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Listeria monocytogenes is a foodborne pathogen that can cause invasive disease with high mortality in immunocompromised individuals and can survive in a variety of food-associated environments for a long time. L. monocytogenes clonal complex (CC) 87 is composed of ST87 and three other STs and has been identified as the most common subgroup associated with both foods and human clinical infections in China. Therefore, the persistence of CC87 L. monocytogenes in food-associated environments poses a significant concern for food safety. In this study, 83 draft genomes of CC87 L. monocytogenes, including 60 newly sequenced genomes, were analyzed with all isolates from our previous surveillance in Zigong, Sichuang, China. Sixty-eight of the studied isolates were isolated from one retail market (M1 market), while the others were from seven other markets (M2-M8 markets) in the same city. Whole-genome multilocus sequence typing (wg-MLST) and the whole-genome single nucleotide polymorphism (wg-SNP) analysis were performed. Three persistent contamination routes were identified in the M1 market, caused by 2 clusters (A and B) and a wgST31 type. Cluster A isolates were associated with the persistent contamination in a raw meat stall (M1-S77), while Cluster B isolates caused a persistent contamination in aquatic foods stalls. Five wgST31 isolates caused persistent contamination in a single aquatic stall (M1-S65). A pLM1686-like plasmid was found in all Cluster A isolates. A novel plasmid, pLM1692, a truncated pLM1686 plasmid without the cadmium, and other heavy metal resistance genes were conserved in all wgST31 isolates. By comparing persistent and putative non-persistent isolates, four genes that were all located in the prophage comK might be associated with persistence. These findings enhanced our understanding of the underlying mechanisms of contamination and assist in formulating targeted strategies for the prevention and control of L. monocytogenes transmission from the food processing chain to humans. IMPORTANCE Contamination of food by Listeria monocytogenes at retail level leads to potential consumption of contaminated food with high risk of human infection. Our previous study found persistent contamination of CC87 L. monocytogenes from a retail market in China through pulsed-field gel electrophoresis and multilocus sequence typing. In this study, whole-genome sequencing was used to obtain the highest resolution inference of the source and reasons for persistent contamination; meat grinders and minced meat were the major reservoir of persistent contamination in meat stalls, whereas fishponds were the major reservoir in seafood stalls, with different L. monocytogenes isolates involved. These isolates carried different properties such as plasmids and prophages, which may have contributed to their ability to survive or adapt to the different environments. Our findings suggest that whole-genome sequencing will be an effective surveillance tool to detect persistent L. monocytogenes contamination in retail food markets and to design new control strategies to improve food safety.

Background: Listeria monocytogenes consists of four lineages that occupy a wide variety of ecological niches. Sequence type (ST) 87 (serotype 1/2b), belonging to lineage I, is one of the most common STs isolated from food products, food associated environments and sporadic listeriosis in China. Here, we performed a comparative genomic analysis of the L. monocytogenes ST87 clone by sequencing 71 strains representing a diverse range of sources, different geographical locations and isolation years., Results: The core genome and pan genome of ST87 contained 2667 genes and 3687 genes respectively. Phylogenetic analysis based on core genome SNPs divided the 71 strains into 10 clades. The clinical strains were distributed among multiple clades. Four clades contained strains from multiple geographic regions and showed high genetic diversity. The major gene content variation of ST87 genomes was due to putative prophages, with eleven hotspots of the genome that harbor prophages. All strains carry an intact CRISRP/Cas system. Two major CRISPR spacer profiles were found which were not clustered phylogenetically. A large plasmid of about 90 Kb, which carried heavy metal resistance genes, was found in 32.4% (23/71) of the strains. All ST87 strains harbored the Listeria pathogenicity island (LIPI)-4 and a unique 10-open read frame (ORF) genomic island containing a novel restriction-modification system., Conclusion: Whole genome sequence analysis of L. monocytogenes ST87 enabled a clearer understanding of the population structure and the evolutionary history of ST87 L. monocytogenes in China. The novel genetic elements identified may contribute to its virulence and adaptation to different environmental niches. Our findings will be useful for the development of effective strategies for the prevention and treatment of listeriosis caused by this prevalent clone.

