TCBS Agar Research Articles

Pufferfish (Tetraodon cutcutia) Sampled from a Freshwater River Serves as an Intermediate Reservoir of a Sucrose Nonfermenting Variant of Vibrio cholerae PS-4.

ABSTRACTWe describe the genomic characteristics of Vibrio cholerae strain PS-4 that is unable to ferment sucrose on a thiosulfate citrate bile salt sucrose (TCBS) agar medium. This bacterium was isolated from the skin mucus of a freshwater pufferfish. The genome of strain PS-4 was sequenced to understand the sucrose nonfermenting phenotype. The gene encoding the sucrose-specific phosphotransferase system IIB (sucR) was absent, resulting in the defective sucrose fermenting phenotype. In contrast, genes encoding the glucose-specific transport system IIB (ptsG) and fructose-specific transport system IIB (fruA) showed acid production while growing with respective sugars. The overall genome relatedness indices (OGRI), such as in silico DNA-DNA hybridization (isDDH), average nucleotide identity (ANI), and average amino acid identity (AAI), were above the threshold value, that is, 70% and 95 to 96%, respectively. Phylogenomic analysis based on genome-wide core genes and the nonrecombinant core genes showed that strain PS-4 clustered with Vibrio cholerae ATCC 14035T. Further, genes encoding cholera toxin (ctx), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), toxin-coregulated pilus (tcp), and lipopolysaccharide biosynthesis (rfb) were absent. PS-4 showed hemolytic activity and reacted strongly to the R antibody. Therefore, the Vibrio cholerae from the pufferfish adds a new ecological niche of this bacterium.IMPORTANCE Vibrio cholerae is native of aquatic environments. In general, V. cholerae ferments sucrose on thiosulfate citrate bile salt sucrose (TCBS) agar and produces yellow colonies. V. cholerae strain PS-4 described in this study is a sucrose nonfermenting variant associated with pufferfish skin and does not produce yellow colonies on TCBS agar. Genes encoding sucrose-specific phosphotransferase system IIB (sucR) were absent. The observed phenotype in the distinct metabolic pathway indicates niche-specific adaptive evolution for this bacterium. Our study suggests that the nonfermenting phenotype of V. cholerae strains on TCBS agar may not always be considered for species delineation.

Simple method devised for rapid isolation and identification of Vibrio cholerae from water resources of Sunsari District, Nepal

Cholera is a gastrointestinal disease caused by pathogenic strain of Vibrio cholerae, the disease clinically manifested by rice-water diarrhea, nausea and vomiting. This study aimed to study the incidence of Vibrio species and employ simple method for rapid detection of Vibrio cholerae from water samples of Sunsari, Nepal. Identification of V. cholerae through biochemical tests requires extensive labor and costs. In resource limited laboratories, isolation and identification of V. cholerae often becomes difficult. Therefore, this study also aimed for selecting scope of this methodology as a scientific outcome for rapid isolation and identification of Vibrio cholerae. A total of 100 water samples were collected from Sunsari district in which 25 samples were collected from sewage, 25 from pond, 25 from tap and 25 from tube well. The samples of collected water were sent to the microbiology laboratory of Central Campus of Technology maintained in ice cold box and were enriched in Alkaline Peptone water and selectively isolated from TCBS agar and NA agar without NaCl. Pathogens were isolated and identified by conventional microbiological techniques. Out of 100 water samples collected, sucrose fermenting Vibrio species were isolated only from 16 water samples. Further the selective isolation of V. cholerae from nutrient agar without NaCl isolated 6 isolates from sewage samples and 3 isolates from pond samples. The distribution of Vibrio cholera in the water sample was found to be 9%, distribution of V. alginolyticus was found to be 4% and distribution of V. fluvialis was found to be 3%. In this study, non-sucrose fermenting Vibrio species were not isolated from the water samples. However, sucrose fermenting Vibrio species was obtained with yellow pigmentation in TCBS agar medium. The yellow pigmented colonies of Vibrio isolates recovered from TCBS and even from Nutrient Agar devoid of sodium chloride provided sufficient evidence of V. cholerae after series of other biochemical tests. This study concludes that yellow colonies (sucrose-fermenting) of Vibrio from TCBS agar medium that can grow on nutrient agar without added NaCl and which exhibit a positive oxidase reaction can be confidently identified as presumptive V. cholerae. In resource-constrained environments, this simple method can reduce the labor cost, chemicals and time-consuming procedure of performing multiple biochemical and molecular assays for identification.

