Thiosulfate Citrate Bile Salts Sucrose Research Articles

Prevalence and antimicrobial susceptibility pattern of Vibrio cholerae isolates from cholera outbreak sites in Ethiopia

BackgroundCholera is an acute infectious disease caused by ingestion of contaminated food or water with Vibrio cholerae. Cholera remains a global threat to public health and an indicator of inequity and lack of social development. The aim of this study was to assess the prevalence and antimicrobial susceptibility pattern of V. cholerae from cholera outbreak sites in Ethiopia.MethodsAcross-sectional study was conducted from May 2022 to October 2023 across different regions in Ethiopia: Oromia National Regional State, Amhara National Regional State and Addis Ababa City Administration. A total of 415 fecal samples were collected from the three regions. Two milliliter fecal samples were collected from each study participants. The collected samples were cultured on Blood Agar, MacConkey Agar and Thiosulfate Citrate Bile Salt Sucrose Agar. A series of biochemical tests Oxidase test, String test, Motility, Indole, Citrate, Gas production, H2S production, Urease test were used to identify V. cholerae species. Both polyvalent and monovalent antisera were used for agglutination tests to identify and differentiate V. cholerae serogroup and serotypes. In addition, Kirby-Bauer Disk diffusion antibiotic susceptibility test method was done. Data were registered in epi-enfo version 7 and analyzed by Statistical Package for Social Science version 25. Descriptive statistics were used to determine the prevalence of Vibrio cholerae. Logistic regression model was fitted and p-value < 0.05 was considered as statically significant.ResultsThe prevalence of V. cholerae in the fecal samples was 30.1%. Majority of the isolates were from Oromia National Regional State 43.2% (n = 54) followed by Amhara National Regional State 31.2% (n = 39) and Addis Ababa City Administration 25.6% (n = 32). Most of the V. cholerae isolates were O1 serogroups 90.4% (n = 113) and Ogawa serotypes 86.4% (n = 108). Majority of the isolates were susceptible to ciprofloxacin 100% (n = 125), tetracycline 72% (n = 90) and gentamycin 68% (n = 85). More than half of the isolates were resistant to trimethoprim-sulfamethoxazole 62.4% (n = 78) and ampicillin 56.8% (n = 71). In this study, participants unable to read and write were about four times more at risk for V. cholerae infection (AOR: 3.8, 95% CI: 1.07–13.33). In addition, consumption of river water were about three times more at risk for V. cholerae infection (AOR: 2.8, 95% CI: 1.08–7.08).Conclusionour study revealed a high prevalence of V. cholerae from fecal samples. The predominant serogroups and serotypes were O1 and Ogawa, respectively. Fortunately, the isolates showed susceptible to most tested antibiotics. Drinking water from river were the identified associated risk factor for V. cholerae infection. Protecting the community from drinking of river water and provision of safe and treated water could reduce cholera outbreaks in the study areas.

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.

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

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%.

Use of Common Herbs to Control Microbial Aerosols in Toilet Rooms

Four distinct plant species were used in an investigation into the capacity of plant active volatile essence to eradicate microorganisms in the atmosphere (bioaerosols) in restrooms: Allium sativum (garlic), Allium cepa (onion), Zingiber officinalis (ginger), and Ocimum gratissimum (scent leaf). The study was conducted in the student restrooms for microbiology at Rivers State University Nkpolu Oroworukwu in Port Harcourt. Sedimentation method of aerosol sampling was used to collect the air samples. Artificial culture media were exposed to air at different times, including nutrient agar for total heterotrophic bacteria, Mac Conkey agar for enteric bacteria, sadour and dextrose agar for aerobic fungi, thiosulfate citrate bile salt sucrose agar for vibrio, Cystine lactose electrolyte deficient agar for urinary pathogens, and mannitol salt agar for staphylococcus.&#x0D; Samples was taken from the toilet without any mashed plant and served as a control. The THB isolates identified were Bacillus spp, Pseudomonas sp, Staphylococcus sp, Lactobacillus sp, Enterococcus sp, Corynobacterium sp, Escherichia coli, Klebsiella pneumonia. While the fungi isolated were Aspergillus niger, pennicillum sp, Rhizopus sp, Saccharomyces sp, Mucor Sp, Candida sp, Asperillus flavus, Fusarium sp. It can be deduced that these medicinal herbs has the ability in reducing microbial aerosol using ginger, onion, scent leaf, garlic also by combination of the herbs which are Onion &amp; garlic, Onion &amp; garlic, Onion &amp; ginger, Garlic &amp; scent leaf, Garlic &amp; ginger, Scent leaf &amp; ginger, Onion, garlic &amp; scent leaf, Onion, garlic &amp; ginger, Garlic, Scent leaf &amp; ginger, Onion, garlic, scent leaf &amp; ginger. Specifically the microbial aerosols in this investigation including bacteria and fungi were Bacillus species, Pseudomonas species, Staphylococcus species, Lactobacillus species, Enterococcus species, Corynobacterium, Escherichia coli, Klebsiella pneumonia. Aspergillus niger, pennicillum species, Rhizopus species, Saccharomyces species, Mucors species, Candida species, Asperillus flavus, Fusarium species. This work proved that microbial load can be reduced using natural means (plant extract).

