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1.
Anal Bioanal Chem ; 412(1): 93-101, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31797016

RESUMO

The aim of this study was to develop an effective and specific visual method to rapidly detect and identify Vibrio parahaemolyticus (V. parahaemolyticus) based on the polymerase spiral reaction (PSR). The method utilized only two pairs of primers designed specifically to target the conserved tlh gene sequence of V. parahaemolyticus. Nucleic acid amplification can be achieved under isothermal conditions using DNA polymerase. The reaction could be accomplished in < 40 min with high specificity and sensitivity. The limits of detection of V. parahaemolyticus in purified genomic DNA and pure culture were 300 fg/µL and 2.4 CFU/mL per reaction, respectively, which were 100-fold more sensitive than with conventional PCR. The model food samples showed consistent specificity and sensitivity to the pure bacterial culture. With these encouraging results, it is expected that the novel, effortless and reliable isothermal nucleic acid testing assay developed in this study has potential to be applied to screening for V. parahaemolyticus in seafood samples.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Vibrio parahaemolyticus/isolamento & purificação , Primers do DNA , DNA Bacteriano/análise , Genes Bacterianos , Limite de Detecção , Vibrio parahaemolyticus/genética
2.
Zhonghua Liu Xing Bing Xue Za Zhi ; 40(8): 889-894, 2019 Aug 10.
Artigo em Chinês | MEDLINE | ID: mdl-31484249

RESUMO

Objective: To understand the seasonality and etiological characteristics of infectious diarrhea in adults from Shanghai. Methods: Adult patients with diarrhea who had visited the enteric disease clinics in 22 hospitals that carrying on the Diarrhea Comprehensive Surveillance sentinel programs in Shanghai during 2014-2017, were surveyed. Stool specimens were collected according to the different intervals of sampling and detected for 12 bacteria and 5 viruses. Concentration ratio and circular distribution method were used for data analysis. Results: From 2014 to 2017, a total of 9 573 stool specimens were collected from the targeted diarrhea patients ≥18 years old (n=96 067), through the Shanghai Diarrhea Comprehensive Surveillance program. The positive rate of detection was 46.44%. Seasonal peaks of infectious diarrhea were both seen in summer (bacteria peak, diarrheagenic Escherichia coli and Vibrio parahaemolyticus, etc.) and in winter (virus peak, Norovirus, etc.). Both bacterial and viral infections presented seasonal concentration (Raleigh's test P<0.001) but more obvious with bacterial infection. Viral infection accounted for 60.19% of the cause of infectious diarrhea. The top five predominant pathogens appeared as Norovirus, Rotavirus, diarrheagenic Escherichia coli, Vibrio parahaemolyticus, and Salmonella spp.. Conclusions: Among the adult outpatients with infectious diarrhea in Shanghai, obvious seasonality was seen, with peaks in both summer and winter. Viral infection with Norovirus in particular, appeared as the predominant source of infection. Active, continuous and comprehensive diarrhea-related surveillance programs would be able to monitor the changing dynamic of pathogen spectrum, and lead to the adoption of targeted preventive measures.


Assuntos
Bactérias/isolamento & purificação , Diarreia/diagnóstico , Diarreia/etiologia , Disenteria/diagnóstico , Disenteria/etiologia , Fezes , Pacientes Ambulatoriais/estatística & dados numéricos , Vigilância da População/métodos , Vírus/isolamento & purificação , Adolescente , Adulto , Bactérias/classificação , Criança , Pré-Escolar , China/epidemiologia , Diarreia/epidemiologia , Disenteria/epidemiologia , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Fezes/virologia , Humanos , Pessoa de Meia-Idade , Norovirus/isolamento & purificação , Rotavirus/isolamento & purificação , Salmonella/classificação , Salmonella/isolamento & purificação , Estações do Ano , Vibrio parahaemolyticus/isolamento & purificação , Vírus/classificação
3.
Int J Food Microbiol ; 308: 108290, 2019 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-31442712

