Prevalence and Antimicrobial Resistance Diversity of Salmonella Isolates in Jiaxing City, China.

Nontyphoidal Salmonella (NTS) is a cause of foodborne diarrheal diseases worldwide. Important emerging NTS serotypes that have spread as multidrug-resistant high-risk clones include S. Typhimurium monophasic variant and S. Kentucky. In this study, we isolated Salmonella in 5019 stool samples collected from patients with clinical diarrhea and 484 food samples. Antibiotic susceptibility testing and whole-genome sequencing were performed on positive strains. The detection rates of Salmonella among patients with diarrhea and food samples were 4.0% (200/5019) and 3.1% (15/484), respectively. These 215 Salmonella isolates comprised five main serotypes, namely S. Typhimurium monophasic variant, S. Typhimurium, S. London, S. Enteritidis, and S. Rissen, and were mainly resistant to ampicillin, tetracycline, chloramphenicol, and trimethoprim/sulfamethoxazole. The MDR rates of five major serotypes were 77.4%, 56.0%, 66.7%, 53.3%, and 80.0%, respectively. The most commonly acquired extended-spectrum ß-lactamase-encoding genes were blaTEM-1B, blaOXA-10, and blaCTX-M-65. The S. Typhimurium monophasic variant strains from Jiaxing City belonged to a unique clone with broad antibiotic resistance. S. Kentucky isolates showed the highest drug resistance, and all were MDR strains. The discovery of high antibiotic resistance rates in this common foodborne pathogen is a growing concern; therefore, ongoing surveillance is crucial to effectively monitor this pathogen.

3D reconstruction of light-field images based on spatiotemporal correlation super-resolution.

In this paper, we make full advantage of the information correlation of subaperture images and propose a new super-resolution (SR) reconstruction method based on spatiotemporal correlation to achieve SR reconstruction for light-field images. Meanwhile, the offset compensation method based on optical flow and spatial transformer network is designed to realize accurate compensation between adjacent light-field subaperture images. After that, the obtained light-field images with high resolution are combined with the self-designed system based on phase similarity and SR reconstruction to realize accurate 3D reconstruction of a structured light field. Finally, experimental results demonstrate the validity of the proposed method to perform accurate 3D reconstruction of light-field images from the SR data. Generally, our method makes full use of the redundant information between different subaperture images, hides the upsampling process in the convolution, provides more sufficient information, and reduces time-consuming procedures, which is more efficient to realize the accurate 3D reconstruction of light-field images.

Development and Application of a Multiplex Fluorescent PCR for Shigella Detection and Species Identification.

This study was to develop a multiplex fluorescent PCR for Shigella detection and species identification. Five primer pairs for Shigella detection and species identification were designed by Primer Premier 5.0. The multiplex fluorescent PCR was optimized by varying single parameter while other parameters were maintained. The multiplex fluorescent PCR assay could correctly detect Shigella and identify four Shigella species with a detection limits of 10 pg genomic DNA per reaction. Testing different strains and clinical samples confirmed the sensitivity and specificity of the multiplex fluorescent PCR. The newly developed multiplex fluorescent PCR assay is simple, sensitive and specific for Shigella detection and species identification. It has a potential to be used in routine Shigella detection and species identification in clinical laboratories.

First Identification and Limited Dissemination of mcr-1 Colistin Resistance in Salmonella Isolates from Jiaxing.

ABSTRACT: Salmonella, a major foodborne pathogen, causes severe gastrointestinal disease in people and animals worldwide. Plasmid-borne mcr-1, which confers colistin resistance in Salmonella, has significant epidemiological interest for public health safety. Here, we report the first evidence of mcr-1-mediated colistin resistance in one multidrug-resistant strain, 16062, from 355 Salmonella isolates collected for Jiaxing foodborne pathogen monitoring in Zhejiang Province from 2015 to 2019. In addition to colistin, 16062 displayed multidrug resistance to various antimicrobials (ß-lactams, quinolone, sulfonamide, florfenicol, ampicillin, streptomycin, nalidixic acid, aminoglycoside, and trimethoprim-sulfamethoxazole). The mcr-1-carrying IncX4 plasmid (p16062-MCR) in this study shares a conserved structure with other mcr-IncX4 plasmids. We found that other antimicrobial-resistance genes (aac(6')-Ib-cr, aadA1, aadA2, aph(3')-Ia, oqxA, oqxB, sul1, and cmlA1) are located on p16062-cmlA, an atypical IncHI2 plasmid, in isolate 16062. This is the first identification of transferable colistin resistance in a foodborne Salmonella isolate collected in Jiaxing City, the 5-year monitoring of which revealed limited dissemination. By determining the genetic features of the plasmid vehicle, the characteristics of transferable mcr genes circulating in isolates from Jiaxing are now clearer.

