Thiosulphate Citrate Bile Salt Sucrose Research Articles

Study on Antibiotic Resistance Profiles Exhibited by Vibrio Species Isolated from Landfill Soils in Zaria Metropolis, Northern Nigeria

Study’s Novelty/Excerpt This study investigates the occurrence and antibiotic resistance profiles of Vibrio species isolated from landfill soils in Zaria Metropolis, with a focus on environmental reservoirs of antibiotic resistance. The research uniquely identifies a high prevalence of Vibrio cholerae non-O1 among the isolates and reveals significant multidrug resistance (MDR), particularly against Ampicillin, while also showing complete susceptibility to Gentamicin and Chloramphenicol. These findings highlight the potential for landfills to serve as environmental reservoirs of antibiotic resistance, underscoring the need for further research to understand the implications for public health and environmental management. Full Abstract Antibiotic resistance in bacteria presents a major risk to public health and the environment. Infections caused by Vibrio species continue to be a serious public health concern. This study investigated the occurrence and antibiotic resistance profiles of Vibrio species isolates from landfill soils in Zaria Metropolis. A total of one hundred and twenty (120) soil samples were collected from designated landfills in four locations in Sabon-Gari, Samaru, Tudun-Wada, and Zaria City. Vibrio species were isolated using Thiosulphate citrate bile salt sucrose (TCBS) agar. Bacteriological analysis of the soil samples revealed 9(7.50%) Vibrio species isolates with Vibrio cholerae non-O1 exhibiting the highest prevalence of 4 (3.33%) among all isolates. Using the Kirby-Bauer method, the isolates were tested for susceptibility against ten commonly used antibiotics belonging to three different classes. The highest resistance was to Ampicillin (88. 89%), while all the isolates (100%) showed susceptibility to Gentamicin and Chloramphenicol. Five isolates (55.56%) were Multidrug Resistance (MDR). The highest Multidrug Resistance (MDR, 60%) was observed in Vibrio cholerae non-O1. The isolate resistant to the highest number of antibiotics was obtained from the Tudun-Wada sample location. Three isolates (33.33%) showed resistance to 4 antibiotics, while 2 isolates, 1(11.11%) each were resistant to 5 and 6 antibiotics. The isolates were Multiple Antibiotic Resistance (MAR) to 4-6 antibiotics, and four different phenotypic resistance profiles were observed among them. The origin and varying levels of resistance to multiple antibiotics indicated could be traced to the faecal constituent of the waste in landfills produced by people or animals that have been treated indiscriminately with various antibiotics or items containing residual antimicrobial agents disposed of in dump soils, highlighting the potential environmental reservoirs of antibiotic resistance and call for further research to understand the implications for public health and environmental management.

Las Bolitas Syndrome in Penaeus vannamei Hatcheries in Latin America.

In September 2023, several hatcheries in Latin America experienced significant mortality rates, up to 90%, in zoea stage 2 of Penaeus vannamei. Observations of fresh mounts revealed structures resembling lipid droplets, similar to those seen in a condition known as "las bolitas syndrome". Routine histopathological examinations identified detached cells and tissues in the digestive tracts of affected zoea, contrasting with the typical algal cell contents seen in healthy zoea. Polymerase chain reaction (PCR) testing for over 20 known shrimp pathogens indicated minimal differences between diseased and healthy batches. Both groups tested negative for acute hepatopancreatic necrosis disease (AHPND) but positive for Vibrio species and Rickettsia-like bacteria in the diseased samples. Histological analyses of the affected zoea revealed characteristic tissue degeneration in the hepatopancreas, forming spheres that eventually migrated into the upper gut, midgut, and midgut caeca, a pathology identified as bolitas syndrome (BS). Microbiological assessments revealed Vibrio species at concentrations of 106 CFU zoea/g in affected zoea, approximately two orders of magnitude higher than in healthy zoea. Bacterial isolation from both healthy and BS-affected zoea on thiosulphate-citrate-bile salts-sucrose (TCBS) agar and CHROMagar™ (Paris, France), followed by identification using API 20E, identified six strains of Vibrio alginolyticus. Despite similarities to "las bolitas syndrome" in fresh mounts, distinct histopathological differences were noted, particularly the presence of sloughed cells in the intestines and variations in hepatopancreatic lobes. This study highlights the critical need for further research to fully understand the etiology and pathology of bolitas syndrome in zoea stage 2 of P. vannamei to develop effective mitigation strategies for hatchery operations.

