Mechanism of resistance

Published 27.1.22

Enterococci 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 which is occasionally isolated from the clinical specimen is – Enterococcus casseliflavus, Enterococcus gallinarum, Enterococcus durans, Enterococcus raffinosus etc.

Enterococcus gallinarum and Enterococcus casseliflavus are intrinsically resistant to vancomycin.

Low affinity PBP

PBP5 (which is PBP4 in Enterococcus faecalis) have low affinity for beta-lactams. Hence, it can continue peptidoglycan synthesis even when other PBPs are saturated. The intrinsic resistance is highest in cephalosporins, then in carbapenems. It is lowest in penicillin including aminopenicillins.

Acquired resistance to aminopenicillin

85-90% of Enterococcus faecium and a small number of Enterococcus faecalis are resistant to amoxicillin. In it is because of:
1. Sequantial mutation in the PBP5 gene.
2. 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.

Beta-lactamase production

This is more common in Enterococcus faecalis, however has been reported in Enterococcus faecium occasionally. It is plasmid-mediated and similar to Staphylococcal beta-lactamase. These organisms are resistant to penicillin, amoxicillin but not to beta-lactam beta-lactamase combinations, for example, co-amoxiclav.

Penicillin-resistant but ampicillin-susceptible E. faecalis (PRASEF)

a point mutation in the PBP4 is responsible for this phenotype.

Glycopeptide resistant Enterococcus are becoming a major antibiotic resistance issue worldwide. These strains are known as 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 the 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 result in the prevention of peptidoglycan biosynthesis.

Van gene medicated resistance

There are at least 6 types of van gene which cause glycopeptide resistance. The van gene mutation convert changes the substitution of 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 7 fold lower affinity.

There are at least 9 van genes, classified based on their level of resistance, transferability and inducibility.

L,d-transpeptidase

This is a similar mechanism that has been described in the beta-lactam section above. The crosslinking by this enzyme use precursors without the terminal d-alanine, so glycopeptides are unable to prevent this reaction. This is mainly seen in Ent faecium.

Vancomycin-variable enterococci (VVE)

This is an emerging resistance pattern, seen in both E faecium and E faecalis. These strains present with a vancomycin susceptible phenotype but develop resistance rapidly within a few days on exposure to Vancomycin or Teicoplanin. There are different mechanisms that result in the expression of vanHAX genes. Outbreaks have been reported from Canada and European countries.

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 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 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

Target site modification due to mutations in domain V of 23S rRNA

There could be multiple mutation and in Enterococcus the critical step in development of resistance is the forst mutation. After that sequencial 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 cfr gene. These resistance is transferrable and may give rise to a phenotype called PhLOPSA (resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A). Presence of cfr mutation may not alsways 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 has been described PoxtA.
These conferes resistance to phenicols, linezolid/tedizolid and tetracyclines.

Other mechanisms

Cell wall thickening, biofilm formation and efflux pumps have been suggested or being investigated.

• Upregulated efflux pump – tetL genemediated. It increases the numebr of MFS superfamily efflux pump.

• Target protection – ribosomal protection protein meiated by TetM gene.

• Target modification – mutation in ribosomal protein S10, mediated by rpsJ gene

Moon et al, The structures of penicillin-binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into β-lactam resistance, J Biol Chem, . 2018 Nov 30;293(48):18574-18584. doi: 10.1074/jbc.RA118.006052. Epub 2018 Oct 24.

Gagetti et al, Resistance to β-lactams in enterococciResistencia a los β-lactámicos en enterococos, Revista Argentina de Microbiología, Volume 51, Issue 2, April–June 2019, Pages 179-183.

Sacco et al, Serine/Threonine Protein Phosphatase-Mediated Control of the Peptidoglycan Cross-Linking l,d-Transpeptidase Pathway in Enterococcus faecium, ASM Journals, mBio, Vol. 5, No. 4

Ahmed and Baptiste, Vancomycin-Resistant Enterococci: A Review of Antimicrobial Resistance Mechanisms and Perspectives of Human and Animal Health, Microbial Drug ResistanceVol. 24, No. 5, 1 Jun 2018https://doi.org/10.1089/mdr.2017.0147

Bender et al, Update on prevalence and mechanisms of resistance to linezolid, tigecycline and daptomycin in enterococci in Europe: Towards a common nomenclature, Drug Resistance Updates, Volume 40, September 2018, Pages 25-39

Fiedler et al, Tigecycline resistance in clinical isolates of Enterococcus faecium is mediated by an upregulation of plasmid-encoded tetracycline determinants tet(L) and tet(M), J Antimicrob Chemother, 2016 Apr;71(4):871-81.