Loss of a Major Enterococcal Polysaccharide Antigen (Epa) by Enterococcus faecalis Is Associated with Increased Resistance to Ceftriaxone and Carbapenems

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.

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.