Molecular characterisation of Vibrio parahaemolyticus carrying tdh and trh genes using ERIC-, RAPD- and BOX-PCR on local Malaysia bloody clam and Lala

Molecular typing methods have been widely applied for many purposes. In this study, such methods were adopted as DNA fingerprinting tools to determine the origin and divergence of virulent Vibrio parahaemolyticus strains found in local seafood. Although not all strain carry virulent tdh and trh gene, increasing prevalence demands an effective fingerprinting scheme which can constantly monitor and trace the sources of such emerging food pathogens. By using ERIC-, RAPD-, and BOX-PCR methods, 33 Vibrio parahaemolyticus isolates from local Malaysia bloody clam (Anadara granosa) and Lala (Orbicularia orbiculata) with confirmed presence of tdh and trh gene were characterised, followed by determination of clonal relatedness among virulent strains using cluster analysis and discriminatory index. This study also involved application of Immunomagnetic Separation (IMS) Method which significantly improved the specificity of strain isolation. Cluster analysis using Unweighted Pair Group Mathematical Averaging (UPGMA) and Dice Coefficient shown clustering according to isolation food source, IMS level and haemolysin gene possessed. Nevertheless, different DNA fingerprinting methods generated different clustering at different similarity cut-off percentage, regardless as individual or as composite dendrograms. ERIC- and RAPD-PCR composite fingerprinting relatively shown the highest discriminatory index at following similarity cut- off percentage: 0.68 at 50%; 0.83 at 65%; and 0.93 at 75%. Discriminatory power increased with similarity cut-off percentage. However, result also suggested that BOX-PCR might be an effective fingerprinting tool, as it generated three clusters with no single-colony isolate at 70% similarity cut-off. This study not only achieved its objective to determine clonal relatedness among virulent strains from local seafood via characterisation, but also speculated the best possible combination of molecular typing methods to effectively do s

Efficiency of four Malaysian commercial disinfectants on removing Listeria monocytogenes biofilm

Listeria monocytogenes(L. monocytogenes) is a gram positive food-borne pathogen that is able to form biofilm on food factory surfaces. Formation of biofilm makes the bacteria much more resistance to environmental stresses such as disinfectant. The extracellular polymeric matrix (biofilm structure) which is mostly comprised of sticky extracellular polysaccharides (EPS) and proteins can protect bacteria in a harsh condition. The efficiency of four disinfectants on removing L. monocytogenes biofilm was investigated. Five concentration levels (100, 50,25, 12.5, and 6.25%) of disinfectants were tested. In the microtitre assay, the optical density at 595 nm CV-OD 595 value, was used to measure the amount of remained biofilm after 24 h. Results showed that disinfectants did not have significant effect on removing L. monocytogenes biofilm. Formation of L. monocytogenes biofilm significantly decreased the efficiency of disinfectants. Biofilm produced by strain number 9 showed higher resistance to disinfectant. Low concentrations (<50%) of disinfectants did not show significant effect on removing L. monocytogenes biofilm

Molecular characterization of Escherichia coli isolated from different food sources

Escherichia coli and Escherichia coli O157 were identified from "selom" (Oenanthe stolonifera), "pegaga" (Centella asiatica), beef, chicken, lamb, buffalo, "ulam Raja" (Cosmos caudatus) and "tenggek burung" (Euodia redlevi). The bacteria were recovered using chromagenic agar. Isolated Escherichia coli and Escherichia coli 0157 were further characterized by plasmid profiling and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The virulence genes of the isolates (VT1, VT2, LT, ST, eaeA, inV) that produces pathogenic Escherichia coli and 16S rRNA gene were screened by a multiplex PCR assay. The plasmid profiling analysis showed that out of 176 isolates, only 103 isolates contained plasmids. ERIC-PCR analysis generated amplified products in the range of ~150 bp to > 1000 bp categorizing isolates into a total of 52 different profiles. Multiplex PCR showed that 20 (32.3%) of the isolates carried eaeA gene, 6 (9.7%) isolates possessed inV genes, only 1 (1.6%) have VT2 genes and 1 (1.6%) as well carried VT1 genes, 2 (3.2%) of the isolates harboured LT genes, and only 1 (1.6%) isolate possessed ST genes. There were no correlation between plasmid, ERIC-PCR and virulence genes profiles

Konfirmasi Gen tdh dan trh Secara PCR pada Bakteri V. parahaemolyticus dari Sampel Telur Chelonia mydas (penyu hijau) dan Eretmochelys imbricata (penyu sisik)

Detection of toxR, tdh and trh genes in Vibrio parahaemolyticus from turtle eggs have been conducted. The confirmation test were done by using PCR methods. The presences of tdh and trh genes were considered as virulence factor. The results showed that turtle eggs from Chelonia mydas and Eretmochelys imbricata contained toxR gene 18.6 %, where eighteen of them contained tdh gene 8.4 % and none of them contained trh gene. KeyWord : Vibrio parahaemolyticus, gen tdh, gen trh, PC

Transfer of Campylobacter jejuni from raw to cooked chicken via wood and plastic cutting boards.

