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1.
PLoS Pathog ; 17(1): e1009194, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33439894

RESUMO

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.


Assuntos
Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/metabolismo , Viabilidade Microbiana , Proteoma/metabolismo , Vibrio parahaemolyticus/crescimento & desenvolvimento , Vibrio parahaemolyticus/patogenicidade , Virulência , Células Cultivadas , Meios de Cultura , Regulação Bacteriana da Expressão Gênica , Proteoma/análise , Vibrioses/metabolismo , Vibrioses/microbiologia , Vibrio parahaemolyticus/metabolismo
2.
Nat Commun ; 11(1): 5777, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33188170

RESUMO

Vibrio parahaemolyticus is the leading cause of seafood-borne diarrheal diseases. Experimental overproduction of a type 3 secretion system (T3SS1) in this pathogen leads to decreased intestinal colonization, which suggests that T3SS1 repression is required for maximal virulence. However, the mechanisms by which T3SS1 is repressed in vivo are unclear. Here, we show that host-derived nitrite modifies the activity of a bacterial histidine kinase and mediates T3SS1 repression. More specifically, nitrite activates histidine kinase sensor VbrK through S-nitrosylation on cysteine 86, which results in downregulation of the entire T3SS1 operon through repression of its positive regulator exsC. Replacement of cysteine 86 with a serine (VbrK C86S mutant) leads to increased expression of inflammatory cytokines in infected Caco-2 cells. In an infant rabbit model of infection, the VbrK C86S mutant induces a stronger inflammatory response at the early stage of infection, and displays reduced intestinal colonization and virulence at the later stage of infection, in comparison with the parent strain. Our results indicate that the pathogen V. parahaemolyticus perceives nitrite as a host-derived signal and responds by downregulating a proinflammatory factor (T3SS1), thus enhancing intestinal colonization and virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina Quinase/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Vibrio parahaemolyticus/metabolismo , Vibrio parahaemolyticus/patogenicidade , Anaerobiose , Animais , Sequência de Bases , Sítios de Ligação , Células CACO-2 , Citocinas/metabolismo , Regulação para Baixo/genética , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Modelos Biológicos , Nitratos/metabolismo , Nitritos/metabolismo , Nitrosação , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Coelhos , Transcrição Genética , Vibrio parahaemolyticus/genética , Virulência/genética
3.
Food Microbiol ; 90: 103498, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32336378

RESUMO

This study was aimed at characterizing microbiologically Gilthead sea bream (Sparus aurata) and Sea bass (Dicentrarchus labrax) produced in two estuarine ecosystems in Andalusia (Spain): the estuary of the river Guadalquivir (La Puebla del Río, Sevilla) (A), and the estuary of the river Guadiana (Ayamonte, Huelva) (B). The collected fish individuals and water were analysed for hygiene indicator microorganisms and pathogens. The statistical analysis of results revealed that microbial counts for the different microbiological parameters were not statistically different for fish type. On the contrary, considering anatomic part, viscera showed significantly higher concentrations for Enterobacteriaceae, total coliforms and for Staphylococcus spp. coagulase +. Furthermore, location A showed in water and fish higher levels for lactic acid bacteria, aerobic mesophilic bacteria, Enterobacteriaceae, total coliforms and Staphylococcus spp. coagulase +. Neither Listeria monocytogenes, nor Salmonella spp. were detected, though Vibrio parahaemolyticus was identified, molecularly, in estuarine water in location B. The predictive analysis demonstrated that the initial microbiological quality could have an impact on product shelf-life, being longer for location B, with better microbiological quality. Results stress the relevance of preventing the microbiological contamination of water in estuary production systems in order to assure the quality and safety of Gilthead sea bream and Sea bass.


Assuntos
Aquicultura , Bactérias/isolamento & purificação , Bass/microbiologia , Doenças dos Peixes/microbiologia , Dourada/microbiologia , Animais , Bactérias/classificação , Bactérias/patogenicidade , Ecossistema , Enterobacteriaceae/isolamento & purificação , Enterobacteriaceae/patogenicidade , Estuários , Doenças dos Peixes/epidemiologia , Armazenamento de Alimentos , Prevalência , Alimentos Marinhos/microbiologia , Espanha/epidemiologia , Staphylococcus/isolamento & purificação , Staphylococcus/patogenicidade , Vibrio parahaemolyticus/isolamento & purificação , Vibrio parahaemolyticus/patogenicidade
4.
Artigo em Inglês | MEDLINE | ID: mdl-32178325

