The success of acinetobacter species; genetic, metabolic and virulence attributes

Abstract

An understanding of why certain Acinetobacter species are more successful in causing nosocomial infections, transmission and epidemic spread in healthcare institutions compared with other species is lacking. We used genomic, phenotypic and virulence studies to identify differences between Acinetobacter species. Fourteen strains representing nine species were examined. Genomic analysis of six strains showed that the A. baumannii core genome contains many genes important for diverse metabolism and survival in the host. Most of the A. baumannii core genes were also present in one or more of the less clinically successful species. In contrast, when the accessory genome of an individual A. baumannii strain was compared to a strain of a less successful species (A. calcoaceticus RUH2202), many operons with putative virulence function were found to be present only in the A. baumannii strain, including the csu operon, the acinetobactin chromosomal cluster, and bacterial defence mechanisms. Phenotype microarray analysis showed that compared to A. calcoaceticus (RUH2202), A. baumannii ATCC 19606(T) was able to utilise nitrogen sources more effectively and was more tolerant to pH, osmotic and antimicrobial stress. Virulence differences were also observed, with A. baumannii ATCC 19606(T), A. pittii SH024, and A. nosocomialis RUH2624 persisting and forming larger biofilms on human skin than A. calcoaceticus. A. baumannii ATCC 19606(T) and A. pittii SH024 were also able to survive in a murine thigh infection model, whereas the other two species were eradicated. The current study provides important insights into the elucidation of differences in clinical relevance among Acinetobacter species.

Figure 1. A. baumannii core genome.

Functional distribution of the genes found in all six A. baumannii strains included in this study.

Figure 2. Distribution of genes in individual strains of the A. calcoaceticus – A. baumannii complex.

(A) Venn diagram showing the number of overlapping genes between the four strains that make up the A. calcoaceticus – A. baumannii complex. (B) Pairwise comparisons of the number of genes present in A. baumannii ATCC 19606^T but absent in each of A. calcoaceticus (blue), A. pittii (green) and A. nosocomialis (red).

Figure 3. Genetic organisation and conservation of the siderophore clusters found in A. baumannii ATCC 19606^T and not in A. calcoaceticus.

(A) Siderophore cluster 1 (operons 36–39) is known as the acinetobactin chromosomal cluster, and (B) siderophore cluster 2 (operon 17) (See Table 2 for details about the operons). The presence of homologues for each gene in A. pittii, A. nosocomialis, and A. calcoaceticus is shown.

Figure 4. Metabolic diversity of specific strains of the A. calcoaceticus – A. baumannii complex.

(A) Phenotype Microarray (PM) comparative results showing the number of compounds used (green) or not used (red) by A. baumannii ATCC 19606^T [A], A. nosocomialis [B], A. pittii [C] and A. calcoaceticus [D]. The external circle and PM number represent the Biolog plate number. (B) A. baumannii ATCC 19606^T is able to metabolise the carbon source, D–glucarate and produce α–Ketoglutarate through the functional enzymes, D–glucarate dehydrogenase and KDG dehydratase. α–Ketoglutarate is then utilized in the citrate cycle. These enzymes are not found in A. calcoaceticus.

Figure 5. Virulence attributes of individual strains belonging to the A. calcoaceticus – A. baumannii complex.

(A) Adherence of A. baumannii ATCC 19606^T, A. pittii, A. nosocomialis and A. calcoaceticus to human bronchial epithelial cells after 1 hour. Results are expressed as mean number of bacteria per 100 epithelial cells ± standard deviation (SD) of two independent experiments performed in duplicate. The asterisk signifies statistical significance (P<0.05) between A. pittii and A. baumannii. The comparison between A. pittii and A. calcoaceticus was also significant (P<0.05). (B) Levels of IL–6 and (C) IL–8 in the culture medium of human bronchial epithelial cells after 24 hour stimulation with specific strains of the A. calcoaceticus – A. baumannii complex. Results are expressed as mean levels of IL–6 and IL–8 (in ng/ml) ± SD of three independent experiments. Asterisk signifies statistical significance (P<0.05) between A. pittii and A. baumannii. The comparison between A. pittii and A. calcoaceticus was also significant (P<0.05).. (D) Persistence and biofilm formation of A. baumannii ATCC 19606^T (squares), A. pittii (upward triangles), A. nosocomialis (downward triangles) and A. calcoaceticus (diamonds) on three–dimensional human skin constructs. Results are expressed as mean CFU per skin construct ± SD of three independent experiments. Dotted line represents the lower limit of detection. Asterisk signifies statistical significance comparing A. calcoaceticus with A. baumannii ATCC 19606^T (P<0.05) (E) Alcian–blue PAS staining shows biofilm formation (black arrow) on human skin constructs by A. baumannii ATCC 19606^T but not by (F) A. calcoaceticus. Scale bar is equivalent to 20 µm. (G) Approximately 1×10⁴ CFU were injected in the thigh muscles of neutropenic mice and the number of viable bacteria was determined after 48 hrs. Results are expressed as mean number of bacteria (in CFU/muscle) ± SD from three animals. Dotted line represents lower limit of detection. Asterisk signifies statistical significance (P<0.05) between A. baumannii and A. nosocomialis or A. calcoaceticus.