[Genetic characterization and drug resistance analysis of Salmonella Kentucky ST314 in Shenzhen in 2010-2021].

OBJECTIVE: To understand the prevalence, genetic characteristics and drug resistance features of Salmonella Kentucky ST314 in Shenzhen. METHODS: Whole genome sequencing of 14 strains of Salmonella Kentucky ST314 collected from 2010-2021 by the Foodborne Disease Surveillance Network of Shenzhen Center for Disease Control and Prevention for phylogenetic evolutionary analysis, drug resistance gene and plasmid detection; drug susceptibility experiments were performed by micro-broth dilution method. RESULTS: A total of 57 strains of Salmonella Kentucky were collected from the foodborne disease surveillance network, 14 of which were ST314. The Shenzhen isolates were clustered with isolates from Southeast Asian countries such as Vietnam and Thailand on clade 314.2, and the single nucleotide polymorphism distance between local strains in Shenzhen was large, indicating dissemination. In this study, a total of 17 drug resistance genes/mutations in 9 categories were detected in the genome of Salmonella Kentucky ST314, carrying 3 extended spectrum beta-lactamases(ESBLs), including bla_(CTX-M-24)(14.3%, 2/14), bla_(CTX-M-55)(7.1%, 1/14), and bla_(CTX-M-130)(14.3%, 2/14), all located on plasmids. Regarding quinolone resistance factors, two plasmid-mediated quinolone resistance(PMQR) genes were identified in the genome: qnrB6(71.4%, 10/14) and aac(6')Ib-cr(78.6%, 11/14), a quinolone resistance quinolone resistance-determining regions(QRDR) mutation T57 S(100%, 14/14). The multi-drug resistance rate of Salmonella Kentucky ST314 in Shenzhen was 92.86%(13/14)with the highest rate of resistance to tetracycline and cotrimoxazole(100%, 14/14), followed by chloramphenicol(92.86%, 13/14), cefotaxime and ampicillin(78.57%, 11/14), ciprofloxacin and nalidixic acid(71.43%, 10/14), and ampicillin-sulbactam had the lowest resistance rate(21.43%, 3/14). CONCLUSION: ST314 is the second most prevalent ST type among Salmonella Kentucky in Shenzhen, mainly isolated from food, especially poultry; phylogenetic analysis suggests that ST314 is a disseminated infection and the genome shows a highly genetically conserved phenotype. Drug resistance of Salmonella Kentucky ST314 is very serious, especially QRDR mutation, PMQR gene co-mediated quinolone resistance and plasmid-mediated cephalosporin resistance are prominent and deserve extensive attention.

Pathogenetic detection, retrospective and pathogenicity analysis of a fatal case of Vibrio vulnificus in Shenzhen, China.

We report a 36-year-old male patient died of V. vulnificus-induced septicaemia and multiple organ failure syndrome after oyster consumption at a restaurant. We isolated and identified V. vulnificus vv16015 from the patient's blood sample and antibiotic susceptibility tests indicated sensitivity to all 21 antibiotics. Oyster samples were subsequently collected from the restaurant's supplier and three strains of V. vulnificus were isolated. Whole genome sequencing and analysis revealed vv16015 to be distantly related to these strains and confirmed that V. vulnificus contamination was present in the seafood of the restaurant and supplier. Using a Galleria mellonella larvae infection model, the virulence of vv16015 was determined to be higher than that of comparison strains isolated from a surviving patient (vv15018) and an oyster (vv220015). The human and environment distribution of V. vulnificus in Shenzhen is sporadic and heterogeneous, and vv16015 is highly virulent compared to other strains.

Emergence of chromosomally located blaCTX-M-14b and qnrS1 in Salmonella enterica serotype Kentucky ST198 in China.