Combined TCBS and CHROMagar Analyses Allow for Basic Identification of Vibrio vulnificus within a 48 h Incubation Period in the Coastal Baltic Sea.

With rising infection rates in recent years, Vibrio vulnificus poses an increasing threat to public safety in the coastal brackish Baltic Sea. It is therefore important to monitor this organism and assess the V. vulnificus infection risk on a more regular basis. However, as the coastline of the Baltic Sea is 8000 km long and shared by nine nations, a convenient, fast, inexpensive, yet efficient V. vulnificus identification method is essential. We evaluated the effectiveness of a two-step agar-based approach consisting of successive Vibrio isolation and cultivation on thiosulphate-citrate-bile salt sucrose (TCBS) agar and CHROMagar™ Vibrio for V. vulnificus in comparison with V. cholerae, V. parahaemolyticus, and V. alginolyticus. Our study contains isolates from water and sediment across a broad expanse of the Baltic Sea including 13 locations and two different summers, the time of year during which Vibrio infections are usually much more frequent. Confirmation of isolate species identity was carried out using molecular analyses. The two-step agar plating method performed well across different locations and timeframes in correctly identifying V. vulnificus by more than 80%, but the sensitivity in other Vibrio species varied. Thus, our approach yielded promising results as a potential tool for early V. vulnificus detection across a broad timeframe and transect of the Baltic Sea and potentially other brackish environments.

Bacteriological assessment of kunu-zaki sold in selected communities in Port Harcourt, Nigeria

Kunu-zaki is a fermented beverage sold in Nigeria; often prepared under unsafe conditions, predisposing it to pathogen contaminations. The bacteria associated with Kunu-zaki sold in the different area of Port Harcourt and an antibiogram of the pathogens was determined using standard methods involving nutrient agar, Salmonella-Shigella agar, Thiosulphate citrate bile salt sucrose agar, MacConkey agar, Mannitol salt agar, Eosin-Methylene blue agar and Mueller Hinton agar. The counts for kunu-zaki obtained from Choba, Mile 1, Rumuodara and Rumuokoro ranged from 2.87±0.11 to 5.39±0.19, 4.50±0.27 to 5.46±0.38, 4.02±0.54 to 5.96±0.27 and 4.24±1.13 to 5.94±0.22 log10cfu/ml, respectively. The confirmation of the isolates as: Staphylococcus spp. (27%), Lactobacillus spp. (20%), Streptococcus spp. (17%), Bacillus spp. (17%), E. coli (10%), and Shigella at (10%) was based on cultural and biochemical characteristics. The Gram-negative bacteria were 100% susceptible to pefloxacin, gentamycin, ciprofloxacin and tarivid while the Gram-positive bacteria isolates showed varying resistance to the antibiotics with Staphylococcus being the most susceptible. The occurrence of the antibiotic resistance isolates possess danger for potential consumers, hence the need to improve the production process.

Phenotypic characterization and antimicrobial susceptibility profiles of <i>Vibrio cholerae</i> isolates during the October 2022 and January 2023 outbreak in North-Kivu province, The Democratic Republic of Congo

