Objective To analyze the molecular characteristics of Salmonella paratyphi A ( S. pa-ratyphi A) strains prevailing in Hangzhou area in recent years. Methods Pulse field gel electrophoresis ( PFGE) and multiple-locus variable-number tandem repeat analysis ( MLVA) were performed for molecular typing and epidemiological analysis of 72 S. paratyphi A strains isolated in Hangzhou area during 2002 to 2013. Results The 72 S. paratyphi A strains were divided into 11 PFGE ( by using restriction enzymes of Xba Ⅰ and Bln Ⅰ) and 6 MLVA types. Among the selected 34 variable number tandem repeat ( VNTR) sites, 4 sites (1188K, 2075K, 2201K and 4346K) showed high polymorphism, in which PFGE displayed a higher resolution than MLVA. Except for the 5 PFGE types of X4B5, X7B7, X8B8, X9B9 and X10B10, the other 6 PFGE types belonged to a same clone sharing a similarity of greater than 95%, and the S. Paratyphi A strains in that clone accounted for 93. 1% of the total strains isolated in Hangzhou. Conclu-sion The occurrence of paratyphoid A in Hangzhou area from 2002 to 2013 was mainly caused by S. para-typhi A strains belonging to the same clone. Combination of PFGE with MLVA was conducive to epidemiolog-ical investigation of paratyphoid A.

Objective To determine the molecular characteristics of predominant Salmonella typhi and Salmonella paratyphi A strains prevalent in Hangzhou area from 2002 to 2008.Methods Pulse field gel electrophoresis (PFGE),multi-locus variable-number tandem repeat analysis (MLVA) and multi-locus sequence typing (MLST) were applied for typing as well as analysis of the molecular characteristics of 31 S.typhi isolates and 404 S.paratyphi A isolates from Hangzhou area during 2002 to 2008.Results The 404 S.paratyphi A isolates could be divided into six PFGE types (P1-P6).99.0％ of the S.paratyphi A isolates (400/404) belonged to the same one clone family (P1 and P2 types),in which P1 strains occupied 93.3％ (373/400) of the isolates.The 31 S.typhi isolates displayed a high diversity,which could be classified into 14 PFGE types,28 MLVA types with 90.3％ resolving power and 3 MLST types.The S.typhi strains prevalent in Hangzhou area were similar to those in Southeast Asia but different from those in Europe.The variable number tandem repeat (VNTR) sites with high polymorphism,TR1,TR2 and Sal02,could be used to the markers for diagnosis of S.typhi isolates in the area.The MLST types of 31 S.typhi isolates included all the three types currently found in the world but the ST2 type of S.typhi strains was predominant (23/31,74.2％).Conclusion The paratyphoid A prevalence in Hangzhou area in the recent years is caused by infection of the same clone family of S.paratyphi A whereas the S.typhi strains prevalent in the area display a high diversity.

Objective:To analyze the epidemiological and etiological characteristics of dengue fever in Hangzhou in 2018.Methods:RT-PCR was used to detect the nucleic acids and analyze the serotypes of dengue viruses (DENV) in serum samples collected from dengue fever cases. Phylogenetic trees based on the E gene sequences of DENV isolated from the serum samples were then constructed and analyzed. Epidemiological characteristics of these dengue fever cases were analyzed. Results:A total of 80 cases of dengue fever were detected in Hangzhou in 2018 with 55 imported cases and 25 indigenous cases (24 caused by DENV-1 and one by DENV-3). These indigenous cases mainly occurred during late July to early October with people above 50 years old accounting for 68%. Phylogenetic analysis showed that DENV-1 strains isolated from the indigenous cases in Yuhang, Jianggan-Shangcheng and Qiantang districts all belonged to genotype Ⅰ, and were respectively closely related to the strains from Indonesia in 2015, Myanmar in 2017, Ningbo in 2018 and Hangzhou imported cases from Thailand in 2018. The indigenous DENV-3 strain belonged to genotype Ⅲ, and shared 99.5% homology with the Singapore strain in 2013.Conclusions:Imported cases accounted for a large fraction of the dengue fever cases in Hangzhou, which brought a high risk to indigenous outbreak. Due to multiple imported cases, the current epidemic presented a characteristic of multiple small-scale outbreaks.

