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IntroductionListeria monocytogenes is a globally distributed bacterium that exhibits genetic diversity and trait heterogeneity. The alternative sigma factor SigB serves as a crucial transcriptional regulator essential for responding to environmental stress conditions and facilitating host infection.MethodWe employed a comprehensive genetic analysis of sigB in a dataset comprising 46,921 L. monocytogenes genomes. The functional attributes of SigB were evaluated by phenotypic experiments.ResultsOur study revealed the presence of two predominant SigB factors (SigBT1 and SigBT2) in L. monocytogenes, with a robust correlation between SigBT1 and lineages I and III, as well as SigBT2 and lineage II. Furthermore, SigBT1 exhibits superior performance in promoting cellular invasion, cytotoxicity and enhancing biofilm formation and cold tolerance abilities under minimally defined media conditions compared to SigBT2.DiscussionThe functional characteristics of SigBT1 suggest a potential association with the epidemiology of lineages I and III strains in both human hosts and the natural environment. Our findings highlight the important role of distinct SigB factors in influencing the biological traits of L. monocytogenes of different lineages, thus highlighting its distinct pathogenic and adaptive attributes.

Listeria monocytogenes (L. monocytogenes) is a pathogen that is transmitted through contaminated food and causes the illness known as listeriosis. The virulence factor InlA plays a crucial role in the invasion of L. monocytogenes into the human intestinal epithelium. In addition, InlA enhances the pathogenicity of host strains, and different strains of L. monocytogenes contain varying variations of InlA. Our study analyzed a total of 4393 published L. monocytogenes genomes from 511 sequence types (STs) of diverse origins. We identified 300 unique InlA protein sequence types (PSTs) and revealed 45 highly mutated amino acid sites. The leucine-rich repeat (LRR) region was found to be the most conserved among the InlA, while the protein A (PA) region experienced the highest mutation rate. Two new types of mutations were identified in the B-repeat region of InlA. Correspondence analysis (CA) was used to analyze correlations between the lineages or 10 most common sequence types (STs) and amino acid (aa) sites. ST8 was strongly correlated with site 192_F, 454_T. ST7 exhibited a strong correlation with site 51_A, 573_E, 648_S, and 664_A, and it was also associated with ST6 and site 544_N, 671_A, 738_B, 739_B, 740_B, and 774_Y. Additionally, a strong correlation between ST1 and site 142_S, 738_N, ST2 and site 2_K, 142_S, 738_N, as well as ST87 and site2_K, 738_N was demonstrated. Our findings contribute significantly to the understanding of the distribution, composition, and conservation of InlA in L. monocytogenes. These findings also suggest a potential role of InlA in supporting molecular epidemiological tracing efforts.

Listeria monocytogenes is an important pathogen that can cause listeriosis. Despite the growing recognition of Listeria spp. as a foodborne and environmental pathogen, the understanding of its prevalence and characteristics of Listeria spp. in the marine environment remains unknown. In this study, we first investigated the genetic and phenotypic characteristics of Listeria species isolated in a coastal city in China. The findings revealed that the sequence type 87 (ST87) L. monocytogenes, a prevalent clinical and seafood strain in China, dominates in recreational beach sands and possesses a notable biofilm-forming capacity in seawater. The presence of ST87 L. monocytogenes in coastal environments indicates the potential health risks for both recreational activities and seafood consumption. Moreover, the ST121 isolates from sand had a versatile plasmid encoding multifunctional genes, including uvrX for UV resistance, gbuC for salt resistance, and npx for oxidative resistance and multiple transposases, which potentially aid in survival under natural environments. Black-headed gulls potentially facilitate the spread of L. monocytogenes, with similar ST35 strains found in gulls and beach sand. As a reservoir of microbes from marine environments and human/animal excrement, coastal sand would play an important role in the spread of L. monocytogenes and is an environmental risk for human listeriosis.