RESUMO

The plasmid-mediated colistin resistance gene mcr-1 has been identified in various Enterobacteriaceae species, which poses a great challenge to the public health. The present study aimed to investigate the prevalence of mcr-1 in Vibrio parahaemolyticus isolated from food samples in China, and to conduct a comprehensive analysis on the molecular characterization of V. parahaemolyticus isolate carrying mcr-1-harboring plasmid. A total of 646 V. parahaemolyticus strains isolated from 2531 food samples collected in retail markets in 34 different cities in China were screened for colistin resistance. Of the 646 V. parahaemolyticus isolates tested, 25 (2.5%) exhibited colistin resistance. The mcr-1 gene was detected in one colistin-resistant V. parahaemolyticus isolate, VP181, obtained from a shrimp sample collected in Hong Kong. The mcr-1 gene was located on a transferable IncX4 plasmid with size of ~40 kb. A Class A ß-lactamase gene, blaCARB-17 and the plasmid-mediated quinolone resistance (PMQR) gene qnrVC5 were detected in the mcr-1-positive V. parahaemolyticus isolate VP181. Virulence gene assays indicated that tdh was detected in VP181 by PCR. This is the first report of the occurrence of plasmid-encoded mcr-1 in virulent V. parahaemolyticus strain. Our findings indicate horizontal transfer of this gene to non-Enterobacteriaceae gram-negative bacteria, which warrants further investigation because of the public health threat it poses.


Assuntos
Antibacterianos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana/genética , Vibrio parahaemolyticus/genética , beta-Lactamases/genética , China , Plasmídeos/genética , Plasmídeos/isolamento & purificação , Vibrio parahaemolyticus/efeitos dos fármacos , Vibrio parahaemolyticus/isolamento & purificação
4.
Food Microbiol ; 84: 103270, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31421783

RESUMO

Vibrio parahaemolyticus is the leading bacterial cause of seafood-associated gastroenteritis worldwide. Moreover, infections and outbreaks caused by V. parahaemolyticus has kept increasing over the last two decades. In this study, we investigated the genetic diversity, virulence factors and farm-to-table spread pattern of V. parahaemolyticus by analyzing 383 genomes of food-associated isolates. These strains were isolated from diverse sample types from six provinces of China in 2014, being classified into three tiers of the farm-to-table spread process: food production, circulation and consumption. The genetic diversity of V. parahaemolyticus in different classifications, including geographical location, sample type, source and spread tier, was similar, as the median number of pairwise SNPs within each classification was between 33,013 and 33,659. Specifically, there was no clear boundaries in genetic diversity of the isolates from inland vs. coastal provinces, as well as of those from freshwater vs. seawater products. Moreover, the virulence genes and genomic islands were only found in a small number of isolates, indicating a low disease risk of the food-associated isolates in this study. By further exploring 28 recently emerged clonal groups, we identified seven farm-to-table spread events, showing a common pattern of single-source radial spread accompanied with occasional gene gain/loss events. Generally speaking, our work highlighted the colonization of V. parahaemolyticus in inland provinces and freshwater environment, and provided a snapshot of the farm-to-table spread pattern of V. parahaemolyticus food-associated isolates. Our results showed the feasibility of tracking the farm-to-table spread of foodborne pathogen, which would help construct the whole genome sequencing-based molecular tracking network in the future.


Assuntos
Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Variação Genética , Vibrio parahaemolyticus/genética , Fatores de Virulência/genética , Animais , Técnicas de Tipagem Bacteriana , China , Surtos de Doenças , Ilhas Genômicas , Humanos , Tipagem de Sequências Multilocus , Filogenia , Frutos do Mar/microbiologia , Vibrioses/microbiologia , Vibrio parahaemolyticus/classificação , Vibrio parahaemolyticus/isolamento & purificação , Virulência/genética
5.
Mikrochim Acta ; 186(7): 401, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31183576

RESUMO

A method is described for single-step detection of V. parahaemolyticus in seafood via aptamer-based SERS. A gold-coated polydimethylsiloxane (PDMS) film was used for the enhancement of Raman scattering. The Raman reporter 4-mercaptobenzoic acid was linked to aptamer modified gold nanoparticles (AuNPs) served as a signalling probe. The negatively charged signalling probe was assembled onto the cysteamine-modified Au-PDMS film through electrostatic adsorption. On addition of V. parahaemolyticus, it will be bound by the aptamer as a biorecognition element, and this leads to the dissociation of the signalling probe from the Au-PDMS film. Hence, the Raman signal (at 1592 cm-1) decreases. The assay has a wide linear response that covers the 1.2 × 102 to 1.2 × 106 cfu·mL-1 V. parahaemolyticus concentration range. The detection limit is 12 cfu·mL-1. The method was successfully applied to the determination of V. parahaemolyticus in oyster and salmon samples. Graphical abstract Schematic presentation of a surface-enhanced Raman spectroscopic method for single step detection of Vibrio parahaemolyticus using gold coated polydimethylsiloxane as the active substrate and aptamer modified gold nanoparticles. This solid substrate simplified the analysis procedures and enhanced the sensitivity.