Objective To develop a microsphere-based suspension array for simultaneous detec-tion and identification of Salmonella H antigens by using Luminex xTAG technology and to evaluate its capa-bility in serotyping Salmonella strains. Methods The fliC and fljB genes, encoding the H antigen of Salmo-nella, were selected as the target genes. Universal upstream primers were designed based on the highly con-served regions of fliC and fljB genes, and the corresponding specific reverse primers were designed based on the variable regions. While synthesizing, the 5′end of each upstream primer was labeled with biotin and the 5′end of each specific reverse primer was modified with its certain TAG sequence. After amplified and la-beled with biotin and TAG sequence, the PCR products of specimens were hybridized with the mixture of va-rious MagPlex-xTAGTM microspheres. Each set of microspheres contained its unique anti-TAG sequences. The results of hybridization were analyzed by using Luminex MagPix reader system and the median fluores-cence intensity ( MFI) was reported. The H antigens of 145 Salmonella strains were identified with this de-veloped xTAG suspension array, and the results were compared with those obtained by using traditional ser-um agglutination test. Results The PCR products of different H antigens ranged from 94 bp to 245 bp and could be identified by hybridizing with MagPlex-xTAGTM microspheres. There was no cross-reaction between different H antigens or with DNAs derived from Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus and Shigella flexneri. Compared with the traditional serum agglutination test, the sensitivity and specificity of the xTAG suspension array in the identification of H antigens of 145 Salmonella strains were 95. 1% and 100%, respectively. Conclusion The developed xTAG suspension array was a specific, accurate and effective method for simultaneous detection and identification of 31 H antigens of common Salmonella serovars strains. It could be used for determining the H antigens of more than 90 Salmonella strains within 5 hours.

We determined molecular characteristics of Listeria monocytogenes foodborne isolates in Hangzhou and investigated the characterization of local strains.Multi-locus sequence typing(MLST) and pulsed-field gel electrophoresis(PFGE) were applied to identify molecular types of Listeria monocytogenes isolates.Results showed that a total of 133 strains of 6 serotypes were divided into 19 MLST types including a new type ST767.ST9 and ST121 were the major ST types.There were 33 and 45 PFGE patterns characterized by AscⅠ and ApaⅠ.The molecular types of Listeria monocytogenes strains were widely distributed in Hangzhou.It is indicated that the major clusters were Lineage Ⅰ and Lineage Ⅱ which will cause listeriosis.The contamination of Listeria monocytogenes in food is serious in Hangzhou and the surveillance and management should be strengthened to prevent the food borne diseases.

Objective To understand the genomic epidemiology of Salmonella paratyphi A strains circulating in Hangzhou area in recent years. Methods Next generation sequencing(NGS) technology was used to obtain genomes of 60 Salmonella paratyphi A strains isolated in Hangzhou area from 2002 to 2013. Genomes of 391 Salmonella paratyphi A strains were downloaded from the Sequence Read Archive (SRA) and Assembly database. After removing recombinations, the phylogenetic tree of all 451 genomes based on single nucleotide polymorphisms(SNP) was constructed using ATCC9150 as the reference. SRST2 and mul-tilocus sequence typing (MLST) were used to analyze sequence types(ST). The Salmonella In Silico Typ-ing Resource (SISTR) was used for core genome multilocus sequence typing (cgMLST). Resistant genes were screened out with SRST2 and BLASTN. Seven kinds of antibiotics were selected to conduct drug sus-ceptibility test in the 60 strains isolated in Hangzhou. Results A total of 19 258 SNP loci were found in 451 genomes. The average distance in the phylogenetic tree between strains was 0.007 0 and the distance less than 0.05 accounted for 96.73%, indicating a little difference in the 451 Salmonella paratyphi A ge-nomes. Fifty-eight Hangzhou strains of ST85 were highly similar in genomic sequences,which suggested that the clonal spread of ST85 strains caused the epidemic of paratyphoid A in Hangzhou area during 2002-2013. Salmonella paratyphi A strains isolated in Hangzhou were distantly related to five domestic strains (average distance:0.057),but close to 15 Yunnan strains(average distance:0.003 2) and closest to strains isola-ted in Cambodia (average distance: 0.001 8), suggesting the possibility of transnational spread of ST85 strains. Among two Hangzhou strains of ST129,HZ333 was closely related to two strains isolated in Jiangsu Province(average distance:0.009 7),suggesting the possibility of domestic transmission of ST129 strains. Except for two untyped strains,the other 58 strains were divided into nine cgMLST types. Except for 57 un-typed strains,the other 334 strains obtained from public databases were classified into 165 cgMLST types. Fifty-six out of the 60 Hangzhou strains carried the resistant gene aac6-Iy and the other four carried aac6-Iaa gene. Thirteen out of the 391 strains obtained from public databases didn′t carry resistant genes, while the other 378 strains carried the resistant gene aac6-Iy. Among the 60 Hangzhou strains,56 were sensitive to all of the seven kinds of antibiotics;three were resistant to cotrimoxazole and one was resistant to ampicillin and tetracycline. Conclusion The epidemic of paratyphoid A fever in Hangzhou from 2002 to 2013 was mainly caused by the clonal spread of ST85 strains that had the possibility of transnational spread. Some Hangzhou strains of ST129 had the possibility of domestic transmission. SNPs analysis had an advantage over pulse field gel electrophoresis(PFGE) technology in resolution and could be used to accurately trace the cause of paratyphoid A fever. Resistant gene aac6-Iy was generally carried. NGS technology had a promising prospect in the control and prevention of infectious diseases.