This study aimed to investigate the prevalence and molecular characteristics of Listeria monocytogenes in cooked products in Zigong City, China. The overall occurrence of the L. monocytogenes in the ready-to-eat (RTE) shops and mutton restaurants surveyed was 16.2% (141/873). An occurrence of 13.5% was observed in RTE pork, 6.5% in RTE vegetables, and more than 24.0% in either cooked mutton or cooked haggis. Serotype 1/2b (45.4%), 1/2a (33.3%), and 1/2c (14.2%) were the predominant types. By comparing the clonal complexes (CCs) based on multilocus sequence typing (MLST) of the L. monocytogenes from cooked foods in Zigong City and 33 listeriosis cases from different districts of China, CC87, CC9, CC8, and CC3 were showed to be prevalent in cooked products and CC87 and CC3 were the first two frequent types in the 33 clinic-source strains. All CC87 stains harbored the newly reported Listeria pathogenicity island 4 (LIPI-4) gene fragment ptsA, and all CC3 strains possessed the Listeria pathogenicity island 3 (LIPI-3) gene fragment llsX. These may increase the occurrence of the strains belonging to CC87 and CC3 in listeriosis cases in China and also underline the risk of infection owing to the consumption of the cooked products from Zigong. ST619 (serotype 1/2b) harbored both llsX and ptsA, indicating a potential hypervirulent sequence type in Zigong.

Listeria monocytogenes , one of most problematic foodborne pathogens, is responsible for listeriosis in both humans and animals and mainly transmitted through the food chain. In this report, we propose a simple, rapid, and nearly instrument-free molecular technique using multiple cross displacement amplification (MCDA) label-based gold nanoparticles lateral flow biosensor (LFB) for specific, sensitive, and visual detection of L. monocytogenes . The MCDA-LFB method was carried out at a constant temperature (61°C) for only 20 min during the reaction stage, and then the amplification mixtures were directly detected by using LFB, eliminating the use of an electrophoresis instrument, special reagents, or amplicon analysis equipment. The whole procedure, from sample processing to result indicating, was finished within 1 h. The analytical specificity of MCDA-LFB method was successfully determined by distinguishing the target bacterium from other pathogens. The analytical sensitivity of the MCDA-LFB assay was 10 fg of genomic templates per reaction in pure culture, which was in complete accordance with MCDA by gel electrophoresis, real-time turbidity, and colorimetric indicator. The assay was also successfully applied to detecting L. monocytogenes in pork samples. Therefore, the rapidity, simplicity, and nearly equipment-free platform of the MCDA-LFB technique make it possible for food control, clinical diagnosis, and more. The proof-of-concept assay can be reconfigured to detect various target sequences by redesigning the specific MCDA primers., Competing Interests: Yi Wang and Changyun Ye have filed for a patent from the State Intellectual Property Office of the People’s Republic of China, which covers the novel method and sequences included in this manuscript (application number: CN201610892015.1). The other authors report no conflicts of interest in this work.

Here, a novel model of loop-mediated isothermal amplification (LAMP), termed multiple inner primers-LAMP (MIP-LAMP), was devised and successfully applied to detect Listeria monocytogenes. A set of 10 specific MIP-LAMP primers, which recognized 14 different regions of target gene, was designed to target a sequence in the hlyA gene. The MIP-LAMP assay efficiently amplified the target element within 35 min at 63 °C and was evaluated for sensitivity and specificity. The templates were specially amplified in the presence of the genomic DNA from L. monocytogenes. The limit of detection (LoD) of MIP-LAMP assay was 62.5 fg/reaction using purified L. monocytogenes DNA. The LoD for DNA isolated from serial dilutions of L. monocytogenes cells in buffer and in milk corresponded to 2.4 CFU and 24 CFU, respectively. The amplified products were analyzed by real-time monitoring of changes in turbidity, and visualized by adding Loop Fluorescent Detection Reagent (FD), or as a ladder-like banding pattern on gel electrophoresis. A total of 48 pork samples were investigated for L. monocytogenes by the novel MIP-LAMP method, and the diagnostic accuracy was shown to be 100% when compared to the culture-biotechnical method. In conclusion, the MIP-LAMP methodology was demonstrated to be a reliable, sensitive and specific tool for rapid detection of L. monocytogenes strains.