IntroductionListeria monocytogenes is a foodborne bacterium that could persist in food and food processing environments for a long time. Understanding the population structure and genomic characterization of foodborne L. monocytogenes is essential for the prevention and control of listeriosis.MethodsA total of 322 foodborne L. monocytogenes isolates from 13 geographical locations and four food sources in China between 2000 and 2018 were selected for whole-genome sequencing.ResultsIn silico subtyping divided the 322 isolates into five serogroups, 35 sequence types (STs), 26 clonal complexes (CCs) and four lineages. Serogroup IIa was the most prevalent serogroup and ST9 was the most prevalent ST of foodborne L. monocytogenes strains isolated in China. The in-depth phylogenetic analysis on CC9 revealed that ST122 clone might be original from ST9 clone. Furthermore, 23 potentially relevant clusters were identified by pair-wised whole-genome single nucleotide polymorphism analysis, indicating that persistent- and/or cross-contamination had occurred in markets in China. ST8 and ST121 were the second and third top STs of L. monocytogenes in China, which had heterogeneity with that of L. monocytogenes isolates from other countries. The antibiotic resistance genes aacA4, tetM, tetS, dfrG carried by different mobile elements were found in L. monocytogenes strains. One lineage II strain carrying Listeria Pathogenicity Island 3 was first reported. In addition, a novel type of premature stop codon in inlA gene was identified in this study.DiscussionThese findings revealed the genomic characteristics and evolutionary relationship of foodborne L. monocytogenes in China on a scale larger than previous studies, which further confirmed that whole-genome sequencing analysis would be a helpful tool for routine surveillance and source-tracing investigation.

Rapid and accurate identification of foodborne pathogenic bacteria is of great importance because they are often responsible for the majority of serious foodborne illnesses. The confocal Raman microspectroscopy (CRM) is a fast and easy-to-use method known for its effectiveness in detecting and identifying microorganisms. This study demonstrates that CRM combined with chemometrics can serve as a rapid, reliable, and efficient method for the detection and identification of foodborne pathogenic bacteria without any laborious pre-treatments. Six important foodborne pathogenic bacteria including S. flexneri, L. monocytogenes, V. cholerae, S. aureus, S. typhimurium, and C. botulinum were investigated with CRM. These pathogenic bacteria can be differentiated based on several characteristic peaks and peak intensity ratio. Principal component analysis (PCA) was used for investigating the difference of various samples and reducing the dimensionality of the dataset. Performances of some classical classifiers were compared for bacterial detection and identification including decision tree (DT), artificial neural network (ANN), and Fisher’s discriminant analysis (FDA). Correct recognition ratio (CRR), area under the receiver operating characteristic curve (ROC), cumulative gains, and lift charts were used to evaluate the performance of models. The impact of different pretreatment methods on the models was explored, and pretreatment methods include Savitzky–Golay algorithm smoothing (SG), standard normal variate (SNV), multivariate scatter correction (MSC), and Savitzky–Golay algorithm 1st Derivative (SG 1st Der). In the DT, ANN, and FDA model, FDA is more robust for overfitting problem and offers the highest accuracy. Most pretreatment methods raised the performance of the models except SNV. The results revealed that CRM coupled with chemometrics offers a powerful tool for the discrimination of foodborne pathogenic bacteria.

Listeria monocytogenes is a ubiquitous foodborne pathogen causing both invasive and non-invasive listeriosis. Sequence type (ST) 9 strains is common in food and food processing environments. In this study, the whole-genome sequences (WGS) of 207 ST9 isolates from different sources, geographical locations (14 countries), and isolated years were analyzed. The ST9 isolates were divided into three clusters after phylogenetic analysis; 67.63% of ST9 isolates contained putative plasmids with different sizes and genomic structure, the putative prophages inserted in the chromosome at ten hotspots, and seven types of premature stop codon (PMSC) mutations in inlA were found in 81.86% of the ST9 isolates. In addition, 78.26% of ST9 isolates harbored Tn554-like elements carrying arsenic resistance genes. All the ST9 isolates conservatively contained environment-resistance genes on the chromosome. This analysis of population structures and features of ST9 isolates was aimed to help develop effective strategies to control this prevalent pathogen in the food chain.

ABSTRACT 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.

