This study demonstrates the usefulness of comparative genome sequencing for a rapid survey of all putative resistance mechanisms in A. baumannii. The determination and detailed comparison of the genome sequences of the MDR strain AYE, and the strain SDF, which is free from most resistance acquired by A. baumannii over recent decades, allowed the identification of many genes associated with antibacterial resistance at once. We identified 52 genes associated with resistance in the AYE strain, including 17 genes not, to our knowledge, previously described in A. baumannii. The clustering of 45 (86.5%) of them within an 86-kb AbaR1 resistance island was unexpected.
The detailed sequence analysis of the AbaR1 island, the largest genomic island identified to date, showed that it was built through the recursive insertion of broad host-range mobile genetic elements (transposons, gene cassettes from class 1 integrons), with gene cassettes (sometimes chimeric) mostly originating from the genera Pseudomonas, Salmonella, and Escherichia. Together with A. baumannii, many members of these genera are commonly found in aqueous environments of healthcare facilities, where, under antimicrobial pressure in these settings, genetic exchange among them may be promoted.
Another unexpected finding was the presence of a similar structure in the genome of susceptible strain SDF, identically inserted in the homologous ATPase-like ORF. This genomic island was found in an “empty” state, exhibiting mobility-associated genes but no resistance markers. This coincidental genetic insertion in the two strains strongly suggests that this ATPase ORF constitutes a specific hotspot of genomic instability in the A. baumannii
genome. This prompted us to investigate whether this feature was common to all A. baumannii
strains. Using a polymerase chain reaction assay based on the conserved ATPase ORF flanking sequences (Protocol S1
), 17 (77%) out of the 22 clinical A. baumannii
isolates were found to exhibit an intact ATPase ORF. These 17 isolates included 11 isolates resistant to several antibiotic families, including β-lactams, and six susceptible to β-lactams. Among the five isolates exhibiting an interrupted ATPase ORF, four were resistant to most β-lactams except imipenem, including some that were also resistant to other antibiotic families, and one was susceptible to β-lactams but resistant to cotrimoxazole and rifampin (Protocol S1
). The presence of a genomic island within the ATPase ORF is thus not a conserved feature among isolates, and its absence at this location is not predictive of the observed pattern of antibiotic susceptibility. This suggests a particular flexibility of the A. baumannii
genome, in line with its exceptional ability in gathering foreign genetic material. Whole-genome sequencing of additional A. baumannii
strains will be needed to assess the full range of mechanisms through which clinical isolates can so efficiently acquire new resistance genes.
Finally, our last surprise was the identification of several putative resistance genes in a strain not exhibiting the associated phenotype. This was the case for two putative β-lactamases (present in the AYE and SDF), including the class D β-lactamase bla
OXA-69. The expression of OXA-69 in E. coli
resulted in low levels of carbapenem resistance [35
], and its sequence is 97% identical to the OXA-51 carbapenemase, found associated with full carbapenem resistance [41
]. However, the presence of bla
OXA-69 in the susceptible AYE strain suggests that it might only be a step away from acquiring this new resistance through subtle changes in expression level or a specific mutation or series of mutations that alter the substrate profile and enhance catalytic activity. Besides the direct acquisition of genetic material from resistant bacterial species, the maintenance of spare copies of “ready-to-optimize” resistance genes in the genome of A. baumannii,
perhaps selected by exposure to subinhibitory levels of the drug in the environment, might also play a role in its rapid adaptation to new derivatives of the major antibiotic classes.