Heterogeneity, Characteristics, and Public Health Implications of Listeria monocytogenes in Ready-to-Eat Foods and Pasteurized Milk in China

Listeria monocytogenes is a foodborne pathogen with a high mortality rate in humans. This study aimed to identify the pathogenic potential of L. monocytogenes isolated from ready-to-eat (RTE) foods and pasteurized milk in China on the basis of its phenotypic and genotypic characteristics. Approximately 7.7% (44/570) samples tested positive for L. monocytogenes among 10.8% (39/360) RTE and 2.4% (5/210) pasteurized milk samples, of which 77.3% (34/44) had < 10 MPN/g, 18.2% (8/44) had 10–110 MPN/g, and 4.5% (2/44) had > 110 MPN/g. A total of 48 strains (43 from RTE foods and five from milk samples) of L. monocytogenes were isolated from 44 positive samples. PCR-serogroup analysis revealed that the most prevalent serogroup was II.2 (1/2b-3b-7), accounting for 52.1% (25/48) of the total, followed by serogroup I.1 (1/2a-3a) accounting for 33.3% (16/48), serogroup I.2 (1/2c-3c) accounting for 12.5% (6/48), and serogroup II.1 (4b-4d-4e) accounting for 2.1%. All isolates were grouped into 11 sequence types (STs) belonging to 10 clonal complexes (CCs) and one singleton (ST619) via multi-locus sequence typing. The most prevalent ST was ST87 (29.2%), followed by ST8 (22.9%), and ST9 (12.5%). Virulence genes determination showed that all isolates harbored eight virulence genes belonging to Listeria pathogenicity islands 1 (LIPI-1) (prfA, actA, hly, mpl, plcA, plcB, and iap) and inlB. Approximately 85.4% isolates carried full-length inlA, whereas seven isolates had premature stop codons in inlA, six of which belonged to ST9 and one to ST5. Furthermore, LLS (encoded by llsX gene, representing LIPI-3) displays bactericidal activity and modifies the host microbiota during infection. LIPI-4 enhances neural and placental tropisms of L. monocytogenes. Results showed that six (12.5%) isolates harbored the llsX gene, and they belonged to ST1/CC1, ST3/CC3, and ST619. Approximately 31.3% (15/48) isolates (belonging to ST87/CC87 and ST619) harbored ptsA (representing LIPI-4), indicating the potential risk of this pathogen. Antimicrobial susceptibility tests revealed that > 95% isolates were susceptible to 16 antimicrobials; however, 60.4 and 22.9% isolates were intermediately resistant to streptomycin and ciprofloxacin, respectively. The results show that several isolates harbor LIPI-3 and LIPI-4 genes, which may be a possible transmission route for Listeria infections in consumers.


INTRODUCTION
Listeria monocytogenes is a foodborne pathogen with a high mortality rate in humans. The vulnerable groups, such as the elderly, pregnant women, fetuses and immunocompromised individuals are the main population infected by L. monocytogenes (Cartwright et al., 2013). This pathogen has high tolerance against stressors associated with food processing, including refrigerating temperatures, low moisture content, high salinity, and a wide pH range (Hain et al., 2007). Furthermore, sequence type (ST) 8, ST5, ST87, ST2, ST121, ST14, and ST9 subtypes of L. monocytogenes are present and grow in some specific niches, resulting in longterm contamination during food production and processing (Fagerlund et al., 2016;Chen et al., 2017;Chau et al., 2017;Pasquali et al., 2018;Melero et al., 2019).
Listeria infections potentially result from ingestion of L. monocytogenes-contaminated foods. In 2017-2018, the largest listeriosis outbreak worldwide occurred in South Africa, resulting from ingestion of ready-to-eat (RTE) meat contaminated with L. monocytogenes ST6 (Smith et al., 2019). Previous studies reported an incidence of 0.46 cases per 100,000 individuals in Europe in 2015 (European Food Safety Authority and European Centre for Disease Prevention and Control, 2016). According to previous study, 147 clinical cases, 479 Listeria isolates and 82 outbreak-related cases were reported from 28 provinces between 1964 and 2010 in China, respectively (Feng et al., 2013). Fan et al. (2019) conducted a systematic review for the listeriosis in mainland China, wherein 136 records were identified, and reporting 562 patients with listeriosis from 2011 to 2017, indicating a drastic increase in the number of patients over the past decade. Listeriosis patients were primarily reported in developed cities (Beijing city and the coastal cities), probably owing to dietary habits and the high population density in these areas. Risk identification of L. monocytogenes in RTE foods is important because they provide critical information regarding food sources of listeriosis. Therefore, comprehensive surveillance of L. monocytogenes in RTE foods throughout China is of utmost importance.
This study aimed to detect and enumerate L. monocytogenes in RTE foods and pasteurized milk products and to determine their heterogeneity, characteristics, and public health implications in Chinese retail outlets.