RESUMO

Expression of the regulatory stress rpoS gene controls the transcription of cspA genes, which are involved in survival and adaptation to low temperatures. The purpose of this study was to assess the growth kinetics of naturally occurring V. parahaemolyticus in shellstock oysters and in vitro and the cold-shock-induced expression of the rpoS and cspA gene response in vitro during postharvest refrigeration. Naturally contaminated eastern oysters (Crassostrea virginica) and pathogenic (Vp-tdh) and nonpathogenic (Vp-tlh) isolates were stored at 7 ± 1 °C for 168 h and 216 h, respectively. The regulatory stress (rpos) and cold-shock (cspA) gene expressions were determined by reverse transcription PCR. At 24 h, the (Vp-tdh) strain grew faster (p < 0.05) than the (Vp-tlh) strain in oysters (λ = 0.33, 0.39, respectively) and in vitro (λ = 0.89, 37.65, respectively), indicating a better adaptation to cold shock for the (Vp-tdh) strain in live oysters and in vitro. At 24 h, the (Vp-tdh) strain rpoS and cspA gene expressions were upregulated by 1.9 and 2.3-fold, respectively, but the (Vp-tlh) strain rpoS and cspA gene expressions were repressed and upregulated by -0.024 and 1.9-fold, respectively. The V. parahaemolyticus strains that were isolated from tropical oysters have adaptive expression changes to survive and grow at 7 °C, according to their virulence.


Assuntos
Temperatura Baixa , Crassostrea , Regulação da Expressão Gênica , Ostreidae , Vibrio parahaemolyticus , Animais , Refrigeração , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/patogenicidade
5.
Int J Mol Sci ; 21(4)2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-32069894

RESUMO

Kuruma prawn, Marsupenaeus japonicus, has the third largest annual yield among shrimp species with vital economic significance in China. White spot syndrome virus (WSSV) is a great threat to the global shrimp farming industry and results in high mortality. Pellino, a highly conserved E3 ubiquitin ligase, has been found to be an important modulator of the Toll-like receptor (TLR) signaling pathways that participate in the innate immune response and ubiquitination. In the present study, the Pellino gene from Marsupenaeus japonicus was identified. A qRT-PCR assay showed the presence of MjPellino in all the tested tissues and revealed that the transcript level of this gene was significantly upregulated in both the gills and hemocytes after challenge with WSSV and Vibrio parahaemolyticus. The function of MjPellino was further verified at the protein level. The results of the three-dimensional modeling and protein-protein docking analyses and a GST pull-down assay revealed that the MjPellino protein was able to bind to the WSSV envelope protein VP26. In addition, the knockdown of MjPellino in vivo significantly decreased the expression of MjAMPs. These results suggest that MjPellino might play an important role in the immune response of kuruma prawn.


Assuntos
Proteínas de Artrópodes/genética , Penaeidae/genética , Ubiquitina-Proteína Ligases/genética , Vibrioses/genética , Sequência de Aminoácidos/genética , Animais , Proteínas de Artrópodes/isolamento & purificação , China , Perfilação da Expressão Gênica/métodos , Hemócitos/microbiologia , Hemócitos/virologia , Humanos , Imunidade Inata/genética , Penaeidae/microbiologia , Penaeidae/virologia , Receptores Toll-Like/genética , Ativação Transcricional/genética , Vibrioses/microbiologia , Vibrio parahaemolyticus/patogenicidade , Vírus da Síndrome da Mancha Branca 1/genética , Vírus da Síndrome da Mancha Branca 1/patogenicidade
6.
Biosens Bioelectron ; 151: 111968, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31999578

RESUMO

In the world wide, food poisoning accidents related to Vibrio spp. are on the rise, even numbers of food poisoning by other foodborne pathogens are decreasing. Therefore, the requirement of the rapid, sensitive and convenient detection method for V. parahaemolyticus has been grown. The objective of this study is to develop a colorimetric loop-mediated isothermal amplification (LAMP) assay using a molecular beacon (HRPzyme connected with complementary oligonucleotides at the 5' and 3' ends) for the rapid, sensitive, and convenient detection of V. parahaemolyticus. The colorimetric LAMP assay optimized at 58.8°C showed a detection limit of 1 × 100 CFU/mL and was confirmed to be specific to V. parahaemolyticus. The colorimetric LAMP assay can be finished within 1 h including DNA extraction step. The method was successfully applied to flatfish samples artificially inoculated with known amount of V. parahaemolyticus, and its cut-off value for the flatfish samples was 1 × 101 CFU/g. In addition, the colorimetric LAMP assay developed in the study was found to be able to correct false-positive results, which are known to be a disadvantage of conventional LAMP assays. Therefore, these results indicated that the colorimetric LAMP method is a useful tool for the rapid, sensitive and convenient detection of V. parahaemolyticus in fishes and can also be used as a point-of-care molecular diagnostic technique since it does not require any expensive equipment such as a thermocycler and detectors.


