A total of 49 isolates of V. parahaemolyticus and 8 isolates of V. cholerae isolated from freshwater fish of patin (Pangasius hypopthalmus) and red tilapia (Oreochromis sp.) were purchased from different retail level in Selangor, Malaysia. All of the isolates showed a multiple resistances towards all 15 antibiotics tested. Some of the isolates show a high resistance to different antibiotics including bacitracin, vancomycin, tetracycline, furazididone, cephalothin and erythromycin. However, both species was susceptible towards imipenem. Overall antibiotics resistance patterns of all isolates were resistant from 2 to 14 resistance patterns with multiple antibiotic resistance (MAR) index ranging from 0.13 to 0.93 respectively. As the results obtained in the dendrogram produced from both species had indicates that these antibiotics were intensively used whether in the aquaculture farm through feeds during culture or at the hatchery production of seed. Thus, this study will provides an essential information of the MAR index and also the clustering analysis in order to determine the biosafety of Vibrio spp. in freshwater aquaculture fish sold at different retail level in Malaysia.
This study was conducted to evaluate antimicrobial properties of ethanolic extracts of the leaves of Nephelium lappaceum, Curcuma longa, Cinnamomun cassia, Durio zibethinus, Vitex trifolia, Amaranthus tricolor, Syzygium samarangense and Manihot esculenta. Antibacterial properties of the extracts were studied against fifteen strains of different gram positive and gram negative pathogenic bacteria, including Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Vibrio para, and Escherichia coli using the agar disk diffusion method. Among the tested extracts, only Amaranthus tricolor exhibited specific inhibition of one of the tested bacteria; Bacillus cereus. Using the microdilution method, its minimum inhibitory concentration (MIC) value was determined to be 20 mg/mL.
Vibrio parahaemolyticus is a foodborne pathogen and their human infection is regularly associated with the consumption of raw or undercooked seafood and contaminated water supplies. Many conventional biochemical identification and confirmation procedures are performed to detect the presence of this pathogen, both from seafood or environmental samples. However, these procedures not only require two or more days to complete, they do not have the capabilities to determine the number of V. parahaemolyticus cells in any given samples. Thus, in this study we describe the development of a rapid SYBR green based real-time PCR assay, targeting the thermo labile (tl) gene of V. parahaemolyticus for the detection and enumeration of this bacterium from seafood and environmental samples. We report that the real-time PCR assay and the primers designed are highly specific, and only generated the desired amplicons with V. parahaemolyticus DNA samples against other bacteria and fungi species. Our assay is also highly sensitive, and, is able to detect V. parahaemolyticus with high coefficient values in concentrations as low as 1.0 pg/μl DNA for pure genomic DNA solutions and 10 cells/ml in serially diluted cell suspension and spiked samples. This assay can be completed in less than 3 hours and may be used as a tool for rapid determination of V. parahaemolyticus densities in the food industries, environmental risk assessment and for clinical diagnostics purposes.
Pathogenic Vibrio parahaemolyticus is one of the leading causes of bacterial gastroenteritis in many countries. Among the strains examined, 36 RAPD-types were found when amplified with primers OPA8 and OPA10. The analysis shows the majority of V. parahaemolyticus isolates originated from seafood were branched into four major clusters at 18.2%, 20.7% 34% and 3.4% similarity levels. This suggests that there is potential for a single strain to be distributed widely within a population and there also potential for multiple contaminating strains of different clonal lineages to be present within the same population. Optimum temperature (37ºC) was the highest and stable formation of biofilm. The total percentage of biofilm formation at 37ºC was 33.33% for each of weak, moderate and strong biofilm producers. Room temperature produces 61.1% of weak biofilm producer, while 13. 89% for moderate biofilm producers and produce 25% of strong biofilm. While a total of 91.67% weak biofilm producers at 4ºC and 8:33% for room temperature and no growth of strong biofilm. Upon analysis, strong biofilm was tracked from the largest group at 37°C and room temperature which produce 27.27% of strong biofilm producer respectively. Interestingly, they are derived from cockles.
