Analysis of Multilocus Sequence Typing and Virulence Characterization of Listeria monocytogenes Isolates from Chinese Retail Ready-to-Eat Food

Eighty Listeria monocytogenes isolates were obtained from Chinese retail ready-to-eat (RTE) food and were previously characterized with serotyping and antibiotic susceptibility tests. The aim of this study was to characterize the subtype and virulence potential of these L. monocytogenes isolates by multilocus sequence typing (MLST), virulence-associate genes, epidemic clones (ECs), and sequence analysis of the important virulence factor: internalin A (inlA). The result of MLST revealed that these L. monocytogenes isolates belonged to 14 different sequence types (STs). With the exception of four new STs (ST804, ST805, ST806, and ST807), all other STs observed in this study have been associated with human listeriosis and outbreaks to varying extents. Six virulence-associate genes (inlA, inlB, inlC, inlJ, hly, and llsX) were selected and their presence was investigated using PCR. All strains carried inlA, inlB, inlC, inlJ, and hly, whereas 38.8% (31/80) of strains harbored the listeriolysin S genes (llsX). A multiplex PCR assay was used to evaluate the presence of markers specific to epidemic clones of L. monocytogenes and identified 26.3% (21/80) of ECI in the 4b-4d-4e strains. Further study of inlA sequencing revealed that most strains contained the full-length InlA required for host cell invasion, whereas three mutations lead to premature stop codons (PMSC) within a novel PMSCs at position 326 (GAA → TAA). MLST and inlA sequence analysis results were concordant, and different virulence potentials within isolates were observed. These findings suggest that L. monocytogenes isolates from RTE food in China could be virulent and be capable of causing human illness. Furthermore, the STs and virulence profiles of L. monocytogenes isolates have significant implications for epidemiological and public health studies of this pathogen.


INTRODUCTION
Listeria monocytogenes is a gram-positive, facultative intracellular bacterium that is responsible for listeriosis. Listeriosis can cause meningitis, newborn septicemia, encephalomyelitis, or even death in humans, especially in the elderly, pregnant women, or newborn. Every year, ∼1591 cases of listeriosis in humans are report, with a 19% case-fatality rate in the United States (Scallan et al., 2011). In the European Union, a total of 1763 confirmed human cases of listeriosis (notification rate of 0.44 cases per 100,000 population) were reported in 2013 (EFSA, 2015). As an important foodborne pathogen, it is widespread in nature and lives naturally in plants and soil environments. Its ability to survive and grow over a wide range of environmental conditions, including refrigeration temperatures, high salt concentration and low pH, makes it a potential hazard in foods (Ryser and Marth, 2007).
Differences in virulence between L. monocytogenes strains may also influence infection and clinical outcome. Some strains are highly pathogenic and sometimes deadly, whereas others are less virulent or even avirulent and produce little harm in the host (Olier et al., 2002). Several methods have been used to differentiate L. monocytogenes strains. Based on somatic (O) and flagellar (H) antigens represents a conventional approach to understanding L. monocytogenes isolates ecological distribution and epidemiology. However, there are 13 serotypes and only four serotype (1/2a, 1/2b, 1/2c, and 4b) cause almost all cases of listeriosis in human. To further discriminate these strains, numerous molecular methods have been developed for epidemiological investigations and of help for the surveillance and control of listeriosis. Multilocus sequence typing (MLST) method is one of the most robust tools for investigating the global epidemiology of microbial populations (Sullivan et al., 2005). Based on the sequencing of seven housekeeping genes, it is highly discriminatory and provides unambiguous results that can be comparable directly among laboratories via the internet. In recent years, MLST has evolved to the reference method for global epidemiology and population biology (Maiden, 2006;Ragon et al., 2008).
Certain virulence and virulence-associated genes also play very important roles in intracellular survival, cell-to-cell spread, and virulence of L. monocytogenes. The presence of a number of virulence factors such as surface-associated internalins, listeriolysin O, and listeriolysin S (LLS) in L. monocytogenes significantly regulates its pathogenicity (Cotter et al., 2008;Shen et al., 2013). Of which, internalin A (InlA), encode by inlA, is responsible for facilitating the entry of L. monocytogenes into nonprofessional phagocytic cells expressing the human isoform of E-cadherin (Lecuit et al., 2001). It has been shown that mutations in inlA leading to a premature stop codon (PMSC) significantly reduce the invasion of the strain to human epithelial cells (Nightingale et al., 2005a). In addition, a small number of closely related L. monocytogenes clones have caused multiple outbreaks worldwide. These epidemic clones (EC) are divided into seven groups: ECI, ECII and ECIV within serotype 4b, ECIII, ECV and ECVII in serotype 1/2a, and ECVI within serotype 1/2b (Kathariou, 2003;Knabel et al., 2012;Lomonaco et al., 2013).
Since the main food linked to listeriosis outbreaks was readyto-eat (RTE) food, the consumer had limit opportunities to destroy the pathogen before the food consumed. Therefore, the risk associated with strains isolated from RTE food may be more severe. In our previous study, 80 RTE L. monocytogenes isolates were examined (Wu et al., 2015a). In order to determine the potential pathogenic profile and relative risk of these RTE L. monocytogenes isolates, this study provide a phylogenetic framework based on MLST analysis of L. monocytogenes isolates and evaluate their virulence-genes and EC-specific markers. In addition, we analyzed the full-length sequences of important virulence factor (inlA) to investigate correlations among the MLST types, amino acid sequence of inlA, and virulence potential.

