Characterization of Vibrio Parahaemolyticus Isolated From Local Cockles (Anadara Granosa) From Tanjung Karang, Kuala Selangor

Vibrio parahaemolyticus is widespread in occurrence and has been recognized as a cause of gastroenteritis related to consumption of raw or improperly cooked seafood. Outbreaks of V. parahaemolyticus food poisoning are most common in Taiwan, Japan and Southeast Asia. In this study, V. parahaemolyticus was isolated from 62 of 100 (62%) samples of cockles (Anadara granosa) collected from Tanjong Karang, Kuala Selangor. A total of 62 strains were studied for the presence or absence of regulatory gene (toxR), virulence genes (tdh and trh), their antibiotic resistance, the occurrence of plasmids and their molecular fingerprints by Randomly Amplified Polymorphic DNA – Polymerase Chain Reaction (RAPD-PCR) and Enterobacterial Repetitive Intergenic Consensus - Polymerase Chain Reaction (ERIC-PCR) assays. All 62 strains were positive for the regulatory gene (toxR) of V. parahaemolyticus. The PCR analysis for the detection of tdh or trh genes showed two (3.2%) positive strains carrying tdh gene and eleven (17.7%) strains had trh gene. The MPN value for all samples was more than 1100 MPN/g. This study has shown that all strains were multiple resistant to three or more of the seventeen antibiotics tested with the MAR indices ranging from 0.58-0.94. All isolates of V. parahaemolyticus were highly resistant towards the antibiotics tested, except one strain that was sensitive towards norfloxacin. Plasmids were found in 80% of the strains analyzed and 18 different plasmid profiles were observed. The plasmid size ranged from 2.7 to more than 54 kb. Two molecular typing methods were used in this study to examine the genetic relatedness among the V. parahaemolyticus strains. In the analysis by RAPD-PCR and ERIC-PCR, the size of RAPD and ERIC fragments ranged from 0.25 to 10.0 kb with an average number of ten and eight bands, respectively. Sixty-two genotypes among the 62 V. parahaemolyticus strains were generated using RAPD and ERIC-PCR which indicates that the strains were very diverse. Hence, this study, demonstrated that the local cockles are potential source for pathogenic V. parahaemolyticus

Isolation and Molecular Characterisation of Listeria Monocytogenes and Listeria Innocua from Poultry Meat

Thirty isolates of Listeria monocytogenes (12) and Listeria innocua (18) were isolated from poultry meat. All isolates were confirmed by Microbact (Medvet, Australia) identification kits. All the isolates were subjected to chromosomal and plasmid DNA screening and antibiotic resistance test. Based on the antibiotic resistance profiles, Listeria monocytogenes and Listeria innocua were differentiated into 10 and 9 profiles respectively. The antibiotyping procedure discriminated the Listeria monocytogenes and Listeria innocua into 10 and 3 different groups respectively. Most of the isolates were resistant to nalidixic acid (100%), c1indamycin (97%), spectinomycin (97%), cefuroxime (93%), cefriaxone (80%), cephalothin (73%), cefotaxime (67%), novobiocin (37%), chloramphenicol (27%), kanamycin (20%), rifampicin (20%), tobramycin (17%), norfloxacin (13%), netilmicin(10%) and imipenem (3%). The results of the plasmid profiles and antibiotyping show that there is no correlation between them. RAPD-PCR has been used to generate polymorphic genomic fingerprints to discriminate the Listeria isolates. Primer GEN15009 was chosen whereby it produced reproducible and typeable results in all isolates examined with the bands ranging from 0.25 to 3.0 kilobase pairs. From the dendrogram generated L. monocytogenes were separated from L. innocua and the strains in each species were differentiated as well. The data indicate that RAPD-PCR based approaches is a valid means of discriminating strain differences among isolates of L. monocytogenes and L. innocua and as an adjunct to differentiate among Listeria spp

Detection of Bacillus cereus in formula milk and ultra high temperature (UHT) treated milk products

Ultra high temperature (UHT) treated milk products and formula milk are known to be frequently contaminated with Bacillus cereus. Presence of B. cereus in these milk products is of particular concern considering the majority of consumers are infants and children. Possible sources of contamination are contaminated raw milk, cross-contamination during processing, under-processing and mishandling of milk products. This study was conducted to detect the presence of B. cereus in both formula milk (n=12) and UHT milk (n=20) sold in selected retail markets. The approach consisted of enumerating by MPN/g followed by PCR assay aimed at detecting gyrB gene in B. cereus, that encode for the subunit B protein of DNA gyrase (topoisomerase type II). Contamination level of B. cereus in both types of samples examined ranged from 1100 MPN/g. The contamination level of B. cereus was found to be highest in full cream UHT milk (> 1100 MPN/g) and formula milk (> 1100 MPN/g). The PCR analysis showed that 41.7% (5/12) formula milk and 30% (6/20) UHT milk samples were detected with B. cereus, respectively. This is the first report of such study demonstrating the presence of B. cereus in formula milk from Malaysia. Therefore, constant surveillance of these milk products would reduce the potential risk of B. cereus-linked outbreaks