Background: Cholera is an infectious disease characterized by severe watery diarrhea, frequently occurring in outbreaks which affects many communities in the Democratic Republic of the Congo (DRC). At the end of October 2022, a cholera outbreak was declared in the camp of internally displaced people (IDP) of Kanyaruchinya, 20 kilometers north of Goma, the provincial capital of the North-Kivu province in DRC, as well as in other IDP camps and settlements around the city of Goma. The aim of this study was to phenotypically characterize Vibrio cholerae isolates associated with this outbreak, and to determinate their antimicrobial susceptibility profiles.
 Methodology: Between October 31, 2022 and January 31, 2023, faecal swab samples were collected into Cary-Blair medium from 1604 and 538 patients with clinical signs of cholera at the Kanyaruchinya IDP, and IDPs camps and settlements around the city of Goma, respectively. After enrichment in 1% alkaline peptone water, the samples were cultured on thiosulphate-citrate-bile salt-sucrose (TCBS) agar for isolation and phenotypic characterization of V. cholerae O1 using conventional biochemical tests and serotyping technique. Antimicrobial susceptibility of selected isolates was peformed to a panel of 8 antibiotics by the disk diffusion method in accordance with EUCAST and CLSI guidelines.
 Results: Vibrio cholerae was cultured from 807 samples (50.3%) of 1604 patients from the Kanyaruchinya IDP, and from 206 samples (38.3%) of 538 patients around the city of Goma (p<0.01). All the V. cholerae isolates from the Kanyaruchinya IDP (807/807, 100.0%) were serotyped as V. cholerae O1 Inaba whereas 136 (66.0%), 67 (32.5%), and 3 (1.5%) V. cholerae O1 isolates from around Goma were serotyped as V. cholerae O1 Ogawa, V. cholerae O1 Inaba, and Hikojima respectively. Antimicrobial susceptibility test on 174 and 62 isolates selected randomly from the 807 and 206 V. cholerae isolates from the Kanyaruchinya IDP camp, and from around the city of Goma respectively, showed that all the tested V. cholerae O1 isolates were resistant to polymyxin and cotrimoxazole, while being susceptible to tetracycline and azithromycin. All tested V. cholerae O1 isolates from Kanyaruchinya IDP camp displayed a unique antimicrobial susceptibility profile characterized by resistance to ampicillin, cotrimoxazole and chloramphenicol, and susceptiblility to ciprofloxacin, norfloxacin, azithromycin, tetracycline and doxycycline. Their counterparts from settlements around the city of Goma displayed a more variable antimicrobial susceptibility profile.
 Conclusion: Our results suggest that a single V. cholerae O1 Inaba clone probably caused the cholera outbreak in the Kanyaruchinya IDP camp, whereas during the same period, several V. cholerae clones (Ogawa, Inaba and Hikojima) were associated with the cholera outbreak around the city of Goma.

Isolation of Bacteria From Seahorse (Hippocampus barbouri)

Captive breeding of seahorses (Hippocampus barbouri) which is a follow-up of their activities in the exploitation of natural large - scale does not escape from the problems of the disease. Gills and kidneys are the main target of the inspection. Wet mount is one way of checking the disease. This study aims to look at other forms of bacteria isolated from the gills and kidney seahorse (H. barbouri) morphologically. Media used are media Zobell Marine Agar (ZMA) and Thiosulphate Citrate Bile Salts Sucrose Agar (TCBSA) then be isolated bacteria with a pillowcase technique spread to see koloi dominant bacteria. more dominant bacteria grow dimurinikan back with scratch method (streak). The result of five isolates (isolates A, B, C, D and E) were predominantly found growing on media ZMA, two isolates (isolates B and C) not found growing on media TCBSA. Overall bacteria in media and TCBSA ZMA has the form round colonies and smooth-edged. TCBSA media can not be found on the bacterial colonies that fluoresce.

Distribution and antibiogram of Vibrio species from hospital wastewater in Southwest, Nigeria.

the continuous generation of wastewater and its release into the environment with little or no treatment remains a threat to the environment and public health. We examined the prevalence and antibiotic susceptibility profiles of Vibrio species isolated from untreated wastewater samples from Ondo State Specialist Hospital Okitipupa, Nigeria, as part of the global efforts to provide information for containing the spread of resistant infections. twelve hospital wastewater samples were collected aseptically and transported to the laboratory for analysis. The samples were processed on thiosulphate citrate bile salt sucrose agar and colonies typical of Vibrio species were selected for further identification. All isolates were confirmed by polymerase chain reaction (PCR) using Vibrio-specific primers and the PCR products were sequenced for species identification. The susceptibility profiles of the isolates were determined by the Kirby-Bauer disc diffusion method. twenty-nine (58%) of 38 presumptive isolates were confirmed as Vibrio by PCR, while 23 (60.5%) isolates were screened up to species level by sequencing. Six different species following the trend: 26.1% V. fortis and V. algivorus, 17.4% V. cholerae, 13.0% V. panuliri, 8.7% V. stylophorae and V. parahaemolyticus were identified. The isolates were commonly resistant (73.9%-91.3%) to doxycycline, tetracycline, erythromycin and meropenem. The least resistance rate (17.4%) was observed against amikacin and cotrimoxazole. All isolates were multidrug-resistant, with multiple antibiotic resistance (MAR) indices exceeding the 0.2 recommended limit. this study has shown that untreated hospital wastewater is a reservoir for diverse strains of multiply resistant Vibrio species. Therefore, it is essential to adequately treat hospital wastewater to eliminate these emerging pollutants and set up a monitoring scheme to evaluate the treatment plants' effectiveness to reduce the pollutants' impact on the environment and the population.

