Genomic Characterization of mcr-1- carrying Salmonella enterica Serovar 4,[5],12:i:- ST 34 Clone Isolated From Pigs in China

Abstract

Salmonella enterica serovar 4,[5],12:i:-, so-called Typhimurium monophasic variant, has become one of the most frequently isolated serovars both in humans and in animals all over the world. The increasing prevalence of mcr-1-carrying Salmonella poses significant global health concerns. However, the potential role of Salmonella 4,[5],12:i:- in mcr-1 gene migration through the food chain to the human remains obscure. Here, we investigated 337 Salmonella isolates from apparently healthy finishing pigs, which is rarely studied, obtained from pig farms and slaughterhouses in China. The mcr-1 gene was found in four colistin-resistant S. enterica 4,[5],12:i:- isolates. Notably, all four isolates belonged to sequence type 34 (ST34) with multidrug resistance phenotype. Further genomic sequencing and antimicrobial resistance characterization confirmed that mcr was responsible for the colistin resistance, and the conjugation assay demonstrated that three of four isolates carried mcr-1 in IncHI2 plasmid. Importantly, mcr-1 and class-1 integron were found to co-localize in two strains with IncHI2 plasmid. By collecting all the mcr-1-carrying Typhimurium and monophasic variant strains across the food chain (farm animals, animal-origin food, and humans), our phylogenomic analysis of available 66 genomes, including four strains in this study, demonstrated an independent phylogenetic cluster of all eight Chinese swine-originated isolates and one human isolate. Together, this study provides direct evidence for clonal and pork-borne transmission of mcr-1 by Salmonella 4,[5],12:i:- ST34 in China and highlighted a domestication pathway by acquisition of additional antimicrobial resistance determinants in Chinese ST34 isolates.

Keywords: 12:i:-; Colistin; ST34; Salmonella enterica serovar 4; [5]; food chain; mcr-1.

FIGURE 1

The serovar and antibiotic resistance diversity of Salmonella across farm and slaughterhouses. (A) Spatial variation of the distribution of multiple Salmonella serovars isolated from fecal samples analyzed in this study. (B) Distribution of the average resistance (in percent) of various serovars regardless of the source of isolation toward multiple antibiotics. The names of the antibiotics are abbreviated as ampicillin (AMP), amoxicillin (AMC), cefotaxime (TAX), ceftriaxone (CRO), gentamicin (GEN), kanamycin (KAN), streptomycin (STR), tetracycline (TET), doxycycline (DOX), ciprofloxacin (CIP), enrofloxacin (ENR), sulfa-trimethoprim (COT), sulfamethoxazole (SMX), colistin (CST), chloramphenicol (CHL), and florfenicol (FLO).

FIGURE 2

Sequence comparison of reconstructed mcr-1-positive plasmid from whole-genome sequence of SAL_13.3, SAL_13.5, SAL_13.6 with pLS44712 (NZ_CP035918), pS61394 (NZ_CP035916), pWW012 (NZ_CP022169), and pCFSA122-1 (NZ_CP033224.2) from a Chinese pork sample as a reference strain. Black arrows refer to pCFSA122-1, purple color refers to pSAL_13.3, pink color refers to pSAL_13.5, light blue color refers to SAL_13.6, dark blue color refers to pLS44712, blue color refers to pS61394, and yellow color refers to pWW012.

FIGURE 3

Genetic comparison of mcr-1 gene context between SAL_13.3, SAL_13.5, SAL_13.6 with pLS44712 (NZ_CP035918), pS61394 (NZ_CP035916), pWW012 (NZ_CP022169), and pCFSA122-1 (NZ_CP033224.2). N.B. Blue color in mcr-1 gene context refers to 90–100% identity and SAL_15.5 carries mcr-1 gene on chromosome.

FIGURE 4

Global phylogenomic analysis of mcr-1-positive S. Typhimurium and monophasic isolates from different hosts retrieved from NCBI database (sample ID). Green color refers to human strains, blue color refers to animal strains, brown color refers to pork strains, gray color refers to environmental strains, and orange color refers to the isolated strains in this study. N refers to the numbers of resistant genes. ST refers to the sequence type.