Bacterial Isolates
A total of 80 L. monocytogenes isolates were collected from retail ready-to-eat food samples in 24 Chinese cities were analyzed, comprising 27 isolates from cold vegetable dishes in sauce, 7 isolates from cold noodles dishes in sauce, 17 isolates from roast chicken, 12 isolates from roast duck, 11 isolates from roast pork, and 5 isolates from pasteurized milk ( Table 1). The isolates were obtained between September 2012 and January 2014 according to the GB 4789.30-2010 of food microbiological examination of L. monocytogenes (National Food Safety Standards of China) with slight modifications and the most probable number (MPN) method (Gombas et al., 2003). They were identified by Gram stain, catalase, and oxidase tests and Micro ID Listeria identification system (Microgen, Camberley, UK). Serovars and antibiotic susceptibility were confirmed in previous study (Wu et al., 2015a). Additionally, 35 isolates were selected for the sequencing analysis of inlA, based on MLST profile, molecular serogroup, food origin, and antimicrobial resistance. Each isolate was incubated at 37 • C overnight on TSB-YE (Tryptic soy agar with yeast extract). Genomic DNA was extracted using a Genomic DNA Extraction kit (Dongsheng Biotech, Guangzhou, China) according to the manufacturer's instructions. The concentration of genomic DNA was determined at 260 nm using a NanoDrop-ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, MA, USA).

Multilocus Sequence Typing
The MLST scheme used to characterize L. monocytogenes isolates is based on the sequence analysis of the following seven housekeeping genes: abcZ (ABC transporter), bglA (betaglucosidase), cat (catalase), dapE (succinyl diaminopimelate desuccinylase), dat (D-amino acid aminotransferase), ldh (Llactate dehydrogenase), and lhkA (histidine kinase) (Salcedo et al., 2003). The PCR amplification conditions were as follow: an initial cycle of 94 • C for 4 min; 35 cycles of 94 • C for 30 s, 52 • C for 30 s (45 • C for bglA), 72 • C for 2 min, and a final extension at 72 • C for 10 min. The DNA fragments were purified using a PCR purification kit (Qiagen, Genman) and were sequenced in each direction with Big Dye fluorescent terminators on an ABI3730XL sequencer (Applied BioSystems).

Determination of Virulence Genes and Epidemic Clone
The isolates were identified using duplex PCR detection containing hly (707 bp) and inlB (367 bp) genes, which are specific to L. monocytogenes, as previously described (Xu et al., 2009). Multiplex PCR (Liu et al., 2007) was used to determine the presence of the virulence genes inlA, inlC, and inlJ. The llsX gene was detected by PCR assays to identify the LLS-positive L. monocytogenes strains (Clayton et al., 2011). Presumptive major ECs (ECI, ECII, and ECIII) were found in the isolates as described previously (Chen and Knabel, 2007).
All primers and PCR conditions are presented in Supplementary  Table 1. inlA Gene Sequencing The 2400 bp long inlA gene was sequenced in 35 isolates representing the clonal diversity of L. monocytogenes. External primers were used to amplification covering the whole inlA ORF and internal primers for sequencing (Supplementary Table 2). The inlA sequences were assembled using Seqman (DNASTAR, Lasergene). Mutation types were determined according to the site of mutation that leads to PMSC in inlA (Nightingale et al., 2005a) and by comparing the obtained inlA sequence data to that of the L. monocytogenes EGDe reference strain (Glaser et al., 2001).