Culture-dependent examination of the bacteriological quality of ready-to-eat African salads in Enugu metropolis, Nigeria and antibiotic resistance profile of associated bacteria

This study investigated the bacteriological quality of ready-to-eat (RTE) African salads in Enugu metropolis, Enugu, Nigeria. A total of 10 samples of African salad were purchased from 10 different vendors in Enugu metropolis. The samples were purchased from Agbani Road, Ogbete, Mayor, Uwani, Kenyatta, Achara Layout, Obiagu and Timber. Isolation and enumeration of bacterial isolates were done using Nutrient agar, Eosin Methylene Blue (EMB) agar, Thiosulphate-citrate-bile salts-sucrose (TCBS) agar, Salmonella-Shigella Agar (SSA) and MacConkey agar, following standard methods. Identification of the bacterial isolates were done through biochemical tests and the Analytical Profile Index (API 20E) test kit. The antibiotic sensitivity of the bacterial isolates was also done using the Kirby Bauer disc diffusion method. Total culturable heterotrophic count was above 300 colonies across the samples. The highest bacterial counts recorded on EMB, SSA and TCBS across the samples were 6.3 × 106 CFU/g, 7.4 × 106 CFU/g and 1.21 × 107 CFU/g respectively. The identities of the organisms were; Salmonella spp., Staphylococcus aureus, Escherichia coli, Vibrio mimicus, Vibrio fluvialis, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio hollisae. The prevalent organism across the samples was Vibrio spp. The antibiotic sensitivity test suggested that Vibrio spp. was resistant to Ampiclox and Amoxycillin but sensitive to Erythromycin, Pefloxacin and Septrin. From this study, it was discovered that consumers of RTE African salad from majority of the vendors across Enugu metropolis are at risk of severe food poisoning.

Identification of Vibrio parahaemolyticus by PCR Method in Fish and Water Samples from Tigris River, Turkey

Climate change has caused increasing Vibrio parahaemolyticus infections in areas unaffected by this pathogen. Water temperature is an important ecological factor. It is consequently of high importance to follow V. parahaemolyticus in aquatic environments and foods. The aim of this work was to detect the presence of V. parahaemolyticus in Tigris River from eight different sites namely Malabadi Bridge, Fiskaya, Ongözlü Bridge, Bağıvar, Pamukçay, Ambarçay, Seyrantepe and Silvan. Total 480 samples of water and fish were used for V. parahaemolyticus detection. Thiosulphatecitrate‐bile salt sucrose (TCBS) Agar was employed for bacteria isolation for 24 h at 37◦C. The dominant uniform bacterial colonies were purified by streaking onto the TCBS agar plates three times. Identification of this pathogen was made by PCR. A total of 480 water and fish samples were analyzed. According to the results, V. parahaemolyticus was detected in 40 (6.6%) fish samples taken from the Seyrantepe region.

EVALUATION ON METHODS FOR IDENTIFICATION OF VIBRIO SP.: KAWAKAWA (EUTHYNNUS AFFINIS) BRINING SHREDDED AND CHITOSAN ADDITION AS PRESERVATIVE

The kawakawa (Euthynnus affinis) brine salting that used to make shredded were preservative with salt. Salt addition has aim to inactivate of bacterial contamination. Obviously, bacteria is still capable of growing in kawakawa brine shredding Therefore, in this study chitosan was added as antibacterial in shredded processing. Vibrio sp., is one of common halophilic bacteria found in seafood. If this bacteria is consumed, it can cause serious problems in human health. The objective of this study was to evaluate the factors that affect in identifying Vibrio sp., on kawakawa brine shredding (C), shredded non-chitosan (FC) and shredded contain of chitosan (FC+). The methods evaluated were steps in enriching bacteria, culturing bacterial in selective media and analysing bacterial by API 20E kit. Enriching and incubation periods were needed by halophilic bacteria to adaptation in new environment. It was required to observe the bacteria characteristics' that would be isolated. Bacterial colonies were growth on Thiosulphate citrate bile salt sucrose (TCBS) were not Vibrio sp., but confirmed as Pseudomonas luteola and Proteus vulgaris based on API 20E analysis. It was showed that TCBS media had some advantages in identifying Vibrio sp. In conclusion, to get the best result in identifying bacteria, at least two or more methods were used to avoid misidentification.

