Using the size fractionation method, we measured the decay rates of Escherichia coli, Salmonella Typhi and Vibrio parahaemolyticus in the coastal waters of Peninsular Malaysia. The size fractions were total or unfiltered, <250 μm, <20 μm, <2 μm, <0.7 μm, <0.2 μm and <0.02 μm. We also carried out abiotic (inorganic nutrients) and biotic (bacterial abundance, production and protistan bacterivory) measurements at Port Dickson, Klang and Kuantan. Klang had highest nutrient concentrations whereas both bacterial production and protistan bacterivory rates were highest at Kuantan. We observed signs of protist-bacteria coupling via the following correlations: Protistan bacterivory-Bacterial Production: r = 0.773, df = 11, p < 0.01; Protist-Bacteria: r = 0.586, df = 12, p < 0.05. However none of the bacterial decay rates were correlated with the biotic variables measured. E. coli and Salmonella decay rates were generally higher in the larger fraction (>0.7 μm) than in the smaller fraction (<0.7 μm) suggesting the more important role played by protists. E. coli and Salmonella also decreased in the <0.02 μm fraction and suggested that these non-halophilic bacteria did not survive well in seawater. In contrast, Vibrio grew well in seawater. There was usually an increase in Vibrio after one day incubation. Our results confirmed that decay or loss rates of E. coli did not match that of Vibrio, and also did not correlate with Salmonella decay rates. However E. coli showed persistence where its decay rates were generally lower than Salmonella.
The mechanisms through which brown-marbled grouper accomplishes resistance to infection, particularly against Vibrios, are not yet fully understood. In this study, brown-marbled grouper fingerlings were experimentally infected with Vibrio parahaemolyticus, to identify disease resistance grouper, and the serum proteome profiles were compared between resistant and susceptible candidates, via two-dimensional gel electrophoresis (2-DE). The results showed that putative parvalbumin beta-2 subunit I, alpha-2-macroglobulin, nattectin and immunoglobulin light chain proteins were among proteins that significantly overexpressed in the resistant fish as compared to the susceptible group of fish, whereas apolipoprotein E and immunoglobulin light chain proteins were observed to be differentially overexpressed in the susceptible fish. Further analysis by peptide sequencing revealed that the immunoglobulin light chain proteins identified in the resistant and susceptible groups differed in amino acid composition. Taken together, the results demonstrated for the first time that putative parvalbumin beta-2 subunit I, alpha-2-macroglobulin, nattectin and immunoglobulin light chain are among important proteins participating to effect disease resistance mechanism in fish and were overexpressed to function collectively to resist V. parahaemolyticus infection. Most of these molecules are mediators of immune response.
Vibrio parahaemolyticus has long been known pathogenic to shrimp but only recently it is also reported pathogenic to tropical cultured marine finfish. Traditionally, bacterial diseases in aquaculture are often treated using synthetic antibiotics but concern due to side effects of these chemicals is elevating hence, new control strategies which are both environmental and consumer friendly, are urgently needed. One promising control strategy is the bacteriophage therapy. In this study, we report the isolation and characterization of a novel vibriophage (VpKK5), belonging to the family Siphoviridae that was specific and capable of complete lysing the fish pathogenic strain of V. parahaemolyticus. The VpKK5 exhibited short eclipse and latent periods of 24 and 36 min, respectively, but with a large burst size of 180 pfu/cell. The genome analysis revealed that the VpKK5 is a novel bacteriophage with the estimated genome size of 56,637 bp and has 53.1% G + C content. The vibriophage has about 80 predicted open reading frames consisted of 37 complete coding sequences which did not match to any protein databases. The analysis also found no lysogeny and virulence genes in the genome of VpKK5. With such genome features, we suspected the vibriophage is novel and could be explored for phage therapy against fish pathogenic strains of V. parahaemolyticus in the near future.
A simple but promising electrochemical DNA nanosensor was designed, constructed and applied to differentiate a few food-borne pathogens. The DNA probe was initially designed to have a complementary region in Vibrio parahaemolyticus (VP) genome and to make different hybridization patterns with other selected pathogens. The sensor was based on a screen printed carbon electrode (SPCE) modified with polylactide-stabilized gold nanoparticles (PLA-AuNPs) and methylene blue (MB) was employed as the redox indicator binding better to single-stranded DNA. The immobilization and hybridization events were assessed using differential pulse voltammetry (DPV). The fabricated biosensor was able to specifically distinguish complementary, non-complementary and mismatched oligonucleotides. DNA was measured in the range of 2.0×10(-9)-2.0×10(-13)M with a detection limit of 5.3×10(-12)M. The relative standard deviation for 6 replications of DPV measurement of 0.2µM complementary DNA was 4.88%. The fabricated DNA biosensor was considered stable and portable as indicated by a recovery of more than 80% after a storage period of 6 months at 4-45°C. Cross-reactivity studies against various food-borne pathogens showed a reliably sensitive detection of VP.
