TCBS Agar Research Articles

ANTIBACTERIAL EFFICACY OF BACTERIOPHAGES AGAINST VIBRIO CHOLERAE

This study investigates the use of bacteriophages as an alternative antibacterial approach to combat antibiotic-resistant Vibrio cholerae, a major public health concern. Vibrio cholerae strains were identified through culture on TCBS agar, microscopy, and biochemical tests (catalase, oxidase, indole, and citrate). Antibiotic susceptibility was assessed using the Kirby-Bauer disk diffusion method across five antibiotics: tetracycline, norfloxacin, ampicillin, ciprofloxacin, and erythromycin. Results showed resistance to ampicillin, tetracycline, and erythromycin, while strains remained susceptible to norfloxacin and ciprofloxacin Five bacteriophages against Vibrio cholerae were isolated from 50 wastewater samples, and only two (F1 and F2) showed lytic activity, forming clear plaques. These two lytic phages were further purified and characterized for stability across different temperatures, pH levels, and host range. The isolated phages were tested for host range against six Vibrio cholerae strains, with F1 lysing five strains and F2 lysing three, and demonstrated stability across temperatures (20°C to 55°C) and pH levels (4 to 9). They remained active across a wider temperature and pH range demonstrating their potential for therapeutic use. The study shows that these phages are highly specific, targeting only Vibrio cholerae strains from various sources without affecting other bacterial species. This specificity makes them safer for therapeutic use, as they are less likely to disrupt the body’s natural bacterial flora. Further genomic studies are recommended to characterize the genetic makeup of these phages, optimizing their application in cholera treatment. This study provides critical insights into the potential of bacteriophages as an alternative treatment for antibiotic-resistant Vibrio cholerae.

Mass Mortality of the Invasive Sea Urchin Diadema setosum in Türkiye, Eastern Mediterranean Possibly Reveals Vibrio Bacteria Infection

ABSTRACTThe invasive Diadema setosum (Leske, 1778) long‐spined sea urchin has been in the Mediterranean Sea since 2006, and then, it has been known that its population density has reached high values on the southern Aegean coasts of Türkiye. This study aimed to report the mass mortality event of D. setosum with the determined agent on the Aegean coast of Türkiye. In addition, it was targeted to provide information on the current status of its density based on seasonal SCUBA diving observations for the period between January 2023 and November 2023. The results showed that the mass mortality of D. setosum was determined in Muğla, Aegean Sea, in August 2023. Spine loss, the mucoid layer at the bottom of the appendages, and the outer body surface were observed as the clinical symptoms of the diseased samples. Bacterial growth was detected on Vibrio‐selected TCBS agar plates, while no other parasitic agents were determined in the coelomic fluid of sea urchins. The results of the biochemical (API 20E) and molecular tests confirmed the isolated bacteria as Vibrio spp. The density of D. setosum showed statistically significant temporal changes, and the highest and lowest mean density values were recorded in autumn and spring, respectively. Environmental stressors, such as increasing sea temperature levels, affect the marine ecosystem and lead to opportunistic pathogens. Long‐term monitoring of disease outbreaks is necessary to understand the interactions between species and the ecosystem.

Isolation and Identification of Vibrio Species from Different Types of Water Sources Along with Their Drug Susceptible Pattern

