Genomic epidemiology of Vibrio parahaemolyticus from acute diarrheal patients in Shenzhen City from 2013 to 2021 / 中华预防医学杂志

Objective: To characterize the prevalence and genomic epidemiology of Vibrio parahaemolyticus from acute diarrheal patients in Shenzhen City from 2013 to 2021. Methods: Based on the Shenzhen Infectious Diarrhea Surveillance System, acute diarrheal patients were actively monitored in sentinel hospitals from 2013 to 2021. Whole-genome sequencing (WGS) of Vibrio parahaemolyticus isolates was performed, and the genomic population structure, serotypes, virulence genes and multilocus sequence typing were analyzed. Outbreak clusters from 2019 to 2021 were explored based on single-nucleotide polymorphism analysis. Results: A total of 48 623 acute diarrhea cases were monitored in 15 sentinel hospitals from 2013 to 2021, and 1 135 Vibrio parahaemolyticus strains were isolated, with a positive isolation rate of 2.3%. Qualified whole-genome sequencing data of 852 isolates were obtained. Eighty-nine serotypes, 21 known ST types and 5 new ST types were identified by sequence analysis, and 93.2% of strains were detected with toxin profile of tdh+trh-. 8 clonal groups (CGs) were captured, with CG3 as the absolute predominance, followed by CG189. The CG3 group was dominated by O3:K6 serotype and ST3 sequence type, while CG189 group was mainly O4:KUT, O4:K8 serotypes and ST189a and ST189 type. A total of 13 clusters were identified, containing 154 cases. About 30 outbreak clusters with 29 outbreak clusters caused by CG3 strains from 2019 to 2021. Conclusion: Vibrio parahaemolyticus is a major pathogen of acute infectious diarrhea in Shenzhen City, with diverse population structures. CG3 and CG189 have been prevalent and predominant in Shenzhen City for a long time. Scattered outbreaks and persistent sources of contamination ignored by traditional methods could be captured by WGS analysis. Tracing the source of epidemic clone groups and taking precise prevention and control measures are expected to significantly reduce the burden of diarrhea diseases caused by Vibrio parahaemolyticus infection in Shenzhen City.

Molecular characterization of Vibrio parahaemolyticus collected from human infections in Shenzhen, between 2002 and 2008 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To determine the occurrence and distribution of specific clones of pathogenic Vibrio parahaemolyticus(VP)isolated in Shenzhen and to assess the relationship between serotype O3:K6 and the globally distributed pandemic clone.</p><p><b>METHODS</b>A total of 1005 VPs isolated from diarrhea patients in 2002-2008 were sero-typed. Real-time PCR was used to detect the virulence genes tlh, toxR, tdh, trh and orf8 in 281 isolates from 68 different serotypes. The main serotypes were typed by pulsed field gel electrophoresis(PFGE). Strains with dominant serotypes and PFGE patterns were assayed by GS-PCR and toxRS sequencing for the identification of pandemic clone. Multilocus sequence typing(MLST)analysis was reserved for exemplary 41 O3 : K6 and O1 : K25 isolates.</p><p><b>RESULTS</b>Seventy-nine serotypes were observed among the 1005 isolates, including O3 : K6(57.9%), O4 : K8(8.16%), O1 : KUT(5.87%), O1 : K25(5.27%), O4 : K68(1.39%), O1 : K56(1.39%) and O9 : K44(0.99%). Most of the strains(99.36%)showed PCR positive to tlh, toxR, and tdh but eleven strains were tdh negative. MLST showed that all the 36 O3 : K6 isolates belonged to ST3 and all the 5 O4 : K8 strains were ST189. These results matched the description of the pandemic VP clone.</p><p><b>CONCLUSION</b>A recognizable burden of diarrheal illness caused by VP had been seen in Shenzhen. Results from serotyping indicated that although there existing a large variety of diversities, the dominant serotype appeared to be O3 : K6. VP isolates identified in Shenzhen mainly showed as tdh positive but trh negative, in consistent with the current pandemic O3 : K6 clone. The pandemic O3 : K6 clone did appear to co-exist with other clones of O3 : K6, as well as O4 : K8,O1 : K25. Potential outbreak of VP could be monitored through the laboratory-based surveillance programs, suggesting that the strategies related to prevention and control of VP should be prioritized in Shenzhen.</p>

