Introduction

Enterococci are non-obligatory anaerobes oval in morphology, appear as a single cell, pairs or short chains, on a smear. E. faecalis had been reported more prevalent than E. faecium in humans. The most frequently caused infection by these organisms includes Urinary tract infections (UTI’s), wound infections, followed by bloodstream infection (Sood, Malhotra, Das, & Kapil, 2008). The rarely encountered infections include neonatal, central nervous system and respiratory tract infections. These organisms display intrinsic low-level resistance to Penicillins, Cephalosporins, Clindamycin, Nalidixic acid and Aminoglycosides (Arias & Murray, 2012; Kristich, Rice, & Arias, 2014).

The primary threat involved in the selection of therapeutic agents against severe infection caused by these agents is their ability to exhibit innate resistance to antibiotic groups. The basis towards the appearance of decreased susceptibility to multiple drugs in Enterococci are due to

• Innate potential to display decreased susceptibility to various antibiotics.

• Acquisition of antimicrobial resistance genes through translocation on plasmids and chromosomes.

• The ability for an interchange of low susceptibility genes (Shantala & Nagarathnamma, 2015).

Glycopeptides group of antibiotics

These group of antibiotics integrate with peptides and develop complexes, which ends in Acyl D-alanyl alanine. This combination impedes cell wall biosynthesis, which results in a defect of cell wall integrity.

Isolates of Enterococci resistant to Glycopeptides develops modified mucopeptide precursor in which dipeptide ends are converted to either lactate or serine which results in decreased affinity to bind to mucopeptide precursors and make them incapable of preventing cell wall biosynthesis (Miller, Munita, & Arias, 2014).

Based on phenotypic and genotypic characters, nine distinct gene clusters from Van A to Van N are reported in Enterococci (Shantala et al., 2015; Sujatha & Praharaj, 2012).

Vancomycin-Resistant Phenotype A

These phenotypes are linked with Minimum Inhibitory Concentration of ≥ 128 microgram/ml for vancomycin and ≥ 8 microgram/ml for Teicoplanin. Isolates of Enterococcus from a urine sample with MIC of ≥ 32µg/ml for glycopeptides have been observed in our study (Rajan, Amirtha, Soundaram, & Anandi, 2017).

The following genes define the generation of modified mucopeptide precursors:

• Van gene A: This ligase generates the depsipeptide ending in D-alanyl lactate which binds Vancomycin with decreased affinity.

• Van gene H: This Dehydrogenase generates D- lactate from pyruvate.

• Van gene X: This dipeptidase splits dipeptide, of Chromosomal origin and creates glycopeptides –sensitive pentapeptides termini to incorporate into growing murein chain.

• Van gene Y: This carboxypeptidase removes alanine units from dipeptide termini and supplement dipeptidase action.

• Van gene R / S: This system detects glycopeptide presence and prompts van genes expression. Van S serves as a detector of vancomycin presence and transfers a signal to response regulator R thereby activating generation of van H, van A and van X involved in resistance mechanism (Depardieu, Podglajen, Leclercq, Collatz, & Courvalin, 2007).

• Van gene Z: Contributes to low Teicoplanin susceptibility.

Vancomycin-resistant phenotype B

Enterococci with resistant phenotype display Minimum Inhibitory Concentration of ≥8 microgram/ml to vancomycin but not activated by glycopeptide, Teicoplanin. This resistant phenotype is acquired on extrachromosomal genetic elements or host chromosome. This phenotype encodes Van R_B and Van S_B-a two-component system, homologs of Van H, D –Alanyl alanine ligase (Van X) and carboxypeptidases (Van Y).

Vancomycin dependent Enterococci

Resistant Isolates of Enterococci dependent on vancomycin for growth have evolved in Phenotypes A and B. These strains shut off dipeptide synthesis and needs dipeptide alternative for growth. Dipeptide, D-alanyl-D-alanine is not required for the biosynthesis of the cell wall in resistant isolates while vancomycin is available. Additionally, in the absence of dipeptide, D-alanyl-D-alanine or D-alanyl-D-lactate, the cell cannot resume to develop or reproduce (Kristich et al., 2014).

Aminoglycosides

In E. faecium, inherent resistance is amplified by rRNA methyltransferase encoded on a chromosome, EfmM gene that uses S-adenosyl methionine, methylation of specific residue on 16S rRNA by methyl donor (Galimand et al., 2011). The deactivation of efmM enhances the ability of these organisms to become susceptible to Aminoglycosides, Tobramycin and Kanamycin. Low-level intrinsic resistance to aminoglycosides is chromosomally encoded on ^’N –acetyltransferase.