Resistance genes and plasmid profile of bacteria Isolated from selected water bodies in Owo Metropolis

Aquatic samples from three water bodies were collected following laid down procedures. The bacteria were isolated using, Eosin Methylene Blue agar (EMB), Mannitol salt agar (MSA), Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) Agar, Muller Hinton (MH). The isolates were subjected to colonial, morphological and biochemical characterization. Pathogenicity test was carried out on the isolates using blood agar to determine their hemolytic activities. Antibiotic resistance status was also investigated against standard antibiotics using the Kirby Bauer disk diffusion method and the multiple antibiotic resistance indices of the isolates were also determined. The plasmid profiles were determined and the resistance genes were identified using molecular means. The bacteria species isolated were Veillonella Spp, Vibrio orientalis and Micrococcus luteus. Veillonella Spp showed Beta hemolysis while Micrococcus luteus showed alpha hemolysis and Vibrio orientalis showed gamma hemolysis. Antibiotic susceptibility test showed all isolates to be antibiotic resistant to varying degrees and multiple antibiotic resistance indices (MARi) depending on their level of resistance. Veillonella. spp. had MARi of 0.3 while M. luteus and V. orietalis had MARi of 0.5. Plasmid profiling showed all isolates to have plasmids with bandwidth of 10,000 bp. Vibrio spp and Veillonella Spp were positive for beta lactamase genes (bla SHV). Only Vibrio was positive for resistance to aminoglycosides (aac(3)-IV). Vibrio orientalis and Micrococcus luteus were found to have the resistance genes for quinolones (qnrA gene) while all the isolates possess genes against the sulphonamides (sul1 gene) used. The result of this work establishes the antibiotic resistance status of bacteria isolated from aquatic habitat and suggests their important role in the spread of antimicrobial resistance AMR, and antibiotic resistance genes (ARGs)

Growth Performance, Feed Utilisation, Endogenous Digestive Enzymes, Intestinal Morphology, and Antimicrobial Effect of Pacific White Shrimp (Litopenaeus vannamei) Fed with Feed Supplemented with Pineapple Waste Crude Extract as a Functional Feed Additive.

This study used pineapple waste crude extract (PWCE) to increase the potential of Pacific white shrimp (Litopenaeus vannamei) production for food sustainability and stability. The objective was to investigate the appropriate technique to increase the yield production and quality of shrimp and decrease waste from shrimp culture. Pacific white shrimp (average body size: 0.51 g) were fed with commercial feed supplemented with PWCE at various concentrations of 0 (control), 90, 170, and 250 ppt. Shrimp were fed five times per day for 80 days. At the end of the trial, the results showed that shrimp fed with the PWCE 250 ppt supplementation provided the highest growth rate and the best feed utilisation and yield (P < 0.05). The protein content of whole shrimp in all shrimp fed with the PWCE supplementation diet was higher than that in the control group (P < 0.05). On the contrary, the variation of endogenous digestive enzymes, including protease, trypsin, and the T/C ratio, was significantly lower in shrimp fed a diet supplemented with PWCE 250 ppt (P < 0.05). While in this group, the number of microorganisms on thiosulfate-citrate-bile salt-sucrose (TCBS), blood agar, and trypticase soy agar (TSA) was lowest (P < 0.05). Furthermore, the dietary PWCE at 250 ppt increased the volume of microvilli in the hindgut of shrimp, but the supplementation at 170 ppt improved the number of F-cells in the epithelial cells of the hepatopancreas. Nevertheless, the supplementation of PWCE in the diet did not affect the water quality (P > 0.05). Therefore, pineapple waste crude extract supplementation improves both quantitative and qualitative yields and tends to reduce waste.