Abstract Background: Human activities are almost usually the cause of contaminated water supplies and places with large population densities and intensive land usage are particularly prone to pollution of drinking water sources. It is challenging to detoxify polluted water sources. Cholera is an acute form of diarrheal disease that plagued human civilization over the centuries. Vibrio spp., commonly found in aquatic environments, are the most common cause of bacterial gastroenteritis in the world, those may spread to humans through the ingestion of contaminated drinking water or exposure to seawater. Methods: In the present study, a total of 12 samples were collected from four types of water sources for the isolation of Vibrio spp. along with their drug-resistant pattern through conventional, biochemical, and antibiotic disc diffusion methods. Results: The samples were enriched into alkaline peptone water and then inoculated into culture media such as nutrient agar, MacConkey, and TCBs agar medium. After incubation for 24 h at 37°C on TCBS agar, yellow and green colonies were screened out for biochemical identification. Nonsucrose-fermenting Vibrio parahaemolyticus, Vibrio mimicus, and Vibrio vulnificus showed green colonies, and sucrose-fermenting Vibrio cholera and Vibrio alginolyticus showed yellow colonies on TCBS. The antibiotics ERY (15 μg), PCN (10 μg), CEX (30 μg), and VAN (30 μg) were completely resistant while the more effective antibiotics against Vibrio spp. were SXT (25 μg), CHL (30 μg), and GEN (30 μg). However, ciprofloxacin and nitrofurantoin showed moderate efficacy. Conclusion: To expand current knowledge on the occurrence, ecological niche, and persistence of potential human pathogenic Vibrio spp. in aquatic environments, the extensive laboratory experiment is required. To sum up, this kind of information is crucial for bringing attention to the existence of Vibrio spp. and enabling prompt implementation of preventive measures.

Detection and quantitative microbial risk assessment of pathogenic Vibrio cholerae in a river used for drinking, domestic, fresh produce irrigation and recreational purposes

Cholera infection results from the ingestion of water or food contaminated with toxigenic Vibrio cholerae. This study evaluated the occurrence of toxigenic V. cholerae in Asata River, Enugu Metropolis, Nigeria, and estimated V. cholerae infection risks from use of the River water for drinking, domestic and recreational purposes. Vibrio was detected and quantified using membrane filtration technique and thiosulfate–citrate–bile salts–sucrose agar. Isolates were screened by PCR, using specific primers targeting the internal transcribed spacer (its) region between 16 and 23S rRNA and the cholera toxin (ctx) gene. Sequenced 16SrRNA gene amplicons of two selected isolates were used for phylogenetic analysis. Quantitative microbial risk assessment (QMRA) was conducted using the β-Poisson dose–response model. About 81% (58/72) of Asata River samples recorded Vibrio counts above 1.0 ×\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\ imes$$\\end{document} 103 cfu/100 mL. Of the fifty Vibrio isolates screened, its was detected in 54% (27/50), out of which 74% (20/27) had the ctx gene of toxigenic V. cholerae. Evolutionary relatedness of the sequenced isolates to V. cholerae was revealed. The estimated risks of cholera infection in persons exposed to untreated Asata River water were above 0.5 for all the exposure scenarios, for both the rainy and dry seasons. The risks were highest (~ 0.9) for exposure via drinking water and annual risk of infection was deduced to have a probability of 1.0. Therefore, dependence on the untreated Asata River water for drinking, recreational, domestic and irrigation purposes may present a potential public health risk of cholera outbreak. We recommend increased monitoring and surveillance of River water for Vibrio abundance and that Asata River be protected from further degradation.

IN VITRO ANTIBACTERIAL ACTIVITY OF ETHANOLIC EXTRACT OF DRUMSTICK (Moringa olifera) LEAF AGAINST SHRIMP PATHOGENIC BACTERIA VIBRIO PARAHAEMOLYTICUS

Aim of the present study was to evaluate the antibacterial effect of ethanolic extract of Moringa olifera leaf against shrimp pathogenic bacteria Vibrio parahaemolyticus. The bacteria were cultured on TCBS agar media and enumerated. The ethanolic M. olifera leaf extract (1 mg/mL) was prepared and its antibacterial activity at different dose (400 µL, 300 µL and 200 µL) was evaluated against V. parahaemolyticus by using disk-diffusion method. The bacteria load of 1.75×104 CFU/mL, 1.63×104 CFU/mL and 1.54×104 CFU/mL was employed for each concentration. It was observed that 400 µL of ethanolic extract of M. olifera showed the highest inhibition zones (11.97 mm, 13 mm and 15.13 mm) but not equal to the inhibition zones (17.23 mm, 18.20 mm and 19.70 mm) by standard antibiotic (oxytetracycline). The present study revealed that ethanolic extract of moringa leaf (1mg/ml) had significant positive effect (P value = 0.003) on different V. parahaemolyticus load at 5% level of significance though effect of different doses with same concentration (1mg/ml) had not significantly differ from each other. Therefore, the ethanolic extract of M. olifera might be used as alternative way to prevent diseases which are caused by V. parahaemolyticus.

