Whole-Genome Sequencing-Based Characterization of Clinical Listeria monocytogenes Isolates in China, 2013-2019.

Invasive listeriosis is a rare but serious foodborne disease that causes maternal-neonatal, central nervous system, and bloodstream infections. The aim of this study was to assess the whole-genome sequencing (WGS)-based genetic diversity of clinical Listeria monocytogenes isolates over a 7-year period and prove the effect of WGS application in food vehicle investigation. A total of 360 isolates were recovered during 2013 and 2019 through the national listeriosis special surveillance program. Two hundred twenty-six isolates (62.8%) were associated with pregnancy. All isolates belonged to lineage I (214 isolates) or lineage II (146 isolates), with 4 serogroups (46.9% IIb, 39.7% IIa, 12.5% IVb, and 0.8% IIc). All isolates were in 25 clonal complexes (CCs) and 3 singletons, with CC87, CC8, and CC5 being the most common causes of human listeriosis. All clinical isolates were positive for Listeria pathogenicity island 1 (LIPI-1), LIPI-3 was present in 21.4% of isolates and LIPI-4 was detected in 29.2% of isolates. LIPI-4-positive isolates, including CC87, sequence type (ST)619, ST382, CC4, and CC2, have been shown to confer hypervirulence. Fifteen isolates harbored at least one antimicrobial encoding gene, including tet (M), mef (A), msr (D), and dfr (G). The sublineage designations were consistent with CC designations, and 215 distinct cgMLST types (CTs) were classified, the most abundant being CT58 and CT750. In summary, there is a high level of genetic diversity among the clinical isolates. WGS has strengthened listeriosis surveillance and will be implemented for other foodborne bacteria in the National Molecular Tracing Network for Foodborne Disease.

Characteristics of Settings and Etiologic Agents of Foodborne Disease Outbreaks - China, 2020.

INTRODUCTION: Foodborne diseases are a growing public health problem and have caused a large burden of disease in China. This study analyzed epidemiological characteristics of foodborne diseases in China in 2020 to provide a scientific basis for prevention and control measures. METHODS: Data were collected from 30 of 31 provincial-level administrative divisions (PLADs) in the mainland of China, excluding Xizang (Tibet) Autonomous Region, via the National Foodborne Disease Outbreaks Surveillance System. The number and proportion of outbreaks, illnesses, hospitalizations, deaths by setting, pathogen-food category pairs and etiology were calculated. RESULTS: In 2020, 7,073 foodborne disease outbreaks were reported, resulting in 37,454 illnesses and 143 deaths. Among the identified pathogens, microbial pathogens were the most common confirmed etiology, accounting for 41.7% of illnesses. Poisonous mushrooms caused the largest proportion of outbreaks (58.0%) and deaths (57.6%). For venues where foodborne disease outbreaks occur, household had the highest number of outbreaks (4,140) and deaths (128), and catering service locations caused the largest proportion of illnesses (59.9%). Outbreaks occurring between June and September accounted for 62.8% of total outbreaks. CONCLUSIONS: Foodborne disease outbreaks mainly occurred in households. Microbial pathogens remained the top cause of outbreak-associated illnesses. Poisonous mushrooms were ranked the top cause of deaths in private homes in China. The supervision and management of food safety and health education should be strengthened to reduce the burden of foodborne diseases. Publicity should be increased to reduce the incidence of mushroom poisonings in families, and supervision and management of food should be strengthened to reduce microbial contamination.

Mushroom Poisoning Outbreaks - China, 2010-2020.

