الفهرس 
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Abstract
A total of 150 imported frozen raw fish samples consisting of 20 sardine, 30 mackerel, 40 horse mackerel and 60 shrimp were collected and analyzed by bacteriological, antimicrobial and molecular methods. The main objectives of the present work were to study the occurrence and characteristics of Vibrio spp. in frozen marine fish. Firstly Bacteriological examination was done by traditional method according to ISO/TS 21872-1& 21872-2. Two steps method for enrichment of Vibrio spp. were performed. Briefly, 25 g of each tissue sample was added to 225 ml alkaline saline peptone water-ISO (ASPW-ISO) as a first selective enrichment to form 1/10 initial suspension, then incubated at 370C for 6±1h. After that, 1ml of the initial suspension was transferred to a tube containing 10 ml ASPW-ISO representing the second selective enrichment, then incubated at 41.50c for 18 ±1h. Isolation and identification were started by streaking the surface of Thiosulphate Citrate Bile Sucrose agar (TCBS) and Vibrio parahaemolyticus Sucrose Agar (VPSA) plates with a loopful from the incubated ASPW-ISO to permit the development of the isolated colonies. Agar plates were then inverted and put in an incubator at 37 0C. After 24±3h of incubation, plates were examined for presence of typical colonies of presumptive pathogenic Vibrio spp. Suspected colonies were isolated and purified by streaking at least five typical colonies on saline nutrient agar plates and incubated at 370c for 24h ±3h. Pure colonies were stained with gram stain and tested for oxidase and microscopic motility.
Obtained gram negative, oxidase positive and motile isolates were selected for further biochemical confirmatory tests . Nine isolates of Vibrio spp. were identified. They were divided into 3 isolates of V.parahaemolyticus , one isolate of V. vulnificus and 5 isolates of V. fluvialis. Biochemical confirmatory tests were performed using two different methods, The first was manual method for biochemical confirmation including saline TSI agar slopes test , detection of ornithine decarboxylase, detection of L-lysine decarboxylase, detection of arginine dihydrolase, detection of β-galactosidase, detection of indole and halotolerance test . The second was an automated phenotypic microbiology identification system utilizing colorimetric reagent cards (VITEK2C) that performed about 47 biochemical reactions (Table 7) giving a final report of all applied biochemical reactions and the final identification of isolated colonies with its probability percentage after 6 hours.. The presence of virulence associated genes of pathogenic Vibrio.spp was investigated using standard PCR. The 16SrRNA gene specific for the genus Vibrio was used to confirm the genus in all isolates. All the isolates, irrespective of the species, were positive for 16S rRNA gene confirming the genus in all the isolates studied. V. parahaemolyticus isolates were positive for tdh, trh and tlh genes. On the other hand, V. vulnificus isolates were positive for virulence genes; vvhA, SerE and Bt2. Also, V. fluvialis isolates were positive for virulence genes toxR, vflu and vfh. Susceptibility of Vibrio spp. to antibiotics was studied firstly using the standard Kirby Bauer disk diffusion method . Susceptibility testing was performed and interpreted in accordance with Clinical and Laboratory Standard and Institute (CLSI) guidelines (CLSI, 2011).
Then antimicrobial susceptibility was performed using VITEK2C system that includes an Advanced Expert System (AES) that analyzes MIC patterns giving results in accordance to clinical and laboratory standards institute (CLSI guide) in MIC report. The high percentage of ampicillin-resistant Vibrio spp isolates suggests low efficiency of Ampicillin in empirical treatment of infections caused by this organism. It could be concluded that there is a need for appropriate food safety practices when consuming these products. Moreover, continued monitoring of both the prevalence and antimicrobial susceptibility profile is needed to better safety.