Assuntos
Técnicas Biossensoriais , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Vibrioses/diagnóstico , Vibrio parahaemolyticus/isolamento & purificação , Colorimetria , Microbiologia de Alimentos/tendências , Doenças Transmitidas por Alimentos/diagnóstico , Doenças Transmitidas por Alimentos/microbiologia , Humanos , Limite de Detecção , Alimentos Marinhos , Vibrioses/microbiologia , Vibrio parahaemolyticus/patogenicidade
7.
Microbiol Immunol ; 64(3): 167-181, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31850542

RESUMO

Vibrio parahaemolyticus is a leading cause of seafood-borne bacterial gastroenteritis in humans. Since its discovery in 1950, this bacterium has been isolated in widespread outbreaks and in sporadic cases of gastroenteritis worldwide. Although the exotoxin, thermostable direct hemolysin, had been the focus of extensive research on the pathogenicity of V. parahaemolyticus, the whole-genome sequencing of a clinical isolate, RIMD2210633 strain, was a breakthrough in this field. The possession of two sets of gene clusters for type III secretion systems (T3SS1 and T3SS2) was unveiled by that genome project. T3SS is a protein export apparatus that delivers bacterial proteins, called effectors, directly into the host's cytosol, to disrupt host cell function. The subsequent studies have established that T3SS2, which is encoded in an 80 kb pathogenicity island called V. parahaemolyticus pathogenicity island (Vp-PAI), is closely related to enteropathogenicity. Recent functional analyses of Vp-PAI-encoded genes revealed the sophisticated mechanisms in V. parahaemolyticus for sensing the intestinal environment and host cell contact, and a dozen T3SS2-exported proteins encoded in Vp-PAI. In this review, we summarize recent advances in V. parahaemolyticus research regarding the control of the expression of Vp-PAI-encoded genes, structural components and the secretory regulation of T3SS2, and the biological activities of T3SS2-exported effectors. Thus, Vp-PAI-encoded T3SS2 becomes an important key in the postgenomic era to shed light on the enteropathogenic mechanism of V. parahaemolyticus.


Assuntos
Ilhas Genômicas/genética , Sistemas de Secreção Tipo III , Vibrioses/microbiologia , Vibrio parahaemolyticus , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Interações entre Hospedeiro e Microrganismos , Humanos , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo , Vibrio parahaemolyticus/patogenicidade
8.
Int J Food Microbiol ; 317: 108461, 2020 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-31794931

RESUMO

Vibrio parahaemolyticus is the leading cause of foodborne bacterial poisoning in China. The aim of this research is to conduct a study on the prevalence, virulence, and antimicrobial resistance of V. parahaemolyticus from different types of food samples in 12 different cities of China. Since fluoroquinolones are the major choice of treatment for V. parahaemolyticus infections, the genetic basis for fluoroquinolone resistance in V. parahaemolyticus were also investigated. V. parahaemolyticus was detected in 163 of the 784 food samples collected from 12 different cities in China, resulting in a prevalence of 20.79%. The prevalence of V. parahaemolyticus in ready-to-eat (RTE) food (4.96%) was much lower than those of shrimp (32.62%) and fish (22.00%). Virulence gene screening showed that 44 (27.00%) V. parahaemolyticus strains carried at least one virulence gene. Four isolates from shrimp and three isolates from fish contained both the virulence genes tdh and trh. In addition, the trh was firstly detected in one isolate collected from RTE food. All isolates exhibited relatively high resistance rates to ampicillin (82.21%), gentamicin (19.63%), and tetracycline (14.11%), while <10% of strains were resistant to ciprofloxacin (4.91%), levofloxacin (4.91%), and tetracycline (4.29%). Eight fluoroquinolone-resistant V. parahaemolyticus were selected to determine the molecular basis for fluoroquinolone resistance. These eight isolates belonged to three different types according to enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). A Ser83Ile substitution in GyrA was deteted in seven fluoroquinolone-resistant strains, except V209 which harbored a Ser83Phe substitution in GyrA. Moreover, A Ser85Leu substitution in ParC was found in five isolates (V52, V53, V61, V163, and V209). Plasmid-mediated quinolone resistance (PMQR) genes were detected in all eight fluoroquinolone-resistant V. parahaemolyticus strains. This is the first report of Ser83Phe substitution in GyrA, qnrD and qnrS1 in V. parahaemolyticus. The information generated in this study will provide valuable information for risk assessment of V. parahaemolyticus infections and future control of antibiotic-resistant V. parahaemolyticus species in China.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Doenças Transmitidas por Alimentos/epidemiologia , Vibrio parahaemolyticus/efeitos dos fármacos , Vibrio parahaemolyticus/genética , Substituição de Aminoácidos/genética , Ampicilina/farmacologia , Animais , China/epidemiologia , DNA Girase/genética , Fast Foods/microbiologia , Doenças Transmitidas por Alimentos/microbiologia , Gentamicinas/farmacologia , Humanos , Levofloxacino/farmacologia , Plasmídeos/genética , Prevalência , Alimentos Marinhos/microbiologia , Tetraciclina/farmacologia , Vibrio parahaemolyticus/isolamento & purificação , Vibrio parahaemolyticus/patogenicidade , Virulência/genética
9.
Fish Shellfish Immunol ; 97: 421-431, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31846777