Samples
Between March 2014 and June 2016, 360 RTE foods and 210 pasteurized milk samples were collected from 21 cities in China, including cold vegetable dish in sauce (n = 60 samples), duck (n = 77), fried rice (n = 15), chicken (n = 139), pork (n = 61), goat meat (n = 8), and pasteurized milk (n = 210). All of RTE foods were loose-packed in different markets. All samples were immediately placed in sterile bags, kept in an insulated shipping cooler with frozen gel packs placed on the sides, middle, and above the samples to maintain below 4 • C. All the samples were transferred back to the laboratory immediately and analyzed within 4 h of receiving the samples.

Qualitative and Quantitative Analysis
Qualitative detection of L. monocytogenes was performed on the basis of the guidelines of the National Food Safety Standard of China (4789.30-2010) (Anonymous, 2010), with minor modifications. Briefly, 25 g (mL) of homogenized samples were added to 225 mL Listeria enrichment broth 1 (LB1) (Guangdong Huankai, Co. Ltd., Guangzhou, China). The cultures in LB1 media were incubated at 30 • C for 24 h. After incubation, 100 µL of the LB1 enrichment culture was transferred to 10 mL Listeria enrichment broth 2 (LB2) (Guangdong Huankai, Co. Ltd.) and incubated at 30 • C for 24 h. A loopful (about 10 µL) of the LB2 enrichment culture was streaked onto Listeria CHROMagar plates (CHROM-agar, Paris, France) and incubated at 37 • C for 48 h. At least three (when possible) presumptive colonies were selected for the identification of L. monocytogenes, using the Microgen ID Listeria identification system (Microgen, Camberley, United Kingdom) in accordance with the manufacturer's instructions.
For quantitative detection, the most probable number (MPN) method using a nine-tube was followed, as reported previously (Gombas et al., 2003). Briefly, nine tubes were divided into three sets of three tubes each. Homogenized samples (25 g) were added to 225 mL half Fraser Broth (Guangdong Huankai, Co. Ltd.). The first set of tubes contained 10 mL of the sample homogenate in 225 mL half Fraser Broth, while the second and third sets contained 10 mL of half Fraser Broth inoculated with 1 and 0.1 mL of the homogenate, respectively. Different volumes, i.e., 10, 1, and 0.1 mL, of the sample homogenate represented 1.0, 0.1, and 0.01 g of the original sample, respectively. The nine tubes were incubated at 30 ± 2 • C for 24 ± 2 h. The darkened Fraser tubes were streaked onto Listeria CHROMagar plates. If a Fraser Broth tube did not display darkening, it was reexamined after an additional 26 ± 2 h of incubation. The presumptive colonies were purified again on the Tryptic Soy Agar (TSA) plates (Guangdong Huankai, Co. Ltd.) and then identified using the Microgen ID Listeria identification system. The MPN value was calculated based on the number of positive tube(s) in each sample and the MPN table (U.S. Department of Agriculture, 1998).

MLST Analysis
Using the method of Ragon et al. (2008), MLST analysis of L. monocytogenes was performed on the basis of the following seven housekeeping genes: acbZ (ABC transporter), bglA (betaglucosidase), cat (catalase), dapE (Succinyl diaminopimelate desuccinylase), dat (D-amino acid aminotransferase), ldh (lactate dehydrogenase), and lhkA (histidine kinase) (Supplementary Table S2). A detailed protocol for the present MLST analysis, including primers, PCR conditions, was in accordance with the guidelines of the Pasteur Institute website 1 , and STs and clonal complexes (CCs) of each isolate were assigned on the basis of each variant locus of each housekeeping gene. A phylogenetic tree was generated to analyze relationships among the isolates, using MEGA X (Kumar et al., 2018).

