Salmonella enterica subsp. enterica serovar 1,4,,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,,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,,12:i:-, which was higher than that in Salmonella Typhimurium. Moreover, Salmonella 1,4,,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,,12:i:- isolates demonstrated MLST pattern ST34. Genomic deletions within the fljBA operon and surrounding genes were only found in Salmonella 1,4,,12:i:- isolates, with all isolates containing a deletion of fljB. However, hin and iroB were identified in all Salmonella 1,4,,12:i:- isolates. Three different deletion profiles were observed and two of them were different from the reported Salmonella 1,4,,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,,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.
Citation: Yang X, Wu Q, Zhang J, Huang J, Guo W, Cai S (2015) Prevalence and Characterization of Monophasic Salmonella Serovar 1,4,,12:i:- of Food Origin in China. PLoS ONE 10(9): e0137967. https://doi.org/10.1371/journal.pone.0137967
Editor: Praveen Rishi, Panjab University, INDIA
Received: April 7, 2015; Accepted: August 24, 2015; Published: September 11, 2015
Copyright: © 2015 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: Nucleotide sequences were deposited to the MLST database under PubMLST IDs St1922. All allele sequences of the seven housekeeping genes in our study as well as ST1922 can be found in "Download" at the following URL: http://mlst.warwick.ac.uk/mlst/dbs/Senterica.
Funding: The authors' study was supported by the Science and Technology Projects of Guangdong (2013B050800026, JMZ; 2014A040401055, XJY) and the Science and Technology Projects of Guangzhou (201300000074, JMZ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Salmonella are often acquired from contaminated food, and are important causes of gastroenteritis and bacteremia, posing a worldwide threat to public health . There are estimated to be 94 million cases of gastroenteritis globally per year, with 155,000 deaths attributed to Salmonella [2,3].
Since 2009, Salmonella enterica subsp. enterica serovar 1,4,,12:i:- has ranked among the four most frequent serovars causing human salmonellosis in China . It is also one of the most common serovars isolated from humans and foods in several other countries [4,5]. This atypical serovar, lacking the phase 2 flagellar antigen [6,7], has become increasingly important since the mid-1990s worldwide. Currently, it is designated as a monophasic variant of Salmonella Typhimurium, with antigenic and genotypic similarities [6,8–10].
The mechanisms for the evolution of Salmonella 1,4,,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 . 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 [10,12–14].
Moreover, the public health impact of Salmonella 1,4,,12:i:- infection is increased by the high rate of antimicrobial resistance associated with this serovar. In particular, an epidemic multidrug-resistant (MDR) clonal lineage of Salmonella 1,4,,12:i:-, designated the European clone, which is resistant to ampicillin, streptomycin, sulfonamides, and tetracycline (R-type ASSuT) , has emerged in Europe and has been implicated in several outbreaks [16–18]. Some Salmonella 1,4,,12:i:- isolates from Spain, America, and Canada with additional resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (R-type ACSSuT) [6,19,20] have also been detected.
Several previous studies have examined Salmonella 1,4,,12:i:- from human cases of food poisoning in China [2,21]; however, systematic data for the prevalence of Salmonella 1,4,,12:i:- in the food chain is lacking. Therefore, the objective of this study was to characterize Salmonella 1,4,,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.
Materials and Methods
From July 2011 to May 2014, 2800 food samples were collected from retail markets; these included meat and meat products (639), aquatic products (554), quick-frozen food (316), edible mushrooms (270), vegetables (272), dairy products (186), fruit (10), and ready-to-eat food (553). The sampling sites were distributed throughout the 24 provincial capitals of China (S1 Fig). In total, 524 Salmonella isolates were obtained and identified to serovar level according to the Kauffmann-White scheme . For monophasic Salmonella serovar 1,4,,12:i:-, the absence of phase 2 flagellar antigen was verified using the flagellar phase reversal method as described by Chiou et al . Strains were definitively assigned to Salmonella Typhimurium or Salmonella 1,4,,12:i:- using the duplex-PCR assay developed by Tennant et al  and recommended by the European Food Safety Authority (EFSA) Panel on Biological Hazards . The malic acid dehydrogenase gene (mdh), which is specific to Salmonella Typhimurium and Salmonella 1,4,,12:i:-, was also detected using primers previously described . Ratios of Salmonella 1,4,,12:i:- occurrence were calculated against all serotyped Salmonella or Salmonella Typhimurium isolated from the same batch of food samples in the same period .
