Kajian ini dijalankan untuk mengesahkan kemampuan teknologi DNA mikroaturan cip gen OliproTM FoodPATH bagi mengesan bakteria patogen makanan. Sebanyak 9 jenis DNA bakteria patogen makanan telah digunakan iaitu Bacillus cereus, Escherichia coli O157:H7, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella spp., Shigella spp. dan Campylobacter spp. Sebanyak 36 kombinasi templat DNA bakteria patogen makanan telah digunakan. Pengesahan bagi mengesan bakteria patogen makanan dilakukan dengan menggunakan kaedah reaksi berantai polimerase (PCR) dan penghibridan Southern-blotting di atas cip gen untuk mengesahkan kemampuannya. Keputusan daripada analisis hibridasi di atas cip gen telah dibandingkan dengan hasil gel elektroforesis 2.0% (w/v). Lima saringan diperlukan untuk menghabiskan 36 kombinasi templat DNA bakteria patogen makanan. Setiap saringan, satu cip gen telah digunakan sebagai kawalan negatif tidak diinokulasikan dengan sebarang kombinasi DNA bakteria patogen makanan. Daripada hasil kajian, didapati bahawa semua kombinasi templat DNA bakteria patogen makanan telah dapat dikesan. Cip yang digunakan sebagai kawalan negatif tidak menunjukkan kehadiran DNA. Oleh itu, daripada kajian ini cip gen OliproTM FoodPATH didapati memberikan keputusan yang lebih baik berbanding dengan 2.0% (w/v) gel elektroforesis.
Enterohemorrhagic Escherichia coli (EHEC) especially serotype O157:H7 is one of the important food-borne pathogens because it is able to produce crucial toxins Shiga. However, the outbreak of this organism in Thailand has not been reported. Antibody to O157 antigen was detected in some Thai populations and Shiga toxin-producing E. coli were detected in low numbers of clinical specimens. Interestingly, some E. coli that showed positive to O157 fimbriae probe and lack of virulence gene were isolated from certain patients and one isolate of E. coli O157:H7 which possessed stx1, stx2v was detected in a normal child. In addition, the incidence of E. coli O157:H7 strains were monitored by the samples from cattle and retail beef in Thailand although their inability to produce toxins or produce in a low concentration was demonstrated. This review discusses the incidences of E. coli O157 in clinical and environmental samples of Thailand including the transmission possibility of this bacterium across the Thai border through food trade.
E. coli O157:H7 is associated with life threatening diseases such as hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Raw milk is considered a high risk food as it is highly nutritious and serves as an ideal medium for bacterial growth. The aim of this study was to investigate the prevalence of E. coli O157:H7 in raw cow, goat and buffalo milk samples. MPN-PCR method targeting the major virulence rfbE gene and fliCH7gene of E. coli O157:H7 was used. Total of 177 raw milk samples were collected from local dairy farms in the state of Selangor, Malaysia. The highest prevalence of E. coli O157:H7 was found in raw cow milk (18.75%). E. coli O157:H7 was detected in 7.32% and 3.57% of raw goat and buffalo milk, respectively. The estimated quantity of E. coli O157:H7 in raw cow, goat and buffalo milk ranged from
Fresh raw shrimps were dipped for 10, 20, and 30 min at room temperature (25°C ± 1°C) in lactic acid (LA; 1.5%, 3.0%, v/v) to evaluate their antipathogenic effects against Vibrio cholerae, Vibrio parahaemolyticus, Salmonella entreitidis, and Escherichia coli O157:H7 inoculated at a level of 10(5) CFU/g. Significant reductions in the population of all these pathogenic bacteria were recorded after dipping treatments, which were correlated to the corresponding LA concentrations and treatment time. With respect to the microbial quality, 3.0% LA treatment for 10 min was acceptable in reducing the pathogenic bacteria. Additionally, sensory evaluation results revealed a 10-min dip in 3.0% LA to be more acceptable organoleptically compared with 20 and 30 min of treatments. Results of the present study are envisaged to be useful for commercial applications for effective decontamination of shrimp.