Using bacterial whole genome sequencing to identify toxin genes associated with disease outbreaks in black tiger shrimp (Penaeus monodon) aquaculture production

Vibrio-related diseases cause significant economic impact on global shrimp production. Although toxin expression is a well-established mechanism of Vibrio-related disease in humans, the role of toxin expression in Vibrio disease in aquaculture generally is not well defined. The current study investigated a sustained, rapid onset, mass mortality at a commercial tiger prawn, Penaeus monodon, hatchery. Affected shrimp were between mysis-2 and post-larvae-15. Disease outbreaks were prevented through prophylactic treatment with oxytetracycline, suggesting a bacterial-related causative agent. Conversely, mortality was not reduced with erythromycin treatment. Whole genome sequencing (WGS) using the Illumina Nextera XT protocol performed on mixed microbial cultures from erythromycin and non-erythromycin infused thiosulphate-citrate-bile salts-sucrose (TCBS) TCBS agar on a MiSeq, and postlarval samples on a HiSeq. The WGS revealed several Vibrio-related sequences dominated by reads with highest homology to Vibrio harveyi and Vibrio alginolyticus. In addition, a range of putative virulence genes encoding for antibiotic resistance and toxin production were identified. Taqman qPCR assays were designed to specifically quantify four of the putative toxin-genes found, namely the Zonula occludens toxin (ZOT), hemolysin d toxin (HylD), YaFO toxin-antitoxin and Repeats–in-Toxin (RTX) genes. The qPCR assays confirmed the presence of the toxin target genes with low cycle threshold value (Ct) in the bacterial cultures and postlarval samples. Specifically, the ZOT and HylD genes were detected in high copy number in the non-erythromycin and erythromycin-treated cultures. Although the detection of toxin-genes does not demonstrate causation of mortality, the application of WGS combined with qPCR analysis has revealed the presence of a range of virulence factors within the hatchery system and allowed a rapid implementation of management protocols to prevent entry of genetically toxin-armed Vibrio spp. until more conclusive studies into disease mechanisms could be conducted.

Prevalence of Vibrio Cholerae and Vibrio Species from Different Sources in Bayelsa State, Nigeria

The distribution of Vibrio cholerae and non-cholera Vibrio species from different sources from five localities in Bayelsa State, Nigeria was investigated in this study. A total of 44 stool samples, 22 freshwater samples, 60 brackish water samples and 64 seafood samples (crabs, shrimps and fishes) were collected from January to April, 2019 for the purpose of V. cholerae prevalence study. Samples were transported to the laboratory using Car-Blair’s medium. This was followed by samples enrichment in 1% alkaline peptone water and pour plating on thiosulphate citrate bile-salt sucrose (TCBS) agar. Characteristic yellow colonies were subjected to further biochemical and physiological characterization to further identify V. cholerae. Antibiotics susceptibility patterns for isolated V. cholerae strains were investigated. Furthermore, water samples (fresh and brackish) and seafood samples were collected on a monthly basis to ascertain the effect of seasons (dry and wet months) on the distribution of Vibrio spp. A total of 16 (36.36%) stools samples were positive for V. cholerae. In addition, 12 (54.55%) of freshwater samples, 28 (46.67%) of brackish water samples and 22 (34.38%) of seafood samples were contaminated with V. cholerae. The monthly mean values of Vibrio spp. from environmental sources showed statistically significant difference (P<0.05) between the dry months (low rainfall) and wet months (frequent rainfall). Higher average values were observed during the dry months. The result of the antibiotics sensitivity test showed all V. cholerae strains were susceptible to ciprofloxacin, ofloxacin and pefloxacin while varying degree sensitivities were observed in tetracycline and augmentin. Cholera and other non-cholera Vibrio spp gastrointestinal infections are still a major concern to the health of the public. Local and regional governments should enforce and promote the need for personal and communal hygienic practices.

Development of enhanced selective media for detection of Vibrio parahaemolyticus in oysters.

The online version contains supplementary material available at (10.1007/s10068-021-00877-0).

Geographical distribution and antibiotics susceptibility patterns of toxigenic Vibrio cholerae isolates from Kisumu County, Kenya.