Highly fluoroquinolone-resistant Salmonella enterica serotype Kentucky has become widespread in recent years, largely associated with the spread of sequence type 198 (ST198), which often leads to multidrug resistance. Research on the genomic epidemiology of Salmonella Kentucky in China is currently uncommon. In this study, we analysed the genomic epidemiology and antimicrobial resistance characteristics of Salmonella Kentucky ST198 collected from foodborne disease surveillance in Shenzhen, China, during 2010-2021, using whole-genome sequencing and antibiotic susceptibility testing. In addition, 158 global Salmonella Kentucky ST198 genomes were included for comparison. Among 8559 Salmonella isolates, 43 Salmonella Kentucky ST198 isolates were detected during 2010-2021. The global Salmonella Kentucky ST198 evolutionary tree was divided into five clades, with Shenzhen isolates distributed in clades 198.1, 198.2-1 and 198.2-2, mainly clustered with Chinese strains. Strains in clade 198.2 dominated in Shenzhen and all of them showed multidrug resistance. Nine strains showed high resistance to ceftriaxone, which was associated with blaCTX-M-14b in clade 198.2-1, which was demonstrated to be located on the chromosome. Fifteen strains showed high resistance to ciprofloxacin, which was associated with carriage of qnrS1 in clade 198.2-2. qnrS1 was first located on an IncHI2 plasmid and then transferred into the chromosome. Here we report the genomic and antimicrobial resistance characterisation of Salmonella Kentucky ST198 in Shenzhen. Of particular concern, we identified for the first time a clade 198.2-1 isolate carrying blaCTX-M-14b as well as chromosomally located qnrS1 in clade 198.2-2 of Salmonella Kentucky ST198 in China, highlighting the necessity of surveillance of clade 198.2.

Epidemiological and genomic analyses of human isolates of Streptococcus suis between 2005 and 2021 in Shenzhen, China.

Streptococcus suis (S. suis) is an important food-borne zoonotic pathogen that causes swine streptococcosis, which threatens human health and brings economic loss to the swine industry. Three-quarters of human S. suis infections are caused by serotype 2. A retrospective analysis of human S. suis cases in Shenzhen, a megacity in China, with high pork consumption, between 2005 and 2021 was conducted to understand its genomic epidemiology, pathogen virulence, and drug resistance characteristics. The epidemiological investigation showed that human cases of S. suis in Shenzhen were mainly associated with people who had been in close contact with raw pork or other swine products. Whole-genome sequence analysis showed that 33 human isolates in Shenzhen were dominated by serotype 2 (75.76%), followed by serotype 14 (24.24%), and the most prevalent sequence types (STs) were ST7 (48.48%) and ST1 (39.40%). ST242 (9.09%) and ST25 (3.03%), which were rarely reported, were also found. Phylogenetic analysis showed that the Shenzhen human isolates had close genetic relatedness to isolates from Guangxi (China), Sichuan (China), and Vietnam. We found a new 82 KB pathogenicity island (PAI) in the serotype 2 isolate that may play a role in sepsis. Similarly, a serotype 14 isolate, containing 78 KB PAI, was isolated from a patient presenting with streptococcal toxic shock syndrome (STSLS) who subsequently died. Multi-drug resistance (MDR) was high in human isolates of S. suis from Shenzhen. Most human isolates were resistant to tetracycline, streptomycin, erythromycin, and clindamycin, and 13 isolates had intermediate resistance to penicillin. In conclusion, swine importation from Guangxi, Sichuan, and Vietnam should be more closely monitored, and the use of antibiotics limited to reduce the potential for antimicrobial resistance (AMR).

Outbreak dynamics of foodborne pathogen Vibrio parahaemolyticus over a seventeen year period implies hidden reservoirs.