Introduction: Mushroom poisoning was the leading cause of foodborne disease outbreaks and outbreak-associated deaths in China. Mushroom poisoning outbreak surveillance can provide insight into the epidemiological characteristics of mushroom poisonings and guide policymaking and health education to reduce illnesses and deaths.Methods: Foodborne Disease Outbreak Surveillance System was upgraded in 2011 to collect foodborne disease outbreaks in China. Mushroom poisoning outbreaks during 2010-2020 were selected to analyze geographical distribution, seasonal distribution, and setting of food preparation.Results: A total of 10,036 outbreaks, which resulted in 38,676 illnesses and 788 deaths, were reported in this period. Mushroom poisonings occurred all over the country, but with highest incidence in the southwest and central China. Overall, 84.6% outbreaks were associated with food prepared in households, followed by 8.7% in street stalls, and 2.5% in canteens. Mushroom poisoning outbreaks clearly exhibited seasonality, and the peak season was summer through autumn. Outbreaks occurring between May and October accounted for 94.1% of total outbreaks, 92.4% illnesses, and 97.2% deaths.Conclusions: Mushroom poisoning is a food safety issue of higher concern in China. Targeted health education is essential to reduce mushroom poisoning, especially in southwest China. Citizens are advised to not collect or eat wild mushrooms.

Prevalence of Antimicrobial Resistant of Vibrio parahaemolyticus Isolated from Diarrheal Patients - Six PLADs, China, 2016-2020.

WHAT IS ALREADY KNOWN ON THIS TOPIC?: Vibrio parahaemolyticus (V. parahaemolyticus) is frequently resistant to common antimicrobials such as ampicillin and generally highly susceptible to most clinically used antimicrobials. WHAT IS ADDED BY THIS REPORT?: V. parahaemolyticus were highly resistant to cefazolin and ampicillin: 94.4% and 37.0%, respectively. However, it was below 3% resistance to all 10 other antimicrobials including clinically relevant agents and even imipenem. The overall levels of antimicrobial resistance and multidrug resistance were 95.1% and 3.3%, respectively. The distribution of antimicrobial resistance and the multidrug resistance had regional, temporal, sexual, and isolated source strain variation. WHAT ARE THE IMPLICATIONS FOR PUBLIC HEALTH PRACTICE?: This study provides data on drug resistance of V. parahaemolyticus in Chinese clinical settings, which will help develop a public health strategy.

Application of Whole-Genome Sequencing in the National Molecular Tracing Network for Foodborne Disease Surveillance in China.

National Molecular Tracing Network for Foodborne Disease Surveillance (TraNet) was launched in 2013, which is the only real-time whole-genome sequencing (WGS)-based subtyping network in China for effective foodborne disease surveillance. TraNet covers three levels of public health laboratories, national, provincial, and municipal. The TraNet national databases have a total of more than 54,000 entries representing seven common foodborne bacteria from humans, food, and environments. Raw sequence data are uploaded to TraNet by Data Delivery Center. Assembled sequence data, pulsed-field gel electrophoresis (PFGE) profiles, antibiotic resistance patterns, and epidemiological data are submitted to national pathogen-specific databases managed by China National Center for Food Safety Risk Assessment. PFGE patterns and WGS-based subtyping are compared for rapid differentiation of clusters of geographically diverse foodborne infections. WGS-based TraNet has played significant roles in improving foodborne disease surveillance in China for rapid outbreak investigation, source tracking, and cluster analysis of particular pathogens across the country.

[Attribution analysis of food borne disease outbreaks in canteen from 2002 to 2016 in China].

OBJECTIVE: Based on the angle of the "pathogenic bacteria-food", to analyze the cause of events in canteen from 2002 to 2016 in China, and to provide scientific basis for prevention and control of food borne diseases. METHODS: Collect and finish the food borne disease outbreaks events in canteen from 2002 to 2016, do the descriptive analysis of the number of events, cases and deaths by the different cause food, pathogenic factors and link, in order to do the multi-dimensional attribution analysis. RESULTS: From 2002 to 2016, there were 2129 food poisoning events in canteen in China. By the analysis of single dimensional attribution, the main cause food were vegetables, meat and fungi, respectively, the total events of 46. 09%, 16. 91% and 10. 53%. Bio-contaminants was the primary cause of pathogenic factor, accounting for 34. 05%. Production and processing were the main trigger links, accounting for 43. 31%. By the multidimensional attribution analysis, the highest number of cases(426) caused by green beans contain saponi, there were 404 incidents caused by improper production and processing. Poisonous mushrooms caused 125 toxic incidents, among them, some incidents(75) were caused by accidental consumption and misuse. CONCLUSION: It is suggested to regard green bean as the main cause food, focus on the aspects of production and processing and strengthen supervision, in order to reduce the occurrence of disease.