Presence of Bacillus cereus s.l. from ready-to-eat cereals (RTE) products in Sarawak

Bacillus cereus is a soil inhabitant gram positive bacterium, and is known to cause severe food poisoning. The objective of this study was to isolate and identify the presence of Bacillus cereus s.l. from selected ready to eat cereals purchased randomly from local supermarkets in Kuching and Kota Samarahan, Sarawak. The result showed that four of the 30 food samples were detected to be contaminated by B. cereus s.l.. Our findings suggested that it is important for the public to be aware of the safety of RTE cereals consumption, as it is possible that B. cereus s.l. may be present in high count number and pose hazardous health effects to the consumers

Characterization of Escherichia coli isolated from cultured catfish by antibiotic resistance and RAPD analysis

Antibiotic susceptibility and genetic diversity of E. coli isolated from cultured catfish and their surrounding environment were determined. The levels of resistance of the E. coli isolates towards six different antibiotics tested differed considerably. Though the isolates displayed resistance towards some of the antibiotics tested, none of the isolates showed resistant towards norfloxacin, sulphametoxazole/trimethoprim and chloramphenicol. RAPD-PCR analysis using single primer and primers combination clustered the E. coli isolates into 3 and 5 groups, respectively. The results of this study suggest that the E. coli isolates from the catfish and their surrounding environment derived from a mixture of sensitive and resistant strains with diverse genetic contents. The use of the RAPD analysis is sufficiently discriminatory for the typing of the E. coli isolates

Identification of Vibrio parahaemolyticus isolates by PCR targeted to the toxR gene and detection of virulence genes

Vibrio parahaemolyticus is a gram negative bacterium and causes gastrointestinal illness in humans. In this study, twenty five out of fifty cockle samples from Padang, Indonesia produced purple colonies when they were grown on selective medium, CHROMagarTM Vibrio. Specific–PCR for toxR gene detection gave positive results in which a band with 368 base pairs size appeared on the gel for all the isolates that confirmed the presence of V. parahaemolyticus. In the virulence properties test, all the isolates showed negative results for tdh and trh genes detection. The results indicate that the isolates under this study do not contain virulence properties that correlate to the ability of infection and diseases, which means that they are nonpathogenic

Antibiotic resistance and plasmid profiling of Vibrio parahaemolyticus isolated from cockles (Anadara granosa) at Tanjung Karang, Kuala Selangor

A total of sixty V. parahaemolyticus strains isolated from local cockles (Anadara granosa) were investigated by their antibiotic resistance patterns and plasmid profiles. The isolates showed multiple resistances towards most of the antibiotics tested. All strains of V. parahaemolyticus isolated harbored 1-3 plasmids, with sizes ranging from 2.7 to 54 kb. All V. parahaemolyticus strains showed high multiple antibiotics in frequencies of 0.58 - 0.94 indicating that the strains were derived from high-risk sources. In addition, no particular plasmid profile was predictive of a particular pattern of antibiotic susceptibility. These findings are essential because of the suggested involvement of seafood especially shellfish and environment in transmission of this pathogen to human. Thus, indicating that seafood may be a source of food- acquired antibiotic resistant bacteria to consumer

Rapid detection and enumeration of pathogenic Vibrio parahaemolyticus in raw vegetables from retail outlets

This study aims to determine the frequency and density of potentially pathogenic Vibrio parahaemolyticus, defned 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 trh-positive 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

Detection of Vibrio parahaemolyticus in cockle (Anadara granosa) by PCR

This study aimed to determine the occurrence of Vibrio parahaemolyticus in cockles (Anadara granosa) at a harvesting area and to detect the presence of virulent strains carrying the thermostable direct hemolysin (tdh) and TDH-related hemolysin genes (trh) using PCR. Of 100 samples, 62 were positive for the presence of V. parahaemolyticus with an MPN (most probable number) value greater than 3.0 (>1100 MPN per g). The PCR analysis revealed 2 samples to be positive for the tdh gene and 11 to be positive for the trh gene. Hence, these results demonstrate the presence of pathogenic V. parahaemolyticus in cockles harvested in the study area and reveal the potential risk of illness associated with their consumption

Synthesis and Characterization of Cordierite and Mullite composite

Cordierite-mullite composite has been prepared from rice husk derived silica; Magnesium sulphate derived magnesium oxide, and calcined alumina powder. Phase formation behavior of the sample has been studied from the XRD pattern of the sintered sample. The densification behaviour of the composite has been studied using dilatometer. The densification behavior of the composite as a function of sintering temperature has also been reported. Flexural strength as a function of sintering temperature and mullite content in the composite has also been studied