Antimicrobial resistance, virulence and genetic characterization of ESBL-producing Shigella spp isolated from fecal samples of men who have sex with men

Shigella spp. are major causative agents of diarrheal disease worldwide, with increasing reports of antimicrobial resistance associated with global dissemination, particularly among men who have sex with men (MSM). Antimicrobial resistance in commonly isolated Shigella spp. represents a growing international concern, especially given the rising prevalence of multidrug-resistant (MDR) lineages. This study aimed to characterize the phenotypic and genomic features of Shigella flexneri and Shigella sonnei isolates exhibiting extended-spectrum β-lactamase (ESBL)-producing phenotypes, as well as non-ESBL-producing controls, collected in Madrid between 2017 and 2024. A total of 30 isolates were analyzed using antimicrobial susceptibility testing and whole-genome sequencing. ESBL production was primarily associated with bla CTX−M−27 , although bla CTX−M−15 was also detected, predominantly in isolates from MSM individuals. All ESBL genes were located on IncFII plasmids, except in one S. flexneri isolate, in which the gene was carried on an IncB/O/K/Z plasmid. To our knowledge, this is the first report of S. flexneri harboring CTX-M-15 in MSM populations and one of the few reports of S. sonnei with similar characteristics. Resistance to additional antibiotic classes was mediated by multiple acquired genes and chromosomal mutations in gyrA and parC . Phylogenetic analysis revealed distinct clades within each serogroup, with S. sonnei exhibiting greater genetic diversity than S. flexneri. Nevertheless, lineage 3 predominated among S. sonnei isolates, particularly genotypes 3.6.1.1 and 3.6.1.1.2. Virulence profiling identified variability in both plasmid- and chromosome-encoded determinants, including differences in the presence of the pINV plasmid. Overall, these findings highlight the co-occurrence of multidrug resistance, virulence factors, and mobile genetic elements, underscoring the adaptive potential and epidemiological success of circulating Shigella lineages.