ISAba1 targets a specific position upstream of the intrinsic ampC gene of Acinetobacter baumannii leading to cephalosporin resistance.

Sir, Resistance to third-generation cephalosporins such as ceftazidime and cefotaxime in Acinetobacter baumannii is often due to increased expression of an intrinsic ampC gene. Expression increases when the ISAba1 insertion sequence (IS) is present in the appropriate orientation upstream of the ampC gene, providing an outward-facing promoter that appears to be stronger than the intrinsic promoter because it increases transcription. – 3 This mechanism has been found to be widespread, and ISAba1 is generally found upstream of the ampC gene in isolates that are resistant to third-generation cephalosporins. – 8 We have previously shown that most of the isolates in our collection that belong to global clone 1 (GC1) or global clone 2 (GC2), and are resistant to ceftazidime and cefotaxime, carry ISAba1 upstream of ampC. – 7 The precise position of ISAba1 in the isolates examined was determined by sequencing a PCR amplicon that includes the IS and ampC, as described previously. Sequence types were determined using the Oxford multilocus sequence typing scheme as described previously. Initially, two Australian GC2 isolates were examined. The ampC genes of A91, an ST92 isolate from Sydney, 2005 (GenBank accession number JN968483), and RBH44, an ST69 isolate from Brisbane, 2002, differed by a single base substitution, and ISAba1 was in the same orientation and 9 bp away from the ATG initiation codon of ampC in both. ISAba1 is in exactly the same position in the GC1 isolate AYE (GenBank accession number CU459141). However, the insertion events seem to be independent because the ampC alleles, here named allele 1 for GC1 and allele 2 for GC2, differed at 38 or 37 positions in the 1152 bp ampC gene (Table 1). We showed recently that the ampC sequence in GC1 isolates without ISAba1 is identical to that of AYE, which carries ISAba1-ampC. To verify that allele 2 is usually associated with GC2, ampC from A320/RUH134 (isolated in the Netherlands in 1982), which is the oldest GC2 isolate susceptible to third-generation cephalosporins in our collection, was sequenced; the allele (GenBank accession number JN247441) was the same as in the later ISAba1-containing isolates (identical to RBH44 ampC). The shared location of the IS was unexpected, as the promoter identified experimentally by mapping the 5′-end of the transcript is completely contained within ISAba1, and consequently there should be no constraint on the position of the IS to ensure highlevel transcript initiation. The location of this promoter, which includes an extended 210 (TGnCATTAT) that should increase its strength, is shown in Figure S1 (available as Supplementary data at JAC Online). Furthermore, ISAba1 has been shown to transpose to multiple locations. Nonetheless, it appeared that ISAba1 had independently inserted into the same position in both the GC1 and a GC2 background. To further examine the possibility that ISAba1 can target this position, the location of ISAba1 relative to ampC in other sequences available in the GenBank non-redundant DNA database was examined. Two additional cases of ISAba1 associated with distinct ampC alleles were found in accession numbers EU604835 and AY325306. In both of them, the IS was again in the same orientation and separated from the ATG initiation codon of ampC by 9 bp. The number of single nucleotide differences between the various ampC alleles is shown in Table 1.