Twelve strains of Escherichia coli O157:H7 were isolated from 9 of 25 beef samples purchased from retail stores in Malaysia. These strains produced Shiga toxin 2 with or without Shiga toxin 1 and had the eae gene and a 60-MDa plasmid. The antibiograms and the profiles of the arbitrarily primed PCR of the strains were diverse, suggesting that the strains may have originated from diverse sources.
Surface acoustic wave mediated transductions have been widely used in the sensors and actuators applications. In this study, a shear horizontal surface acoustic wave (SHSAW) was used for the detection of food pathogenic Escherichia coli O157:H7 (E.coli O157:H7), a dangerous strain among 225 E. coli unique serotypes. A few cells of this bacterium are able to cause young children to be most vulnerable to serious complications. Presence of higher than 1cfu E.coli O157:H7 in 25g of food has been considered as a dangerous level. The SHSAW biosensor was fabricated on 64° YX LiNbO3 substrate. Its sensitivity was enhanced by depositing 130.5nm thin layer of SiO2 nanostructures with particle size lesser than 70nm. The nanostructures act both as a waveguide as well as a physical surface modification of the sensor prior to biomolecular immobilization. A specific DNA sequence from E. coli O157:H7 having 22 mers as an amine-terminated probe ssDNA was immobilized on the thin film sensing area through chemical functionalization [(CHO-(CH2)3-CHO) and APTES; NH2-(CH2)3-Si(OC2H5)3]. The high-performance of sensor was shown with the specific oligonucleotide target and attained the sensitivity of 0.6439nM/0.1kHz and detection limit was down to 1.8femto-molar (1.8×10(-15)M). Further evidence was provided by specificity analysis using single mismatched and complementary oligonucleotide sequences.
The role of Escherichia coli H antigens in hydrophobicity and attachment to glass, Teflon and stainless steel (SS) surfaces was investigated through construction of fliC knockout mutants in E. coli O157:H7, O1:H7 and O157:H12. Loss of FliC(H12) in E. coli O157:H12 decreased attachment to glass, Teflon and stainless steel surfaces (p<0.05). Complementing E. coli O157:H12 ΔfliC(H12) with cloned wildtype (wt) fliC(H12) restored attachment to wt levels. The loss of FliCH7 in E. coli O157:H7 and O1:H7 did not always alter attachment (p>0.05), but complementation with cloned fliC(H12), as opposed to cloned fliCH7, significantly increased attachment for both strains compared with wt counterparts (p<0.05). Hydrophobicity determined using bacterial adherence to hydrocarbons and contact angle measurements differed with fliC expression but was not correlated to the attachment to materials included in this study. Purified FliC was used to functionalise silicone nitride atomic force microscopy probes, which were used to measure adhesion forces between FliC and substrates. Although no significant difference in adhesion force was observed between FliC(H12) and FliCH7 probes, differences in force curves suggest different mechanism of attachment for FliC(H12) compared with FliCH7. These results indicate that E. coli strains expressing flagellar H12 antigens have an increased ability to attach to certain abiotic surfaces compared with E. coli strains expressing H7 antigens.
Escherichia coli strains isolated from patients with diarrhea or hemolytic uremic syndrome (HUS) at Pusan University Hospital, South Korea, between 1990 and 1996 were examined for traits of the O157:H7 serogroup. One strain isolated from a patient with HUS belonged to the O157:H7 serotype, possessed a 60-MDa plasmid, the eae gene, and ability to produce Shiga toxin 1 but not Shiga toxin 2. Arbitrarily primed PCR analysis suggested that this strain is genetically very close to a O157:H7 strain isolated in Japan.
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.
Foodborne illness is a global burden that impacts a country politically, economically and
socio-economically. The severity of the burden can be unmeasurable as foodborne illness
is often an underestimated problem. In order to enlighten the burden, appropriate food
safety control measures should be taken. This study aimed to optimize a multiplex
Polymerase Chain Reaction (mPCR) detection method to identify foodborne pathogens
simultaneously. Six foodborne pathogens namely, Salmonella spp., Escherichia coli O157,
Vibrio parahaemolyticus, Vibrio cholerae, Listeria monocytogenes and Campylobacter
spp., were targeted in the mPCR detection method. Each mPCR parameter was tested and
the outcome was analysed to obtain a successful mPCR protocol to detect the targeted
foodborne pathogens. The amplified PCR products showed that the optimized mPCR
protocol will be a potential rapid diagnostic tool in foodborne pathogen detection.