RESUMO

During the immune defense reaction of invertebrate, a plenty of reactive oxygen species (ROS) could be induced to product. Though ROS can kill foreign invaders, the accumulation of these reactive molecules in animals will cause serious cell damage. Carotenoids could function as scavengers of oxygen radicals. In this research, cDNA and genomic DNA of one carotenoid isomerooxygenase gene (named EcNinaB-X1) were cloned from Exopalaemon carinicauda. EcNinaB-X1 gene was composed of 12 exons and 11 introns. EcNinaB-X1 knock-out (KO) prawns were produced via CRISPR/Cas9 technology and the change of their phenotypes were analyzed. Of the 400 injected one-cell stage embryos with cas9 mRNA and one sgRNA targeting the first exon of EcNinaB-X1 gene, 26 EcNinaB-X1-KO prawns were generated and the mutant rate reached 6.5% after embryo injection. The EcNinaB-X1-KO prawns had significant lower mortality than those in wild-type group when the prawns were challenged with Vibrio parahaemolyticus or Aeromonas hydrophila. In conclusion, we first demonstrate the function of the carotenoid isomerooxygenase gene in immune defense of E. carinicauda by performing directed, heritable gene mutagenesis.


Assuntos
Proteínas de Artrópodes/genética , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes/métodos , Oxigenases/genética , Palaemonidae/enzimologia , Palaemonidae/genética , Aeromonas hydrophila/patogenicidade , Animais , Carotenoides/química , Deleção de Genes , Imunidade Inata , Mutagênese , Palaemonidae/microbiologia , Vibrio parahaemolyticus/patogenicidade
10.
J Food Prot ; 83(1): 155-162, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31860395

RESUMO

Vibrio parahaemolyticus is a leading seafood-borne pathogen that causes gastroenteritis, septicemia, and serious wound infections due to the actions of virulence-associated proteins. We compared the extracellular proteins of nonvirulent JHY20 and virulent ATCC 33847 V. parahaemolyticus reference strains. Eighteen extracellular proteins were identified from secretory profiles, and 11 (68.75%) of the 16 proteins in ATCC 33847 are associated with virulence and/or protection against adverse conditions: trigger factor, chaperone SurA, aspartate-semialdehyde dehydrogenase, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, glutamate 5-kinase, alanine dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, outer membrane protein OmpV, ribosome-associated inhibitor A, chaperone protein Skp, and universal stress protein. Two nontoxic-related proteins, amino acid ABC transporter substrate-binding protein and an uncharacterized protein, were identified in JHY20. The results provide a theoretical basis for supporting safety risk assessment of aquatic foods, illuminate the pathogenic mechanisms of V. parahaemolyticus, and assist the identification of novel vaccine candidates for foodborne pathogens.


Assuntos
Proteínas de Bactérias/análise , Vibrio parahaemolyticus/patogenicidade , Fatores de Virulência/análise , Proteínas Hemolisinas , Virulência
11.
Jpn J Infect Dis ; 73(2): 102-110, 2020 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-31666496

RESUMO

The annual number of outbreaks of Vibrio parahaemolyticus and Salmonella food poisoning and that of patients in Japan, from 2000 to 2018, decreased exponentially even though the size of the individual outbreaks (the number of patients per outbreak) tended to become larger. For food poisonings caused by Campylobacter, the annual number of outbreaks increased exponentially while outbreak size became smaller and the annual number of patients remained almost unchanged. For food poisoning caused by norovirus, both the number of outbreaks and that of patients remained high throughout. Over time, the geographical and seasonal distribution of food poisonings became narrower for Vibrio parahaemolyticus and Salmonella, while they became wider for Campylobacter and norovirus. Further analyses using the attack rate-patient number plots suggested that the number of the outbreaks was determined mainly by the levels of microbial contamination of foods before they were brought into the facilities for consumption.