Assuntos
Aptâmeros de Nucleotídeos/química , Dimetilpolisiloxanos/química , Ouro/química , Nanopartículas Metálicas/química , Vibrio parahaemolyticus/química , Sequência de Bases , Benzoatos/química , Técnicas Biossensoriais/métodos , Cisteamina/química , Escherichia coli/química , Limite de Detecção , Listeria monocytogenes/química , Salmonella typhimurium/química , Sensibilidade e Especificidade , Análise Espectral Raman , Staphylococcus aureus/química , Compostos de Sulfidrila/química , Vibrio parahaemolyticus/isolamento & purificação
6.
ISME J ; 13(10): 2578-2588, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31235840

RESUMO

Humans have profoundly affected the ocean environment but little is known about anthropogenic effects on the distribution of microbes. Vibrio parahaemolyticus is found in warm coastal waters and causes gastroenteritis in humans and economically significant disease in shrimps. Based on data from 1103 genomes of environmental and clinical isolates, we show that V. parahaemolyticus is divided into four diverse populations, VppUS1, VppUS2, VppX and VppAsia. The first two are largely restricted to the US and Northern Europe, while the others are found worldwide, with VppAsia making up the great majority of isolates in the seas around Asia. Patterns of diversity within and between the populations are consistent with them having arisen by progressive divergence via genetic drift during geographical isolation. However, we find that there is substantial overlap in their current distribution. These observations can be reconciled without requiring genetic barriers to exchange between populations if long-range dispersal has increased dramatically in the recent past. We found that VppAsia isolates from the US have an average of 1.01% more shared ancestry with VppUS1 and VppUS2 isolates than VppAsia isolates from Asia itself. Based on time calibrated trees of divergence within epidemic lineages, we estimate that recombination affects about 0.017% of the genome per year, implying that the genetic mixture has taken place within the last few decades. These results suggest that human activity, such as shipping, aquatic products trade and increased human migration between continents, are responsible for the change of distribution pattern of this species.


Assuntos
Vibrioses/microbiologia , Vibrio parahaemolyticus/classificação , Vibrio parahaemolyticus/isolamento & purificação , Variação Genética , Genoma Bacteriano , Humanos , Filogenia , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/genética
7.
Mar Pollut Bull ; 144: 105-116, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31179975

RESUMO

Vibrio species are widely distributed in the estuarine and coastal waters that possess the greatest threat to human health worldwide. In this study it is aimed to isolate and observe the abundance of Vibrio sp. and prevalence of biomarker genes and antibiotic resistance profile of V. cholerae isolated from the Port Blair bays of South Andaman. A total of 56 water samples were collected from the seven sampling stations of Port Blair bays in which maximum number of Vibrio sp. population density (1.78 × 104) was recorded in Phoenix Bay. Among the 786 isolates 57.38% of the isolates were confirmed as Vibrio sp., Vibrio cholerae and Vibrio parahaemolyticus. PCR results revealed that the prevalence of biomarker genes was recorded maximum in the isolates from Phoenix Bay and Junglighat Bay samples. Upon further analysis, it was observed that the prevalence of hlyA gene (215 bp), was found to be the most widespread biomarker determinant in 84.17% of isolates. Major virulence determinants; ctxA, ompU and toxR genes were not detected in V. cholerae isolates from Port Blair bays. Maximum antibiotic resistance pattern was observed in Phoenix Bay isolates and maximum number of V. cholerae isolates was resistance to tetracycline (60.76%). Cluster and Principal Component Analysis were employed to understand the diversity and distribution of Vibrio isolates and its biomarker genes. Upon PCA analysis seasonal influence was not much perceived in Vibrio species diversity in Port Blair bays and the lack of significant difference in the detection of species diversity in this study is due to resemblance in geographical conditions and sources of pollution.


