Detection, Enumeration, and Isolation of Vibrio parahaemolyticus and V. vulnificus from Seafood: Development of a Multidisciplinary Protocol

2017 ◽  
Vol 100 (2) ◽  
pp. 445-453 ◽  
Author(s):  
Swapan K Banerjee ◽  
Jeffrey M Farber
Keyword(s):  
Foodborne Pathogens ◽  
Vibrio Parahaemolyticus ◽  
Cost Effective ◽  
Molecular Methods ◽  
Biochemical Tests ◽  
Selective Media ◽  
Isolation And Characterization ◽  
Species Specific ◽  
Pcr Targeting ◽  
Vibrio Spp

Abstract Vibrio parahaemolyticus and V. vulnificus are bacterial foodborne pathogens that can cause illnesses in humans after ingestion or exposure to contaminated seafood or coastal waters. Aprocedure that combines microbiological, biochemical, and molecular methods was designed and optimized for thedetection, enumeration, isolation, and characterization of these clinically significant Vibrio spp. Initially, microbiological culturing is used to resuscitate and isolate presumptive Vibrio spp. from chilled seafood samples. Biochemical tests are then used to analyze and select presumptive isolates at the species level, and, lastly, molecular methods, such as PCR targeting species-specific hemolysin genes, are used to confirm identification and assess the potential pathogenicity of presumptive isolates. By using artificially contaminated molluscan homogenates with known numbers of V. parahaemolyticus,this method yielded, on average, 90% recovery on complete agar media and 88% recovery on selective media. For V. vulnificus, the recovery rates were 86% (complete media) and 84% (selective media). Linearity of recovery of Vibrio spp. from artificially contaminated seafood homogenates supportedthe applicability of this method. Overall, this performance-tested protocol is easy to use, cost-effective, and fit-for-purpose, with potential for routine use in basic microbiological facilities.

Use of Oligonucleotide Array for Identification of Six Foodborne Pathogens and Pseudomonas aeruginosa Grown on Selective Media

2005 ◽  
Vol 68 (11) ◽  
pp. 2278-2286 ◽  
Author(s):  
MIAO CHU LIN ◽  
AY HUEY HUANG ◽  
HAU YANG TSEN ◽  
HIN-CHUNG WONG ◽  
TSUNG CHAIN CHANG
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Foodborne Pathogens ◽  
Intergenic Spacer ◽  
Oligonucleotide Array ◽  
Universal Primers ◽  
Morphological Observation ◽  
Biochemical Tests ◽  
Nylon Membrane ◽  
Selective Media ◽  
Species Specific

Identification of presumptive foodborne pathogens grown on selective media may take one to several days and requires a different battery of biochemical tests for each microorganism. A molecular identification method was developed in which universal primers were used to amplify the 16S to 23S rDNA intergenic spacer of target microorganisms, and PCR products were hybridized to a panel of species-specific oligonucleotides that were immobilized on a nylon membrane. The seven target microorganisms were Bacillus cereus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, and Vibrio parahaemolyticus. After testing a large collection of target bacteria (29 to 51 strains) and nontarget bacteria (>500 strains), the performances (sensitivity and specificity) of the oligonucleotide array were as follows: B. cereus (100 and 77%), E. coli (100 and 100%), L. monocytogenes (100 and 90%), P. aeruginosa (100 and 100%), Salmonella (100 and 100%), S. aureus (100 and 100%), and V. parahaemolyticus (100 and 94.2%). Other species in the B. cereus group cross-hybridized to the probes used for identification of B. cereus, and positive results should be confirmed by additional morphological observation of colonies. Listeria innocua cross-reacted with probes used to identify L. monocytogenes, but a simple hemolysis test was used to differentiate the two species. Some strains of Vibrio harveyi and Vibrio mimicus cross-hybridized with probes used for identification of V. parahaemolyticus and caused false-positive reactions. The advantage of the array is that a common protocol was used to identify the seven target microorganisms and multiple different microorganisms could be simultaneously identified on a single array.

scholarly journals Isolation and Characterization of Vibrio parahaemolyticus in the hepatopancreas of cultured white pacific shrimp - Litopenaeus vannamei

2020 ◽  
Vol 11 (1) ◽  
pp. 797-805
Author(s):  
Kolli Guna Ranjan ◽  
Girija Sankar G ◽  
Satyanarayana Raju DVV
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Bacterial Flora ◽  
Distance Matrix ◽  
Common Species ◽  
Genetic Identification ◽  
Molecular Technique ◽  
Microbial Culture ◽  
Biochemical Tests ◽  
Isolation And Characterization

