ISAba825, a Functional Insertion Sequence Modulating Genomic Plasticity and bla _OXA-58 Expression in Acinetobacter baumannii

Acinetobacter baumannii is an important opportunistic pathogen responsible for a variety of nosocomial infections (6, 21, 22, 26). It can rapidly evolve multidrug resistance (MDR) when confronted with antibiotic therapy (4, 6, 21, 26), and in particular, the emerging resistance to carbapenems among nosocomial strains represents a major concern worldwide (4, 20, 22). One of the mechanisms proposed to play a significant role in carbapenem resistance in A. baumannii is the expression of OXA-type carbapenemases (6, 7, 15, 16, 21, 26). Other mechanisms, such as alterations in outer membrane (OM) permeability, can also contribute to carbapenem resistance in this organism (5, 18). In this context, we recently reported that the loss of the OM channel CarO as a result of the natural insertional inactivation of its coding gene by insertion sequence (IS) ISAba825 or ISAba125 correlated with reduced susceptibility to carbapenems (18). One of these ISs, ISAba825, recently assigned to the IS982 family (http://www-is.biotoul.fr), is composed of an 876-bp open reading frame (ORF) coding for a DDE-type transposase bounded by a perfect 17-bp inverted repeat (IR) (18). ISAba825 generated a 7-bp duplication (ATCGTTA) at the insertion site within carO (18). It is well known that ISs can cause insertion mutations and genome rearrangements and enhance the spread of resistance and virulence determinants within pathogenic species (2, 9, 11, 13, 17, 19, 23). In this work, we evaluated the impact of ISAba825 in modulating A. baumannii genome plasticity and carbapenem resistance.

To follow ISAba825 transposition, we tagged the element with a kanamycin (Kn) resistance cassette (Fig. 1 A) and subcloned it into the plasmid pKNOCK, a suicide vector in A. baumannii (1). To evaluate the ability of ISAba825::Kn to transpose in A. baumannii, we first transformed an A. baumannii ATCC 17978 strain containing the Amp^r plasmid pWH1266 (which can replicate in A. baumannii and Escherichia coli [8]) with pKNOCK ISAba825::Kn. The rationale was that, as pKNOCK is unable to replicate in A. baumannii, Kn^r bacteria would arise, among other possible events, from ISAba825::Kn transposition to the chromosome, to ATCC 17978 endogenous plasmids pAB1 and pAB2 (27), or to pWH1266 (step 1). Step 2 involved plasmid extraction from these Kn^r colonies, transformation of E. coli DH5α or ATCC 17978 competent cells, and selection in LB agar plates containing Kn or Kn and Amp to isolate plasmids containing ISAba825::Kn insertions. At this step, we observed Kn^r transformants only when ATCC 17978 was used as the recipient for transformation (albeit none of them were Amp^r), suggesting that in A. baumannii there was effective transposition into plasmids other than pWH1266, which appeared to be nonreplicative in E. coli.

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FIG. 1.

(A) Construction of ISAba825::Kn. First, we generated an EcoRV site immediately downstream of the transposase (Tpase) gene and upstream of the right inverted repeat (IRR) of ISAba825 by PCR using primers 825TF and 825TR (Table 1). As the Tpase stop codon TAA is within the IRR, we introduced another TAA codon upstream of the EcoRV site to maintain both the original transposase sequence and the IRR structure. Afterwards, a kanamycin resistance cassette (Kn^R) bounded by two EcoRV sites was generated by PCR using primers KnEcoRVF and KnEcoRVR (Table 1), employing plasmid pBBR1MCS5 (10) as a template, and was inserted into the equivalent site present in ISAba825-EcoRV. The single TAA site in ISAba825 and the two TAA sites in ISAba825::Kn are underlined. The Tpase gene is boxed and the direction of transcription indicated by an arrow. The IRL and IRR are also underlined. The scheme is not drawn to scale. (B) Target sites of ISAba825::Kn insertions in linearized representations of ATCC 17978 endogenous plasmids pAB1 (GenBank accession number NC_009083) and pAB2 (GenBank accession number NC_009084). Genes and their corresponding directions of transcription are indicated by horizontal arrows. The locus tag of each gene is indicated in parentheses below the gene description. Dark arrowheads indicate ISAba825::Kn insertions at the corresponding nucleotide positions in the plasmids. Arrowheads above and below lines indicate IS insertions in opposite orientations. The scheme is not drawn to scale. (C) ISAba825::Kn and ISAba825 target sequences. Target site duplications (bold letters) and flanking regions of ISAba825::Kn insertions in pAB1 and pAB2 and ISAba825 insertions in carO and tnpA are shown. IR regions are boxed, and arrows indicate the Tpase transcription direction.