Assuntos
Baías/microbiologia , Farmacorresistência Bacteriana/genética , Genes Bacterianos , Vibrio cholerae/genética , Vibrio parahaemolyticus/genética , Fatores de Virulência/genética , Biodiversidade , Humanos , Índia , Prevalência , Especificidade da Espécie , Vibrio cholerae/isolamento & purificação , Vibrio parahaemolyticus/isolamento & purificação
8.
Environ Sci Pollut Res Int ; 26(20): 21034-21043, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115814

RESUMO

Vibrio parahaemolyticus is the most common pathogen causing seafood-borne illnesses in Korea. The present study evaluated the occurrence, virulence, and antimicrobial resistance of V. parahaemolyticus in seawater and bivalves obtained in 2016 from the southern coast of Korea, an important region for commercial aquaculture industries, especially the Korean raw seafood culture. V. parahaemolyticus was detected in 87 of 160 (54.4%) bivalve samples and in 32 of 130 (24.5%) seawater samples. Especially high levels were detected during summer to early autumn. All the seawater and bivalves contained less than 2 and 5% of the tdh and trh genes of the isolates, respectively, and seawater isolates possessed two fewer genes than the bivalve isolates. Of 23 antimicrobials tested, three agents (ofloxacin, norfloxacin, and trimethoprim/sulfamethoxazole) effectively treated V. parahaemolyticus illness due to the sensitivity of the isolates. The isolates were highly resistant to ampicillin, however, excluding it as a treatment option. More than half of the isolates exhibited resistance to at least three antimicrobials. These findings indicate the importance of an integrated monitoring and surveillance program noting the occurrence, virulence, and antimicrobial resistance patterns of V. parahaemolyticus in various aquatic sources for preventing human health risks from seafood consumption.


Assuntos
Aquicultura/estatística & dados numéricos , Bivalves/microbiologia , Farmacorresistência Bacteriana , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/patogenicidade , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Humanos , República da Coreia , Estações do Ano , Água do Mar/microbiologia , Vibrio parahaemolyticus/efeitos dos fármacos , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/isolamento & purificação , Virulência/genética
9.
Mem Inst Oswaldo Cruz ; 114: e190053, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31038542

RESUMO

A multi-resistant strain of Vibrio parahaemolyticus was isolated from a tropical estuary in Rio de Janeiro, Brazil. Genome sequencing was conducted to establish the molecular basis of antibiotic resistance in this organism. The genetic content of this strain revealed it to be a non-virulent lineage that nevertheless possesses several antibiotic resistance determinants.


Assuntos
Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/genética , Vibrio parahaemolyticus/efeitos dos fármacos , Vibrio parahaemolyticus/genética , Microbiologia da Água , Genômica , Testes de Sensibilidade Microbiana , Vibrio parahaemolyticus/isolamento & purificação
10.
Biosens Bioelectron ; 136: 132-139, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31078871

RESUMO

We present a fully integrated portable centrifugal microsystem for multiplex detection of food poisoning bacteria with a large volume of sample up to 1 mL. The microsystem consists of a portable genetic analyzer and a fully integrated centrifugal microdevice. The centrifugal microdevice is designed with two units: a 3D printed solution-loading cartridge and a centrifugal microfluidic disc. All the essential solutions for loop-mediated isothermal amplification (LAMP) reaction are stored inside the cartridge, and orderly released into centrifugal microdevice by a rotation program. Each unit of the device is designed with 20 reaction chambers for simultaneous detection of food-borne bacteria in one test. To increase the amount of a sample to 1 mL, we incorporated the super absorbent polymer (SAP) in the waste chamber to absorb the sample and the washing solution during the device operation. The whole process was automatically conducted including designated solution release, bead-based DNA extraction, isothermal gene amplification by Eriochrome Black (EBT)-mediated LAMP reaction, and colorimetric and UV-visible detection of amplicons. The ratio between Abs640nm and Abs570nm was used as a criterion to confirm the positive result, and the result was positive upon the condition of Abs640/Abs570 ≥ 1.0. To demonstrate the pathogenic bacteria detection on our proposed microsystem, we targeted three kinds of bacteria (Escherichia coli O157:H7, Salmonella typhimurium, and Vibrio parahaemolyticus) for monoplex and multiplex detection. The whole process from sample to result was completed within 1 h with a low limit of detection (LOD) of 102 cells/mL.


