Molecular analysis of the integrons of metallo-β-lactamase-producing Pseudomonas aeruginosa isolates collected by nationwide surveillance programs across Japan

We investigate the evolving molecular epidemiology of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa isolates collected in a 100 institution, nationwide surveillance study in Japan from 2004 to 2006. MBL-producers were detected in 23/996 isolates (2.3%) in 2004 and 21/992 (2.1%) in 2006. Antimicrobial resistance (specifically, carbapenem resistance) rates between two periods did not differ significantly. MBL-producers were more prevalent in urinary tract isolates. blaIMP-1 group was the most predominant (38 isolates, 80%), followed by 3 blaIMP-7, 2 blaIMP-11 group, and 1 blaVIM-1. All MBL genes were identified in 16 different class 1 integrons, most of which were novel to INTEGRALL database. A total of 17 isolates of sequence type (ST) 235, a recognized worldwide drug-resistant lineage, were distributed in 5 geographic regions across Japan. ST235 isolates included a sublineage associated with In113-like integron. ST357 was identified in 14 isolates, 9 of which harboring a sole blaIMP-1 gene cassette (In994) were recovered from Chugoku region in 2004. ST357 isolates with blaIMP-11 group or ST235 with blaIMP-7 emerged in 2006. We also report for the first time the presence of novel fosI gene cassette in strains other than Mycobacterium spp. Our data give an important “snapshot” of the molecular characteristics and dynamics of MBL-producing lineages in P. aeruginosa in Japan. The significant association of specific genotypes and integrons implies that dissemination and transmission of the preexisting resistant lineage, rather than horizontal gene transfer in situ, might largely explain their endemicity.


Background
As carbapenems are among the most reliable therapeutic options for treating Pseudomonas aeruginosa infection, the finding of carbapenem resistance is an ominous development that challenges this "last resort antibiotic". Production of metallo-β-lactamases (MBLs) is an important mechanism of carbapenem resistance, not only because MBLs can hydrolyse most β-lactams including carbapenems, but MBL-producing P. aeruginosa are frequently also multidrug-resistant (resistant to ≥ 3 classes) [1]. More importantly, bla MBL genes are often located in the gene cassette (GC) arrays in integrons, where multiple resistant determinants against antimicrobials or disinfectants are accumulated, and can be horizontally transferred [2].
Recently, the INTEGRALL database was developed for providing a systematic nomenclature for integrons [12]; these genetic elements can be potentially shared by a wide range of microorganisms as a result of horizontal gene transfer [13]. Moreover, GC arrays of integrons could be excised or integrated by the action of integrases and therefore be more diverse [2]. Therefore, INTE-GRALL is expected to facilitate our comprehensive understanding of the complexity and development of integron-mediated drug resistance.
In the current study, we conducted a molecular epidemiological survey of the genetic context of MBLproducing P. aeruginosa isolates collected by nationwide surveillance. Our data not only provide an important "snapshot" of the molecular characteristics and dynamics of MBL-producing predominant lineages including ST235 and ST357, but also provide important baseline information for future surveillance programs. (This work was presented in part at the 51st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2011.)