This study was undertaken to characterize the antibiotic resistance and randomly amplified polymorphic DNA (RAPD) profiles of Vibrio parahaemolyticus isolates from raw vegetable samples. A total of 46 isolates of V. parahaemolyticus recovered from raw vegetables samples and were confirmed by PCR were analyzed in this study. Most of the isolates were resistant to nalidixic acid (93.48%) and were the least resistant towards imipinem (4.35%). The MAR index results also demonstrated high individual and multiple resistances to antibiotics among the isolates. From the RAPD analysis, the size for RAPD fragments generated ranged from 250 bp to 1,500 bp, with most of the strains contained three major gene fragments of 350, 1,000 and 1,350 bp. The RAPD profiles revealed a high level of DNA sequence diversity within the isolates. Antibiotic resistance and RAPD proved to be effective tools in characterizing and differentiating the V. parahaemolyticus strains.
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.
Aimed of this study was to determine the presence of Vibrio cholerae in cockles (Anadara granosa)
from different coasts in Malaysia and to measure the biosafety of V. cholerae in raw cockles at wet market in Malaysia using the polymerase chain reaction (PCR) in combination with the most probable number (MPN) method. A total of 100 samples from 4 different wet markets in the West and East were examined for the presence of V. cholerae. The prevalence of V. cholerae between the two coasts was not significant different. In fact, the 74% of samples from West coast area was found positive while the 69% for samples collected in the East coast. West coast samples showed a prevalence of 60% for the wet market A=, 64% for B=, 88% for C= and 84% for the market D); East coast samples showed the same percentage with 72% for the wet markets E, F and H, followed by wet market G with 60%.With the MPN-PCR method, using 80 samples of raw cockles obtained from 4 wet markets, the occurrence of V. cholerae detected was of 95%. The frequency of V. cholerae in raw cockles obtained from wet market I and L was higher (100%) compared to other wet market (Wet market B=, 90%; Wet market C=, 95%).The density of V. cholerae detected in all samples ranged from 24000 MPN/g, but most of the samples (24 samples) were in category >24000 MPN/g concentration. V. cholerae was present in raw cockles in higher number. Hence, these results demonstrate the presence of pathogenic V.cholerae in cockles harvested and reveal the potential risk of illness associated with their consumption. This study will be the first biosafety assessment of V. choleare in raw cockles in Malaysia and it will provide significant insights about Malaysian scenario.
Unprocessed ‘budu’ is a mixture of anchovies and salt that has been fermented for a period of time, and has not been heat-treated nor formulated with additional ingredients. This study analyzed Malaysian
unprocessed ‘budu’ from 12 producers for microbiological, salt, protein, histamine and 3-MCPD contents.
The results demonstrated that Malaysian unprocessed ‘budu’ were free from pathogenic Coliform, E. coli,
V. parahaemolyticus and V. cholerae contaminations. Carcinogenic 3-MCPD was below detection level of 2 ppb for all 12 samples tested. However, 58% of the unprocessed ‘budu’ had histamine content greater than the hazardous levels of 50 mg/100 g sample.
A total of 32 clinical strains of Vibrio cholerae, including members of the 01 and 0139 serogroup
were collected from Klang, Selangor; Penang Island; Samarahan, Sarawak and Miri, Sarawak in Malaysia. In general, all the isolates except the 0139 serotype expressed low resistance to all the antibiotics tested with their Multiple Antibiotic Resistance (MAR) indices ranged from 0.10 to 0.48. The presence of ctx gene that encoded the cholera toxin was confirmed in all these clinical isolates by polymerase chain reaction. The results from the RAPD-PCR were analyzed using the RAPDistance software (Version 1.04). From the dendrogram generated, two main groups were observed which were subdivided into two clusters each. The Selangor’s isolates and the 0139 Penang’s isolates formed one group whereas the Samarahan, Sarawak isolates and the Miri, Sarawak isolates made up the other group, thus delineating their different sources of origin based on their geographical location.
