Strain: A cross sectional study was designed to analyse sixteen whole genome sequences of Enterococcus faecalis retrieved from the NCBI database [14] as of Feb’ 2018, scaffolds and contigs were excluded. Also, the size of the genome, replicons, GC percentage, number of genes and proteins encoded were derived from the same database [14] [Table/Fig-1]. Assembled sequences were submitted to MLST (Multilocus sequence typing) 1.8 algorithm from Center for Genomic Epidemiology (CGE) [15]. MLST was performed including seven housekeeping genes (aroE, gdh, gki, gyd, pstS, xpt, yqiL) of E. faecalis to classify the strains based on the sequence types.

Resistant Gene Profiling:Resfinder 3.0 is a user-friendly, computational tool [16] from the CGE. The whole genome sequence in the FASTA format was used as an input file to obtain facts about drug resistant genes present in the pathogen. The test was conducted for acquired antimicrobial resistance including all available antimicrobial drugs of six major classes such as aminoglycoside, beta-lactam, colistin, fluoroquinolone, fosfomycin, fusidic acid, glycopeptide, macrolide, lincosamide, streptogramin, nitroimidazole, oxazolidinone, phenicol, rifampicin, sulphonamide, rifampicin, trimethoprim and run using default parameters [Table/Fig-2].

Plasmid Profiling: PlasmidFinder 1.3 is yet another effective tool from CGE to probe the presence of plasmids in the genomes selected [17]. The algorithm ran using default parameters and the reference database selected was that of Enterococcus, Streptococcus and Staphylococcus. The data obtained from the study was used to correlate between the presence of plasmids and the drug resistant phenotypes recorded earlier [Table/Fig-3].

Multiple Sequence Alignment (MSA): The protein sequences of predominant drug resistant phenotypes coding for lsa(A), tet(M) and erm(B) were further analysed across several genus and species. Since lsa(A) was mostly present in Enterococcus spp. alone MSA was performed using FASTA sequence of different strains of E. faecalis. However, tet(M) and erm(B) were found to occur in diverse microbial population and hence, was subjected to MSA using MUSCLE programme of MEGA v.7.0 to ascertain the diversity of these proteins [18]. UPGMB was the clustering method applied with default parameters. Non-duplicate isolates exhibiting 100% query coverage with 95-100% identity were selected for the analysis [Table/Fig-4]. The subcellular localisation of the protein was deduced by PSORTb v3.0 [19].

[Table/Fig-4]:

Multiple sequence alignment demonstrating diversity of (a) Lsa(A), (b) Tet (M) protein and (c) Erm (B) protein among different bacterial genus and species.

Phylogenetic Analysis of Erm(B) AND Tet(M) Protein: The diversity in course of evolution of LsaA, Erm(B) and Tet(M) proteins [Table/Fig-5] were inferred by using the Maximum Likelihood method based on the Poisson correction model [20]. Evolutionary analyses were conducted in MEGA v7.0 [21]. The bootstrap consensus tree inferred from 500 replicates was taken to represent the evolutionary history of the protein analysed [22].

[Table/Fig-5]:

Phylogenetic tree demonstrating the diversity of (a) Lsa (A), (b) Tet (M) and (c) Erm (B) protein among different bacterial genus and species.

The principal objective of the present study was to acquire a basic understanding on the drug resistant profiles exhibited by E. faecalis strains submitted in public domain from across the globe. Sixteen whole genome sequences of E. faecalis returned resistance profiles as detected by ResFinder 3.0 corresponding to: 1) aminoglycoside; 2) glycopeptides; 3) macrolide, lincosamide streptogramin B (MLS); 4) tetracycline; 5) phenicol; and (6) trimethoprim. Among the six antimicrobial drug groups gene lsa(A) encoding MLS resistance was found to be the most predominant (n=16; 100%), followed by tet(M) (n=6; 37.5%) and erm(B) (n=4; 25%). Three strains out of sixteen harboured more than four resistance encoding genes (25%) [Table/Fig-6]. The multi-locus sequence typing showed that each of the strains belongs to a specific sequence type and they may not be clustered into the same sequence type.

Plasmid profiling of the sequences retrieved were consistent with the data acquired from NCBI database. The plasmid type and % identity to the known plasmid sequences are provided in [Table/Fig-3]. Interestingly, aminoglycoside, glycopeptide and trimethoprim resistant phenotypes were found to be occurring only in strains harbouring plasmids, whilst the other phenotypes like tetracycline, phenicol and MLS were found in both plasmid bearing and non-plasmid containing strains [Table/Fig-3].

