Genomic analysis of Acinetobacter baumannii prophages reveals remarkable diversity and suggests profound impact on bacterial virulence and fitness

Abstract

The recent nomination by the World Health Organization of Acinetobacter baumannii as the number one priority pathogen for the development of new antibiotics is a direct consequence of its fast evolution of pathogenicity, and in particular of multidrug resistance. While the development of new antibiotics is critical, understanding the mechanisms behind the crescent bacterial antibiotic resistance is equally relevant. Often, resistance and other bacterial virulence elements are contained on highly mobile pieces of DNA that can easily spread to other bacteria. Prophages are one of the mediators of this form of gene transfer, and have been frequently found in bacterial genomes, often offering advantageous features to the host. Here we assess the contribution of prophages for the evolution of A. baumannii pathogenicity. We found prophages to be notably diverse and widely disseminated in A. baumannii genomes. Also remarkably, A. baumannii prophages encode for multiple putative virulence factors that may be implicated in the bacterium's capacity to colonize host niches, evade the host immune system, subsist in unfavorable environments, and tolerate antibiotics. Overall our results point towards a significant contribution of prophages for the dissemination and evolution of pathogenicity in A. baumannii, and highlight their clinical relevance.

Figure 1

Prevalence of prophages in Acinetobacter baumannii genomes. (a) Whiskers plot of prophage frequency per bacterial genome. The horizontal line at the center of the whiskers plot represents the median. The bottom and top of the plot represent the first and third quartiles. The external edges of the whiskers represent the minimum and maximum number of prophages per genome. Significant differences (Tukey’s test) of P < 0.05 are represented by*. (b) Prevalence of total prophages, “intact prophages”, defective prophages, more than one “intact prophage”, and more than one defective prophage. Prevalence was determined considering a dataset of 795 A. baumannii genomes.

Figure 2

Distribution of prophages among Acinetobacter baumannii strains considering the size of the bacterial genome. (a) Average number of prophages (“intact” and defective) per bacterial genome size. (b) Average number of “intact prophages” per bacterial genome size. (c) Average number of defective prophages per bacterial genome size.

Figure 3

Distribution of genome size of the 109 “intact prophages” integrating Acinetobacter baumannii genomes. (a) Prevalence of prophages in A. baumannii genomes by family; (b) Whiskers plot of average genome size of prophages according to family. Horizontal line at the center represents the median, bottom and top of the plot represent the first and third quartiles, and external edges of the whiskers represent the minimum and maximum genome size of prophages per family. Significant differences (Tukey’s test) of P < 0.05 are represented by*.

Figure 4

Dot plot matrices of whole sequences of 109 prophages from Acinetobacter baumannii. (a) Whole genome analysis; and (b) Whole proteome analysis. Darker zones indicate higher identity. Clusters of prophages with identities higher than 50% are indicated and numbered. Graphics summarize the frequency of genome identity levels found in the analysis. For dot plot matrices with values of identity see Supplementary Tables S5 and S6. Matrices were adapted from the identity matrices retrieved from the phylogenetic trees constructed using Geneious Tree Builder.

Figure 5

Phylogenetic tree of prophage genomic sequences. Tree was constructed using the Tamura-Nei genetic distance model and the neighbor-joining tree building method in Geneious Tree Builder (Geneious version 9.1.8), with boostrapping set to 100 and tree rooted using Acinetobacter baumannii plasmid pNaval18-231 as the outgroup. Tree branches are proportional to branch length, and branch labels represent bootstrap percentages. Clusters of prophages with genome identities above 90% are indicated in the tree. Red: Siphoviridae; Green: Myoviridae; Blue: Podoviridae; Grey: family unknown.

Figure 6

Phylogenetic tree of prophage proteomic sequences. Tree was constructed using the Jukes-Cantor genetic distance model and the neighbor-joining tree building method in Geneious Tree Builder (Geneious version 9.1.8), with boostrapping set to 100 and tree rooted using Acinetobacter baumannii plasmid pNaval18-231 as the outgroup. Tree branches are proportional to branch length, and branch labels represent bootstrap percentages. Clusters of prophages with proteome identities above 90% are indicated in the tree. Red: Siphoviridae; Green: Myoviridae; Blue: Podoviridae; Grey: family unknown.

Figure 7

Putative virulence genes identified in the genomic sequences of 109 “intact” prophages of Acinetobacter baumannii. (a) Distribution of putative virulence genes per prophage; (b) Prevalence of potential virulence factors grouped by class.