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 prevalent in tropical marine environment in all seasons and can cause seafood-borne gastroenteritis. A total of 251 suspected isolates were tested including 60 from frozen shrimp, 50 from cultured live shrimp, 67 from sediments of culture ponds and 74 from water were subjected to polymerase chain reaction (PCR) targeting the toxR gene for confirmation as V. parahaemolyticus. Of the 128 toxR positive isolates, 15% of the isolates from culture environment (from live shrimp, sediments and water) and 7% of frozen shrimp samples were positive for the tdh and trh genes. Since urease production could be a marker of trh but not tdh in V. parahaemolyticus, a total of 189 of the 251 suspected V. parahaemolyticus isolates were tested for urease production and 41% of the isolates were found to be positive for urease production. However not all urease positive strains of V. parahaemolyticus were positive for either tdh or trh genes. Detection of virulent strains in shrimp culture environment in Malaysia suggests a probable risk for health of people consuming raw shrimp.
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.
Vibrio parahaemolyticus is a main foodborne disease in seafood and generally seafood is
easily deteriorates in quality of color and flavor. In this study, clove (Syzygium aromaticum)
extract shows potent antibacterial activity against growth of antibiotics resistant Vibrio
parahaemolyticus on seafood samples (cockles and shrimps). Vibrio parahaemolyticus was
artificial contaminates on the samples with 106 CFU/ml. The samples were treated with different
concentration of cloves extract with 10 mg/ml which are 0.5%, 5% and 10% concentration
from methanol food grade extraction in 0 hr, 5 min, 10 min, 15 min, 30 min, 60 min and
120 min. Tab water and deionized water were selected as a negative control. As a result, the
amount of 10 % cloves managed to mitigates the number of V. parahaemolyticus on seafood
samples in 5 minutes and 15 min on both samples. Therefore, our results signify the fact that
cloves can be apply as natural sanitizer which could meet consumer demands for safe and
traditionally consumed either raw without any undesirable effect when applied in the seafood
system industries.
Vibrio parahaemolyticus is well known to be abundantly distributed in marine, coastal and
estuarine environments. Since 1951, V. parahaemolyticus had been the source of numerous
outbreaks related to contaminated or mishandled seafood. However, V. parahaemolyticus
had been detected on other types of food. This issue has prompted this study to investigate
on the prevalence of V. parahaemolyticus in various food samples and determine the risk
associated with it. The results of the MPN-plating technique of the study indicated that V.
parahaemolyticus was detected in seafood (33.3%, 95% Confidence Interval [CI] 31.9 – 34.8 ,
94 – 290 MPN/g) and vegetables (10.0%, 95% CI 9.7 – 10.3 , 9.2 – 23 MPN/g) while negative
V. parahaemolyticus was detected in fruits (0.0%, 95% CI 0 – 1,
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.
Detection of enterotoxin by targeting entFM and hblA genes in Bacillus cereus BC1 strain inoculated into ready to eat food (RTF) and drink samples using polymerase chain reaction (PCR) was conducted. The B. cereus BC1 strain was confirmed as a Bacillus diarrhoeal enterotoxin (BDE) when tested by a commercially available Enzyme-linked immunosorbent assay-BDE immunoassay (ELISA-BDE immunoassay, TECRA). In the specificity study, both enterotoxin genes were detected on chromosomal DNA of B. cereus BC1 strain by showing a specific band of 1269 bp (entFM) and 874 bp (hblA), respectively. However, none of the target genes were detected for the other 15 genomic DNA bacteria (B. cereus (ATCC 11779), B. subtilis (ATCC 6633), Campylobacter jejuni (ATCC 29428), C. coli (Jabatan Kimia Malaysia, JKM), Clostridium perfringen (ATCC 13124), Enterobacter sakazaki (ATCC 51329), Escherichia coli (ATCC 43888), E. coli (ATCC 11735), Legionella pneumophila (ATCC 33152), Listeria monocytogenes (ATCC 35967), Salmonella typhi (IMR), S. enteritidis (ATCC 13076), S. typhimurium (ATCC 14028), Shigella flexeneri (ATCC 12022) and Vibrio cholerae bengal (Institute Medical Research (IMR), Malaysia) examined. The detection limit of both genes was 0.1 ng of genomic DNA. Thus, in the presence study it is evidence that the PCR analysis targeting enterotoxin of entFM and hblA genes are suitable and useful in detecting enterotoxic B. cereus in RTFs and drinks contaminated sample.
