Genomic survey of Indian vancomycin-resistant E. faecium maps resistance genes and mobile elements

Vancomycin-resistant Enterococcus faecium, one of the pathogens the World Health Organization classifies as high priority because of rising antibiotic resistance, has now been mapped in unusual genomic detail for India. A research team led by Shalini Mathpal, Avani Panickar, Tushar Joshi, Sudha Ramaiah and Anand Anbarasu analyzed 63 whole-genome sequences of E. faecium retrieved from public databases, all originally collected from patients in Indian healthcare facilities between January 2017 and December 2021. The isolates were confirmed as vancomycin-resistant E. faecium, and the resulting survey, published in Current Genetics, offers one of the most comprehensive whole-genome views of this hospital pathogen from the Indian subcontinent to date.

The motivation is straightforward. VREfm is a prominent cause of healthcare-associated infections, including urinary tract infections, intra-abdominal infections and bloodstream infections, and it poses the greatest threat to hospitalized and immunocompromised patients, in whom it contributes to increased morbidity, mortality and limited treatment options. In Asia, the prevalence of VREfm is estimated at around 22.4 percent, higher than in many European countries though still lower than rates in the United States. Yet despite this clinical burden, comprehensive genomic studies on Indian isolates have remained limited; most available reports from the country rest on small sample sizes and often lack whole-genome sequencing-based analyses. The new study set out to fill that gap by characterizing population structure, resistance mechanisms and the mobile genetic machinery that spreads them.

The team drew on datasets from thirteen NCBI BioProject accessions, quality-filtered the raw reads, annotated the genomes and then applied a battery of comparative tools: pan-genome analysis with Roary, multilocus sequence typing, single-nucleotide polymorphism phylogeny, and systematic screening for resistance genes, plasmids, insertion sequences, integrons, transposons and prophages. Of three candidate reference genomes evaluated, the DO genome showed the highest similarity to the clinical isolates and was selected for downstream analyses. The pan-genome of the collection totaled 29,196 gene clusters, but only 1,660 of these formed the conserved core present in 99 to 100 percent of isolates. The overwhelming majority, 25,283 genes, were rare cloud genes found in fewer than 15 percent of strains, underscoring how genetically fluid this species is.

That fluidity showed up sharply in the population structure. Multilocus sequence typing identified eight distinct sequence types among the 63 isolates, with ST80 dominating at 52 isolates, or 82.5 percent of the collection, followed by ST18 with four isolates and ST17 with two. ST31, ST117, ST1380, ST1693 and ST1886 each appeared once. ST80 has been described in earlier reports as a globally disseminated, hospital-associated lineage linked to vancomycin resistance, but pairwise SNP analysis here revealed a wide distribution of distances, ranging from roughly 5,000 to 8,000 SNPs with a median near 6,300. That means the apparent dominance of ST80 reflects several related yet distinct sublineages rather than a single recent clonal expansion sweeping through Indian hospitals.

The resistome was equally striking. ResFinder identified 87 distinct antimicrobial resistance genes spanning aminoglycosides, macrolides, glycopeptides, sulfonamides, tetracyclines and beta-lactams. The most prevalent gene was msr(C), an ATP-binding cassette transporter conferring resistance to erythromycin, telithromycin and streptogramins, found in almost all isolates. It was followed by aac(6′)-Ii, linked to high-level aminoglycoside resistance, and erm(B), which encodes an rRNA methyltransferase behind macrolide-lincosamide-streptogramin B resistance. The vanHAX operon, the engine of vanA-type vancomycin resistance, was detected in 45 of 63 isolates, or 71.4 percent, a proportion that was statistically significant against an expected 50 percent in a binomial test. Linezolid resistance genes optrA and poxtA appeared in 14 and 2 isolates respectively, a notable finding given that linezolid and daptomycin are the last-line treatments for VREfm infections.

Plasmid profiling revealed 32 unique replicon types, a heterogeneous plasmidome in which many isolates carried multiple replicon types simultaneously. The most frequent was repUS15_2_repA from pNB2354p1, present in 10 percent of isolates, followed by rep2_1_orf1 from pRE25 at 9.3 percent; pRUM-like plasmids, often associated with vancomycin resistance in clinical strains, were also common. Small Col-type plasmids lacking canonical rep genes were found as well, typically mobilized by co-resident conjugative elements. Crucially, several resistance genes, including the aminoglycoside determinants, beta-lactamases such as blaTEM-104 and blaOXA-232, tet genes, erm(B), the vanHAX operon and even the plasmid-mediated quinolone resistance gene qnrS1, were detected on plasmid replicons, suggesting a concrete role for plasmids in disseminating resistance across the population.