Clostridium botulinum is the causative pathogen of botulism. Laboratory detection of C. botulinum is essential for clinical therapy treatment of botulism due to the difficulty in diagnosis, especially in infant botulism. The extreme toxicity of botulinum neurotoxin (BoNT) requires a sensitive detection method. Due to the detection limit of real-time quantitative PCR (q-PCR), a more sensitive detection method, micro-drop digital PCR (ddPCR) was applied in C. botulinum main serotypes A and B. The following performance criteria were evaluated by ddPCR: analytical sensitivity; repeatability; and diagnostic specificity. The limit of detection (LOD) was 0.84 and 0.88 copies/μl for BoNT A and B genes, respectively, by ddPCR with high specificity, compared to 5.04×102 and 6.91×102 copies/μl by q-PCR. It was increased 10 times compared with q-PCR in spiked stool samples. This improvement in sensitivity was especially important in clinical samples as more positive samples were detected by digital PCR compared with q-PCR. Meanwhile, enrichment time for low bacteria content samples was shortened by four hours both in serotypes A and B C. botulinum by ddPCR compared with q-PCR, which are important for laboratory diagnosis and epidemiology work.

Abstract 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.

Abstract Background Neonatal listeriosis is a rare but severe disease manifesting as septicemia and central nervous system (CNS) infections with a high fatality rate of around 20 to 30%. Whole genome sequencing (WGS) is a promising technique for pathogen identification and infection source tracing with its high resolution. Case presentation A case of neonatal sepsis with listeriosis was reported with positive blood culture for Listeria monocytogenes. The case was investigated to confirm the vertical transmission of the infection and identify the potential food source of the maternal L. monocytogenes infection using WGS. L. monocytogenes was isolated from the neonate’s blood sample the day after caesarean delivery and from the mother’s genital and pudenda swab samples 5 days and 13 days after caesarean delivery. WGS showed that the isolate from the neonate was identical to the genome type of the isolates from the mother, with only one of the 4 isolates from the mother differing by one single nucleotide polymorphism (SNP). By WGS, one L. monocytogenes isolate from a ready-to-eat (RTE) meat sample in the patients’ community market shared the same sequence type but was ruled out as the cause of infection, with 57 SNP differences to the strain causing the maternal-neonatal infection. The food isolate also carried a novel plasmid pLM1686 that harbored heavy metal resistance genes. After caesarean section, the mother was treated with a third generation cephalosporin which L. monocytogenes is naturally resistant to, which may explain why genital and pudenda swabs were still culture-positive for L. monocytogenes 13 days after delivery. Conclusions Genital swab culture for L. monocytogenes had been informative in the diagnosis of maternal listeriosis in this case. The high resolution of WGS confirmed the maternal-neonatal transmission of L. monocytogenes infection and ruled out the L. monocytogenes contaminated RTE meat from the local market as the direct source of the mother’s infection.

ABSTRACTListeria monocytogenes is a high risk pathogen which can cause invasive diseases in humans. We previously reported that black-headed gulls from Dianchi Lake of Kunming carrying L. monocytogenes, while the characteristics of these isolates and the relationship with habitats of migratory gulls have not been explored. In this study, we investigated the prevalence and molecular characteristics of Listeria monocytogenes from black-headed gulls in Dianchi Lake, and phylogenetic analysis based on core genome SNPs was used to determine the genetic relationship of the strains from Dianchi Lake and other regions. Occurrence of L. monocytogenes in black-headed gull feces in 2016, 2017 and 2018 was 1.0%, 1.0% and 0.6% respectively. The predominant serotype of 28 isolates was 4b, while the predominant sequence types were ST145 and ST201. Based on their prevalence and genomic relationships, ST5 and ST87 were likely to be sourced locally while ST145 and ST201 were likely to be non-local. L. monocytogenes may travel along the bird migration route leading to transmission over a large geographical span carried by black-headed gull. Although the prevalence of L. monocytogenes was low, its carriage by the migratory black-headed gulls poses potential public health risks in regions where the migratory birds passage and reside.