Controlling foodborne diseases requires robust outbreak detection and a comprehensive understanding of outbreak dynamics. Here, by integrating large-scale phylogenomic analysis of 3,642 isolates and epidemiological data, we performed 'data-driven' outbreak detection and described the long-term outbreak dynamics of the leading seafood-associated pathogen, Vibrio parahaemolyticus, in Shenzhen, China, over a 17-year period. Contradictory to the widely accepted notion that sporadic patients and independent point-source outbreaks dominated foodborne infections, we found that 71% of isolates from patients grouped into within-1-month clusters that differed by ≤6 single nucleotide polymorphisms, indicating putative outbreaks. Furthermore, we showed that despite the long time spans between clusters, 70% of them were genomically closely related and were inferred to arise from a small number of common sources, which provides evidence that hidden persistent reservoirs generated most of the outbreaks rather than independent point-sources. Phylogeographical analysis further revealed the geographical heterogeneity of outbreaks and identified a coastal district as the potential hotspot of outbreaks and as the hub and major source of cross-district spread events. Our findings provide a comprehensive picture of the long-term spatiotemporal dynamics of foodborne outbreaks and present a different perspective on the major source of foodborne infections, which will inform the design of future disease control strategies.

Population dynamics and antimicrobial resistance of Salmonella Derby ST40 from Shenzhen, China.

Salmonella enterica subsp. enterica serovar Derby (S. Derby) is one of the most common serotypes responsible for salmonellosis in humans and animals. The two main sequence types (ST) observed in China are ST40 and ST71, with ST40 presently being the most common in Shenzhen. Recent years have seen an increasing number of cases of salmonella caused by ST40 S. Derby, but the epidemiology is not clear. We gathered 314 ST40 S. Derby isolates from food and patient samples for 11 years in Shenzhen; 76 globally prevalent representative strains were also collected. Whole-genome sequencing (WGS) combined with drug resistance phenotyping was used to examine population structural changes, inter-host associations, drug resistance characteristics, and the food-transmission risks of ST40 S. Derby in Shenzhen over this period. The S. enterica evolutionary tree is divided into five clades, and the strains isolated in Shenzhen were primarily concentrated in Clades 2, 4, and 5, and thus more closely related to strains from Asian (Thailand and Vietnam) than European countries. Our 11-year surveillance of S. Derby in Shenzhen showed that Clades 2, 4, and 5 are now the dominant epidemic branches, and branches 2 and 5 are heavily multi-drug resistant. The main resistance pattern is ampicillin-tetracycline-ciprofloxacin-chloramphenicol-nalidixic acid-streptomycin-sulfamethoxazole/trimethoprim. This may lead to a trend of increasing resistance to ST40 S. Derby in Shenzhen. Using a segmentation of ≤3 SNP among clone clusters, we discovered that Clades 2 and 4 contained multiple clonal clusters of both human- and food-derived strains. The food-derived strains were mainly isolated from pig liver, suggesting this food has a high risk of causing disease outbreaks in Shenzhen.

A Novel Molecular Method for Simultaneous Identification of Vibrio parahaemolyticus 57 K-Serogroups Using Probe Melting Curve Analysis.

The serotyping of Vibrio parahaemolyticus, which is crucial to the surveillance and detection of outbreaks of vibriosis infection, has been widely used in many countries. In this study, we developed a molecular assay, named multiplex ligation reaction based on probe melting curve analysis (MLMA), for simultaneous identification of V. parahaemolyticus 57 K-serogroups. Based on the previous genomes of 418 strains including 39 K-serogroups and the 18 K-serogroups sequences from public databases, we obtained 57 K-serogroups specific gene sequences for designing primers and probes. The developed MLMA assay for identifying the V. parahaemolyticus 57 K-serogroups showed high reproducibility, with the intra- and inter-assay standard deviations and coefficients of variation of no more than 1°C and 1%, respectively. The limit of detection for all gene targets ranged from 0.1 to 1.0 ng/µl. We validated the MLMA assay with a double-blind test identifying 595 V. parahaemolyticus isolates using conventional serotyping methods for comparison. The results showed the kappa value between the MLMA assay and the traditional serological method was 0.936 and that there was a 96.97% consistency rate with conventional serotyping methods for all detected isolates. Additionally, five rare K-serogroups were identified using the MLMA assay, as well as 18 strains that could not be identified using the traditional serotyping method. Thus, the MLMA assay provides a rapid, robust, and promising tool for the molecular serotyping of V. parahaemolyticus K-serogroups and has the potential application to the detection of outbreaks and surveillance of V. parahaemolyticus infection.