Data Analysis
For each MLST locus, an allele number was given to each distinct sequence variant, and a distinct sequence type (ST) number was attributed to each distinct combination of alleles among the seven genes. Sequence types (STs) were determined by using the L. monocytogenes MLST database ( (Thompson et al., 1997), which was followed by phylogenetic analysis using the maximum likelihood algorithm in MEGA 6 (version 6.05) (Tamura et al., 2011).
The evolutionary characteristics of the seven housekeeping genes among the isolates were analyzed using the software dnasp5, which can distinguish between randomly (i.e., neutrally) and non-randomly evolving DNA sequences. Tajima's D test indicated that abcZ, cat, dapE, dat, ldh, and lhkA evolved randomly, whereas bglA evolved non-significantly (0.9151, P > 0.10) ( Table 3). However, seven gene portions harbored a total of 195 polymorphisms (5.93%; range 3.51-12.1% per gene). The average nucleotide diversity π was 2.6%, ranging from 0.9 to 6.2% per gene. The GC% observed in all alleles ranged from 36.4 to 42.9%, which was consistent with the 39% value observed across the entire L. monocytogenes EGDs genome (Glaser et al., 2001).

Prevalence of Virulence Associated Genes and EC Markers
Eighty isolates of L. monocytogenes from retail RTE food were detected for the presence of virulence genes. The result showed that all isolates harbored listeriolysin O genes (hly) and internalin genes (inlA, inlB, inlC, and inlJ), and 38.8% (31/80) of isolates harbored listeriolysin S genes (llsX) ( Table 4). The llsX-positive isolates including twenty-one 4b-4d-4e isolates, eleven 1/2b-3b-7 isolates. EC markers were identified in 26.3% (21/80) of isolates by multiplex PCR. ECI was the only EC marker identified and was observed in 4b-4d-4e isolates from roast meat (roast chicken/duck/pork) in this study. These EC isolates were also found to be positive for llsX.

Analysis of Selected Isolates for inlA Sequence
Sequencing the full 2400 bp inlA ORF in 35 L. monocytogenes isolates yielded 14 different inlA allelic types, indicating 88.6% of gene diversity. A total of 126 (5.2%) sites were polymorphic, including 85 sites with synonymous substitutions and 41 sites with non-synonymous substitutions. The average number of nucleotide differences per site between two sequences (π) was 0.01907; the average number of nucleotide differences (k) was 45.834. The GC% observed in inlA was 37.1%. The phylogenetic tree generated using the inlA sequences of 35 RTE isolates and the L. monocytogenes EGDe reference strain  Table 1). This tree was generated using the S.T.A.R.T (version 2).
Comparison of the amino-acid sequences of inlA between 35 L. monocytogenes isolates and the homologous sequence type strain EGDs, 13 internalin A variants were grouped. Each STs showed same variants, excluding CC1 (ST1, ST807), CC3 (ST3, ST804), and ST5 and ST805. Isolates from ST1 divided into two variants. In total, 40 amino acids were substituted (40/800, 5%). *Indicate statistical differences of P < 0.05. **Indicate statistical differences of P < 0.01. ***Statistical differences of P < 0.001.  Most of the substitutions (31/40, 77.5%) occurred in the leucinerich repeats (LLR), the inter-repeat (IR) domain and the B-repeat domain (Figure 3). The most conserved region of InlA among the 35 RTE L. monocytogenes isolates was the LRR domain. In parallel, a previous study (Ragon et al., 2008) reported the high constraint of the LRR-region and the moderate constraint of the IR-and B-repeat regions.

