TCBS Agar Research Articles

Phenotypic and Genotypic characterization of Vibrio species isolated from marine fishes

Vibriosis is considered the most important threatening disease problem facing aquaculture. The bacteria occur widely in aquatic environment and are part of the normalflora of coastal seawater and are opportunistic pathogens in marine animals.A total number of (311 ) fish ,(97 )Seabream , ( 118 ) Seabass, (96 ) Mugil capito were collected from Kafr Hamedo and Ezzbet El Borg marine water fish farm (Damietta governorate), El-Manzala and Mansoura Fish Market (EL-Dakahlia governorate)in the period from March 2016 to April 2017 during the four seasons. The clinical examination of diseased fish revealed that, presenceof redness at base of anal fin and erosion of caudal fin, presence of ulcersin skin, darkness of the skin, detached scales, fin erosion, corneal opacity and palegills. Post mortem examination of infected fishes revealed that, liver appearedenlarged, congested or pale with engorged gall bladder, splenomegaly, congestedkidney and hemorrhage in abdominal cavity. Congested gills with excessiveamount of mucous and enlarged liver with hemorrhagic patches on itsedges.The isolated bacteria on thiosulfate citratebile salt sucrose agar (TCBS) gave yellow colonies for Vibrio alginolyticus and gave green colonies for Vibrio parahemolyticus. The strains were tested according to their susceptibility as resistant, intermediate or sensitive for each antibiotic group. Vibrio alginolyticus and Vibrio parahemolyticus revealed sensitivity to ciprofloxacin (Cipro5µg) and novobiocine (NV30µg). Moreover intermediate sensitivity was found to oxytetracycline (OT 30 µg). While resistance was observed to amoxicillin (Aml 10 µg) and cholistine sulphate (CT 10 µg).The isolated bacteria (Vibrio alginolyticus and Vibrio parahemolyticus) were 171/311 (54.98%) from infected fish where the total number of Seabass 72/118 (61%), Seabream 47/97 (48.45%) and Mugil capito 52/96 (54.17%). The highest infection rate was recorded by summer (100% , 82.61% , 80%), winter (73% , 58.33% , 54.54%) , spring (56% , 42.11% , 69.23%) and autumn (36% , 30.23% , 40.38%) for Seabass, Seabream and Mugil capito respectively.PCR yielded a single specific and clear amplificated band ofexpected size 737bp for collagenase gene and 387bp for pR72H gene for V. alginolyticus and V. parahemolyticus respectively.

The Diversity of Vibrios Associated with Vibriosis in Pacific White Shrimp (Litopenaeus vannamei) from Extensive Shrimp Pond in Kendal District, Indonesia

Vibriosis out breaks frequently occur in extensive shrimps farming. The study were commenced to find out the clinical signs of white shrimp that was infected by the Vibrio and to identify the bacterial associated with vibriosis in the pacific white shrimp, Litopenaeus vannamei. Bacterial isolates were gained from hepatopancreas and telson of moribund shrimps that were collected from extensive shrimp ponds of Kendal District, Indonesia and cultured on Thiosulfate Citrate Bile Salts Sucrose Agar (TCBSA). Isolates were clustered and identified using repetitive sequence-based polymerase chain reaction (rep-PCR). Three representative isolates (SJV 03, SJV 05 and SJV 19) were amplified with PCR using primers for 16S rRNA, and sequence for further identification. The clinical signs of shrimps affected by vibrio were pale hepatopancreas, weak of telson, dark and reddish coloration of smouth, patches of red colour in part of the body on the carapace, periopods, pleuopods, and telson. A total of 19 isolates were obtained and belong to three groups of genus Vibrios. Result of the 16S DNA sequence analysis, the vibrio found in this study related to vibriosis in white shrimps from extensive shrimp ponds of Kendal were closely related to Vibrio harveyi (SJV 03); V. parahaemolyticus (SJV 05) and V. alginolyticus (SJV 19).