COMPARATIVE STUDY ON THE INVITRO ANTIBACTERIAL ACTIVITY OF SELECTED MEDICINAL PLANTS AGAINST PATHOGENIC VIBRIO SPECIES FROM DISEASED PENAEUS MONODON (FAB)

The pathogenic Vibrio sp. were isolated from diseased shrimp of Penaeus monodon (FAB) using TCBS agar medium by streak plate method. Acalipha indica (L), Vinca rosea (L), Embilica officinalis (G), Ocimum sanctum(L) and Lucas aspera (L) medicinal plants are one of the most commonly used natural antimicrobial agents used traditionally. Hence an attempt has been made to screen the antivibrio activity of some selected medicinal plants against pathogenic Vibrio sp by disk diffusion method. Among the five plants tested, water extract of Lucas aspera showed the maximum zone of inhibition against Vibrio cholerae (18±3.4 mm in diameter) and the alcohol extract showed the maximum zone of inhibition against Vibrio harveyi (NL) (16±3 mm in diameter).

First report of the presence of Vibrio vulnificus in the Gulf of Gdansk.

Vibrio infections are becoming more frequent in the Baltic Sea region, which is caused by an increase in the sea surface temperature. Climate change creates the conditions for the emergence of new environmental niches that are beneficial for Vibrio spp., especially in the summer months. Vibrio vulnificus, which causes wound infections and septicaemia, represents a particularly dangerous species of Vibrio spp. There are numerous publications on the prevalence of V. vulnificus in various regions of the Baltic Sea, but there is a lack of such data for the Polish coast. This prompted us to conduct a pilot study into the prevalence of the bacteria in the Gulf of Gdansk. The study aimed to detect Vibrio spp. in the coastal waters and the wet sand at the beaches and bathing areas in the Gulf of Gdansk. During the period from June 16th to September 23rd 2020, 112 samples of seawater and 105 samples of wet sand were collected at 16 locations along the coast of the Gulf of Gdansk and Hel peninsula. Isolation of Vibrio spp. was conducted by filtering method and the isolated bacteria was cultured on CHROM agar Vibrio and TCBS agar. Final genus identification was performed by the MALDI TOF technique. In the present study, 10 isolates of Vibrio spp. were obtained from seawater and wet sand samples collected in the Gulf of Gdansk and Hel peninsula coast. Three of the isolates were identified as V. vulnificus; the presence of the species was confirmed in the seawater samples which had been collected in Hel (1 isolate), Jastarnia (1 isolate), and Chalupy (1 isolate). One strain of Vibrio alginolyticus was isolated from the seawater sample collected in Hel. Moreover, identification was incomplete for 6 of the isolated strains, these were identified as Vibrio cholerae/mimicus These strains were collected in Jastarnia (1 isolate), Kuznica (1 isolate), Gdansk-Brzezno (1 isolate), Puck (2 isolates), Chalupy (1 isolate). Our preliminary research study confirmed the presence of potentially pathogenic V. vulnificus in the Gulf of Gdansk in the summer months. Therefore, further monitoring of the presence of Vibrio spp. in the Baltic coast area is necessary.

Improved isolation and detection of toxigenic Vibrio parahaemolyticus from coastal water in Saudi Arabia using immunomagnetic enrichment.

