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.
To date, cholera has cycle the world seven times through the seven pandemic cycles that has
affected tens of millions of people. The objective of this study was to determine the presence
and density as well as the antibiotic resistance profile of Vibrio cholerae isolated from catfish
(Pangasius hypohthalamus). From the combination of the Most Probable Number-Polymerase
Chain Reaction-plating on TCBS agar methods, V. cholerae was detected in 32 samples and
V. cholerae O139 was detected in 7 samples, with a density ranging between
Thirty-six strains of Escherichia coli isolated from animals in Bario, a remote area in Sarawak, Malaysia, were examined for presence of plasmid DNA and their susceptibility to nine antimicrobial agents. Of the total 36 isolates, five bovine and six canine isolates were found to contain plasmid DNA ranging in sizes from 2.6 to 70 kilobases. All were susceptible to chloramphenicol, erythromycin, gentamicin, nalidixic acid and neomycin but resistance to ampicillin (47%), erythromycin (19%), streptomycin (25%) and tetracycline (11%) was observed. Resistance was associated with carriage of a 47 kb (SC98), 70 kb, (SC133) and 56 and 4.6 kb (SC119) plasmids which were transmissible to the Escherichia coli K12 recipient. It is concluded that animals form a potential reservoir of R plasmids carrying E. coli in the study area.
Pennywort (Centella asiatica) is a herbaceous vegetable commonly consumed raw as ‘ulam’ or salad. Consumption of raw leafy green vegetables is one of the pathogenic mechanisms that could cause foodborne outbreaks. The aim of the present work was therefore to investigate the effect of pulsed light (PL) treatment at fluences of 1.5, 4.2, 6.9, 9.6, and 12.3 J/cm² on the microbiological and physical quality of pennywort stored at 4 ± 1°C. Escherichia coli (E. coli) were inoculated onto the pennywort leaves before being exposed to PL and viewed using scanning electron microscopy (SEM). PL fluences of 6.9, 9.6, and 12.3 J/cm² significantly reduced the microbial count; however, the highest inactivation was obtained by using fluences of 9.6 and 12.3 J/cm². The color of pennywort was not significantly affected by PL treatment applied at lower fluences of 1.5, 4.2, and 6.9 J/cm²; however, at higher fluence, 9.6 and 12.3 J/cm², the color was affected. PL at 1.5, 4.2, 6.9, and 9.6 J/cm² was able to retain the texture appearance of the leaves. To conclude, PL at 6.9 J/cm² showed the best fluence to reduce total aerobic mesophilic count while retaining the physical properties of pennywort leaves and extend the shelf life to about four days. The inactivation of E. coli population was significantly higher at PL fluence of 6.9 J/cm². It was observed that PL caused the destruction to the surface of E. coli’s cell membrane. The reductions of samples inoculated with E. coli were better than those achieved in native microbiota. Furthermore, the present work also demonstrated that PL treatment was able to reduce the microbial count on pennywort leaves.
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.
Fifty-nine isolates of Salmonella enterica subsp. enterica (S. enterica) isolated from indigenous vegetables, ‘selom’ (Oenanthe stolonifera) associated with 13 different serovars were obtained from Chemistry Department of Malaysia. The isolates encompass the common serovar, Salmonella enterica subsp. enterica serovar Weltevreden (S. Weltevreden) (39%) and Salmonella enterica subsp. enterica serovar Agona (S. Agona) (8.5%). Frequencies of the other 11 Salmonella serovars were ranged from 1.7% to 5.1%. All isolates were characterized by Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR), random amplified polymorphic DNA (RAPD), plasmid profiling and antimicrobial susceptibility testing. The results demonstrated ERIC-PCR, RAPD and composite analysis of both are suitable typing methods for S. enterica by demonstrating good discriminative ability and can be utilize as a rapid approach of comparing S. enterica isolates for epidemiological investigation. From this study, ERIC-PCR is exhibited lower discriminatory power when compare with RAPD. On the other hand, plasmid profiles yielded 32 profiles with molecular size ranging from 1129 bp to 17911 bp. Thirteen antimicrobial agents were included in this study and all isolates showed 100% (59/59) resistant to erythromycin and showed Multiple Antimicrobial Resistance (MAR) indexes ranging from 0.08 to 0.68. Dendrogram generated from antimicrobial resistance profiling exhibited poor discriminatory capability at serovar level. Although poultry still remain as the common reservoir for multidrug resistant (MDR) Salmonella. The isolation of 13 Salmonella serovars from selom that showed high MDR in this study is alarming. These results supported the notion that indigenous vegetable (selom) are gaining more antimicrobial resistance and could be potential health hazards.