In this study, antibacterial characteristic of silver/poly (lactic acid) nanocomposite (Ag/PLA-NC) films was investigated, while silver nanoparticles (Ag-NPs) were synthesized into biodegradable PLA via chemical reduction method in diphase solvent. Silver nitrate and sodium borohydride were respectively used as a silver precursor and reducing agent in the PLA, which acted as a polymeric matrix and stabilizer. Meanwhile, the properties of Ag/PLA-NCs were studied as a function of the Ag-NP weight percentages (8, 16, and 32 wt% respectively), in relation to the use of PLA. The morphology of the Ag/PLA-NC films and the distribution of the Ag-NPs were also characterized. The silver ions released from the Ag/PLA-NC films and their antibacterial activities were scrutinized. The antibacterial activities of the Ag/PLA-NC films were examined against Gram-negative bacteria (Escherichia coli and Vibrio parahaemolyticus) and Gram-positive bacteria (Staphylococcus aureus) by diffusion method using Muller-Hinton agar. The results indicated that Ag/PLA-NC films possessed a strong antibacterial activity with the increase in the percentage of Ag-NPs in the PLA. Thus, Ag/PLA-NC films can be used as an antibacterial scaffold for tissue engineering and medical application.
There are relatively little data on bacteria with antimicrobial activities from Antarctic, especially from the South Shetland Islands when compared to the other parts of the world. Hence, this project was set to isolate and characterize bacteria that produce anti-microbial compounds from Greenwich Island (one of the South Shetland Islands), Antarctica. A total of 356 strains of bacteria were isolated from Greenwich Island. They were screened for antimicrobial activities against 13 Gram-negative and one Gram-positive indicator food-borne pathogens. Two out of the 356 Antarctic bacterial strains exhibited an antagonistic effect on the indicator strains, Escherichia coli, Salmonella spp., Klebsiella pneumoniae, Enterobacter cloacae, Vibrio parahaemolyticus and Bacillus cereus. The two Antarctic bacterial strains were designated as SS157 and SR13. Biochemical and 16S rDNA analysis indicated that the strain SS157 was closely related to Pseudomonas congelans while the strain SR13 was closely related to Pseudomonas tremae. The anti-microbial compounds produced by the two Antarctic bacteria were not sensitive to temperature and were not degraded by trypsin or pronase indicating that they were likely to be chemical compounds or antibiotics. Antimicrobial compounds from strains SS157 and SR13 were broad spectrum, and targeted both Gram-positive and negative pathogens.
Vibrio parahaemolyticus is one of the most widely recognized pathogenic Vibrio species due to numerous outbreaks and its’ wide occurrence in marine environment. In this study, 32 isolates of V. parahaemolyticus isolated from cockles were tested for sensitivity to 16 antibiotics and the presence of plasmids. All the isolates were multi-resistance, defined as resistant to atleast three different antibiotics with multiple antibiotic resistance indexes ranging from 0.31 to 0.69, indicating the isolates originate from high risk sources of contamination where antibiotics are often used. In the plasmid profiling test, only 15 isolates (47%) harbored plasmid DNA, which ranged in size from 2.7 to 56.2 kb, separating the isolates into 14 plasmid profiles. Hence, food contaminated with antibiotic resistant V. parahaemolyticus could be a major threat to public health due to the distinct possibility that they can be a significant reservoir of genes encoding antibiotic resistance determinants that can be transferred intra or interspecies. As in many developing countries, raw food hygiene and antimicrobial resistance epidemiology is still in the infancy stage in the locality of the study and thus our data provide a current baseline profile of antimicrobial resistance and plasmid of V. parahaemolyticusfrom cockles in Padang, Indonesia.
This study aims to determine the frequency and density of potentially pathogenic Vibrio parahaemolyticus, defined as those possessing thermostable-direct hemolysin (tdh) and/or tdh-related hemolysin (trh) genes, in raw salad vegetables at retail level in Selangor, Malaysia. A combination of Most Probable Number - Polymerase Chain Reaction (MPN-PCR) method was applied to detect the presence of tdh and/or trh gene-possessing V. parahaemolyticus and to enumerate their density in the samples. A total of 276 samples of vegetables commonly eaten raw in Malaysia (Cabbage = 30; Carrot = 31; Cucumber = 28; Four winged bean = 26; Indian pennywort = 17; Japanese parsley = 21; Lettuce = 16; Long bean = 32; Sweet potato = 29; Tomato = 38; Wild cosmos = 8) were analyzed. The samples were purchased from two supermarkets (A and B) and two wet markets (C and D). With the MPN-PCR technique, about 12.0% of the samples were positive for the presence of V. parahaemolyticus tdh-positive, with maximum densities of up to 39 MPN/g. The total frequency of V. parahaemolyticus trh-positive in the samples was 10.1%, with maximum concentration 15 MPN/g. V. parahaemolyticus tdh-positive was most prevalent in samples from Wet Market C (20.78%) and also in vegetable type Oenanthe stolonifera (Japanese parsley) with 19.0%, while V. parahaemolyticus trhpositive was predominant in samples from Wet Market D (16.7%) and was most frequent in both Oenanthe stolonifera (Japanese parsley) and Cucumis sativus (Cucumber) with 14.3% prevalence for each type. The results highlighted the fact that raw vegetables could be contaminated with virulent V. parahaemolyticus and could act as a transmission route, thus poses risk to consumers from the consumption of raw vegetables. To the author’s knowledge, this is the first assessment of V. parahaemolyticus carrying tdh and trh genes in raw
vegetables from retail outlets in Malaysia.