DISSEMINATION OF DIARRHEAGENIC BACTERIA IN COMMUNITY-BASED NATURAL WATER SOURCES AND WASTEWATER ENVIRONMENTS: A COMPREHENSIVE ANALYSIS IN UNIVERSITY OF NIGERIA, NSUKKA AND ITS METROPOLIS.

The presence of pathogenic strains abounds in our environment, arising from many sources. The study was aimed at identifying Vibrio species, E. coli, and other coliforms in various water sources within a university setting and its metropolis. Approximately 20 samples from roof-collected rainwater, municipal piped tap water, borehole water and 60 samples of sewage effluent from the central effluent water of University of Nigeria, Nsukka were systematically collected at intervals and subjected to bacteriological analysis. The samples underwent dilutions, and aliquots were inoculated on chromocult and thiosulfate-citrate-bile salts-sucrose (TCBS) agar. Simultaneously, 100 ml of each natural water sample underwent membrane filtration, followed by the inoculation of TCBS agar plates in duplicates. Both sets of culture plates were incubated at 37 °C for 24 h. Further biochemical characterization was performed after microscopy. Susceptibility profile was done using disk diffusion assay. Descriptive statistics were used in the aggregation of the results where necessary. Results revealed that each sample, when plated on chromocult agar, yielded a substantial number of colonies of E. coli (bluish colour) and other coliforms (pink-red colonies). Notably, wastewater gave a maximum yield of Vibrio cholera. Conversely, all significant pathogenic Vibrio species were recovered on TCBS agar inoculated with natural water samples, wastewater and sewage effluent, with isolation rates appearing independent of both time (in weeks) and collection points. Antimicrobial susceptibility studies disclosed that some of the isolates exhibited antibiotic resistance. The study underscores the need to install appropriate measures to mitigate the contamination of the environment. Keywords: Wastewater, Vibrio, Antimicrobial, Chromocult, Pathogen, Sewage

Diversity of Culturable Bacteria from the Coral Reef Areas in the South China Sea and Their Agar-Degrading Abilities.

The South China Sea (SCS) is abundant in marine microbial resources with high primary productivity, which is crucial for sustaining the coral reef ecosystem and the carbon cycle. Currently, research on the diversity of culturable bacteria in the SCS is relatively extensive, yet the culturable bacteria in coral reefs has been poorly understood. In this study, we analyzed the bacterial community structure of seawater samples among Daya Bay (Fujian Province), Qionghai (Hainan Province), Xisha Islands, and the southern South China Sea based on culturable methods and detected their abilities for agar degradation. There were 441 bacterial strains, belonging to three phyla, five classes, 43 genera, and 101 species, which were isolated by marine agar 2216E (MA; Becton Dickinson). Strains within Gammaproteobacteria were the dominant group, accounting for 89.6% of the total bacterial isolates. To investigate vibrios, which usually correlated with coral health, 348 isolates were obtained from TCBS agar, and all isolates were identified into three phylum, three classes, 14 orders, 25 families, and 48 genera. Strains belonging to the genus Vibrio had the greatest number (294 strains), indicating the high selectivity of TCBS agar for vibrios. Furthermore, nineteen strains were identified as potentially novel species according to the low 16S rRNA gene similarity (<98.65%), and 28 strains (15 species) had agar-degrading ability. These results indicate a high diversity of culturable bacteria in the SCS and a huge possibility to find novel and agar-degrading species. Our study provides valuable microbial resources to maintain the stability of coral ecosystems and investigate their roles in the marine carbon cycle.