Results and discussion
Antimicrobial susceptibility of MBL-producing P. aeruginosa isolates In the present study, 44 MBL-producing P. aeruginosa isolates were identified (Table 1); these were recovered from 6 out of 8 geographic regions across Japan. The number of isolates is comparable in 2004 and in 2006. MBLproducers were isolated more frequently from urinary tract specimens than from other specimens (24/408 vs. 20/1580; P < 0.0001). Although this trend was consistent with the previous reports in Japan [23], the reason for preference of MBL-producers for urinary tract was not addressed in this study where we did not analyse non-MBL-producers.
All MBL-producers met the international criteria for MDRP (Table 2). Overall, the antibiotic resistance patterns of MBL-producers did not differ significantly among the isolates between 2004 and 2006, except in cases where piperacillin was tested (4/23 resistant to piperacillin in 2004 vs 16/21 in 2006; P = 0.0002). Although this result reinforces the impact of the MBL production in MDRP, their limited susceptibility to the entire armamentarium of β-lactams including piperacillin or aztreonam, which MBLs are expected to hydrolyse less efficiently, suggests the involvement of multiple factors contributing to their drug resistance [24].
Acknowledging that CLSI breakpoints were not available, the majority of MBL-producers showed increased susceptibility to arbekacin (MIC >8 μg/ml in only 5 strains, 11%). Although further investigation is needed, this agent might be a candidate for consideration as a therapeutic agent [25]. For more than two decades in Japan, arbekacin is a commercially available aminoglycoside effective against methicillin-resistant Staphylococcus aureus infections and its use in Gram negative infections is being explored .
All MBL-producing isolates possessed intI1, and all MBL genes were located in the GC arrays of class 1 integron (Table 2); 16 distinct MBL-harboring integrons were identified. Complete GC arrays of all integrons except one were successfully sequenced, as many as 13 of which were novel to INTEGALL database. Fifteen isolates harbored In113-like integrons, which consisted of 3 GCs, namely bla IMP-1 group cassette (bla IMP-1 in In113, shown as a number of metallo-β-lactamase-producing P. aeruginosa isolates divided by a number of all P. aeruginosa isolates.  bla IMP-6 in In915, and bla IMP-10 in In829) followed by aacA28 and aacA1a GCs. Similarly, thirteen isolates harbored In994-like integrons which contains a sole bla IMP-1 group cassette (bla IMP-1 in In994 and bla IMP-10 in In995). The family of bla MBL genes and their position in integron GC arrays did not show significant differences in resistance profile ( Table 2).
The significant association of specific genotypes and integrons implies that, in a relatively short period of time, dissemination and transmission of the preexisting resistant lineage, rather than horizontal gene transfer or recombination in situ, might largely explain their endemicity. Moreover, the genotypes of MBL-producers were unexpectedly similar to those in the central Europe with different resistant determinants, suggesting their possible advantage beyond other genotypes in the clinical settings [4]. Although the isolate number was small, the present study elucidated the emergence of ST357 isolates with novel integrons in distant geographic regions in 2006, as well as an emergence of the ST235/ pulsotype C with partial integron harboring bla IMP-7 in Kanto region, which might suggest the increased complexity of endemic lineages. In addition, isolates with ST175 was recovered in Kyushu with significantly higher frequency (3/3 vs 5/41, P = 0.0042).
One of the limitations of the present study is that this is retrospective in nature and therefore the results are not expected to be directly applied to the current clinical situation. There are also several reports in Japan addressing a variety of integron structures of P. aeruginosa [15][16][17][18][19][20], and they seemed to contain identical or similar integrons to ones identified in the current study. Nevertheless, the findings here are important, since most of the integron structures were novel to INTEGRALL database [12]; this could be explained by the lack of comprehensive platform for integron structure and nomenclature before the development of INTEGRALL database until recently. Alternatively, this might reflect true heterogeneity and diversity of the integrons among clinical isolates, especially among non-predominant lineages. Moreover, MLST comparison of cross-sectional nationwide survey is much informative for understanding of the dynamics of drug-resistant organisms. In addition, this is the first report of the presence of novel fosI gene cassette other than Mycobacterium spp. [26], and further analysis regarding the actual transfer route beyond the boundary of the species is warranted. As genotyping including MLST and INTEGRALL has been increasingly applied for tracking the development of the drug resistance rationally, the data in the present study would provide a robust baseline to estimate the current trend and future diversification of the drug-resistant lineages [20]. Another shortcoming of this study is that horizontal gene transfer of bla MLB genes, possibly located on plasmid or chromosome, was not experimentally addressed.

Conclusions
The current study offers an important "snapshot" of the molecular characteristics and dynamics of MBL-producing P. aeruginosa lineages including ST235 and ST357 across Japan and provide critical information for the design and implementation of surveillance programs targeted to the discovery of multidrug resistant P. aeruginosa.