BackgroundVibrio parahaemolyticus is recognized globally as a cause of foodborne gastroenteritis and its widely disseminated in marine and coastal environment throughout the world. The main aim of this study was conducted to investigate the presence of toxigenic V. parahaemolyticus in costal water in the Eastern Province of Saudi Arabia by using immunomagnetic separation (IMS) in combination with chromogenic Vibrio agar medium and PCR targeting toxR gene of species level and virulence genes.MethodsA total of 192 seawater samples were collected from five locations and enriched in alkaline peptone water (APW) broth. One-milliliter portion from enriched samples in APW were mixed with an immunomagnetic beads (IMB) coated with specific antibodies against V. parahaemolyticus polyvalent K antisera and separated beads with captured bacteria streaked on thiosulfate citrate bile salts sucrose (TCBS) agar and CHROMagar Vibrio (CaV) medium.ResultsOf the 192 examined seawater samples, 38 (19.8%) and 44 (22.9%) were positive for V. parahaemolyticus, producing green and mauve colonies on TCBS agar and CaV medium, respectively. Among 120 isolates of V. parahaemolyticus isolated in this study, 3 (2.5%) and 26 (21.7%) isolates of V. parahaemolyticus isolated without and with IMB treatment tested positive for the toxin regulatory (toxR) gene, respectively. Screening of the confirmed toxR gene-positive isolates revealed that 21 (17.5%) and 3 (2.5%) were positive for the thermostable direct hemolysin (tdh) encoding gene in strains isolated with IMB and without IMB treatment, respectively. None of the V. parahaemolyticus strains tested positive for the thermostable related hemolysin (trh) gene. In this study, we found that the CaV medium has no advantage over TCBS agar if IMB concentration treatment is used during secondary enrichment steps of environmental samples. The enterobacterial repetitive intergenic consensus (ERIC)-PCR DNA fingerprinting analysis revealed high genomic diversity, and 18 strains of V. parahaemolyticus were grouped and identified into four identical ERIC clonal group patterns.ConclusionsThe presented study reports the first detection of tdh producing V. parahaemolyticus in coastal water in the Eastern Province of Saudi Arabia.

Efficiency and specificity of CARD-FISH probes in detection of marine vibrios.

Vibrio is a bacterial genus widely distributed in natural aquatic systems. Some Vibrio species can cause severe diseases in both marine organisms and humans. Previous studies revealed a link between the current climate change and increased incidence of the Vibrio-associated diseases recently causing sanitary, economic and/or ecological problems worldwide. The conventional culture-based methods (e.g. selection on TCBS agar) used to monitor the presence of Vibrio spp. in environmental samples are not always straightforward and can underestimate the number of cells, especially in microbial populations containing a fraction of 'dormant' cells (e.g. cells in the Viable but Non Culturable [VBNC] state). This problem can be overcome by using alternative culture-free approaches such as Catalysed Reporter Deposition-Fluorescence In situ Hybridization (CARD-FISH). To select an efficient CARD-FISH probe for detection of Vibrio spp. in environmental samples, we have assessed the most promising probes described in the literature by using both computer-assisted and experimental approaches. Our results demonstrate that the use of the optimized protocol along with a very specific probe, ViB572a, can offer the high sensitivity and selectivity of CARD-FISH detection of marine vibrios in natural seawater.

Prevalence and antimicrobial susceptibility patterns of Vibrio cholerae in dairy excreta

Vibrio cholerae is a major etiological agent of human diarrhoea and has become epidemic across the world in the recent past. A cross-sectional study was conducted to assess the prevalence of V. cholerae from dairy excreta along with antimicrobial resistant status of the isolates. A total 50 samples were collected from 50 different household manure pit located at Bangladesh Agriculture University (BAU) surrounding area, Mymensingh. Alkaline peptone water was used for enrichment of the samples followed by inoculation onto thiosulfate citrate bile salt sucrose (TCBS) agar media for the isolation of Vibrio spp., which were further confirmed via Vibrio genus specific molecular assay. Biochemical tests were performed to identify V. cholerae from the isolates of Vibrio spp. Out of 50 samples 17 (34%) were confirmed as Vibrio spp. as they produced characteristic yellow colonies on TCBS agar and had found to possess recombinase A gene that confirmed the identity of Vibrio spp. From this 17 Vibrio isolates, 6 (12% in total from 50 samples) were identified as V. cholerae based on different biochemical tests. All the isolates fermented glucose, maltose, sucrose and mannitol with the production of only acid. The isolates were positive in oxidase, gelatinase, methyl-red (MR) and indole test, but negative in case of voges-proskaure (VP) test. In antimicrobial susceptibility testing, V. cholerae isolates showed 100% sensitivity to gentamycin, chloramphenicol and tetracycline with moderate sensitivity to ciprofloxacin and co-trimoxazole. A high level of resistance was observed to ampicillin (100%) followed by moderate resistance to erythromycin and imipenem. In the present study about 33.33% (n = 2) of 6 isolated V. cholerae were found to be multidrug resistant (MDR) as they demonstrated resistant against 3 antimicrobial agents. The findings of this study substantiate the presence of MDR V. cholerae in the dairy excreta, which indicates the role of domestic animals to serve as a reservoir that might pose a health risk to human. Hygienic management of animal waste is needed to reduce the burden of human illness.
 Asian Australas. J. Biosci. Biotechnol. 2021, 6 (1), 40-49

Phylogenetic characterization and multidrug resistance of bacteria isolated from seafood cocktails.