Identification of Vibrio Cholerae using Analytical Profile Index (API 20E)

Cholera outbreak have resulted in the deaths of millions of people globally. The cholera-causing Vibrio cholerae, has multiple serogroups, and produces epidemics when two strains, O1 and O139, are present. With the exception of recent, rare instances, V. cholerae O139 caused prior outbreaks; however, V. cholerae O1 was responsible for all following epidemics. Hence, identification of the V. cholerae is an important step in the control of cholera outbreak especially in the local community where inadequate facilities for prognosis limit prompt treatment of patients. A total of 15 isolates previously identified with molecular method were subjected to TCBS Agar and Analytical Profile Index. All the sucrose-fermenting colonies on TCBS agar were correctly identified with API kit. The study concluded that API kit can be substituted for molecular method in prognosis of Cholera outbreak especially in resource-limited Countries due cost implication.

Molecular identification and antibiotic resistance profiling of Vibrio spp. in diarrhoeal patients from Dinajpur, Bangladesh

This research aimed to characterize Vibrio species from cholera-suspected patients in three hospitals in Dinajpur. We collected 36 stool samples and identified Vibrio spp. using cultural, morphological, and biochemical tests. The highest prevalence was observed in children (18.18%), compared to adolescents (0%), adults (16.67%), and senior adults (16.67%). Cultural identification revealed yellow colonies on TCBS agar, hemolytic colonies on blood agar, pale non-lactose fermenting colonies on MacConkey agar, and white, opaque colonies on nutrient agar. Gram staining showed gram-negative, curved rod-shaped bacteria. Biochemical tests indicated positive reactions for oxidase, catalase, MR, TSI, citrate utilization, MIU, and indole, while VP was negative. Molecular characterization by PCR and gel electrophoresis using 16S rRNA primers confirmed the presence of Vibrio spp. Antibiotic sensitivity testing showed that isolates were sensitive to cotrimoxazole-25µg (21 mm to 30 mm), neomycin-30µg (22 mm), gentamycin-10µg (15 mm), streptomycin-10µg (20 mm), and tetracycline-30µg (17 mm), but resistant to penicillin, mecillinam, and amoxicillin. These findings suggest streptomycin and cotrimoxazole as effective drugs for controlling cholera.

Detection of multi-antibiotic resistant Vibrio parahaemolyticus isolated from fresh produce in Dhaka, Bangladesh

The demand for fresh produce is rising daily as it's an excellent way to maintain a healthy lifestyle. The current work fixed the target employing culture-based and molecular techniques for isolating and identifying the pathogenic Vibrio parahaemolyticus from fresh produce. The colonial appearance on TCBS agar and CHROMagar culture plates revealed the prevalence of Vibrio spp. and V. parahaemolyticus in all examined samples, which were 45/112 (40.2%) and 40/112 (35.7%), with the highest load of up to 2.0x105cfu/g and 6.4x103cfu/g, respectively. Sixty isolates in all were chosen for further molecular characterization. Multiplex PCR results exhibited that all tested isolates were positive for tlh gene and six for tdh gene but none of isolate for trh gene. According to obtained data on antimicrobial susceptibility, the major portion of tested strains was found to be resistant to ampicillin (90%), tetracycline (80%), and streptomycin (70%). The range of the multiple antibiotic index was 0-0.33. This study finding revealed that the existence of potential pathogenic V. parahaemolyticus in tested fresh produce samples constitutes a risk to public health and consumer safety thus urging continued surveillance.

Vision Technique for Vibrio Detection using Shrimp Cultivation Isolated Water in TCBS Agar Plates

Maintaining water quality is a crucial factor for shrimp cultivation. Detecting the vibrio bacteria distribution in the ponds could prove to be a crucial for successful cultivation. Thiosulfate-citrate-bile salts-sucrose (TCBS) agar is a commonly used medium to stimulate the Vibrio colony presence. In this research, water samples from shrimp cultivation were collected, isolated in TCBS agar plates, and incubated for 24 hours, afterwards exported as an image dataset. The YOLOv7 computer vision technique was implemented to detect and count Vibrio colonies with faster and higher precision compared to the tedious manual counting technique done by laboratory researchers. Additionally, this technique is applicable and scalable to large sets of data. From the execution done so far, the proposed system has achieved an average accuracy of 78.99%.