Low susceptibility to aminoglycosides are acquired and are encoded on aminoglycoside modifying enzymes. The bifunctional gene encoding, acetylase and phosphotransferase enzyme confer high-level resistance aminoglycosides excluding Streptomycin (Hollenbeck & Rice, 2012). Our study reported more than 70% urinary isolates with MIC≥500 µg/ml for High-level Gentamicin and 30% isolates with MIC≥2000 µg/ml for High-level Streptomycin (Rajan, Amirtha, Soundaram, & Anandi, 2017).

Resistance to Streptomycin is either ribosomal or due to adenyl transferase enzyme. Most isolates of E. faecium produce 6^’ acetylase transferase enzyme, thereby these isolates develop inborn resistance to aminoglycosides (Hollenbeck et al., 2012).

In isolates with MIC of 62 - 500 microgram/ml (moderate level resistance) to Streptomycin, the low permeability observed may overcome with the use of cell wall acting agents. In strains with MIC of more than 2000 microgram/ml, cell wall acting agents enhance absorption of Streptomycin.

In isolates that produce inactivating enzymes, high-level resistance to Streptomycin is controlled at a concentration of 16, 000 microgram /ml. In contrast, ribosomal resistance is not affected at higher concentration of 128, 000 microgram/ml of Streptomycin (Miller et al., 2014).

In isolates with MIC of 256 microgram/ml for Gentamicin, and <500 micrograms/ml for Streptomycin, combination with cell wall acting agent can benefit therapy. Prevalence of acetylase transferase enzyme and subclass APH - (3') - III an encoding gene contributes to Kanamycin resistance (High level) and nullify Amikacin synergism. Nephrotoxic potential of these drugs restricts its use in severely affected patients (Hollenbeck et al., 2012).

Beta-lactam antibiotics

The decreased affinity of Penicillin Binding Proteins (PBP's) is accountable to diminished Penicillin susceptibility in Enterococci. Consequently, minimum inhibitory concentration is higher in Enterococci than in Streptococci. Thus cessation of PBP’s does not progress to antibacterial activity in isolates. Exposure of susceptible isolates of E.faecalis to Penicillin lead to mutant strains with enhanced production of Penicillin Binding Protein 5.Strains of E.faecium display enhanced intrinsic resistance to Penicillin with MIC of 16-32 microgram/ml than isolates of E.faecalis with MIC of 2-4 microgram /ml.

In E. faecalis plasmid-mediated bla genes encoding β lactamases production have been reported Enterococci isolates display inherent resistance to beta-lactam antibiotics as a result of low tendency penicillin-binding proteins, belong to class B. Due to less inclination for beta-lactam drugs, chromosomal Penicillin Binding Protein accomplishes mucopeptide biosynthesis at drug concentration which saturates other Penicillin Binding Proteins. In E. faecium clinical isolates, decreased susceptibility to β lactamase enzyme is connected with a genetic variation or Penicillin Binding Protein 5 hyper-production with Minimum Inhibitory Concentration of more than 256 milligram/L for Ampicillin in few isolates. In E. faecalis β lactamase enzyme producers will respond to the inhibitor of β lactams and aminoglycoside combination. E. faecium isolates may respond to Ampicillin with Minimum Inhibitory Concentration of ≤64 mg/L (Arias, Contreras, & Murray, 2010).

The genetic determinants responsible for inherent resistance to cephalosporins in isolates of Enterococcus faecalis are less affinity Penicillin Binding Proteins (Kristich & Little, 2012; Miller et al., 2014).

Penicillin Binding Protein₅ modifications are linked with enhanced beta-lactam resistance such as ampicillin. Conjugative transfer of chromosomally coded PBP₅ genetic determinants is transmitted among E. faecium strains. In E. faecalis strains, the mutation in Penicillin Binding Protein₅ may result in augmented Imipenem and Ampicillin resistance (Kristich et al., 2014). In Enterococci, the Minimum Inhibitory Concentration of amoxicillin or ampicillin is one dilution lesser than Penicillin.

Macrolides, Lincosamide and Streptogramin

Acquired resistance to macrolides, due to erm B genotype lessen the ability of these drugs to bind 50S ribosomal subunit. Low-level resistance to Macrolide is due to efflux gene,mefA which drive macrolides of the cell (Garrido, 2014)

E. faecalis is inherently resistant to Quinupristin-Dalfopristin (Q-D), due to the presence of chromosomal genetic determinant ABC transporter (Lsa). Quinupristin drug has been proved as an effective therapeutic agent for resistant isolates of E. faecium. Combination therapy of Quinupristin-Dalfopristin (Q-D) with Doxycycline, Gentamicin, Rifampicin and Ampicillin is utilised for E. faecium endocarditis and Vga genes, in E. faecium, an ABC transporter which exports the antibiotic from the cell (Miller et al., 2014).