Assuntos
Contaminação de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Escherichia coli O157/isolamento & purificação , Doenças Transmitidas por Alimentos/prevenção & controle , Dispositivos Lab-On-A-Chip , Limite de Detecção , Sistemas Automatizados de Assistência Junto ao Leito , Salmonella typhimurium/isolamento & purificação , Vibrio parahaemolyticus/isolamento & purificação
11.
Anal Chim Acta ; 1062: 124-130, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-30947988

RESUMO

Vibrio parahaemolyticus (VP), as a kind of harmful bacterium to human beings, its rapid and accurate detection is particularly important. Herein, we developed a simple and efficient dual-modal Faraday cage-type immunosensor for ultrasensitive detection of VP based on electrochemiluminescence (ECL) and anodic stripping voltammetry (ASV). After being captured by capture unit on magnetic glassy carbon electrodes, VP was recognized by detector unit, achieving ECL and ASV dual-modal detection. Compared with traditional sandwich-type immunosensors, this proposed immunosensor has the advantages of significant signal amplification effects, due to its distinctive Faraday cage-type structure. Benefited from it, all Ru(bpy)22+ and AgNPs labeled on the detector unit could participate in the electrode reaction and generate stronger ECL and electrochemical signals respectively. Under the optimized conditions, the linear range was from 102 to 107 CFU mL-1 with a limit of detection (LOD) of 33 CFU mL-1, and the selectivity, stability and reproducibility were satisfactory. In addition, the results of two detection methods can mutually validate each other to avoid false positive results. Moreover, this Faraday cage-type immunosensor with dual-modal detection can also be used for detection of VP in seawater samples with recoveries in the range of 96.1-107.9%, which would be a promising tool for pathogenic bacteria analysis.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Imunoensaio , Medições Luminescentes , Vibrio parahaemolyticus/isolamento & purificação , Eletrodos , Humanos
12.
Lett Appl Microbiol ; 68(6): 537-545, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30933376

RESUMO

Viable but nonculturable (VBNC) Vibrio parahaemolyticus cannot be detected by the standard cultivation-based methods. In this study, commonly used viability assessment methods were evaluated for the detection of V. parahaemolyticus in a VBNC state. Vibrio parahaemolyticus cells exposed to nutrient deficiency at cold temperature were used for epifluorescence microscopy with SYTO9 and propidium iodide (PI) staining and real-time polymerase chain reaction (qPCR) with propidium monoazide (PMA), and its resuscitative ability was determined by a temperature upshift in freshly prepared artificial sea water (ASW; pH 7) fluids. Viable cells with intact membranes always exceeded 5·0 log CFU per ml in ASW microcosms at 4°C. After 80 days, cycle thresholds for V. parahaemolyticus ATCC 27969 were 16·15-16·69. During cold-starvation, PMA qPCR selectively excluded DNAs from heat-killed cells. However, there may be some penetration of PMA into undamaged cells that persisted in ASW for 150 days, as evidenced by their ability to resuscitate from a VBNC state after a temperature upshift (25°C); V. parahaemolyticus ATCC 33844 and V. parahaemolyticus ATCC 27969 were successfully reactivated from a VBNC state in ASW microcosms containing <5% NaCl, following enrichment in ASW medium (pH 7). SIGNIFICANCE AND IMPACT OF THE STUDY: Few studies have evaluated the characteristics of and detection methods for viable but nonculturable (VBNC) Vibrio parahaemolyticus induced by cold-starvation. Currently, VBNC cells are routinely detected by SYTO9 and propidium iodide double staining. However, viable cell counts might be overestimated by this approach, suggesting that the fluorescence dyes may be ineffective for accurately determining the viability of bacterial cells. We demonstrated that quantitative real-time polymerase chain reaction with propidium monoazide, which selectively permeates damaged cell membranes, can be used to obtain viable cell counts of V. parahaemolyticus after its evolution to a VBNC state under cold-starvation conditions.


Assuntos
Azidas/química , Microscopia de Fluorescência/métodos , Propídio/análogos & derivados , Reação em Cadeia da Polimerase em Tempo Real/métodos , Vibrio parahaemolyticus/isolamento & purificação , Temperatura Baixa , Viabilidade Microbiana/efeitos dos fármacos , Propídio/química , Vibrio parahaemolyticus/genética
13.
Mol Genet Genomics ; 294(4): 1007-1022, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30968246

