LAUSR

Sir, The gene encoding NPS-1 b-lactamase (blaNPS-1) was first reported on a self-transmissible plasmid of clinical isolates of Pseudomonas aeruginosa. The enzyme can hydrolyse carbenicillin, azlocillin, cefoperazone and cefsulodin, but cannot effectively hydrolyse more stable b-lactams (cefotaxime, ceftazidime, ceftriaxone, monobactam and imipenem). Furthermore, blaNPS-1 was encoded on a mobile element of a large conjugative plasmid (pB4) isolated from an activated sludge, which shows that blaNPS-1 may rapidly transfer between different species from different sources. In the current study, we report an NPS-1-associated Tn3-like complex transposon in a microbial keratitis isolate of P. aeruginosa. An MDR P. aeruginosa strain (PA34) was isolated from a microbial keratitis patient in India (MIC in Table S1, available as Supplementary data at JAC Online). WGS was performed using MiSeq (Illumina, San Diego, CA, USA) generating 300 bp paired-end reads and a Nextera XT DNA library preparation kit (Illumina) was used to prepare the library. CLC Genomics Workbench 10.0 (QIAGEN Bioinformatics, Aarhus, Denmark) was used to assemble the raw reads. A total of 2 365 558 trimmed reads were assembled into 75 contigs .10 kb with an average coverage of 85%. The nucleotide sequence of the transposon is available under the GenBank R accession number MF487840. Itinerary BLASTn R has revealed that the draft genome of PA34 carries a class 1 integron that is located within a Tn3-like transposon. The integron has a locus containing two gene cassettes comprising genes for trimethoprim (dfrA15) and chloramphenicol (cmlA1) resistance. Analysis of the nucleotide sequence flanking these two cassettes revealed important characteristics of gene cassettes which included attC sequences downstream of the dfrA15 and cmlA1 genes. See Figure 1(a and b). The dfrA15 of PA34 is identical to that of many enteric bacteria, suggesting this may be their source of origin. Furthermore, the cmlA1 gene was similar to a chromosomal chloramphenicol efflux transporter (U12338.3). This indicates that acquisition of a chromosomal gene may have occurred in the integron as a gene cassette. As trimethoprim and chloramphenicol are not a choice in treatment regimens for P. aeruginosa infections owing to its natural resistance, the presence of dfrA15 and cmlA1 gene cassettes may be of less concern. However, their presence in isolates from the eye, which is a site of interaction of a heterogeneous microbial community, may indicate the potential source of transferable resistance traits. This integron has a new array of gene cassettes and was named In1427, a novel class I integron. A characteristic orf5 of the integron was truncated by IS6100, which has also been reported to disrupt an integron in Klebsiella oxytoca. This suggests that IS6100 may be responsible for introduction of In1427 into the transposon of PA34. The transposon also possesses three other antibiotic resistance genes: two aminoglycoside-modifying enzymes (AMEs) and one b-lactamase (NPS-1) (Figure 1a). The first AME is APH(3’’)-Ib, identical to strA from Pseudomonas sp. B13, and the second AME is APH(6)-Id, identical to strB from Mycobacterium abscessus subsp. bolletii F1725 plasmid BRA100. Tauch et al. have described that the strA–strB gene pair in Tn5393c was highly transferable among bacteria isolated from plants, animals and humans. However, the original structure of Tn5393c, which was found first in the R plasmid pRAS2 from the fish pathogen Aeromonas salmonicida subsp. salmonicida (AF262622.1), was interrupted in PA34 by a 5735 bp region that also included another resistance gene (blaNPS-1). The segment was inserted between the tnpR and strA–strB pairs (Figure 1c). An ISPsy42 (Tn3 family) was found in the region between 9711 and 15 439 bp of the transposon. This suggests that Tn5393c may have the capacity to be integrated by other mobile elements that may be incorporated into resistance genes (blaNPS-1 in this case). A putative mobile element (Tn6205) was found between two resolvase genes and was associated with the strA, strB and blaNPS-1 genes (Figure 1a). The Tn6205 also disrupted the second tnpA. This genetic organization of a Tn3-like transposon of PA34 suggests that insertion and recombination of antibiotic resistance genes may have occurred in multiple steps and dissemination of antibiotic resistance may have occurred between distantly related bacteria. blaNPS-1 was inserted between tnpR and strA and its genetic environment is different in PA34 than that previously reported in the plasmid pB4 (Figure 1d). In PA34, blaNPS-1 is associated with Tn3like transposons along with aminoglycoside resistance genes. This suggests that blaNPS-1 can be found in different genetic environments and may have different sources in the environment. The nucleotide sequence of blaNPS-1 was 100% similar to plasmid pMLH50