The Epidemiology of Listeria monocytogenes in China.

Listeria monocytogenes, a ubiquitous bacterium in nature, can lead to human listeriosis through food consumption. Listeriosis is a rare, preventable, and treatable foodborne disease but can cause hospitalizations and fatalities. We reviewed the literature published in China to better understand the prevalence of L. monocytogenes in food products, incidence of human listeriosis, and characteristics of L. monocytogenes strains in China. The average prevalence of L. monocytogenes in Chinese food products in 28 provinces was 4.42%, with the highest prevalence of 8.91% in meat-poultry products, followed by aquatic animals, Chinese salad and salad, rice and flour products, and so on. Two hundred fifty-three invasive listeriosis cases were reported from 2011 to 2016 in 19 provinces, and the overall case-fatality rate was 25.7% with no deaths reported of pregnant women and children. L. monocytogenes strains were generally susceptible to most antibiotics, with ampicillin and penicillin G still effective in treatment. The predominant sequence types (STs) in food were ST9 and ST8, while clinically ST87 was most common ST in China. The national human listeriosis pilot surveillance started in 2013, and a total of 133 listeriosis cases have been collected until now. On the basis of the surveillance program, further research should be conducted to uncover the reason for the prevalence and pathogenic mechanism of the highly epidemiological hypervirulent ST87 strains in China.

Trends of foodborne diseases in China: lessons from laboratory-based surveillance since 2011.

Foodborne disease is one of the most important public health issues worldwide. China faces various and unprecedented challenges in all aspects of the food chain. Data from laboratory-based foodborne disease surveillance systems from 2013 to 2016, as well as different regions and ages, can be found along with differences in the patterns of pathogens detected with diverse characteristics. Vibrio parahaemolyticus has been the leading cause of infectious diarrhea in China, especially among adults in coastal regions. Salmonella has been a serious and widely distributed pathogen responsible for substantial socioeconomic burden. Shigella was mostly identified in Northwest China and the inland province (Henan) with less-developed regions among children under 5 years. Data from foodborne disease outbreak reporting system from 2011 to 2016 showed that poisonous animals and plant factors responsible for most deaths were poisonous mushrooms (54.7%) in remote districts in southwest regions. The biological hazard that caused most cases reported (42.3%) was attributed to V. parahaemolyticus, the leading cause of foodborne outbreaks. In this review, we summarize the recent monitoring approach to foodborne diseases in China and compare the results with those in developed countries.

[Quality control results of microbiology of provincial laboratories in China in 2014].

OBJECTIVE: To get a baseline of 32 provincial center for disease control and prevention( CDC) microbiology laboratory in the aspect of quantitative test ability of Staphylococcus aureu, qualitative test ability of diarrheagenic Escherichia coli and unknown intestinal pathogen, and to comprehend the quality of microbiology testing. METHODS: Two different concentration samples of Staphylococcus aureus I and II were made. Diarrheagenic Escherichia coli and unknown intestinal pathogen samples were transported using the special semi-solid AGAR transport medium, in the form of four pure different serotypes each. The results of Staphylococcus aureu were logarithmic transformed, and evaluated with Z-score method using the average and standard deviation. The reporting results of diarrheagenic Escherichia coli and unknown intestinal pathogen were evaluated as right or wrong, and non-reporting were evaluated as missing. RESULTS: The satisfaction rate of Staphylococcus aureus was 82. 81%. The accuracy rate of diarrheagenic Escherichia coli was 45. 16%. Of the 32 results of unknown intestinal pathogen, 29 CDCs reported correctSalmonella serotypes, and the accuracy rate was 90. 63%. Combined the three quality control results, 9 CDCs reached the accurate results, with the accuracy rate of 28. 13%. CONCLUSION: 32 provincial CDCs have the identification ability of Salmonella serotyping and qualitative test ability of Staphylococcus aureu, while they need to strength diarrheagenic Escherichia coli identification and serotyping ability.