Antimicrobial susceptibility testing
Antimicrobial susceptibility was evaluated using the Kirby-Bauer disk diffusion method with 20 antimicrobial agents, in accordance with the Clinical Laboratory Standards Institute guidelines . The antimicrobials used were ampicillin, amoxicillin-clavulanic acid, cephalothin, cefazolin, cefoxitin, ceftriaxone, cefotaxime, ceftazidime, cefoperazone, cefepime, chloramphenicol, tetracycline, nalidixic acid, ciprofloxacin, amikacin, gentamicin, streptomycin, kanamycin, trimethoprim-sulfamethoxazole, and sulfonamides (Oxoid, Basingstoke, UK).
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.
Multilocus sequence typing (MLST)
Salmonella isolates were characterized by MLST using previously reported primers specific for seven housekeeping genes: aroC, dnaN, hemD, hisD, purE, sucA, and thrA. The sequence type (ST) of each isolate was assigned according to the MLST database (http://mlst.ucc.ie/mlst).
PCR-based characterization of gene deletion profiles of Salmonella isolates
For all Salmonella Typhimurium and Salmonella 1,4,,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  and García et al . fljA, fljB, hin, and iroB are located at the 3′ end of cluster V, while STM2757 and STM2758 flank the 5′ 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,,12:i:- isolates .
As shown in S1 Table and S2 Fig, serotyping, including flagellar phase reversal, identified 13 Salmonella isolates lacking phase 2 flagellar antigen. These Salmonella 1,4,,12:i:- isolates originated from beef (n = 6), pork (n = 5), chicken (n = 1), and pigeon (n = 1). All isolates produced a single 1,000-bp amplicon corresponding to a fragment of the IS200 element, typical of Salmonella 1,4,,12:i:-. The control group of 58 Salmonella Typhimurium isolates were also confirmed, with 55 isolates producing two amplicons (1,000-bp and 1,389-bp), which is specific for Salmonella Typhimurium, and the remaining three being positive for the 1,389-bp phase 2 flagellar gene but negative for the 1,000-bp IS200 fragment. Positive detection of mdh confirms the presence of Salmonella Typhimurium or its monophasic variant Salmonella 1,4,,12:i:-. In the current study, mdh was detected in all Salmonella 1,4,,12:i:- and Salmonella Typhimurium isolates. The overall Salmonella 1,4,,12:i:- occurrence ratio reached 2.5% (13/524), whereas Salmonella 1,4,,12:i:-/Salmonella Typhimurium ratio was 22.4% (13/58) in the period of 2011 to 2014.
All 71 of the Salmonella isolates examined in this study showed some level of antibiotic resistance. In total, 76.9% of Salmonella 1,4,,12:i:- and 58.6% of Salmonella Typhimurium isolates were resistant to three or more antimicrobials (Table 1). Among these antibiotics, high rates of resistance were observed for tetracycline, sulfonamides, ampicillin, nalidixic acid, streptomycin, kanamycin, trimethoprim-sulfamethoxazole, and chloramphenicol in both Salmonella 1,4,,12:i:- and Salmonella Typhimurium isolates (S2 and S3 Tables). The main antimicrobial resistance patterns of the 13 Salmonella 1,4,,12:i:- and 58 Salmonella Typhimurium isolates are shown in Table 1.
The 13 Salmonella 1,4,,12:i:- isolates showed highly similar virulence gene profiles. All isolates contained gipA (encoding a Peyer’s patch-specific virulence factor) and sodC1 (putative Cu/Zn superoxide dismutase), carried by the Gifsy-1 and Gifsy-2 lambdoid prophages, respectively, while sspH1 (encoding a Salmonella type III effector protein), located in Gifsy-3, and three genes associated with the Salmonella virulence plasmid (spvC, pefA, and rck) were absent. However, three Salmonella 1,4,,12:i:- isolates shared a different virulence gene profile, and were positive for sopE1 (Salmonella type III effector protein). Overall, the prevalence of each of the virulence genes was similar between the Salmonella 1,4,,12:i:- and Salmonella Typhimurium isolates, except for the absence of the three plasmid genes and presence of gipA in all of the Salmonella 1,4,,12:i:- isolates (Tables 2 and 3).
Among the 13 Salmonella 1,4,,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,,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).
Genomic deletions within the fljBA operon were only found in Salmonella 1,4,,12:i:- isolates. The 13 Salmonella 1,4,,12:i:- isolates showed three different deletion profiles for cluster V, namely ΔfljAB-I, ΔfljAB-II, and ΔfljAB-III. ΔfljAB-I and ΔfljAB-II were the most common deletion profiles. fljB was absent and hin was present in all Salmonella 1,4,,12:i:- isolates. However, fljA was present in only one isolate (deletion profile ΔfljAB-III). The deletion profiles ΔfljAB-I and ΔfljAB-II were marked by the presence and absence of STM2757 and STM2758, respectively (Table 5 and S3 Fig).