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%).

Identification and genomic analysis of a Vibrio cholerae strain isolated from a patient with bloodstream infection

Vibrio cholerae is a bacterium ubiquitous in aquatic environments which can cause widespread infection worldwide. V. cholerae gradually became a rare species of bacteria in clinical microbiology laboratories with the control of the cholera epidemic. In this study, we isolated a V. cholerae strain, named VCHL017, from the blood of an elderly patient without gastrointestinal symptoms. The patient had a history of hookworm infection and multiple myeloma. Furthermore, she was immunocompromised, and received long-term chemotherapy and antimicrobial agents. VCHL017 was inoculated on blood agar and thiosulfate citrate bile salt sucrose plates (TCBS) to observe morphological characteristics. Then this isolate was identified by matrix-assisted laser desorption/ionization time-of-flight spectrometry (MALDI-TOF MS). The minimum inhibitory concentrations (MICs) for cefazolin, ceftazidime, cefepime, meropenem, tetracycline, ciprofloxacin, chloramphenicol, and gentamicin of VCHL017 were determined by the microbroth dilution method. PCR and serum agglutination tests were used to determine whether the serogroups of the isolate belonged to the O1/O139 and cholera toxin encoding genes. Finally, the genomic features and phylogeny of VCHL017 were analyzed by whole genome sequencing (WGS). VCHL017 was a non-O1/O139 V cholerae strain that did not carry the ctxA gene. Antimicrobial susceptibility tests revealed that VCHL017 was susceptive to chloramphenicol and tetracycline. Although it did not carry the genes encoding the cholera toxin, WGS indicated that VCHL017 carried a variety of other virulence factors. By calculating the average nucleotide identity (ANI), we precisely identified the species of VCHL017 as V. cholerae. There are also A171S and A202S missense mutations in gyrA of VCHL017. The phylogenetic analysis indicated that VCHL017 was closely related to V. cholerae strains isolated from aquatic environments. Our results suggest that continuous monitoring is necessary for non-O1/O139 V cholerae strains isolated from outside the digestive tract, which could be pathogenic through multiple virulence factors.

S2079 Not Just Another Case of Diarrhea: A Case of Vibrio Cholera During COVID-19 Era

Introduction: Vibrio cholerae is a gram negative, comma-shaped, toxin producing bacteria, which causes large volumes of watery stool leading to dehydration, hypovolemic shock, and metabolic acidosis. Stool culture with PCR is the gold standard for diagnosis. It is pertinent that if cholera is a differential, treatment with isotonic oral rehydration and antibiotics are not delayed. When traveling to endemic or epidemic areas vaccination should be considered. Here we discuss a case of Vibrio cholera infection diagnosed in Farmington, Connecticut in a patient with recent travel back from Pakistan in mid-April 2022. Case Description/Methods: 61-year-old female with no relevant past medical history presented to the hospital complaining of fatigue and 3 days of profuse, watery, dark diarrhea occurring 8 times daily, starting the day she returned home to Connecticut after traveling in Pakistan. She drank primarily store-bought bottled water but reported some cooking with tap water. She was lethargic and hypovolemic with a diffusely tender abdomen and hyperactive bowel sounds. Lab work was pertinent for leukocytosis, AKI, hypokalemia, and mild metabolic acidosis. Hemoccult stool, Clostridium difficile toxins A&B, Salmonella, Shigella, and fecal leukocyte count were negative. Her course was complicated by altered mental status, worsening AKI, hypokalemia, and metabolic acidosis requiring a bicarbonate drip. Eight days into her admission stool study resulted positive for cholera (Figure 1). Her metabolic derangements were aggressively treated, and her symptoms improved. Discussion: During the COVID-19 pandemic and ongoing travel restrictions, cholera is not typically the first infection thought of for patients with diarrhea and fatigue. Still, we are seeing cases rise in locations where cholera case-fatality rates were previously dropping in a pre-pandemic COVID-era, thought to be from reduced health promotion activities.1 In 2019, the Center for Disease Control and Prevention reported a total of 14 cases of Vibrio cholera within the United States,2 most from overseas travel. Thus, it is important to keep a broad differential diagnosis regardless of location even during a time of travel restrictions.Figure 1.: Vibrio culture on thiosulfate-citrate-bile salts-sucrose (TCBS) agar (left) and on blood agar (right).