Integrated Whole-Genome Sequencing Infrastructure for Outbreak Detection and Source Tracing of Salmonella enterica Serotype Enteritidis.

As an important foodborne pathogen, Salmonella enterica serotype Enteritidis is recognized as one of the most common causes of human salmonellosis globally. Outbreak detection for this highly homogenous serotype, however, has remained challenging. Rapid advances in sequencing technologies have presented whole-genome sequencing (WGS) as a significant advancement for source tracing and molecular typing of foodborne pathogens. A retrospective analysis was conducted using Salmonella Enteritidis isolates (n = 65) from 11 epidemiologically confirmed outbreaks and a collection of contemporaneous sporadic isolates (n = 258) during 2007-2017 to evaluate the performance of WGS in delineating outbreak-associated isolates. Whole-genome single-nucleotide polymorphism (SNP)-based phylogenetic analysis revealed well-supported clades in concordance with epidemiological evidence and pairwise distances of ≤3 SNPs for all outbreaks. WGS-based framework of outbreak detection was thus proposed and applied prospectively to investigate isolates (n = 66) from nine outbreaks during 2018-2019. We further demonstrated the superior discriminatory power and accuracy of WGS to resolve and delineate outbreaks for pragmatic food source tracing. The proposed integrated WGS framework is the first in China for Salmonella Enteritidis and has the potential to serve as a paradigm for outbreak detection and source tracing of Salmonella throughout the stages of food production, as well as expanded to other foodborne pathogens.

[Rapid identification of 15 common S. enterica Salmonella serovars by multiplex real-time PCR].

OBJECTIVE: Multiplex real-time PCR for the identification of 15 Salmonella serovars was developed. METHODS: Through the Salmonella genome comparison, 12 membrane proteins STM4497 gene can be used to identify 15 Salmonella serovars, and these 12 genes were respectively listed as A-L genes. Then primers were designed according to A-L gene conserved sequences, and then multiplex real-time PCR was established assessed with the evaluation of the limit detection, sensitivity, specificity, and repeatability. The 206 Salmonella strains were identified using multiplex real-time PCR with the comparison of the serum slide agglutination assay. RESULTS: The limit detection of multiplex PCR ranged from 1. 1×10~(-3)-1. 2×10~(-3) ng/µL. The target genes were 100% specificity, and the relative standard deviation was lower than 2. 97%. Compared with the serum slide agglutination assay, Kappa ranged 0. 92-1. 00. CONCLUSION: The multiplex real-time PCR can be used to identify 15 Salmonella serovars, which is rapid, accurate and specific.

Synthesis of FePcS-PMA-LDH Cointercalation Composite with Enhanced Visible Light Photo-Fenton Catalytic Activity for BPA Degradation at Circumneutral pH.

(1) Background: Iron tetrasulfophthalocyanine with a large nonlinear optical coefficient, good stability, and high catalytic activity has aroused the attention of researchers in the field of photo-Fenton reaction. Further improvement of the visible light photo-Fenton catalytic activity under circumneutral pH conditions for their practical application is still of great importance. (2) Methods: In this paper, iron tetrasulfophthalocyanine (FePcS) and phosphomolybdic acid (PMA) cointercalated layered double hydroxides (LDH) were synthesized by the ion-exchange method. All samples were fully characterized by various techniques and the results showed that FePcS and PMA were successfully intercalated in layered double hydroxides and the resulted compound exhibited strong absorption in the visible light region. The cointercalation compound was tested as a heterogeneous catalyst for the visible light photo-Fenton degradation of bisphenol A (BPA) at circumneutral pH. (3) Results: The results showed that the degradation and total organic carbon removal efficiencies of bisphenol A were 100% and 69.2%, respectively. (4) Conclusions: The cyclic voltammetry and electrochemical impedance spectroscopy measurements demonstrated that the main contribution of PMA to the enhanced photo-Fenton activity of FePcS-PMA-LDH comes from the acceleration of electron transfer in the reaction system. Additionally, the possible reaction mechanism in the photo-Fenton system catalyzed by FePcS-PMA-LDH was also proposed.