Listeria is ubiquitous in natural environments and can be isolated from animal hosts, including rodents. Listeria ivanovii and Listeria monocytogenes are the main pathogenic species that can cause serious listeriosis in animals and human. In this study, we investigated the prevalence of Listeria in wild rodents from six regions in China, and analyzed the molecular characteristics and antibiotics resistance of the L. ivanovii isolates. A total of 702 fecal samples of 25 different species of wild rodents were examined, and 75 were Listeria positive, including 26 L. ivanovii strains, 2 L. monocytogenes strains, and 47 Listeria innocus strains. The 26 L. ivanovii isolates (including 2 subspecies) were divided into 5 different sequence types by multilocus sequence typing with ST6 being the dominant type, and 5 different pulsotypes by pulsed-field gel electrophoresis. The results of antimicrobial susceptibility revealed that all L. ivanovii isolates were sensitive to rifampin, chloramphenicol, gentamicin, erythromycin, tetracycline, vancomycin, penicillin G, imipenem, trimethoprim–sulfamethoxazole, but some isolates (including ST1, ST7, and ST8) were resistant to clindamycin. The results suggest that the prevalence of L. ivanovii (3.7%, 26/702) in wild rodents was higher in some regions, and the genetic diversity of L. ivanovii isolates in the wild rodents is relatively low and most belong to one lineage. These wild rodents may act as the natural host for L. ivanovii and possibly transmit the pathogen between wild animals and humans. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a foodborne pathogen frequently isolated from raw pork meat. This study was designed to investigate the prevalence and molecular characteristics of L. monocytogenes in raw pork from open markets in China. The survey was conducted monthly over a 12-month period in Zigong, China. L. monocytogenes was isolated from 262 of 1641 samples collected (16.0%) including minced meat samples (131/608, 21.5%), pork pieces samples (111/857, 13.0%) and environmental swabs (20/176, 11.4%). The isolation rates in spring and winter were significantly higher than those in summer and autumn ( X 2 = 68.85, P < 0.05). All isolates were subjected to serotyping, multi-locus sequence typing (MLST) and AscI pulsed-field gel electrophoresis (PFGE). The 262 isolates were subtyped into five serotypes: 1/2b (43.1%), 1/2c (35.5%), 1/2a (19.1%), 4b (1.1%), 3a (1.1%); 20 sequence types (STs) with four most frequent STs, being ST9 (35.9%), ST87 (19.8%), ST3 (16.0%) and ST8 (14.1%); and 39 pulsotypes (PTs) with PT4 (26.3%), PT30 (14.5%) and PT11 (12.6%) being most frequent. Two primary pulsotypes from pork pieces were previously isolated from clinical listeriosis cases in the local hospitals. The six markets from different districts differed in the level of contamination and strain types. Persistent contamination of L. monocytogenes was found in the markets especially in meat mincers, which were found to be one likely source of continuous cross contamination. These findings will help develop strategies to reduce L. monocytogenes contamination in open markets for better public health control and prevention of foodborne L. monocytogenes infections. [ABSTRACT FROM AUTHOR]

• Nisin was encapsulated with 99.8% efficiency in soy soluble polysaccharides (SSPS). • Nisin-loaded SSPS nanoparticles exhibited a desirable particle size (112 nm). • Nisin has high stability and antimicrobial activity after being loaded by SSPS. • Nisin-loaded SSPS nanoparticles increases the shelf-life of the fresh tomato juice. Nisin is a natural antimicrobial agent and food-grade material, while the poor stability and short duration of antimicrobial activity limit its widespread use in the food industry. In the present work, soluble soybean polysaccharide (SSPS)-based nanoparticles have been developed to improve the stability and sustained antimicrobial activity of nisin. The encapsulation efficiency (EE) of nisin-loaded SSPS nanoparticles (Nisin-SSPS-NPs) prepared under the optimized conditions can be up to 99.8%, and the particle size is about 112 nm. The formation of Nisin-SSPS-NPs was mainly mediated by the electrostatic interactions and hydrogen bonding, which was evidenced by the results of zeta potential and Fourier Transform infrared spectroscopy (FTIR). Agar diffusion assay exhibited that Nisin-SSPS-NPs had confirmed antimicrobial activity against Gram-positive bacteria, such as Listeria monocytogenes , Bacillus subtilis , and Staphylococcus aureus. The sustained release of nisin in Nisin-SSPS-NPs endows nisin with a long-lasting antimicrobial activity, which increases the shelf-life of the fresh tomato juice. [ABSTRACT FROM AUTHOR]