Specific Escherichia coli isolates lysogenised with prophages that express Shiga toxin (Stx) can be a threat to human health, with cattle being an important natural reservoir. In many countries the most severe pathology is associated with enterohaemorrhagic E. coli (EHEC) serogroups that express Stx subtype 2a. In the United Kingdom, phage type (PT) 21/28 O157 strains have emerged as the predominant cause of life-threatening EHEC infections and this phage type commonly encodes both Stx2a and Stx2c toxin types. PT21/28 is also epidemiologically linked to super-shedding (>103 cfu/g of faeces) which is significant for inter-animal transmission and human infection as demonstrated using modelling studies. We demonstrate that Stx2a is the main toxin produced by stx2a+/stx2c+ PT21/28 strains induced with mitomycin C and this is associated with more rapid induction of gene expression from the Stx2a-encoding prophage compared to that from the Stx2c-encoding prophage. Bacterial supernatants containing either Stx2a and/or Stx2c were demonstrated to restrict growth of bovine gastrointestinal organoids with no restriction when toxin production was not induced or prevented by mutation. Isogenic strains that differed in their capacity to produce Stx2a were selected for experimental oral colonisation of calves to assess the significance of Stx2a for both super-shedding and transmission between animals. Restoration of Stx2a expression in a PT21/28 background significantly increased animal-to-animal transmission and the number of sentinel animals that became super-shedders. We propose that while both Stx2a and Stx2c can restrict regeneration of the epithelium, it is the relatively rapid and higher levels of Stx2a induction, compared to Stx2c, that have contributed to the successful emergence of Stx2a+ E. coli isolates in cattle in the last 40 years. We propose a model in which Stx2a enhances E. coli O157 colonisation of in-contact animals by restricting regeneration and turnover of the colonised gastrointestinal epithelium.
Amongst 107 diarrheal cases studied a bacterial agent was isolated from 71 (66%) cases of which 60 (85%) were due to a single agent and the remaining 11 (15%) were of mixed infections. Enterotoxigenic Escherichia coli (ETEC) was isolated from 65 cases. Other pathogens isolated included Salmonella spp, Shigella spp and rotavirus. There was a higher isolation rate of ETEC from females and rotavirus from males. The infection rate was found to higher for the 0-2 year age group as compared to the 3-5 year age group. Amongst the ETEC isolated the STa 2 toxotype was the predominant type.
The epidermal mucus of fish contains antimicrobial agents that act as biological defence against disease. This study aims to identify antibacterial activity and protein concentration of epidermal mucus of Barbodes everetti, a Bornean endemic freshwater fish. The epidermal mucus was extracted with 3% acetic acid, 0.85% sodium chloride and crude solvents. The mucus activity against eight strains of human pathogenic bacteria, including Bacillus cereus ATCC 33019, Escherichia coli O157:H7, Listeria monocytogenes ATCC 7644, Pseudomonas aeruginosa ATCC 27853, Salmonella braenderup ATCC BAA 664, Salmonella typhimurium, Staphylococcus aureus ATCC 25933, and Vibrio cholerae, were tested. The acetic acid mucus extract of B. everetti was able to inhibit five strains of bacteria and show no activity toward E. coli O157:H7, B. cereus ATCC 33019 and L. monocytogenes ATCC 7644. Moreover, the highest protein concentration was quantified in crude extract, followed by aqueous and acetic acid extracts. This study provides a preliminary knowledge on the activity of epidermal mucus of B. everetti towards five out of the eight human pathogens tested, therefore it may contain potential sources of novel antibacterial components which could be further extracted for the production of natural antibiotics towards human-related pathogenic bacteria.