Assuntos
Infecções por Caliciviridae/epidemiologia , Infecções por Campylobacter/epidemiologia , Doenças Transmitidas por Alimentos/epidemiologia , Gastroenterite/epidemiologia , Intoxicação Alimentar por Salmonella/epidemiologia , Vibrioses/epidemiologia , Campylobacter/patogenicidade , Surtos de Doenças , Doenças Transmitidas por Alimentos/microbiologia , Doenças Transmitidas por Alimentos/virologia , Gastroenterite/virologia , Humanos , Incidência , Japão/epidemiologia , Norovirus/patogenicidade , Salmonella/patogenicidade , Infecções por Salmonella/epidemiologia , Vibrio parahaemolyticus/patogenicidade
12.
Nihon Saikingaku Zasshi ; 75(4): 215-225, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-33390409

RESUMO

Vibrio parahaemolyticus, one of the Gram-negative common enteric pathogens, was first isolated in Japan in 1950. Since its discovery, this bacterium has been a major cause of food-poisoning in Japan, and its infection has recently undergone a global expansion. V. parahaemolyticus possesses a classical exotoxin, thermostable direct hemolysin, and two sets of type III secretion systems (T3SSs) that are able to inject effectors directly into host cells, which are its key virulence factors. Exotoxin/effector is exploited by many Gram-negative pathogens, and plays critical roles in pathogenesis by damaging host cells or by modulating host cell functions, through its activity on/in host cells. In recent years, functional activities of T3SS effectors produced by V. parahaemolyticus have been extensively studied, which has substantially increased our understanding of the pathogenic mechanisms of the bacterium. In paricular, some T3SS effectors of V. parahaemolyticus act as cytotoxins and thereby damage host cells. Here, I focus on these cytotoxic effectors of V. parahaemolyticus and describe recent advances in our understanding of their mechanisms of action.


Assuntos
Citotoxinas/toxicidade , Exotoxinas/toxicidade , Sistemas de Secreção Tipo III , Vibrio parahaemolyticus/patogenicidade , Virulência , Doenças Transmitidas por Alimentos/microbiologia , Proteínas Hemolisinas , Interações entre Hospedeiro e Microrganismos , Humanos
13.
BMC Genomics ; 20(1): 988, 2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31847806

RESUMO

BACKGROUND: Vibrio spp. is the major infection-producing marine bacteria in commercially important bivalve Paphia undulata. The host resistance is the major determining factor for the development of pathogenesis. To explore defense mechanisms, researchers have focused primarily on the study of differential expression of individual or specific groups of host immune genes during pathogen-challenge. RESULTS: We compared the expression profile in the surf clams infected with avirulent V. alginolyticus and virulent V. parahaemolyticus to mark the possible molecular mechanisms of pathogenesis. Comparison of the differentially expressed genes between the two groups of Vibrio-infected clams revealed that the number of down-regulate genes in V. parahaemolyticus injected clams (1433) were significantly higher than the other group (169). Based on Gene Ontology classification, a large proportion of these down-regulate genes were found to be associated with cellular and molecular mechanisms for pathogen recognition, and immunity development thereby explaining the low survival rate for the V. parahaemolyticus-treated clams and suggesting a higher virulence of this bacterium towards the surf clams. Quantitative real-time PCR of 24 candidate genes related to immunity involving the JAK-STAT signaling pathway, complementary cascade, cytokine signaling pathway, oxidative stress, phagocytosis and apoptosis down regulated under V. parahaemolyticus infection, indicating compromised host defense. Furthermore, we could demonstrate a central role of JAK-STAT pathway in bacterial clearance. dsRNA mediated depletion of a clam STAT homolog gene results in dramatic increase in the infection by V. alginolyticus, a mildly pathogenic strain under control conditions. CONCLUSIONS: The difference in gene expression profiles in surf clams treated with two Vibrio species with a differential pathogenicity to P. undulate and downstream molecular analysis could enlighten on the probable molecular mechanisms of the Vibrio pathogenesis and the virulence of V. parahaemolyticus in surf clams, which also benefits to develop new strategies for disease control in surf calm aquaculture.


