Sal(E) resistance gene widespread in emerging Staphylococcus nepalensis

An emerging Staphylococcus species carries a resistance determinant that can reduce susceptibility to several antibiotics used against Gram-positive infections. A phylogeographic analysis of Staphylococcus nepalensis, including two isolates recovered from a hypersaline lagoon in Rio de Janeiro, found that the sal(E) gene was present in all 36 publicly available genomes examined. The results suggest that sal(E) is a deeply conserved, species-associated feature rather than a recently acquired resistance gene, although other antimicrobial-resistance determinants vary substantially among lineages and ecological sources.

S. nepalensis is a coagulase-negative staphylococcus first described in 2003 from the respiratory tract of goats in the Himalayan region of Nepal. Retrospective evidence indicates that it circulated earlier: isolates collected in the United Kingdom during the 1960s were originally classified as Staphylococcus saprophyticus and Staphylococcus xylosus but were reidentified as S. nepalensis in 2020. The species has since been reported in human, animal, food and environmental samples, and its capacity to occupy such different niches has prompted growing concern that it may be an overlooked opportunistic pathogen with zoonotic relevance.

For the new study, researchers sampled water and sediment from the Brejo do Espinho lagoon system at Araruama, an anthropogenically impacted hypersaline environment in Rio de Janeiro. They isolated two staphylococcal strains, COLB in 2016 and AM1 in 2018, and identified them initially by MALDI-TOF mass spectrometry before confirming their taxonomy through whole-genome sequencing. Average nucleotide identity values of 99.60% and 99.69% against the S. nepalensis reference strain established both isolates as members of the species. The findings show that S. nepalensis was present in South America at least four years before its first published report in Brazil, which followed its detection in the oral microbiota of domestic cats in 2020.

Phenotypic susceptibility testing found that both Brazilian isolates were resistant to quinupristin-dalfopristin, lincomycin, tetracycline and doxycycline, while AM1 showed intermediate susceptibility to clindamycin, with a minimum inhibitory concentration of 2 micrograms per millilitre. The two strains remained susceptible to the other agents tested. Genome analysis identified sal(E) in both isolates, as well as tet(K), which encodes an efflux pump associated with tetracycline and doxycycline resistance. AM1 additionally carried lnu(A), a lincosamide nucleotidyltransferase associated with resistance to lincomycin and clindamycin. These findings broadly matched the measured phenotypes, although the authors cautioned that gene predictions in public genomes indicate potential resistance rather than experimentally confirmed phenotypes.

Phylogenomic analysis placed COLB and AM1 within a well-supported lineage associated with strains from Korean fermented foods. The two Brazilian isolates differed by only one core-genome single-nucleotide polymorphism despite having been collected two years apart, supporting their classification as clonally related and indicating persistence of the lineage in the lagoon. They were separated from other globally sampled strains by at least 1,648 core-genome SNPs. The analysis used 2,348 core genes and 20,043 recombination-corrected variable positions, and most nodes in the maximum-likelihood tree received full bootstrap support.

Across the broader dataset of 36 genomes, researchers identified S. nepalensis in environmental, food, animal-associated and human-associated sources on several continents. The available genomes revealed a heterogeneous resistome that included blaZ and mecA, associated with beta-lactamase production and methicillin resistance, respectively. Other detected determinants included lnu(A), tetK, catA, dfrE, dfrG, aac(6′)-Ie, mphC and ermB. The two human clinical isolates from Viet Nam carrying mecA were especially significant because methicillin resistance could complicate treatment and cause a clinically recognizable staphylococcal infection to be misclassified. The authors also identified heavy-metal tolerance genes, including arsR, arsB, arsC, cadD, mco, merA, merB and merT, as well as qacG, which is linked to resistance to quaternary ammonium disinfectants.

The two lagoon isolates carried the arsenic-resistance operon genes arsR, arsB and arsC, which can contribute to tolerance of arsenate, arsenite and antimonite found in polluted environments. AM1 also carried mco, associated with copper tolerance and protection against oxidative stress, and merA, which encodes a mercuric reductase. Both strains contained the plasmid replicons rep7a and rep19c, while AM1 carried the additional replicon rep21, previously associated with a plasmid carrying lnu(A). These replicons occur frequently in staphylococci from human, animal and food sources, but the available data cannot establish when or how AM1 acquired them. The findings therefore point to the potential mobility of additional resistance determinants without proving that a specific transfer event occurred.

