Bacteriophage (phage) can interact with bacteria in complex ways: they can eradicate bacterial populations via cell lysis, provide genetic traits to bacteria via horizontal gene transfer and chromosome integration, and block infection by other phages. Phage are highly abundant in the bladder, they rival bladder bacteria in terms of diversity, and their presence correlates with horizontal acquisition of genetic content by bladder bacteria. Bacteria can affect the health of the bladder and urinary tract, and phage can affect the abundance and fitness of bacteria. Potentially phage could affect the dynamics of the urinary microbiome in the asymptomatic state, or be a factor in dysbiosis and infection of the urinary tract. To assess the effect of phage on the bladder microbiota, our aim is to determine the host range of bladder phage and understand genetic determinants associated with that host range. We hypothesize that genetic elements (e.g. genes) in E. coli strains will be associated with resistance to phage infection.

Previously the phages Greed and Lust were isolated from the bladder of four women with urge urinary incontinence. In this study we utilize E. coli phages Greed and Lust as a model system to identify genetic content differences between E. coli strains susceptible and resistant to infection by these bladder coliphages. Using a standard phage lysis assay, we screened E. coli urine bladder isolates (UMB), standard lab strains (B, C, K-12), and uropathogenic E. coli (UPEC). We then analyzed the genomes of the screened E. coli strains using a novel algorithm that identifies genetic content associated with a difference in a binary phenotype (e.g. phage susceptible and resistant). The core of the algorithm matches a query sequence to genomes of the susceptible and resistant strains. If a hit is shared between susceptible and resistant strains, it is eliminated from further consideration; if a hit is not shared, then its open reading frame (ORF) is outputted. This ORF is then analyzed for sequence homology to known genes to assess if it is relevant to the phenotype of interest (e.g., phage host range).

All lab E. coli strains (K-12, B, C) and three UMB strains were susceptible to both Greed and Lust. All three UPEC and 47 UMB strains were resistant to both Greed and Lust. We identified PemIK, a toxin-antitoxin (TA) module, that is present in some resistant strains but not in any of the susceptible strains.

TA modules consist of a toxin that arrests bacteria cell growth under stress conditions and eventually kills the cell. Under unstressful conditions, the antitoxin binds and neutralizes the toxin; under stressful conditions, the antitoxin is degraded by proteases, the toxin is released and the cell dies. PemIK is known to maintain low copy plasmids in E. coli, functioning as an addiction mechanism that prevent loss of the plasmid. Little is known about PemIK in terms of phage infection; however, its homologue MazEF has been associated with phage cycle abortion. Our interpretation is that the PemIK module protects against Greed and Lust infection by limiting phage propagation in cells and spread to the population.

TA modules may be a factor that limits phage infection of bacteria in the bladder. The TA profile of the urobiome could affect the lysis of host bacteria or the horizontal transfer of fitness genes in bacteria, thus having an impact on the health of the bladder. In the future we plan to clone the PemIK module into the susceptible E. coli strain K-12, as we hypothesize this will result in a K-12 strain resistant to Greed and Lust infections. Identification of phage resistance factors in the bladder could lead to a better understanding of bladder health and beneficial traits to include in probiotics.