MOLECULAR CHARACTERIZATION OF VIBRIO PARAHAEMOLYTICUS ISOLATED FROM SOME MOLLUSKS, SHELLFISH AND THEIR HARVESTING WATER FROM SUEZ CANAL AREA

The study included 410 samples of shellfish (164 clams, 86 mussels, and 160 shrimps) collected from Suez Canal area as the following: 156 from Port Said, 164 from Ismailia and 90 from Suez governorates. The harvesting water samples (48) were collected from the three sites. All samples were collected during the warmest season (June, July, and August) through two years. After extraction of muscle tissue and homogenization, enriched samples were identified by plating onto TCBS agar medium. Presumptive V. parahaemolyticus colonies were selected, purified, and further identified by API20 and PCR techniques targeting toxRgene. The pathogenicity of the isolates was examined by detection of Thermostable Direct Haemolysin (TDH) and related Haemolysin (TRH) genes. Results revealed that the overall prevalence of V. parahaemolyticus in shellfish was 38/410 (9.27%), where as in water was 12/48 (25%). Higher contamination rate was detected in shrimp (15%), and the highest prevalence was recorded in Ismailia governorate (12.2%). The detection rate of TDH and TRH genes was 21.05% and 5.26% consequently indicating its health hazards to the consumers. This study concluded that the examined shellfish may have the potential human health risk associated with the presence of pathogenic V. parahaemolyticus. The preventative measures should be considered.

English

Various biodiversity indices revealed that Microcystis aeruginosa is a major bloom forming colonial cyanobacterium dominantly present in the examined two districts of Eastern Madhya Pradesh. Microcystis viridis, Microcystis panniformis and Microcystis botrys along with filamentous cyanobacteria Anabaena spp., Arthrospira major and Oscillatoria limosa/O. laetevirens were the other species present. Amplification of VCO1 and VCO139 choleragenic Vibrio cholerae strains in phytoplankton material revealed their association with cyanobacteria. VCO1 gene was amplified in five water bodies, and among them, one reservoir also displayed amplification of VCO139 gene. VCO1 and VCO139 genes were not amplified in three water bodies. All of them were infested with Anabaena spp. as the second largest phytoplankton constituent. It was hypothesized that Anabaena spp. produced some antibacterial metabolites with antagonistic property against V. cholerae. To prove this, colonies of V. cholerae on TCBS agar were isolated from those water bodies which displayed VCO1 and VCO139 gene amplification. Methyl Red test, Voges-Prauskaur test and arginine dehydrolase tests confirmed Vibrio. Further identification of V. cholerae was carried out by amplification of VCO1 and VCO139 genes in genomic DNA isolated from V. cholerae colonies. A hexane extractable metabolite extracted from lab culture of Anabaena fertilissima CCC597, a native of these lakes, was tested for its antagonizing effect on growth of V. cholerae strains O1 and O139. A “closed water system” was used to examine the effect of A. fertilissima cell mass on time-dependent population size of Vibrio. Upon such incubation, there was a steady decrease in the viable colony counts of V. cholerae. Key words: Antibacterial effect, cyanobacterial population, Vibrio cholerae O1 and 139, important value index.

ANTIBIOGRAM AND MOLECULAR CHARACTERIZATION OF VIBRIO CHOLERAE ISOLATED FROM MARINE FISH

Objective: Molecular identification and antibiotic susceptibility evaluation of Vibrio cholerae from marine fish available in local fish market Thanjavur, Tamil nadu, India.Methods: inoculation was done by using nutrient agar as general media and TCBS agar as selective media and confirmed as V.cholerae by Gram stain (Microscopic Observation), Growth characteristics of different media, Biochemical tests like Methyl Red Test, Nitrate Reduction Tests, Indole Test etc. Sensitivity (drug sensitivity) was done in Muller Hinton Agar (MH Agar) using disc diffusion method ten different antibiotics were used to evaluate the antibiogram profile, molecular detection was done by targeting 16S rRNA gene by using a universal primer.Results: V.cholerae is present in marine fish samples, as showed by culture method and microscopic observation as well biochemical tests. PCR amplification of 16S rRNA gene showed the amplification of targeted gene and antibiogram profile showed that isolates are more sensitive to Ampicillin in comparison with others antibiotics used in this study. Ampicillin can be used for V.cholerae infection by the physicians and amoxicillin must be avoided which is resistant.Conclusion: Molecular detection is safe and rapid methods for bacteria identification as revealed by PCR amplification of 16S rRNA gene. As the isolates are more sensitive to Ampicillin in comparison with others antibiotics used in this study. Ampicillin can be used for V.cholerae infection by the physicians and amoxicillin and nitrofurantoin must be avoided.

