Enterococcus faecalis Countermeasures Defeat a Virulent Picovirinae Bacteriophage

Abstract

Enterococcus faecalis is an opportunistic pathogen that has emerged as a major cause of nosocomial infections worldwide. Many clinical strains are indeed resistant to last resort antibiotics and there is consequently a reawakening of interest in exploiting virulent phages to combat them. However, little is still known about phage receptors and phage resistance mechanisms in enterococci. We made use of a prophageless derivative of the well-known clinical strain E. faecalis V583 to isolate a virulent phage belonging to the Picovirinae subfamily and to the P68 genus that we named Idefix. Interestingly, most isolates of E. faecalis tested—including V583—were resistant to this phage and we investigated more deeply into phage resistance mechanisms. We found that E. faecalis V583 prophage 6 was particularly efficient in resisting Idefix infection thanks to a new abortive infection (Abi) mechanism, which we designated Abiα. It corresponded to the Pfam domain family with unknown function DUF4393 and conferred a typical Abi phenotype by causing a premature lysis of infected E. faecalis. The abiα gene is widespread among prophages of enterococci and other Gram-positive bacteria. Furthermore, we identified two genes involved in the synthesis of the side chains of the surface rhamnopolysaccharide that are important for Idefix adsorption. Interestingly, mutants in these genes arose at a frequency of ~10−4 resistant mutants per generation, conferring a supplemental bacterial line of defense against Idefix.

Parallel Genomics Uncover Novel Enterococcal-Bacteriophage Interactions

“…Tn-Seq identified numerous E. faecalis genes that govern VPE25 susceptibility. Mutations in epa variable genes conferred VPE25 resistance by preventing phage adsorption, similar to other E. faecalis phages (23,(35)(36)(37). We discovered that phage infection of mismatch repair gene mutants results in the emergence of phage adsorption deficiencies; thus, the mismatch repair system likely fails to correct DNA damage of epa genes during phage infection.…”

“…The epa genes encode proteins involved in the formation of a cell surface-associated rhamnose-containing polysaccharide (34). We and others have previously demonstrated that enterococcal phages unrelated to VPE25 utilize Epa to adsorb and infect E. faecalis (23,(35)(36)(37). Initial work from our group showed that mutation of the VPE25 receptor Pip EF prevented VPE25 DNA entry into E. faecalis V583; yet, phages could still adsorb to receptor mutants (32), suggesting that the factors that promote phage infection via surface adsorption remained to be identified.…”

Bacteriophage resistance alters antibiotic mediated intestinal expansion of enterococci

“…4A, 4B, and Table S1). Notably, epaX was recently found to aid in the adsorption of the Podoviridae phage Idefix (18).…”

“…Specifically, mutations in the core gene epaR and/or variable region genes including epaS, epaW, epaX, epaY and epaAC were sufficient to abrogate phage infection. Recently, it was discovered that mutation of epaR and epaX in E. faecalis prevented phage infection by excluding phage adsorption (16, 18). Here, we found that modifications made by epa variable genes are important for initial phage adsorption, although adsorption does not always lead to successful phage infection.…”

Virulent coliphages in 1-year-old children fecal samples are fewer, but more infectious than temperate coliphages

“…Coliphage genome sequencing. For 50 of the coliphages isolated, DNA was extracted from crude phage stocks 64 . Crude lysates were PEG precipitated, followed by a DNaseI/RNase treatment to remove external contamination, and DNA was extracted with the Wizard DNA clean-up system (Promega).…”