3D reconstruction of structured light fields based on point cloud adaptive repair for highly reflective surfaces.

In this paper, a novel method, to the best of our knowledge, of structured light fields based on point cloud adaptive repair is proposed to realize 3D reconstruction for highly reflective surfaces. We have designed and built a focused light field camera whose spatial and angular resolution can be flexibly adjusted as required. Then the subaperture image extraction algorithm based on image mosaic is deduced and presented to obtain multidirectional images. After that, the 3D reconstruction of structured light field imaging based on point cloud adaptive repair is presented to accurately reconstruct for highly reflective surfaces. In addition, a method based on smoothness and repair rate is also proposed to objectively evaluate the performance of the 3D reconstruction. Experimental results demonstrate the validity of the proposed method to perform high-quality depth reconstruction for highly reflective surfaces. Generally, our method takes advantage of the multidirectional imaging of the light field camera and can ensure good modulation effect of structured light while avoiding hardware complexity, which makes it application more convenient.

Population Structure and Multidrug Resistance of Non-O1/Non-O139 Vibrio cholerae in Freshwater Rivers in Zhejiang, China.

To understand the environmental reservoirs of Vibrio cholerae and their public health significance, we surveyed freshwater samples from rivers in two cities (Jiaxing [JX] and Jiande [JD]) in Zhejiang, China. A total of 26 sampling locations were selected, and river water was sampled 456 times from 2015 to 2016 yielding 200 V. cholerae isolates, all of which were non-O1/non-O139. The average isolation rate was 47.3% and 39.1% in JX and JD, respectively. Antibiotic resistance profiles of the V. cholerae isolates were examined with nonsusceptibility to cefazolin (68.70%, 79/115) being most common, followed by ampicillin (47.83%, 55/115) and imipenem (27.83%, 32/115). Forty-two isolates (36.52%, 42/115) were defined as multidrug resistant (MDR). The presence of virulence genes was also determined, and the majority of the isolates were positive for toxR (198/200, 99%) and hlyA (196/200, 98%) with few other virulence genes observed. The population structure of the V. cholerae non-O1/non-O139 sampled was examined using multilocus sequence typing (MLST) with 200 isolates assigned to 128 STs and 6 subpopulations. The non-O1/non-O139 V. cholerae population in JX was more varied than in JD. By clonal complexes (CCs), 31 CCs that contained isolates from this study were shared with other parts of China and/or other countries, suggesting widespread presence of some non-O1/non-O139 clones. Drug resistance profiles differed between subpopulations. The findings suggest that non-O1/non-O139 V. cholerae in the freshwater environment is a potential source of human infections. Routine surveillance of non-O1/non-O139 V. cholerae in freshwater rivers will be of importance to public health.

Quantification and confirmation of four aflatoxins using a LC-MS/MS QTRAP system in multiple reaction monitoring, enhanced product ion scan, and MS3 modes.

A simple, rapid, and efficient liquid chromatography tandem mass spectrometry (LC-MS/MS) method, operated in electrospray ionization and quadrupole linear ion trap modes, has been developed for the identification and structural characterization of aflatoxins in peanuts and its derivative products or bean sauce. Samples (5 g) were extracted with acetonitrile/water/formic acid (79:20:1, v/v). After centrifugation and dilution, the extracts were separated on a C18 analytical column by gradient elution (acetonitrile/0.2% formic acid) and analyzed by UPLC-MS/MS. External calibration was used for qualification. The developed multiple reaction monitoring-information-dependent acquisition-enhanced product ion method enabled quantification and confirmation of the analytes in a single run. Enhanced product ion mode was used for qualitative analysis, while multiple reaction monitoring mode was used for quantitative analysis. An in-house library was constructed for identification. Calibration curves showed good linearity with correlation coefficients (r) higher than 0.994. Limits of detection were determined to be below 0.26 µg kg-1 for most analytes. The recoveries for those substances were in the acceptable range of 80.2%-119.1%. A new LC-MS3 method was established for further confirmation. One pickled pepper peanut was found to contain aflatoxins B1, B2, and G1 with contents of 90.93, 26.64, and 1.92 µg kg-1, respectively.

MALDI-TOF mass spectrometry-based serotyping of V. parahaemolyticus isolated from the Zhejiang province of China.