Pathogenic Vibrio species isolated from estuarine environments (Ceará, Brazil) - antimicrobial resistance and virulence potential profiles.

Detection of virulent strains associated with aquatic environment is a current concern for the management and control of human and animal health. Thus, Vibrio diversity was investigated in four estuaries from state of Ceará (Pacoti, Choró, Pirangi and Jaguaribe) followed by antimicrobial susceptibility to different antimicrobials used in aquaculture and detection of main virulence factors to human health. Isolation and identification were performed on TCBS agar (selective medium) and dichotomous key based on biochemical characteristics, respectively. Nineteen strains of genus Vibrio were catalogued. Vibrio parahaemolyticus (Choró River) and V. alginolyticus (Pacoti River) were the most abundant species in the four estuaries. All strains were submitted to disk diffusion technique (15 antimicrobials were tested). Resistance was found to: penicillin (82%), ampicillin (54%), cephalotin (7%), aztreonan (1%), gentamicin, cefotaxime and ceftriaxone (0.5%). Five pathogenic strains were chosen to verification of virulence factors. Four estuaries showed a high abundance of species. High number of tested positive strains for virulence is concerning, since some of those strains are associated to human diseases, while others are known pathogens of aquatic organisms.

Morphological and Biochemical Characterization of Bacteria Isolated from Fresh and Salted Hilsa, Tenualosa ilisha (Hamilton, 1822)

Morphological and biochemical characterization of different bacteria isolated from eight fresh and salted hilsa collected from four fish markets namely Palashi Bazaar, Karwan Bazaar, Anando Bazaar and New Market of Dhaka city was carried out. Five types of bacteriological culture media namely nutrient agar as generalized complex medium, EMB agar medium for coliform, SS agar for Salmonella-Shigella, MSA for Staphylococcus and TCBS for Vibrio were used to isolate the bacteria associated with the hilsa. In salted hilsa, no microbial growth was observed on SS and TCBS agar plate. A total of 66 bacterial isolates were obtained in the study where detail study of 24 strains (12 Gram positive and 12 Gram negative) using morphological and biochemical characterization was conducted. Variable morphological and biochemical properties of the isolated strains were observed. Gram positive bacteria such as Listeria monocytogenes, Bacillus megaterium, B. marinus, Planococcus citreus, Micrococcus varians, M. nishinomiyaensis, Staphylococcus lentus, S. epidermidis, S. aureus and Gram negative bacteria such as Salmonella paratyphi, Salmonella spp., Proteus morganii, P. vulgaris, Vibrio sp., Halobacterium sp. were presumptively identified in the current study. Presence of these bacteria in fish indicates the possibility of occurrence of food poisoning. So, care should be taken during handling and consumption of hilsa whether fresh or salted as it carries pathogenic strains of bacteria of public health concern.Bangladesh J Microbiol, Volume 32, Number 1-2,June-Dec 2015, pp 7-13

Detection, Identification, and Prevalence of Pathogenic Vibrio parahaemolyticus in Fish and Coastal Environment in Jordan.

Vibrio parahaemolyticus is widely distributed in the marine environments and considered the leading cause of human gastroenteritis in Asian countries. A total of 150 marketed fish and 50 water and sediment samples from the Gulf of Aqaba were examined for the prevalence of pathogenic strains of V. parahaemolyticus. A total of 132 typical isolates obtained from the primary selective medium (thiosulfate-citrate bile salt sucrose agar) and showed positive biochemical properties were subjected to confirmation by polymerase chain reaction targeting the gyrB and toxR genes. These genes were confirmed at rates of 82% (108 isolates) and 72% (95 isolates), respectively. The toxR positive isolates were tested for the presence of thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) virulence genes. Accordingly, the prevalence rates of pathogenic V. parahaemolyticus were 4%, 8%, and 12% in sediment, water, and fish samples, respectively. The 16S rRNA amplification and sequences were conducted for confirmation of the isolates and showing the relatedness among these isolates. The results showed that both 16S rRNA and toxR assays had same sensitivity and tested isolates had high nucleotide similarity irrespective of their sources.