Central to the analysis was sal(E), which occurred in every genome and was surrounded by a conserved chromosomal neighborhood containing genes associated with tRNA modification, translation fidelity and DNA repair. This conserved organization argues against sal(E) having been deposited recently on an independently acquired plasmid or other mobile element. Sal(E) belongs to the ABC-F family of antibiotic target-protection proteins, which interfere with the binding of antimicrobial agents to the bacterial ribosome. The study used AlphaFold3 to predict the three-dimensional structure of the S. nepalensis protein and found an overall architecture similar to the experimentally characterized Sal(B) protein from Staphylococcus aureus. The predicted model had a pTM score of 0.8.

Although some residues in the Sal(E) model differed from the corresponding region of Sal(B), none of the observed sequence changes among five representative S. nepalensis proteins occurred in the antibiotic-resistance domain loop, a region associated with antibiotic displacement. The proteins shared 97.8% amino-acid conservation, with 531 of 543 positions conserved. The authors proposed that the equivalent interhelical loop in Sal(E) could help block antibiotic binding, but emphasised that the functional effect of this prediction still requires experimental validation. The conservation of the protein sequence, chromosomal position and predicted fold collectively supports predominantly vertical inheritance of sal(E) within S. nepalensis.

Despite its resistance profile, the new isolates showed no clear evidence of acute virulence in the Galleria mellonella larval infection model. All larvae injected with COLB or AM1 survived throughout the 96-hour observation period, and no known virulence-associated genes were identified in any of the 36 genomes using the databases consulted. These results do not rule out human or animal pathogenicity: S. nepalensis has been associated with bacteraemia and urinary tract infection, and virulence varies among staphylococcal species and strain backgrounds. The authors instead argue that accurate species identification and continued genomic surveillance are essential for recognising infections that might otherwise be missed or attributed to a better-known coagulase-negative staphylococcus.

Recombination analysis indicated that the population structure of S. nepalensis is predominantly clonal. Gubbins detected 211 recombination blocks, accounting for 5.88% of the SNPs in the core-genome alignment, while ClonalFrameML produced a low overall recombination-to-mutation ratio of 8.09 × 10⁻⁷. The mean recombinant tract length was 97.58 base pairs, consistent with localised events rather than large-scale chromosomal replacement. By contrast, the species possesses an open pangenome: across the 36 genomes, researchers identified 2,348 core genes, 485 shell genes and 1,880 cloud genes, with accessory genes making up 51% of the total. The combination of a mostly clonal backbone and a flexible accessory genome may allow S. nepalensis lineages to acquire clinically important resistance determinants while retaining stable, vertically inherited characteristics such as sal(E). The dataset remains limited and geographically biased, however, because it reflects opportunistic availability of genomes rather than systematic surveillance. Broader sampling across humans, animals, food and environmental settings will be needed to define the species’ true distribution, transmission pathways and public-health significance.

Subject of Research: Phylogeographic and resistome analysis of Staphylococcus nepalensis

Article Title: Phylogeographic analysis of Staphylococcus nepalensis reveals global occurrence of antimicrobial-resistant lineages carrying the sal(E) resistance gene

Article References: Becerra, J., Gaião, I., Vincenzi, R. A., Merighi, D. G. S., Vásquez-Ponce, F., Arevalo, S. J., Pariona, J., Melocco, G., Esposito, F., Rodrigues, F., & Lincopan, N. (2026). Phylogeographic analysis of Staphylococcus nepalensis reveals global occurrence of antimicrobial-resistant lineages carrying the sal(E) resistance gene. Molecular Biology Reports, 53(1), Article 1525. https://doi.org/10.1007/s11033-026-12601-4

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12601-4

Keywords: Staphylococcus nepalensis, sal(E) resistance gene, antimicrobial resistance, phylogeography, whole-genome sequencing, ABC-F proteins, One Health surveillance, environmental microbiology, methicillin resistance, resistome, Phylogeographic, analysis