IntroductionShigella flexneri (S. flexneri) is an important zoonotic pathogen. Infections caused by this bacterium in nonhuman primates, especially cynomolgus monkeys (Macaca fascicularis), represent major health risks and biosecurity concerns for laboratory animal facilities. Characterizing S. flexneri isolates originating from captive cynomolgus monkeys is essential for implementing targeted disease prevention and control strategies.MethodsIn this present study, strain MF041021 was isolated from fecal samples of a dysenteric cynomolgus monkey. Species identification was accomplished using 16S rRNA gene sequencing. Additional phenotypic and genotypic characterizations included detection of virulence genes, antimicrobial susceptibility testing against clinically important agents, and screening for antimicrobial resistance genes. Pathogenicity of the isolate was evaluated via intraperitoneal inoculation in C57BL/6 mice.ResultsThe isolate MF041021 (GenBank accession: PZ779204) was identified as S. flexneri by 16S rRNA sequencing. Sequence analysis revealed that the 16S rRNA gene of this isolate shared over 97.1% similarity with those of ten S. flexneri strains derived from various hosts deposited in GenBank, with the highest similarity (99.9%) observed with a human-derived strain (CP050985). Virulence gene profiling demonstrated that the isolate harbored four virulence-associated genes (ipaC, ipaH, ial, and shet1A), while the sen gene was absent. Pathogenicity was pronounced in C57BL/6 mice. Antimicrobial susceptibility testing showed that the isolate was resistant to ampicillin but susceptible to ciprofloxacin, imipenem, meropenem, levofloxacin, cotrimoxazole, and ofloxacin. Resistance gene analysis identified tetB, ampC, ant (3′)-Ia, and aadA1.DiscussionThe cynomolgus monkey-derived S. flexneri isolate MF041021 demonstrated substantial pathogenic potential, harboring multiple virulence factors and antimicrobial resistance genes. Its close phylogenetic relationship with human-derived strains, as evidenced by 16S rRNA gene analysis, underscores the need for enhanced biosecurity, standardized husbandry practices, and prudent antimicrobial stewardship in laboratory macaque colonies. These findings from this single clinical isolate provide valuable case-level reference data for the diagnosis, prevention, and control of S. flexneri infection in captive non-human primates.