Isolation and Identification of <i>Vibrio</i> sp. in the Hepatopancreas of Cultured White Pacific Shrimp (<i>Litopenaeus vannamei</i>)

The Vibrio is the most common genera with crustaceans often causing various diseases in Aquaculture and significant economic losses. Many Vibrio species are pathogenic to human and have been implicated in food borne diseases. The present study was carried out, the isolation and identification of pathogenic bacterial flora were isolated from infected in hepatopancreas of vannamei. The SPDS Oceanic farm, RS Aqua farm of and Valli vilas Aqua farm Vellar estuary, Cuddalore District, Tamil Nadu, during the period of (September 2013 to November 2013). The collected samples were plated on TCBS- (Thiosulfate-Citrate-Bile salt-Sucrose) agar medium. The present study, totally 253 green colonies were isolated from TCBS agar plates and among these, 175colonies were identified by using the biochemical tests showed the V. parahaemolyticus, V. mimicus, V. vulnificus, V.damsela and P.shigelloides. The maximum species was recorded in V.parahaemolyticus (83.4 %) and minimum was observed in V.mimicus (1.7 %).

Isolation and Identification of <i>Vibrio</i> sp. in the Hepatopancreas of Cultured White Pacific Shrimp (<i>Litopenaeus vannamei</i>)

The Vibrio is the most common genera with crustaceans often causing various diseases in Aquaculture and significant economic losses. Many Vibrio species are pathogenic to human and have been implicated in food borne diseases. The present study was carried out, the isolation and identification of pathogenic bacterial flora were isolated from infected in hepatopancreas of vannamei. The SPDS Oceanic farm, RS Aqua farm of and Valli vilas Aqua farm Vellar estuary, Cuddalore District, Tamil Nadu, during the period of (September 2013 to November 2013). The collected samples were plated on TCBS- (Thiosulfate-Citrate-Bile salt-Sucrose) agar medium. The present study, totally 253 green colonies were isolated from TCBS agar plates and among these, 175colonies were identified by using the biochemical tests showed the V. parahaemolyticus, V. mimicus, V. vulnificus, V.damsela and P.shigelloides. The maximum species was recorded in V.parahaemolyticus (83.4 %) and minimum was observed in V.mimicus (1.7 %).

Microbial and proximate composition of fresh and salted Hilsa, Tenualosa ilisha

Bacterial and nutritional quality of fresh and salted hilsa collected from four markets of Dhaka city were investigated. Five different culture media viz. nutrient agar, EMB agar for coliform, SS agar for Salmonella-Shigella, MSA agar for Staphylococcus and TCBS for Vibrio were used. The highest heterotrophic bacterial count 1.22 ± 0.12 × 106 cfu/g was recorded in the fresh fish sample of Karwan Bazar. Maximum coliform count, 1.20 ± 0.10 × 106 cfu/g was detected in the fresh fish sample of the same market. No bacterial colony was found on SS agar and TCBS agar plate in salted fish. Proximate composition of raw hilsa of dorsal and ventral part was 56.49 ± 0.13% and 55.45 ± 0.06% moisture, 23.62 ± 0.28% and 22.99 ± 0.36% protein, 18.01 ± 0.39% and 18.96 ± 0.43% fat and 1.71 ± 0.04% and 2.26 ± 0.09% ash, respectively. In salted T. ilisha, the proximate composition of dorsal and ventral part was 45.13 ± 0.54% and 40.20±0.20% moisture, 20.79 ± 0.17% and 21.48 ± 0.15% protein, 16.89 ± 0.47% and 19.54 ± 0.26% lipid and 16.65 ± 0.41% and 18.35 ± 0.08% ash. The fresh fish samples were associated with high bacterial loads than that of salted fish. The protein, lipid, moisture contents decreased and ash content increased after salting condition.Bangladesh J. Zool. 42(2): 227-236, 2014