Listeria ivanovii subsp. ivanovii is an intracellular bacterium distributed widely in nature, causing the listeriosis in ruminants and humans. Previous researches had isolated 116 strains of L. ivanovii subsp. ivanovii from wild rodents and pikas of different regions in China, and the predominant sequence types were ST1 and ST2. In this study, we first investigated the biological characteristics and virulence of these two clonal strains including motility, metabolism and virulence in cells and mouse model. The results demonstrated the ST1 strains exhibited motility, wide metabolic activity and hypervirulence, whereas the ST2 strains showed non-motility, relative lower metabolic activity and virulence. Considering the transmissible ability from wild rodents and pikas to ecological environment, the L. ivanovii subsp. ivanovii with potential pathogenicity to humans and ruminants should be monitored.

MraW is a 16S rRNA methyltransferase and plays a role in the fine-tuning of the ribosomal decoding center. It was recently found to contribute to the virulence of Staphylococcus aureus. In this study, we examined the function of MraW in Escherichia coli O157:H7 and found that the deletion of mraW led to decreased motility, flagellar production and DNA methylation. Whole-genome bisulfite sequencing showed a genome wide decrease of methylation of 336 genes and 219 promoters in the mraW mutant including flagellar genes. The methylation level of flagellar genes was confirmed by bisulfite PCR sequencing. Quantitative reverse transcription PCR results indicated that the transcription of these genes was also affected. MraW was furtherly observed to directly bind to the four flagellar gene sequences by electrophoretic mobility shift assay (EMSA). A common flexible motif in differentially methylated regions (DMRs) of promoters and coding regions of the four flagellar genes was identified. Reduced methylation was correlated with altered expression of 21 of the 24 genes tested. DNA methylation activity of MraW was confirmed by DNA methyltransferase activity assay in vitro and repressed by DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza). In addition, the mraW mutant colonized poorer than wild type in mice. We also found that the expression of mraZ in the mraW mutant was increased confirming the antagonistic effect of mraW on mraZ. In conclusion, mraW was found to be a DNA methylase and have a wide-ranging effect on E. coli O157:H7 including motility and virulence in vivo via genome wide methylation and mraZ antagonism.

Listeria monocytogenes is among the most important foodborne pathogens, which can be isolated from bird feces. The aim of this study was to examine the prevalence, genotypes, virulence factors and drug-resistance characteristics of L. monocytogenes isolated from the black-headed gull in Kunming, China. Serotypes and virulence genes were identified by polymerase chain reaction. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) methods were used to describe the genetic characteristics of all positive isolates. Furthermore, all L. monocytogenes isolates were tested for antimicrobial susceptibility by disk diffusion method. All isolates were positive for inlA, inlB, plcB, prfA, iap, actA and hly genes. Serotyping showed the isolates belonged to serotype 1/2b (2/9, 22%), 1/2c (3/9, 33%), and 4a (4/9, 44%). PFGE showed 4 different pulsotypes (PTs), and MLST differentiated the 9 isolates into 4 sequence types (STs), ST3 (1), ST5 (1), ST35 (3) and ST201 (4). The result of antimicrobial susceptibility revealed that all isolates were naturally sensitive to most antibiotics. The L. monocytogenes isolates had pathogenic potential. There is a potential infection risk for people who come to frequent contact with black-headed gulls in Kunming. Hence, it is necessary to conduct surveillance of L. monocytogenes in the migratory black-headed gulls, which would be valuable to prevent listeriosis in local residents and tourists. Keywords: Listeria monocytogenes, Virulence gene, PFGE, MLST, Antibiotic resistance

Streptococcus suis sequence type 7 emerged and caused 2 of the largest human infection outbreaks in China in 1998 and 2005. To determine the major risk factors and source of the infections, we analyzed whole genomes of 95 outbreak-associated isolates, identified 160 single nucleotide polymorphisms, and classified them into 6 clades. Molecular clock analysis revealed that clade 1 (responsible for the 1998 outbreak) emerged in October 1997. Clades 2–6 (responsible for the 2005 outbreak) emerged separately during February 2002–August 2004. A total of 41 lineages of S. suis emerged by the end of 2004 and rapidly expanded to 68 genome types through single base mutations when the outbreak occurred in June 2005. We identified 32 identical isolates and classified them into 8 groups, which were distributed in a large geographic area with no transmission link. These findings suggest that persons were infected in parallel in respective geographic sites.