The continual increase in resistance to antibacterial drugs has become a major public health problem, and their indiscriminate use in agriculture, aquaculture, and the treatment of human and animal diseases has severely contributed to the occurrence and spread of multidrug resistance genes. This study phylogenetically characterized multidrug-resistant bacteria isolated from seafood cocktails. Seafood cocktail dishes from 20 establishments on public roads were sampled. Samples were grown on TCBS agar and blood agar. Forty colonies with different macro- and microscopic characteristics were isolated. The 16S rRNA gene V4 and V6 hypervariable regions were amplified, sequenced and phylogenetically analyzed. Antibacterial drug resistance was determined by disk diffusion assay. Isolated bacteria were identical to species of the genera Enterococcus, Proteus, Vibrio, Staphylococcus, Lactococcus, Vagococcus, Micrococcus, Acinetobacter, Enterobacter, and Brevibacterium, with 75-100% presenting resistance or intermediate resistance to dicloxacillin, ampicillin, and penicillin; 50-70% to cephalosporins; 30-67.5% to amikacin, netilmicin and gentamicin; 40% to nitrofurantoin and other antibacterial drugs; 25% to chloramphenicol; and 2.5% to trimethoprim with sulfamethoxazole. In general, 80% of the bacteria showed resistance to multiple antibiotics. The high degree of bacterial resistance to antibacterial drugs indicates that their use in producing raw material for marine foods requires established guidelines and the implementation of good practices.

Isolation of Vibrio cholerae from clinical and drinking water samples during Cholera Outbreak in Khairpur Sindh Pakistan

Background: Cholera is a diarrheal disease that is caused by the bacterium Vibrio cholerae that transmits through contaminated water, food, oral-fecal route and spread through poor sanitation system. This study aims to investigate the reports of clinical and drinking water samples during an epidemic in Pakistan.
 Methodology: This study was conducted in District Khairpur including 660 samples (360 from clinical and 300 from drinking water) from 2014-2016. All samples were enriched in Alkaline peptone water for 6 hours and then streaked on TCBS agar, incubated at 35ºC for 24 hours. The next day standard microbiological, biochemical, serological techniques were used for the identification of V. cholerae and further identification was performed using PCR.
 Results: Out of 360 clinical samples, 76(21.11%) were positive for V. cholerae. The species-specific Outer membrane protein W precursor (ompW) gene was amplified and shown at the correct size (588 bp) through agarose gel electrophoresis. The serotyping revealed that the isolates belonged to serogroup Inaba, O1. cholerae O1 strains shown typical El Tor phenotype similar to V. cholerae El Tor strain N16961 (PBR VP+) used as the reference strain in this study. All age groups were affected where the highest onset was seen among those aged 19 years and above. The culture of drinking water (n=300), observed negative on TCBS agar.
 Conclusion: As far as our knowledge, this is the first time when the presence of V. cholerae El Tor peak has been reported for causing cholera outbreaks in Khairpur Sindh, Pakistan. However, these findings can be used for further investigations and recognizing control measures.

Essential oil blend as a safe and effective disinfectant strategy for shrimp hatcheries

The Vibrio group of bacteria is considered highly pathogenic to shrimp larvae. Shrimp larvae are susceptible to Vibrio. It is difficult to eradicate Vibrio from the hatchery environment completely. Efforts were made to design an effective disinfectant with almost no side effects or residues. A formulation was developed by combining essential oils with antimicrobial properties to fight against pathogenic Vibrios, such as Vibrio harveyi and V. parahaemolyticus, in a hatchery environment. The anti-Vibrio formulation is a blend of Eucalyptus globulus, jasmine, and gardenia oils, designated as essential oil blend formulation (EOBF). The effective dose of EOBF was optimized on bacteria using TSA and on Vibrio using TCBS agar media. The optimized dose of 40 ppm EOBF was sprayed on the tank and culture area dry surfaces. The trial sites selected were the water filter tank, maturation tank, fry reservoir tank, quarantine and maturation tank, hatchery tank, spawning tank, rinsing tank, plankton tank, larvae rearing tank, packing tank, artemia tank, polychaetes (bucket and floor), squid (equipment and table), and freshwater tank of an experimental hatchery. The swab samples were collected after 3 hours, and a significant decrease in Vibrio, mostly green colonies, such as Vibrio harveyi and V. parahaemolyticus, was found after EOBF application. The obtained results showed that EOBF is an effective disinfectant against pathogenic Vibrios in shrimp hatcheries