Antibacterial activity of the methanolic extracts of Eclipta alba against bacteria in intestinal tract of black tiger shrimp (Penaeus monodon)

Antibacterial activity of the methanolic extracts of Eclipta alba were carried out by the disc diffusion method on 12 strains of bacteria isolated from 30 samples of tiger shrimp (intestines) collected from shrimp farms in Tan Thanh hamlet, An Loi commune, Tran Van Thoi district, Ca Mau province. All strains isolated were grown for 24h on TCBS agar plates at 30°C in yellow colony or green colony. Antibacterial activity assay of the extract was carried out in the concentration of 8 µg/mL, 16 µg/mL, 32 µg/mL, 64 µg/mL and 128 µg/mL. The extract of Eclipta alba were tested against 10 bacterial strains were isolated. The highest antibacterial potentiality was exhibited by the methanolic extract of E. alba with G5 strain, means of zones of bacterial growth inhibition are 30,3mm. Three of twelve isolates (Y1, G2, G5) were chosen to examine the biochemical characteristics with the API 20E Kit and identified by two method: Maldi toff mass and sequencing 16S rDNA using primers pair 27F (5¢-AGAGTTTGATCCTGGCTCAG-3¢) and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The results showed that Y1 isolate was similarity of 97% with E. Cloacae; G2 isolate was similarity of 98% with V. brasiliensis and G5 isolate was similartty of 99% with V. Parahaemolyticus.

Application of a Rapid Method for Detecting Vibrio Parahaemolyticus in Seafood

AbstractA double‐layer agar plate (DLAP) method was used to detect Vibrio parahaemolyticus in culture broth and seafood samples, which were added with certain amount of V. parahaemolyticus and then subjected to freezing treatment. The results of DLAP, which were compared with the most probable number (MPN) method and direct‐plating methods such as Bio‐chrome Vibrio medium (BCVM) and thiosulfate–citrate–bile salts–sucrose agar (TCBS), showed that they were not significantly different (P > 0.05) from each other under the normal experimental conditions. However, if the bacterium was inactive or injured, the results of DLAP were as effective as MPN and more accurate than those of BCVM and TCBS. Furthermore, DLAP showed the total agreement compared with the MPN method by examining the presence of V. parahaemolyticus in naturally contaminated seafood samples. Because of its sensitivity and exactness, DLAP offers a broad application for fast screening and detecting of V. parahaemolyticus in seafood.Practical ApplicationsConsumption of raw or undercooked seafood, particularly shellfish, contaminated with Vibrio parahaemolyticus may lead to the development of acute gastroenteritis characterized by diarrhea, headache, vomiting, nausea, abdominal cramps and low fever. Now, this bacterium is recognized as an important seafood‐borne pathogen throughout the world. Usually, 5 to 7 days are required for V. parahaemolyticus detection by the most commonly used procedure – the most probable number method, which is very time‐consuming and labor‐intensive. Some time‐saving alternatives such as polymerase chain reaction or DNA methods are not economically appealing. The considerations demand for a rapid method for V. parahaemolyticus detection that is at least as sensitive and accurate as the methods used currently. A double‐layer agar plate method described here can satisfy these criteria and can be used for rapid detection of V. parahaemolyticus in artificially and naturally contaminated samples in laboratory testing.