Antibiotic resistance and molecular identification of dominant bacteria associated with loose shell syndrome in mangrove crabs (Scylla spp.)

The emergence of loose shell syndrome (LSS) in Philippine mangrove crabs poses a serious threat to both the aquaculture industry and the delicate ecosystems it relies on. This study aimed to identify dominant bacteria associated with LSS and assess their antibiotic resistance to guide treatment and prevention strategies while safeguarding public health. Mangrove crab samples with loose carapace were collected from the province of Capiz, Philippines. Dominant bacteria that grew on Thiosulfate– Citrate–Bile Salts–Sucrose (TCBS) and modified chitin agar were isolated and identified by sequencing of the16S rRNA. Antibiotic susceptibility of the dominant bacterial isolates was performed using the agar disk diffusion method. Dominant bacteria that grew on TCBS agar included putative Vibrio, Proteus, Shewanella, and Stutzerimonas. The isolates had antibiotic resistance indices (ARI) ranging 0.8 to 1.2. Chitinolytic bacteria were dominated by Vibrio, Shewanella, and Brevundimonas. Both Vibrio and Brevundimonas exhibited resistance to gentamicin, ciprofloxacin, and norfloxacin, while Shewanella showed resistance to ciprofloxacin. The presence of multi-drug resistant bacteria in LSS-affected crabs highlights the urgent need for effective health management strategies in the industry while mitigating the risks associated with antibiotic resistance to protect both the sustainability of mangrove crab aquaculture and the health of consumers.

Study of the population Vibrio Sp. on the health of vanname shrimp (Litopenaeus vannamei) in Cv. Reksa Bumi Situbondo

Vannamei shrimp is one of the commodities of excellence in aquaculture. The production system is carried out mass with high stocking density causes vannamei shrimp culture susceptible to disease. One type of disease that can cause mass mortality in cultured shrimp is vibriosis. This activity aims to study the abundance of bacterial populations Vibrio sp. and its effect on the health of Vanname Shrimp (Litopenaeus vannamei) at CV. Reksa Bumi Situbondo East Java. Identification of Vibrio sp. performed on TCBS agar media. Clinical symptoms of shrimp that are attacked by vibriosis. Besides the attack vibrio sp. produce clinical symptoms such as body flushing, red swimming legs and red tail. The abundance of Vibrio sp. that exceeds the threshold causes WFD disease attacks and IMNV. The high abundance of Vibrio sp. caused by conditions unfavorable aquatic environment due to entering the transition season resulting in sudden changes in water quality and increase the abundance of bacteria.

Characterization and Genetic Relationships of Vibrio spp. Isolated from Seafood in Retail Markets, Yala, Thailand

Vibrio spp. including Vibrio cholerae and V. parahaemolyticus are the major causative agent of diarrhea involved in seafood consumption. Yala is a southern border province in Thailand where no seafood sources are provided. Thus, it is necessary to import several seafood products from neighboring provinces. The risk of Vibrio spp. replication is increased during seafood transport. Therefore, the objectives of this study were to isolate and characterize V. cholerae and V. parahaemolyticus from seafood in Yala and to investigate their genetic relationships with virulent and clinical strains. A total of 100 Vibrio isolates were obtained from four different sample types in retail markets. It was found that two-thirds of all isolates were yellow colonies on TCBS agar. Using PCR with specific primers, 34 % of V. cholerae and 8 % of V. parahaemolyticus were identified. To classify the virulence strains, Vibrio spp. were positive for hlyA and tdh, 17 and 1 %, respectively. Moreover, 54 % of Vibrio isolates were able to produce biofilm and 28 % of those isolates showed a high level of biofilm production. All representative strains of V. cholerae presented ß-hemolysin on blood agar correlated to hlyA while representative strains of V. parahaemolyticus presented γ-hemolysin and ß-hemolysin on blood agar. In addition, ERIC-PCR provided different DNA profiles of representative V. cholerae and V. parahaemolyticus which some strains showed a correlation with clinical strains and virulent strains. Therefore, these findings might indicate the risk of Vibrio infections for seafood consumers, especially in Yala province where seafood needs to be imported from other provinces. HIGHLIGHTS The first report showed high risk of Vibrio cholerae and V. parahaemolyticus spread in non-seafood source area: Yala province The majority of Vibrio cholerae and V. parahaemolyticus isolated from seafood in retail markets, Yala, Thailand was classified as virulent strains which possess major virulence genes, hemolytic activity, and biofilm formation Several Vibrio isolates obtained from seafood showed genetic similarities with virulent and clinical strains GRAPHICAL ABSTRACT