Multiple sequence alignment of Tet(M) and Erm(B) proteins showed conserved sequences among a diverse group of organisms tested [Table/Fig-4]. Single amino acid variations were dispersed along the protein sequences which indicates the process of evolution of genes encoding these proteins. The MSA for Lsa(A) protein which was species specific and conserved among Enterococci with variations dispersed throughout the protein sequence [Table/Fig-4]. The subcellular localisation of proteins as deduced by PSORTb v3.0 showed Lsa(A) to be in the transmembrane region and Tet(M) and Erm(B) proteins in the cytoplasm. Phylogenetic analysis performed using the protein sequences of Tet(M) and Erm(B) showed the evolution and diversification of protein among several genus and species analysed. Phylogenetic analysis of Lsa(A) protein showed diversity among the E.faecalis species studied, demonstrating rapid evolution of this protein at the species level [Table/Fig-5]. Thus, the present study provides an insight about the genetic basis of drug resistance exhibited by the strains of E. faecalis investigated.

Antimicrobials are an integral part of the health care system and find its use in various fields of medicine including dentistry. The emergence of drug resistant pathogens has turned the treatment options towards the dark side. The World Health Organization (WHO) has reiterated the fact that treatment failure due to resurgence of drug resistant organisms will be the challenge faced in this millennium [23]. The commonly used drugs in dentistry are erythromycin, vancomycin, tetracycline, doxycycline, metronidazole etc., Although, local and systemic modes of delivery are used, local administration of antibiotics is preferred since the target tissue can be reached easily. This method also creates a selective pressure on the organisms infecting tissues, thus paving way for acquisition of drug resistant genes from similar pathogens. Since most of the drug resistant genes are carried on mobile elements like plasmids, transposons and integrons, the horizontal dissemination of drug resistant genes becomes undemanding.

The present study unravels the antibiotic resistant profiles of E. faecalis which is considered to be one of the vital pathogens related to endodontic and periodontal infections. Resistance towards vancomycin and tetracycline have been extensively studied in this pathogen. Interestingly, we could observe a few rare genes such as fex(A) and dfr(G) in two of the strains of E. faecalis (E.faecalis L12, E.faecalis CLB21560). Most of the Gram positive organisms exhibit resistance towards MLS class of drugs via two major mechanisms viz., drug efflux and demethylation of 23S rRNA. The gene lsa(A) is responsible for intrinsic resistance to lincosamides and streptogramins A in E. faecalis [24]. BLAST analysis and several other reports confirmed the species specificity of this protein. Although the role of Lsa remains ill-defined it may be involved in drug efflux pathway, as the protein resembles ATP-Binding Cassette (ABC)-efflux pumps. Clindamycin and quinupristin-dalfopristin resistance was reported in 100% of the strains exhibiting this phenotype. Targeted inactivation of lsa(A) gene produced susceptible phenotypes, while recombinant plasmid carrying lsa gene mediated complementation restored the resistant phenotype [25].

Another best understood mechanism of streptogramin resistance is through the demethylation of 23S rRNA [26]. The Erm proteins disrupt the binding between macrolides and rRNA by the transfer of methyl group at A2058 of 23S rRNA rendering the strains resistant to MLS. erm(A) and erm(B) which confers MLS(A) and MLS(B) phenotypes are carried on broad host range plasmid such as pAMβ1 [27]. This report is in agreement with the present study where we could observe erm (B) gene in plasmid bearing strains of E. faecalis. Hospital strains of E. faecalis have recorded highest prevalence of resistance to erythromycin (34.1%) which is mostly encoded by erm(B) gene (70.9%) [28]. Erm type of protein shows diversity across different genus and species, thus elucidating an underlying genetic evolution of these species. Since erythromycin is routine drug of choice in dental clinics, periodical monitoring and investigations into resistance genes encoding Erm protein should be performed in E. faecalis isolated from tissue or oral site which is non-compliant with treatment. This may reduce further complications by resistant species and also prevent horizontal gene transfer among other organisms in oral cavity.

Tetracycline resistance in Enterococci dates back to early 1960, soon after its introduction in clinical settings. The most prevalent resistant genes isolated from asymptomatic apical periodontitis were reported to be tet(M) (42%), tet(W) (29%) and erm(C) (24%) [29]. In vitro resistance studies on plaque samples demonstrated multidrug resistance in E. faecalis strains. The resistance profile in decreasing order was clindamycin and metronidazole (100%), erythromycin (80.8%) and tetracycline (53.2%) [30]. The Tet(M) protein mediates resistance via ribosomal protection while, Tet(L) confers resistance through drug efflux mechanisms which are energy dependent [31]. The present study also reports an overall prevalence of 43.8% of tetracycline encoded genes which is in close association with other in vitro studies.

