LETTER
The enterococcal polysaccharide antigen (Epa) locus of Enterococcus faecalis encodes enzymes and transporters involved in E. faecalis cell wall polysaccharide metabolism (1–3). We previously showed that deletion of epaB (which encodes a putative glycosyl transferase and possibly mediates transfer of rhamnose to cell wall polysaccharides [1]) and disruption of several other epa genes resulted in reduced virulence in murine peritonitis (1, 2) and urinary tract infection (4) models. In addition, OG1RFΔepaB and disruption of epaB were impaired in colitogenic activity in IL-10–/– mice (5) and showed lysozyme susceptibility (5), attenuation in nonvertebrate models (5), translocation (1), biofilm formation, resistance to polymorphonuclear leukocyte killing, susceptibility to NPV-1 phage (1), and marked alteration in cell morphology, suggesting a role for Epa in cell wall synthesis or function (1).
A recent study on Enterococcus hirae LcpA (Psr) (6) postulated that LcpA catalyzes the attachment of “rhamnose-containing polysaccharides” onto the cell’s peptidoglycan (6). Reasoning that altering rhamnose-containing polysaccharides of E. faecalis could alter its peptidoglycan and thus the activity of beta-lactams, we examined our E. faecalis epa mutants, including those lacking cell wall rhamnose, to determine whether the MICs of select beta-lactams against these mutants differ versus OG1RF.
The strains used included wild-type (WT) OG1RF (7, 8), OG1RFΔepaB, reconstituted OG1RFΔepaB::epaB (5, 9), and several epa disruption mutants (1, 2) [OG1RFepaA::aph(3′)-IIIa, OG1RFepaB::aph(3′)-IIIa, OG1RFepaE::aph(3′)-IIIa, OG1RFepaL::aph(3′)-IIIa, and OG1RFepaN::aph(3′)-IIIa] representing different transcripts of the epa gene cluster.
MICs were determined with Etest strips of ceftriaxone (CRO), ampicillin (AMP), doripenem (DOR), meropenem (MEM), and daptomycin (DAP; which acts via unrelated mechanisms) (AB Biodisk, Solna, Sweden; Liofilchem, Inc., Waltham, MA) on cation-adjusted Mueller-Hinton II (Oxoid, UK) agar plates. MICs were read according to the manufacturer’s guidelines, and MIC determinations were repeated at least twice with reproducible results. Table 1 shows the MICs of test antibiotics against WT OG1RF and epa mutants.
TABLE 1.
MICs against WT and epa locus mutants of E. faecalis OG1RF
| Organism | E-test MIC (μg/ml)a |
||||
|---|---|---|---|---|---|
| CRO | AMP | DOR | MEM | DAP | |
| WT E. faecalis OG1RF | 24 | 0.5 | 6 | 4 | 1–2 |
| OG1RFepaA::aph(3′)-IIIa | >256 | 0.5 | >32 | 6 | 0.25–0.38 |
| OG1RFepaE::aph(3′)-IIIa | >256 | 1 | >32 | 12 | NT |
| OG1RFepaL::aph(3′)-IIIa | >256 | 1 | >32 | 12 | NT |
| OG1RFepaN::aph(3′)-IIIa | >256 | 1 | >32 | 24 | NT |
| OG1RFepaB::aph(3′)-IIIa | >256 | 1.5 | >32 | >32 | 1.5–2 |
| OG1RFΔepaB | >256 | 2 | >32 | >32 | 1–2 |
| OG1RFΔepaB::epaB | 24 | 1 | 4 | 4 | 1–2 |
CRO, ceftriaxone; AMP, ampicillin; DOR, doripenem; MEM, meropenem; DAP, daptomycin; NT, not tested.
The CRO MIC increased from 24 μg/ml (OG1RF and OG1RFΔepaB::epaB) to >256 μg/ml with OG1RFΔepaB and the disruption mutants, including OG1RFepaA::aph(3′)-IIIa, which has the most growth impairment (1). The DOR and MEM MICs were also considerably higher with epa mutants than with OG1RF and OG1RFΔepaB::epaB.