Bacterial isolates
A total of 1,988 P. aeruginosa isolates (996 isolates in 2004 and 992 in 2006), were retrospectively collected as a part of nationwide surveillance programs in Japan (Japan Antimicrobial Resistance Surveillance Participants Group) [27,28]. Briefly, up to 10 non-duplicated patient isolates of 10 organism groups including P. aeruginosa were collected for three months in 2004 and in 2006 from a total of 100 institutions in 8 geographic regions. MBLproducers were phenotypically identified by the doubledisk synergy test using ceftazidime (30 μg) and sodium mercaptoacetate (30 μg) disks (Eiken Chemical, Inc., Tokyo, Japan) following the manufacturer's instructions, and were subjected to further molecular epidemiological investigation [29]. Ethical approval has been obtained from Toho University School of Medicine Ethics Committee (2511925030).
MDRP was defined as strains with non-susceptibility to at least 1 agent in ≥3 antimicrobial categories according to the international expert proposal [32]; amikacin as aminoglycosides, imipenem as antipseudomonal carbapenems, ceftazidime or cefepime as antipseudomonal cephalosporins, aztreonam as monobactams.

Analysis of MBL genes and integron by PCR amplification and nucleotide sequencing
Genomic DNA was prepared by the Wizard genomic DNA preparation kit (Promega, Madison, WI, USA) according to the manufacturer's recommendations. PCR was performed with Takara ExTaq (Takara Bio, Shiga, Japan). Nucleotide sequencing was performed by a commercial DNA sequencing service (Greiner Bio-one, Tokyo, Japan).
bla MBL genes were detected and identified by PCR and nucleotide sequencing using primers for bla IMP and bla VIM [33]. bla IMP-1 , bla IMP-10 , and bla IMP-6 are referred as bla IMP-1 group because the deduced amino acid sequences of bla IMP-1 differ by single amino acid from those of bla IMP-10 and bla IMP-6 . Similarly, bla IMP-11 and bla IMP-41 are as bla IMP-11 group because the deduced amino acid sequences differ by single amino acid. Integrase genes specific for class 1, 2, or 3 integrons were detected by PCR using primers by Shibata et al. [33]. Integron GC arrays were determined by PCR and nucleotide sequencing by primer walking [34]. According to INTEGRALL nomenclature, novel completely sequenced GCs, namely from a part of 5′-conserved segment (5′CS) to a part of 3′-conserved segment (3'CS), were submitted to INTEGRALL for the attribution of new integron numbers [12].
Genotyping MLST was performed as described by Curran et al. [3]. Pulsed-field gel electrophoresis (PFGE) of SpeI-digested DNA fragments from each strain was performed on a CHEF-MAPPER II apparatus (Bio Rad Laboratories, Rockland, Maine, USA) at 6 V/cm, for 19.7 h with pulse times running from 5.3 to 34.9 s in 0.5 × TBE buffer at 14°C. PFGE patterns in the dendrogram were analyzed with Molecular Analyst Software Fingerprinting II (Bio-Rad Laboratories). Only restriction fragments larger than 50 kb were used for analysis. PFGE genotypes were distinguished by the unweighted pair-group method using average linkages (UPGMA) and Dice coefficient, and isolates with ≥80% similarity were assigned into the same pulsotype.

Statistical analyses
Statistical calculations were made using Graph-Pad Prism 5.01 (GraphPad Software Inc.). P values were calculated from two-tailed Fisher's exact test for categorical variables, and P value <0.01 was considered as a statistically significant difference.

Nucleotide sequences accession numbers for novel integrons
Genbank/EMBL/DDBJ accession numbers for novel nucleotide sequences in the present study are as follows: AB901034-AB901041 for In826-In833, and AB901042-AB901046 for In991-995, and AB901047 for partial integron of isolate TUM3995.

Competing interest
The authors declare that they have no competing interests.
Authors' contribution YM participated in the design of the study, carried out the experiments, and drafted the manuscript. TS participated in the design of the study, interpreted data, and revised the manuscript. YI conceived and designed the study, and revised the manuscript. AY participated in the design of the study, carried out the experiments, and drafted the manuscript. RAB, KY, and KT designed the study, and revised the manuscript. All authors read and approved the final manuscript.