The Complex Genetic Context of bla PER-1 Flanked by Miniature Inverted-Repeat Transposable Elements in Acinetobacter johnsonii

On a large plasmid of Acinetobacter johnsonii strain XBB1 from hospital sewage, bla PER-1 and ISCR1 were found in a complex Tn402-like integron carrying an arr3-aacA4 cassette array. The integron was truncated by the same 439-bp miniature inverted-repeat transposable element (MITE) at both ends. bla PER-1 and its complex surroundings might have been mobilized by the MITEst into an orf of unknown function, evidenced by the presence of the characteristic 5-bp direct target repeats. The same 439-bp MITEs have also been found flanking class 1 integrons carrying metallo-β-lactamases genes bla IMP-1, bla IMP-5 and bla VIM-2 before but without ISCR1. Although the cassette arrays are different, integrons have always been truncated by the 439-bp MITEs at the exact same locations. The results suggested that MITEs might be able to mobilize class 1 integrons via transposition or homologous recombination and therefore represent a possible common mechanism for mobilizing antimicrobial resistance determinants.

Introduction bla PER-1 encodes the extended-spectrum b-lactamase (ESBL) PER-1 conferring resistance to penicillins, cephalosporins and monobactams [1] and has been found in Aeromonas spp., Acinetobacter baumannii, Alcaligenes faecalis, Pseudomonas aeruginosa and the Enterobacteriaceae in Asia and Europe [2,3]. The production of PER-1 by Gram-negative bacilli of clinical significance compromises the option for antimicrobial chemotherapy. It has been well established that the mobilization of antimicrobial resistance determinants such as ESBL-encoding genes is largely mediated by mobile genetic elements including insertion sequences, transposons, integrons and gene cassettes. Recently, miniature inverted-repeat transposable elements (MITEs), which are small non-autonomous mobile elements containing repeated sequences and are present in diverse bacteria [4], have also been found mediating the mobilization of antimicrobial resistance determinants [5]. bla PER-1 has been found in diverse genetic contexts and is generally located downstream of the insertion sequence ISPa12 [2]. However, bla PER-1 had not been found associated with MITEs before [2].
Acinetobacter johnsonii (Acinetobacter genospecies 7) is a bacterial species that has usually been found in the aquatic environment [6] but occasionally colonizes humans [7] or causes clinical infections [8]. A strain of A. johnsonii was recovered from hospital sewage in western China and was found carrying bla PER-1 . Here the genetic context of bla PER-1 was examined in detail.

Materials and Methods
Strain and the Detection of bla PER A. johnsonii XBB1 was obtained from sewage collected at the influx of the wastewater treatment plant in West China Hospital, Chengdu, China, on October 2010 and was identified as A. johnsonii by partially sequencing the 16S rRNA gene [9]. No specific permissions were required for the activities to obtain the sewage as the wastewater treatment plant is a closed unit in the hospital and the field studies did not involve endangered or protected species. A. johnsonii XBB1 was resistant to ceftriaxone and ceftazidime [9] and the presence of bla PER gene was detected using PCR with self-designed primers (PER-UF, 59-CCTGAC-GATCTGGAACCTTT; PER-UR, 59-TCATCGASGTC-CAGTTTTGA). PCR was performed using ExTaq premix (Takara, Dalian, China) and the following conditions: 94uC for 5 min; 30 cycles of 94uC for 30 s, 55uC for 30 s, 72uC for 1 min; and a final elongation step at 72uC for 7 min.

Genome Sequencing and Assembly
A. johnsonii XBB1 was subjected to the whole genome sequencing. The genomic DNA of XBB1 was prepared using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany) and was then sequenced using the Roche 454 GS FLX+ (Roche 454 Life Sciences, Branford, CT, USA) and the Illumina Paired-end (Illumina, San Diego, CA, USA) platforms at the Beijing Genomics Institute (Beijing, China). Reads containing at least 50-bp non-adapter non-primer sequence generated by the 454 platform were assembled using the Newbler program (version 2.6) using 40 bp as the minimum overlap length and 90% as the minimum overlap identity. The assembled sequences were further assembled with reads generated by the Illumina platform using the Sspace program (version 2.0). The sequence gap of the plasmid carrying bla PER-1 was filled in using PCR with primers designed based on sequences available and Sanger sequencing using an ABI 37306l DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Similarity searches of sequences obtained were carried out using BLAST programs (http://www.ncbi.nlm.nih.gov/BLAST/) and the function of proteins was predicted using the InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/).