BACKGROUND: Vibrio parahaemolyticus is as an important food-borne pathogen circulating in China. Since 1996, the core serotype has become O3:K6, which has specific genetic markers. This serotype causes the majority of outbreaks worldwide. Until now, nearly 21 serotypes were considered as serovariants of O3:K6. Among these, O4:K68, O1:K25 and O1:KUT have caused pandemic outbreaks. O4:K8, a serovariant of O3:K6, has become the second most dominant serotype circulating in China after O3:K6. In this study, we report the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze and characterize 146 V. parahaemolyticus isolates belonging to 23 serotypes. RESULTS: Upon mass spectral analysis, isolates belonging to O4:K8 formed a distinct group among the five main pandemic groups (O3:K6, O4:K8, O4:K68, O1:K25 and O1:KUT). Two major protein peaks (m/z 4383 and 4397) were significantly different between serotype O4:K8 and the four other pandemic strains. Both of these peaks were present in 32 out of 36 O4:K8 isolates, but were absent in 105 out of 110 non-O4:K8 isolates. These peaks were also absent in all 74 pandemic serotypes (O3:K6, O4:K68, O1:K25 and O1:KUT). CONCLUSION: Our results highlight the threat of O4:K8 forming a distinct group, which differs significantly from pandemic serotypes on the proteomic level. The use of MALDI-TOF MS has not been reported before in a study of this nature. Mass spectrum peaks at m/z 4383 and 4397 may be specific for O4:K8. However, we cannot conclude that MALDI-TOF MS can be used to serotype V. parahaemolyticus.

Genetic and population analyses of Vibrio parahaemolyticus isolates from three major coastal regions in China.

AIM: This study aims to evaluate the genetic and population structure of Vibrio parahaemolyticus in the major coastal regions of China. MATERIALS & METHODS: Multilocus sequence typing was performed. RESULTS: Insertion of large sequence into recA happened in nearly 30 strains, which were untypeable by multilocus sequence typing. A collection of 307 V. parahaemolyticus isolates were typed into 160 sequence types, including 117 novel ones. eBURST analysis revealed five clonal complexes, 11 doublets, and 108 singletons. The 160 sequence types formed two main lineages in the phylogenetic analysis. CONCLUSION: V. parahaemolyticus along the Chinese coastal regions exhibits high levels of genetic diversity and has undergone significant purifying selection and frequent recombination. A deeper understanding of V. parahaemolyticus genetic diversity could be obtained at the level of genome sequences.

A Real-Time PCR with Melting Curve Analysis for Molecular Typing of Vibrio parahaemolyticus.

Foodborne disease caused by Vibrio parahaemolyticus is a serious public health problem in many countries. Molecular typing has a great scientific significance and application value for epidemiological research of V. parahaemolyticus. In this study, a real-time PCR with melting curve analysis was established for molecular typing of V. parahaemolyticus. Eighteen large variably presented gene clusters (LVPCs) of V. parahaemolyticus which have different distributions in the genome of different strains were selected as targets. Primer pairs of 18 LVPCs were distributed into three tubes. To validate this newly developed assay, we tested 53 Vibrio parahaemolyticus strains, which were classified in 13 different types. Furthermore, cluster analysis using NTSYS PC 2.02 software could divide 53 V. parahaemolyticus strains into six clusters at a relative similarity coefficient of 0.85. This method is fast, simple, and conveniently for molecular typing of V. parahaemolyticus.

A multiplex liquid-chip assay based on Luminex xMAP technology for simultaneous detection of six common respiratory viruses.

We utilized one-step multiplex reverse transcription-PCR (RT-PCR) and Luminex xMAP technology to develop a respiratory multiplex liquid-chip assay (rMLA) for simultaneous detection of 6 common respiratory viruses, including influenza virus type A (FluA) and type B (FluB), para-influenza virus type 3 (PIV-3), respiratory syncytial virus (RSV), human metapneumovirus (MPV) and a threatening virus to China, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Performance of rMLA was evaluated by comparing with real-time RT-PCR. Detection data from clinical specimens showed that the rMLA had diagnostic sensitivities of 97.10% for FluA, 94.59% for FluB, 98.68% for PIV-3, 94.87% for RSV and 95.92% for MPV (No Data for MERS-CoV due to the lack of positive specimens). Data of analytical sensitivities showed that the detection limits of the rMLA assay were 5-25 viral RNA copies per µl for FluA, FluB, PIV-3 and MERS-CoV, approximate to the real-time RT-PCR assay; while the values were 8 and 22copies/µl for MPV and RSV, lower than the real-time RT-PCR(78 and 114 copies/µl respectively). The results indicated that the rMLA is a sensitive, specific detection tool and comparable to real-time RT-PCR, especially suitable for high-throughput detection of respiratory specimens.

