Multiple prophage acquisition events over the course of an outbreak drive lysogenic conversion of capsular polysaccharides produced by carbapenem-resistant Acinetobacter baumannii isolates.

Prophages are important drivers of bacterial evolution and can provide pathogens with additional factors that enhance virulence and/or resistance properties. However, they are not considered when assessing the dynamics of outbreak populations using phylogenetic approaches. Recently, we showed that BAL114, an extensively antibiotic-resistant Acinetobacter baumannii isolate from Vietnam that carries KL58 at the capsular polysaccharide (CPS) biosynthesis locus, produced CPS with two types of linkage between KU58 tetrasaccharide units that were both different from the linkage formed by the WzyKL58 polymerase. Here, we determined the complete genome of BAL114 and identified two wzy genes, named wzy1Ph and wzy2Ph, in separate prophage that have driven lysogenic conversion by changing the BAL114 CPS. These prophages appear to suppress the action of WzyKL58. Comparison of the BAL114 chromosome with that of BAL062 from the same outbreak sublineage revealed that they shared 99.98% identity over common regions (96% coverage) but differed in prophage content and by recombination-mediated events. Further combinations of prophages with CPS-modifying genes were identified in other members of the same lineage, and a time-calibrated, recombination-free phylogeny revealed multiple prophage acquisitions that could drive structural changes in the CPS had occurred over the short course of the outbreak. The wzy1Ph and wzy2Ph genes were detected in several different prophage sequences found in diverse Acinetobacter genomes, suggesting that they are being transferred between prophages. This study revealed that prophages may use CPS modification for their own defense and highlighted the importance of considering prophage and recombination when tracing the evolution of bacterial populations.IMPORTANCECapsular polysaccharide (CPS) is a major virulence factor for the critical priority pathogen Acinetobacter baumannii and is also the primary receptor for a number of bacteriophages and monoclonal antibodies. Hence, changes in CPS structure may influence virulence potential and substantially impact patient outcomes using novel therapies. Here, we have shown that acquired genes found in prophage can alter the structure of the CPS produced by the chromosomal CPS biosynthesis locus by supplying alternate Wzy polymerases to link the K unit oligosaccharides together. This highlights the need to examine the accessory genome, which we found to be rapidly evolving, as it differed between the two isolates that are closely related based on single-nucleotide polymorphism phylogeny. Other members of the same lineage from the same outbreak carried various prophages that included CPS-modifying genes. Detecting these acquired genes will be needed to underpin the application of alternative therapies.