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.

Simultaneous isolation and enumeration of virulent Vibrio cholerae and Vibrio vulnificus using an advanced MPN-PCR method.

Vibrio cholerae and Vibrio vulnificus are critical foodborne pathogens that need to be intensively controlled for their infection due to the intake and distribution of seafood, especially raw oysters. For this reason, various methods have already been developed for the detection and enumeration of these bacteria. The most probable number (MPN)-PCR (polymerase chain reaction) method is commonly used with the selective-differential medium for the efficiency and convenience of cell enumeration. One of the most frequently used for detecting Vibrio spp. is thiosulfate-citrate-bile salts-sucrose (TCBS) agar. But this selective-differential medium can fail to distinguish between V. cholerae, V. vulnificus, and Vibrio alginolyticus. For this reason, the conventional MPN-PCR method with TCBS medium for the detection of Vibrio spp. has a problem with processing PCR two times. This study suggests a simple and minimized detection method using one-time PCR and non-NaCl Luria-Bertani (LB-0) medium culture. This detection method is based on the difference in salt requirement between V. cholerae and V. vulnificus. Employing the developed methodology, the simultaneous cell enumeration of V. cholerae and V. vulnificus can be possible at a low cost. Furthermore, this study proposes a new specific primer to detect virulence-related genes from V. cholerae and V. vulnificus. This advanced MPN-PCR method was verified using bioaccumulated pacific oysters (Crassostrea gigas) by V. cholerae and V. vulnificus.

Identification of Vibrio spp. causing vibriosis in spiny lobsters (Panulirus homarus L.) in Bengkulu marine temporary shelter ponds

Spiny lobster (Panulirus homarus) is one of the export commodities of the Indonesian fisheries subsector and an important component for shrimp fisheries in Indonesia. In the development of lobster cultivation, there are several obstacles, the presence of vibriosis infection caused by the pathogenic Vibrio bacteria. This study aimed to identify Vibrio spp. bacteria in spiny lobsters (P. homarus) reared in the marine cultivation ponds, Bengkulu, Indonesia. Clinical symptoms of lobsters infected with vibriosis are red spots on the uropod, pleopod, and abdominal parts. Bacterial isolation was conducted by isolated some internal organs in spiny lobsters, that are, gills, stomachs, haemolymph, and hepatopancreas. The result showed there are 5 isolates of Vibrio bacteria that coded by IN3, ST2, HA1, HP2, and HP3. These bacteria isolates were identified through their colony morphology and biochemical tests. Characterization on the Thiosulfate Citrate Bile Salt Sucrose (TCBS) medium showed that lobsters were infected with Vibrio species. Based on Bergey’s Manual of Determinative Bacteriology, Austin and Austin, the identification results showed that HA1 isolate was identified as Vibrio algynolyticus, IN3 isolate was identified as V. anguillarum, ST2 was identified as V. ordalii, HP2 in first lobster was identified as with V. algynolyticus that mostly in the hepatopancreas, and HP3 was identified as V. splendidus

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.

Prevalence of Bacteria and Antimicrobial Resistance Genes in Hospital Water and Surfaces.