Development and Application of A Duplex Real-Time RT-PCR Assay for Simultaneous Detection of Coxsackievirus A6 and Coxsackievirus A10.

Hand, foot and mouth disease (HFMD) is a serious public health problem. Generally, it is considered that HFMD is mainly caused by enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). Nevertheless, the incidence of HFMD caused by coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) has increased significantly and CVA6 and CVA10 have become major causes of HFMD epidemic. This study develops a duplex real-time reverse transcript polymerase chain reaction (RT-PCR) assay for simultaneous detection of CVA 6 and CVA 10. The specificity, sensitivity, and reproducibility of this assay were analyzed. No cross-reactions with other viruses or false positives were observed. The detection limit of this assay was as low as 11.935 copies for CVA6 and 17.591 copies for CVA10 per reaction (concentration giving a positive duplex real-time RT-PCR result in 95% of samples). The coefficients of variation of the intra- and inter-assay reproducibility for CVA 6 and CVA 10 were both lower than 2%. Our results showed that this duplex real-time RT-PCR assay was a simple, rapid, and cost-effective method for simultaneous identification of CVA6 and CVA10.

Genetic characterization and antiretroviral resistance mutations among treatment-naive HIV-infected individuals in Jiaxing, China.

The aim of this study was to characterize HIV-1 genotypes and antiretroviral resistance mutations among treatment-naive HIV-infected individuals in Jiaxing, China. The HIV-1 partial polymerase (pol) genes in 93 of the 99 plasma samples were successfully amplified and analyzed. Phylogenetic analysis revealed the existence of five HIV-1 genotypes, of which the most prevalent genotype was CRF01_AE (38.7%), followed by CRF07_BC (34.4%), CRF08_BC (16.1%), subtype B/B' (5.4%), and CRF55_01B (2.1%). Besides, three types of unique recombination forms (URFs) were also observed, including C/F2/A1, CRF01_AE/B, and CRF08_BC/CRF07_BC. Among 93 amplicons, 46.2% had drug resistance-associated mutations, including 23.7% for protease inhibitors (PIs) mutations, 1.1% for nucleoside reverse transcriptase inhibitors (NRTIs) mutations, and 20.4% for non-nucleoside reverse transcriptase inhibitors (NNRTIs) mutations. Six (6.5%) out of 93 treatment-naive subjects were identified to be resistant to one or more NNRTIs, while resistance to NRTIs or PIs was not observed. Our study showed the genetic diversity of HIV-1 strains circulating in Jiaxing and a relative high proportion of antiretroviral resistance mutations among treatment-naive patients, indicating a serious challenge for HIV prevention and treatment program.

Dynamic quantification of avian influenza H7N9(A) virus in a human infection during clinical treatment using droplet digital PCR.

This study involved a human infection with avian influenza H7N9(A) virus in Zhejiang province, the first one after implementing the closure measures of living poultry markets in China. The clinical symptoms, epidemiological and virological characteristics of the case were described briefly, and as the emphasis, H7N9 virus was detected quantitatively and continuously from the collected samples in 10 different periods of the patient's treatment in order to reveal changes of viral load in patient's body during the treatment. This study first used reverse-transcription droplet digital PCR (RT-ddPCR) assays to monitor viral load dynamically for human H7N9 infection, synchronously performing real-time RT-PCR as a reference technology to obtain more comprehensive data for comparison. Our results indicated that RT-ddPCR compared to real-time RT-PCR is more sensitive and accurate for quantifying H7N9 viral load without the use of standard curves. Furthermore it can provide reference data for clinical policies including infectivity judgement, ward transferring and therapy adjustment for the patient during treatment.

Living poultry markets in rural area: Human infection with H7N9 virus re-emerges in Zhejiang Province, China, in winter 2014.