AMP MIC differences were modest between OG1RF and the epa mutants and the growth impaired OG1RFepaA::aph(3′)-IIIa mutant did not show any change. The DAP MIC was the same for all strains except OG1RFepaA::aph(3′)-IIIa, likely due to its growth impairment.
Our previous data on polysaccharide (PS) content showed that a major polysaccharide band was missing in epa disruption mutants and was restored in the epaB complemented strain (1). In addition, our analyses of purified polysaccharide (Epa) from WT OG1RF and the OG1RFepaB::aph(3′)-IIIa mutant showed that Epa is composed of glucose, rhamnose, N-acetylglucosamine, N-acetylgalactosamine, and galactose, while there was no rhamnose present in Epa purified from the OG1RFepaB::aph(3′)-IIIa but, instead, mannose was present (1).
Our results indicate that altered cell wall polysaccharide content, such as lack of rhamnose and/or the presence of mannose in epa mutant cell walls, is associated with marked resistance to CRO, DOR, and, MEM. The mechanism for resistance remains to be determined.
ACKNOWLEDGMENT
We thank Karen Jacques-Palaz for technical assistance.
REFERENCES
- 1.Teng F, Singh KV, Bourgogne A, Zeng J, Murray BE. 2009. Further characterization of the epa gene cluster and Epa polysaccharides of Enterococcus faecalis. Infect Immun 77:3759–3767. doi: 10.1128/IAI.00149-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Xu Y, Singh KV, Qin X, Murray BE, Weinstock GM. 2000. Analysis of a gene cluster of Enterococcus faecalis involved in polysaccharide biosynthesis. Infect Immun 68:815–823. doi: 10.1128/IAI.68.2.815-823.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Hancock LE, Gilmore MS. 2002. The capsular polysaccharide of Enterococcus faecalis and its relationship to other polysaccharides in the cell wall. Proc Natl Acad Sci U S A 99:1574–1579. doi: 10.1073/pnas.032448299. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Singh KV, Lewis RJ, Murray BE. 2009. Importance of the epa locus of Enterococcus faecalis OG1RF in a mouse model of ascending urinary tract infection. J Infect DIS 200:417–420. doi: 10.1086/600124. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Ocvirk S, Sava IG, Lengfelder I, Lagkouvardos I, Steck N, Roh JH, Tchaptchet S, Bao Y, Hansen JJ, Huebner J, Carroll IM, Murray BE, Sartor RB, Haller D. 2015. Surface-associated lipoproteins link Enterococcus faecalis virulence to colitogenic activity in IL-10-deficient mice independent of their expression levels. PLoS Pathog 11:e1004911. doi: 10.1371/journal.ppat.1004911. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Marechal M, Amoroso A, Morlot C, Vernet T, Coyette J, Joris B. 2016. Enterococcus hirae LcpA (Psr), a new peptidoglycan-binding protein localized at the division site. BMC Microbiol 16:239. doi: 10.1186/s12866-016-0844-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Bourgogne A, Garsin DA, Qin X, Singh KV, Sillanpaa J, Yerrapragada S, Ding Y, Dugan-Rocha S, Buhay C, Shen H, Chen G, Williams G, Muzny D, Maadani A, Fox KA, Gioia J, Chen L, Shang Y, Arias CA, Nallapareddy SR, Zhao M, Prakash VP, Chowdhury S, Jiang H, Gibbs RA, Murray BE, Highlander SK, Weinstock GM. 2008. Large-scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF. Genome Biol 9:R110. doi: 10.1186/gb-2008-9-7-r110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Murray BE, Singh KV, Ross RP, Heath JD, Dunny GM, Weinstock GM. 1993. Generation of restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function. J Bacteriol 175:5216–5223. doi: 10.1128/jb.175.16.5216-5223.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Panesso D, Montealegre MC, Rincon S, Mojica MF, Rice LB, Singh KV, Murray BE, Arias CA. 2011. The hylEfm gene in pHylEfm of Enterococcus faecium is not required in pathogenesis of murine peritonitis. BMC Microbiol 11:20. doi: 10.1186/1471-2180-11-20. [DOI] [PMC free article] [PubMed] [Google Scholar]