BackgroundMultiple drug resistance has become a major threat to the treatment of cholera. Recent studies in Kenya have described the epidemiology, especially the risk factors, of cholera; however, there is little information on the phenotypic and drug susceptibility patterns of Vibrio cholerae (V. cholerae) in outbreaks that in the recent past have occurred in western Kenya.AimTo characterise and determine the antibiotics’ susceptibility profiling of toxigenic V. cholerae isolates from Kisumu County.SettingThe project was conducted in Kisumu County, Kenya.MethodsA total of 119 V. cholerae O1, biotype El Tor, isolates collected during 2017 cholera outbreak in Kisumu County were used for this study. The samples were cultured on thiosulphate-citrate-bile salts sucrose (TCBS) agar and biochemical tests were carried out using standard procedures. Susceptibility tests were conducted by using various conventional antibiotics against standard procedures.ResultsOf the 119 isolates, 101 were confirmed to be V. cholerae belonging to serotypes Inaba and Ogawa, with Inaba being the predominant serotype (73.95%). The isolates were susceptible to ciprofloxacin (100%), ofloxacin (100%), gentamycin (100%), doxycycline (99%), ceftriaxone (99%) and streptomycin (96.04%) antimicrobials, and resistant to erythromycin (53.47%), amoxicillin (64.4%), nalidixic acid (83.2%) and ampicillin (89.11%), with high resistance to cotrimoxazole (99%) and tetracycline (97%).ConclusionVibrio cholerae was resistant to multiple antibiotics, including those commonly used in the management of cholera. Taken together, there is a need to carry out regular surveillance on antimicrobial drug resistance during outbreaks.

A Comparative In Vivo and In Vitro Study on the Effectiveness of Homoeopathic Medicines on Aquarium Zebrafish Infected with Vibrio parahaemolyticus

Abstract Background Human infections associated with Vibrio parahaemolyticus rank high among the pathogenic bacteria associated with fishes which causes septicaemia and gastroenteritis in humans. Humans acquire such infections by handling of infected fishes. Therefore, rather than curing the diseases in humans, it can be prevented in fishes by homoeopathic medicines. Objectives The main objective of the study is to show the effectiveness of homoeopathic medicines in curing aquarium zebrafish infected with Vibrio parahaemolyticus. Materials and Methods Fifteen pairs of healthy aquarium zebrafish were chosen for the study. They were inoculated with Vibrio parahaemolyticus after the acclimatisation period of 1 month. The signs were noted down in chronological order. homoeopathic medicines Sulphur 200, Arsenicum album 200 and Pyrogenium 200 was administered based on the totality obtained. An in vitro study was also done by the serial dilution of water from each tank and also by the serial dilution of the fish gut which was plated in thiosulphate-citrate-bile salts-sucrose (TCBS) agar media. A growth sensitivity study to show the growth inhibition was done on Muller Hinton agar media. Results Although the incubation period of vibriosis was 4 days, the condition was completely cured within 10 days of administration of homoeopathic medicines. But in the case of tetracycline, the signs disappeared after 15 days. Sulphur being deep acting remedy showed cure in fishes as well as in the water. Arsenicum album showed therapeutic as well as prophylactic characters. Conclusion From the study, it is proved that the Sulphur 200 was the most effective remedy. Homoeopathic medicines do not produce any resistant bacteria but improves the immunity of the living organism. Sulphur, the deep acting remedy ensures 100% cure, i.e., both the water and the fishes are cured of Vibrio parahaemolyticus.

Profiles of Vibrio and heterotrophic bacteria in the intensive Vanamae shrimp culture using bioremediation technique in Karawang

Vibrio sp. group is one of the parameters of the health level of a shrimp culture. The aim of this study is to learn the population of vibrio and heterotrophic bacteria in Vaname shrimps’ cultivation under the use of probiotic bacteria. The profile analysis of the Vibrio sp. and heterotrophic bacteria were done in the Vanname shrimp culture with implementation of selected bacteria as bioremediation agents in Balai Layanan Usaha Budidaya Perikanan, Karawang. The studies were conducted in 110 days using the culture density of 60 shrimps/m2. Three ponds of 2,500 m2 namely A5, A7 and B5 ponds were set up for the observation and sample-taking for nine times. The Vibrio sp. and heterotrophic bacteria were analyzed using a plate-count technique with Thiosulphate Citrate Bile-salt Sucrose (TCBS) agar and Sea-Water Complete (SWC) media respectively. The shrimp’s growth and survival rate, and the water quality parameters were also monitored periodically. The result showed that Vibrio sp. population were fluctuated. The population was between 1.4-29.0 x 102 CFU/mL. The result showed that Vibrio sp. population were fluctuated. The highest Vibrio population was 103 CFU/mL while the highest profile of heterotrophic bacteria around 105 CFU/mL in average. The shrimp weight in final day of culture showed positive increase with the average body weight of 22.34±0.68 g/indv, while the average daily growth rate was 0.31±0.01 % g/day and the average of survival rate in the final experiment was 58.42±1.50%. The water quality was fluctuated by increasing days of culture. The temperature of water during culture periode ranged between 26.46 ± 1.87°C to 26.97 ± 0.87°C, with the pH value ranged from 7.31 ± 0.36 to 7.59 ± 0.28 and the salinity ranged between 24.23 ± 43.87 to 24.61 ± 4.15 ppt.