The Old World vulture may carry and spread pathogens for emerging infections since they feed on the carcasses of dead animals and participate in the sky burials of humans, some of whom have died from communicable diseases. Therefore, we studied the precise fecal microbiome of the Old World vulture with metataxonomics, integrating the high-throughput sequencing of almost full-length small subunit ribosomal RNA (16S rRNA) gene amplicons in tandem with the operational phylogenetic unit (OPU) analysis strategy. Nine vultures of three species were sampled using rectal swabs on the Qinghai-Tibet Plateau, China. Using the Pacific Biosciences sequencing platform, we obtained 54 135 high-quality reads of 16S rRNA amplicons with an average of 1442±6.9 bp in length and 6015±1058 reads per vulture. Those sequences were classified into 314 OPUs, including 102 known species, 50 yet to be described species and 161 unknown new lineages of uncultured representatives. Forty-five species have been reported to be responsible for human outbreaks or infections, and 23 yet to be described species belong to genera that include pathogenic species. Only six species were common to all vultures. Clostridium perfringens was the most abundant and present in all vultures, accounting for 30.8% of the total reads. Therefore, using the new technology, we found that vultures are an important reservoir for C. perfringens as evidenced by the isolation of 107 strains encoding for virulence genes, representing 45 sequence types. Our study suggests that the soil-related C. perfringens and other pathogens could have a reservoir in vultures and other animals.Emerging Microbes & Infections (2017) 6, e9; doi:10.1038/emi.2016.137; published online 22 February 2017

Listeria is ubiquitous in a variety of environments and can be isolated from a wide range of animal hosts. Rodents are capable of carrying pathogenic bacteria in their intestines, such as Listeria, and can disseminate those pathogens into the natural environment and to where human activity occurs. In this study, we investigated the occurrence and antimicrobial susceptibility of Listeria spp. isolated from wild rodents found in natural environments in China. We collected 341 intestinal fecal samples of rodents from five different regions of China, all representing different rodent habitats. The antimicrobial susceptibility of the Listeria spp. isolates obtained were firstly assessed using the Kirby–Bauer disk diffusion method. Thirty-one samples were positive for Listeria spp., of which 11 were positive for Listeria monocytogenes and seven were positive for Listeria ivanovii. Other species identified include Listeria innocua, Listeria fleischmannii and Listeria floridensis. All Listeria spp. isolates were sensitive to the majority of the antimicrobials tested, but largely resistant to oxacillin (94.1%) and cefuroxime (70.6%). All L. monocytogenes isolates were further characterized by serotyping, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). L. monocytogenes strains were grouped into three serotypes, five sequence types and five pulsotypes (PTs) by serotyping, MLST and PFGE, respectively. Almost half of the isolates (five of 11) belonged to serotype 1/2b, ST87 and PT1. This study determined that Listeria is carried in the intestinal tracts of wild rodents from multiple regions at a low rate, filling an epidemiological data gap on Listeria in natural environments in China.Emerging Microbes & Infections (2017) 6, e44; doi:http://10.1038/emi.2017.28; published online 7 June 2017

Escherichia coli is both of a widespread harmless gut commensal and a versatile pathogen of humans. Domestic animals are a well-known reservoir for pathogenic E. coli. However, studies of E. coli populations from wild animals that have been separated from human activities had been very limited. Here we obtained 580 isolates from intestinal contents of 116 wild Marmot Marmota himalayana from Qinghai–Tibet plateau, China, with five isolates per animal. We selected 125 (hereinafter referred to as strains) from the 580 isolates for genome sequencing, based on unique pulse field gel electrophoresis patterns and at least one isolate per animal. Whole genome sequence analysis revealed that all 125 strains carried at least one and the majority (79.2%) carried multiple virulence genes based on the analysis of 22 selected virulence genes. In particular, the majority of the strains carried virulence genes from different pathovars as potential 'hybrid pathogens'. The alleles of eight virulence genes from the Marmot E. coli were found to have diverged earlier than all known alleles from human and other animal E. coli. Phylogenetic analysis of the 125 Marmot E. coli genomes and 355 genomes selected from 1622 human and other E. coli strains identified two new phylogroups, G and H, both of which diverged earlier than the other phylogroups. Eight of the 12 well-known pathogenic E. coli lineages were found to share a most recent common ancestor with one or more Marmot E. coli strains. Our results suggested that the intestinal E. coli of the Marmots contained a diverse virulence gene pool and is potentially pathogenic to humans. These findings provided a new understanding of the evolutionary origin of pathogenic E. coli.Emerging Microbes & Infections (2016) 5, e122; doi:10.1038/emi.2016.122; published online 7 December 2016

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.