Aims: We quantified Campylobacter jejuni transferred from naturally contaminated raw chicken fillets and skins to similar cooked chicken parts via standard rubberwood (RW) and polyethylene cutting boards (PE). Methods and Results: RW and PE cutting boards (2·5×2·5cm2) were constructed. RW surfaces were smooth and even, whereas PE was uneven. Scoring with scalpel blades produced crevices on RW and flaked patches on the PE boards. Raw chicken breast fillets or skin pieces (10g) naturally contaminated with Camp. jejuni were used to contaminate the cutting boards (6·25cm2). These were then briefly covered with pieces of cooked chicken. Campylobacter jejuni on raw chicken, the boards, and cooked chicken pieces were counted using a combined most-probable-number (MPN)-PCR method. The type of cutting board (RW, PE; unscored and scored) and temperature of cooked chicken fillets and skins were examined. Unscored PE and RW boards were not significantly different in regards to the mean transfer of Camp. jejuni from raw samples to the boards. The mean transfer of Camp. jejuni from scored RW was significantly higher than from scored PE. When the chicken fillets were held at room temperature, the mean transfer of Camp. jejuni from scored RW and PE was found to be 44·9 and 40·3%, respectively. Conclusions: RW and PE cutting boards are potential vehicles for Camp. jejuni to contaminate cooked chicken. Although cooked chicken maintained at high temperatures reduced cross-contamination via contaminated boards, a risk was still present. Significance and Impact of the Study: Contamination of cooked chicken by Camp. jejuni from raw chicken via a cutting board is influenced by features of the board (material, changes caused by scoring) and chicken (types of chicken parts and temperature of the cooked chicken). © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology

Consumption of raw oysters: a risk factor for Vibrio parahaemolyticus infection

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 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 × 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

Prevalence of Listeria monocytogenes in frozen burger patties in Malaysia

A total of 112 burger patties (35 beef burger patties, 39 chicken burger patties and 38 fish burger patties) which are commercially available at retail level were investigated for the presence and number of Listeria monocytogenes. These samples were analyzed using MPN-PCR method and conventional culturing methods. L. monocytogenes was detected in 33.3% of chicken burger patties, 22.9%of beef patties, and 10.5% of fish patty samples. From all contaminated raw burger patties, the estimated count of L. monocytogenes was ranged from 3 to 75 MPN/g. The results suggest that burger act as a potential source of listeriosis if the contaminated burger patty is consumed without adequate cooking. The risk associated with consumption of these samples was found to be high particularly for processed food at retail level in Malaysia. Therefore, food manufacturers play an importantrole in monitoring the manufacturing process and conduct a periodical surveillance on microbiological quality assessment on the processing plants. Besides, there is a need to increase awareness ofconsumers and food handlers to practice proper cooking of the burger patties before the point of consumption, to reduce the risk of listeria infection

Biofilm assessment of vibrio parahaemolyticus from seafood using random amplified polymorphism DNA-PCR

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 (37°C) was the highest and stable formation of biofilm. The total percentage of biofilm formation at 37°C 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 4°C 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

Phenotypic MicroArray (PM) profiles (carbon sources and sensitivity to osmolytes and pH) of Campylobacter jejuni ATCC 33560 in response to temperature

The present study aimed to provide an insight of C. jejuni ATCC 33560 phenotype profiles (carbon sources and sensitivity to osmolytes and pH) using Phenotypic MicroArray (PM) system in response to optimal and suboptimal temperature. C. jejuni ATCC 33560 showed utilization carbon sources from amino acids and carboxylates but not from sugars. C. jejuni ATCC 33560 is sensitive to NaCl at 2% and above but showed survival in a wide range of food preservatives (sodium lactate, sodium phosphate, sodium benzoate, ammonium sulphate and sodium nitrate). When incubated at suboptimal temperature, no phenotype loss was observed in carbon source plates. Phenotype loss of C. jejuni ATCC 33560 was observed in sodium chloride (1%), sodium sulphate (2-3%), sodium formate (1%), sodium lactate (7-12%), sodium phosphate pH7 (100mM and 200mM), ammonium sulphate pH8 (50mM), sodium nitrate (60mM, 80mM and 100mM), sodium nitrite (10mM), and growth in pH5. The phenotypic profile from present study will provide a better insight related to survival of C. jejuni ATCC 33560

Investigation of stx2 eae+ Escherichia coli O157:H7 in beef imported from Malaysia to Thailand

To gain insight into the microbiological safety of food products routinely traded across international borders in Southeast Asian countries, beef imported from Malaysia to southern Thailand was examined for contamination with Escherichia coli O157 and its subsequent spread into the imported areas. We screened 31 samples exported from Malaysia and 36 domestic Thai samples. Isolation methods including an O157 antigen-targeted immunomagnetic separation technique, screening on CHROMagar O157 medium, and serotype confirmation of E. coli isolates by specific agglutination tests were employed. Fourteen strains of E. coli O157:H7 were isolated from eight Malaysian samples (25.8%) and six strains from four Thai samples (11.1%). These strains were of the stx1- stx2+ eae+ genotype except one Malaysian strain which was of the stx1 - stx2-eae+ genotype. All 19 O157:H7 strains possessing the stx2 gene produced little or no Stx2 (reversed passive latex agglutination titer ≤ 4). Of the 19 strains, five Malaysian (38.5%) and two Thai (33.3%) strains exhibited resistance to a set of antibiotics. Finally, the results of two DNA fingerprinting analyses (O157 IS-printing targeted to IS629 and pulsed-field gel electrophoresis, PFGE) of the O157:H7 strains possessing the stx2 gene, indicated that the Malaysian and Thai strains are closely related. Therefore, E. coli O157:H7 might be transferred from Malaysia to southern Thailand through beef trade