[Analysis of antimicrobial resistance results of Vibrio parahaemolyticus isolated from shellfish and its habitat in 2013].

OBJECTIVE: To evaluate the antimicrobial resistance of Vibrio parahaemolyticus isolated from shellfish and its habitat in Sichuan, Fujian and Guangxi. METHODS: The susceptibility of 186 isolates of Vibrio parahaemolyticus to 8 antibiotics were determined by broth microdilution susceptibility test. The antibiotics of ampicillin, cefotaxime, ceftazidime, gentamicin, tetracycline, chloramphenicol, ciprofloxacin and trimethoprim/sulfamethoxazole were used. RESULTS: The antibiotic resistance rate were 69.35% in which ampicillin resistance was the most prevalent. The geometric mean of ampicillin MIC value of all isolates was greater than the interpretation value of resistance. Among the 186 tested isolates, the multiple antibiotic resistance and/or intermediate resistance was 4. All strains were 100% sensitive to cefotaxime, ceftazidime, chloramphenicol and ciprofloxacin. The ampicillin susceptibility spectrum of aquaculture farm was the highest among the three sectors as 73.68% and Sichuan was the highest among the three provinces as 70.94% although there were no significant differences. There were 44 samples out of which 2 and above strains were isolated, and the susceptibility spectrum polymorphism rate of strains isolated from the same sample was 77.27% (34/44). CONCLUSION: The ampicillin resistance rate is relatively high, and shellfish habitat may be the main source of antimicrobial resistance of Vibrio parahaemolyticus. There is an urgent need to strengthen the surveillance and management of Vibrio parahaemolyticus in the shellfish breeding process.

Temporal and Spatial Variation in the Abundance of Total and Pathogenic Vibrio parahaemolyticus in Shellfish in China.

We investigated the abundance of total and pathogenic Vibrio parahaemolyticus in shellfish sampled from four provinces in China during May 2013 and March 2014 using the most probable number-polymerase chain reaction (MPN-PCR) method. Total V. parahaemolyticus was detected in 67.7% of 496 samples. A total of 38.1% and 10.1% of samples exceeded 1,000 MPN g(-1) and 10,000 MPN g(-1), respectively. V. parahaemolyticus densities followed a seasonal and geographical trend, with Guangxi and Sichuan shellfish possessing total V. parahaemolyticus levels that were 100-fold higher than those of the Liaoning and Shandong regions. Moreover, the levels of V. parahaemolyticus were at least 10-fold higher in the summer and autumn than in the cooler seasons. Pathogenic V. parahaemolyticus levels were generally lower than total V. parahaemolyticus levels by several log units and tended to be high in samples contaminated with high total V. parahaemolyticus levels. The aqua farms had a lower prevalence but higher abundance of total V. parahaemolyticus compared to retail markets. The catering markets showed the lowest levels of total V. parahaemolyticus, but 20.0% of samples exceeded 1,000 MPN g(-1). The levels of both total and pathogenic V. parahaemolyticus in oysters were higher than in clams. The log-transformed abundance of V. parahaemolyticus was significantly correlated with both water temperature and air temperature but not water salinity. These results provide baseline contamination data of V. parahaemolyticus in shellfish in China, which can be applied to local risk assessments to prioritize risk control to key sectors and evaluate the effectiveness of future control measures.

[Analysis on 2011 quality control results on aerobic plate count of microbiology laboratories in China].

