Spontaneous resistance to phage NPV1 confers increased daptomycin susceptibility in Enterococcus faecalis OG1RF

Enterococcus faecalis is a Gram-positive opportunistic pathogen that inhabits the gastrointestinal tract. Because of the high frequency of antibiotic resistance among Enterococcus clinical isolates, interest in using phage to treat enterococcal infections and to decolonize high-risk patients for antibiotic-resistant Enterococcus is rising. Bacteria can evolve phage resistance, but there is little published information on these mechanisms in E. faecalis. In this report, we identified genetic determinants of resistance to phiNPV1 and their effects on E. faecalis susceptibilities to daptomycin and sodium chloride-induced osmotic stress. We found that loss-of-function mutations in epaR confer phiNPV1 resistance by blocking phage adsorption. We attribute the inability of the phage to adsorb to the loss of an extracellular polymer in strains with inactivated epaR. Phage-resistant epaR mutants exhibited increased daptomycin and osmotic stress susceptibility. Our results demonstrate that spontaneous resistance to phiNPV1 comes at a cost in E. faecalis OG1RF, resulting in concomitant increased susceptibilities to daptomycin and sodium chloride-induced osmotic stress.

decreased daptomycin susceptibility (24). Because of these results, we investigated the 137 daptomycin MIC of our OG1RF mutants ( Figure 5). We found that OG1RF-C, OG1RFΔepaR, 138 and OG1RFΔepaR complemented with the OG1RF-C epaR allele were each significantly more 139 susceptible to daptomycin than OG1RF. Interestingly, deletion of bgsB also conferred increased 140 daptomycin susceptibility ( Figure 5). This was complemented by expression of the wild-type 141 bgsB allele in cis, but not by expression of the OG1RF-C bgsB allele in cis ( Figure 5). Finally, 142 daptomycin susceptibility was substantially altered in the OG1RF∆epaR∆bgsB mutant, with 3 of 143 6 experimental trials resulting in an MIC below the level of detection of the E-test strip (<0.016 144 µg/mL; a value of 0.008 µg/mL was used for these data points in statistical analysis). Without 145 complete data regarding the MIC of OG1RF∆epaR∆bgsB, we did not quantitatively determine 146 whether there is a synergistic relationship between bgsB and epaR regarding daptomycin 147 susceptibility.
148 149 epaR protects OG1RF from osmotic stress. The epa gene cluster was up-regulated when E. 150 faecalis V583 was grown with 6.5% sodium chloride supplementation, indicating that the Epa 151 polymer has a role in osmotic stress response (25). As such, we investigated the effect of 152 sodium chloride on our epaR mutants. We tested our mutants for their tolerance for sodium 153 chloride stress using BHI agar supplemented with sodium chloride at concentrations of 0%, 154 2.5%, 5%, and 7.5%. Overnight cultures in stationary phase were serially diluted and spotted on difference in CFU compared to the wild-type ( Figure 6). However, when cells were cultured in 160 fresh BHI broth 1 h prior to spotting, a CFU difference was observed for OG1RF∆bgsB with 161 7.5% sodium chloride ( Figure S2). These data suggest that both epaR and bgsB play roles in 162 sodium chloride-induced osmotic stress response.

164
EpaR constitutes a major mutational pathway for ϕNPV1 resistance. We isolated 9 165 spontaneous ϕNPV1-resistant mutants of OG1RF and sequenced the epaR region of these 166 mutants. All 9 mutants have non-synonymous substitutions in epaR (Table 3). Since we know 167 that mutations in epaR affect daptomycin susceptibility, we also determined the daptomycin MIC 168 of these ϕNPV1-resistant strains and found that all were more significantly more susceptible to 169 daptomycin than the wild-type ( Figure S3).

