Mechanism of resistance in Enterococcus
drsuryabrata@gmail.comEnterococci are intrinsically resistant to many classes of antibiotics – like cephalosporins, Aminoglycoside (low-level resistance), macrolides, clindamycin, quinupristin-dalfopristin (E faecalis), Fusidic acid, Sulfonamide [EUCAST], which limits our options when we try to treat Enterococcal infections.
Most clinical infections are caused by Enterococcus faecalis, followed by Enterococcus faecium.
Other Enterococci occasionally isolated from the clinical specimen are – Enterococcus casseliflavus, Enterococcus gallinarum, Enterococcus durans, Enterococcus raffinosus etc.
Enterococcus gallinarum and Enterococcus casseliflavus are intrinsically resistant to vancomycin.
Beta-lactam resistance
- Low-affinity PBP – PBP5 (PBP4 in Enterococcus faecalis) has a low affinity for beta-lactams. Hence, it can continue peptidoglycan synthesis even when other PBPs are saturated. The intrinsic resistance is highest in cephalosporins than in carbapenems. It is lowest in penicillins, including aminopenicillins.
2. Acquired resistance to aminopenicillin – 85-90% of Enterococcus faecium and a small number of Enterococcus faecalis are resistant to amoxicillin. It is because of:
- Sequential mutation in the PBP5 gene
- Bypassing the PBP – ampicillin-insensitive l,d-transpeptidase, (Ldtfm) – an alternative enzyme is used instead of PBP in cell wall synthesis. [Sacco, 2014].
These mechanisms are common in Enterococcus faecium.
3. Beta-lactamase production – This is more common in Enterococcus faecalis; however, it has been reported in Enterococcus faecium occasionally. It is plasmid-mediated and similar to Staphylococcal beta-lactamase. These organisms are resistant to penicillin and amoxicillin but not to beta-lactam beta-lactamase combinations, for example, co-amoxiclav.
4. Penicillin-resistant but ampicillin-susceptible E. faecalis (PRASEF) – a point mutation in the PBP4 is responsible for this phenotype.
Glycopeptide
Glycopeptide-resistant Enterococcus are becoming a major antibiotic resistance issue worldwide. These strains are Glycopeptide Resistant Enterococcus (GRE) or Vancomycin-resistant Enterococcus (VRE). Although a specific resistance mechanism may make a strain resistant to vancomycin and not to teicoplanin, the acronym VRE is often used loosely for glycopeptide-resistant Enterococcus.
Certain clones of VRE are specially adapted to the hospital environment, multidrug-resistant or associated with outbreaks e.g. clonal complex E. faecium 17 (CC17).
The mechanism of action of the glycopeptides is inhibiting cell wall formation. The cell wall is composed of a rigid peptidoglycan layer, which is a polymer of two compounds, N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). Glycopeptides bind to the D-alanine-D alanine residue of the NAM and prevent cross-linking between two NAM molecules. This results in the prevention of peptidoglycan biosynthesis.
The mechanisms of resistance are –
- Van gene-mediated resistance – The van gene mutation converts D-Alanine-D-Alanine (D-Ala-D-Ala) to either D-Alanine-D-Lactate (D-Ala-D-Lac) or D- Alanine-D-Serine (D-Ala-D-Ser).
D-Ala-D-Lac has 1000-fold less affinity for glycopeptide, whereas D-Ala-D-Ser has a 7-fold lower affinity.
There are at least nine van genes, classified based on their level of resistance, transferability and inducibility.
| Genotype | Can be found in | Resistant to (phenotype) | Transferrable? |
| Van A | E faecalis, E faecium | Vancomycin, Teicoplanin | Chromosomal, Transferrable |
| Van B | E faecalis, E faecium | Vancomycin only (However, an additional mutation conferring teicoplanin resistance has been reported) | Chromosomal, Transferrable |
| Van C | E casseliflavus, E gallinarum | Vancomycin only (intrinsic resistance in these bacteria) | Chromosomal |
| Van D | E faecium E faecalis (also found in some E gallinarum) | Vancomycin, Teicoplanin (variable) | Chromosomal |
| Van E, Van L | E faecalis | Vancomycin only | Chromosomal |
| Van G | E faecalis | Vancomycin only | Chromosomal, Transferrable |
| Van M | E faecium | Vancomycin, Teicoplanin | Transferrable |
| Van N | E faecium | Vancomycin only | Plasmid, transferrable |
2. L,d-transpeptidase – This is a similar mechanism described in the beta-lactam section above. The crosslinking by this enzyme uses precursors without the terminal d-alanine, so glycopeptides cannot prevent this reaction. This is mainly seen in Ent faecium.
3. Vancomycin-variable enterococci (VVE) – This is an emerging resistance pattern in E faecium and E faecalis. These strains present with a vancomycin susceptible phenotype but develop resistance rapidly within a few days of exposure to Vancomycin or Teicoplanin. There are different mechanisms that result in the expression of vanHAX genes. Outbreaks have been reported in Canada and European countries.
Daptomycin
Epidemiological cut-off (ECOFF) value for E. faecium and E. faecalis to ≤4 μg/ml for daptomycin. The major mechanisms of resistance are –
- Activation of liaFSR regulatory system – This is a cell envelope stress response leading to a decrease in the phosphatidylglycerol content of the cell membrane. It also causes thickening of the cell wall and aberrant septum placement. Cross-resistance has been noted with vancomycin and bacitracin. This system may play a role in diverting the daptomycin from the preferred target on the septum to other areas of the cell membrane.
- Increase the thickness of the cell wall; altered homeostasis – yycFG [walKR] and vraSR mutation – thickening of the cell wall, altered homeostasis.
In addition to these, multiple other mutations may contribute to daptomycin resistance. These are gdpD (Glycerophosphoryl diester phosphodiesterase) and cls (Cardiolipin synthetase). These mutations often act together or in a sequential manner in conferring resistance.
The resistance is due to these phenotypic changes –
- generating a positive charge on the cell membrane to repulse daptomycin
- cell envelope stress response – decrease in PG content of cell membrane, thickening of cell wall, aberrant septum formation, diverting the daptomycin from its preferred site of action
- Increasing the fluidity of the cell membrane
Linezolid
- Target site modification due to mutations in domain V of 23S rRNA: There could be multiple mutations, and in Enterococcus, the critical step in the development of resistance is the first mutation. After that, sequential mutation may happen by recombination. Some of the mutations are – rplC mutation (affecting ribosomal protein L3), rplD gene mutation (affecting L4), and rplV (affecting L22). These mutations are chromosomal.
- Target site modification by Cfr methyltransferase. It is mediated by the cfr gene. This resistance is transferrable and may give rise to a phenotype called PhLOPSA (resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A). The presence of cfr mutation may not always confer resistance or may confer resistance to linezolid but spare tedizolid.
- OptrA gene-mediated target protection: optrA encodes for an ATP-binding cassette (ABC)-F protein and mediates resistance through target protection. It is transferrable. There is another similar gene that has been described PoxtA.
These confer resistance to phenicols, linezolid/tedizolid and tetracyclines. - Other mechanisms like cell wall thickening, biofilm formation and efflux pumps have been suggested or investigated.
Tigecycline
- Upregulated efflux pump – tetL gene-mediated. It increases the number of MFS superfamily efflux pumps.
- Target protection – ribosomal protection protein mediated by TetM gene.
- Target modification – mutation in ribosomal protein S10, mediated by rpsJ gene