Shiga toxin-producing E. coli (STEC) is an important foodborne pathogen causing diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome in humans. STEC is an implicated in the vast majority of outbreaks, widely via consumption of STEC contaminated beef, as important vehicle of transmission of this organism to human. The E. coli O157:H7 serotype is traditionally identified by serological identification of the somatic antigen (O157) and structural flagella (H7). In this study, the bacteria were identified as STEC serotype O157:H7 with three primer pairs that amplified fragments of secD, rfbE and fliC genes in PCR assays. These primer pairs specifically amplified different sizes of target genes: a 244bp region of the E. coli diagnostic marker gene (secD); a 317bp region of the O157 lipopolysacharide (LPS) gene (rfbE); and a 381bp region of the H7 flagellin gene (fliC). The singleplex, duplex and triplex PCR assay developed in this study have a sensitivity limit at 2.8 x 103, 2.8 x 105 and 2.8 x 107 CFU/ml of E. coli O157:H7, respectively. Sensitivity to detect trace amount of E. coli O157:H7 DNA was reduced as the number of primer used was increased for competing to the same DNA template.
Antibacterial effect of modified sago starch-alginate edible film incorporating lemongrass oil at various concentrations was studied. Edible films were prepared from a mixture of modified sago starch and alginate. Lemongrass oil (0.1 - 0.4%, v/w) and glycerol (0 and 20%, w/w) were incorporated in the films to act as natural antimicrobial agent and plasticizer, respectively. The films were characterized for antibacterial activity against food pathogenic bacteria such as Escherichia coli O157:H7, Salmonella Enteritidis and Staphylococcus aureus. The edible film exhibited antibacterial activity against Escherichia coli O157:H7 and Salmonella Enteritidis by using agar diffusion assay method. For films tested against Escherichia coli O157:H7, the zone of inhibition increased significantly (p < 0.05) with addition of lemongrass oil at all levels both in the presence and absence of glycerol. The films also significantly (p < 0.05) inhibited the growth of Salmonella enteritidis only with 0.4% lemongrass oil (in the presence and absence of glycerol). However, the films containing lemongrass oil did not show any inhibition effect on Staphylococcus aureus.
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.
The antibacterial activity of solvent-extracted oil of noni (Morinda citrifolia L.), spinach (Spinacia oleracea L.), lady’s finger (Abelmoschus esculentus (L.) Moench), bitter gourd (Momordica charantia Linn.), and mustard (Brassica nigra L.) seed oils, and coconut (Cocos nucifera L.) oil, palm (Elaeis guineensis L.) mesocarp in hydrolyzed and unhydrolyzed form were determined in order to explore their potential usage as antibacterial agent. The hydrolysis process that was catalyzed by immobilized lipase of Rhizomucor miehei (RMIM) showed highest hydrolytic activity with 1.0 ml of added water volume except bitter gourd seed oil and palm mesocarp oil which has maximum hydrolytic activity with added water volume of 5 ml and 2.5 ml respectively. Before hydrolysis, all oil samples did not show inhibition ring zones (IRZ) on any of the tested bacteria strains (Salmonella typhimurium, Listeria monocytogenes and Escherichia coli O157:H7). Hydrolyzed lady’s finger and bitter gourd seed oil showed IRZ on all tested bacteria strains; hydrolyzed mustard seed oil on S. typhimurium and L. monocytogenes; hydrolyzed spinach seed oil and coconut oil on L. monocytogenes; hydrolyzed noni seed oil and palm mesocarp oil did not exhibit IRZ on any of the tested bacteria strains. Most of the hydrolyzed oil exhibit an inhibition activity that was different from their respective dominant fatty acids except noni seed oil and palm mesocarp oil.