Antibiotic resistance of emerging pathogenic bacteria of hybrid grouper farming in Indonesia

Abstract. Istiqomah I, Isnansetyo A, Murwantoko, Handayani DP, Lestari YN, Taslihan A, Permana IGN, Wijayanti E. 2023. Antibiotic resistance of emerging pathogenic bacteria of hybrid grouper farming in Indonesia. Biodiversitas 24: 2493-2501. A hybrid grouper is among the main coastal farming species in Asian countries, yet vibriosis is still a significant issue. Therefore, a knowledge of pathogenic strains responsible for recent cases is essential for developing appropriate control measures. The purpose of the study was to describe emerging bacterial pathogens associated with hemorrhagic disorder, skin wounds, and mortality in cantang hybrid grouper farms in Indonesia. A total of 30 bacterial strains were recovered from the kidney of diseased grouper on TCBS agar. Based on the ability to induce clinical signs and mortality of grouper juveniles owing to intraperitoneal bacterial infection at 106 CFU/fish, the bacteria were divided into three groups: pathogenic (n=11), low-pathogenic (n=2), and non-pathogenic (n=17). The pathogenic strains were putatively identified as Photobacterium damselae subsp. Damselae (n=8), Vibrio alginolyticus (n=1), Vibrio harveyi (n=2), and Vibrio azureus (n=2) based on the biochemical characteristics and 16S rRNA and DNA gyrase B subunit gene sequences. The pathogenic bacteria were sensitive to erythromycin, gentamycin, oxytetracycline, and kanamycin. However, it still has various resistant patterns to ampicillin, fosfomycin, chloramphenicol, streptomycin, rifampicin, and enrofloxacin. This study demonstrated the emergence of pathogenic P. damselae subsp. damselae, Vibrio alginolyticus, V. harveyi, and V. azureus in grouper aquaculture in Indonesia with multiple antibiotic resistance, which needed to further studies.

Pathogenicity of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) in shrimp (Litopanaeus vannamei) in Serang, Banten, Indonesia

Abstract. Saputra A, Maftuch, Andayani S, Yanuhar U. 2023. Pathogenicity of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) in shrimp (Litopanaeus vannamei) in Serang, Banten, Indonesia. Biodiversitas 24: 2365-2373. Vannamei shrimp target EMS (Early Mortality Syndrome)/AHPND (Acute Hepotopancreatic Necrosis Disease) cases in several Asian nations, including China, Vietnam, Malaysia, Thailand, India, and Indonesia. AHPND cases on shrimp farms have been reported in several Indonesian regions. The aim of this study was to evaluate the histopathological pathogenicity of Vibrio parahaemolyticus in different shrimp tissues exposed to different doses and durations of exposure. PCR amplification of V. parahaemolyticus was performed using Vp.flaE-79F (5?-GCA GCT GAT CAA AAC GTT GAG T-3?) and Vp.flaE-934R (5?-GATACCTTGTTACGACTT -3?) primers for molecular identification of bacteria isolated from shrimp exhibiting AHPND symptoms. V. parahaemolyticus positive target band at 897 bp. Bacterial isolates with green colonies on TCBS agar media were chosen for other test. Challenge tests with healthy shrimp were conducted using the V. parahaemolyticus strain. The challenge test on shrimp demonstrated the same clinical and pathological symptoms as the signs of infected shrimp in rivers, including swimming into the container’s corner and changes in the carapace’s pale color and empty midgut. The results showed that challenged shrimp displayed necrosis, atrophy, granulomas, cell sloughing, and had significantly considerable hemocyte infiltration that led to melanization. The highest percentage (63.33%) of damage was recorded in the treatment with a concentration of 108 CFU mL-1. It was also observed that virulence of V. parahaemolyticus strain varied with different concentrations. At concentrations of 108 CFU mL-1 and 106 CFU mL-1, virulence of V. parahaemolyticus strain caused 100% mortality in 73 hour post-infection (p.i). The lowest concentration limit for pathogenicity was 104 CFU mL-1 with a mortality of 48% at 73 p.i. The challenge test results showed that V. parahaemolyticus strain that causes AHPND was the main pathogen affecting vannamei shrimp in Serang, Banten, Indonesia.