Vibrio parahaemolyticus is the most common species among crustaceans, often causing various diseases and significant losses in aquaculture. Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease that has severely damaged the global shrimp industry. This species of bacteria is associated with gastrointestinal illness in humans and has been implicated in foodborne disease. The present study carried out, isolation and characterization of pathogenic bacterial flora isolated from the infected hepatopancreas of vannamei, obtained from various aquafarms in Andhra Pradesh, India, on 11th June 2018. The collected samples were plated on TCBS- (Thiosulfate-Citrate-Bile salt-Sucrose) agar medium and Hi -Chrome vibrio, as described in Bergey's manual of systematic bacteriology. Isolated colonies were subjected to the following tests- microscopic examination, growth at different temperatures, growth at different NaCl concentrations, and biochemical tests. Further purity, maintenance, and propagation of purified cultures were done. The microbial culture was identified using 16s rRNA molecular technique. Phylogenetic Evolutionary analyses and distance matrix were conducted in MEGA7.In the present study, different samples were screened, a total of three green colonies (V44, V45, V46) were isolated, identified by biochemical tests and genetic identification as Vibrio parahaemolyticus. A systematic methodology has been developed to isolate and characterize Vibrio sp. from diseased shrimp and identify them by genetic analysis

scholarly journals Rapid detection of Vibrio species using liquid microsphere arrays and real-time PCR targeting the ftsZ locus

2007 ◽  
Vol 56 (1) ◽  
pp. 56-65 ◽  
Author(s):  
Dobryan M. Tracz ◽  
Paul G. Backhouse ◽  
Adam B. Olson ◽  
Joanne K. McCrea ◽  
Julie A. Walsh ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Rapid Detection ◽  
Vibrio Vulnificus ◽  
Comparative Sequence Analysis ◽  
Molecular Techniques ◽  
Array Technology ◽  
Species Specific ◽  
Pcr Targeting

The development of rapid and sensitive molecular techniques for the detection of Vibrio species would be useful for the surveillance of sporadic infections and management of major outbreaks. Comparative sequence analysis of the ftsZ gene in the predominant Vibrio species that cause human disease revealed distinct alleles for each examined species, including Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Light Upon eXtension (LUX) real-time PCR assays were developed to target these species-specific polymorphisms, and were successful in rapidly differentiating the major pathogenic Vibrio species. Luminex liquid microsphere array technology was used to develop a comprehensive assay capable of simultaneously detecting V. cholerae, V. parahaemolyticus and V. vulnificus. These assays permitted the identification of a presumptive V. parahaemolyticus isolate as Vibrio alginolyticus, which was verified using additional molecular characterization.

NGHIÊN CỨU ĐẶC ĐIỂM GÂY BỆNH VÀ DỊCH TỄ CỦA BỆNH TÔM CHẾT SỚM (EMS) TẠI TỈNH QUẢNG BÌNH NHẰM ĐỀ XUẤT QUY TRÌNH CHẨN ĐOÁN VÀ PHÒNG TRỊNGHIÊN CỨU ĐẶC ĐIỂM DỊCH TỄ CỦA BỆNH TÔM CHẾT SỚM (EMS) TẠI TỈNH QUẢNG BÌNH NHẰM ĐỀ XUẤT QUY TRÌNH CHẨN ĐOÁN VÀ PHÒNG TRỊ

2015 ◽  
Vol 104 (5) ◽  
Author(s):  
Trần Thị Linh Giang ◽  
Dương Viết Phương Tuấn
Keyword(s):  
Bile Salt ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Spp