PCR-Serogroup Analysis
The ERIC-PCR fingerprinting was used to screen isolates from the same sample in order to remove the duplicate isolates (data not shown). L. monocytogenes isolates from the same sample with > 90% similarity was considered as clonal. Clonal isolates from individual sample were excluded. At least one L. monocytogenes isolate from each known source sample was submitted to further analysis. A total of 48 strains of L. monocytogenes were isolated from 44 L. monocytogenespositive samples, including four samples (Cold vegetable dish in sauce, Pickled pig ear, Roasted chicken wing, and Salt baked chicken), each containing two different isolates. L. monocytogenes isolates recovered from RTE foods (43 isolates) and pasteurized milk samples (five isolates) were subjected to serogroup assessment via multiplex PCR analysis. As shown in Figure 1, 33.3% (16/48) isolates belonged to serogroup I.1 (1/2a-3a); 12.5% (6/48), serogroup I.2 (1/2c-3c); 2.1% (1/48), serogroup II.1

DISCUSSION
Listeria monocytogenes is a prominent facultative foodborne pathogen with a worldwide prevalence. Numerous listeriosis outbreaks and sporadic cases have resulted from ingestion of L. monocytogenes-contaminated foods. In particular, RTE foods are considered vehicles of Listeria spp. owing to the absence of further heat treatment before consumption. The occurrence of listeriosis varies among different countries and usually occurs at 0.1-11.3 cases per million individuals (FAO/WHO, 2004).
In this study, we assessed 570 food samples in China from 2014 to 2016, 39 RTE foods and 5 pasteurized milk samples (7.7%) tested positive for L. monocytogenes, concurrent with contamination of L. monocytogenes in RTE foods reported in Beijing (Wang W. et al., 2015) but significantly greater than that of RTE foods in Estonia (Koskar et al., 2019) and Tokyo, Japan (Shimojima et al., 2016). However, Wang et al. (2018) reported that 16.2% of samples were contaminated with L. monocytogenes in retail outlets selling RTE foods and restaurants serving mutton in Zigong, Sichuan Province. Among the pasteurized milk samples, only 2.4% (5/210) samples were positive for L. monocytogenes. Nonetheless, the contamination rate in other countries is markedly greater than that reported herein in China, amounting to 20% among pasteurized milk in Ethiopia (Seyoum et al., 2015) and 4.9% in milk and milk products in Tamil Nadu, India (Karthikeyan et al., 2015). This discrepancy is potentially attributed to differences in the sample size, sample constitution, hygiene conditions, or geographical locations. Risk assessment of a L. monocytogenes infection through ingestion of RTE foods depended not only on the contamination rate of RTE foods, but also on their level of contamination. To limit the occurrence of listeriosis, several countries have formulated a series of food microbiological standards for L. monocytogenes for different foods. The United States Department of Agriculture (USDA) has adopted a zero-tolerance policy (absence of the organism in 25 g food sample) for L. monocytogenes in RTE foods (Orsi et al., 2011). The EU food safety regulations limit L. monocytogenes to < 100 CFU/g at the end of the shelf-life of a food product (Ziegler et al., 2019). Furthermore, China has adopted a zero-tolerance policy for RTE meat products (GB 29921-2013) (Anonymous, 2013); however, such regulations have not been adopted for pasteurized milk. In many European countries, L. monocytogenes counts must do not exceed 100 CFU/g during the shelf-life of RTE foods (Castro et al., 2017). Herein, 77.3% (34/44) samples were contaminated with L. monocytogenes below 10 MPN/g, 18.2% (8/44) were between 10 and 110 MPN/g, and only two (4.5%) samples were over 110 MPN/g. Concurrently, Koskar et al. (2019) reported that 0.3% of positive samples exceeded the food safety criterion of 100 CFU/g. The food matrix, storage time, and temperature were significant factors influencing the growth of L. monocytogenes (Ziegler et al., 2019). Results showed that the most common STs were ST8 strains in fried rice/noodles (100%, 3/3) and duck (50%, 3/6) isolates, while the ST87 strains (carried ptsA gene) were most found in chicken (44.4%, 8/18) and pork (75%, 3/4) isolates. The contamination rate of positive samples collected from Nanchang and Chengdu city were the highest among 43 cities, both being 50% (5/10), followed by Xi'ning (25%, 5/20). The count of L. monocytogenes in these positive samples may have drastically increased owing to the presence of numerous nutrients and an ambient storage temperature. The present results indicate that RTE foods are potentially an important transmission route for L. monocytogenes infections. Further surveillance of L. monocytogenes in RTE foods is warranted for risk assessment.
