according to Fig. 1 The hypothesis connecting structure between the ISCR1 and the 3′-CS of the integron. a Integrase were amplified by PCR using primer pairs intI1-F and intI-R. class 1 integrons were amplified by 5′CS and 3′CS.ISCR1 were amplified by PCR using primer pairs ISCR1-F and ISCR1-R. ISCR1-linked genes were amplified by PCR using primer pairs ISCR1-F and sul1-R. b Lengths of the PCR products obtained with primers A and B. c Lengths of the PCR products obtained with a series primers (I-X)   the systematic search in PubMed and GenBank. The structure type K is similar to a structure In37::ISCR1::qnrA1 (accession No. AY259086) [8]. A genetic structure (type B), [aadB + aadA2]:ISCR1:[qnrA1 + ampR], is the same as a structure already reported In293::ISCR1::qnrA1 in E. cloacae, [9] which was also found in E. coli, K. pneumoniae, Acinetobacter spp., P. aeruginosa and S. maltophilia in this study. We detected the structure D type, [dfrA25]:ISCR1:[sapA-like + qnrB2], that was previously found and described in Salmonella isolates [10], but which has never before been described in E. coli or K. pneumoniae. The structure G has been detected in clinical K. pneumoniae (accession No. JF775516) isolates in a previous study [11] [13]. About structure H, I and J, Ruirui Xia et al. described similar structures carrying bla PER-1 and qnrVC-like genes and made an exhaustive study [14].
Enterobacteriaceae strains carrying the structures are becoming more common [15][16][17]. In this study, nine distinct structures were identified among Enterobacteriaceae strains, including seven distinct structures connecting the ISCR1 and the class 1 integron that have never been described in any species. It should be noted that only four distinct structures were identified in MDR nonfermenting isolates. One possible explanation is that chromosomal resistance mechanisms, such as efflux pumps, are more common than laterally transferred genetic resistance factors in these genera in this bacterial population.

Conclusions
This is the first study to describe the structure connecting the ISCR1 and the class 1 integron in clinical MDR Gram-negative bacterial isolates in a large-scale study. In total, 12 distinct structures were described. Several types of these structures are similar with the structure of other reports, but not entirely same. This structure is a powerful gene-capturing tool that can mobilize antibioticresistance genes. Therefore, the structural analysis of the structure connecting the ISCR1 and the class 1 integron could guide treatment strategies and provide directions for future research into the mechanisms of bacterial antibiotic resistance.

Nucleotide sequence accession number
The nucleotide sequences of the structure A to L in this work have been submitted to the GenBank database and assigned accession No. JX880393, JX880383, KM111274, JX880388, KM111278, KM111276, KM111280, JX880386, KM111273, KM111272, KM111271 and KM111275.