Prevalence and Characterization of Monophasic Salmonella Serovar 1,4,[5],12:i:- of Food Origin in China

Salmonella enterica subsp. enterica serovar 1,4,[5],12:i:- is a monophasic variant of Salmonella Typhimurium, which has recently been recognized as an emerging cause of infection worldwide. This bacterium has also ranked among the four most frequent serovars causing human salmonellosis in China. However, there are no reports on its contamination in Chinese food. Serotyping, polymerase chain reaction, antibiotic resistance, virulotyping, and multilocus sequence typing (MLST) assays were used to investigate the prevalence of this serological variant in food products in China, and to determine phenotypic and genotypic difference of monophasic isolates and Salmonella Typhimurium isolated over the same period. Salmonella 1,4,[5],12:i:- was prevalent in various food sources, including beef, pork, chicken, and pigeon. The study also confirmed the high prevalence (53.8%) of resistance to ampicillin, streptomycin, sulfonamides, and tetracycline in Salmonella 1,4,[5],12:i:-, which was higher than that in Salmonella Typhimurium. Moreover, Salmonella 1,4,[5],12:i:- isolates in our study were different from Salmonella Typhimurium isolates by the absence of three plasmid-borne genes (spvC, pefA, and rck) and the presence of gipA in all isolates. All Salmonella 1,4,[5],12:i:- isolates demonstrated MLST pattern ST34. Genomic deletions within the fljBA operon and surrounding genes were only found in Salmonella 1,4,[5],12:i:- isolates, with all isolates containing a deletion of fljB. However, hin and iroB were identified in all Salmonella 1,4,[5],12:i:- isolates. Three different deletion profiles were observed and two of them were different from the reported Salmonella 1,4,[5],12:i:- clones from Spain, America, and Italy, which provided some new evidence on the independent evolution of the multiple successful monophasic clones from Salmonella Typhimurium ancestors. This study is the first report of Salmonella 1,4,[5],12:i:- in food products from China. The data are more comprehensive and representative, providing valuable information for epidemiological studies, risk management, and public health strategies.


Introduction
Salmonella are often acquired from contaminated food, and are important causes of gastroenteritis and bacteremia, posing a worldwide threat to public health [1]. There are estimated to be 94 million cases of gastroenteritis globally per year, with 155,000 deaths attributed to Salmonella [2,3].
The mechanisms for the evolution of Salmonella 1,4, [5],12:i:-remain unknown. Several genes, including fljB, fljA, and hin, located within the fljBA operon, are involved in expression of phase 2 flagellar antigen. fljB encodes the phase 2 flagellar antigen, while fljA (encoding a negative regulator of fliC, coding for the phase 1 flagellar antigen) and hin (encoding a DNA invertase) are responsible for flagellar phase variation, a regulatory mechanism involving switching between the production of FliC or FljB [5]. Thus, mutations, deletions, or insertions of fljB itself, along with disorder in the regulatory mechanism of phase variation, could result in failure to express the phase 2 flagellar antigen [7,[10][11]. Interestingly, various fljB deletion profiles, as well as deletions within other genes of the fljBA operon and surrounding genes (iroB and STM2757), have been detected [10,[12][13][14].
Several previous studies have examined Salmonella 1,4,[5],12:i:-from human cases of food poisoning in China [2,21]; however, systematic data for the prevalence of Salmonella 1,4, [5],12: i:-in the food chain is lacking. Therefore, the objective of this study was to characterize Salmonella 1,4, [5],12:i:-isolates collected from food samples across China from 2011-2014, and compare these isolates with Salmonella Typhimurium isolated in the same period to better understand the prevalence, phenotypic and genetic diversity, and susceptibility to antimicrobials of this serovar in China. Such data will provide a scientific basis for the development of food safety regulations.

Detection of virulence-related genes
Seven genes with reported contributions to virulence were selected. Four targets (gipA, sodC1, sopE1, sspH1) were located on prophages, while three (spvC, pefA, rck) were located on a virulence plasmid. Amplification was performed using primers and conditions described previously [26,27]. Salmonella Typhimurium ATCC14028 and Salmonella Typhimurium CMCC50115 were used as positive controls.