Vibrio parahaemolyticus em carcinicultura marinha

Vibrio parahaemolyticus pode causar vibriose em camarão cultivado e também gastrenterites no consumidor. Neste trabalho foram verificadas as condições higiênico-sanitárias em camarões (Litopenaeus vannamei) e na água de viveiros de carcinicultura no litoral piauiense quanto a V. parahaemolyticus. Foram coletadas 148 amostras de água e 72 amostras de camarão em três fases de cultivo: pós-larva, juvenil e engorda em quatro propriedades. Foram transferidos 25g de cada amostra para frasco contendo 225mL de água peptonada com 3% de sal para o preparo da diluição 10-1, a partir dessa foram preparadas as diluições 10-2 e 10-3. Para o teste presuntivo, alíquotas de 1,0mL dessas diluições foram semeadas em três tubos com caldo Horie Arabinose Violeta de Etila (37ºC/24 horas). Repicou-se para agar TCBS (37ºC/24 horas). As colônias típicas foram semeadas em: agar motilidade sal, agar nutritivo sal, caldo peptonado sal e ágar TSI sal (incubados a 37ºC/24 horas), testes bioquímicos: halofilismo (0; 3; 6; 8 e 10%), crescimento a 42ºC, teste de Kanagawa, prova de Hugh-Leifson, descarboxilação da lisisna e arginina e fermentação de manitol e sacarose. V. parahaemolyticus foi semelhante estatisticamente nas amostras de água e das fases pós-larva e engorda em todas as propriedades estuário e viveiros. Nas amostras de camarões também não houve diferença significativa estatística entre propriedades e fases de cultivo As águas do sistema produtivo e os camarões cultivados no litoral piauiense apresentam condições higiênico-sanitárias satisfatórias quanto à enumeração de Vibrioparahaemolyticus.

Toxigenic Potential of Vibrio cholerae O1and O139 Serotypes Isolated from Cases of Diarrhea in Baghdad hospitals

Cholera is most important water borne pathogen. The public health significance of a V. cholerae isolate is routinely assessed by two critical properties: the production of cholera toxin CT and the possession of either the O1 or O139 antigen, which acts as a marker of epidemic potential. The objective of this study is to detect V. cholerae serotypes directly from stools and determines their toxiginicity potential. Sixty four stool samples were collected from four hospitals in Baghdad from November 2010 to February 2011. The age of patients was ranging from two months to 12 years, 26 females and 38 males. Immunochromatographic test used for qualitative detection of O1and /or O139 serotypes was used in addition to routine culture for isolation of V. cholerae. Using specific primer cholera toxin gene, ctxA2-B, was amplified and the PCR product was detected by agarose gel electrophoresis. Out of 64 stool samples only 16 (25%) was positive. Fifeen 93.7% of these samples were positive for O1serogroup and just one 6.3% was positive for O139 serogroup. Stool sample culture on alkaline peptone water and then on TCBS agar enhance the growth of 11(17.2%) V. cholerae isolates, 10 (90.9%) were belong to O1 serotype and one 9.1% belong to O139. The results of ctxA2-B gene amplification show that, 9 (90%) out of 10 O1serotypes was positive. While the only one 100% O139 serotype was positive. As conclusion, the incidence of cholera caused by V. cholerae O1 is more than that caused by V. cholerae O139 in Baghdad hospitals. Immunochromatographic test is a rapid and sensitive test in recover V. cholerae O1 and O139 serotypes. PCR is a simple molecular tool to determine the toxigenicity of V. cholerae isolates.

A Survey of a Cholera Epidemic in Aran va Bidgol City in Summer 2011

Background and purpose: Cholera is an endemic disease in Iran and in some cases each year from around the country report. The aim of this study was to evaluate the prevalence of the disease cholera among people suffering from acute diarrhea in the Aran va Bidgol. Materials and Methods: In this study, patients with acute diarrhea were examined for cholera detection in the city of Aran va Bidgol in summer 2011. All 294 patients with acute diarrhea referred to rectal swabs in the laboratory of health centers and in the specific microbial culture medium of thiosulfate citrate bile sucrose agar. Furthermore, some information about health behaviors, such as drinking water supply, status of food preservation, toilets status, drinks, and food consumption in the outdoors was collected from all participants in this study. Results: Laboratory results showed that Vibrio cholerae isolated from 21 patient stool cultures and these patients were to have cholera disease. About 23.81% of the patients were male and 76.19% were female. Fifteen patients (71.43%) were Nag form of cholera and 6 patients (28.57%) were diagnosed with Eltor form of cholera. According the completed questionnaire by patients the origin of V. cholerae has been announced consumption of raw vegetables (42.85%) and springs and subterranean water (38.59%). Conclusion: Consumption of raw vegetables and water (springs and subterranean) are the important factors for mobility to cholera and it should be more considered prevention and control program and monitoring methods about using of safe water and food. [Ahmadabadi Arani M, *Jahed Khaniki Gh, Moallemi A, Sabahibidgoli M, Shariatifar N, Ghaffarian M. A Survey of a Cholera Epidemic in Aran va Bidgol City in Summer 2011. IJHS 2014; 2(2): 19-24] http://jhs.mazums.ac.ir