Morpho-physiological-based selection criteria for chili (Capsicum annuum) under drought stress during vegetative to generative phase

Abstract. Istiqomah I, Isnansetyo A, Murwantoko, Handayani DP, Lestari YN, Taslihan A, Permana IGN, Wijayanti E. 2023. Antibiotic resistance of emerging pathogenic bacteria of hybrid grouper farming in Indonesia. Biodiversitas 24: 2493-2501. A hybrid grouper is among the main coastal farming species in Asian countries, yet vibriosis is still a significant issue. Therefore, a knowledge of pathogenic strains responsible for recent cases is essential for developing appropriate control measures. The purpose of the study was to describe emerging bacterial pathogens associated with hemorrhagic disorder, skin wounds, and mortality in cantang hybrid grouper farms in Indonesia. A total of 30 bacterial strains were recovered from the kidney of diseased grouper on TCBS agar. Based on the ability to induce clinical signs and mortality of grouper juveniles owing to intraperitoneal bacterial infection at 106 CFU/fish, the bacteria were divided into three groups: pathogenic (n=11), low-pathogenic (n=2), and non-pathogenic (n=17). The pathogenic strains were putatively identified as Photobacterium damselae subsp. damsela (n=8), Vibrio alginolyticus (n=1), Vibrio harveyi (n=2), and Vibrio azureus (n=2) based on the biochemical characteristics and 16S rRNA and DNA gyrase B subunit gene sequences. The pathogenic bacteria were sensitive to erythromycin, gentamycin, oxytetracycline, and kanamycin. However, it still has various resistant patterns to ampicillin, fosfomycin, chloramphenicol, streptomycin, rifampicin, and enrofloxacin. This study demonstrated the emergence of pathogenic P. damselae subsp. damselae, Vibrio alginolyticus, V. harveyi, and V. azureus in grouper aquaculture in Indonesia with multiple antibiotic resistance, which needed to further studies.

Detection of Vibrio sp. on vaname shrimp (Litopanaeus vannamei) cultivation pond in Gorontalo, Indonesia

Abstract. Ulfiani P, Hasan AM, Retnowati Y. 2022. Detection of Vibrio sp. on vaname shrimp (Litopanaeus vannamei) cultivation pond in Gorontalo, Indonesia. Nusantara Bioscience 14: 340-346. Vibrio is a pathogenic bacteria that can attack cultured shrimp. Therefore, it is necessary to increase the intensity of water changes and efforts to control water quality to maintain dissolved oxygen levels and manage organic matter content in ponds to prevent the increase of pathogenic bacteria, especially Vibrio. In addition, water physicochemical factors can influence the presence and population of Vibrio bacteria in ponds. This study aims to determine the presence of Vibrio sp. in vaname shrimp culture ponds in Gorontalo, Indonesia. Descriptive quantitative analysis was used to analyze the data collected from this study. Shrimp pond water samples were obtained from shrimp ponds in Molosipat Village, West Popayato Sub-district, Pohuwato District, Gorontalo, Indonesia, using a purposive sampling technique with an interval of three days from the first water change to the last before the water change in one period (approximately 10 days). The water samples were subjected to physicochemical analysis, including temperature, pH, salinity, ammonia, nitrate, and dissolved oxygen. The results showed that there were Vibrio bacteria in the Thiosulfate Citrate BileSalt Sucrose Agar (TCBSA) medium with a population that tends to increase in the range of 1.0 × 103 - 2.7 × 104 CFU/mL. The isolation results showed that two isolates were named ISL1 and ISL2, with the colony morphology characters of ISL1 isolates being green and ISL2 isolates being yellow. The results of microscopic observations of the two isolates showed comma-shaped and gram-negative cells. Molecular identification using the 16S rRNA gene and reconstruction of the phylogenetic tree using the Neighbor-Joining algorithm showed that ISL1 isolate is very closely related to Vibrio parahaemolyticus strain 11-1 (relationship level 99%) and ISL2 is closely related to V. parahaemolyticus strain B2-1 (relationship level 86%).