Quantitative detection of gold nanoparticles in soil and sediment.

Ever-growing application of engineering nanoparticles in many sectors of the society requires efficient methods to extract them from soil and sediment, for the sake of environmental protection. In this study, we develop a new method which uses sodium pyrophosphate solution (TSPP, Na4P2O7) as extratant to extract gold nanoparticle (AuNPs) from soil and sediment under optimized parameters through vortexing, water bath oscillation, ultrasonic bath and precipitation. SP-ICP-MS was used for the detection of number concentration, mass concentration and size distribution of AuNPs in soil. UV irradiation was innovatively used to directly degrade soil organic matter to improve the recovery of AuNPs due to their low recovery rate in rich organic soils. It could be found that the mass fraction recovery increased from 36% (without UV digestion) to 83% (with 48h UV digestion). The extraction method is versatile for different coating layers and wide-ranging particle sizes in real soil and sediment. Therefore, the rapid and efficient characterization and quantification of AuNPs in soil and sediment are achieved, and the researches on the extraction method of AuNPs and their behavior and toxicity assessment in soil environment can be enriched.

Whole-Genome Analysis of Salmonella enterica Serovar Enteritidis Isolates in Outbreak Linked to Online Food Delivery, Shenzhen, China, 2018.

In July 2018, an outbreak of 10 cases of Salmonella enterica serovar Enteritidis infection occurred in Shenzhen, China. Outbreak investigation complemented by whole-genome sequencing traced the source to food ordered online. Our investigation highlights the role of online food delivery platforms as a new mode of foodborne disease transmission.

Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovars.

BACKGROUND: While Salmonella serotyping is of paramount importance for the disease intervention of salmonellosis, a fast and easy-to-operate molecular serotyping solution remains elusive. We have developed a multiplex ligation reaction based on probe melting curve analysis (MLMA) for the identification of 30 common Salmonella serovars. METHODS: Serovar-specific primers and probes were designed based on a comparison of gene targets (wzx and wzy encoding for somatic antigen biosynthesis; fliC and fljB for flagellar antigens) from 5868 Salmonella genomes. The ssaR gene, a type III secretion system component, was included for the confirmation of Salmonella. RESULTS: All gene targets were detected and gave expected Tm values during assay evaluation. Cross reactions were not demonstrated between the 30 serovars (n = 211), or with an additional 120 serovars (n = 120) and other Enterobacteriaceae (n = 3). The limit of identification for all targets ranged from using 1.2 ng/µL to 1.56 ng/µL of DNA. The intra- and inter-assay standard deviations and the coefficients of variation were no more than 0.5 °C and less than 1% respectively, indicating high reproducibility. From consecutive outpatient stool samples (n = 3590) collected over a 10-month period at 11 sentinel hospitals in Shenzhen, China, we conducted a multicenter study using the traditional Salmonella identification workflow and the MLMA assay workflow in parallel. From Salmonella isolates (n = 496, 13.8%) derived by both workflows, total agreement (kappa = 1.0) between the MLMA assay and conventional serotyping was demonstrated. CONCLUSIONS: With an assay time of 2.5 h, this simple assay has shown promising potential to provide rapid and high-throughput identification of Salmonella serovars for clinical and public health laboratories to facilitate timely surveillance of salmonellosis.

Simultaneous Identification of Clinically Common Vibrio parahaemolyticus Serotypes Using Probe Melting Curve Analysis.