PurposeAntimicrobial resistance (AMR) has become a worldwide environmental and public health problem, causing more than 250,000 deaths per year. Unregulated usage, unsafe hospital practices, and misuse in veterinary contribute to the development of multidrug resistance in various bacteria. Hospital water was hypothesized to be a hotspot for AMR transmission because of (1) increased exposure to antibiotic load, (2) poor drainage and sanitation system, (3) interaction between environmental and clinical microbes. The purpose of the research was to assess the biodiversity and AMR in hospital tap waters.MethodologyIn this study, the microflora of the hospital tap water and hospital surfaces was observed by obtaining water samples from the intensive care unit (ICU), surgical wards, and washrooms. These were processed through membrane filtration and spread on seven different media (Aeromonas Medium, Azide Dextrose Agar, MacConkey Agar, Mannitol Salt Agar, Pseudomonas Cetrimide Agar, Salmonella Shigella Agar, and Thiosulfate Citrate Bile Salts Sucrose Agar). Surface samples were collected from the faucet, basin, and drain and directly spread on the media plates. Isolates were identified using standard bacteriological and biochemical tests.Kirby-Bauer disk diffusion method was performed using 21 antibiotic disks from 10 different antibiotic classes. They included ampicillin (AMP), amoxicillin (AML), piperacillin-tazobactam (TZP), cefipime (FEP), cefoxitin (FOX), ceftazidime (CAZ), ceftriaxone (CRO), imipenem (IMP), meropenem (MEM), ciprofloxacin (CIP), moxifloxacin (MXF), levofloxacin (LEV), amikacin (AK), gentamicin (CN), tigecycline (TGC), aztreonam (ATM), erythromycin (E), clindamycin (DA), rifampicin (RD), colistin (CT), and chloramphenicol (C). The results were interpreted according to EUCAST guidelines for the antibiogram of the isolates; 38 isolates were selected out of 162 based on different parameters for genotyping and detection of six beta-lactamase genes (blaSHV, blaTEM, blaCTX-M, blaOXA, blaKPC, blaNDM).ResultsAmong these 162 isolates, 82 were obtained from water sources and 80 were collected from surfaces (faucet, basin, drain). The isolates included a variety of bacteria including Aeromonas spp. (20%), Klebsiella spp. (13%), Staphylococcus aureus (13%), Pseudomonas spp.(10%), Escherichia coli (9%), Vibrio spp. (8%), Enterococcus spp. (6%), Shigella spp. (6%), Salmonella spp. (4%), Acinetobacter spp. (3%), Staphylococcus epidermitis (3%), Streptococci spp. (2%), Proteus spp. (1%), Citrobacter spp. (1%), and Serratia spp. (1%). A diverse range of microbes were identified including clinically relevant bacteria, which shows that the urban water cycle is already contaminated with multidrug-resistant microflora of the hospital settings. Macrolide and lincosamide showed the highest resistance followed by penicillin, monobactam, and cephalosporins. blaSHV and blaTEM were prevalent in samples. blaNDM was also found which manifests as a real threat since it causes resistance against carbapenems and colistin, antibiotics reserved as a last resort against infections.ConclusionsThis study presented the ground reality of antibiotic resistance in Pakistan and how its subsequent spread poses a great threat to the strides made in the field of medicine and public health. Strict regulations regarding antibiotic usage, hospital effluent, and urban water sanitation must be imposed to curb the devastating effects of this increasing phenomenon.

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.&#x0D; Asian Australas. J. Biosci. Biotechnol. 2021, 6 (1), 40-49

Prevalence and Characterization of Food-Borne Vibrio parahaemolyticus From African Salad in Southern Nigeria.

The demand for minimally processed vegetables (African salad) has increased partly due to its inclusion in ready-to-eat foods. Nevertheless, the associated risk of the presence of emergent foodborne pathogens, such as Vibrio parahaemolyticus might be underestimated. The present study was designed to isolate and characterize foodborne V. parahaemolyticus from minimally processed vegetables using culture-based methods and molecular approach. A total of 300 samples were examined from retail outlets between November 2018 and August 2019 from Southern Nigeria. The prevalence of vibrios from the overall samples based on the colonial proliferation of yellow, blue-green and/or green colonies on thiosulfate citrate bile salts sucrose agar was 74/300 (24.6%). An average of two green or blue-green colonies from respective plates was screened for V. parahaemolyticus using analytical profile index (API) 20 NE. Polymerase chain reaction further confirmed the identity of positive V. parahaemolyticus. The counts of V. parahaemolyticus ranged from 1.5 to 1,000 MPN/g. A total of 63 recovered V. parahaemolyticus were characterized further. The resistance profile of the isolates include ampicillin 57/63 (90.5%), cefotaxime 41/63 (65.1%), ceftazidime 30/63 (47.6%), amikacin 32/63 (50.8%), kanamycin 15/63 (23.8%), and oxytetracycline 16/63 (25.4%). The multiple antibiotic index ranged from 0–0.81. The formation of biofilm by the isolates revealed the following: strong formation 15/63 (23.8%), moderate formation 31/63 (49.2%), weak formation 12/63 (19.1%), and no formation 5/63 (7.9%). A total of 63/63 (100%), 9/63 (14.3%), and 20/63 (31.8%) of the isolates harbored the tox R gene, TDH-related hemolysin (trh) and thermostable direct hemolysin (tdh) determinants respectively. The isolates with O2 serogroup were most prevalent via PCR. Isolates that were resistant to tetracycline, kanamycin, and chloramphenicol possessed resistant genes. The presence of multidrug-resistant vibrios in the minimally processed vegetables constitutes a public health risk and thus necessitates continued surveillance.

