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An Innovative Method for Rapid Identification and Detection of Vibrio alginolyticus in Different Infection Models.

ABSTRACT: Vibrio alginolyticus is one of the most common pathogenic marine Vibrio species, and has been found to cause serious seafood-poisoning or fatal extra-intestinal infections in humans, such as necrotizing soft-tissue infections, bacteremia, septic shock, and multiple organ failures. Delayed accurate diagnosis and treatment of most Vibrio infections usually result to high mortality rates. The objective of this study was to establish a rapid diagnostic method to detect and identify the presence of V. alginolyticus in different samples, so as to facilitate timely treatment. The widely employed conventional methods for detection of V. alginolyticus include biochemical identification and a variety of PCR methods. The former is of low specificity and time-consuming (2-3 days), while the latter has improved accuracy and processing time. Despite such advancements, these methods are still complicated, time-consuming, expensive, require expertise and advanced laboratory systems, and are not optimal for field use. With the goal of providing a simple and efficient way to detect V. alginolyticus, we established a rapid diagnostic method based on loop-mediated Isothermal amplification (LAMP) technology that is feasible to use in both experimental and field environments. Three primer pairs targeting the toxR gene of V. alginolyticus were designed, and amplification was carried out in an ESE tube scanner and Real-Time PCR device. We successfully identified 93 V. alginolyticus strains from a total of 105 different bacterial isolates and confirmed their identity by 16s rDNA sequencing. We also applied this method on infected mouse blood and contaminated scallop samples, and accurate results were both easily and rapidly (20-60 min) obtained. Therefore, the RT-LAMP assay we developed can be conveniently used to detect the presence of V. alginolyticus in different samples. Furthermore, this method will also fulfill the gap for real-time screening of V. alginolyticus infections especially while on field.

SUBMITTER: Fu K

PROVIDER: S-EPMC4858747 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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An Innovative Method for Rapid Identification and Detection of Vibrio alginolyticus in Different Infection Models.

Fu Kaifei K Li Jun J Wang Yuxiao Y Liu Jianfei J Yan He H Shi Lei L Zhou Lijun L

Frontiers in microbiology 20160506

Vibrio alginolyticus is one of the most common pathogenic marine Vibrio species, and has been found to cause serious seafood-poisoning or fatal extra-intestinal infections in humans, such as necrotizing soft-tissue infections, bacteremia, septic shock, and multiple organ failures. Delayed accurate diagnosis and treatment of most Vibrio infections usually result to high mortality rates. The objective of this study was to establish a rapid diagnostic method to detect and identify the presence of V ...[more]

PMID: 27199971

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Project description:BACKGROUND:Vibrio alginolyticus is an important pathogen that has to be closely monitored and controlled in the mariculture industry because of its strong pathogenicity, quick onset after infection and high mortality rate in aquatic animals. Fast, simple and specific methods are needed for on-site detection to effectively control outbreaks and prevent economic losses. The detection specificity towards the pathogenic strains has to be emphasized to facilitate pointed treatment and prevention. Polymerase chain reaction (PCR)-based molecular approaches have been developed, but their application is limited due to the requirement of complicated thermal cycling machines and trained personnel. RESULTS:A fast, simple and highly specific detection method for V. alginolyticus pathogenic strains was established based on isothermal recombinase polymerase amplification (RPA) and lateral flow dipsticks (LFD). The method targeted the virulence gene toxR, which is reported to have good coverage for V. alginolyticus pathogenic strains. To ensure the specificity of the method, the primer-probe set of the RPA system was carefully designed to recognize regions in the toxR gene that diverge in different Vibrio species but are conserved in V. alginolyticus pathogenic strains. The primer-probe set was determined after a systematic screening of amplification performance, primer-dimer formation and false positive signals. The RPA-LFD method was confirmed to have high specificity for V. alginolyticus pathogenic strains without any cross reaction with other Vibrio species or other pathogenic bacteria and was able to detect as little as 1 colony forming unit (CFU) per reaction without DNA purification, or 170?fg of genomic DNA, or 6.25?×?103?CFU/25?g in spiked shrimp without any enrichment. The method finishes detection within 30?min at temperatures between 35?°C and 45?°C, and the visual signal on the dipstick can be directly read by the naked eye. In an application simulation, randomly spiked shrimp homogenate samples were 100% accurately detected. CONCLUSIONS:The RPA-LFD method developed in this study is fast, simple, highly specific and does not require complicated equipment. This method is applicable for on-site detection of V. alginolyticus pathogenic strains for the mariculture industry.

Project description:Penaeus monodon is highly susceptible to vibriosis disease. Aims of the study were to identify the pathogen causing vibriosis in P. monodon through molecular techniques and develop a biocontrol method of the disease by application of herbal extracts. Shrimp samples were collected aseptically from the infected farm and the bacteria were isolated from the infected region of those samples. Based on phenotypic identification, several isolates were identified as Vibrio sp. 16S rRNA gene sequences of the selected isolates exhibited 100% homology with V. alginolyticus strain ATCC 17749. An in vivo infection challenge test was performed by immersion method with V. alginolyticus where these isolates caused high mortality in juvenile shrimp with prominent symptoms of hepatopancreatic necrosis. Antibiogram profile of the isolates was determined against eleven commercial antibiotic discs whereas the isolates were found resistant to multiple antibiotics. A total of twenty-one herbal extracts were screened where Emblica officinalis, Allium sativum, and Syzygium aromaticum strongly inhibited the growth of V. alginolyticus in in vitro conditions. In in vivo conditions, the ethyl acetate extracts of E. officinalis and A. sativum successfully controlled the vibriosis disease in shrimp at a dose of 10 mg/g feed. This is the first report on molecular identification and biocontrol of V. alginolyticus in shrimp in Bangladesh.Penaeus monodon is highly susceptible to vibriosis disease. Aims of the study were to identify the pathogen causing vibriosis in P. monodon through molecular techniques and develop a biocontrol method of the disease by application of herbal extracts. Shrimp samples were collected aseptically from the infected farm and the bacteria were isolated from the infected region of those samples. Based on phenotypic identification, several isolates were identified as Vibrio sp. 16S rRNA gene sequences of the selected isolates exhibited 100% homology with V. alginolyticus strain ATCC 17749. An in vivo infection challenge test was performed by immersion method with V. alginolyticus where these isolates caused high mortality in juvenile shrimp with prominent symptoms of hepatopancreatic necrosis. Antibiogram profile of the isolates was determined against eleven commercial antibiotic discs whereas the isolates were found resistant to multiple antibiotics. A total of twenty-one herbal extracts were screened where Emblica officinalis, Allium sativum, and Syzygium aromaticum strongly inhibited the growth of V. alginolyticus in in vitro conditions. In in vivo conditions, the ethyl acetate extracts of E. officinalis and A. sativum successfully controlled the vibriosis disease in shrimp at a dose of 10 mg/g feed. This is the first report on molecular identification and biocontrol of V. alginolyticus in shrimp in Bangladesh.

Dataset Information

An Innovative Method for Rapid Identification and Detection of Vibrio alginolyticus in Different Infection Models.

Publications

An Innovative Method for Rapid Identification and Detection of Vibrio alginolyticus in Different Infection Models.

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