Isolation and Genomic Characterization of phiVibrioH1 a Myoviridae Phage for Controlling Pathogenic Vibrio parahaemolyticus from Seafood and Human

Vibrio parahaemolyticus is a zoonotic pathogen causing vibriosis in marine fish and associated with food poisoning outbreaks in humans. This study aimed to isolate and characterize bacteriophage against multidrug resistant (MDR) Vibrio parahaemolyticus from seafood and humans, and also to assess the lytic efficacy of phage on growth. Vibrio Parahaemolyticus was isolated from flesh of 80 white shrimp, 70 blue crabs and 50 mullets, and from 50 hand swabs of humans (fish handlers) on Thiosulphate Citrate bile salts sucrose agar media. The suspected colonies were biochemically identified. Pathogenic V. parahaemolyticus carrying thermostable direct hemolysin (tdh+) gene was molecularly detected . The pathogenic tdh+V. parahaemolyticus (n=25) isolates were explored for antimicrobial vulnerability resistant to 12 antimicrobials employing the disc diffusion method. Fifteen isolates of tdh+V. parahaemolyticus were 100% resistant to five antimicrobials. Bacteriophage was isolated from sewage water using spot test and double over layer agar assay. The phiVibrioH1 was belonged to family Myoviridae according to transmission electron microscopy. This study revealed a polyvalent phage infecting wide host range of MDR V. parahaemolyticus, V. vulnificus, V. fluvialis, V. alginolyticus, Pseudomonas aeruginosa, E. coli O26 and Proteus vulgaris. The genome of phage was sensitive to digestion with BamH1 and ECoR1 restriction enzymes indicating double stranded DNA. From 24 to 42 hours post-treatment of pathogenic V. parahaemolyticus with the PhiVibrioH1 phage showed complete lysis of bacterial cells. This study confirmed that the phiVibrioH1 is a lytic phage and has a high potential to control pathogenic V. parahaemolyticus strains recovered from seafood and humans.

AMOUNT AND TYPE OF MICROBES ON THE COMMON CARP (Cyprinus carpio) IN JATILUHUR RESERVOIR WEST JAVA

This research was conducted in Microbiology Laboratory of Biology Department, Faculty of Mathematics and Natural Sciences, Padjadjaran University in May 2017. The purpose of the research was to identify the number and types of microbes contained in carp (Cyprinus carpio) in Jatiluhur Reservoir, West Java. The method used in this research was sample survey method or field survey. The water and common carp sampling stations are divided into 3 stations, station 1 is located in Zone 1 (low density), station 2 is located in Zone 3 (medium density), and station 3 is located in Zone 5 (high density). Determination of sampling location water and fish is done based on Data Recapitulation of Density of Karamba Floating Net from the Department of Animal Husbandry and Fisheries of West Java.Water samples and fish mucus were tested using Total Plate Count (TPC) method using different specific media that is Trypticase Soy Agar (TSA), deMann Ragosa Sharpe Agar (MRSA), Kliger Iron Agaar (KIA), Thiosulphate Citrate Bile salts Sucrose Agar (TCBS), and Xylose Lysine Deoxycholate (XLD). Microbes can be known by species because they just grown on specific media that can directly showed the specific characteristics of certain microbial species based on the color of the colony. The parameter observed were microbiological test (TPC). The result of the research showed that there was Bacillus sp, Escherichia coli, Lactobacillus sp, Vibrio cholerae, Vibrio parahemoliticus, Vibrio spp, dan Shigella sp on the water samples and carp mucus. The result of research on water samples and fishery products in Jatiluhur Reservoir showed that water quality in Zone 1, Zone 3, and Zone 5 is still below the threshold of microbial contamination based on SNI 7388: 2009, while fish located in Zone 3 and Zone 5 is contaminated by Escherecia coli and Vibrio cholerae, exceed the threshold of SNI 7388: 2009 on microbial contamination in fresh fish.