Characterization of Vibrio spp. in environmental water samples collected from flood prone areas of Bangladesh and their antibiotic resistance profile.

Last cholera epidemic has been recorded in Bangladesh between 1992–1993, while few sporadic localized outbreaks have been reported as recent as 2005. Serotype O1 of Vibrio cholera is considered as the principal causative agent which transmits through contaminated drinking water resulting that epidemic. Therefore, the objective of this research was to isolate V. cholera in 3 different water sources; River, pond and tube-well, in 5 different locations of Gazipur, Bangladesh, and to analyze their antibiogram study. A total of 45 water samples were randomly collected for the isolation and identification of Vibrio spp. Samples are then serially diluted in alkaline peptone water and streak on Thiosulfate Citrate Bile Salt Sucrose-TCBS agar for quantification of V. spp. For V. cholera isolation water samples were first enriched in nutrient broth at 37 °C for 16 hours followed by cultivation in selective media; TCBS agar at 37 °C for 24 hours. Yellow colonies on TCBS agar were screed as V. cholera and was confirmed by analyzing their biochemical characteristics like Catalase, Oxidase, MR, VP, Indole, Sugar fermentation. Following isolation antibiotic sensitivity test was performed on each V. cholera isolates to determine their antibiotic sensitivity profile. The results showed, out of 45 samples 12 contained V. cholera. Tube-well water has significantly lower concentration (log CFU/mL) of V. spp. than river and pond water (P < 0.05). Bacterial concentration doesn't deviate (P > 0.05) significantly in 5 different location the sample was collected from. All the 12 isolates were sensitive to Gentamicin and ciprofloxacin (100%), while Chloramphenicol (91.67%), Sulfamethoxazole (91.67%), Azithromycin (66.67%) showed high sensitivity. Isolates showed marginal sensitivity towards Tetracycline (33.33%), and Cephalexin (16.67%) and 100% resistance against antibiotics like Vancomycin, Penicillin, Erythromycin, and Nalidixic Acid. Based on these data we recommend using tube-well water instead of river and pond water for drinking purposes. Furthermore, we suggest selective use of sensitive antimicrobials listed here for therapeutics of cholera outbreak.

The Numerical Phenetic of Taxonomy Vibrio in Shellfish (Meretrix meretrix) at Edu-Tourism Mangrove Cengkrong Beach Trenggalek

HighlightIdentify the Vibrio bacteriaDiversity of shellfish Vibrio bacteria (Meretrix Meretrix)Relationship similarity of Vibrio bacteria with MVSP 3.1 Program produces DendogramAbstract Meretrix meretrix is one type of shellfish that is potential, and economic value, which high animal protein content as well as seafood that is widely consumed in Indonesia. This study aims to determine the diversity and similarity of Vibrio in M. meretrix at eduwisata mangrove Cengkrong Beach, Trenggalek. Isolation and identification of Vibrio was conducted by using the media TCBSA (Thiosulfate Citrate Bile Salt Sucrose Agar) streat plate through morphological observation, physiological and biochemical tests based Bergey’s Manual of Systematic Bacteriology conducted in the laboratory of Microbiology Faculty of Biology, Universitas Gajah Mada. Data analysis was performed using the MVSP (Multivariate Statistical Package) 3.1 in the form of a dendrogram showing the similarity relationship Vibrio bacteria. The results of the identification of Vibrio bacteria in M. Meretrix are V. herveyi, V. parahemolyticus, V. nereis and V. proteolyticus. V. herveyi has similarities with V. parahemolyticus 66% and 62% with V. proteolyticus while with V. nereis bacterium 54%.