The present study was conducted to assess the rapid molecular identification and characterization of 45 Vibrio parahaemolyticus isolates from 15 samples of 3 different types of fish (Kembung, Bawal and Sangeh) in the Kuching-Samarahan district. Polymerase chain reaction (PCR) based confirmation was done targeting the 450 bp fragment of the thermolabile (tl) gene, while DNA fingerprinting was performed using Randomly Amplified Polymorphic DNA (RAPD) PCR with the primer GEN15008. All the 45 V. parahaemolyticus isolates were positive for the tl gene, however, only 34 were typable via RAPD-PCR with bands sizes ranging from slightly over 250 bp to 2.5 kbp. The degree of diversity was then determined via the Simpson Index which showed a value of 0.891, indicating high diversity among the isolates. Data from the RAPD-PCR fingerprints were later used to construct a dendrogram for clustal analysis. From the dendrogram, the 34 isolates were grouped into 2 major clusters containing 26 and 8 isolates, respectively. Further analyses of the dendrogram also indicated that the 34 isolated were clustered according to the period of sampling. This is an interesting observation as it shows the high discriminatory capability of RAPD-PCR to be used as molecular epidemiological tool to study the temporal distribution of V. parahaemolyticus.
Little is known on the biosafety level of Vibrio spp. in freshwater fish in Malaysia. The purpose of this study was to investigate the prevalence and concentration of Vibrio spp. and V. parahaemolyticus in
freshwater fish using the Most Probable Number-Polymerase Chain Reaction (MPN-PCR) method. The study was conducted on 150 samples from two types of freshwater fish commonly sold at hypermarkets, i.e. Pangasius hypophthalmus (catfish) and Oreochromis sp. (red tilapia). Sampling was done on the flesh, intestinal tract and gills of each fish. The prevalence of Vibrio spp. and V. parahaemolyticus was found to be 98.67% and 24% respectively with higher percentages detected in samples from the gills followed by the intestinal tract and flesh. Vibrio spp. was detected in almost all red tilapia and catfish samples. V. parahaemolyticus was detected in 25% of the catfish samples compared to 22.6% of red tilapia fish. The density of Vibrio spp. and V. parahaemolyticus in the samples ranged from 0 to 1.1x107 MPN/g. Although the maximum value was 1.1x107 MPN/g, most samples had microbial loads ranging from 0 to >104 MPN/g. The outcome on the biosafety assessment of Vibrio spp. and V. parahaemolyticus in freshwater fish indicates another potential source of food safety issues to consumers.
Young and mature leaves of Terminalia catappa of alcoholic and aqueous extracts were evaluated for in vitro antibacterial activity against Vibrio sp. isolated from aquatic animals. Young leaves of T. catappa showed higher antibacterial activity when compared to mature leaves against Vibrio parahemolyticus, with methanolic and aqueous extracts exhibited the largest inhibition zones, 23 and 24 mm, respectively as determined by disc diffusion technique. Ethanolic extract of young leaves showed the lowest MIC and MBC at 3.13 mg/ml and 6.25 mg/ml, respectively. Both alcoholic and aqueous extracts of young and matures leaves exhibit variations in protein, RNA as well as pyrine and pyrimidines leakage of Vibrio sp. Cell membrane disruption is proposed as the mechanism of action of T. catappa leaves extract against Vibrio sp.
Foodborne disease has been associated with microorganisms like bacteria, fungi, viruses and parasites. Most commonly, the outbreaks take place due to the ingestion of pathogenic bacteria like Salmonella Typhi, Escherichia coli, Staphylococcus aureus, Vibrio cholera, Campylobacter jejuni, and Listeria monocytogenes. The disease usually happens as a result of toxin secretion of the microorganisms in the intestinal tract of the infected person. Usually, the level of hygiene in the food premises reflect the quality of the food item, hence restaurant or stall with poor sanitary condition is said to be the contributor to food poisoning outbreak. In Malaysia, food poisoning cases are not rare because the hot and humid climate of this country is very suitable for the growth of the foodborne bacteria. The government is also implementing strict rules to ensure workers and owners of food premises prioritize the cleanliness of their working area. Training programme for food handlers can also help them to implement hygiene as a routine in a daily basis. A lot of studies have been done to reduce foodborne diseases. The results can give information about the types of microorganisms, and other components that affect their growth. The result is crucial to determine how the spread of foodborne bacteria can be controlled safely and the outbreak can be reduced.