Carbapenem and amikacin resistance on a large conjugative Acinetobacter baumannii plasmid

Sir, Worldwide, most multiply antibiotic-resistant (MAR), and particularly carbapenem-resistant, Acinetobacter baumannii causing infections belong to global clone 1 (GC1) or global clone 2 (GC2). Though there have been incidences where a different clonal group was predominantly responsible for an epidemic outbreak, MAR isolates that are not members of these clones have been observed to cause infections in hospitals but are not as common. Isolate D46, collected at Royal North Shore Hospital, Sydney in 2010, was shown to be resistant to ampicillin, imipenem, meropenem, ticarcillin/clavulanate, ceftazidime, cefotaxime, streptomycin, spectinomycin, sulfamethoxazole, tetracycline, trimethoprim, chloramphenicol, florfenicol, kanamycin, neomycin, gentamicin, amikacin, tobramycin, nalidixic acid and ciprofloxacin, making it extensively antibiotic resistant. D46 was previously shown to be ST110 (Oxford MLST) and to harbour the aphA6 amikacin resistance gene within TnaphA6 and an aadB gene cassette (gentamicin, kanamycin and tobramycin resistance) in the small plasmid pRAY. An ISAba1 upstream of the chromosomal ampC confers resistance to third-generation cephalosporins, ceftazidime and cefotaxime. Here, we have further examined the causes of resistance. Using PCR as described previously, D46 was shown to contain the strA-strB, sul2 and tetA(B) genes, responsible for streptomycin, sulfamethoxazole and tetracycline resistance, respectively. These genes were in the same configuration as in AbGRI1-2 (Tn6167), but D46 does not have an island in comM. Carbapenem and ticarcillin/clavulanate resistance was due to the blaOXA-23 carbapenemase gene, which was within Tn2006. To better understand how Tn2006 was acquired, conjugation was performed as described previously, using D46 as a donor and a rifampicin-resistant mutant of A. baumannii ATCC 17978 as a recipient. Transconjugants resistant to meropenem, imipenem and ticarcillin/clavulanate, indicative of blaOXA-23, and resistant to kanamycin, neomycin and amikacin, indicative of aphA6, were recovered. Hence, both Tn2006 and TnaphA6 were located on a conjugative plasmid. The whole genome sequence of D46 was determined using Illumina HiSeq and assembled as described previously. The draft genome comprised 115 contigs with an average read depth of 88.7× coverage and D46 was determined to be ST25 according to the Pasteur MLST scheme. Three plasmids were detected. The sequence of the smallest plasmid, pD46-1 (6078 bp), contained aadB and was almost identical to pRAY* (a single base difference). pD46-2 is an 8731 bp cryptic plasmid that was identical to p1ABTCDC0715 (GenBank accession number CP002523) from a GC2 isolate. To determine whether D46 harboured Tn2006 together with TnaphA6 on a plasmid related to pAb-G7-2 and pD72-2, contigs that matched them were retrieved from the draft genome. Seven contigs were recovered and assembled using PCR. The amplicons were sequenced, finalizing the assembly. Plasmid pD46-3 has a 67027 bp backbone sharing 99.99% identity (five singlebase changes) with that of pD72-2 (Figure 1a). pD46-3 also harboured TnaphA6 in the same position as pD72-2 (Figure 1a). One end of two backbone contigs had inversely oriented fragments of ISAba1 and they were linked with blaOXA-23 in Tn2006 using PCR (Figure 1a). The complete 74 916 bp sequence of