Phytochemicals belonging to the group’s phenols, terpenes, betalains, organosulfides, indoles and protein inhibitors are important components in fruits, vegetables, legumes, whole grains and nuts that have health promoting benefits and a variety of applications in food and pharmaceutical industries. Initially only a few of these important phytochemicals are produced commercially by chemical synthesis. However, recent developments in the field of biotechnology have provided metabolic engineering strategies that use microorganisms as cell factories for high production of these products. This review will discuss the general biosynthetic pathways, metabolic engineering and optimization strategies of functional phytochemicals that have received a lot of attention from investigators.
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 important role 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 of consumers and food handlers to practice proper cooking of the burger patties before the point of consumption, to reduce the risk of listeria infection.
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.
An awareness of Escherichia coli as a foodborne pathogen and illness causing bacterium has been increased among consumers. Moreover, there is demand for natural product in order to reduce synthetic product that can cause toxic to the human. In this study, antibacterial activity, in term of MIC, MBC and killing-time curve of methanolic extract of Boesenbergia rotunda have been tested against a standard E. coli ATCC 25922 and two E. coli isolated from milk products using Clinical and Laboratory Standard Institute (CLSI) methods. The results show that B. rotunda extract was susceptible to all E. coli strains. The MIC and MBC values of B. rotunda extract against E. coli ranged 0.019 mg/mL 2.5 mg/mL and 0.039 mg/mL – 5.0 µg/mL, respectively. Killing-time curves were constructed at concentrations of 0x MIC, 1/2x MIC, 1x MIC, and 2x MIC. All E. coli strains can be killed with concentration of 2x MIC after 2 hours. The results show that B. rotunda extract has potential antibacterial activity against E. coli.
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 cutoff 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 so.
The aim of this study is to compare the occurrence of thermophilic Campylobacter spp. in chicken retail at wet markets and hypermarkets. Campylobacter contaminations in chicken samples from wet market (70.7%) were comparatively lower than chicken samples sold in hypermarket (91.4%). Of the 77 Campylobacter isolates, 59 (76.6%) were identified as Campylobacter jejuni and 18 (23.4%) isolates were identified as C. coli. All Campylobacterisolates are multi-resistant to the antimicrobial agents. Most of the isolates were resistant to tetracycline (92.2%) and erythromycin (98.7%). This study concluded that chicken samples from both wet market and hypermarket were contaminated with Campylobacter, most of which are antimicrobial-resistant strains.
Several Norovirus cases due to consumption of green onions have been reported during recent years but reports on red onions are not found. Onions are one of the major tastes in Malaysian food which are sometimes consuming raw especially the green onion. Viral contamination in onions can occur due to planting condition and not properly prepared food. This situation can pose the human health risk. A method was developed to detect the Norovirus that might present on different type of onions. In this study, 60 samples were collected from local market. Elution by Tryptose Phosphate Glycine broth and concentration steps using negatively charge filter were applied to enhance the detection of virus in food due to low copies of virus on food surface. The viral RNA was extracted using Qiagen Rneasy Mini kit before further detection using One-step RT-PCR. The total incidence of Norovirus in green onion and red onion was 13.33% (4/30) and 3.33 % (1/30) respectively. This is the first report of the detection of Norovirus in red and green onions in Malaysia. Based on the results, it is concluded that this method is reliable to detect Norovirus on onions and vegetables surface and hence can be applied in the laboratories for routine or food borne outbreak investigation.
Legislation concerning the safety assessment and labelling of foodstuffs has been implemented in many countries. Consequential to a number of cases of food adulteration reported globally, a fast and reliable detection method for the food traceability is required in ensuring effective implementation of food legislation in a country. In this study, PCR-RFLP technique based on cyt b gene has been tested for its suitability for these purposes. This method combines the use of a pair of universal primer that amplifies a 359 bp fragment on the cyt b gene from meat muscle DNA and restriction enzyme analysis. Analysis of experimental beef frankfurter, minced beef, pork frankfurter and pork cocktail samples demonstrated the suitability of the assay for the detection of the beef (Bos taurus) and pork (Sus scrofa), but not applicable for some processed food, particularly detection of mackerel (Rasterelliger brachysoma), sardine (Saedinella Fimbriata) and tuna (Thunnus tonggol) origin in canned food. Commercial frauds through species mislabelling or misdescribed were not detected. The assay is demonstrated applicable for routine analysis of meat traceability of foodstuffs and legislation purposes, if sufficient availability of detectable mtDNA in the foodstuffs is ensured.