The dynamic nature of Vibrio parahaemolyticus epidemiology has presented a unique challenge for disease intervention strategies. Despite the continued rise of disease incidence and outbreaks of vibriosis, as well as the global emergence of pandemic clones and serovariants with enhanced virulence, there is a paucity of molecular methods for the serotyping of V. parahaemolyticus strains to improve disease surveillance and outbreak investigations. We describe the development of a multiplex ligation reaction based on probe melting curve analysis (MLMA) for the simultaneous identification of 11 clinically most common V. parahaemolyticus serotypes spanning a 10-year period. Through extensive sequence analyses using 418 genomes, specific primers and probes were designed for a total of 22 antigen gene targets for the O- and K- serogroups. Additionally, the toxR gene was incorporated into the assay for the confirmation of V. parahaemolyticus. All gene targets were detected by the assay and gave expected Tm values, without any cross reactions between the 11 clinically common serotypes or with 38 other serotypes. The limit of identification for all gene targets ranged from 0.1 to 1 ng/µL. The intra- and inter-assay standard deviations and the coefficients of variation were no more than 1°C and <1% respectively, indicating a highly reproducible assay. A multicenter double-blind clinical study was conducted using the traditional V. parahaemolyticus identification workflow and the MLMA assay workflow in parallel. From consecutive diarrheal stool specimens (n = 6118) collected over a year at 10 sentinel hospitals, a total of 153 V. parahaemolyticus isolates (2.5%) were identified by both workflows. A total agreement (kappa = 1.0) between the serotypes identified by the MLMA assay and conventional serological method was demonstrated. This is the first molecular assay to simultaneously identify multiple clinically important V. parahaemolyticus serotypes, which satisfies the acute need for a practical, rapid and robust identification of V. parahaemolyticus serotypes to facilitate the timely detection of vibriosis outbreaks and surveillance.

Variation in rhizosphere microbiota correlates with edaphic factor in an abandoned antimony tailing dump.

The distribution pattern of root-associated bacteria in native plant growth in tailing dumps with extreme conditions remains poorly understood and largely unexplored. Herein we chose a native plant, Bidens bipinnata, growing on both an Sb tailing dump (WKA) and adjacent normal soils (WKC) to in-depth understand the distribution pattern of root-associated bacteria and their responses on environmental factors. We found that the rhizosphere microbial diversity indices in the tailing dump were significantly different from that in the adjacent soil, and that such variation was significantly related with soil nutrients (TC, TOC, TN) and metal(loid) concentrations (Sb and As). Some dominant genera were significant enriched in WKA, suggesting their adaption to harsh environments. Notably, these genera are proposed to be involved in nutrient and metal(liod) cycling, such as nitrogen fixing (Devosia, Cellvibrio, Lysobacter, and Cohnella), P solubilizing (Flavobacterium), and Sb and As oxidation (Paenibacillus, Bacillus, Pseudomonas, and Thiobacillus). Our results suggest that certain root-associated bacteria in tailing dump were governed by soil edaphic factors and play important ecological roles in nutrient amendments and metal cycling for the successful colonization of Bidens bipinnata in this tailing dump.

Characterization of a Novel Diarrheagenic Strain of Proteus mirabilis Associated With Food Poisoning in China.