PCR-based characterization of gene deletion profiles of Salmonella isolates
For all Salmonella Typhimurium and Salmonella 1,4, [5],12:i:-isolates, the presence of fljA, fljB, and hin, as well as iroB, which flanks the fljBA operon, and the closely located STM2757 and STM2758 genes, were analyzed using gene-specific primers and PCR conditions as previously described by Soyer et al [10] and García et al [12]. fljA, fljB, hin, and iroB are located at the 3 0 end of cluster V, while STM2757 and STM2758 flank the 5 0 end. This region spans 16 genes (from STM2758 to iroB) that were previously reported to be present in Salmonella Typhimurium LT2, but absent in a selection of Spanish Salmonella 1,4, [5],12:i:-isolates [28].
Among the 13 Salmonella 1,4, [5],12:i:-and 58 Salmonella Typhimurium isolates, five distinct STs were identified based on a seven-gene MLST scheme ( Table 4). The number of alleles for the seven housekeeping genes ranged from 2-554. hisD554 and ST1922 were identified as a novel allele type and a novel MLST pattern, respectively. Two STs (ST19 and ST34) were predominant, and together accounted for 94.4% of isolates tested. ST19 was the most common Salmonella Typhimurium ST, accounting for 44 of 58 isolates. All the Salmonella 1,4, [5],12:i:isolates were assigned to ST34, a single-locus variant of ST19 (with dnaN19 instead of dnaN7); 10 Salmonella Typhimurium isolates were also designated ST34. One Salmonella Typhimurium isolate was identified as ST36, with two further isolates assigned to ST1544. These three isolates also differed from other Salmonella Typhimurium isolates based on duplex-PCR results (positive for the 1,389-bp phase 2 flagellar gene, but negative for the 1,000-bp IS200 fragment).

Discussion
This study confirmed the presence of Salmonella 1,4, [5],12:i:-in food products in China. The Salmonella isolates examined in this study were collected from a nationwide food investigation covering most provincial capitals of China. Thus, the results were fairly comprehensive and representative of China as a whole. The occurrence of Salmonella 1,4, [5],12:i:-was higher in our study compared with other countries [11,29].
Previous studies have suggested that the vast majority of these monophasic Salmonella strains are from pigs and pork products [30,31], followed by isolates obtained from poultry and cattle. Isolation from other sources was believed to be rare [9,32]. In our study, although Salmonella 1,4,[5],12:i:-isolates were obtained from various sources, beef and pork products provided 84.6% of all tested isolates, proving to be the most important reservoirs in China.
All Salmonella 1,4, [5],12:i:-isolates examined in the current study exhibited deletions in the fliBA operon and flanking genes, although variations were identified. In previous studies, various deletion profiles affecting the fljBA operon and surrounding genes have been observed in Salmonella 1,4, [5],12:i:-strains. Most Salmonella 1,4, [5],12:i:-isolates from Spain appear to be characterized by deletions of fljA, fljB, hin, and iroB and conservation of STM2757 [12,13,28], whereas some of the American Salmonella 1,4, [5],12:i:-isolates seem to contain deletions that eliminate fljA, fljB, and STM2757 but maintain hin and iroB [7,10]. The deletion profile of Salmonella 1,4, [5],12:i:-strains from Italy is characterized by the absence of the fljA, fljB, and hin genes and the presence of STM2757, as in Spanish strains, but a conserved iroB, as with the American strains [14]. Among the isolates described in this work, hin and iroB were always conserved. The ΔfljAB-II deletion profile was identical to the American Salmonella 1,4, [5],12: i:-strains, whereas the ΔfljAB-I and ΔfljAB-III profiles were newly described for Salmonella 1,4, [5],12:i:-strains. This observed variation supported the independent evolution of multiple successful monophasic clones from Salmonella Typhimurium ancestors [5]. Sequencing of the entire fljBA operon and flanking genes in future studies could give better insight into the genetic background of such variants.