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.
The mode of action and activities of guava leaf extracts against various food pathogens were studied. The killing kinetics, viability and cell leakage of Kocuria rhizophila, Salmonella typhimurium, Listeria monocytogenes and Escherichia coli O157:H7, measured after exposure to guava methanolic extracts (GME) revealed a significantly higher (p≤0.05) release of bacterial nucleic acids, K+ ions and protein than that of untreated microbes, indicating disruption of the bacterial membrane. GME caused a significantly higher (p≤0.05) release of RNA in gramnegatives compared to gram-positives. GME caused a relatively small but significant release of pyrines and pyrimidines in all organisms investigated. GME probably disrupted the integrity of the Gram-negative microorganism lipopolysaccharide (LPS) layer. Unlike all the other microorganisms tested, E. coli O157:H7, demonstrated the lowest protein leakage, the highest K+ leakage, the highest pyrines and pyrimidines leakage within the first 10 min of extract exposure, but the lowest after 30 minutes, which may indicate their good homeostasis ability or adaptability. Understanding the mode of action of this flavonoid rich guava leaf extract, would help develop it as an alternative biodegradable and safe, antimicrobial for food and medicine, and as a by-product of the guava industry.
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.
Foodborne diseases are mainly caused by bacterial contamination which can lead to severe diarrhea. This study aimed to detect the presence of Shiga toxin-Producing Escherichia coli O157, Escherichia coli non-O157 and virulence gene in raw vegetables. The samples were purchased from wet market and hypermarket in Selangor. The detections were carried out by using the combination methods of Most Probable Number-Polymerase Chain Reaction (MPNPCR). A total of 37(18.5%) samples were found to be contaminated by STEC. Out of these 37 isolates, four (10.8%) of the isolates were E. coli O157 while 33(89.2%) were E. coli nonO157. However, there was no E. coli O157:H7 detected in all the samples. The occurrence of Shiga toxin-Producing E. coli in edible raw vegetables samples suggests the importance of this pathogen in vegetables. Therefore, more studies are required to remove this pathogen from vegetables.
The main source of E. coli 0157:H7 is cattle, but recent studies showed high percentage of outbreaks
contributed by contaminated water. The occurrence of E. coli O157:H7 in environmental water samples poses a potential threat to human health. The aim of this study was to establish a protocol for the detection of the pathogen E. coli O157:H7 and E. coli virulence genes (eaeA, rfbE, hly, stx1, and stx2) in a multiplex PCR protocol using six specific primer pairs. The target genes produced species-specific amplicons at 625 bp, 397 bp, 296 bp, 166 bp, 210 bp and 484 bp for E. coli O157:H7 (fliCh7 gene) and virulence genes (eaeA, rfbE, hly, stx1, and stx2) respectively. The results obtained show that the established PCR protocol is suitable for a rapid and specific analysis of the pathogenic E. coli O157:H7 in environmental water samples for the assessment of microbiological risks.
The use of polyclonal antibody (IgG) has recently been applied to the detection of bacteria. We developed a lateral flow assay (LFA) strip using a specific IgG in combination with colloidal gold on a nitrocellulose membrane. A conjugate, gold-anti Escherichia coli (E. coli) O157:H7 IgG was developed in this study for the detection of E. coli O157:H7 in food. The 40 nm in size of colloidal gold nanoparticles was used to conjugate the anti-E. coli O157:H7 IgG. The optimal concentration, 12.0 µg/ml of the anti-E. coli O157:H7 IgG was determined by standard curve generated in titration method. The serially diluted of E. coli O157:H7 was detected and clearly visualized on the LFA strip as low as 106 CFU/ml (result not shown). The IgG raised in rabbit have shown specific binding capacity against E. coli O157:H7. No other genus of bacteria, including Salmonella typhimurium, Listeria monocytogenes and Campylobacter jejuni reacted to the IgG. The LFA strip could also detect E. coli O157:H7 in different food samples matrices after 18 h-enrichment and this result were in accordance with the results of the polymerase chain reaction (PCR) and colony count.
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.
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
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
Ludwigia octovalvis is an aquatic plant widely distributed throughout the tropical and sub-tropical regions. It is commonly consumed as a health drink and traditionally used for treating various ailments such as dysentery, diarrhea, diabetes, nephritisn and headache. No information is available on its in vivo antibacterial activity against an important foodborne pathogen, Shiga toxin producing Escherichia coli O157:H7.