Identification of Vibrio harveyi, Vibrio ichthyoenteri, and Photobacterium damselae isolated from olive flounder Paralichthys olivaceus in Korea by multiplex PCR developed using the rpoB gene

Major fish bacterial diseases in Korea are edwardsiellosis, streptococcosis, and vibriosis. Among vibrionaceae, Listonella anguillarum, Vibrio harveyi, V. ichthyoenteri, and Photobacterium damselae were identified as causative organisms of vibriosis in flounder. In this study, we developed a multiplex PCR method using the RNA polymerase β subunit (rpoB) gene, known as a housekeeping gene for identification of Vibrio spp. causing vibriosis in flounder. Three pairs of PCR primers were designed based on the rpoB sequence of three species, V. harveyi, V. ichthyoenteri, and P. damselae. The PCR assay, using a mixture of six primers, yielded amplicons of 601, 434, and 533 bp in V. harveyi, V. ichthyoenteri, and P. damselae. None of the untargeted species yielded an amplicon. The detection limits for pure culture in kidney were 2.5 × 104 cfu/g kidney for V. harveyi, 2.5 × 105 cfu/g kidney for V. ichthyoenteri, and 2.5 × 106 cfu/g kidney for P. damselae. From the colonies on TCBS agar plates of different samples, 632 Vibrio spp. isolated from aquacultured flounder between 2004 and 2010 were identified by the multiplex PCR method. As a result, 265 strains (41.9 %) were V. ichthyoenteri; 115 strains (18.2 %) were V. harveyi and 72 strains (11.4 %) were P. damselae.

Survival of Vibrio cholerae O1 and Vibrio parahaemolyticus in fried and boiled Malaysian fish sausage

Survivability of Vibrio cholerae O1 and Vibrio parahaemolyticus in a popular Malaysian fish sausage snack, keropok lekor (either boiled or fried) in two conditions (closed or opened) was investigated by spiking ca. 8.00 log CFU/ml of each bacterial culture. Spread plate method onto TCBS agar was used to determine the numbers of inoculated vibrios that had survived as per tested condition. V. parahaemolyticus was more sensitive than V. cholerae O1 and lost viability in saline (control, closed condition). Both vibrios were not detected in the opened condition control after 1 h but keropok lekor proved to significantly support the viability of vibrios for up to 6 h. No significant growth or reduction (p > 0.05) was detected in an enclosed container but a significant reduction (p < 0.05) was observed in the opened condition over a 6 h incubation period. After 6 h incubation, V. cholerae survived at 7.95 ± 0.05 log CFU/g (boiled, enclosed), 6.11 ± 0.18 log CFU/g (fried, enclosed), 4.23 ± 0.48 log CFU/g (boiled, opened) and 3.08 ± 0.46 log CFU/g (fried, opened), respectively. Though V. parahaemolyticus showed poor viability in saline, they persisted well in keropok lekor with numbers recorded at 5.75 ± 0.09 log CFU/g (boiled, enclosed), 5.49 ± 0.59 log CFU/g (fried, enclosed), 2.85 ± 0.45 log CFU/g (boiled, opened) and 2.13 ± 0.52 log CFU/g (fried, opened), respectively. While vibrios are known to be easily killed by heat, post-cooking contamination might lead to food poisoning as a result from vibrios persistence in keropok lekor.

