Horizontal transfer of an ISAba125-activated ampC gene between Acinetobacter baumannii strains leading to cephalosporin resistance.

Sir, In Acinetobacter baumannii, resistance to third-generation cephalosporins such as ceftazidime and cefotaxime is known to arise as a consequence of acquisition of an insertion sequence, ISAba1, upstream of the chromosomal ampC gene. Indeed, ISAba1 is frequently found upstream of the ampC gene in isolates that are resistant to third-generation cephalosporins, and the promoter that directs transcription of ampC is located within ISAba1. Other insertion sequences have also been detected upstream of ampC. ISAba125 is present upstream of ampC in ACICU, a global clone 2 (GC2) isolate from Italy, and it has been suggested that it may also increase ampC expression, leading to cephalosporin resistance. We have examined resistance to third-generation cephalosporins in ACICU, kindly supplied by Dr Alessandra Carottoli, Istituto Superiore di Sanità, Rome, Italy, and in A388, a global clone 1 (GC1) isolate from Greece described previously. Both ACICU and A388 were found to be resistant to ceftazidime and cefotaxime (MIC.128 mg/L), but in A388 ISAba1 was not detected upstream of ampC using a PCR assay that links them (Figure 1a). To determine whether a different IS was present in A388, a PCR was designed to detect the ampC gene linked to the folE gene (Figure 1), as it is in its normal location on the chromosome. An amplicon of 1667 bp, the predicted size when no IS is present (Figure 1c), was obtained with GC1 isolates 3208 and D2, which were susceptible to ceftazidime and cefotaxime (MIC 4–16 mg/L). For A388, this amplicon was larger, consistent with the presence of an IS, and the sequence of this folE–ampC amplicon revealed a copy of ISAba125 located 57 bp upstream of the initiation codon of the ampC gene. The amplicons from 3208 and D2 were also sequenced and the sequence of the region surrounding ISAba125 in A388 was identical to those sequences, and to that found in the published genome of the antibiotic-susceptible GC1 isolate AB307-0294 (GenBank accession number CP001172). Because the sequence of ampC found in another GC1 isolate, AB0057 (GenBank accession number CP001182), differed over a short span of 214 bp, the sequence of this region was re-determined and found to be identical to those described here. Hence, it appears that ISAba125 in A388 has inserted into the standard GC1 genome. Examination of the sequence surrounding ISAba125 upstream of the ampC gene in the genome of ACICU (GenBank accession number CP000863), which is from a different clonal complex, namely GC2, revealed that it was almost identical to the GC1 sequence. This suggests that a region derived from a GC1 strain carrying ISAba125 has been incorporated into the ACICU genome. To determine the size of the segment potentially derived from a GC1 strain, the sequence of ACICU in this region was compared with the corresponding region from AB307-0294 and AB0057, representing GC1, and MDR-ZJ06 (GenBank accession number CP001937), representing GC2. A segment of about 9 kb surrounding ISAba125 in ACICU differs from that of other GC2 isolates by about 2%, but is nearly identical to the standard GC1 sequence. Surrounding this segment, the sequences of ACICU and other GC2 strains were the same, but diverged from the GC1 sequence, due to multiple single-nucleotide polymorphisms. On average, GC1 and GC2 sequences differed by 3%. Together these findings indicate that a 9 kb segment of the ACICU genome is likely to have been incorporated into the GC2 lineage from a GC1 strain via homologous recombination. To determine whether the presence of ISAba125 increased the expression of the ampC gene, the RH581–RH582 PCR amplicons from 3208 and from A388 were cloned into pCR2.1TOPO. The MICs of ceftazidime and cefotaxime conferred by the plasmid containing the fragment from 3208 (8 mg/L and 16 mg/L) were 16-fold and 32-fold higher, respectively, than for the vector control (0.5 mg/L). When the cloned fragment contained ISAba125, the MICs were further elevated, to .256 mg/L. To compare the level of ampC transcripts in A388 and ACICU with those in the susceptible isolates, 3208 and D2, quantitative reverse transcription PCR (qRT– PCR) was also performed using primers RH1388, 5′-TGGCTGTGGGT GTTATTCAA-3′, and RH1389, 5′-ACCTGCTGTCGCGGTAAATA-3′, for ampC and RH1384, 5′-GGAGAAAGCAGGGGATCTTC-3′, and RH1385, 5′-ATCCTCTCAGACCCGCTACA-3′, for 16S rRNA. In both A388 and ACICU, the transcript level was 20-fold higher than in 3208 and D2, confirming that the presence of ISAba125 also increased expression of ampC, leading to increased resistance to third-generation cephalosporins.