Genomics Revealed Novel Chromosomal Integration of Antimicrobial Resistance Cluster tet(B), sul2, blaTEM-1B, aph(3″)-Ib, and aph(6)-Id in Salmonella Typhimurium.

Antimicrobial-resistant Salmonella has posed a huge threat to food safety and public health, and tetracycline is the commonly used antibiotic for treating salmonellosis. In this study, eight Salmonella Typhimurium isolates from diarrhea patients (n = 7) and clam (n = 1) exhibited high-level tetracycline resistance (minimum inhibitory concentration = 128 μg/mL), and these isolates were further resistant to ampicillin and sulfonamides, formatting the R-type ASuT (ampicillin, sulfonamides, and tetracycline). Then, these eight isolates were sequenced using PacBio platform, revealing the presence of tetracycline resistance gene tet(B), along with sulfonamide-resistance gene sul2, extended-spectrum-β-lactamase gene bla_TEM-1B, and aminoglycoside resistance genes aph(3″)-Ib and aph(6)-Id on the chromosome, which was associated with R-type ASuT. Four types (A, B1, B2, and C) of genetic arrangement for chromosomally encoding tet(B) were found, inserted into fljBA operon. Type C (fljBA operon-tet(B)-sul2-bla_TEM-1B-aph(3″)-Ib-aph(6)-Id-merACDEPTR) was the most common type and was accompanied by various insertion sequences (ISs) (IS26, IS1, and ISVsa5) and recombinases. Pairwise sequence alignment showed that type C arrangement likely resulted from stepwise acquisitions and rearrangements facilitated by the actions of ISs, followed by integration into the chromosome by prophages. Phylogenomic analysis showed that all eight Salmonella Typhimurium isolates from China in this study, along with a human-borne Salmonella Typhimurium isolate (DA34821) from Germany and a foodborne Salmonella Typhimurium isolate (CFSA629) from China, clustered into a single clade, sharing ≤67 SNPs, which suggested that clone spread occurred. These findings underline the emergence of R-type ASuT in Salmonella Typhimurium, which is attributed to the presence of an antimicrobial resistance gene cluster (tet(B), sul2, bla_TEM-1B, aph(3″)-Ib, and aph(6)-Id) encoded on the chromosome.