| Summary: | <i>Salmonella</i> enterica subsp. enterica serovar Typhimurium (<i>S</i>. <i>typhimurium</i>) is an important zoonotic pathogen with important public health significance. To understand <i>S. typhimurium</i>’s epidemiological characteristics in China, multi-locus sequence typing, biofilm-forming ability, antimicrobial susceptibility testing, and resistant genes of isolates from different regions and sources (human, food) were investigated. Among them, ST34 accounted for 82.4% (243/295), with ST19 ranking second (15.9%; 47/295). ST34 exhibited higher resistance levels than ST19 (<i>p</i> < 0.05). All colistin, carbapenem, and ciprofloxacin-resistant strains were ST34, as were most cephalosporin-resistant strains (88.9%; 32/36). Overall, 91.4% (222/243) ST34 isolates were shown to have multidrug resistance (MDR), while 53.2% (25/47) ST19 isolates were (<i>p</i> < 0.05). Notably, 97.8% (45/46) of the MDR-ACSSuT (resistance to Ampicillin, Chloramphenicol, Streptomycin, Sulfamethoxazole, and Tetracycline) isolates were ST34, among which 69.6% (32/46) of ST34 isolates were of human origin, while 30.4% (14/46) were derived from food (<i>p</i> < 0.05). Moreover, 88.48% (215/243) ST34 showed moderate to strong biofilm-forming ability compared with 10.9% (5/46) ST19 isolates (<i>p</i> < 0.01). This study revealed the emergence of high-level antibiotic resistance <i>S. typhimurium</i> ST34 with strong biofilm-forming ability, posing concerns for public health safety.
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