DISCUSSION
As an important foodborne pathogen, L. monocytogenes remains a signification public health and food safety threat worldwide.
In China, many studies of the prevalence of L. monocytogenes in food have been reported (Chen et al., 2009(Chen et al., , 2014, but the outbreaks and infection of listeriosis in food are very limited. However, not all L. monocytogenes isolates have an equal capacity to cause disease. Therefore, it is important to investigate the molecular characteristics and virulence potential of L. monocytogenes isolates for designing and implementing more effective prevention strategies. The RTE isolates analyzed in this study were isolated from most of provincial capitals of China (Wu et al., 2015a), which could be better understanding the characterization of this important pathogen in China.
To enable increased characterization, MLST can be complemented by the nucleotide sequence determination of one or more highly diverse genes, such as those encoding antigens or antibiotic resistance determinants (Sullivan et al., 2005). In this study, 80 L. monocytogenes RTE isolates belonged to limited number of major clones. Except four new STs (ST804,805,806,807), the other detected STs were already described in the L. monocytogenes Institute Pasteur MLST database (http://bigsdb.web.pasteur.fr/perl/bigsdb/bigsdb.pl? db=pubmlst_listeria_isolates_public&page=profiles): 327 strains of ST1 (51% from human isolates), 253 strains of ST3 (37% from human and 55% from food and environment), 143 strains of ST7 (63% from animal and feed), 37 strains of ST8 (including 15 human and 10 food), 130 strains of ST9 (35% from human and 21% from food), 52 strains of ST5 (50% from human and 23% from food), 22 strains of ST87 (55% from human and 23% from food), 70 strains of ST155 (50% from human), 6 strains of ST224 (2 isolates from human and 4 from environment), and 74 strains of ST121 (54% from food). Strains of all STs observed in our study have been associated to various extents with human listeriosis and outbreaks, indicating that L. monocytogenes strains of these STs have at least a theoretical pathogenic potential. It is worth nothing that ST1, comprising 20% of the isolates in this study, have the same sequences as F2365 (a serotype 4b isolates from a soft cheese outbreak in California in 1985), CLIP12848 (a serotype 4b isolates from outbreak in France in 1989), and CLIP68868 (a serotype 4b isolates from outbreak in Sweden 1995) which should the worthy of the attention of the food hygiene supervision department. Interestingly, novel STs (ST805, ST806) were found in multidrug resistant strains (Figure 1), showing some allele numbers had exchanged. Multiple resistance in L. monocytogenes was linked to the presence of a self-transferable plasmid that was proposed to originate in Enterococcus-Streptococcus (Charpentier and Courvalin, 1999). Therefore, the self-transferable plasmid may also impact the homologous recombination of housekeeping genes. However, further research is needed to determine the reason underlying this correlation. The presence of hly, inlA, inlB, inlC, inlJ, and llsX was evaluated in L. monocytogenes isolates recovered from patients, food and the environment (Kathariou, 2003;Wieczorek et al., 2012;Wu et al., 2015b). In this study, PCR based analysis of the 80 RTE isolates showed that most of these isolates possessed virulence genes similar to those of clinical isolates (Mammina et al., 2009). The hly gene is encoding the listeriolysin O (LLO), the determinant that is required for the disruption of the phagocytic vacuole and the release of bacteria into the cytoplasm, a prerequisite for their intracellular proliferation. Therefore, LLO is an essential virulence factor and its absence leads to total avirulence (Portnoy et al., 1992). L. monocytogenes adheres to and actively through actions of internalins, a complex family of LRR-containing proteins (Bierne et al., 2007). In this study, we selected inlA, inlB, inlC, and inlJ for tested internalins in L. monocytogenes isolates. These genes are claimed to play a role in pathogenesis of human listeriosis. Besides, the llsX gene (encoding LLS) was employed as a genetic marker to detect L. monocytogenes with Listeria pathogenicity island 3 (LIPI-3) (Cotter et al., 2008), and strains from the present study exhibited 38.8% (31/80) positivity. The LLS, which is present in a subset of strains of lineage I, is induced only under oxidative stress conditions and contributes to murine virulence and in survival in polymorphonuclear neutrophils (Cotter et al., 2008). Overall, our result showed that the positive-llsX strains were found exclusively in lineage I (shown Table 3 and Figure 1), the evolutionary lineage of L. monocytogenes that contributes to the majority of spontaneous and epidemic outbreaks of listeriosis (Jeffers et al., 2001). Besides, most of positive-llsX strains involving ECI (21/32, 65.6%) have been related to several major outbreaks in the US and Europe in the past (Kathariou, 2003;Liu, 2008). In general, ECs is defined as a small number of isolates of a presumably common ancestor (Chen and Knabel, 2007). Specific ECs continue to be associated with sporadic cases around the world (Neves et al., 2008;Mammina et al., 2009;Lomonaco et al., 2013). Recently, Cantinelli et al. (2013) conclude that the "epidemic clone" denominations represent a redundant but largely incomplete nomenclature system for MLST-defined clones, which must be regarded as successful genetic groups that are widely distributed across time and space. In our study, L. monocytogenes isolates of MLST-CC1, including ST1 and ST807 were both ECI which was consistent with this conclusion. However, the high occurrence of multiple virulence-associated genes and ECI markers in L. monocytogenes isolates from Chinese RTE food could pose a significant health risk as these isolates can be pathogenic and potentially capable of causing an epidemic.