171
Most of the EpaR sequence (amino acid positions 35-458 of 484 total) is a predicted sugar 172 transferase domain (TIGR03025; E-value: 2.45e -137 ). This domain consists of a conserved C-173 terminal region responsible for the sugar transferase activity (pfam02397) and a variable N-174 terminal region with predicted flippase activity. All epaR mutations in spontaneously ΦNPV1-175 resistant OG1RF strains occur in the C-terminal region of the sugar transferase domain (Table   176 3). We identified other proteins containing the same predicted sugar transferase domain as is indispensable for ϕNPV1 adsorption to E. faecalis OG1RF, and that inactivating mutations in 187 epaR constitute a major pathway for ϕNPV1 resistance in this strain background. We also found 188 that inactivating mutations in epaR and bgsB result in increased susceptibilities to daptomycin 189 and sodium chloride stress. Our results show that resistance to ϕNPV1 comes at a cost.

191
Adsorption to the host is the first step to a productive phage infection. For a tailed phage particle

203
The epa gene cluster is involved in the synthesis of a cell wall rhamnose polysaccharide 204 referred to as the enterococcal polysaccharide antigen (Epa). There is precedence for cell wall cluster consists of 18 core genes (epaA to epaR) and a set of strain-variable genes that occur 208 downstream (20, 29). Unfortunately, the Epa structure has not been determined (30), which is a 209 critical gap in knowledge about the enterococcal cell surface.

211
The connection between the epa gene cluster and ϕNPV1 resistance was first investigated by

242
In summary, in this study we characterized a mechanism for spontaneous ϕNPV1 resistance in 243 E. faecalis OG1RF. The major advance of this study is that we determined that phage 244 resistance comes at a cost to E. faecalis OG1RF; specifically, increased daptomycin 245 susceptibility. E. faecalis OG1RF is a natively daptomycin-sensitive strain that became more 246 sensitive as a result of phage resistance. We did not assess whether ϕNPV1 treatment could 247 reverse resistance in daptomycin-resistant clinical isolates. This is an exciting area for future 248 research.

251
Bacterial strains, media, and bacteriophages. A complete list of bacterial strains and 252 bacteriophage used in this study can be found in Table 1. E. faecalis strains were cultured in 253 brain heart infusion (BHI; Difco) at 37°C without agitation. E. coli strains were cultured in LB 254 broth at 37°C with shaking at 225 rpm unless otherwise stated. Plates of the appropriate media 255 were made by adding 1.5% agar to the broth prior to autoclaving. For MIC testing, Muller Hinton 256 media supplemented with 1.5% agar (MHA) was used. Phages were stored in phage buffer as when required for selection. 5-bromo 4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) was 259 used at 120 µg/mL and 40 µg/ml for E. faecalis and E. coli, respectively. The upper soft agar for 260 phage assays was M17 medium supplemented with 0.75% agar, while the lower layer was BHI 261 supplemented with 1.5% agar.  Table S1.  Table S1. The approximately 2.0 kb product was purified and digested with BamHI 292 and EcoRI. The digested product was ligated to plasmid pLT06 through restriction sites added 293 on the primers (highlighted in red in Table S1). The ligation product was then purified and 294 electroporated and propagated in E. coli EC1000. OG1RF was made electrocompetent using 295 the glycine method (36) (3% glycine) and transformed with 1 µg of the plasmid. OG1RF 296 transformants were screened for successful transformation and subsequently inoculated in BHI 297 supplemented with Cm at 30°C. The culture was diluted 1:100 in BHI and incubated at 30°C for

327
For some experiments, overnight cultures were subcultured into fresh BHI broth for 1 h prior to 328 serial dilution and spotting.

330
Phage adsorption assay. An overnight bacterial culture was diluted 1:5 in fresh BHI broth. The 331 culture was then equilibrated at 37°C for 20 min in a water bath. ϕNPV1 was added at an M.O.I 332 of 10 -3 . After 15 min, a 1 mL aliquot was centrifuged at 16.6 x g for 1 min at room temperature. determined as follow:     being phage buffer rather than 50% acetic acid. phiNPV1 was added at a PFU of 10 5 and the 556 mixture was incubated for 15 minutes at 37°C. The PFU was evaluated with phage spot assay.