Vibrio cholerae still represents a significant threat to human health worldwide despite the advances in hygiene, consumer knowledge, food treatment and food processing. In Malaysia, statistics in year 2009 have shown that among the food and water borne diseases, food poisoning has the highest incidence rate of 36.17 per 100,000 populations and with a mortality rate of 0.01 per 100,000 populations. In this study, 22 seafood samples comprising of fish, squid, crustacean and mollusks purchased from wet market and supermarket were analyzed. The Most Probable Number (MPN) and real time PCR was used to enumerate the Vibrio cholerae in seafood sample. The results showed that MPN-real time PCR of the samples from wet market had a maximum of >1100 MPN/g compare to 93 MPN/g enumerated from the MPN plate. The MPN-real time PCR in the samples from supermarket indicated 290 MPN/g as compared to 240 MPN/g enumerated from the MPN plate. The standard curves showed that there was a good linear correlation between the Ct values. The minimum level of detection of Vibrio cholerae standard DNA at targeted gene was 3 x 10-5 ng/μl.
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.
Bacteriophages are ubiquitous in our world, mainly in the oceans, soil, the water and food
we consume. They can be used efficiently in modern biotechnology, as well as alternatives
to antibiotics for many antibiotic resistant bacterial strains. Phages can be used as vehicles
for vaccines both DNA and protein, for the detection of pathogenic bacterial strain, as biocontrol
agents in agriculture and food industry. This review outlines the properties as well
as the influence of different external physical and chemical factors like temperature and
acidity on phage persistence. A better understanding of the complex problem of phage
sensitivity to external factors may be useful for other researchers working with phages.
Furthermore, the applications of bacteriophages were described in this paper as well.
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.
The prevalence of Salmonella in chicken and beef sold in retails outlets in Malaysia was
determined by analysing 312 raw beef and chicken meat samples including their processed
products. Samples purchased from supermarkets, butcher shops and wet market, which being
classified into raw, minced and processed chicken and beef. A total of 86 (27.6%) samples were
found positive for Salmonella spp., with chicken meat samples (40.4%) showed greater presence
compared to beef (15.4%). Highest presence of Salmonella were detected from wet market
samples (35.4%), followed by supermarket (26.9%) and butcher shop (21.3%). The prevalence
of Salmonella were higher in unpacked chicken meat (84.8%), followed by unpacked beef
(27.8%). Salmonella serovars were identified as S. Enteritidis, S. Hadar, S. Dublin, S. Anatum,
S. Stanley, S. Gallinarum, S. Choleraesuis and S. Typhimurium. Detection of 8 Salmonella
serovars showed possibilities of cross contamination in various sources either at slaughtering
house, processing plant or until storage at retails level. Improper cooking method on meats and
hygiene practices prior to consume should be avoided in order to ensure food safety before
ingestion.
Bacteriophages are the viruses of bacteria and are widely distributed in the biosphere, exhibiting
dramatic manifestations both in liquid cultures and on solid media. In this study, bacteriophages
were isolated from different types of food (beef, chicken meats, cucumber, lettuce, clam,
cockles and shrimp) and sewage samples using 6 reference pathogen strains (Salmonella
Enteritidis, Salmonella Typhimurium, Campylobacter jejuni, Vibrio parahaemolyticus, Listeria
monocytogenes and Escherichia coli). A total of 29 bacteriophage isolates were obtained and
further examined for titer via agar overlay assay. The titers were determined within the range
of 108
to 1011 PFU/mL. Our results showed that diverse of bacteriophages are naturally present
in a variety of foods.
Salmonella has caused foodborne illnesses globally and it has been a rising threat on fresh produce. The objective of this study was to determine the prevalence and concentration of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium in freshly prepared fruit juice sold at hawker stalls. Analysis was conducted by employing most probable number-polymerase chain reaction (MPN-PCR). A total of 50 freshly prepared fruit juices were examined and the prevalence of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium in the fruit juices were 34%, 20% and 10%, respectively, with an estimated microbial load varying from 0 to 42 MPN/g. Of the five different fruits, carrot juice had the highest prevalence of Salmonella spp. (60%) and Salmonella Typhi (40%). However, Salmonella Typhimurium was detected in apple (30%), orange (10%) and starfruit juice (10%). Factors contributing to the presence of Salmonella were cross-contamination and poor sanitation practice. Besides, negligence on temperature and storage time also led to the growth of Salmonella. Proper monitoring and risk assessment are needed in order to establish control measures to ensure the quality and safety of fruit juices in Malaysia.
This study aimed to determine the biofilm formation ability by Salmonella Typhi on cucumber, mango and guava surface, as well as to determine the relationship between time contact and biofilm formation. Crystal violet assay was performed to quantify the biofilm formation based on the value of optical density at 570 nm of the destaining crystal violet at the specific interval time. The result showed that the attachment of the bacterial cells on the fresh produce surface increased with the contact time. The readings of OD570at time 12 h for cucumber, mango and guava surfaces were 0.824, 0.683 and 0.598, respectively, indicating that the biofilm formation by Salmonella Typhi on different fresh produce surface varied with time. Since the result showed that Salmonella Typhi formed biofilm on fresh produce surfaces, hygienic practice from farm to fork including handling, processing, distribution and storage of the fresh produce should be of concern.
Klebsiella pneumoniae (K. pneumoniae) is one of the most important members of Klebsiella genus in Enterobacteriacae family, which is responsible for pneumonia (the destructive lung inflammation disease). Vegetables are known as source of contamination with K. pneumonia. Raw vegetables are usually consumed in salads and other dishes. The aim of this study was to investigate the occurrence of K. pneumoniae in raw vegetables marketed in Malaysia. Two hundred commonly used salad vegetables (lettuces, parsley, cucumber, tomato and carrot) from hypermarkets and wet markets were investigated for presence of K. pneumoniae using Most Probable Number-Polymerase Chain Reaction (MPN-PCR). K. pneumoniae was found to be significantly more frequent (100%) and (82.5%) in lettuce and cucumbers, respectively. K. pneumoniae contamination was lowest in carrot samples (30%). All samples were contaminated with K. pneumoniae ranging from
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.
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.
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.
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.