Isolation and Characterization of Resistant Bacterial Species Isolated From Shallow Well Water Situated Close to Graves as a Public Health Menace in Osogbo, Osun State

This study was designed to evaluate the physicochemical and bacteriological qualities of well water situated close to graves. Total heterotrophic bacterial count (THBC), total coliform count (TCC) and fecal coliform count (FC) were done on nutrient agar, MacConkey and Eosine methylene blue agar, respectively, using spread plate method at incubation conditions of 37℃ for 24 hours for THBC and TCC whereas 44.5℃ for 24-48 hours for fecal coliforms. Membrane filtration was used for Vibrio spp. count on TCBS agar while conventional biochemical method was performed for bacterial identification. Isolated bacteria were subjected for antimicrobial resistant testing using 8 families of antibiotics. Bacteria with MAR index value of ≥75 were sent for genomic identification and sequencing. The average value of the ten water samples analyzed over the period of study had high total suspended solids (mg/l), phosphate (mg/l); magnesium ion (mg/l) contents and a very low dissolved oxygen mg/L content. The THB count was (5.0×107-1.35×108 CFU/ml), TCC count (3.55×105- 1.04×106 CFU/ml), FC count (2.10×105-6.90×105 CFU/ml) while Vibrio spp. count was 45-144 CFU/100 ml and the MPN estimated reading for coliform was also high. The percentage occurrence and MAR index value of the bacteria isolated from the well water, respectively were Vibrio spp. 23%; 75, Klebsiella spp. 20%; 100, Bacillus spp. 14%; 100, Staphylococcus spp. 14%; 62.5, E. coli 12%; 62.5, Pseudomonas spp. 12%; 75, Glycomyces spp. 3%; 100 and Proteus spp. 3%; 50. The closeness of the well to the grave makes the decomposing leachates with high organic, inorganic, biological and poisonous metals sink into the underground aquifers. Stamford Journal of Microbiology, Vol.12 (1) 2022: 47-53

Microbiological Quality of Vannamei Shrimp Pond Waters in Rupat Island, Bengkalis District, Riau.

The presence of pathogenic bacteria in shrimp culture water could influence the shrimp growth and production. This research aimed to quantify total coliform, Escherichia coli and total Vibrio in pond water of Vannamei shrimp (Litopenaeus vannamei) culture in Rupat Island. Water samples from inlet water (IW), water from three ponds (PW2, PW3, PW6) and outlet water (OW) were collected for the quantification of total coliform and Escherichia coli by the MPN technique and total Vibrio count on TCBS agar. Coliforms was found in all water samples, the lowest count was in inlet water sample (20 MPN/100 mL), and the highest was contained in outlet water (>2400 MPN/100 mL), however E. coli was not detected in all water samples. Meanwhile, the lowest total Vibrio count was found in PW3 (6.35 × 103), and the highest was in the PW6 (3.10 × 105). In addition to the microbiological quality, some water quality parameters were also observed, those were water temperature in the range of 27.5- 29.4°C, salinity of 18.7-21.5 ppt, pH of 7.17-7.89, DO of 1.3-7.6 mg/L, NH3 content of 0.96-1.26 mg/L and DOM of 26.2–27.6 mg/L. The data indicated that pathogenic bacteria were present in all pond waters which should be decreased for the safety shrimp production.