Putative ISAba825::Kn transposition events were analyzed using plasmid extractions from 40 ATCC 17978 Kn^r colonies, and in each of them the precise insertion site was determined by DNA sequencing (Maine DNA Sequencing Facility) of the IS immediate external neighboring regions by using primers ISout1 and ISout2 (Table 1). Reads of up to 500 bp at either side of ISAba825::Kn revealed 5 different IS insertions, all of them in the indigenous plasmids pAB1 and pAB2 (Fig. 1B and C). AT-enriched short target site duplications bounding the IRs were observed in all these cases, consistent with ISAba825::Kn transposition events (Fig. 1C). It is noteworthy that the target duplications ranged from 6 to 9 bp (Fig. 1C). It is commonly assumed that the length of the duplication is characteristic for a given element. However, certain ISs can generate duplications of atypical length, presumably reflecting small variations in the geometry of the transposition complex (13). Therefore, and although transposition was not tested in a recA-deficient strain, all the evidence described above strongly indicates effective transposition of ISAba825::Kn in A. baumannii ATCC 17978.

We evaluated next whether ISAba825 could be found in close proximity to any member of the different OXA groups in carbapenem-resistant strains of our collection by using combinations of forward and reverse primers specific for ISAba825 (Table 1) and the different oxa genes, respectively (14). From a total of 10 strains screened, a PCR amplification band of approximately 1,500 bp was obtained only for strain Ab880 by using primers 825TR and OXA-58R (Table 1). Sequence analysis revealed that an insertion of ISAba825 truncated the transposase gene (tnpA) of an ISAba3-like element located upstream of a bla_OXA-58 gene (Fig. 2 A). As in other cases, the bla_OXA-58 gene was bounded by two ISAba3 elements (24, 25). Southern blot analysis showed that this arrangement was present in a plasmid (not shown), designated pAb880. Electrotransformation of pAb880 into ATCC 17978 resulted in 32- and 64-fold increments in meropenem (MER) and imipenem (IPM) MICs, respectively, compared to those of nontransformed bacteria (Table 2). This outcome suggested that the ISAba825 bla_OXA-58 arrangement carried in pAb880 most probably contributed to carbapenem resistance. 5′ rapid amplification of cDNA ends (5′ RACE) analysis identified the presence of a transcript initiating 119 bp upstream of the start codon of the bla_OXA-58 gene, thus defining a hybrid promoter (ISAba825-ISAba3-like) in which the −10 region is within the ISAba3-like element and the −35 region is located within the ISAba825 left IR (IRL) (Fig. 2B). In this context, previous reports have also shown that insertion of different ISs within the 5′ ISAba3 element can result in alternate hybrid promoters modulating bla_OXA-58 expression (3, 25). We then tested the contribution of the ISAba825-ISAba3-like hybrid promoter to bla_OXA-58-mediated carbapenem resistance by comparing the IPM and MER MICs of ATCC 17978 bacteria transformed with plasmid pWHP825 (pWH1266 bearing bla_OXA-58 under the control of the ISAba825-ISAba3-like hybrid plus the previously described ISAba3-derived promoter [3]), pWHP3 (pWH1266 bearing bla_OXA-58 lacking the hybrid promoter but preserving the ISAba3-derived promoter), or pWH1266 alone (Fig. 2B). As seen in Table 2, bacteria harboring pWHP825 exhibited MER and IPM MICs 64- and 128-fold higher, respectively, than those of cells harboring pWH1266. In contrast, transformation of bacteria with pWHP3 only doubled the MIC values for these carbapenems. In agreement, transcript levels of bla_OXA-58 in pWH825-containing cells were 7-fold higher than those in pWHP3-containing cells and about 100-fold higher than those in bacteria bearing pWH1266 (Table 2). Thus, the ISAba825-ISAba3-like hybrid promoter-directed bla_OXA-58 overexpression constitutes a likely mechanism leading to carbapenem resistance acquisition in A. baumannii.

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FIG. 2.

ISAba825 insertion generated a hybrid promoter driving bla_OXA-58 overexpression in the clinical A. baumannii strain Ab880. (A) Schematic representation of the genetic structure resulting from ISAba825 insertion within the ISAba3-like element located 5′ upstream of the bla_OXA-58 gene present in plasmid pAb880. Genes and their corresponding transcription orientations are indicated by horizontal arrows. The boundaries of the sequenced fragment are indicated by vertical bars at the edges. The figure is not drawn to scale. (B) Promoter regions for bla_OXA-58 in the arrangement described above. The −35 and −10 motifs inferred for each of the different promoters are boxed, and the transcription initiation site (G in bold) resulting from the hybrid promoter (as determined by 5′ RACE-PCR) is indicated by +1. Promoter prediction was done using BPROM (SoftBerry). The different ATG codons for bla_OXA-58 and tnpA are indicated in bold, and the corresponding directions of transcription are shown by arrows. The inverted repeats of the different IS elements are shaded gray. cont., continued.

Finally, the study of the distribution of ISAba825 by PCR using primer IS (Table 1) showed its presence only in carbapenem-resistant A. baumannii isolates of our collection and failed to detect it in other representatives of the genus Acinetobacter, including 2 different strains of A. radioresistens, A. johnsonii, A. junii, A. lwoffii, A. tandoii, A. bouvetii, A. tjernbergiae, A. grimontii, A. towneri, and A. gerneri. Overall, the results described above, in addition to the inactivation of carO, lead us to propose a role for ISAba825 in the modulation of carbapenem resistance in A. baumannii.

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