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 investigate the characteristics of clinical bacteria and H3N2 influenza A virus coinfection and variations in the hemagglutinin (HA) cleavage site of H3N2 among influenza-like cases.Methods:A total of 12 250 samples were collected from influenza-like cases for real-time PCR detection of H3N2 influenza virus from January 2013 to December 2018. To analyze the characteristics of co-infection, some H3N2-positive samples were selected and analyzed by real-time PCR to detect Staphylococcus aureus, Streptococcus pneumoniae and haemophilus influenzae type B. HA genes of H3N2 isolates were amplified by RT-PCR and sequenced. A phylogenetic tree was constructed based on HA gene sequences. Amino acid variations in cleavage sites were analyzed. Results:H3N2 influenza viruses had been detected every year since 2013, causing 44.69% influenza-positive cases. There were 295 randomly selected H3N2-positive samples, of which 29.2% had clinical bacterial infection. The HA cleavage sites of 210 H3N2 isolates were sequenced and 68 had variations, including 63 carrying K342R (PEKQTR to PERQTR) single-amino acid site variation. The co-infection rate was 31.25% (45/144) in unmutated samples and 23.53% (16/68) in mutated samples (χ 2=1.34, P>0.05). The H3N2 influenza viruses circulating in Hangzhou mainly belonged to two evolutionary clusters of 3c.3a and 3c.2a, and the viruses with K342R mutation at the cleavage site belonged to the evolutionary cluster of 3c.3a. Conclusions:H3N2 influenza virus played an important role in the epidemic of influenza virus in Hangzhou. There were some bacterial co-infections in influenza-positive cases. Cleavage site variations showed regional epidemic characteristics, but had no significant correlation with bacterial co-infection.

Objective: To determine the relationship between dengue virus load and clinical characteristics, so as to provide basis for dengue fever prevention and treatment. Methods : The dengue viral load and typing of 120 patients in Gongshu District of Hangzhou from June to November 2017 were detected by real-time fluorescent quantitative RT-PCR;the clinical indicators of these dengue patients were collected and their correlation with the viral load was analyzed. Results: The DNA detection of dengue virus in 120 patients showed that they were all typeⅡ. The median dengue virus load was 3.91×104 copies/mL. All the patients had fever, the average peak temperature was（38.96 ± 0.69）℃. There were 102（85.00%）cases with asthenia;116（96.67%）cases with white blood cell count（WBC）less than 4× 109/L;119（99.17%）cases with platelet count（PLT）less than 100×109/L;114（95.00%）cases with glutamic oxaloacetate transaminase（GOT）more than 40 U/L;81（67.50%）cases with glutamic pyruvate transaminase（GPT）more than 52 U/L;58（48.33%）cases with creatine kinase（CK）more than 210 U/L. There was no significant correlation of dengue virus load with length of hospitalization, peak temperature,duration of fever, WBC,PLT, GOT, GPT and CK（P>0.05）. There were 75（62.50%）severe patients, and their median viral load was 9.29×104copies/mL, which was higher than 5.33×103copies/mL in non-severe patients（P<0.05）. Conclusion The dengue virus load is not related with length of hospitalization,peak temperature,WBC,PLT,GOT,GPT and CK,but with the severity of the disease.

Objective To investigate the clinical and epidemiological characteristics of Saffold virus (SAFV)infection in outpatient and hospitalized children with acute respiratory tract infections(ARI). Methods A total of 1060 clinical specimens were collected from children with ARI in the Affiliated Children's Hospital,Zhejiang University School of Medicine from March 2011 to February 2014, including 256 samples of throat swabs from outpatients,and 804 samples of trachea suctions from hospitalized patients. Real time PCR(RT-PCR)was performed to detect 5'UTR segment of SAFV.SPSS 17.0 software was used to analyze the test results and clinical data.Results The positive detection rates of SAFV in outpatients and hospitalized children with ARI were 2.3%(6/256)and 13.2%(106/804), respectively(χ2=24.147, P<0.01).Among the hospitalized children,the positive detection rates of SAFV in children <1 year,1-<3 years,3-<6 years and 6-12 years were 14.0%,11.2%,11.1% and 8.3%, respectively(χ2=1.845,P>0.05).The positive rates of SAFV in males and females were 12.7% and 17.7%(χ2=0.279,P>0.05).The detection rate of SAFV in autumn was highest(21.2%), followed by that in spring (14.6%),winter(9.5%)and summer(8.8%)(χ2=15.625, P<0.01).The co-infection rates with other respiratory pathogens of hospitalized and outpatients children were 76.4%(81/106)and 66.7% (4/6).Among the hospitalized patients, the rate of co-infection with respiratory syncytial virus was the highest(36.8%),followed by rhinovirus(27.4%), metapneumovirus(10.4%)and parainfluenza virus (10.4%).Among children with ARI,the fever rate of SAFV-positive cases was lower than that of SAFV-negative cases(χ2=4.069,P<0.05).Conclusions The detection rate of SAFV in hospitalized children with ARI is significantly higher than that in outpatients,and SAFV infection was dominated by co-infection. The prevalence of SAFV in the Hangzhou area presents a certain local epidemic pattern.

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.