BACKGROUND: Avian influenza A H7N9 virus, previously undetected in humans, has caused infections in many areas in China since February 2013. Here we report the re-emergence of a case of H7N9 in rural Jiaxing city, Zhejiang Province, in the winter of 2014. OBJECTIVES: To understand (1) the clinical syndrome, epidemiological and virological characteristics of this case; (2) the importance of controlling live poultry markets (LPMs) in rural areas. STUDY DESIGN: There is one patient and 16 contacts, including 4 family members living in the same household, and 12 medical personnel. Pharyngeal swabs and serum samples were collected from the patient and her contacts. Environment samples were also obtained from the local LPMs. We conducted detailed clinical and epidemiological investigations and laboratory work, including viral RNA extraction, RT-PCR detection and sequencing. Characteristic and phylogenetic analyses were performed using the obtained sequences. RESULTS: H7N9s were detected in environmental samples collected in LPMs in Jiaxing, Zhejiang. Unknown mutations were discovered in amino acids in the sample from the patient. The strain from the patient was in a clade different from isolates obtained in 2013 in phylogenetic trees of HA, NA and PB2. CONCLUSIONS: A severe case of H7N9 was identified in early winter, 2014. Epidemiological and clinical tests were consistent with patterns reported previously, while laboratory findings showed the virus to be different. Live poultry markets in rural Zhejiang Province are in need of closer supervision and enhanced management.

Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains.

Foodborne disease caused by pathogenic Vibrio parahaemolyticus has become a serious public health problem in many countries. Rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. In this study, an EvaGreen-based multiplex real-time PCR assay was established for the detection of pathogenic V. parahaemolyticus. This assay targeted three genetic markers of V. parahaemolyticus (species-specific gene toxR and virulence genes tdh and trh). The assay could unambiguously identify pathogenic V. parahaemolyticus with a minimum detection limit of 1.4 pg genomic DNA per reaction (concentration giving a positive multiplex real-time PCR result in 95% of samples). The specificity of the assay was evaluated using 72 strains of V. parahaemolyticus and other bacteria. A validation of the assay with clinical samples confirmed its sensitivity and specificity. Our data suggest the newly established multiplex real-time PCR assay is practical, cost-effective, specific, sensitive and capable of high-throughput detection of pathogenic V. parahaemolyticus.

A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV.

A one-step multiplex real-time reverse transcription-PCR (RT-PCR) assay was developed for one-tube and simultaneous detection of three genogroups of human norovirus, genogroup I, II and IV (GI, GII and GIV). The specificity and sensitivity of the assay were evaluated and 50 samples were tested by using this assay. The results showed that the multiplex assay had high sensitivity and specificity. The amplification efficiencies of the assay were 91.3%, 90.1%, 88.9% and the detection limits were up to 16.9, 6.3, 43.0 copies/reaction respectively for norovirus GI, GII and GIV detection. No cross-reaction with the other examined RNA viruses was observed, and the qualitative analysis of samples showed that the multiplex assay had a good consistency with its corresponding monoplex assays for the detection of norovirus GI, GII and GIV (Kappa values were 0.848, 0.876 and 0.812 respectively).

[Protective effects of uric acid on nigrostriatal system injury induced by 6-hydroxydopamine in rats].

OBJECTIVE: To investigate the protective effects of uric acid on nigrostriatal system injury induced by 6-hydroxydopamine in rats. METHODS: Thirty male SD rats were divided into four groups. Uric acid of 100 mg/kg, 200 mg/kg, 250 mg/kg were injected intraperitoneally (ip) into 5, 10, 5 rats twice daily at a 2-hour interval for five days and saline was injected ip into 10 rats as controls. At Day 6, 6-hydroxydopamine was injected into striatum to establish Parkinson's disease (PD) model in rats. Then uric acid was injected ip into three groups and saline into controls for five days. Locomotion test, amphetamine-induced rotation and forepaw adjusting step test were performed at Weeks 3 and 4 respectively after injection of 6-hydroxydopamine. HPLC-MS/MS was performed to detect the contents of dopamine and its metabolite homovanillic acid (HVA) in striatum at Week 5. RESULTS: The scores of locomotion in 2 minutes of 200 mg/kg uric acid group (14 +/- 4/2 min) was higher significantly than saline group (4 +/- 5/2 min, P < 0.01). The amphetamine-induced rotation number in the 200 mg/kg uric acid group (10.8 +/- 7.5) was lower significantly that in the saline group (19.3 +/- 5.2, P < 0.01). Forepaw adjusting step test scores of 200 mg/kg uric acid group were higher significantly than those in the saline group (9.89 +/- 3.41 vs 4.36 +/- 3.72, P < 0.01). HPLC-MS/MS showed that the contents of DA (0.29 +/- 0.19) and HVA (1.22 +/- 0.5) in injured striatum of 200 mg/kg uric acid group were higher significantly than those in the saline group (0.05 +/- 0.03, P < 0.01; 0.24 +/- 0.13, P < 0.05). CONCLUSION: An appropriately elevated level of uric acid may protect the dopamine neuron of nigrostriatal system from injury of 6-hydroxydopamine in rats.