Successful DNA amplification is vital for the detection of specific DNA targets in feeds, and this in return depends on the ability of DNA extraction methods to produce good quality DNA. In this study, seven methods were compared for DNA extraction from feeds using quantitative polymerase chain reaction (PCR) of single copy maize (Zea mays) endogenous hmg (high mobility group) gene. Relative levels of hmg were used to evaluate the DNA quality. Spectrophotometer determination of DNA was also carried out to assess DNA yield and DNA purity, while electrophoretic analysis of genomic DNA extracts was carried out to investigate DNA integrity. The findings illustrate that the DNA extraction methods have a significant effect on DNA quality. Statistically, the Epicentre method extracted the highest DNA yield while the Wizard method had the lowest DNA yield with high DNA purity and integrity. However, the Wizard method recovered the most amplifiable DNA per reaction, indicating that template quality and integrity had greater influence over hmg amplification than DNA yield.
Caenorhabditis elegans (C. elegans) have been widely used as an infection model for mammalian related pathogens with promising results. The bacterial factors required for virulence in non-mammalian host C. elegans play a role in mammalian systems. Previous reported that Salmonella found in vegetable and poultry meat could be potential health hazards to human. This study evaluated the pathogenicity of various serovars of Salmonella enterica (S. enterica) that recovered from local indigenous vegetables and poultry meat using C. elegans as a simple host model. Almost all S. enterica isolates were capable of colonizing the intestine of C. elegans, causing a significant reduction in the survival of nematodes. The colonization of Salmonella in C. elegans revealed that the ability of S. enterica in killing C. elegans correlates with its accumulation in the intestine to achieve full pathogenicity. Using this model, the virulence mechanisms of opportunistic pathogenic S. enterica were found to be not only relevant for the interactions of the bacteria with C. elegans but also with mammalian hosts including humans. Hence, C. elegans model could provide valuable insight into preliminary factors from the host that contributes to the environmental bacterial pathogenesis scenario.
The aim of this study was to assess the most probable number-polymerase chain reaction (MPNPCR) technique for detection of Listeria monocytogenes in salad vegetables in comparison with reference EN ISO 11290-2 and Food Drug Administration Bacteriological Analytical Manual method using artificial and naturally contaminated samples. Based on recovery of L. monocytogenes from artificially contaminated samples, MPN-PCR showed a moderate correlation (R=0.67) between spiking concentration and microbial levels which was better than the FDA-BAM method (R=0.642) and ISO 11290-2:1998 method (R=0.655). With naturally contaminated samples, it was found that L. monocytogenes was detected in 25% of the vegetable samples using MPN-PCR; 15% of the samples by the FDA-BAM method and 8% of samples using ISO 11290-2:1998 method. Overall, MPN-PCR was found to be a rapid and reliable method that could facilitate the enumeration of L. monocytogenes in vegetables.
Bacillus cereus (B. cereus) isolates are toxigenic and can cause food poisoning. Cooked rice is
a potentially hazardous food, especially in tropical countries. The aim of this study was to determine the prevalence of B. cereus and B. thuringiensis in raw and cooked rice marketed in Selangor, Malaysia. In this research combination of Most Probable Number - Polymerase Chain Reaction (MPN-PCR) was used to detect gyrB gene in B. cereus and B. thuringiensis. Five local varieties of raw rice samples were negative for B. thuringiensis but all (100%) were positive for B. cereus. A total of 115 cooked rice samples (nasi lemak, nasi briyani, nasi ayam and nasi putih) were studied for the presence of B. cereus and B. thuringiensis. Nasi ayam was found to have the highest prevalence (100%) of B. cereus compared to nasi putih (76.2%) and nasi lemak (70.4%). Nasi briyani had the lowest prevalence (50%) of B. cereus. The frequencies of B. thuringiensis were found to be 10, 30 and 35.2 % in nasi putih and nasi ayam, nasi briyani and nasi lemak, respectively. The range of B. cereus and B. thuringiensis in the samples was from < 3 to 1100 MPN/g in different samples. Maximum number of B. cereus was observed in nasi lemak, nasi briyani and nasi putih ( > 1100 MPN/g) while nasi ayam showed less contamination (460 MPN/g) with B. cereus which was significantly different (P < 0.05 ) from others. The number of B. thuringiensis in nasi lemak, nasi briyani, nasi putih and nasi ayam were found to be >1100, 93, 9.2 and 3.6 MPN/g, respectively.
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.
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.
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.