OBJECTIVE: To test the aerobic plate count examining capability of microbiology laboratories, to ensure the accuracy and comparability of quantitative bacteria examination results, and to improve the quality of monitoring. METHODS: The 4 different concentration aerobic plate count piece samples were prepared and noted as I, II, III and IV. After homogeneity and stability tests, the samples were delivered to monitoring institutions. The results of I, II, III samples were logarithmic transformed, and evaluated with Z-score method using the robust average and standard deviation. The results of IV samples were evaluated as "satisfactory" when reported as < 10 CFU/piece or as "not satisfactory" otherwise. Pearson χ2 test was used to analyze the ratio results. RESULTS: 309 monitoring institutions, which was 99.04% of the total number, reported their results. 271 institutions reported a satisfactory result, and the satisfactory rate was 87.70%. There was no statistical difference in satisfactory rates of I, II and III samples which were 81.52%, 88.30% and 91.40% respectively. The satisfactory rate of IV samples was 93.33%. There was no statistical difference in satisfactory rates between provincial and municipal CDC. CONCLUSION: The quality control program has provided scientific data that the aerobic plate count capability of the laboratories meets the requirements of monitoring tasks.

[Molecular genetic makers for Vibrio parahaemolyticus--a review].

Vibrio parahaemolyticus is an important foodborne pathogen, of which the 03:K6 serotype caused many outbreaks in different countries since 1996. Based on the 10 years data (1992-2001) from China, gastroenteritis caused by Vibrio parahaemolyticus accounted for 31.1% of foodborne disease outbreaks that were resulted from microorganisms. Most environmental strains of Vibrio parahaemolyticus are non-pathogenic strains. However, clinical strains can producethermostable direct hemolysin (TDH), TDH-related hemolysin, and other virulence factors. Here we reviewed three commonly used molecular markers for Vibrio parahaemolyticus, including species-specific genes, the virulence genes and pandemic group-specific genes, so that to provide references for the rapid detection of Vibrio parahaemolyticus and the identification of its pathogenic factor.

Extended MLST-based population genetics and phylogeny of Vibrio parahaemolyticus with high levels of recombination.

A collection of 174 global isolates of Vibrio parahaemolyticus were analyzed by multilocus sequence typing (MLST) on the basis of ten conserved genes. The results showed a high level of nucleotide and allelic diversity with the evidence of purifying selection and of frequent recombination. Recombination played a much greater role than mutation in generating genetic heterogeneity. The 174 strains could be assigned into 89 different sequence types, which could be further separated into six clonal complexes (CCs; CC1 to CC6) plus 71 singletons. The three major CCs, namely CC1 to CC3, corresponded to the groups of pre-1996 clinical old-O3:K6 strains (trh(+), T3SS2ß(+), tdh(-), T3SS2α(-), and GS-PCR(-)), post-1996 pandemic strains (trh(-), T3SS2ß(-), tdh(+), T3SS2α(+), and GS-PCR(+)) and non-clinical isolates (trh(-), T3SS2ß(-), tdh(-), T3SS2α(-), and GS-PCR(-)), respectively. The MLST data enable the construction of a phylogenetic structure from the allelic profiles rather the nucleotide sequences, so as to reduce the affect of frequent recombination. The six CCs arose on a background of mutation and recombination, and according to the previously reported data, this bacterium could be evolved fast due to lateral acquisition of foreign genes especially including those encoding virulence determinants. V. parahaemolyticus had a typical epidemic population structure that is driven by mutation, recombination and lateral gene transfer.

[Development and evaluation of whole-genome DNA microarray of Vibrio parahaemolyticus].

OBJECTIVE: We developed and evaluated a whole-genome DNA microarray of Vibrio parahaemolyticus. METHODS: Based on the genomic sequences of V. parahaemolyticus, we chose a total number of 4770 genes, amplified them by PCR with specific primers, purified the PCR products and printed them onto glass slides. We performed two sets of hybridizations by the method of two-fluorescence comparative hybridization to evaluate the microarray quality, followed by PCR method to validate parts of microarray results. RESULTS: Microarray hybridization results were completely consistent with theory expectations and PCR verification results. CONCLUSION: We successfully developed a batch of good quality whole-genome DNA microarrays of V. parahaemolyticus, built up a method of microarray-based comparative genomic hybridization of V. parahaemolyticus and a set of microarray data analysis standard method.