Proteus mirabilis is commonly considered to be an opportunistic pathogen causing urinary tract infections (UTIs) in humans. However, some strains of P. mirabilis were found to be associated with food poisoning outbreaks, with the pathogenic mechanism still unclear. In our study, we described a novel strain of P. mirabilis C02011 isolated from patients' specimens in a food poisoning in China. In order to determine its gastrointestinal pathogenicity, experiments were performed to compare P. mirabilis B02005 strain (isolated from healthy people) and P. mirabilis American Type Culture Collection (ATCC) 29906 strain both in vitro [Caco-2 cells: bacterial adhesion and invasion assays, Giemsa staining, and transmission electron microscopy (TEM)] and in vivo [BALB/c mouse model: fecal character, colon injury, histological examination, immunochemistry, and western blotting (WB)]. According to the results, C02011 strain exhibited almost identical characteristics with B02005 strain in bacterial appearance and proliferation. In vitro, Caco-2 cells were infected with P. mirabilis C02011, B02005, and P. mirabilis ATCC 29906 strains. After that, Giemsa staining and TEM were used for observing the infection process of C02011 strain. Meanwhile, the adhesive abilities of different strains were rated as follows: P. mirabilis B02005 > P. mirabilis C02011 > P. mirabilis ATCC 29906 (P < 0.01). Invasive abilities of different strains were rated as follows: P. mirabilis C02011 > P. mirabilis B02005 > P. mirabilis ATCC 29906 (P < 0.01). In vivo, BALB/c mice were infected with P. mirabilis C02011 and B02005 strains. C02011 strain shows more virulence than B02005 strain in terms of the following indicators: (1) feces water content and fecal character; (2) colon length of mice; (3) histological examination on mouse intestine tissues; (4) ELISA for detecting TNF-α level in the colon; and (5) WB and immunohistochemistry (IHC) for detecting occludin protein expression in the colon. On the basis of these results, we firstly validated that the novel strain of P. mirabilis C02011 shows more gastrointestinal pathogenicity than the other strains isolated from a healthy individual. In addition, type IV secretion system (T4SS) was preliminarily confirmed to play an important role in the pathogenesis of diarrheal P. mirabilis isolated from the food poisoning incident.

Clonal Expansion of Biofilm-Forming Salmonella Typhimurium ST34 with Multidrug-Resistance Phenotype in the Southern Coastal Region of China.

To disclose the antibiotics susceptibility and wide adaptability of commonly occurring genotypes of Salmonella Typhimurium, the antibiotic resistance and biofilm formation of different multi-locus sequence typing (MLST) types of a collection of 240 S. Typhimurium isolates (33 food and 207 clinical ones) during 2010-2014 in Shenzhen were analyzed. Among these strains, 167 was ST34 (69.58%), and 57 was ST19 (23.75%), respectively. A total of 159 (95.21%) ST34 strains displayed the multidrug resistant phenotype (≥ three classes of antibiotic), whereas only 23 (40.35%) ST19 ones did (P < 0.01). Moreover, a relative high proportion (72.46%) of ST34 isolates was classified as moderate to strong biofilm-producers, while only 15.79% of ST19 (P < 0.01) was. Among the food isolates, more than half (51.52%) were from livestock products, among which 41.18% classified as moderate to strong biofilm-producers. In summary, this study highlights the expansion of S. Typhimurium ST34 of strong biofilm-forming ability and multidrug resistance in the southern coastal region of China. Therefore, monitoring the occurrence of ST34 S. Typhimurium in food sources, especially in livestock products, and taking appropriate measures to control Salmonella spp. infections via decreasing biofilm formation should be addressed.

Simultaneous Identification of Ten Bacterial Pathogens Using the Multiplex Ligation Reaction Based on the Probe Melting Curve Analysis.

Pathogenic Vibrio spp., Aeromonas spp. and Plesiomonas shigelloides are associated with human gastroenteritis and wound infections, as well as fish diseases. The comprehensive and accurate identification of these pathogens is crucial for the current public health. The present study describes the development of a multiplex assay for the simultaneous identification of ten bacterial pathogens in a single reaction by using a multiplex ligation reaction based on probe melting curve analysis (MLMA). The specificity for target genes was 100%, as assessed with a panel of 67 bacterial pathogens, which indicated no cross-reactions. The detection limit of this assay ranged from 0.8 × 107 CFU/mL to 1.5 × 108 CFU/mL at the pure bacterial culture level and from 0.1 ng to 1.0 ng at the DNA level. The MLMA assay was used to detect ten species of pathogens in 269 clinical and seafood samples, and for further validation, the results were compared with the conventional culture method. The results indicated greater than 90% sensitivity and 100% specificity for each bacterial pathogen tested, and the kappa correlation for all the pathogens ranged from 0.95 to 1.00. Overall, this assay is well suited for public health laboratories for its high throughput, accuracy, and low cost.