This study confirmed the presence of Salmonella 1,4,,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,,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,,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.
The prevalence of MDR isolates observed in our surveillance of Salmonella Typhimurium (58.6%) and Salmonella 1,4,,12:i:- (76.9%) is similar to results from previous studies conducted in humans in China [2,21], and to rates of resistance reported in other countries [11,33]. The current study confirmed the prevalence of R-type ASSuT Salmonella Typhimurium isolates, with an even higher prevalence of this phenotype amongst Salmonella 1,4,,12:i:- isolates. In addition, several isolates displayed an R-type ACSSuT phenotype (Table 1). R-type ASSuT was also the most common profile among Salmonella 1,4,,12:i:- strains isolated in Germany from pigs, pork products, and humans , as well as among strains isolated from humans and foodstuffs in England, France, Germany, Italy, Spain, Switzerland, and the Netherlands [1,34]. The emergence and spread of the R-type ASSuT Salmonella 1,4,,12:i:- clonal lineage deserves further attention as it has been responsible for several foodborne Salmonella outbreaks, such as in Luxembourg in 2006  and in Italy in 2010 , which can be associated with severe diseases, even deaths.
Most of the virulence genes with reported contributions to pathogenicity are located in Salmonella pathogenicity islands (SPIs), fimbrial clusters, plasmids and prophages. SPIs and fimbrial clusters are highly conserved regions, while prophages and plasmids are variable within the Salmonella genome. Some differences were observed in the genes located on prophages (gipA, sodC1, sopE1, sspH1) and plasmids (spvC, pefA, rck) when comparing virulence gene profiles from Salmonella Typhimurium and Salmonella 1,4,,12:i:- in previous studies [12,26]. In our study, three plasmid-borne virulence genes (spvC, pefA, and rck) were highly prevalent amongst Salmonella Typhimurium isolates, while none of the Salmonella 1,4,,12:i:- isolates contained these genes. This is consistent with the findings of Capuano et al . The absence of these plasmid-borne virulence genes has been reported previously in R-type ASSuT Salmonella 1,4,,12:i:- strains of the European clone. In contrast, these genes have been identified in some Spanish clone Salmonella 1,4,,12:i:- strains, which are characterized by plasmid-mediated resistance to up to seven antimicrobial drugs, including ampicillin, chloramphenicol, gentamicin, streptomycin, sulfonamides, tetracyclines, and trimethoprim (R-type ACGSSuTTp) . In addition, Capuano et al  also detected gipA in all Salmonella 1,4,,12:i:- isolates of food origin, but in only 76.9% of Salmonella Typhimurium isolates. Another aspect to consider is that the detection of the target virulence genes showed no relationship to the serotype (Salmonella Typhimurium or Salmonella 1,4,,12:i:-), while some correlation with the MLST pattern was observed. All ST34 Salmonella Typhimurium isolates, and a large number (76.9%) of the ST34 Salmonella 1,4,,12:i:- isolates, exhibited a virulence gene profile of “gipA; sodC1”.
All Salmonella 1,4,,12:i:- isolates in the present study were assigned to ST34, sharing identical STs with Salmonella Typhimurium isolates, which further confirmed that Salmonella 1,4,,12:i:- and Salmonella Typhimurium were genetically closely related. Furthermore, these data suggested that Salmonella Typhimurium strains of ST34 might be ancestral candidates for serotype 1,4,,12:i:- from China. ST34 has previously been associated with R-type ASSuT Salmonella 1,4,,12:i:- strains belonging to the European clone [15,35]. In contrast, R-type ACGSSuTTp Salmonella 1,4,,12:i:- strains of the Spanish clone have been assigned to ST19 [12,35]. In the current study, Salmonella 1,4,,12:i:- isolates represented only one ST, while Salmonella Typhimurium isolates belonged to five different STs. This indicated considerably lower sequence diversity amongst Salmonella 1,4,,12:i:- than Salmonella Typhimurium. This observation has also been reported by Guerra et al  and Soyer et al .
All Salmonella 1,4,,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,,12:i:- strains. Most Salmonella 1,4,,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,,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,,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 . 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,,12:i:- strains, whereas the ΔfljAB-I and ΔfljAB-III profiles were newly described for Salmonella 1,4,,12:i:- strains. This observed variation supported the independent evolution of multiple successful monophasic clones from Salmonella Typhimurium ancestors . Sequencing of the entire fljBA operon and flanking genes in future studies could give better insight into the genetic background of such variants.