Verification of the Genetic Context of bla PER-1
To confirm the genetic context of bla PER-1 , two overlapping long-range PCR were employed with primer pair PER-UF/ DWMITE-F1 and PER-UR/UPMITE-R1. Self-designed primers DWMITE-F1 (59-TGGCTCAATGTCTGATTGCT) and UP-MITE-R1 (59-TTGTTTGGGATTTGGTCCTC) were located 141 and 166 bp away from each end of the 15.3 kb large region containing bla PER-1 , respectively ( Figure 1). The conditions of longrange PCR (Fermentas, Burlington, ON, Canada) were 94uC for 2 min; 10 cycles of 94uC for 10 s, 55uC for 30 s, 68uC for 12 min; then 25 cycles of 94uC for 15 s, 55uC for 30 s, 68uC for 6 to 12 min plus 10 s cycle elongation for each successive cycle; and a final elongation step at 68uC for 7 min. The amplicons were sequenced at both directions (see above).
Nucleotide sequences accession number. Sequence of the genetic context of bla PER-1 in A. johnsonii XBB1 has been deposited in GenBank as KF017283.

Results and Discussion
The genetic region containing bla PER-1 in XBB1 generated by assembling whole genome sequencing reads was verified by the overlapping long-range PCR and Sanger sequencing. In XBB1, bla PER-1 was carried by a 399 kb large plasmid, which had been completely sequenced, assembled and circularized. bla PER-1 was found downstream of the insertion sequence ISCR1 in a 5.7 kb region that included gts (encoding a glutathione S-transferase), abct (encoding an ABC-type transporter) and four genes of unknown function in a complex Tn402-like class 1 integron with the arr3 and aacA4 cassettes ( Figure 1). The ISCR1-bla PER-1 structure was bracketed by two copies of 39 conserved segment (39CS), which comprised a truncated qacE gene encoding resistance to quaternary ammonium compounds and the sulphonamide resistance gene sul1. The 59 conserved segment (59CS) and the Tn402-like tni transposition module (tniBD1-tniA) were truncated by the same 439-bp MITE) (Figure 1). The two MITEs, which were in the same direction, might have formed a composite transposon-like mobile element mediating the transposition of the complex Tn402-like class 1 integron containing ISCR1 and bla PER-1 into a gene of unknown function as evidenced by the presence of characteristic 5 bp (GTTGC) direct target repeats (DR, Figure 1).
This MITE is AT-rich (AT content, 72.2%), has no orf and possesses terminal sequences without significant matches to inverted repeats of any known insertion sequences and transposons. The same (100% nucleotide identity) 439-bp MITEs have also been found flanking Tn402-like class 1 integrons carrying different cassette arrays but without ISCR1 in several Acinetobacter strains (Figure 1). In A. johnsonii strain NFM2 that was recovered from a prawn in Australia [10], the 439-bp MITEs flanked an unusual class 1 integron that carried methionine sulfoxide reductase genes and this MITE-flanked element was inserted into a location exactly the same as that seen in the present study. In A. baumannii clinical strain 65FFC, MITEs flanked a class 1 integron containing a single bla IMP-5 cassette and this MITE-flanked structure was transposed into the transposase-encoding tnpA gene of an ISAba14-like element, generating 5-bp (TCCAT) DR [5]. In Acinetobacter bereziniae strain 118FFC (GenBank accession number JX235356), a class 1 integron with the aacA7-bla VIM-2 -aacC1 cassette array was flanked by MITEs, which were mobilized into the transposase-encoding tnpA gene of IS26 evidenced by the presence of 5-bp (TCAAT) DR (Figure 1). Partial MITEs have also been found to truncate the 59CS of a class 1 integron carrying the bla IMP-1 -aac(69)-31-aadA1 cassette array in A. baumannii strain 48-501A (AM283490) and strain 48-696D (AJ640197). Only partial MITE sequence is provided for strain 48-696D, while MITE is truncated by IS1006 in strain 48-501A. No sequences downstream of 39CS are available for both strains and therefore it remains unknown whether there is another copy of MITE at the other end of In86. bla IMP-1 , bla IMP-5 and bla VIM-2 are genes encoding class B metallo-b-lactamases. Of note, the MITE-flanked structures containing Tn402-like integrons have been seen interrupting different genes with the presence of DR, suggesting that the MITEs indeed mediated the mobilization of integrons. Interestingly, MITE always truncated the 59CS and tniA at the same locations in all known sequences regardless of the cassette array and host species or strains, suggesting that the truncation of Tn402-like integrons by MITEs at either ends might have only occurred