RESUMO

Acute hepatopancreatic necrosis disease (AHPND) is a recently discovered shrimp disease that has become a severe threat to global shrimp-farming industry. The causing agents of AHPND were identified as Vibrio parahaemolyticus and other vibrios harboring a plasmid encoding binary toxins PirAvp/PirBvp. However, the epidemiological involvement of environmental vibrios in AHPND is poorly understood. In this study, with an aim to reveal the possible transmission route of AHPND-causing V. parahaemolyticus, we sequenced and analyzed the genomes of four pairs of V. parahaemolyticus strains from four representative regions of shrimp farming in China, each including one strain isolated from diseased shrimp during an AHPND outbreak and one strain isolated from sediment before AHPND outbreaks. Our results showed that all the four shrimp-isolated and three of the sediment-isolated strains encode and secret PirAvp/PirBvp toxins and, therefore, are AHPND-causing strains. In silico multilocus sequence typing and high-resolution phylogenomic analysis based on single-nucleotide polymorphisms, as well as comparison of genomic loci in association with prophages and capsular polysaccharides (CPSs) consistently pointed to a close genetic relationship between the shrimp- and sediment-isolated strains obtained from the same region. In addition, our analyses revealed that the sequences associated with prophages, CPSs, and type VI secretion system-1 are highly divergent among strains from different regions, implying that these genes may play vital roles in environmental adaptation for AHPND-causing V. parahaemolyticus and thereby be potential targets for AHPND control. Summing up, this study provides the first direct evidence regarding the transmission route of AHPND-causing V. parahaemolyticus and underscores that V. parahaemolyticus in shrimp are most likely originated from local environment. The importance of environmental disinfection measures in shrimp farming was highlighted.


Assuntos
Toxinas Bacterianas/genética , Penaeidae/microbiologia , Vibrioses/veterinária , Vibrio parahaemolyticus/classificação , Animais , China , Hibridização Genômica Comparativa , Evolução Molecular , Genômica , Tipagem de Sequências Multilocus , Filogenia , Polimorfismo de Nucleotídeo Único , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/isolamento & purificação , Sequenciamento Completo do Genoma
14.
Dis Aquat Organ ; 132(3): 241-247, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31019129

RESUMO

In June 2017, mass mortalities were reported at whiteleg shrimp Penaeus vannamei farms in Texas, USA. PCR testing for OIE-listed and non-listed pathogens detected the pirA and pirB toxin genes associated with acute hepatopancreatic necrosis disease (AHPND). DNA sequence analyses of cloned pirA and pirB genes showed them to be identical to those detected in other AHPND-causing Vibrio sp. Amplicons generated using PCR tests targeted to the toxR gene showed the Pir toxin genes to be associated with a V. parahaemolyticus type more similar to a genotype found in Mexico compared to that found in Asia. Histology detected masses of bacteria and hemocytic infiltrations as well as extensive necrosis and sloughing of epithelial cells in hepatopancreatic tubules pathognomonic of AHPND. The data support AHPND as the cause of the mortalities. Given that US companies produce shrimp broodstock for farms in Asia and Latin America, the further spread of AHPND in the USA needs to be prevented to avoid serious economic consequences to these industries.


Assuntos
Hepatopâncreas/patologia , Vibrioses/diagnóstico , Vibrio parahaemolyticus/isolamento & purificação , Doença Aguda , Animais , Humanos , Necrose , Penaeidae/microbiologia , Texas , Vibrio parahaemolyticus/genética
15.
J Nanobiotechnology ; 17(1): 37, 2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30841927

RESUMO

BACKGROUND: Magnetosomes (also called bacterial magnetic nanoparticles; BMPs) are biomembrane-coated nanoparticles synthesized by magnetotactic bacteria (MTB). Engineered BMPs fused to protein A (termed ∆F-BMP-FA) bind antibodies (Abs) automatically, and thus provide a series of potential advantages. However, no report so far has systematically evaluated functional applicability of genetically engineered BMPs. RESULTS: We evaluated properties of ∆F-BMP-FA, and developed/optimized culture methods for host strain Magnetospirillum gryphiswaldense ΔF-FA, ∆F-BMP-FA extraction conditions, conditions for Ab conjugation to ∆F-BMP-FA surface, and procedures for antigen detection using ∆F-BMP-FA/Ab complexes (termed BMP-A-Ab). Fed-batch culture for 36 h in a 42-L fermentor resulted in yields (dry weight) of 2.26 g/L for strain ΔF-FA and 62 mg/L for ∆F-BMP-FA. Optimal wash cycle number for ∆F-BMP-FA purification was seven, with magnetic separation following each ultrasonication step. Fusion of protein A to BMPs resulted in ordered arrangement of Abs on BMP surface. Linkage rate 962 µg Ab per mg ∆F-BMP-FA was achieved. BMP-A-Ab were tested for detection of pathogen (Vibrio parahaemolyticus; Vp) surface antigen and hapten (gentamicin sulfate). Maximal Vp capture rate for BMP-A-Ab was 90% (higher than rate for commercial immunomagnetic beads), and detection sensitivity was 5 CFU/mL. ∆F-BMP-FA also bound Abs from crude mouse ascites to form complex. Lowest gentamicin sulfate detection line for BMP-A-Ab was 0.01 ng/mL, 400-fold lower than that for double Ab sandwich ELISA, and gentamicin sulfate recovery rate for BMP-A-Ab was 93.2%. CONCLUSION: Our findings indicate that engineered BMPs such as ∆F-BMP-FA are inexpensive, eco-friendly alternatives to commercial immunomagnetic beads for detection or diagnostic immunoassays, and have high Ab-conjugation and antigen-adsorption capacity.