This study is the first report of Salmonella 1,4,,12:i:- in food products from China. The main sources of Salmonella 1,4,,12:i:- in China appear to be pork and beef. The examined Salmonella 1,4,,12:i:- strains were mainly ST34, confirming the domination of the tetra-resistant ASSuT clone. The observed genomic deletion of the fljBA operon and its flanking genes in Salmonella 1,4,,12:i:- isolates revealed that multiple clones of this serovar are circulating in China, and several new clones were identified. Further studies are needed to assess these Salmonella 1,4,,12:i:- clones in more detail to obtain a better picture of the genetic background and relationship with Salmonella Typhimurium clones circulating in China.
S1 Fig. Map of China illustrating provinces and cities included in the study.
S2 Fig. Identification of 13 monophasic Salmonella 1,4,,12:i:- and 58 biphasic Salmonella Typhimurium isolates by duplex-PCR.
The isolates produced a single 1,000-bp amplicon corresponding to a fragment of the IS200 element were assigned to Salmonella 1,4,,12:i:- (lanes 35–39, 46, 51–53, 55, 58, 59, 62). The isolates produced two amplicons (1,000-bp and 1,389-bp) (lanes 2–23, 26–28, 30–34, 40–45, 47, 50, 54, 56, 57, 60, 63, 64, 66–70, 73–74, 76–79) or a single 1,389-bp amplicon (lanes 29, 61, 75) were assigned to Salmonella Typhimurium. M, DL2,000 DNA Marker. Lanes 1, 25, 49, 65, 72, Salmonella Typhimurium ATCC14028 reference strain; lanes 24, 48, 71, 80, water-only control.
S3 Fig. Genomic deletion analysis of cluster V by PCR using two reference strains and 13 Salmonella 1,4,,12:i:- isolates.
M, DL2,000 DNA Marker. C, water-only control. Lane 1, Salmonella Typhimurium ATCC14028 reference strain; lane 2, Salmonella Typhimurium CMCC50115 reference strain; lane 3, isolate 2011–33; lane 4, isolate 2011–34; lane 5, isolate 2012–35; lane 6, isolate 2012–36; lane 7, isolate 2012–37; lane 8, isolate 2013–45; lane 9, isolate 2013–50; lane 10, isolate 2013–51; lane 11, isolate 2013–52; lane 12, isolate 2014–54; lane 13, isolate 2013–57; lane 14, isolate 2013–58; lane 15, isolate 2013–62. a) fljB, DNA fragments of the expected sizes for fljB (lanes 1 and 2), no amplicon (lanes 3–15); b) hin, DNA fragments of the expected sizes for hin (lanes 1–15); c) fljA, DNA fragments of the expected sizes for fljB (lanes 1, 2, and 10), no amplicon (lanes 3–9, 11–15); d) iroB, DNA fragments of the expected sizes for iroB (lanes 1–15); e) STM2757, DNA fragments of the expected sizes for STM2757 (lanes 1–4, 6, 7, 9–10), no amplicon (lanes 5, 8, 12–15); f) STM2758, DNA fragments of the expected sizes for STM2758 (lanes 1–4, 6, 7, 9–10), no amplicon (lanes 5, 8, 12–15).
S1 Table. Results of serotyping, antimicrobial resistance, virulotyping and MLST analysis of Salmonella isolates in this study.
aFor antimicrobial abbreviation, ampicillin (AMP), amoxicillin-clavulanic acid (AMC), cephalothin (KF), cefazolin (KZ), cefoxitin (FOX), ceftriaxone (CRO), cefotaxime (CTX), ceftazidime (CAZ), cefoperazone (CFP), cefepime (FEP), chloramphenicol (C), tetracycline (TE), nalidixic acid (NA), ciprofloxacin (CIP), amikacin (AK), gentamicin (CN), streptomycin (S), kanamycin (K), trimethoprim-sulfamethoxazole (SXT), and sulfonamides (Su). Names of antimicrobials with capital letters means resistance; Names of antimicrobials with lowercase letters mean intermediate resistance; [tetra], a tetra-resistant pattern including resistance to ampicillin, streptomycin, sulfonamide, and tetracycline (ASSuT R-type); [penta], a penta-resistant pattern including resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline (ACSSuT R-type). bVP1, virulence gene profile: gipA; sodC1; spvC; pefA; rck.
S2 Table. Antimicrobial resistance profiles of Salmonella 1,4,,12:i:- isolates examined in this study.
Conceived and designed the experiments: XJY QPW JMZ. Performed the experiments: XJY JHH. Analyzed the data: XJY JHH. Contributed reagents/materials/analysis tools: XJY JMZ WPG SZC. Wrote the paper: XJY.
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