Objective To investigate the genomic characteristics and virulence factors of emetic-type Bacillus cereus strains isolated from food in Hangzhou for better understanding their pathogenic potential. Methods Real-time PCR was performed to detect the ces gene cluster ( cereulide) in 132 Bacillus cereus strains isolated from food from 2015 to 2017. Genomes of cereulide-positive strains were sequenced using Illumina MiSeq sequencing platform. Genome annotation, virulence factor detection, comparative and evolu-tionary analysis were performed after the sequences of genomes were assembled. Results Twelve strains (9. 09％) carried the ces gene. Their genome sizes ranged from 5. 35 to 5. 75 Mb and GC contents from 35. 25 to 35. 43 mol％. All of them harbored the full cereulide biosynthesis gene cluster, nonhemolytic ente-rotoxin ( NHE)-encoding gene cluster ( nheA, nheB and nheC) and hemolysinⅢ( hlyⅢ) . The average nu-cleotide identity ( ANI ) between the 12 isolates and the reference strain NC7401 ( Accession number:AP007209) was over 99. 35％. Phylogenetic analysis demonstrated these strains were clustered into the same branch with local clinical isolates and the emetic-type Bacillus cereus strains of NC7401 and AH187. Con-clusions The genomic sequences of the emetic-type Bacillus cereus strains isolated from food in Hangzhou area were highly similar to that of the reference strain NC7401. Results of the genomic analysis suggested that these isolates carried many virulence factors that were related to pathogenicity.

Objective: To analyze genomic features of pathogens based on next generation sequencing technique in a food-borne disease event. Methods: A total of 11 blood samples, stomach contents before gastric lavage from the death and patients' foods were collected. S. aureus, B. cereus and toxic substances were detected. B. cereus detected in foods were counted. The conserved region of 16 S rDNA gene and ces gene(cereulide) of B. cereus isolates were detected by real-time PCR. Next Generation Sequencing (NGS) technology was applied to acquire genome sequences of isolates. Different plasmids distribution and comparative genomics analysis with reference sequences in public databases were analyzed. Results: Only B. cereus tested positive in all samples. The counts of B. cereus in Egg fried rice, one food samples, were 1.9×10⁷ CFU/g, and the counts of B. cereus in dried and fried fish and brine pork head meat samples were 3.0×10³ CFU/g both. Ten isolates were carrying hlyⅢ, nheA, nheB, inlA and inhA genes, and nine isolates carried the plcR gene and nine isolates carried the nheC gene. The PCR result of 16 S rDNA gene and ces gene of all isolates were positive. All carried the complete ces genes cluster sequence which were identical to the sequence of plasmid pCER270 (NC_01 0924.1) from strain AH187 in United Kingdom and pNCcld (NC_016792.1) from NC7401 in Japan. The alignment of plasmids turned out the sequence of the isolate differed from the pXO1 and pXO2 plasmids of B. anthracis, but carried the pNCcld plasmid containing the ces genes cluster. The phylogenetic tree based on genomic sequences of ten isolates showed high similarity (distances in phylogenetic tree from 2.0×10^-6-9.0×10^-6) to each other and to the B. cereus strains AH187 and NC7401 (MLST ST26 type, distances in phylogenetic tree from 3.8×10^-5-4.5×10^-5). Conclusion The foodborne disease event was caused by vomiting type Bacillus cereus without plasmid pXO1 and pXO2 contaminated egg fried rice. The vomiting-type food poisoning caused by B. Cereus globally is probably associated with ST26, ST164 and other strains harboring ces gene.

Objective:To analyze the antimicrobial resistance and genomic characteristics of Salmonella enterica serovar Derby strains isolated from human and food sources in Hangzhou. Methods:A total of 60 Salmonella enterica serovar Derby strains isolated in Hangzhou during the period from 2015 to 2020 were subjected to antimicrobial susceptibility testing, pulsed field gel electrophoresis (PFGE) typing and whole-genome sequencing. Multilocus sequence typing (MLST), core genome multilocus sequence typing (cgMLST) and the identification of antimicrobial resistance genes were performed using the sequencing data. Phylogenetic tree based on the single nucleotide polymorphism (SNP) sites in the 60 genomes from Hangzhou and 379 genomes from public databases was constructed. Results:No significant difference was observed in the drug resistance rates between the clinical strains and food strains in Hangzhou. The multidrug resistance (MDR) rate was 76.7% (46/60). All of the 60 Salmonella Derby strains were positive for the antimicrobial resistance genes aac(6′)- Iaa and fosA7. The 60 strains were subtyped into 46 molecular types by PFGE and 53 molecular types by cgMLST(HC2). Except for one strain belonging to ST3220, the other Salmonella Derby strains were ST40. The phylogenetic analysis showed that some strains isolated in Hangzhou were close to the strains in Southeast Asia, suggesting the possibility of cross-border transmission of ST40 strains, with the main food sources being pork and fish; other strains were close to those circulating in Beijing, Guangzhou, Hubei, Chongqing and other provinces, suggesting the possibility of cross-province transmission of the strains, with the main food sources being pork, beef and chicken. Conclusions:The epidemic of Salmonella Derby in Hangzhou was mainly caused by the spread of ST40 strains and MDR was common. Clinical infections might be closely related to the consumption of pork, beef, chicken and fish. There was the possibility of cross-border transmission of Salmonella Derby between Hangzhou and Southeast Asia and cross-province transmission in China.