Serotyping, MLST, and Core Genome MLST Analysis of Salmonella enterica From Different Sources in China During 2004-2019

Abstract

Salmonella enterica (S. enterica) is an important foodborne pathogen, causing food poisoning and human infection, and critically threatening food safety and public health. Salmonella typing is essential for bacterial identification, tracing, epidemiological investigation, and monitoring. Serotyping and multilocus sequence typing (MLST) analysis are standard bacterial typing methods despite the low resolution. Core genome MLST (cgMLST) is a high-resolution molecular typing method based on whole genomic sequencing for accurate bacterial tracing. We investigated 250 S. enterica isolates from poultry, livestock, food, and human sources in nine provinces of China from 2004 to 2019 using serotyping, MLST, and cgMLST analysis. All S. enterica isolates were divided into 36 serovars using slide agglutination. The major serovars in order were Enteritidis (31 isolates), Typhimurium (29 isolates), Mbandaka (23 isolates), and Indiana (22 isolates). All strains were assigned into 43 sequence types (STs) by MLST. Among them, ST11 (31 isolates) was the primary ST. Besides this, a novel ST, ST8016, was identified, and it was different from ST40 by position 317 C → T in dnaN. Furthermore, these 250 isolates were grouped into 185 cgMLST sequence types (cgSTs) by cgMLST. The major cgST was cgST235530 (11 isolates), and only three cgSTs contained isolates from human and other sources, indicating a possibility of cross-species infection. Phylogenetic analysis indicated that most of the same serovar strains were putatively homologous except Saintpaul and Derby due to their multilineage characteristics. In addition, serovar I 4,[5],12:i:- and Typhimurium isolates have similar genomic relatedness on the phylogenetic tree. In conclusion, we sorted out the phenotyping and genotyping diversity of S. enterica isolates in China during 2004-2019 and clarified the temporal and spatial distribution characteristics of Salmonella from different hosts in China in the recent 16 years. These results greatly supplement Salmonella strain resources, genetic information, and traceability typing data; facilitate the typing, traceability, identification, and genetic evolution analysis of Salmonella; and therefore, improve the level of analysis, monitoring, and controlling of foodborne microorganisms in China.

Keywords: MLST; Salmonella enterica; cgMLST; serotype; whole genome sequencing.

FIGURE 1

Distribution of S. enterica isolates in China from 2004 to 2019. The locations where the strains were isolated are shown on the map. The isolates obtained from humans, poultry, livestock, and food are labeled in different colors.

FIGURE 2

Distribution of Salmonella enterica isolates of serotypes (A) and STs (B).

FIGURE 3

Core genome MLST-based phylogenetic tree of S. enterica isolates. The neighbor-joining algorithm phylogenetic tree was created by EnteroBase based on cgMLST information and visualized using Evolview. The numbers stand for Branchlength values. The isolates’ serotypes are labeled in color, and most serovars forms serovar-specific clades except Saintpaul, Derby, and I 4,[5],12:i:-, which are signaled by the same color triangles. I 4,[5],12:i:- and Typhimurium may have a close genetic and evolutionary relationship. Further determination of the evolutionary relationship between the abovementioned serovars will be furtherly carried out. Different colors are used in the outermost ring to indicate the separation source to distinguish the specificities of different isolates.

FIGURE 4

GrapeTree of Salmonella serovar Saintpaul, Derby, and I 4,[5],12:i:- isolates. GrapeTree constructed based on the 3002 core genes by EnteroBase GrapeTree using NINJA neighbor-joining algorithm. (A) GrapeTree among the four serovar Saintpaul isolates, where every node means different cgST and the line between two nodes indicates a long distance (≥1000); (B) GrapeTree of the 21 Derby and 1 cgST (cgST236253) isolates with a long genetic distance (≥1000) to other cgSTs; (C) GrapeTree of the nine serovar I 4,[5],12:i:- and nine cgST isolates with nodes indicating they all have a relatively close genetic distance (≤100).

FIGURE 5

GrapeTree of Salmonella serovar I 4,[5],12:i:- and Typhimurium isolates. GrapeTree of nine serovar I 4,[5],12:i:- isolates and 29 serovar Typhimurium isolates constructed based on cgMLST information by EnteroBase GrapeTree function and neighbor-joining algorithm. These 38 isolates belong to four clusters in the tree, and all nine serovar I 4,[5],12:i:- isolates belong to cluster 3 with 12 serovar Typhimurium isolates, showing that Salmonella serovar I 4,[5],12:i:- and Typhimurium have a closer genetic relationship.

FIGURE 6

Comparison of phylogenies by MLST (A) and cgMLST (B) based on genomic sequences of 250 Salmonella isolates. Tanglegram linking tips with the same label to each other via a straight line produced within dendroscope 3 of 250 Salmonella isolates. By comparing of N-J trees by MLST and cgMLST, clustering in the two trees was mostly congruent although a few sections show several inversions. This led to some isolates being clustered at the edge of one tree, moving to the center of the other. In addition, the tree by cgMLST was more detailed than that by MLST.