Nghiên cứu về Hội chứng chết sớm ở tôm thực hiện ở Quảng Bình với mục đích tìm hiểu đặc điểm gây bệnh của vi khuẩn Vibrio paraheamolyticus và đặc điểm dịch tể để khuyến cao cách phòng trị và có dự báo sớm làm giảm rủi roc ho nghề nuôi tôm. Có 120 phiếu và 91 mẫu tôm nghi bệnh được thu và nuôi cấy, tìm hiểu đặc điểm vi khuẩn này và phân tích gen để xác định độc tố, đồng thời nghiên cứu về đặc điểm dịch tễ của bệnh EMS ở 4 huyện, thành phố . Kết quả cho thấy rằng hơn 70% số mẫu nghi bệnh có kết quả dương tính, tần suất nhiễm bệnh cao 10 – 60,6 % và khác nhau ở các tháng và vụ nuôi, cao nhất vào tháng 4 – 7 DL, X2 = 1.60 (df = 4), với P < 0,05. Tôm nhiễm bệnh EMS có các biểu hiện các triệu chứng điển hình gan tuỵ và tỷ lệ chết cao đến 100% nếu không can thiệp kịp thời. Đặc điểm chung các loài vi khuẩn thuộc giống Vibrio: Gram âm, hình que thẳng hoặc hơi uốn cong, kích thước 0,3-0,5 x 1,4-2,6 μm, không hình thành bào tử và chuyển động nhờ một tiên mao hoặc nhiều tiên mao mảnh và yếm khí, hầu hết là oxy hoá và lên men trong môi trường O/F Glucose. Thiosulphate citrate bile salt agar (TCBS) là môi trường chọn lọc của Vibrio spp. Chúng mẫn cảm với Vibriostat 2,4 diamino-6,7 diisopropyl pteridine phosphate (0/129). Tỷ lệ V. parahaemolyticus gây bệnh tôm chết sớm.Vi khuẩn V. parahaemolyticus gây bệnh có đặc điểm cấu trúc gen khác biệt, nhiễm sắc thể tự điều chỉnh nằm ở vị trí 01. Từ những kết quả hình ảnh trên ta thấy được sự sai khác về trình tự gen DNA của mẫu W1, trình tự gen của mẫu này trùng hợp với trình tự gen DNA của Vibrio parahaemolyticus dùng đối chứng trên và sự sai khác về trình tự gen cũng đã thể hiện được khả năng gây bệnh của vi khuẩn V. parahaemolyticus. Các phản ứng với các loại kháng sinh có hiệu quả từ 8 – 45%, đều làm giảm số lượng tôm chết khi nhiễm, cao nhất là Baymet và Osamet, Olimos. Sử dụng chế phẩm Bokashi trầu với hiệu quả tốt nếu dùng từ đầu vụ và đến cuối vụ, với thành phần Eugenol, chavicol và chavibetol đã hạn chế sự phát triển của bệnh, kể cả những ao có mật độ V. parahaemolyticus cao nhưng ít có nguy cơ gây bệnh.

scholarly journals Conversion of sequence-characterized amplified region (SCAR) bands into high-throughput DNA markers based on RAPD technique for detection of the stem nematode Ditylenchus dipsaci in crucial plant hosts

2008 ◽  
Vol 53 (No. 3) ◽  
pp. 97-104 ◽  
Author(s):  
M. Zouhar ◽  
M. Marek ◽  
O. Douda ◽  
J. Mazáková ◽  
P. Ryšánek
Keyword(s):  
Cost Effective ◽  
Healthy Plant ◽  
Host Preferences ◽  
Sequence Characterized Amplified Region ◽  
Detection And Identification ◽  
Plant Hosts ◽  
Cost Effective Technique ◽  
Ditylenchus Dipsaci ◽  
Species Specific ◽  
Template Dna

<i>Ditylenchus dipsaci</i>, the stem nematode, is a migratory endoparasite of over 500 species of angiosperms. The main method of <i>D. dipsaci</i> control is crop rotation, but the presence of morphologically indistinguishable host races with different host preferences makes rotation generally ineffective. Therefore, a sensitive, rapid, reliable, as well as cost effective technique is needed for identification of <i>D. dipsaci</i> in biological samples. This study describes the development of species-specific pairs of PCR oligonucleotides for detection and identification of the <i>D. dipsaci</i> stem nematode in various plant hosts. Designed DIT-2 primer pair specifically amplified a fragment of 325 bp, while DIT-5 primer pair always produced a fragment of 245 bp in all <i>D. dipsaci</i> isolates. Two developed SCAR primer pairs were further tested using template DNA extracted from a collection of twelve healthy plant hosts; no amplification was however observed. The developed PCR protocol has proved to be quite sensitive and able to specifically detect <i>D. dipsaci</i> in artificially infested plant tissues.

scholarly journals Isolation and Characterization of a Bacterial Strain Enterobacter cloacae (Accession No. KX438060.1) Capable of Degrading DDTs Under Aerobic Conditions and Its Use in Bioremediation of Contaminated Soil

2021 ◽  
Vol 14 ◽  
pp. 117863612110242
Author(s):  
Sonal Suman ◽  
Tanuja
Keyword(s):  
Contaminated Soil ◽  
Growth Parameters ◽  
Chemical Properties ◽  
Enterobacter Cloacae ◽  
Maximum Growth ◽  
Bacterial Degradation ◽  
Biochemical Tests ◽  
Isolation And Characterization ◽  
16S Rna ◽  
Degrading Bacteria