Virulence genes (LIPI-1, inlA, and inlB) were present in all L. monocytogenes isolates. Internalin A (InlA, encoded by inlA) is a major virulence factor associated with the invasiveness of L. monocytogenes and binds E-cadherin on host cells and facilitates the penetration of L. monocytogenes into intestinal epithelial cells (Vazquez-Boland et al., 2001). Therefore, PMSCs in the inlA gene may attenuate the virulence of L. monocytogenes strains with a lesser invasive phenotype among human intestinal epithelial cells (Nightingale et al., 2005;Van Stelten and Nightingale, 2008;Van Stelten et al., 2010). Interestingly, six (100%) ST9/CC9 and one (25%) ST5/CC5 isolates harbored PMSCs in the inlA gene, concurrent with previous studies reported in China (Chen et al., 2018a, 2019a. Similarly, the PMSCs in the inlA gene were frequent in ST121/CC121 strains, which were predominant in foods and associated environments (Ciolacu et al., 2015;Palma et al., 2017;Rychli et al., 2017Rychli et al., , 2018. These data indicate that the ST9 and ST121 strains predominant in foods may acquire PMSCs in the inlA gene. However, the ecological significance of specific InlA variants is not yet understood (Ciolacu et al., 2015). Further research should be conducted to elucidate whether the PMSCs of inlA is associated with food-related stress. The bacteriocin LLS (encoded by the llsX and other genes) contributes to intestinal survival and virulence of L. monocytogenes in a murine oral infection model, alters the gut microbiota and increases L. monocytogenes persistence (Quereda et al., 2016). ST1, ST3, and ST619 strains harbored LIPI-3 (representing llsX), consistent with these STs also causing listeriosis both in China and other countries (Lv et al., 2014;Maury et al., 2016;Wang et al., 2018). ST87/CC87 (and ST619) strains harbored LIPI-4 (representing ptsA), responsible for neural and placental infections (Maury et al., 2016). It is noteworthy that one ST619 isolate had all virulence genes tested (especially llsX and ptsA), indicating a potential hypervirulent ST. Multiple reports also shown that ST619 strain was isolated in food and carried many virulence genes, including llsX and ptsA genes (Chen et al., 2018a(Chen et al., ,b, 2019bWang et al., 2018). In addition, to the best our knowledge, ST619 was only reported in clinic cases in China . However, to date, little information is available on the pathogenicity of ST619 strains, it is necessary to explore the virulence of ST619 strains in the future. Our data suggest that RTE foods and pasteurized milk contaminated with the isolates recovered herein may increase the risk of L. monocytogenes infection for consumers in China.
Listeriosis may be more difficult to control in future owing to the emergence of antimicrobial resistance among L. monocytogenes strains isolated from food products. β-lactam (penicillin and ampicillin) alone or in combination with an aminoglycoside (gentamycin) has been the first-line treatment alternative for listeriosis. However, patients presenting an allergic reaction to penicillin, a second-line treatment alternative, usually involve a combination of trimethoprim with a sulfonamide including sulfamethoxazole in co-trimoxazole (Alonso-Hernando et al., 2012). Fortunately, all 48 L. monocytogenes isolates were susceptible to β-lactam and aminoglycoside antimicrobials herein, indicating that these antimicrobials are still effective to treat listeriosis. Furthermore, rifampicin, tetracycline, chloramphenicol, and fluoroquinolones have been used to treat listeriosis (Allerberger and Wagner, 2010). Herein, L. monocytogenes isolates were intermediately resistant, to a certain extent, to fluoroquinolones, although no completely resistant isolate was observed. The mechanisms underlying fluoroquinolones resistance have been previously reported.
Fluoroquinolone resistance may be mediated by mutations in quinolone resistance-determining regions, including GyrA, GyrB, ParC, and ParE (Bertrand et al., 2016). In addition, the expression of efflux pump FepR, MdeL, and Lde contributed to resistance toward fluoroquinolones (Lismond et al., 2008;Guerin et al., 2014;Lim et al., 2016;Wilson et al., 2018). Vancomycin and novel β-lactams constitute last-line therapy for Listeria infections, and no resistant isolate was observed herein. Therefore, future studies are required to focus on the mechanism underlying the acquisition of fluoroquinolone resistance in L. monocytogenes.

CONCLUSION
In summary, 7.7% (44/570) of RTE foods (39/360) and pasteurized milk (5/210) samples collected from 21 cities in China were positive for L. monocytogenes and two samples had a MPN > 110/g. Serogroup I.1 (ST8, 22.9%) and II.2 (ST87, 29.2%) were dominant among the 48 L. monocytogenes isolates, indicating that some specific serogroups and STs of L. monocytogenes may have distinct ecological niches. The nine classical virulence genes and additional llsX or/and ptsA potential hypervirulent genes were present in some specific L. monocytogenes isolates, indicating that the L. monocytogenes contaminated RTE foods and pasteurized milk may be a possible transmission route for Listeria infection in consumers. Although most isolates were susceptible to antimicrobials, except for streptomycin and ciprofloxacin (efflux pump mediated resistance), potential microbial safety issues in RTE foods and pasteurized milk requires close attention.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.