The mobilome analysis added further layers. Integrons were found in 13 isolates, all carrying the intI integrase gene characteristic of class 1 integrons, with three isolates showing complex CALIN structures of attC recombination sites. Genome-wide insertion sequence profiling identified 25 distinct IS families, dominated by IS3 with 1,303 copies, ISL3 with 1,250, IS30 with 968 and IS256 with 944. The vanHAX operon was commonly found adjacent to IS3, ISL3, IS30, IS6, IS256 and IS1380 elements, and several resistance genes co-localized with insertion sequences on the same contigs. Transposons including Tn917, found in the majority of isolates, and Tn1546, the classic vancomycin-resistance transposon, detected in four isolates, rounded out the picture. Prophage analysis identified incomplete, questionable and intact regions across the collection, with PHAGE_Lister_2389 the most frequently detected, and phage-inducible chromosomal islands were also present.

To organize this complexity, the researchers built a co-localization network in which nodes represented resistance genes, insertion sequences or plasmid replicons and edges represented their sharing of a contig. Centrality metrics including betweenness, bottleneck, closeness, degree and maximal clique centrality consistently ranked IS elements such as IS1380, IS3, IS200/IS605, IS6 and ISL3 among the top nodes, alongside plasmid replicons like pRE25 and pNB2354p1 and resistance genes including aac(6′)-Ii, erm(B), msr(C) and blaTEM-1B. The authors stress that this network provides a framework for generating testable hypotheses about horizontal gene transfer rather than proof of physical linkage, which will require long-read sequencing or PCR-based validation.

Beyond acquired genes, the study examined chromosomal mutations. Every isolate carried mutations in gyrA and parC, associated with fluoroquinolone resistance, and in pbp5, associated with ampicillin resistance. SnpEff-based screening also detected previously reported daptomycin resistance-associated substitutions, including W73C in LiaR, T120A in LiaS, T298S in Cls and S491F in RpoB, alongside T450I in EatA and H486Y in RpoB. Three novel substitutions, Cls-R424S, RpoB-M475V and RpoC-T634K, were predicted deleterious by PROVEAN and were further assessed with MutPred2 and DynaMut2, which indicated destabilizing effects on the corresponding proteins. However, formal association testing with Fisher’s exact test and Benjamini-Hochberg correction found that none of these mutations was significantly linked to vancomycin resistance after adjustment, leading the authors to conclude that vancomycin resistance in this group likely arises from a multifactorial genomic background rather than any single mutation.

The authors are careful about the limits of their work. The 63 publicly available genomes may not capture the full genomic diversity of VREfm across India, the lack of associated clinical and phenotypic data prevented correlation between genetic determinants and actual resistance phenotypes or patient outcomes, and the functional implications of the novel mutations rest entirely on in silico prediction intended for hypothesis generation. Future work, they write, will aim to validate the physical associations among resistance genes, plasmids and insertion sequences through long-read sequencing, and to test candidate mutations experimentally through allelic exchange and MIC assays. Even so, the survey delivers a detailed reference map of the resistome, plasmidome and mobilome of Indian VREfm, and it underscores how plasmids, transposons, insertion sequences and integrons together create the genomic context in which resistance genes travel through hospital populations.

Subject of Research: Genomic surveillance of vancomycin-resistant Enterococcus faecium isolates from India

Article Title: Genomic surveillance of vancomycin-resistant Enterococcus faecium: a study on Resistome, Plasmidome, and mobilome profiling

Article References: Mathpal, S., Panickar, A., Joshi, T., Ramaiah, S., & Anbarasu, A. (2025). Genomic surveillance of vancomycin-resistant Enterococcus faecium: a study on Resistome, Plasmidome, and mobilome profiling. Current Genetics, 71(1), Article 26. https://doi.org/10.1007/s00294-025-01331-y

Image Credits: AI Generated

DOI: 10.1007/s00294-025-01331-y

Keywords: vancomycin-resistant Enterococcus faecium, genomic surveillance, antimicrobial resistance, plasmid replicons, mobile genetic elements, whole-genome sequencing, India, vanHAX operon, insertion sequences, integrons, daptomycin resistance, hospital-associated infections