Staphylococcus aureus (S. aureus), including methicillin-resistant S. aureus (MRSA), is one of the most important human pathogens, which is responsible for bacteremia, soft-tissue infections, and food poisoning. Hence, multiple cross displacement amplification (MCDA) is employed to detect all S. aureus strains, and differentiates MRSA from methicillin-sensitive S. aureus. Multiplex MCDA (m-MCDA), which targets the nuc gene (S. aureus-specific gene) and mecA gene (encoding penicillin-binding protein-2′), could detect S. aureus strains and identify MRSA within 85 min. Detection of the m-MCDA products is achieved using disposable lateral flow biosensors. A total of 58 strains, including various species of Gram-positive and Gram-negative strains, are used for evaluating and optimizing m-MCDA assays. The optimal amplification condition is found to be 63°C for 40 min, with detection limits at 100 fg DNA/reaction for nuc and mecA genes in the pure cultures, and 10 CFU/tube for nuc and mecA genes in the blood samples. The analytical specificity of m-MCDA assay is of 100%, and no cross-reactions to non-S. aureus strains are produced according to the specificity testing. Particularly, two additional components, including AUDG enzyme and dUTP, are added into the m-MCDA amplification mixtures, which are used for eliminating the unwanted results arising from carryover contamination. Thus, the m-MCDA technique appears to be a simple, rapid, sensitive, and reliable assay to detect all S. aureus strains, and identify MRSA infection for appropriate antibiotic therapy.

Listeria monocytogenes can contaminate various foods via food processing environments and contamination of raw materials. There is a limited understanding of L. monocytogenes transmission in retail market and the role of insects in L. monocytogenes transmission in the retail environments. To better understand the risk factors of raw pork contamination, the prevalence of L. monocytogenes was examined in raw pork, retail environments and insects in a retail market over a 6-month period from March to August in 2016 in Beijing, China. A total of 2,789 samples were collected, including 356 raw pork samples, 1,392 meat contact surface swabs (MCS), 712 non-meat contact surface swabs (NMCS) and 329 insect samples. Overall, 424 (15.20%) of the samples were found to be contaminated by L. monocytogenes. Analyzed by serotyping, multilocus sequence typing and pulsed-field gel electrophoresis, the 424 L. monocytogenes isolates were divided into three serotypes (1/2c, 1/2a and 3a), 15 pulsotypes (PTs) and nine sequence types (STs), 1/2c/PT4/ST9 (244/424, 58%) was the most prevalent type of L. monocytogenes strains. The raw pork, MCS of the environments and insects were contaminated with higher levels of L. monocytogenes than NMCS of the environments, which suggested that cross contamination of L. monocytogenes between raw pork and the environment existed in the retail market, and long-term contaminated surfaces and vector insects would act as high risk factors to transmit L. monocytogenes to raw pork. Thus more effective strategies are needed to reduce the risk of retail pork meat contamination by L. monocytogenes and prevent foodborne human listeriosis.