Characteristics of Salmonella enterica serovar Senftenberg lacking Salmonella pathogenicity island 1 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the characteristics of the strains of Salmonella enterica (S. enterica) serovar Senftenberg lacking Salmonella pathogenicity island 1 (SPI-1).</p><p><b>METHODS</b>A total of 10 strains of S. enterica serovar Senftenberg were isolated from 10 cases of diarrhea patients. Pulsed field gel electrophoresis (PFGE), PCR, sequencing techniques and cell invasion test were adapted to study the molecular types and invasiveness of the genes and cells; and made a comparison between the 10 strains and the strains (C02013) isolated in Shenzhen in 2002.</p><p><b>RESULTS</b>The 10 Senftenberg isolated (S09007-S09012, S09014-S09017) in Shanghai showed three PFGE patterns, which were significantly different from the strains isolated in Shenzhen. PCR-amplified results indicated the invasion gene (invA), secreted effector protein gene (sipA) and gene fragments as fhlA-hilA, hilA-spaP and spaP-invH in the 10 strains of SPI-1 were all negative. The sequencing results revealed that the 10 strains isolated in Shanghai lacked most parts of SPI-1 genes, as fragments from orgA to invH and parts of orgA gene itself; however, compared with strains isolated in Shenzhen, the sprB-orgC gene existed. The missing parts of genes were replaced by a simple insertion sequence (IS) of 1000 bp in the strains isolated both in Shenzhen in 2002 and in Shanghai in 2006. The invasiveness rates of the 10 strains (S09007-S09012, S09014-S09017) towards Hela cells were (0.0053 ± 0.0024)%, (0.0046 ± 0.0006)%, (0.0047 ± 0.0003)%, (0.0064 ± 0.0012)%, (0.0065 ± 0.0011)%, (0.0070 ± 0.0020)%, (0.0115 ± 0.0030)%, (0.0099 ± 0.0039)%, (0.0180 ± 0.0135)% and (0.0031 ± 0.0012)%, respectively; which were all significantly lower than the rate of invA-positive control strain STM1344 ((5.0800 ± 0.6333)%); lower or close to the rate of invA-lacked artificial-mutated strain STMinvA-((0.0193 ± 0.0045)%).</p><p><b>CONCLUSION</b>SPI-1 genes are not essential for the diarrhea caused by S. enterica serovar Senftenberg.</p>

Etiologic and molecular characteristics of Vibrio parahaemolyticus strains isolated from diarrheal patients in Shenzhen, in 2007-2008 / 中华流行病学杂志

Objective To study the infection status and the molecular characteristics of Vibrio parahaemolyticus isolated from diarrheal patients in Shenzhen, in 2007 to 2008 and to provide evidence for the prevention and control of diarrheal diseases caused by Vibrio parahaemolyticus. Methods More than 80 fecal specimens from four sentinel surveillance hospitals were collected and cultured each month. A total of 361 isolates of Vibrio parahaemolyticus were sero-typed and examined by real-time PCR for the presence of two major virulence genes, tdh and trh. Of 361 strains, 60 O3: K6 strains isolated from six suspected outbreaks in August, 2007 and in September, 2008 were typed by pulsed-field gel electrophoresis (PFGE). Results 4384 stool samples were detected in four sentinel surveillance hospitals and with 361 Vibrio parahaemolyticus strains isolated that belonged to 28 serotypes. Serotype O3:K6, O4:K8 and O1:KUT accounted for 67.90%, 7.50% and 6.10%, respectively. Of 361 strains, 337 strains belonged to tdh + trh- , 11 strains were tdh-trh- and 13 strains were tdh + trh +. The most prevalent serotype which caused diarrheal diseases was tdh + trh-in Shenzhen. The 60 isolates were discriminated into twenty different PFGE patterns, which belonged to three clones. Among the 60 isolates, most of the PFGE patterns of isolates from the suspected outbreak locations were identical and some strains isolated from different year were different. Conclusion Vibrio parahaemolyticus isolates in Shenzhen were dominated by O3:K6 strains. Most of these isolates carried tdh gene and few carried trh gene. Meanwhile, the identical patterns of isolates from 6 suspected outbreaks locations demonstrated that Vibrio parahaemolyticus outbreaks occurred in July 2007 and in September 2008 in Shenzhen. However, the dominated strains' PFGE patterns were different each year, indicating that the sources of Vibrio parahaemolyticus had a multiplex nature and the multiplex sources such as water, sea food and pickled products should be integrated monitored. Laboratory based surveillance of diarrheal diseases could contribute in establishing early warning system for the better prevention and control of diarrheal diseases.