Linezolid

Linezolid impedes bacterial growth by hindering synthesis of protein through translational initiation complex interaction (Bourgeois‐Nicolaos & Massias, 2007; Shantala et al., 2015). Linezolid is accounted as an alternative drug for CNS infections by Vancomycin-resistant isolates of E. faecium. This drug is described as curative for endocarditis and bloodstream infection, Urinary Tract Infection and wound infections caused by vancomycin-resistant isolates. The clinical success rate of Linezolid therapy for Vancomycin-resistant bloodstream infection ranges from 58 % to 78 %. Long term therapy with linezolid can lead to anaemia, neutropenia and thrombocytopenia in VRE patients (Chong, 2010). In our study among clinical isolates of Enterococci, more than 89% isolates showed susceptible MIC of ≤2µg/ml (Rajan, Amirtha, Soundaram, & Anandi, 2017).

Daptomycin

Daptomycin, display extensive spectrum activity against bacteria such as Staphylococcus aureus and Enterococci and have been used for skin and underlying tissue infections. Daptomycin acts by membrane insertion, depolarisation, the release of ions and results in cell death. A high dose therapy with Daptomycin should be considered for endovascular infections by Enterococci (Kristich et al., 2014; Smith et al., 2015). Combination therapy with 8mg/kg of Daptomycin, Gentamicin and Ampicillin/Rifampicin is proved to be effective in cases of endocarditis by resistant isolates of Enterococci (Hindler & Wong-Beringer, 2015).

Quinolones

Fluoroquinolone resistance due to mutation inhibits affinity of enzymes DNA gyrase and topoisomerase IV to Fluoroquinolones which allows DNA replication to continue notwithstanding the presence of quinolone (Leavis, Willems, Top, & Bonten, 2006).

In E. faecalis, inactivation of Qnr homolog contributed to the decline in resistance to quinolones resistance and hyperexpression of Qnr leads to enhanced resistance (Arsène & Leclercq, 2007). Fluoroquinolones are recommended for uncomplicated Urinary Tract Infections due to its in-vitro susceptibility against Enterococci isolates. Nitrofurantoin and Fosfomycin have been considered as alternative drugs useful for uncomplicated lower urinary tract infections management. Though the susceptibility pattern of VRE towards Doxycycline and Fluoroquinolones varies, these drugs have been proven for the treatment of infections of the skin and underlying tissues and urinary tract (López, Tenorio, Campo, Zarazaga, & Torres, 2011; Werner & Fleige, 2010).

Rifampicin

In Enterococci, rifampicin binds to β subunit of RNA replicase enzyme and blocks mRNA transcription (Kristich et al., 2012). Rifampicin resistance emerges by a variety of genetic changes in rpoB protein. In isolates with rpoB mutation, decreased susceptibility to cephalosporins is observed (Kristich et al., 2014). In rare strains of E. faecium, rifampicin resistance can be reversed by including a less inhibitory concentration of Daptomycin.

Tetracyclines and Glycylcycline

In clinical isolates of Enterococci, tetracycline resistance is generally linked to protein tet (M) and efflux proteins, tet (K) and tet (L) (Chopra & Roberts, 2001; Garrido, 2014).

Tigecycline exhibit extensive activity and potency against Gram-positive organisms with Minimum Inhibitory Concentration ₉₀ value of ≤ 12 microgram/ml. The effectiveness of Tigecycline in therapy with antibacterial agents such as glycopeptide, aminoglycoside, Rifampicin or Daptomycin is reported (Shantala et al., 2015). Our study reported 88% isolates with susceptible MIC of ≤0.12µg/ml (Rajan et al., 2017) for Tigecycline.

Lipoglycopeptides

Lipoglycopeptides display activity against Vancomycin susceptible isolates of Enterococcus. Lipoglycopeptides such as oritavancin telavancin and dalbavancin disintegrate membrane potential, enhances the antibacterial activity and cell death.

Telavancin is recommended for the therapy of complicated skin infections. Oritavancin is active against Van A resistant Phenotype of Enterococci (Linden, 2001).

Conclusion

The unique nature of Enterococci to harbour and transfer resistance genes and the evolution of multidrug resistance poses enormous challenges to clinicians. A better knowledge of resistance mechanism and treatment options will aid in the control of epidemiologic spread. Combination therapy with novel antibiotic groups such as Tigecycline and Daptomycin are promising treatment modalities for severe enterococcal infection.

Acknowledgement

I would like to thank Dr V.Anandi, Former Professor, Department of Microbiology, Vinayaka Mission’s Medical College Karaikal, Tamil Nadu, for guiding me in my research work.

Sources of funding

The author declare that there is no funding support for this study.

Conflict of Interest

The author declare that there is no conflict of interest for this study.