Listeria monocytogenes, an important food-borne pathogen, causes listeriosis and is widely distributed in many different environments. In a previous study, we developed a novel enrichment broth containing D-allose that allows better isolation of L. monocytogenes from samples. However, the mechanism of D-allose utilization by L. monocytogenes remains unclear. In the present study, we determined the metabolism of D-allose in L. monocytogenes and found that lineage II strains of L. monocytogenes can utilize D-allose as the sole carbon source for growth, but lineage I and III strains cannot. Transcriptome analysis and sequence alignment identified six genes (lmo0734 to 0739) possibly related to D-allose metabolism that are only present in the genomes of lineage II strains. Recombinant strain ICDC-LM188 containing these genes showed utilization of D-allose by growth assays and Biolog phenotype microarrays. Moreover, lmo0734 to 0736 were verified to be essential for D-allose metabolism, lmo0737 and 0738 affected the growth rate of L. monocytogenes in D-allose medium, while lmo0739 was dispensable in the metabolism of D-allose in L. monocytogenes. This is the first study to identify the genes related to D-allose metabolism in L. monocytogenes, and their distribution in lineage II strains. Our study preliminarily determined the effects of these genes on the growth of L. monocytogenes, which will benefit the isolation and epidemiological research of L. monocytogenes.

Shigella spp., the etiological agent of shigellosis or bacillary dysentery, are responsible for considerable morbidity and mortality in excess of a million deaths globally per year. Although PCR-based techniques (such as PCR-based dipstick biosensors) have been used for the molecular diagnosis of infectious disease, these assays were restricted due to the need for a sophisticated thermal cycling apparatus to denature target templates. To facilitate simple and rapid detection of target pathogens, we successfully devised an inexpensive, reliable and nearly instrument-free molecular technique, which incorporates multiple cross displacement amplification (MCDA) combined with a newly designed lateral flow biosensor (LFB) for visual, sensitive and specific detection of Shigella. The MCDA-LFB assay was conducted at 65 ˚C for only 20 min during the amplification stage, and then products were directly analyzed on the biosensor, alleviating the use of special reagents, electrophoresis equipment and amplicon detection instruments. The entire process, including specimen processing (35 min), amplification (20) and detection (2-5 min), can be finished within 1 h. The MCDA-LFB assay demonstrated high specificity for Shigella detection. The analytical sensitivity of the assay was 10 fg of genomic templates per reaction in pure culture and 5.86 CFU per tube in human fecal samples, which was consistent with MCDA by colorimetric indicator, gel electrophoresis, real time turbidity and fluorescence detection. Hence, the simplicity, rapidity and nearly instrument-free platform of the MCDA-LFB assay make it practical for ‘on-site’ diagnosis, point-of-care testing and more. Moreover, the proof-of-concept approach can be reconfigured to detect a wide variety of target sequences by re-designing the specific MCDA primers.

The article reported a novel methodology for real-time PCR analysis of nucleic acids, termed endonuclease restriction-mediated real-time polymerase chain reaction (ET-PCR). Just like PCR, ET-PCR only required one pair of primers. A short sequence (Ss), which was recognized by restriction enzyme BstUI, was attached to the 5’ end of the forward (F) or reverse (R) PCR primer, and the new F or R primer was named EF or ER. EF/ER was labeled at the 5’ end with a reporter dye and in the middle with a quenching dye. BstUI cleaves the newly synthesized double-stranded terminal sequences (5’ end recognition sequences and their complementary sequences) during the extension phase, which separates the reporter molecule from the quenching dye, leading to a gain of fluorescence signal. This process is repeated in each amplification cycle and unaffected the exponential synthesis of the PCR amplification. ET-PCR allowed real-time analysis of single or multiple targets in a single vessel, and provided the reproducible quantitation of nucleic acids. positive results were generated in a relatively short period. The analytical sensitivity and specificity of ETR-PCR were successfully evaluated, detecting down to 250 fg of genomic DNA per tube of target pathogen DNA examined, and the positive results were generated in a relatively short period. Moreover, the practical application of ET-PCR for simultaneous detection of multiple target pathogens was also demonstrated in artificially contaminated blood samples. In conclusion, due to the technique’s simplicity of design, reproducible data and low contamination risk, ET-PCR assay is an appealing alternative to conventional approaches currently used for real-time nucleic acid analysis.