Three restriction enzymes were used in Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) using the mitochondrial cytochrome b region to establish a differential diagnosis which detect and discriminate between three meat species: pork, cow and chicken. DNA was extracted from samples containing meat of a single animal such as raw pork (Sus scrofa domesticus), chicken (Gallus gallus) and cow (Bos taurus) as well as mixed samples of two species of animals in different ratios. The amplified 359 base pairs (bp) portion of the mitochondrial cyt b gene from pure or mixed samples in different ratios was cut using three different restriction enzymes resulting in species specific restriction fragment length polymorphism (RFLP). This technique proved to be extremely reliable in detecting the presence of low levels of target DNA obtained from a 0.25 mg component in a particular mixed meat sample. This revealed the cyt b region as highly conserved and consequently a good molecular marker for diagnostic studies. Thus, this technique can be applied to food authentication for the identification of different species of animals in food products.
This goal of this study was to investigate the presence of Vibrio cholerae in street food,
namely satar and otak-otak, using Loop-Mediated Isothermal Amplification (LAMP),
multiplex Polymerase Chain Reaction (mPCR) and conventional plating on Thiosulphate
Citrate Bile-Salt Sucrose (TCBS) agar methods. A total of 78 satar and 35 otak-otak were
purchased from different districts of Terengganu (Besut, Setiu, Kuala Terengganu and
Kemaman). V. cholerae was found in satar with LAMP (10.3%), mPCR (10.3%) and
plating (0%). No V. cholerae was found in otak-otak using the three methods. This might
be due to V. cholerae able to survive in satar after grilling due to its thickness which may
contribute to undercooking. This study concluded that low presence of V. cholerae in satar
and otak-otak can be detected by molecular methods but not the conventional plating
method. LAMP assay is a useful tool for rapid detection of pathogens in food due to its
simplicity, highly sensitive and visual interpretation capability. Though the prevalence of
V. cholerae was low in the samples, proper handling of this food will help in reducing the
risk of acquiring infection from V. cholerae in contaminated samples.
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.
Foodborne pathogens have become a constant threat to the consumer and food industry.
Reduce efficacy of antibiotics with emergence of resistant bacteria has limited the opportunities
for controlling pathogenic bacteria in food commodities and treating foodborne infections.
Bacteriophages can be a promising alternative for alleviate the risk of transmitting pathogenic
bacteria via food commodities. Therefore, this research was conducted to find distribution of
bacteriophages in diverse niches in order to identify suitable sources for isolating bacteriophages
to use controlling foodborne pathogens. Firstly bacterial strains were screened for lysogenic and
selected suitable host bacterial strains were used for isolating and determining bacteriophage titer
in fresh raw food and environmental samples. Eighteen different lytic bacteriophages effective
against Campylobacter, S. aureus, L. monocytogenes and E. coli were isolated from this study.
Bacteriophages titer was determined within range of 102
to 1010 PFU/mL and bacteriophages
were most frequently isolated from chicken (60%) samples. The isolated bacteriophages could
be potential candidates for controlling foodborne diseases.
Three strains of verotoxin-producing Escherichia coli isolated from patients with haemorrhagic colitis harboured plasmids ranging in size from 2.7 kb to 91.2 kb. Those plasmids ranging from 2.7 kb to 6.8 kb hybridized to Shiga-like toxin I and Shiga-like toxin II gene probes.
Nineteen strains of vancomycin-resistant Enterococcus faecium isolated from 10 of 75 (13.3%) tenderloin beef samples were examined for resistance to selected antibiotics, presence of plasmids, and genetic diversity by random amplification of polymorphic DNA analysis. All strains showed multiple resistant to the antibiotics tested. Multiple antibiotic indexing of the vancomycin-resistant E. faecium strains showed that all (100%) originated from high risk contamination environments where antibiotics were often used. Plasmids ranging in size from 1.5 to 36 megadalton were detected in 15 of 19 (79%) strains. Thus, three plasmid profiles and eight antibiotypes were observed among the E. faecium strains. A high degree of polymorphism was obtained by combining the results of the two primers used; with the 19 E. faecium strains being differentiated into 19 RAPD-types. These preliminary results suggest that RAPD-PCR has application for epidemiologic studies and that resistance patterns and plasmid profiling could be used as an adjunct to RAPD for the typing of E. faecium in the study area.