Assuntos
Bivalves/genética , Bivalves/microbiologia , Vibrio parahaemolyticus/patogenicidade , Animais , Bivalves/imunologia , Bivalves/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Imunidade Inata/genética , Janus Quinases/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo
14.
BMC Microbiol ; 19(1): 270, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31796006

RESUMO

BACKGROUND: Due to its rapid lethal effect in the early development stage of shrimp, acute hepatopancreatic necrosis disease (AHPND) has been causing great economic losses, since its first outbreak in southeast China in 2009. Vibrio parahaemolyticus, carrying the pirA and pirB toxin genes is known to cause AHPND in shrimp. The overall objective of this study was to sequence the whole genome of AHPND positive V. parahaemolyticus strains isolated from shrimp (Peneaus monodon) of the south-west region of Bangladesh in 2016 and 2017 and characterize the genomic features and emergence pattern of this marine pathogen. RESULTS: Two targeted AHPND positive V. parahaemolyticus strains were confirmed using PCR with 16S rRNA, ldh, AP3 and AP4 primers. The assembled genomes of strain MSR16 and MSR17 were comprised of a total of 5,393,740 bp and 5,241,592 bp, respectively. From annotation, several virulence genes involved in chemotaxis and motility, EPS type II secretion system, Type III secretion system-1 (T3SS-1) and its secreted effectors, thermolabile hemolysin were found in both strains. Importantly, the ~ 69 kb plasmid was identified in both MSR16 and MSR17 strains containing the two toxin genes pirA and pirB. Antibiotic resistance genes were predicted against ß-lactam, fluoroquinolone, tetracycline and macrolide groups in both MSR16 and MSR17 strains. CONCLUSIONS: The findings of this research may facilitate the tracking of pathogenic and/or antibiotic-resistant V. parahaemolyticus isolates between production sites, and the identification of candidate strains for the production of vaccines as an aid to control of this devastating disease. Also, the emergence pattern of this pathogen can be highlighted to determine the characteristic differences of other strains found all over the world.


Assuntos
Evolução Molecular , Genoma Bacteriano , Hepatopâncreas/microbiologia , Penaeidae/microbiologia , Vibrio parahaemolyticus/genética , Doença Aguda , Animais , Proteínas de Bactérias/genética , Bangladesh , Farmacorresistência Bacteriana Múltipla/genética , Genômica , Hepatopâncreas/patologia , Necrose/microbiologia , Necrose/veterinária , Plasmídeos , RNA Ribossômico 16S/genética , Alimentos Marinhos , Vibrio parahaemolyticus/patogenicidade
15.
Sci Rep ; 9(1): 19571, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31862956

RESUMO

We depicted the epidemiological characteristics of infectious diarrhoea in Jiangsu Province, China. Generalized additive models were employed to evaluate the age-specific effects of etiological and meteorological factors on prevalence. A long-term increasing prevalence with strong seasonality was observed. In those aged 0-5 years, disease risk increased rapidly with the positive rate of virus (rotavirus, norovirus, sapovirus, astrovirus) in the 20-50% range. In those aged > 20 years, disease risk increased with the positive rate of adenovirus and bacteria (Vibrio parahaemolyticus, Salmonella, Escherichia coli, Campylobacter jejuni) until reaching 5%, and thereafter stayed stable. The mean temperature, relative humidity, temperature range, and rainfall were all related to two-month lag morbidity in the group aged 0-5 years. Disease risk increased with relative humidity between 67-78%. Synchronous climate affected the incidence in those aged >20 years. Mean temperature and rainfall showed U-shape associations with disease risk (with threshold 15 °C and 100 mm per month, respectively). Meanwhile, disease risk increased gradually with sunshine duration over 150 hours per month. However, no associations were found in the group aged 6-19 years. In brief, etiological and meteorological factors had age-specific effects on the prevalence of infectious diarrhoea in Jiangsu. Surveillance efforts are needed to prevent its spread.


Assuntos
Diarreia/epidemiologia , Adolescente , Adulto , Campylobacter jejuni/patogenicidade , Criança , Pré-Escolar , China/epidemiologia , Diarreia/etiologia , Diarreia/microbiologia , Escherichia coli/patogenicidade , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Salmonella/patogenicidade , Temperatura , Vibrio parahaemolyticus/patogenicidade , Adulto Jovem
16.
Fish Shellfish Immunol ; 95: 519-527, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31683000