Salmonella enterica is one of the major causes of bacterial foodborne infection. The aims of this study were to determine the antibiotic resistance and the genetic diversity of Salmonella enterica isolated from street foods and clinical samples and to understand the correlation between the prevalence of serovars and genotypes with their source (street food and clinical samples) and geographic origin (Negeri Sembilan, Malacca and Selangor in Peninsular Malaysia). The enterobacterial repetitive intergenic consensus (ERIC) PCR analysis distinguished the Salmonella isolates into 19 ERIC types, with one untypable isolate. Dendrograms were specifically constructed for the S. Biafra and S. Typhi isolates. Identical or very similar ERIC types among the S. Biafra isolates from street food samples indicate transmission of the S. Biafra among the street foods, as well as possible cross-contamination of the street foods. In addition, the identical or very similar ERIC types among the S. Typhi isolates from human samples examined suggest possible similarity in their source of infection. All the twenty four isolates were resistant to rifampin and none were resistant to cefuroxime. Most isolates displayed multiple resistances. Dendrogram of antibiotic resistances produced six clusters, with similarity levels between 18.8% and 100%. Generally, street food and clinical isolates tend to cluster apart. Dendrogram to cluster the antibiotic groups showed that they could be grouped according to classes based on mode of inhibition. The findings suggest that street food contaminated with drug-resistant Salmonella enterica can be an important factor in the continuous emergence of antibiotic resistant Salmonella enterica.
Polymerase chain reaction (PCR) technique was used to assay for the detection of specific genes in the genomes of the Aeromonas spp. isolated from environmental and shellfish sources, particularly aero and hlyA genes, responsible for aerolysin and hemolysin toxins production in this genus. The results showed that: (i) the 1500 bp amplicon of the hlyA gene was detected in 20/38 of the Aeromonas hydrophila, 13/38 of the A. caviae and 6/9 of the A. veronii biovar sobria isolates; (ii) the 690 bp amplicon of the aero gene was detected in 20/38 of A. hydrophila, 17/38 of A. caviae and 6/9 of A. veronii biovar sobria isolates; (iii) the nucleotide blast results of aerolysin gene sequences of the representative strains of A. hydrophila, A. caviae and A. veronii biovar sobria revealed a high homology of 94%, 95% and 95% with published sequences, respectively and ; (iv) the protein blast showed 97%, 94% and 96% homology when compared to the published sequences, respectively. The finding of A. hydrophila virulence genes in other members of the genus Aeromonas, may give a new perspective to the significance of these results. The method described here may be a useful detection tool to assist in further investigation of aero and hlyA genes in the genus Aeromonas, especially for food microbiologist.
The objectives highlighted in the present study were to determine the estimates of measurement uncertainty associated with PALCAM and CHROMagarTM Listeria media, to compare the efficacy between both media in relation to their measurement uncertainties. In addition, this study was carried out to assess the performance characteristics of spread and spiral plating procedures based on the comparison of Listeria monocytogenes enumeration between PALCAM and CHROMagarTM Listeria media. This work involved pure culture experiment, artificially contaminated samples experiment and naturally contaminated samples experiment. In pure culture experiment, PALCAM performance was relatively inferior to CHROMagarTM Listeria medium for both plating procedures. From the artificially contaminated samples, the results revealed that the values of repeatability, reproducibility, and measurement uncertainty at 95% confidence interval were comparable between both media under evaluation. However, at the level of naturally contaminated samples, the performance of CHROMagar
TM Listeria medium was refutable as the presence of high number of competitive microorganisms reduced the clarity of the medium. The current emphasis in ensuring microbiological safety which requires use of accredited laboratories has led to measurable need for measurement uncertainty to ensure reliability of test results for global acceptance.
This study aims to determine the presence of extended-spectrum (ESBL) in Klebsiella pneumoniae isolated from raw vegetables by genotypic and phenotypic method. Fifty-three K. pneumoniae isolates that were obtained by plating method were confirmed by PCR. Isolates obtained were screened for their resistance to selected antibiotics. Phenotypic tests for ESBL detection is basically to confirm production of ESBL, in this study two types of antibiotics used which were amoxycillin/clavulanic Acid (AMC, 30 µg) and ceftazidime (CAZ, 30 µg), The resistance were 5/53 (9.4%) and 1/53 (1.9%), respectively. However, it was interesting to observe that none of the K. pneumoniae isolates demonstrated the presence of any of the bla genes by using genotypic method except blaTEM gene has been detected in two isolates out of 53 isolates of K. pneumoniae in this research.
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-OD595 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 (