Gentamycin resistance is generally mediated by Aminoglycoside Modifying Enzymes (AME).

The aac(6’) – aph (2”) possess both adenyltransferase and phosphotransferase activities, which confers resistance to gentamycin, amikacin, tobramycin and kanamycin. The encoded enzyme is carried on transposons accommodated within the plasmid or the chromosomal DNA. Interestingly, aac(6’)-aph (2”) was also observed in strains harbouring plasmids in the present study, which validates the location of this gene in the pathogen. These mobile elements also facilitate transfer of gene clusters to other anaerobic pathogens found in deep seated wounds. An investigation on the distribution of AME phenotype conducted in Japan detected aac(6’)-aph (2”) in about 42.5% of E. faecalis strains [32]. Strains with aac(6’)-aph (2”) +ant(6)-Ia + aph(3’)-IIIa phenotype showed high level resistance to gentamycin and streptomycin [33]. The genome of E. faecalis CLB21560 also demonstrated a similar combination of genes, hence confirming the AME phenotype of this strain.

Vancomycin resistant E. faecalis (VREF) has emerged as major pathogen in nosocomial infections. Rengaraj R et al., reported highest prevalence of van A and B genes in VREF isolated from clinical specimens [34]. The vancomycin resistant phenotype is aided by the presence of a cluster of genes encoding cell wall modification precursors that exhibits poor affinity towards vancomycin [35]. Here, the normal peptidoglycan precursor with D-alanyl-D-alanine is replaced with D-alanyl-D-lactate, which binds to vancomycin with a lower affinity of 0.001 times when compared to the normal precursors. Several van proteins act synergistically to establish the phenotype [36]. A recent study testing efficacy of genetically engineered bacteriophage φEf11/φFL1C(Δ36)PnisA in treatment of vancomycin resistant strain proved to be a success with vancomycinR strain (E. faecalis V583) exhibiting 99% susceptibility in comparison to 18% in sensitive strains (E. faecalis JH2-2) [37]. The VRE phenotype was observed in two strains analysed in the present study. Additionally, these phenotypes were found to co-occur along with MLSR encoding genes and found in plasmid bearing strains.

The fex(A) gene encodes chloramphenicol resistance which was first identified on the transposon Tn558 in Staphylococcus lentus plasmid. This gene is carried on non-conjugative plasmids in E. faecalis. The gene encodes a transmembrane efflux proteincontaining 475 amino acids forming fourteen transmembrane domains [38,39]. Resistance to trimethoprim-sulfamethoxazole is mediated by dfrG, which encodes the enzyme trimethoprim insensitive dihydrofolate reductase [40]. The dfr genes are mostly found in the plasmids and only a few transmissible dfr genes have been marked in Gram positive organisms, dfrG is one among them and is reported to be detected in Streptococcus pyogenes [41]. The present study also reports the presence of dfrG gene in a strain E. faecalis CLB21560 harboring plasmids which substantiates earlier reports. The prevalence of this gene in E. faecalis was also found to be low (n=1; 6.25%). The present study reiterates the fact that acquired drug resistance should not be ignored, as pathogens assimilating novel genes from the environment might complicate the treatment options and recovery in susceptible patients.

Although the present study has certain limitations such as: a) lack of information about the antimicrobial drug resistance exhibited by strains in vitro; b) study restricted towards intrinsic and acquired resistance pertaining to gene clusters excluding point mutations/substitutions; and c) antimicrobial resistance of clinical isolates of E. faecalis from different geographical locations. The present data provides a clear insight about the panel of genes to be detected primarily in case of a treatment failure in suspected E. faecalis infections. A better therapeutic strategy may be adopted for combating such refractory strains.

The trend of antimicrobial drug resistance is creeping into opportunistic and commensal organisms which may in future turn into resistant pathogens making treatments refractory or futile. Hence, meticulous efforts are to be employed to identify, isolate and treat diseases involving resistant species. Alternate treatment modalities such as usage of combinatorial drugs, phage therapy, and probiotics can reduce the incidence of resistant forms. One of the interesting results derived from the study is that E. faecalis harbours one or more resistance encoding genes and that none of the strains were deprived of resistant genotype. To conclude, a vigilant surveillance and focused treatment will help to reduce the load of resistant gene pools in the oral cavity.