The low-salt stimulon in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is the leading cause of seafood-associated bacterial gastroenteritis and is a moderately halophilic, salt-requiring bacterium. Global gene expression profiles of V. parahaemolyticus grown under 2% and 0.66% NaCl were compared to define the low-salt stimulon. The ectABC-lysC operon for synthesis of the compatible solute ectoine, as well as three compatible solute transport systems, namely ProU (glycine betaine), OpuD1 (glycine betaine) and Pot2 (spermidine), was up-regulated under 2% NaCl relative to 0.66% NaCl. The 2% NaCl condition favored the inducible expression of OmpW, OmpN and OmpA2, while repressed the expression of OmpA1, OmpU and VP1008. These results indicated that, to master the hyperosmotic stress of saline environments, V. parahaemolyticus might not only accumulate osmoprotectants through uptake or endogenous synthesis of compatible solutes, but also remodel its profiles of outer membrane protein to restore its cell membrane. The above differentially regulated genes will provide novel candidates for the further investigation of the molecular mechanisms of osmoadaptation in V. parahaemolyticus.

Cold-induced gene expression profiles of Vibrio parahaemolyticus: a time-course analysis.

A whole-genome DNA microarray was constructed to dissect expression profiles of Vibrio parahaemolyticus in response to a sudden temperature downshift from 37 to 10 degrees C. The mRNA level of each gene at each of three time points (20, 40 and 60 min after temperature downshift) was compared with that just before cold treatment. Clustering analysis of time-course data revealed nine gene clusters with different time-dependent expression patterns. Downregulation of metabolism-related genes was obviously dominant over upregulation at all time points. The distinct negative regulation of metabolism-related genes would account for a generally reduced cellular protein pool resulting from the sudden temperature downshift. In contrast, cold shock had a 'neutral and balanced' regulatory action on nonmetabolic cellular pathways, which likely brought about the remodelling of cell envelope structures and transport/binding functions. We identified a 171-bp 5'-untranslated region in the cspA transcript. The cspA gene encoded cold shock protein A (CspA), and CspA was shown to be the major cold shock protein in V. parahaemolyticus. Evident regulatory motifs were conserved within the cspA promoter regions of Escherichia coli and V. parahaemolyticus. These two bacteria likely use the same mechanism to regulate the cold-inducible expression of cspA.

Genome plasticity of Vibrio parahaemolyticus: microevolution of the 'pandemic group'.

BACKGROUND: Outbreak of V. parahaemolyticus infections occurred since 1996 was linked to a proposed clonal complex, the pandemic group. The whole genome sequence provides an unprecedented opportunity for dissecting genome plasticity and phylogeny of the populations of V. parahaemolyticus. In the present work, a whole-genome cDNA microarray was constructed to compare the genomic contents of a collection of 174 strains of V. parahaemolyticus. RESULTS: Genes that present variably in the genome accounted for about 22% of the whole gene pool on the genome. The phylogenetic analysis of microarray data generated a minimum spanning tree that depicted the phylogenetic structure of the 174 strains. Strains were assigned into five complexes (C1 to C5), and those in each complex were related genetically and phylogenetically. C3 and C4 represented highly virulent clinical clones. C2 and C3 constituted two different clonal complexes 'old-O3:K6 clone' and 'pandemic clone', respectively. C3 included all the 39 pandemic strains tested (trh-, tdh+ and GS-PCR+), while C2 contained 12 pre-1996 'old' O3:K6 strains (trh+, tdh- and GS-PCR-) tested herein. The pandemic clone (post-1996 'new' O3:K6 and its derivates O4:K68, O1:K25, O1:KUT and O6:K18) might be emerged from the old-O3:K6 clone, which was promoted by acquisition of toxRS/new sequence and genomic islands. A phylogenetic intermediate O3:K6 clade (trh-, tdh- and GS-PCR+) was identified between the pandemic and old-O3:K6 clones. CONCLUSION: A comprehensive overview of genomic contents in a large collection of global isolates from the microarray-based comparative genomic hybridization data enabled us to construct a phylogenetic structure of V. parahaemolyticus and an evolutionary history of the pandemic group (clone) of this pathogen.