Isolation and Characterization of Infectious Vibrio Species from the Whiteleg Shrimp: Litopenaeus vannamei

Aquaculture shrimp farming has serious problems with diseases caused by viruses and bacteria, which lead to severe economic loss. The genus Vibrio is a group of facultative anaerobic microbes, most frequently found in aquatic environments and marine hosts. The Gram-negative genus Vibrio is one of the most important classes of bacterial pathogens in aquaculture systems and is a key cause of high mortality. The isolation, identification, and molecular characterization of Vibrio spp are studied from infected shrimp Litopenaeus vannamei. The microorganisms were isolated from the tissues of a white gut infected shrimp collected from diseased aquaculture ponds. After subculturing, microbial isolates were obtained using TCBS agar plates for vibrio selection, and the biochemical key was developed. Meanwhile, 11 isolates of bacteria belonging to the Vibrionaceae family have been identified in this study. For these, six Vibrio species-related isolates are responsible for vibriosis in the shrimp. These included Vibrio harveyi, Vibrio campbellii, Vibrio parahaemolyticus, Vibrio owensii, Vibrio rotiferianus, and Vibrio alginolyticus. The extraction of DNA was carried out with a QIAamp DNA Mini Kit (QIAGEN). The extracted DNA was analyzed using an electrophoresis of 1 % agarose gel, the consistency and quantity of Vibrio spp's gDNA. NanoDropTM 2000 spectrophotometer (Thermo Scientific) was used for determination. In addition, the Agilent 2100 Bioanalyzer (Agilent Technologies) has been used to verify the gDNA libraries' validity, purity, concentration, and scale. In addition, Vibrio spp, a phylogenetic tree focused on maximum likelihood and bootstrapping, was performed using the MEGA version to examine the evolutionary phylogenetic relationship between the strains. This study's main objective would be useful in integrating strain variation in predicting microbiology and microbial risk assessment and may provide scientific guidelines for major Vibrio strains that may be responsible for vibriosis infection in the management of shrimp aquaculture disease.

Vibrio parahaemolyticus-specific Halobacteriovorax From Seawater of a Mussel Harvesting Area in the Adriatic Sea: Abundance, Diversity, Efficiency and Relationship With the Prey Natural Level.

This research aimed to study the abundance and molecular diversity of Vibrio parahaemolyticus-specific Halobacteriovorax strains isolated from seawater of the Adriatic Sea and the relationship between predator and prey abundances. Moreover, predator efficiency of the Halobacteriovorax isolates toward V. parahaemolyticus and Vibrio cholerae non-O1/O139 strains was tested. V. parahaemolyticus NCTC 10885 was used as primary host for the isolation of Halobacteriovorax from seawater by the plaque assay. Molecular identification was performed by PCR detection of a fragment of the 16S rRNA gene of the Halobacteriovoraceae family members. Moreover, 700 bp PCR products were sequenced and compared between them and to clones described for other sampling sites. Vibrio counts were performed on TCBS agar from 100 ml of filtered water samples and presumptive colonies were confirmed by standard methods. Predatory efficiency of Halobacteriovorax isolates was tested by monitoring abilities of 3-day enrichments to form clear lytic halos on a lawn of Vibrio preys, by the plaque assay. Out of 12 seawater samples monthly collected from June 2017 to May 2018, 10 were positive for V. parahaemolyticus specific Halobacteriovorax with counts ranging from 4 to 1.4 × 103 PFU per 7.5 ml. No significant relationship was found between Halobacteriovorax and Vibrio abundances. The 16SrRNA sequences of our Halobacteriovorax strains, one for each positive sample, were divided into three lineages. Within the lineages, some sequences had 100% similarity. Sequence similarity between lineages was always <94.5% suggesting that they may therefore well belong to three different species. All Halobacteriovorax isolates had the ability to prey all tested Vibrio strains. Additional research is necessary to assess whether stable strains of Halobacteriovorax are present in the Adriatic Sea and to understand the mechanisms by which Halobacteriovorax may modulate the abundance of V. parahaemolyticus and other vibrios in a complex marine ecosystem.

