pACICU2 is a conjugative plasmid of Acinetobacter carrying the aminoglycoside resistance transposon TnaphA6

Sir, Worldwide, carbapenem resistance is widespread among Acinetobacter causing infections, and most resistant isolates are also resistant to many other antibiotic classes. However, aminoglycosides can be used to treat carbapenem-resistant Acinetobacter baumannii infections that are not resistant to all members of this class. A variety of aminoglycoside resistance phenotypes have been reported in the species due to resistance genes that occur alone or in combinations. Among them, the aphA6 gene, which has been found mainly in Acinetobacter species,1–4 encodes an aminoglycoside (3′) phosphotransferase, which confers resistance to amikacin, kanamycin and neomycin. The aphA6 gene was originally identified in a large plasmid from a clinical A. baumannii isolate recovered some time prior to 1988, and the plasmid was shown to be transferable to other Acinetobacter species, but not to Escherichia coli.5,6 We recently identified a transposon, TnaphA6 (GenBank accession number JF343537), that consists of the aphA6 gene flanked by directly oriented copies of ISAba125, and found that this transposon is present in all of the Australian amikacin-resistant isolates belonging to global clone 2 (GC2; previously European clone II) examined.3 TnaphA6 was also detected in isolates belonging to global clone 1 (GC1; previously European clone I).4 Subsequently, we reported the sequence of a 70.1 kb conjugative plasmid, pAb-G7-2, carrying TnaphA6 (GenBank accession number KF669606) in the GC1 isolate G7 from 2002, and showed that two further GC1 isolates from 2010 contained the same plasmid.7 In the course of that study, it was noted that a significant portion of pAb-G7-2 was most closely related to a cryptic plasmid, pACICU2 (sized as 64 366 bp in GenBank accession number CP000865), with >99.9% identity over the shared regions. pACICU2 was found in the genome of the Italian GC2 isolate ACICU from 2005,8 belongs to the Aci6 Acinetobacter plasmid family9 and encodes transfer functions.8 It also includes a single copy of ISAba125, and we noted that this insertion sequence (IS) was in precisely the same location as TnaphA6 in pAb-G7-2. The possibility that pACICU2 in fact carries TnaphA6 was therefore explored. ACICU was kindly supplied by Alessandra Carattoli (Instituto Superiore di Sanita, Rome, Italy). The aminoglycoside resistance profile of ACICU reported previously8 included resistance to amikacin, gentamicin and tobramycin and these resistances were potentially accounted for by the aacA4 gene in AbaR2 in the chromosome. ACICU was tested for resistance to a larger group of aminoglycosides using disc diffusion and discs (Oxoid Limited) containing amikacin (30 μg), gentamicin (10 μg), kanamycin (30 μg), neomycin (30 μg), tobramycin (10 μg) or netilmicin (30 μg). ACICU was found to be also resistant to amikacin, kanamycin and neomycin, consistent with the presence of aphA6, as well as to gentamicin, kanamycin, netilmicin and tobramycin, potentially associated with the variant of the aacA4 gene conferring gentamicin resistance. Mapping with previously published PCR primers7 that link the internal segment of TnaphA6 to the flanking sequences found in pAb-G7-2 revealed that TnaphA6 is present in pACICU2. To confirm this, pACICU2 was transferred into a rifampicin-resistant derivative of A. baumannii ATCC 17978, which is also resistant to sulphonamides. Transconjugants were selected on L agar containing rifampicin (100 mg/L) and kanamycin (20 mg/L) and screened for growth on ceftazidime (25 mg/L) and nalidixic acid (100 mg/L), to which the donor was resistant and the recipient susceptible, as described previously.7 Transconjugants recovered were resistant to amikacin, kanamycin and neomycin (Table 1), consistent with the presence of the aphA6 gene. Resistance profiles of the donor (ACICU), recipient (ATCC 17978 rifR) and transconjugants AMP, ampicillin; CTX, cefotaxime; CAZ, ceftazidime; Sm, streptomycin; Sp, spectinomycin; Su, sulphonamide; Tc, tetracycline; Tp, trimethoprim; Km, kanamycin; Nm, neomycin; Gm, gentamicin; Ne, netilmicin; Ak, amikacin; Tm, tobramycin; CIP, ciprofloxacin; NAL, nalidixic acid; IPM, imipenem; MEM, meropenem; TIM, ticarcillin/clavulanic acid (Timentin); RIF, rifampicin; SAM, ampicillin/sulbactam. The columns shaded grey indicate the antibiotics to which resistance is conferred by the aphA6 gene in the plasmid. Resistance profiles of the donor (ACICU), recipient (ATCC 17978 rifR) and transconjugants AMP, ampicillin; CTX, cefotaxime; CAZ, ceftazidime; Sm, streptomycin; Sp, spectinomycin; Su, sulphonamide; Tc, tetracycline; Tp, trimethoprim; Km, kanamycin; Nm, neomycin; Gm, gentamicin; Ne, netilmicin; Ak, amikacin; Tm, tobramycin; CIP, ciprofloxacin; NAL, nalidixic acid; IPM, imipenem; MEM, meropenem; TIM, ticarcillin/clavulanic acid (Timentin); RIF, rifampicin; SAM, ampicillin/sulbactam. The columns shaded grey indicate the antibiotics to which resistance is conferred by the aphA6 gene in the plasmid. Plasmid pAb-G7-2 and related cryptic plasmids pABTCDC0715 (GenBank accession numbers CP002524) and ABKp1 (GenBank accession number CP001922) carry a second additional segment that was not found in pACICU2. This segment is 4.7 kb long and lies between two copies of a 423/424 bp segment (see Figure 1 in Hamidian et al.7). The 423/424 bp segment (the copies differ in length) is found only once in the pACICU2 sequence. Using published primers,7 the 4.7 kb segment was shown to be also present in pACICU2. This addition appears to replace two regions that have been duplicated in the available pACICU2 sequence. Furthermore, most of the minor differences between pAb-G7 and pACICU2 represent one too few or one too many As or Ts in runs of As or Ts, and this could have resulted from the sequencing technology used. Hence, it seems likely that pACICU2 is identical or nearly identical to pAb-G7-2. In conclusion, though the available sequence lacks TnaphA6, pACICU2 confers resistance to amikacin, kanamycin and neomycin and carries the aphA6 gene in TnaphA6 in the location found in pAb-G7-2. It also carries an additional 4.7 kb segment found in pAb-G7-2. Hence, segments are missing from the available pACICU2 sequence and pACICU2 is likely to be indistinguishable from pAb-G7-2 except for a small number of single base substitutions/deletions/additions. This study was supported by NHMRC Project Grant APP1026189. M. H. was supported by a University of Sydney Postgraduate Research Award. None to declare.