Antibiotic Resistance of Vibrio parahaemolyticus Isolated from Cockles and Shrimp Sea Food Marketed in Selangor, Malaysia

Introduction: The main aim of this study is to determine the antibiotic profile of V. parahaemolyticus gastroenteritis associated with the consumption of contaminated shrimp and cockles marketed in Selangor Malaysia. V. parahaemolyticus is the leading cause of seafood-associated gastroenteritis in Asian Countries typically is associated with the consumption of raw shellfish and oysters specially shrimp and cockles. Rapid, sensitive and specific detection methods are needed to control V. parahaemolyticus infections. We describe a recognized the pathogenic V. parahaemolyticus in shrimp and cockles that will be the risk of gastroenteritis associated with the consumption of seafood marketed in Malaysia. Methods: This study was carried out between July 2011 and August 2013 at the Center of Excellence for Food Safety Research, Faculty of Food Science and Technology, Faculty of Medicine and Health Sciences, Department of Biomedical Sciences, and Faculty of Biotechnology, Dep. of Cell and Molecular Biology, University Putra Malaysia and other centers as collaboration. The seafood samples were collected from different markets and more than 400 samples from shrimp and cockles were investigated for detection and isolation of V. parahaemolyticus. CHROMagar Vibrio and TCBS agar media were used for fast detection and isolation of V. parahaemolyticus isolates. PCR based methods targeted to toxR regulatory gene, tlh the species and family gene, tdh and trh the virulence genes were extensively used. The antibiotic susceptibility testing of 65 V. parahaemolyticus isolates recovered from retail shrimp and cockles seafood were determined with four types of E-test antibiotic strips. Results: All the 65 isolates were positive to toxR and tlh genes. Out of 65 isolates, only eight isolates (12.31%) were positive for tdh virulence gene isolated form cockles and shrimp (3 isolates from shrimp and 5 isolates from cockles), whereas twenty six (40%) isolates were positive for trh virulence gene isolated from shrimp and cockles (9 from shrimp and 17 from cockles). This result indicates high occurrence of tdh+ and trh+ isolates in shrimp and cockles marketed in Malaysia. None of the isolates tested possess both virulence genes. For the antibiotic E-test susceptibility test, overall, V. parahaemolyticus is remained susceptible to tetracycline (97%). A slight increase in the susceptibility of tetracycline is observed from 2011 to 2013. While reduced susceptibility was detected only in V. parahaemolyticus for ampicillin. The mean of MIC of the isolates toward ampicillin is increased from 64 μg/ml in 2011 to 128 μg/ml in year 2013. The current study demonstrates a high risk of pathogenic V. parahaemolyticus in the shrimp and cockles marketed in Selangor Malaysia. Conclusions: The potential risk of V. parahaemolyticus infection due to the consumption of contaminated seafood in Malaysia should not be neglected. The increased resistance of ampicilin from our studies in Malaysia since 2004 to 2013 could be in indication of antibiotic abuse in clinical and agricultural used of ampicilin in Malaysia.

Karakterisasi Bakteri Genus Vibrio Dari Ikan Kerapu (Plectropomus sp.)

This research is about the isolation and characterization Vibrio sp. from grouper fish (Plectropomus sp.). This research aimed to observe the amount of vibrio bacteria species based on colony morphology and characterization morphological colony of vibrio isolated from grouper fish(Plectropomus sp.). Bacteria isolation was conducted by isolate some external organs (lesion on its body) and internal organ (jejenum) from grouper fish (Plectropomus sp.). Bacteria was grown on selective medium for vibrio, TCBS (Thiosulfate Citrate Bile Sucrose) agar. The incubation time was 24 hours at 370C. The result showed there are 3 isolate vibrio bacteria that was coded by V1, V2, and V3. V1 morphological characteristis was rounded, edge is entire, convex elevation, green colony colour with blue in the center of colony and smooth tekstur. V2 morphological characteristic was rounded, edge is entire, tortous elevation, colony colour yellow, and smooth tekstur. Morphologicalcharacteristic of V3 was Irregular, egde is Undulate, elevation hill, colony colour yellow and own the smooth tekstur.Keywords: characterization, grouper fish (Plectropomus sp.), isolation, Vibrio