once and the different cassette arrays might therefore have resulted from homologous recombination between integrons. The association of MITEs with a complex class 1 integron containing ISCR1 was not seen before and the ISCR1-bla PER-1 region seen in the present study might have also been introduced by homologous recombination with the 59CS or 39CS serving as one homologous region and the tni module serving as the other required for double crossover. There is also evidence that circular molecules created by recombination in the duplicated 39CS flanking regions containing ISCR-antimicrobial resistance gene [11]. Although the MITEs-flanked structures seen in strain NFM2 from Australia and A. johnsonii XBB1 from China had different genetic components, they were present at the same location, suggesting that the acquisition of the ISCR1-bla PER-1 region might have occurred later than the transposition mediated by MITEs. As mobile genetic elements, MITEs might be able to mobilize bla PER-1 into different plasmids within the host strain and then could generate various genetic scaffolds to facilitate the horizontal transfer of the ESBL gene between clinical isolates and those of an environmental origin. The presence of bla PER-1 within the mobile genetic element formed by MITEs from an environment isolate in a hospital setting is therefore of significance.
To date, bla PER-1 has been found in genetic contexts either associated with the insertion sequence ISPa12 or with ISCR1 (Table 1). ISPa12 and its close relative ISPa13 formed a composite transposon termed Tn1213, which realized the mobilization of bla PER-1 and a truncated gts remnant as evidenced by the presence of characteristic 8-bp DR. Several variants of Tn1213 have also been identified (Table 1), including IS6100 or ISPpu17 truncating ISPa12 and ISPrst1 inserting into Tn1213 [2,12,13]. Tn1213 has been found in various species in Europe and Asia and therefore appears to be a common mechanism mediating the mobilization of bla PER-1 . In particular, Tn1213 and its variants were the only type genetic context of bla PER-1 identified in P. aeruginosa so far. Another type of association of ISPa12 with bla PER-1 has been seen in A. baumannii strain C.A. and Salmonella enterica, in which the insertion of ISPa12 at 57 bp upstream of bla PER-1 was independently of the bla PER-1 acquisition as the presence of the characteristic 8-bp DR flanking ISPa12 suggested transposition of ISPa12 on its own [2]. In these cases, the mechanisms for the mobilization of bla PER-1 remain undetermined. The association of ISCR1 with bla PER-1 has been found in two A. baumannii isolates ( Table 1) before but no further sequence data were available to identify the integrons in which ISCR1 and bla PER-1 might be embedded and to demonstrate whether MITEs were also involved like the case seen in the present A. baumannii strains 7 and 8 Belgium 13 [16] Aeromonas media A72 Switzerland 13 [3] Klebsiella pneumoniae CS1711 Korea 13 [17] P. The bla PER gene in Aeromonas punctata strain 169 (GenBank accession number GQ871757) has also been annotated as bla PER-1 but it has two nucleotide differences from bla PER-1 (Z21957), specifying one amino acid substitution compared to PER-1. Therefore, this bla PER gene was not included. b ?, sequence remains unknown. study. Of note, there are two spacer sizes between ISCR1 and bla PER-1 , suggesting that the acquisition of bla PER-1 by ISCR1 might have occurred more than once. As the spacer between ISCR1 and bla PER-1 was longer than that between ISPa12 and bla PER-1 and gts was truncated in Tn1213, it might be reasonable to propose that the acquisition of bla PER-1 by ISPa12 is a more recent event when compared to the acquisition by ISCR1. Both Tn1213 and the ISCR1-bla PER-1 context have been identified in A. baumannii, suggesting that A. baumannii might be the host species in which different platforms for mobilizing bla PER-1 had been formed.

Conclusion
A unique MITE-flanked complex Tn402-like integron carrying ISCR1 and bla PER-1 was revealed in an A. johnsonii strain from hospital sewage. Such a complex integron flanked by a pair of 439bp MITEs has never been reported before. The 439-bp MITEs might be able to mobilize class 1 integrons via direct transposition or homologous recombination. It appears that MITEs could serve as a common mechanism mediating the mobilization of antimicrobial resistance genes. This study has prompted an additional survey for the presence of MITEs in clinical isolates of Acinetobacter spp.