# Genomic insights into the diversity, antibiotic resistance, and virulence potential of staphylococci isolated from pediatric patients with chronic otitis media with effusion (COME)

## TL;DR

This study uses genome sequencing to explore Staphylococcus strains in children with chronic ear infections, revealing antibiotic resistance and virulence traits that could impact treatment.

## Contribution

The discovery of gram-negative resistance genes in all Staphylococcus strains suggests horizontal gene transfer may enhance pathogenicity in COME.

## Key findings

- Three coagulase-positive and 13 coagulase-negative Staphylococcus strains were identified, including a potential new S. aureus subspecies.
- All 16 strains carried previously unreported gram-negative resistance genes, indicating horizontal gene transfer from Enterobacteriaceae.
- Coagulase-positive strains showed higher acute virulence, while coagulase-negative strains like S. epidermidis promoted chronic infection via immune evasion and biofilm formation.

## Abstract

Chronic otitis media with effusion (COME) is a prevalent pediatric condition characterized by persistent middle ear effusion, potentially leading to hearing loss and developmental delays.

We investigated the diversity, antibiotic resistance, and virulence potential of Staphylococcus species in COME through whole genome sequencing of 16 clinically-derived strains isolated from pediatric patients in the United Kingdom. De novo genome assembly and annotation were performed on Illumina reads. Phylogenetic analyses using 16s rRNA gene, multilocus sequence typing (MLST) and core genome single nucleotide proteins (SNPs) elucidated evolutionary relationships. Species identification was confirmed through in silico DNA-DNA hybridization (ANIb and GGDC). Resistance genes were detected using AMRFinderPlus and Comprehensive Antibiotic Resistance Database (CARD), and virulence factors were identified using VFanalyzer. Pangenome analysis identified unique species-specific genes.

Phylogenetic analysis revealed three coagulase positive Staphylococcus (CoPS) and 13 coagulase negative Staphylococcus (CoNS), with identification of a potential new S. aureus subspecies (strain NU84) Twenty-four genes conferred resistance to nine antibiotic classes, particularly beta-lactams commonly used for COME treatment. Notably, all 16 strains harbored blaTEM-116 and aph(3’)-IIa genes, typically associated with gram-negative bacteria and previously unreported in human Staphylococcus isolates, suggesting horizontal gene transfer from Enterobacteriaceae. CoPS strains exhibited higher acute virulence potential contributing to COME onset, whereas CoNS, particularly S. epidermidis, harbored genes promoting persistence through immune evasion and biofilm formation, consistent with the chronic nature of COME.

Our genomic analysis shows that COME-associated Staphylococcus species have significant pathogenic potential due to acquired resistance and virulence genes. The discovery of gram-negative resistance genes in all Staphylococcus strains indicates horizontal gene transfer may enhance pathogenicity. These findings highlight the urgent need for surveillance and targeted therapies against emerging multidrug-resistant strains in COME treatment.