Acquisition of plasmids conferring carbapenem and aminoglycoside resistance and loss of surface-exposed macromolecule structures as strategies for the adaptation of Acinetobacter baumannii CC104O/CC15P strains to the clinical setting

Abstract

Acinetobacter baumannii (Aba) is an emerging opportunistic pathogen associated to nosocomial infections. The rapid increase in multidrug resistance (MDR) among Aba strains underscores the urgency of understanding how this pathogen evolves in the clinical environment. We conducted here a whole-genome sequence comparative analysis of three phylogenetically and epidemiologically related MDR Aba strains from Argentinean hospitals, assigned to the CC104^O/CC15^P clonal complex. While the Ab244 strain was carbapenem-susceptible, Ab242 and Ab825, isolated after the introduction of carbapenem therapy, displayed resistance to these last resource β-lactams. We found a high chromosomal synteny among the three strains, but significant differences at their accessory genomes. Most importantly, carbapenem resistance in Ab242 and Ab825 was attributed to the acquisition of a Rep_3 family plasmid carrying a bla_OXA-58 gene. Other differences involved a genomic island carrying resistance to toxic compounds and a Tn10 element exclusive to Ab244 and Ab825, respectively. Also remarkably, 44 insertion sequences (ISs) were uncovered in Ab825, in contrast with the 14 and 11 detected in Ab242 and Ab244, respectively. Moreover, Ab825 showed a higher killing capacity as compared to the other two strains in the Galleria mellonella infection model. A search for virulence and persistence determinants indicated the loss or IS-mediated interruption of genes encoding many surface-exposed macromolecules in Ab825, suggesting that these events are responsible for its higher relative virulence. The comparative genomic analyses of the CC104^O/CC15^P strains conducted here revealed the contribution of acquired mobile genetic elements such as ISs and plasmids to the adaptation of A. baumannii to the clinical setting.

Keywords: Acinetobacter baumannii; blaOXA-58; carbapenem resistance; comparative genomics; multi-drug resistance; virulence factors.

Fig. 1.

Linear comparison of the genomes of A. baumannii strains ATCC17978, Ab825, Ab242, Ab244 and DSM30011 inferred using Mauve. Each block corresponds to a DNA fragment of the chromosome and is distinctively coloured for clarity. The degree of conservation is indicated by the vertical bars inside the blocks. Their position relative to the genome line denotes co-linear and inverted regions. Putative prophages (Ph/strain-number; seeTable 3 for details) are displayed for all strains. Two regions usually associated with antimicrobials resistance (Tn6022 and Tn10) are shown for strain Ab825 (see text for details). Regions encoding a Tn6018-like element, a CRISPR-cas cluster, two-partner systems (Tps-1 and 2) and type-6 secretion-system-related genes (T6-1 to 3) are indicated for DSM30011 genome [23]. A resistance island inserted into the dusA gene is present in Ab244 and DSM30011 chromosomes.

Fig. 2.

Schematic representations of the genomic islands interrupting comM and dusA in the A. baumannii clinical strains under study. (a) Tn6022 elements found interrupting the comM gene in the three strains under study (see Table 2 for details). The direct repeat generated at the site of integration is shown enlarged and underlined. In the Tn6022 element found in Ab242, an additional ISAba125 element was localized downstream of the uspA gene. The sites of hybridization of the pair of primers (see Table S1) employed to corroborate this insertion by PCR amplification analysis is shown. (b) Genomic island interrupting the dusA gene in the Ab244 strain (see Table 2 for details). Copper and arsenic ion-resistance clusters are underlined by green and orange bars, respectively. Genes encoding hypothetical phage proteins (grey arrows) are underlined by a grey bar. For comparisons, the intact dusA gene and its flanking genes found in the chromosomes of both Ab242 and Ab825 strains are shown below.

Fig. 3.

G. mellonella lethality assays of the different A. baumannii strains under study. (a) Comparative survival analysis of G. mellonella larvae injected with 10⁶ c.f.u. doses of Ab242 or Ab244 strains. The same dose of the Aba ATCC17978 was also tested as a control reference strain (*P<0.05; n.s.: non-significant). PBS was used as a control. (b) Same, but employing 10⁵ c.f.u. doses of Ab825 or ATCC17978. (***P<0.0001). In all cases the data are representative of three separate survival experiments, each performed with 20 larvae. Survival curves were constructed by the Kaplan–Meier method and compared by Log-rank analysis.

Fig. 4.

Schematic representation of the chromosomal location of ISs shared among Ab242, 244 and Ab825 strains. The IS positions are schematically depicted according to their location in the corresponding genomic sequence (Table S8). ISAba42 (magenta), ISAba43 (purple), ISAba31 (orange), ISAba125 (pink) and ISAba26 (green) interrupt different target genes whereas ISAba1 (light grey) falls in an intergenic region upstream of bla _ADC gene (red arrow). Thus generating a strong promoter putatively enhancing the expression of this resistance gene. The IS insertion sites in a given target gene were depicted with a vertical black arrow and dashed lines. Genes are not drawn to scale.