Certainly, the presence of these virulence-associated genes does not mean a particular strain is virulent, as these genes are normally present in L. monocytogenes but if absence of these genes lead to avirulence. Over the past decade, previous studies identified naturally occurring PMSC mutations in inlA and have demonstrated that these mutations are responsible for virulence attenuation (Van Stelten and Nightingale, 2008). L. monocytogenes strains with PMSCs were frequently isolated from RTE foods in the US, although inlA PMSCs were markedly underrepresented among ECs (Van Stelten et al., 2010). In our study, the sequencing of inlA genes from isolates revealed that three mutations resulted in PMSCs: type 6, 11 and a novel type at position 326 (GAA→TAA), which means most of strains (32/35, 91.4%) are capable of producing full-length InlA required for host cell invasion. Generally, PMSCs have been reported 30%-45% of prevalence in food but 5% of prevalence in clinical isolates (Jacquet et al., 2004;Van Stelten et al., 2010;Chen et al., 2011). Three mutations were both harbored by the lineage II strains (2 strains with ST9, one strain with ST121) (Figure 2), which further confirmed that 1/2a and 1/2c (lineage II) more frequently possess PMSCs than 1/2b or 3b serotypes, with 4b strains rarely having PMSCs (Orsi et al., 2011). PMSC seems to be more frequently observed in ST9 and ST121 isolates as previous reports (Ragon et al., 2008;Ciolacu et al., 2014). ST9 and ST121 include isolates from several different countries and from several different environmental, clinical and food source (Ragon et al., 2008;Parisi et al., 2010;Holch et al., 2013). Some L. monocytogenes strains can persist for a longtime in food processing. The repeatedly lost by convergent evolution in the genetically homogeneous of these genotypes, may be attribute to the selective advantage by the loss of a functional InlA protein or a relaxed selective constraint on maintaining InlA function (Ragon et al., 2008). It is worth mentioning that the novel PMSCs type at position 326 (GAA→TAA), indicated that variation occurred in the LLR domain were located in repeats 10 (Figure 3). This domain promotes interaction with human surface receptor, E-cadherin, and has been reported as highly conserved, especially from repeats 7 to 15 (Ragon et al., 2008). To date, at least 18 distinct mutations in inlA leading to PMSCs have been observed in L. monocytogenes isolates worldwide, only four of them (type 5, 16, 17, and 18) were found in LLR (Van Stelten et al., 2010). Compared with the inlA variants, L. monocytogenes retained the ability for localized recombination is clearly provided by the inlA gene coding InlA, in agreement with previous reports (Nightingale et al., 2005b;Orsi et al., 2007). The substitution of some amino acid were consistent between lineage I strains and II, such as lysine replaced into arginine at position 3 in all lineage I strains (Figure 3). Additionally, the close relationship between inlA sequencing and MLST typing (Figure 2) suggested that housekeeping genes and virulenceassociated genes had some similar genetic characterization, representing monophyletic origin. In other words, our data suggest that L. monocytogenes evolved and generated different STs related to some degree of evolutionary consistent.
In summary, the research findings suggest that L. monocytogenes isolated from Chinese RTE food showed genetic relatedness and virulence attribute. Serotype, lineage, MLST types, even antibiotic resistance showed some degree of genetically relationship. Most MLST sequence types (10/14, 71.4%) found in this study have been linked to human listeriosis around the world. All isolates carried several important virulence genes (inlA, inlB, inlC, inlJ, and hly), whereas 38.8% (31/80) of strains harbored listeriolysin S genes (llsX). Besides, all 4b-4d-4e isolates belonged to ECI showed a high potential to cause human diseases. Further study of inlA sequencing showed most of selected strains (32/35, 91.4%) contained PMSC-lacking inlA gene sequences required for the encoding of InlA factor and bacterial invasion of the host cell. Based on the genetic properties observed in L. monocytogenes isolates, it is reasonable to assume L. monocytogenes found in Chinese retail RTE food have potential to cause listeriosis, and is concerning. These data have significant implications for the epidemiological and public health studies of this pathogen.