Conventional and molecular detection of vibrio cholerae isolated from environmental water with the prevalence of antibiotic resistance mechanisms.

Environmental water is an important source for Vibrio cholerae, which is autochthonous to the aquatic environment, monitoring this bacterium in water is important for control of cholera. Vibrio cholerae represents an enormous public health problem around the world, especially in developing countries. One hundred samples were collected and selected. The samples were filtered and transferred to slants containing 2ml of alkaline peptone water, then subcultured on selective medium Thiosulphate Citrate Bile Salt Sucrose agar. All presumptive isolates were confirmed by using a series of biochemical tests including Oxidase test, Simmon Citrate test, DNase test, Indole test, Klingler Iron Agar (KIA) test, MacConkey agar test and motility. Confirmed Vibrio cholera strains were then screening for slide agglutination test by using commercially antisera polyvalent and monovalent O1 and O139 for determining strain serotype. The DNA extracted from pure culture and Polymerase Chain Reaction assay was used for molecular detection of Vibrio cholerae, a specific primer which designed according to ctxA gene sequences. This primer was detection and amplifying 241 base pairs of the ctxA gene. The resistance to antibiotics by Vibrio cholerae was determining by using thirteen standardized disc diffusion including Amikacin, Ceftriaxone, Ceftazidime, Gentamycin, Tetracycline, Streptomycin, Tobramycin, Cephotaxime, Nalidixic Acid, Norfloxacin, Cephalothin, Rifampicin, Cefixime. From one hundred water samples were detected, fifty-six samples were motile and positive for biochemical tests. Fifteen isolates confirmed as Vibrio cholera by Polymerase Chain Reaction (PCR) assay with primers de­signed for ctxA and 241bp band was observed. These fifteen isolates showed agglutination with polyvalent and monovalent O1 antisera, and two strains represented Ogawa from other strains that showed Inaba. The fifteen isolates exhibited an identical response to each antibiotic examined. They showed sensitive to all antibiotics except Amikacin, Streptomycin, Cefixime, Norfloxacin, Cephalothin. the aim of this study was determined the accurate method for detection of Vibrio cholerae in environmental water. In the current study, we found that the molecular method using Polymerase Chain Reaction performance using the ctxA gene-specific primers for detection of Vibrio cholerae was faster and accurate and specific.

Bromoperoxidase Producing Bacillus spp. Isolated from the Hypobranchial Glands of A Muricid Mollusc Are Capable of Tyrian Purple Precursor Biogenesis

The secondary metabolite Tyrian purple, also known as shellfish purple and royal purple, is a dye with historical importance for humans. The biosynthetic origin of Tyrian purple in Muricidae molluscs is not currently known. A possible role for symbiotic bacteria in the production of tyrindoxyl sulphate, the precursor to Tyrian purple stored in the Australian species, Dicathais orbita, has been proposed. This study aimed to culture bacterial symbionts from the purple producing hypobranchial gland, and screen the isolates for bromoperoxidase genes using molecular methods. The ability of bromoperoxidase positive isolates to produce the brominated indole precursor to Tyrian purple was then established by extraction of the culture, and analysis by liquid chromatography–mass spectrometry (LC–MS). In total, 32 bacterial isolates were cultured from D. orbita hypobranchial glands, using marine agar, marine agar with hypobranchial gland aqueous extracts, blood agar, thiosulphate citrate bile salts sucrose agar, and cetrimide agar at pH 7.2. These included 26 Vibrio spp., two Bacillus spp., one Phaeobacter sp., one Shewanella sp., one Halobacillus sp. and one Pseudoalteromonas sp. The two Bacillus species were the only isolates found to have coding sequences for bromoperoxidase enzymes. LC–MS analysis of the supernatant and cell pellets from the bromoperoxidase producing Bacillus spp. cultured in tryptone broth, supplemented with KBr, confirmed their ability to produce the brominated precursor to Tyrian purple, tyrindoxyl sulphate. This study supports a potential role for symbiotic Bacillus spp. in the biosynthesis of Tyrian purple.