The sensitivity test of vibrio cholerae from isolation of diarrhea case to various type of antibiotics

One of the microbes that causes acute diarrhea is bacteria. Vibrio cholerae is one that causes diarrhea called cholera diarrhea. Cholera diarrhea is caused by enterotoxins produced by these bacterial colonies in the small intestine. Giving antibiotics is still the main choice in the treatment and treatment of diarrhea because it is expected to kill bacteria and will usually stop diarrhea. Irrational use and the existence of abuse and excessive use of antibiotics can be a factor that causes bacterial resistance to antibiotics. Tests carried out to determine the sensitivity of bacteria to an antibiotic. Vibrio cholerae isolate was regrowed in alkaline peptone (APW) water medium and incubated at 37ºC for 18-24 hours, then planted in the thiosulfate-citrate-bile-sucrose (TCBS) medium. The sensitivity test was carried out on V. cholerae bacterial colonies growing on TCBS agar medium with the Disk Diffusion Method from Kirby Bauer. Data were analyzed descriptively. The results showed that most V. cholerae isolates were still sensitive to all types of antibiotics used in the test. Vibrio cholerae has shown resistance to Colistin antibiotics that is equal to 88.2%, Ampicillin at 23.5% and Ceftazidime at 5.9%. The level of resistance to antibiotics that are still low indicates that the antibacterial group can still be used as an alternative in the treatment of cholera diarrhea.

Comparative evaluation of three agar media-based methods for presumptive identification of seafood-originated Vibrio parahaemolyticus strains

Vibrio parahaemolyticus has been one of the major bacterial species responsible for summer food poisoning. However, the limitations of current medium-based strategies for its detection necessitates a more effective selective or discriminatory agar media-based method. In this study, we first developed ChromoVP agar for more specific differentiation of V. parahaemolyticus from other bacterial species. Secondly, we compared this new medium with the commercially-available CHROMagar™ Vibrio medium and thiosulfate–citrate–bile salts–sucrose (TCBS) agar medium for the identification of V. parahaemolyticus strains isolated from seafood. The sensitivity, specificity, accuracy, and positive and negative predictive values of each of these agar media-based methods in the identification of V. parahaemolyticus were determined and compared using 38 V. parahaemolyticus strains and 102 other Vibrio and non-Vibrio strains. As a benchmark, a species-specific polymerase chain reaction assay was performed to detect V. parahaemolyticus and four other Vibrio species in seafood samples based on their genetic differences. Our results showed that ChromoVP agar was more sensitive (89.5%), specific (89.2%), and accurate (89.3%) than CHROMagar™ Vibrio and TCBS agar media in differentiating V. parahaemolyticus strains from other organisms. Therefore, ChromoVP agar is an effective medium for the isolation and enumeration of V. parahaemolyticus from seafood samples.

Photobacterium damselae subsp. damselae associated with bacterial myonecrosis and hepatopancreatic necrosis in broodstock Pacific white leg shrimp, Litopenaeus vannamei (Boone, 1931)

This study presents the first report of myonecrosis and hepatopancreatic necrosis in broodstock of Litopenaeus vannamei. Of the 400 individuals examined, 34 displayed myonecrosis and hepatopancreatic necrosis, conditions previously reported to be caused by bacterial, fungal, parasite, and viral infections. Gross signs of infection include passive swimming, anorexic and the abdominal muscle become opaque with extensive whitish discoloration of distal abdominal segments and the posterior appendages. Macroscopic necropsy examination revealed atrophied hepatopancreas and empty stomach and mid-gut. The histopathological investigations revealed that reddened muscular lesions are composed of coagulative and liquefactive necrosis of striated muscle fibers, often with edema. The hepatopancreas showed necrotic lesions displaying disorganized lumen and dispersed tubular epithelial cells scattered on necrotic myoepithelial cells. Gill lamellae in infected shrimp showed disorganized, necrotic filaments infiltrated with inflammatory hemocyte. The RT-PCR analysis using OIE-recommended primer and additional sets of primers used in previous studies revealed the absence of IMNV as well as other emerging pathogens including MrNV, PvNV, WSSV, IHHNV, TSV, YHV, AHPND, and EHP. Basis on these negative results, microbiological tests were performed. A dominant yellow-colored bacterial colony was isolated on TCBS agar and identified as Photobacterium damselae subsp. damselae KPD. Injection of the isolate of P. damselae KPD proved to be pathogenic to L. vannamei. The study concludes that P. damselae KPD is the causative agent of myonecrosis and hepatopancreatic necrosis and represents a threat to the Indian shrimp hatcheries.