RESUMO

Kruppel-like factors (KLFs) belong to a family of zinc finger-containing transcription factors that are widely present in eukaryotes. In the present study, a novel KLF from the giant river prawn Macrobrachium rosenbergii (designated as MrKLF) was successfully cloned and characterized. The full-length cDNA of MrKLF was 1799 bp with an open reading frame of 1332 bp that encodes a putative protein of 444 amino acids, including three conserved ZnF_C2H2 domains at the C-terminus. Multiple alignment analysis showed that MrKLF and other crustacean KLFs shared high similarity. Quantitative real-time PCR analysis revealed that MrKLF mRNA was found in different tissues of prawns and detected in the gills, hepatopancreas, and intestines. After the challenge with Vibrio parahaemolyticus and Aeromonas hydrophila, different expression patterns of MrKLF in the gills, intestines, and hepatopancreas were observed. RNA interference analysis indicated that MrKLF was involved in regulating the expression of four antimicrobial peptides, namely, Crustin (Crus) 2, Crus8, anti-lipopolysaccharide factor (ALF) 1, and ALF3. These results help promote research on M. rosenbergii innate immunity.


Assuntos
Proteínas de Artrópodes/genética , Infecções Bacterianas/veterinária , Imunidade Inata , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/imunologia , Palaemonidae/imunologia , Aeromonas hydrophila/imunologia , Aeromonas hydrophila/patogenicidade , Animais , Proteínas de Artrópodes/imunologia , Infecções Bacterianas/imunologia , Clonagem Molecular , Brânquias/imunologia , Hepatopâncreas/imunologia , Intestinos/imunologia , Fases de Leitura Aberta , Palaemonidae/microbiologia , RNA Mensageiro , Vibrio parahaemolyticus/imunologia , Vibrio parahaemolyticus/patogenicidade
17.
Fish Shellfish Immunol ; 95: 456-463, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31669282

RESUMO

p38 mitogen-activated protein kinases (MAPKs) are involved in the response to various extracellular stimuli via regulating gene expression. In the present study, a p38 MAPK gene (MpP38) was identified from the clam Meretrix petechialis. The full-length cDNA of MpP38 measures 1,720 bp, consisting of a 134-bp 5'-UTR, a 1,095-bp ORF and a 491-bp 3'-UTR. Both the mRNA and protein expression levels of MpP38 increased after Vibrio challenge, implying that MpP38 is involved in clam immunity. Based on our previous study, a transcription factor activated by p38 MAPK, i.e. microphthalmia-associated transcription factor (MITF), participated in clam immunity by regulating the expression of phenoloxidase (PO). Coupled with other related reports, the mechanism underlying the involvement of MpP38 in clam immunity is most likely that pathogen stimuli induce the phosphorylation of p38 MAPK and thus activate MITF to regulate the expression of the immune-related gene PO. The results obtained in this study support this mechanism. First, we found that the MpP38 phosphorylation level increased in response to Vibrio challenge. Second, as revealed by a yeast two-hybrid assay, there was a direct interaction between MpP38 and MITF. Meanwhile, inhibiting the phosphorylation of MpP38 decreased the phosphorylation level of MpMITF, implying that MpP38 phosphorylation is required for MpMITF activation. Additionally, our results showed that there was a regulatory relationship between MpP38 phosphorylation level and PO expression level. With increased MpP38 phosphorylation level, the PO expression level was also increased after Vibrio challenge; when MpP38 phosphorylation was inhibited, the PO expression level was significantly decreased. This study describes the immune function of p38 MAPK in the clam for the first time and analyses its potential underlying mechanism, which will help to elucidate the immune mechanism in the clam M. petechialis.


Assuntos
Bivalves/imunologia , Bivalves/microbiologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Vibrio parahaemolyticus/patogenicidade , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Regulação da Expressão Gênica , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Fosforilação , Técnicas do Sistema de Duplo-Híbrido , Proteínas Quinases p38 Ativadas por Mitógeno/genética
18.
Biomed J ; 42(3): 187-192, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31466712

RESUMO

BACKGROUND: Vibrio parahaemolyticus is a Gram-negative bacterium widely distributed in marine environments and a well-recognized invertebrate pathogen frequently isolated from seafood. V. parahaemolyticus may also spread into humans, via contaminated, raw, or undercooked seafood, causing gastroenteritis and diarrhea. METHODS: A Nuclear Magnetic Resonance (NMR)-based detection system was used to detect pathogenic levels of this microorganism (105 CFU/ml) with Molecular Mirroring using iron nanoparticles coated with target-specific biomarkers capable of binding to DNA of the target microorganism. The NMR system generates a signal (in milliseconds) by measuring NMR spin-spin relaxation time T2, which correlates with the amount of microorganism DNA. RESULTS: Compared with conventional microbiology techniques such as real-time PCR (qPCR), the NMR biosensor showed similar limits of detection (LOD) at different concentrations (105-108 CFU/ml) using two DNA extraction methods. In addition, the NMR biosensor system can detect a wide range of microorganism DNAs in different matrices within a short period of time. CONCLUSION: NMR biosensor represents a potential tool for diagnostic and quality control to ensure microbial pathogens such as V. parahaemolyticus are not the cause of infection. The "hybrid" technology (NMR and nanoparticle application) opens a new platform for detecting other microbial pathogens that have impacted human health, animal health and food safety.