Assuntos
Nanopartículas de Magnetita/química , Magnetossomos/química , Magnetospirillum/química , Proteína Estafilocócica A/química , Animais , Anticorpos/química , Antígenos de Bactérias/análise , Reatores Biológicos , Ensaio de Imunoadsorção Enzimática , Gentamicinas/análise , Haptenos/análise , Limite de Detecção , Camundongos , Engenharia de Proteínas , Propriedades de Superfície , Vibrio parahaemolyticus/isolamento & purificação
16.
Antonie Van Leeuwenhoek ; 112(8): 1199-1211, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30877501

RESUMO

The Gram-negative marine bacterium Vibrio parahaemolyticus has been identified as a major cause of bacterial food poisoning in China. Here, the population structure and genetic diversity of V. parahaemolyticus from Weihai, a coastal city in China, was studied by the multi-locus sequence typing (MLST) method. In this survey, we isolated 40 strains including environmental and clinical samples of patients with acute gastroenteritis or diarrhea; isolates from other countries were also included for comparison. DnaSP Version5, START V2, eBURST version3 and Mega 6 were used to analyse the data. We found that ST3 and ST332 were the most prevalent clones and that they were closely associated with acute diarrhoeal diseases. These STs showed a low dN/dS ratio and significant linkage disequilibrium. All isolates were divided into four clonal complexes, six groups and nine singletons, showing a high degree of genetic diversity. 18 STs, mostly from environmental isolates, were recognised by the MLST analysis for the first time. In conclusion, ST3 and ST332 were the epidemic STs in the coastal area. ST332 might be a region-specific ST, which needs to be confirmed by further analysis. Thus, the long-term monitoring of V. parahaemolyticus plays an important role in preventing and controlling the transmission between environment and people in Weihai.


Assuntos
Gastroenterite/microbiologia , Variação Genética , Tipagem de Sequências Multilocus , Água do Mar/microbiologia , Vibrioses/microbiologia , Vibrio parahaemolyticus/classificação , Vibrio parahaemolyticus/isolamento & purificação , China , Genótipo , Humanos , Vibrio parahaemolyticus/genética
17.
Biosens Bioelectron ; 132: 271-278, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30878727

RESUMO

Vibrio parahaemolyticus is one of the most important foodborne pathogens that cause various life-threatening diseases in human and animals. Here, we present a rapid detection platform for V. parahaemolyticus by combining loop-mediated isothermal amplification (LAMP) and disposable electrochemical sensors based on screen-printed graphene electrodes (SPGEs). The LAMP reactions using primers targeting V. parahaemolyticus toxR gene were optimized at an isothermal temperature of 65 °C, providing specific detection of V. parahaemolyticus within 45 min at the detection limit of 0.3 CFU per 25 g of raw seafood. The LAMP amplicons can be effectively detected using unmodified SPGEs, redox active molecules namely Hoechst-33258 and a portable potentiostat. Therefore, the proposed system is particularly suitable as a point-of-care device for on-site detection of foodborne pathogens.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Análise de Alimentos/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Alimentos Marinhos/microbiologia , Vibrioses/microbiologia , Vibrio parahaemolyticus/isolamento & purificação , DNA Bacteriano/análise , DNA Bacteriano/genética , Eletrodos , Desenho de Equipamento , Grafite/química , Humanos , Limite de Detecção , Sistemas Automatizados de Assistência Junto ao Leito , Vibrio parahaemolyticus/genética
18.
Future Microbiol ; 14: 437-450, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30855189