DDT is one of the most persistent pesticides among all the different types of organo-chlorine pesticides used. Among all the degradation methods, bacterial degradation of DDT is most effective. The present study was conducted to isolate different bacteria present in waste samples which have the ability to degrade DDT present in the soil in the minimum possible period of time and to observe the effect of different physical and chemical properties of the soil samples. Many pesticide degrading bacteria were isolated and identified through cultural, biochemical tests and further identified by 16S RNA sequencing method. The most potent strain DDT 1 growth in mineral salt medium supplemented with DDT as the only source of carbon (5-100 PPM) and was monitored at an optical density of 600 nm. The growth parameters at different physio-chemical conditions were further optimized. The result showed that Enterobacter cloacae had maximum growth in 15 days. FTIR analysis of the residual DDT after 15 days incubation showed that Enterobacter cloacae was able to degrade pesticide into its further metabolites of DDD, DDE, DDNU and other components can be used for biodegradation of DDT present in contaminated soil and water ecosystems.

scholarly journals Isolation and characterization of Pseudomonas syringae isolates affecting stone fruits and almond in Montenegro

2021 ◽  
Author(s):  
Tamara Popović ◽  
Jelena Menković ◽  
Anđelka Prokić ◽  
Nevena Zlatković ◽  
Aleksa Obradović
Keyword(s):  
Pseudomonas Syringae ◽  
Housekeeping Genes ◽  
Molecular Characteristics ◽  
Biochemical Tests ◽  
High Genetic Diversity ◽  
Leaf Petiole ◽  
Molecular Features ◽  
Isolation And Characterization ◽  
Genomic Species

AbstractIn Montenegro, stone fruit species are grown on intensive and semi-intensive commercial plantations. However, almond production is mainly organized on family gardens and for household consumption. During two seasons (2017–2018), we surveyed apricot, peach, nectarine, sweet cherry, Japanese plum, and almond orchards for the presence of bacterial diseases at different geographical locations in Montenegro. From leaf, petiole and fruit lesions, branch or twig cankers, and necrotizing buds, a total of 29 isolates were obtained and subjected to identification based on their morphological, pathogenic, biochemical, and molecular characteristics. Pathogenicity of the isolates was confirmed by reproducing the symptoms on leaves, fruits, and twigs of the corresponding host plants. The biochemical tests indicated that the isolates belong to Pseudomonas syringae. However, isolates’ characterization showed variation in their phenotypic and molecular features. The presence of the syrB gene and ice nucleation activity grouped most of the isolates within pathovar syringae. The results of rep-PCR using the BOX primer revealed high genetic diversity of isolates. Multilocus sequence analysis (MLSA), using four housekeeping genes, showed that 27 isolates belong to the genomic species 1, P. syringae sensu stricto, corresponding to P. syringae phylogroup 2. However, isolates from the same phylogroup 2 did not form a monophyletic group. One strain isolated from apricot was most distinct and similar to members of genomic species 2, phylogroup 3. All tested isolates showed significant levels of resistance to copper sulfate and high level of sensitivity to streptomycin sulfate in vitro.

scholarly journals Isolation and characterization of pathogenic Escherichia coli bacteriophages from chicken and beef offal

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Celosia Lukman ◽  
Christopher Yonathan ◽  
Stella Magdalena ◽  
Diana Elizabeth Waturangi
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
High Efficiency ◽  
Multiplicity Of Infection ◽  
Isolation And Characterization ◽  
Transmission Electron ◽  
Pathogenic Escherichia Coli ◽  
Family Based

Abstract Objective This study was conducted to isolate and characterize lytic bacteriophages for pathogenic Escherichia coli from chicken and beef offal, and analyze their capability as biocontrol for several foodborne pathogens. Methods done in this research are bacteriophage isolation, purification, titer determination, application, determination of host range and minimum multiplicity of infection (miMOI), and bacteriophage morphology. Results Six bacteriophages successfully isolated from chicken and beef offal using EPEC and EHEC as host strain. Bacteriophage titers observed between 109 and 1010 PFU mL−1. CS EPEC and BL EHEC bacteriophage showed high efficiency in reduction of EPEC or EHEC contamination in meat about 99.20% and 99.04%. The lowest miMOI was 0.01 showed by CS EPEC bacteriophage. CI EPEC and BL EPEC bacteriophage suspected as Myoviridae family based on its micrograph from Transmission Electron Microscopy (TEM). Refers to their activity, bacteriophages isolated in this study have a great potential to be used as biocontrol against several foodborne pathogens.

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