This study aimed to investigate the prevalence and antibiogram of Vibrio parahaemolyticus in processed bivalve molluscs in Kuala Terengganu. A total of 80 seafood samples, namely mussels (n=20), carpet clams (n=20), cockles (n=20) and scallops (n=20), were subjected to PCR and conventional plating method for the detection of V. parahaemolyticus. V. parahaemolyticus was found in green mussels (55%), carpet clam (80%), cockles (40%) and scallops (55%). Fifty-five V. parahaemolyticus isolates were subjected to 9 types antibiotic sensitivity test using discs diffusion method. All isolates were susceptible to Tetracycline and Gentamycin. Isolates showed high resistance towards Vancomycin (52.73%), Penicillin (45.45%) and Amplicillin (32.73%). Resistance towards Amikacin, Ciprofloxacin and Norfloxacin were found to be 1.82%. It can be concluded that local bivalve molluscs were contaminated with V. parahaemolyticus and isolates showed resistance towards certain antibiotics. Therefore, consumption of raw or semi-cooked bivalve molluscs is not advisable.
Vibrio parahaemolyticus is recognized as a frequent causal agent of human gastroenteritis due to the consumption of raw, undercooked or mishandled seafood in many Asian countries. The number of V. parahaemolyticus cases reported is on the rise, and this becomes a concern to the Asian countries as seafood is favoured by Asians. This study aimed to detect and quantify V. parahaemolyticus in raw oysters and to determine the risk associated with the consumption of raw oysters. A total of 30 oyster samples were collected and analysed in this study. MPN-PCR and MPN-Plating methods were employed and carried out concurrently to determine the prevalence of V. parahaemolyticus in raw oysters. The results showed that the prevalence of total V. parahaemolyticus in oysters was 50.00% (15/30) where the MPN/g range was < 3 – > 11000 MPN/g for MPN-PCR method, and 40.00% (12/30) where the MPN/g range was < 3 – 4300 MPN/g for MPN-Plating method. MPN-PCR method was able to estimate the level of virulence (tdh+ and trh+) V. parahaemolyticus in the raw oysters where 10.00% (3/30) of samples were identified to be in a range of 3 – 30 MPN/g. A microbial risk assessment was conducted based on the enumeration data obtained from MPN-PCR method using @risk. The probability of illness annually was 1.76 X 10-6 with a prediction of 31 cases to occur with respect to the exposed Malaysian population, while the rate per 100,000 people was estimated to be at 0.104. In addition, the antibiogram of V. parahaemolyticus was determined using Kirby Bauer Disk Diffusion Test and the results indicated that the isolates were highly resistant towards Bacitracin (100.00%), Vancomycin (100.00%) and were least resistant to Chloramphenicol (8.70%). The MAR index of the isolates ranged from 0.17 to 0.50. In accordance with the results from this study, the consumption of raw oysters is a risk factor for V. parahaemolyticus infection and proactive actions should be taken to reduce the risk of the pathogen in order to improve public health.
Bacteria must develop resistance to various inhospitable conditions in order to survive in the human gastrointestinal tract. Bile, which is secreted by the liver, and plays an important role in food digestion also has antimicrobial properties and is able to disrupt cellular homeostasis. Paradoxically, although bile is one of the guts defenses, many studies have reported that bacteria such as Vibrio parahaemolyticus can sense bile and use its presence as an environmental cue to upregulate virulence genes during infection. This article aims to discuss how bile is detected by V. parahaemolyticus and its role in regulating type III secretion system 2 leading to human infection. This bile-bacteria interaction pathway gives us a clearer understanding of the biochemical and structural analysis of the bacterial receptors involved in mediating a response to bile salts which appear to be a significant environmental cue during initiation of an infection.
The bioactivity of R. nasutus leaf extracts was assessed on Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes, Vibrio parahaemolyticus, Enterobacter aerogenes, Proteus mirabilis, and Klebsiella pneumoniae. Crude chloroform, petroleum ether, ethyl acetate, ethanol and methanol extracts were screened by disc diffusion method. Promising crude extract was further subjected to the column fractionation followed by the screening of the antibacterial activity of individual fractions. Biologically active pure fraction was subjected to the advanced analytical studies like HPLC, LC-MS, IR and NMR for characterisation of the bioactive compound. Ethanolic extract exhibited the maximum antibacterial activity against Klebsiella pneumoniae with the maximum of 35±0.42 mm zone of inhibition. The biologically potent column fraction from ethanol extract with 40±0.42 mm zone of inhibition upon subject to the HPLC, LC-MS, IR and NMR revealed that the active compound is rhinacanthin-C, a naphthoquinone.