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.

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 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.