Bacteriophage resistance alters antibiotic mediated intestinal expansion of enterococci

Abstract

27Enterococcus faecalis is a human intestinal pathobiont with intrinsic and acquired resistance to 28 many antibiotics, including vancomycin. Nature provides a diverse and virtually untapped repertoire of 29 bacterial viruses, or bacteriophages (phages), that could be harnessed to combat multi-drug resistant 30 enterococcal infections. Bacterial phage resistance represents a potential barrier to the implementation 31 of phage therapy, emphasizing the importance of investigating the molecular mechanisms underlying the 32 emergence of phage resistance. Using a cohort of 19 environmental lytic phages with tropism against E. 33 faecalis, we found that these phages require the enterococcal polysaccharide antigen (Epa) for 34 productive infection. Epa is a surface-exposed heteroglycan synthesized by enzymes encoded by both 35 conserved and strain specific genes. We discovered that exposure to phage selective pressure favors 36 mutation in non-conserved epa genes both in culture and in a mouse model of intestinal colonization. 37Despite gaining phage resistance, epa mutant strains exhibited a loss of resistance to the cell wall 38 targeting antibiotics, vancomycin and daptomycin. Finally, we show that an E. faecalis epa mutant strain 39 is deficient in intestinal colonization, cannot expand its population upon antibiotic-driven intestinal 40 dysbiosis and fails to be efficiently transmitted to juvenile mice following birth. This study demonstrates 41 that phage therapy could be used in combination with antibiotics to target enterococci within a dysbiotic 42 microbiota. Enterococci that evade phage therapy by developing resistance may be less fit at colonizing 43 the intestine and sensitized to vancomycin preventing their overgrowth during antibiotic treatment. 44 45 Importance 46With the continued rise of multidrug resistant bacteria, it is imperative that new therapeutic options 47 are explored. Bacteriophages (phages) hold promise for the amelioration of enterococcal infections, 48 however, the mechanisms used by enterococci to subvert phage infection are understudied. Here, we 49 demonstrate that a collection of phages require a cell surface exopolysaccharide for infection of E. 50faecalis. E. faecalis develops phage resistance by mutating polysaccharide biosynthesis genes at a cost, 51 as this renders the bacterium more susceptible to cell wall targeting antibiotics. E. faecalis phage resistant 52 3 isolates are also less fit at colonizing the intestine and these mutations mitigate E. faecalis intestinal 53 expansion upon antibiotic selection. This study suggests that the emergence of phage resistance may 54 not always hinder the efficacy of phage therapy and that the use of phages may sensitize bacteria to 55 antibiotics. This could serve as a promising avenue for phage-antibiotic combination therapies. 56 57 Introduction 58 Enterococci are Gram-positive commensal bacteria native to the intestinal tracts of animals, 59 including humans (1). Under healthy conditions, enterococci exist as minority members of the micr...

Parallel genomics uncover novel enterococcal-bacteriophage interactions

“…TnSeq 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, 35–37). 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.…”

“…epa genes encode proteins involved in the formation of a cell surface-associated rhamno-polysaccharide (34). We and others have previously demonstrated that enterococcal phages unrelated to VPE25 utilize Epa to adsorb and infect E. faecalis (23, 35–37). 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.…”

“…3B). Considering an epa mutant strain of E. faecalis is defective in colonization and outgrowth during antibiotic selection in the intestine (23), we believe that future investigation of LytR mediated regulation of epa and potentially other genomic loci could guide the development of effective therapeutics to control E. faecalis colonization and infections.…”

“…The primer sequences and restriction enzymes used for cloning are listed in Table S4. Plasmids were introduced into electrocompetent E. faecalis cells as previously described (23).…”

“…Recent studies have demonstrated the potential for phage-based therapies against systemic and biofilm associated enterococcal infections (18–22). The decolonization of intestinal MDR E. faecalis may be achieved through the action of phage predation which selects for cell wall variants that are rendered sensitive to antibiotic therapy (23). However, a potential barrier to the wide-spread use of phage therapy against E. faecalis is the development of phage resistance.…”