Phenotypic and Genotypic Characterization of Clinical Enterotoxigenic Escherichia coli Isolates from Shenzhen, China.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of infectious diarrhea in developing countries. This study aimed to characterize the prevalence and phenotypic and genotypic features of ETEC isolates from Shenzhen, China. METHODS: ETEC isolates were obtained from acute diarrheal patients and evaluated for enterotoxin, classical colonization factors (CFs), serotypes, antimicrobial susceptibility, and multilocus sequencing typing (MLST). RESULTS: A total of 168 (1.3%) ETEC strains were isolated from 13,324 diarrheal outpatients during 2009 and 2014. A vast majority of ETEC-infected patients (82.1%) belonged to the age ranging 20-59 years and only six patients were children aged <5 years. Heat-stable toxin (ST) was most frequently detected (81.5%), followed by heat-labile toxin (LT) (13.1%). One or multiple colonization factors (CFs) were identified in 91 ETEC strains (54.2%). The most frequently detected CF was CS6 (with or without other CFs) (84/91), followed by CS21 (14/91). The most common serotype was O159:H34 (n = 36), followed by O148:H28 (n = 25) and O27:H7 (n = 17). High resistant rate was observed to nalidixic acid (77.4%), cephalothin (41.7%), ampicillin (34.5%), and tetracycline (21.4%). Antimicrobial resistance profiles differed among different serogroups. Sequence type (ST) 10 complex, integrated with connected ST218, ST48, ST4, and ST1312 subgroups, covered 73 (43.5%) isolates. CONCLUSIONS: ETEC isolates in Shenzhen of China appeared highly diverse, yet some isolates belonged to well-defined clonal groups sharing a unique set of virulence factors, serotypes, and MLST sequence types. Facing the challenge of ETEC antigenic diversity and geographic variation, novel molecules and/or classical antigens designed by novel strategies might contribute to ETEC vaccine development.

Feasibility of Using Multiple-Locus Variable-Number Tandem-Repeat Analysis for Epidemiology Study of Vibrio parahaemolyticus Infections.

Vibrio parahaemolyticus causes foodborne gastroenteritis, which is often associated with the consumption of raw or undercooked shellfish. Molecular typing can provide critical information for detecting outbreaks and for source attribution. In this study, we describe the development and evaluation of an optimized multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) for the characterization of V. parahaemolyticus isolates. The discriminatory power of MLVA was compared to that of pulsed-field gel electrophoresis (PFGE) by typing 73 sporadic isolates. Epidemiologic concordance was evaluated by typing 23 isolates from five epidemiologically well-characterized outbreaks. The optimized MLVA was applied in early warning, epidemiological surveillance, and source tracking for V. parahaemolyticus infections. There was no significant difference in the discriminatory power of PFGE and MLVA with six or eight VNTR loci for the sporadic isolates. All isolates within an outbreak were indistinguishable by MLVA with six loci, except for one outbreak. Typically, the epidemiological survey could be initiated according to PFGE clusters. We applied MLVA with six loci on 22 isolates in two PFGE clusters. Isolates in one PFGE cluster were distinguished by MLVA. Although a follow-up investigation showed that both clusters had no epidemiological concordance, MLVA decreased the frequency of initiation of epidemiological surveys, thereby reducing labor costs. The ability of MLVA to trace the source of infection was evaluated by isolates from two outbreaks and shrimp samples. The isolates from one of outbreaks and a shrimp had the same MLVA type, suggesting that an epidemiological survey was initiated. Data from the epidemiological investigation subsequently indicated that contaminated shrimp from a nearby city (Dongguan) might be the source of the outbreak. In conclusion, these results indicate that the optimized MLVA may be a promising tool for early warning and epidemiological surveillance of V. parahaemolyticus infections.