Analysis of pulsed-field gel electrophoresis molecular subtyping of Shigella strains in Shenzhen / 中华预防医学杂志

<p><b>OBJECTIVE</b>To analyze the genetic relations of Shigella isolated from Shenzhen in 2001-2006 and develop primary molecular subtyping surveillance network of Shigella.</p><p><b>METHODS</b>Chromosomal DNAs from 55 isolated in agarose were digested with the restriction enzyme Xba I, and then were analyzed by pulsed-field gel electrophoresis. Pulsed-field gel electrophoresis (PFGE) patterns were clustered using BioNumerics software.</p><p><b>RESULTS</b>All 41 distinctive PFGE patterns were identified among 55 strains. 32 strains belonged to one cluster. Differences were observed in other strains.</p><p><b>CONCLUSION</b>Both genetic-related clones and non-related clones of Shigella existed in Shenzhen. The development of PFGE molecular subtyping surveillance network would contribute to the active surveillance, outbreak investigation and source tracking for Shigellosis.</p>

Molecular subtyping of Vibrio cholerae isolates by pulsed-field gel electrophoresis in Shenzhen from 1993 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To determine the genetic relationships between different Vibrio cholerae isolates in Shenzhen from 1993 to 2002.</p><p><b>METHODS</b>Chromosomal DNA from 60 isolates was digested in seakem gold agrose with restriction enzyme Not I and plugs were then analyzed by pulsed-field gel electrophoresis. Pulsed-field gel electrophoresis (PFGE) patterns of V. cholerae isolates were clustered using BioNumerics software.</p><p><b>RESULTS</b>39 distinctive PFGE patterns were identified with each pattern having 20 to 30 bands. Most PFGE patterns were divided into cluster A or cluster B.</p><p><b>CONCLUSION</b>The closely related pandemic clone clusters of V. cholerae strains did exist in Shenzhen. PFGE of V. cholerae could be used for active surveillance and tracking for cholerae.</p>

Rapid simultaneous detection of Salmonella and Shigella using modified molecular beacons and real-time PCR / 中华流行病学杂志

<p><b>OBJECTIVE</b>Dual detection of Salmonella and Shigella using modified molecular beacons and real-time PCR was developed. The established method was applied to rapid diagnosis of Salmonella and Shigella' food poisoning, and for routine monitoring programs.</p><p><b>METHODS</b>Two sets of primers were designed based on the core sequence of invA gene and ssaR gene published on GenBank to detect Salmonella, and ipaH gene were selected to detect Shigella. Three corresponding modified molecular beacons labeled with different fluorophors were designed. The molecular beacons and primer sets were tested against numerous strains from 55 different bacterial species. Then the two assays were combined to establish the dual real-time PCR assay, and were applied to the food poisoning diagnosis and surveillance.</p><p><b>RESULTS</b>For the modified molecular beacons-based dual real-time PCR assay, the sensitivity achieved was 69-93 fg/microl, 32-64 CFU/ml or 1-2 CFU/PCR reaction. There was no cross-reaction with other bacteria served as control. The dual real-time PCR assay was used to detect 134 Salmonella strains and 67 Shigella strains but no false signals were observed. 1100 food poisoning samples were tested with 569 Salmonella and 42 were Shigella identified by real time PCR. Among the positive samples, 551 were detected Salmonella and 41 were Shigella by traditional culture method. The overall test could be finished within 2 hours to one day starting from sample preparation.</p><p><b>CONCLUSION</b>The modified molecular beacons-based dual real-time PCR assay was rapid, sensitive, and specific. It could be applied to the rapid diagnosis of Salmonella and Shigella' food poisoning.</p>