RESUMO

Vibrio Parahaemolyticus infections caused by the pandemic clone have become a global public health issue. The pandemic clone includes over ten sequence types and 49 serotypes. Several markers such as toxRS/new, orf8 and genomic islands were considered specific for pandemic strains, but subsequent studies later confirmed a lack of specificity. Thus, identifying stable indicators for the pandemic clone is still an open question. In recent years, several environmental pandemic strains are growing, constituting a new threat to seafood safety and human health. Traditional methods show limited discrimination in studying the microevolution of pandemic strains. For example, multilocus sequence typing divides many pandemic strains into ST3 type, making it difficult to further distinguish the variability within ST3 strains from different contexts. When using a whole genome sequencing-based technique, strains including those with the same sequence type, could be well separated. Whole genome sequencing-based technology also played important roles in dissecting the evolution process and revealing the mechanism underlying rapid serotype conversion within pandemic strains. In addition, the emergence of multiple-antibiotic resistant pandemic strains needs attention. Altogether, we are facing many challenges posed by pandemic V. parahaemolyticus strains, which need to be resolved in future studies.


Assuntos
Vibrioses/microbiologia , Vibrio parahaemolyticus/isolamento & purificação , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Doenças Transmitidas por Alimentos/tratamento farmacológico , Doenças Transmitidas por Alimentos/epidemiologia , Doenças Transmitidas por Alimentos/microbiologia , Humanos , Pandemias , Filogenia , Vibrioses/tratamento farmacológico , Vibrioses/epidemiologia , Vibrio parahaemolyticus/classificação , Vibrio parahaemolyticus/efeitos dos fármacos , Vibrio parahaemolyticus/genética
19.
J Food Sci ; 84(4): 859-870, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30912864

RESUMO

In this study, we evaluated the temperature requirements for home delivery in Taiwan by considering food safety risks of chilled shrimp that may be contaminated with Vibrio parahaemolyticus. Pathogenic V. parahaemolyticus was chosen because it was the main cause of foodborne outbreaks originating from contaminated seafood in Taiwan. The risk of becoming ill due to consumption of raw shrimp was estimated to be 7.3 × 10-3 per serving, estimated based on the real-time temperature profile. Lowering the maximum temperature to 7 °C during transit and cooking shrimps at 100 °C for 5 min could reduce the risk by more than 94%. These interventions, therefore, were suggested to be used as an integral part of temperature management control in the home delivery cold chain. These findings can help food authorities to institute temperature management policies and regulations to prevent broken cold chains and reduce food safety risks. PRACTICAL APPLICATION: This research may be applicable to home delivery services that deliver low-temperature food products, such as seafood products.


Assuntos
Microbiologia de Alimentos , Conservação de Alimentos , Alimentos Marinhos/microbiologia , Animais , Temperatura Baixa , Humanos , Refrigeração , Taiwan , Vibrio parahaemolyticus/isolamento & purificação
20.
Infect Immun ; 87(5)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30804101

RESUMO

Vibrio parahaemolyticus is a human pathogen, and it is a major cause of severe gastroenteritis in coastal areas. OmpU is one of the major outer membrane porins of V. parahaemolyticus Host-immunomodulatory effects of V. parahaemolyticus OmpU (VpOmpU) have not been elucidated yet. In this study, in an effort towards characterizing the effect of VpOmpU on innate immune responses of the host, we observed that VpOmpU is recognized by the Toll-like receptor 1/2 (TLR1/2) heterodimer in THP-1 monocytes but by both TLR1/2 and TLR2/6 heterodimers in RAW 264.7 macrophages. To the best of our knowledge, this is the first report of a natural pathogen-associated molecular pattern (PAMP) recognized by both TLR1/2 and TLR2/6 heterodimers; so far, mainly the synthetic ligand Pam2CSK4 has been known to be recognized by both the TLR1/2 and TLR2/6 heterodimers. We also have shown that VpOmpU can activate monocytes and macrophages, leading to the generation of proinflammatory responses as indicated by tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and NO production in macrophages and TNF-α and IL-6 production in monocytes. VpOmpU-mediated proinflammatory responses involve MyD88-IRAK-1 leading to the activation of mitogen-activated protein (MAP) kinases (p38 and Jun N-terminal protein kinase [JNK]) and transcription factors NF-κB and AP-1. Further, we have shown that for the activation of macrophages leading to the proinflammatory responses, the TLR2/6 heterodimer is preferred over the TLR1/2 heterodimer. We have also shown that MAP kinase activation is TLR2 mediated.


Assuntos
Imunidade Inata/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Receptores Toll-Like/imunologia , Vibrioses/imunologia , Vibrio parahaemolyticus/imunologia , Vibrio parahaemolyticus/isolamento & purificação , Interações Hospedeiro-Patógeno/imunologia , Humanos , Transdução de Sinais/imunologia
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