Overexpression of the Naturally Occurring bla OXA-51 Gene in Acinetobacter baumannii Mediated by Novel Insertion Sequence IS Aba9

Abstract

Resistance to carbapenems in Acinetobacter baumannii is increasingly reported and is mostly associated with expression of carbapenemases (3, 6). Those carbapenemases are most frequently carbapenem-hydrolyzing class D β-lactamases, which have been identified worldwide, including in Greece (4, 10). Four major subgroups of carbapenem-hydrolyzing class D β-lactamases have been identified in A. baumannii: the naturally occurring OXA-51/69-type β-lactamase and the acquired OXA-23, OXA-24/40 and OXA-58-type β-lactamases. Despite a weak catalytic activity, the naturally occurring OXA-51/69-type β-lactamases might contribute to carbapenem resistance by insertion of ISAba1 in the promoter region of the blaOXA-51/69-like genes (1, 8, 11). Twenty nonrepetitive carbapenem-resistant A. baumannii clinical isolates were isolated from August 2001 to November 2006 at the Papageorgiou General Hospital, Thessaloniki, Greece (Table ​(Table1).1). All 20 isolates were resistant to imipenem, among which 8 were additionally resistant to meropenem (MICs ranging from 8 to 64 μg/ml) (Table ​(Table1).1). No metallo-β-lactamase (MBL) production was detected by using Etest MBL strips (AB Biodisk, Solna, Sweden). Pulsed-field gel electrophoresis (PFGE) analysis was done as previously described (Bio-Rad, Ivry-sur-Seine, France) (5, 9) and showed that the 20 A. baumannii isolates exhibited 5 distinct PFGE profiles defining clones I to V (Table ​(Table1).1). PCR experiments performed using primers specific for blaOXA-58-like, blaOXA-23-like, and blaOXA-24/40-like genes (12) showed that 16 isolates belonging to clones I, II, or III possessed the blaOXA-58 gene, whereas no isolate harbored the blaOXA-23 or blaOXA-40 genes. PCR experiments using primers specific for the naturally occurring blaOXA-51-like gene gave positive results for the 20 A. baumannii strains tested. In three clonally related and blaOXA-58-negative A. baumannii isolates, Ab2, Ab5, and Ab23, ISAba1 was identified upstream of the blaOXA-51 gene (11) (Table ​(Table1).1). In isolate Ab5, the ISAba1 copy located immediately upstream of the blaOXA-51 gene was truncated by a novel insertion sequence named “ISAba9” (Fig. ​(Fig.1).1). ISAba9 is 974 bp long and belongs to the IS982 family (http://www-is.biotoul.fr./). It possesses 17-bp inverted repeats and was bracketed by an 8-bp target site duplication (TTGTTTAA), which is likely the signature of a transposition process. ISAba9 was very recently identified in association with the blaRTG-4 gene in A. baumannii (7). We hypothesized that ISAba9 located upstream of the blaOXA-51 gene in isolate Ab5 might contribute to carbapenem resistance by providing promoter sequences leading to the overexpression of the blaOXA-51 gene, as observed with ISAba1 (2, 8, 11). Analysis of the genetic environment of the blaOXA-51 gene in Ab5 showed that ISAba1 provided −35 and −10 promoter sequences, as already described (1). In addition, a hybrid promoter made of a −35 box located inside ISAba9 and a −10 box located in ISAba1 with an optimal 17-bp spacing between the −35 and −10 boxes was identified (Fig. ​(Fig.1).1). Quantitative analysis of the blaOXA-51 expression was performed by reverse transcription-PCR using the two clonally related isolates Ab2 and Ab5, as previously described (1). Transcriptional profile analysis indicated an eightfold increased expression of blaOXA-51 in the genetic structure containing ISAba1 and ISAba9 compared with ISAba1 alone upstream of this gene (mean ± standard deviation, 8 ± 5), using the 16S RNA gene as a housekeeping gene. PCR screening of the 20 A. baumannii isolates using primers specific for ISAba9 gave positive results for isolates Ab2, Ab3, Ab5, and Ab23, but ISAba9 was located upstream of the blaOXA-51-like gene only in isolate Ab5. FIG. 1. Nucleotide sequence of the −35 and −10 putative promoter regions within ISAba1 described from isolates Ab2 (A) and Ab5 (B). The left inverted repeat (IRL) of ISAba1 and the right inverted repeat (IRR) of ISAba9 are shaded in gray, and ... TABLE 1. Characteristics of A. baumannii isolates In conclusion, we identified the novel insertion sequence ISAba9 as being involved in blaOXA-51 gene overexpression, thus contributing to carbapenem resistance in A. baumannii. ISAba9, after ISAba1, might be an additional tool for genetic plasticity of A. baumannii and, in particular, for acquisition of resistance.