Distribution and Public Health Significance of Vibrio Pathogens Recovered from Selected Treated Effluents in the Eastern Cape Province, South Africa

Treated sewage harbours pathogenic microbes, such as enteric bacteria and protozoa, are capable of causing several diseases. Some of these are emerging pathogens sometimes recovered in the absence of common water quality indicator organisms. The possibility of selected treatments plants serving as momentary reservoirs of Vibrio pathogens during a non-outbreak period was assessed. The occurrence and diversity of Vibrio pathogens were monitored for one year (December 2016 to November 2017) in the treated effluents and upstream and downstream areas of the receiving water bodies of two wastewater treatment plants (WWTPs), designated AL and TS. Physicochemical parameters of TS and AL WWTPs’ water samples were analysed using a multi-parameter meter (Hanna, model HI 9828, Padova, Italy) and a turbidimeter (HACH, model 2100P, Johannesburg, South Africa). Water samples were augmented with alkaline peptone water and cultured on thiosulfate citrate bile salts sucrose agar at 37 °C for 24 h. The recovered probable pathogens were confirmed via PCR amplification, using primers specific for Vibrio species of public health significance. The distribution of Vibrio species positively and significantly (p &lt; 0.01) correlated with turbidity (r = 0.630), temperature (r = 0.615), dissolved oxygen (r = 0.615), pH (r = 0.607), biological oxygen demand (r = 0.573), total dissolved solid (r = 0.543), total suspended solid (r = 0.511), electrical conductivity (r = 0.499), residual chlorine (r = 0.463) and salinity (r = 0.459). The densities of Vibrio species were found to be significantly higher (p &lt; 0.05) in effluents from both AL and TS WWTPs than upstream and downstream of the receiving rivers across the sampling regime. Furthermore, the maximum Vibrio species density across the sampling regime were observed during the warmer Summer and Spring season. Moreover, six medically important Vibrio species were detected in the water samples, indicating that the methods employed were efficient in revealing that WWTPs are potential reservoirs of Vibrio pathogens, which could pose a substantial public health risk if the receiving water is used for domestic purposes. Our findings further strengthen existing calls for the inclusion of emerging bacterial pathogens, including Vibrio species, as water quality indicators by the South African Department of Water Affairs. Hence, we recommend regular monitoring of treated effluents and receiving water bodies to ensure early control of potential outbreaks of vibriosis and cholera.

Prevalence of Vibrio mimicus in Fish, Fishery Products, and Environment of South West Coast of Kerala, India.

Vibrio mimicus is a seafood-borne bacterium involved in incidences of human infections following consumption of raw or undercooked seafood. Regular monitoring of seafood for V.mimicus is necessary for risk assessment and to establish mitigation measures. During the period 2017-2020, a total of 250 samples comprising finfish, shellfish, water, ice, and sediment samples were collected from fish markets, fish landing centers, and fish farms in the Ernakulum district on the Southwest coast of Kerala, India. V. mimicus was isolated using enrichment in alkaline peptone water for 18 h followed by plating on thiosulfate citrate bile salts sucrose agar and then incubated at 37°C for 18-24 h. The presumptive V. mimicus isolates were confirmed by biochemical characterization and molecularly with vmh gene-specific for V. mimicus. The study revealed that the prevalence of V. mimicus is 5.6% in the total of samples screened. The highest occurrence was observed in brackish water fish (19%) followed by freshwater fish (18%) and marine fish (2%) samples. The study points out the risk of brackish water fishes as potential carriers of this pathogen. This requires preventive measures to mitigate health hazards associated with V. mimicus entering into the seafood production chain.

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%.