Intrinsic and acquired resistance of enterococci

Intrinsic and acquired resistance

Two main groups of chromosomal mutations cause two mechanisms of resistance in enterococci. The first causes low-level resistance by reduced drug accumulation either by decreasing the uptake or increasing the efflux of the drug. Endogenous efflux pumps seem to be widespread among wild-type strains of enterococci and might be the explanation for the intrinsic low-level resistance of most enterococci to the fluoroquinolones. 

High-level resistance to fluoroquinolones is due to mutations in regions encoding subunits of DNA gyrase and topoisomerase IV (gyrA, gyrB, parC and parE). Fluoroquinolones interact with complexes of each enzyme in DNA by trapping the complex and hinder further DNA replication . This leads to cell death by yet poorly defined mechanisms. In enterococci mutations in gyrA at positions 83 and 87 and parC at position 80 are more extensively studied than mutations in gyrB and parE. Ciprofloxacin resistance seems to be more widespread in E. faecium, may be due to clonal spread of such strains. It should be emphasized that ciprofloxacin-resistance in enterococci also confers cross-resistance to newer quinolones with better Gram-positive activity and that superinfections in patients treated with fluoroquinolones have been reported. 

 Tetracycline resistance

Tetracycline inhibits protein synthesis by interfering with the binding of amionoacyl tRNA to the ribosome. Tetracycline resistance in enterococci is most commonly encoded by tet(M) that usually is carried by Tn916 or related conjugative transposons that has been found in isolates from both animals and humans. Another gene, tetN, was originally identified on a plasmid in Streptocoous agalactiae that was subsequently transferred to and stably maintained in E. faecalis . tetO has also been found in enterococci; it was originally found in Campylobacter spp. and shows about 75% homology with tetM. These various genes confer resistance by two different mechanisms; tetL mediates active efflux of tetracycline from cells, the same mechanism commonly found in gram-negative bacilli , while tetM and tetN mediate resistance by a mechanism that protects the ribosomes from inhibition by tetracycline. An interesting feature of plasmid pAMa.1 (containing tetL) is that the resistance genes duplicate or amplify when the host is grown in sub-inhibitory concentrations of tetracycline. This results in an increase in the size of the plasmid and also results in higher MICs.

 Macrolide resistance

Macrolides is a group of antimicrobials produced by Streptomyces spp. Erythromycin and tylosin have been used in treatment of infections caused by Gram-positive cocci in both animals and humans. Tylosin has also together with spiramycin been used as growth promoting agents given to animals. Resistance to macrolides is very common among enterococci isolated from humans and from pigs and is most commonly encoded by the erm(B) gene, located on the Tn917 in humans, but this transposon has also been found in bacteria from other sources .

 Glycopeptides resistance

The most recent resistance trait to emerge in enterococci is resistance to vancomycin. Glycopeptide antibiotics, vancomycin and teicoplanin, are used in the treatment of serious infections due to enterococci in cases of resistance or allergy to β-lactams. Despite more than 30 years of clinical use of vancomycin, glycopeptide resistance in enterococci has rarely been detected. However, resistant strains responsible for colonization or infection have been isolated with an increasing frequency from patients in the presence or absence of glycopeptide therapy . Vancomycin and teicoplanin inhibit cell wall synthesis by binding to the D-alaninyl-D-alanine terminus of a pentapeptide cell wall precursor.

The glycopeptides are very large hydrophobic molecules that bind to the peptidyl-D-alanyl-D-alanine termini of the peptidoglycan precursors at the cell surface. The mechanism of action is thought to be as simple as steric inhibition of further cell wall synthesis by the presence of these large molecules at the surface of the cytoplasmic membrane alone, and thus forms a steric hinder that inhibits further cell wall synthesis. Resistance to glycopeptides is mediated by synthesis of modified peptidoglycan precursors to which the glycopeptides cannot bind .

 Six types of glycopeptide resistances have been described in enterococci that can be distinguished on the basis of sequence of the structural gene for the resistance ligase (vanA, vanB, vanC, vanD, vanE and vanG). The VanC phenotype is mainly manifested in species that do not yet pose a significant clinical threat, and little is known about VanD and VanE mechanisms of resistance. E. gallinarum, E. flavescens and E. casseliflavus possess vanC that confer an intrinsic low-level resistance to vancomycin MIC (4 –32 µg/L), but is not transferable. The vanA gene cluster has been reported in several species E faecium, E. faecalis , E avium, E. casseliflavus, E. gallinarum  and E. durans.  

vanA is encoded by a transposon, Tn1546, which is either integrated on the bacterial chromosome or located on a plasmid. vanA contains a resolvase and a transposase, two enzymes that regulate the integration of Tn1546 into foreign DNA, as well as seven other genes (vanS, vanR, vanH, vanA, vanX, vanY, and vanZ). vanS is implicated in sensing vancomycin while vanR induces at least some of the other Tn1546-encoded genes. The vanH dehydrogenase produces D-lactate that is attached to D-alanine by the vanA ligase. The resulting D-ala-D-lactate depsipeptide substitutes for the D-alaninyl-D-alanine moiety of the cell wall precursor, thereby inhibiting vancomycin binding and restoring cell wall synthesis. vanX and vanY cleave the remaining D-alaninyl-D-alanine termini, ensuring even higher levels of vancomycin resistance .    

The mechanism of resistance (Figure. 2.2) has been best characterized for the vanA cluster of seven genes found on the transposable (mobile) genetic element Tn1546. In the presence of an inducer like vancomycin, transcription of the genes necessary for resistance to vancomycin is activated as a result of the interactions of a sensory kinase and a response regulator. The transcribed genes are translated into enzymes, some of which make cell-wall precursors ending in D -alanyl- D -lactate (D -Ala- D -Lac), to which Vancomycin binds with very low affinity. Others prevent synthesis of or modify endogenous cell-wall precursors ending in D -alanyl- D–alanine (D -Ala- D -Ala), to which vancomycin binds with high affinity. All but one of the genes in the vanA clusters have homologues in vanB gene clusters that, in turn, have a unique gene not found in the vanA clusters.  Less is known about VanD or VanE types of resistance, but the genes for types A, B, D, and E all appear to be acquired. In contrast, the genes encoding the VanC type of vancomyin resistance are endogenous, species-specific components of E. gallinarum (vanC-1) and E. casseliflavus/E. flavescens (vanC-2/vanC-3), respectively.