BACKGROUND:Shigellosis is a serious problem in Guizhou and Shigella sonnei is an increasingly prevalent etiologic agent of local shigellosis cases. No data, however, are available about the molecular characterization of the local isolates of S. sonnei. We have conducted this study to molecularly characterize the clinical isolates of S. sonnei in Guizhou Province. RESULTS:76 S. sonnei isolates, including four isolates from 1974-1982 and 72 isolates from 2008-2010, were used for analysis in this study. Pulsed-field gel electrophoresis (PFGE) based on XbaI digestion divided the 76 isolates into 38 PFGE patterns (PT) and 15 PTs were represented by more than one isolates with PT31 (N = 8) containing the most number of isolates, followed with PT2 (N = 6). Multiple-Locus Variable number tandem repeat (VNTR) Analysis (MLVA) based on seven VNTR loci discriminated them into 19 different MLVA types (MTs), and four MTs were represented by more than one isolate with MT4 (N = 39) containing the most number of isolates, followed with MT12 (N = 18). 15 Multilocus sequence typing (MLST) base on 15 loci differentiated the isolates into six sequence types (STs), among which four STs were novel. The most common STs are ST76 (N = 43) and ST116 (N = 25), accounting for 92.1%. Correlation between genetic relationships and geographical origins or isolation years was observed among the isolates studied. Majority of isolates were clustered in accordance with the origin of isolation years based on the genetic data, which were also from similar geographical origins. CONCLUSIONS:Our results revealed the molecular characteristics including the specific genotypes such as four novel STs, clonal relationship, and genetic changes of local isolates from different years, which enhances our understanding of molecular characteristics of S. sonnei and contributes to the prevention and control of shigellosis in Guizhou Province.

Shigella flexneri is one of the major etiologic causes of shigellosis in Guizhou Province, China. However, the genetic characteristics of circulating isolates are unknown. Phenotypic and molecular profiles of 60 S. flexneri isolates recovered in Guizhou between 1972 to 1982 and 2008 to 2010 were determined. Nine serotypes (1a, 2a, 3a, 1b, 2b, X, Y, 4av and Yv) were identified. Multi-locus sequence typing differentiated the isolates into 20 sequence types (STs); 18 were novel. Four STs, ST 129, ST 100, ST 126 and ST 18, were most abundant, accounting for 65% of the isolates. Thirty-nine NotI-pulsed field gel electrophoresis patterns (pulsotypes, PTs) were observed; eight PTs were represented by more than one isolate with six isolates sharing the PT 13 profile. Multi-locus variable-nucleotide tandem-repeat analysis recognized 44 different types (MTs); seven MTs were represented by more than one isolate and MT 1 was most commonly encountered. Correlation between genetic relationships and serotypes was observed among the isolates studied; the majority of isolates belonging to the same serotype from different years clustered together based on the molecular data. These clustered isolates were also from similar geographical origins. These results enhance our understanding of genetic relationships between S. flexneri in Guizhou Province and can be used to help understand the changing etiology of shigellosis in China.

Vibrio parahaemolyticus and Vibrio vulnificus are two marine seafood-borne pathogens causing severe illnesses in humans and aquatic animals. In this study, a recently developed novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully developed and evaluated for simultaneous detection of V. parahaemolyticus and V. vulnificus strains in only a single reaction. Two MERT-LAMP primer sets were designed to specifically target toxR gene of V. parahaemolyticus and rpoS gene of V. vulnificus. The MERT-LAMP reactions were conducted at 62 °C, and the positive results were produced in as short as 19 min with the genomic DNA templates extracted from the V. parahaemolyticus and V. vulnificus strains. The two target pathogens present in the same sample could be simultaneously detected and correctly differentiated based on distinct fluorescence curves in a real-time format. The sensitivity of MERT-LAMP assay was 250 fg and 125 fg DNA per reaction with genomic templates of V. parahaemolyticus and V. vulnificus strains, which was in conformity with conventional LAMP detection. Compared with PCR-based techniques, the MERT-LAMP technology was 100- and 10-fold more sensitive than that of PCR and qPCR methods. Moreover, the limit of detection of MERT-LAMP approach for V. parahaemolyticus isolates and V. vulnificus isolates detection in artificially-contaminated oyster samples was 92 CFU and 83 CFU per reaction. In conclusion, the MERT-LAMP assay presented here was a rapid, specific, and sensitive tool for the detection of V. parahaemolyticus and V. vulnificus, and could be adopted for simultaneous screening of V. parahaemolyticus and V. vulnificus in a wide variety of samples.