Assuntos
Imagem por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Reação em Cadeia da Polimerase , Vibrio parahaemolyticus/patogenicidade , Animais , Técnicas Biossensoriais/métodos , Humanos , Imagem por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Reação em Cadeia da Polimerase/métodos , Vibrio parahaemolyticus/citologia
19.
Food Microbiol ; 84: 103258, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31421777

RESUMO

The aim of the present study was to investigate the genetic variability of Vibrio parahaemolyticus strains isolated from naturally contaminated Mediterranean mussels (Mytilus galloprovincialis) and Grooved carpet shells (Ruditapes decussatus) from three harvesting areas of Sardinia (Italy) using a combination of different typing methods: traditional phenotypic systems and molecular techniques. Ninety-nine putative V. parahaemolyticus strains isolated from shellfish collected before and after purification were included in the study. Seventy-two isolates were confirmed as V. parahaemolyticus and were submitted to REP, ERIC and BOX PCRs. The combined dendrogram showed the similarity of the data set of the three typing methods and demonstrates how the different techniques grouped the strains in two clusters in accordance with each singular dendrogram. Several strains rendered a unique pattern regardless of the typing method, which indicates the high discriminatory power of the methods. Moreover, the use of multiple typing methods allowed a more accurate characterization of the genetic profiles of isolates and the identification of clones hardly revealed through the common techniques. The intraspecific typing of environmental V. parahaemolyticus can be of great interest in order to recognize clonal relationships between environmental contamination, foodborne disease, and geographical/temporal distribution of this pathogen. The comparative analysis focusing on the obtained genetic profiles supports the possibility for typing methods to discriminate strains with similar phenotypic profile, identifying the level of genetic correlation among the strains and the presence of genetic clones.


Assuntos
Variação Genética , Mytilus/microbiologia , Alimentos Marinhos/microbiologia , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/genética , Animais , Técnicas de Tipagem Bacteriana , Itália , Vibrio parahaemolyticus/patogenicidade
20.
Food Microbiol ; 84: 103233, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31421792

RESUMO

Globally, V. parahaemolyticus infection is a leading cause of bacterial diarrheal diseases. Pathogenic V. parahaemolyticus strains that produce hemolysins are responsible for these diseases. The composition of pathogenic and non-pathogenic V. parahaemolyticus and the change of the bacterial composition before and after traditional selective enrichment in a single sample associated with disease outbreak remain unclear. We investigated an outbreak by using next generation sequencing and digital PCR to address those questions. NGS showed that the V. parahaemolyticus caused the outbreak belonged to s single clone. In contrast, among the seven non-pathogenic V. parahaemolyticus isolated from the suspected food sample, 4 serotypes and 6 PFGE patterns were identified. And nearly 70,000 SNPs were identified among the non-pathogenic strains. This result confirmed that the outbreak was caused by V. parahaemolyticus. Furthermore, NGS results clearly showed the diversity of non-pathogenic V. parahaemolyticus in a single contaminated food sample. The ratios of non-pathogenic and pathogenic V. parahaemolyticus were 31.41 and 620.11 in the original and enriched food samples respectively showed by digital PCR. Meta-genomic data indicated the top 3 species were Weissella cibaria, Weissella confusa, and Enterobacter cloacae in the original food sample, and Vibrio sp Ex25, Vibrio sp 712i, and V. parahaemolyticus in the enriched sample. Therefore, the combing of NGS and digital PCR results showed that traditional Vibrio selective enrichment media could facilitate the growth of Vibrios, however, it provided no advantages to pathogenic V. parahaemolyticus. Hence, our results indicated that the traditional culture methods alone may lead to wrong conclusions and so improvements in culture methods are needed.


Assuntos
Diarreia/microbiologia , Surtos de Doenças , Doenças Transmitidas por Alimentos/etiologia , Doenças Transmitidas por Alimentos/microbiologia , Vibrioses/diagnóstico , Vibrio parahaemolyticus/genética , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reação em Cadeia da Polimerase , Vibrioses/microbiologia , Vibrio parahaemolyticus/patogenicidade
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