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
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.
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.
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.
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.
Antibiotic resistance in campylobacter is an emerging global public health problem after MRSA and VRE. Fluoroquinolone and macrolide resistance have been found to be more common in this world leading foodborne pathogen. A total of fifty-six isolates of Campylobacter jejuni obtained from raw vegetables
which are consumed as ulam (salad) in Malaysia, were tested with 12 antibiotics used clinically and
agriculturally. The resistance was determined using the disk diffusion method. Results were determined
by hierarchic numerical methods to cluster strains and antibiotics according to similarity profiles. Fifty
five C. jejuni isolates from different isolation sites were all clustered together into ten groups. This indicates that the commodities (raw salad vegetables/ulam) where the isolates originated might share a similar source of cross-contamination along the production route. All antibiotics tested correlated and there were four groupings reflecting their mode of actions. Generally, C. jejuni isolates were found to be highly resistant to erythromycin (91.1%) and tetracycline (85.7%). Both agents are popular antibiotics used clinically to treat bacterial infections. On the other hand, the C. jejuni isolates showed high percentage (80.4%) of resistance towards enrofloxacin, an extensively used antimicrobial agent in agriculture practices. This study showed that C. jejuni isolates were highly multi-resistance to as many as 10 antibiotics. Therefore, in terms of biosafety, the presence of antibiotic resistance strains in the food chain has raised concerns that the treatment of human infections will be compromised.
Salmonella has been reported to be presence both in raw and processed foods worldwide. In this study, the prevalence, quantification and antibiotic susceptibility of Salmonella isolated from raw vegetables or locally known as ulam such as asiatic pennywort (Centella asiatica (L) Urb), water dropwort (Oenanthe javanica (Blume) DC), long bean (Vigna sinensis EndL), and winged bean (Psophocarpus tetragonolobus (L) DC) obtained from retail markets in Selangor, Malaysia were carried out. From 96 samples tested, the overall prevalence of Salmonella spp. was 97.9%, Salmonella Enteritidis was 54.2% and Salmonella Typhimurium was 82.3% respectively. Samples were contaminated with Salmonella ranging from < 3 to 2400 MPN/g. Salmonella Enteritidis and Salmonella Typhimurium isolates obtained from the raw vegetables (ulam) were found to exhibit high resistance against ampicillin (100%), erythromycin (100%), amoxicillin/clavunic acid (81.3%), cephalothin (75%), streptomycin (50%) and ciprofloxacin (50%). All Salmonella isolates showed multi drug resistant (MDR) profile with each isolate being resistant to 3 or more antibiotics. The multiple antibiotic resistance (MAR) index of Salmonella isolates ranged from 0.27 to 0.55 for Salmonella Enteritidis and 0.27 to 0.82 for Salmonella Typhimurium. The presence of Salmonella on raw vegetables (ulam) and high antibiotic resistance isolates indicated that raw vegetables could be contaminated and thus imposes possible health risk to local consumers.
Listeria monocytogenes (L. monocytogenes) is an important foodborne pathogen which can cause foodborne listeriosis with high mortality rates especially in susceptible population groups such as pregnant women, elderly and immunocompromised individuals. The biosafety level of L. monocytogenes in chicken offal has becomes a great concern as chicken offal is a cheap source of protein and it is often served as side dishes in South East Asian countries. In Malaysia, the consumption of chicken offal has almost doubled from 5 g per capita per day in the early 1980s to 9 g per capita per day in 2009. In this study, risk assessment was conducted to estimate the risk of acquiring listeriosis from consumption of chicken offal in Malaysia. A microbial survey on the prevalence and concentration of L. monocytogenes in chicken offal were carried out in Selangor, Malaysia over a one-year period (November 2010 to October 2011). It was assumed that there were no seasonal changes in the prevalence and consumption pattern all year round. Assuming that 5.6 million people in Selangor, Malaysia consume a single serving (125 g) of chicken offal per week, it is estimated that in a year there could be 0.61 cases and 1.98 × 10-4 cases of listeriosis per 100,000 population of pregnant woman and immunocompromised individual, respectively. However, the potential for getting listeriosis among the healthy population was very low, only 1.39 × 10-8 cases per 100,000 population. This study demonstrated risk assessment model not only used as a tool to estimate the risk of acquiring illness but it can influence public health surveillance and providing data in setting appropriate level of protection.