Escherichia coli O157:H7 is a major food-borne pathogen that has resulted in numerous
outbreaks around the world. Widespread distribution of the organism in various ecological
niches impedes the control measures. This study aimed to detect and quantify E. coli O157:H7
in beef sold in wet markets and hypermarkets in Malaysia and to determine the risk of E. coli
O157:H7 infection linked to consumption of beef. The rfbO157 and flicH7 primers targeted on
somatic antigen (O157) and flagellar antigen (H7) respectively of E. coli O157:H7 was used for
the MPN-PCR method. A total of 99 beef samples were collected from local wet markets and
hypermarkets. The highest E. coli O157:H7 contamination rate was observed in beef samples
collected from wet markets (89.50%), whereas the contamination rate in hyper market A and B
were compratively low (35.35 and 20% respectively). However, the microbial load was highest
in the beef samples from hypermarket A (1100 MPN/g) while E. coli O157:H7 bacterial load
in beef samples from hypermarket B and wet market ranged from 3 to 93 MPN/g and 3 to 240
MPN/g, respectively. Using the Quantitative Microbial Risk Assessment (QMRA) approach
the risk was estimated incorporating the findings of the prevalence study and predictions
based on home storage, cooking and consumption patterns. Three different exposure pathways
were investigated to estimate the risk associated with contaminated beef and Monte Carlo
simulation was used to determine the level of uncertainty. The developed model predicated that
consumption of contaminated beef can be accountable for 1.83E+06 E. coli O157:H7 cases per
year in Malaysia. The reliability of the model, data gaps and further research needs, is discussed.
Through continuous improvement Quantitative Microbial Risk Assessment provides valuable
insight into controlling and prevention strategies.
Foodborne illness is recognized as an emerging infectious disease. The incidence of foodborne
infections is common and the majority cases are undiagnosed or unreported. Apart from some
diarrhea or minor gastrointestinal problem, some foodborne pathogenic microbes may cause
death, particularly to those people with weakened immune system. In this study, we have
developed a new fermented papaya beverage using symbiotic culture of yeast and acetic acid
bacteria under controlled biofermentation process. An in-vitro assessment of fermented papaya
beverage against few foodborne pathogenic microorganism was conducted to determine
its minimum bactericidal concentration (MBC>99). Three types of foodborne pathogen:
Escherichia coli O157, Salmonella enterica serovar Typhimurium ATCC 53648, Salmonella
enterica serovar Enteritidis (isolated from infectious chicken) were selected. From minimum
bactericidal concentration (MBC>99) assay, both fermented papaya pulp and leaves beverages
have shown 100% killing rate against three selected foodborne pathogenic microbes. Inversely,
non-fermented papaya pulp and leaves beverages indicated no inhibition at all. In fact, further
dilution of fermented papaya pulp and leaves beverages demonstrated different degree of
MBC>99 and brix value, but the pH value remained less than 3.5. These findings indicated
the combination of soluble solid compounds presents in both fermented papaya beverage and
product acidity play an important role in the inhibition of pathogenic microorganisms. The
preliminary promising results of this work have shown that the great potential of fermented
papaya beverages as a preventive measure to reduce the incidence of foodborne illness.
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.
The organic foods’ market is becoming one of the rapidly growing sections in agricultural economies in the world. During the last two decades, food-borne outbreaks associated with fresh produce have rapidly increased. E. coli O57:H7, the caustic agent of acute hemorrhagic diarrhea and abdominal cramps, is mainly associated with meat and poultry product outbreaks but frequent outbreaks linked to the consumption of vegetables have been reported. The aim of this study was to investigate prevalence of E. coli O157:H7 in some organic foods. A total of 230 organic food samples including four-winged bean, tomato, white radish, red cabbage, chinese cabbage, lettuce, cucumber and chicken form retailed groceries and supermarkets in Malaysia were investigated. Low prevalence of E. coli O157:H7 was detected in organic vegetables and chickens. The estimated quantity of E. coli O157:H7 in all samples ranged from 2400 MPN/g. The overall MPN/g estimate of E. coli O157:H7 in the samples from organic groceries was higher than supermarket with the maximum of >2400 MPN/g. Most of the samples from supermarket showed a minimum of