Use of indigenous beneficial bacteria (Lactobacillus spp.) as probiotics in shrimp (Penaeus monodon) aquaculture

The present study was conducted to study in-vitro the antagonistic effect of Lactobacillus spp. against the pathogenic bacterial vibrio harveyi on shrimp. For this purpose, shrimp samples were collected from three different Ghers at Batiaghata upazilla, Khulna. Gills and intestines were taken out from the samples to identify the load of Lactobacillus spp. and Vibrio spp. The results revealed that the load of Lactobacillus spp. was found more than Vibrio spp. both in gills and intestines; the gills also contained higher load of Vibrio spp. than in the intestines. V. harveyi was separated from the isolated Vibrio spp. with different types of biochemical tests: Gram stain, Motility test, Indole test, VP test, MR test, Arginine dihydrolase, Salt tolerance test, growth at different temperature ranges and colony color on TCBS agar media. The isolated V. harveyi was subjected for in-vitro test. In in-vitro challenge test, the potential antagonistic effect of Lactobacillus spp. against V. harveyi was gradually obtained at 0, 4th, 8th, 12th hour of treatments. Interesting finding was that, with the time, the load of V. harveyi was reduced gradually and the lowest load was obtained after 12 hours of probiotic inoculation. The present study revealed an excellent in-vitro antagonistic probiotic effect of Lactobacillus spp. on V. harveyi. Therefore the result suggested that probiotic treatment might be an effective alternative to the use of antibiotics in treatments of bacterial diseases in shrimp aquaculture.Res. Agric., Livest. Fish.5(1): 127-135, April 2018

Detection of tlh and tdh genes in Vibrio Parahaemolyticus inhabiting farmed water ecosystem used for L. Vannamei aquaculture

Vibrio parahaemolyticus is hallophilic gram-negative bacteria that live as natural inhabitant in aquatic environment. All Vibrio parahaemolyticus strain known to have thermolabile hemolysin encoded by tlh gene as species marker. Thermostable direct hemolysin encoded by tdh gene is responsible for regulating virulence factor in Vibrio parhaemolyticus. Aim of this research is to detect tlh and tdh gene from water of L. vannamei aquaculture in Rembang regency. Colonies of green-blueish bacteria grew from isolation of L. vannamei aquaculture water in CD-VP media which was identified as Vibrio parahaemolyticus. Colonies of V. parahaemolyticus grew to be small and green-blueish bacteria colonies in TCBS agar. Result of molecular analysis showed that bacteria isolated from water sample are specifically identified as Vibrio parahaemolyticus bacteria by the detection of tlh gene. Vibrio parahaemolyticus isolated from water of L. vannamei aquaculture detected as tdh negative that indicates tdh gene is not present in isolated bacteria. Vibrio parahaemolyticus isolate were cultured in Wagatsuma agar for tdh gene confirmation test that showed Kanagawa negative result, which indicated that V. parahaemolyticus did not produce thermostable direct hemolysin. These results showed that Vibrio parahaemolyticus isolated from aquatic environment of L. vannamei aquaculture in Rembang regency did not show virulence factors.

Macroalgal and Seagrass Diets Alter Epibiotic Bacterial Communities on the Blue CrabCallinectes sapidusand the American LobsterHomarus americanus

ABSTRACT Bacteria in the genus Vibrio can be opportunistic pathogens for organisms in marine and estuarine systems. Consumption of seagrass by the blue crab Callinectes sapidus or macroalgal kelp by the lobster Homarus americanus reduced epibiotic bacteria in the Vibrio genus. Bacterial Vibrio spp. densities were estimated by colony growth on thiosulfate citrate bile sucrose agar. Nonspecific epibiotic bacterial densities were estimated by colony growth on nonselective agar or by direct counting under a fluorescence microscope. Consumption of the seagrass Ruppia maritima, but not the aquatic plant Stukenia pectinata or a control diet, reduced Vibrio spp. densities on blue crabs. A diet containing the kelp Saccharina latissima reduced Vibrio spp. densities on lobsters compared with a control diet. Results for nonspecific bacterial densities were mixed. A reduction was observed when cultured colonies were counted but not when cells were counted directly. The results suggest that diets containing seagrass or...