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.
During 2009 to 2010, a total of 408 blood samples collected from malaria patients in Ranong (149) and Yala (259) Provinces, Thailand were investigated for Plasmodium spp using microscopic examination. There are no statistical differences in the prevalence of P. falciparum and P. vivax in samples collected from Ranong and Yala (46% vs 52%, and 54% vs 45%, respectively). Single nucleotide polymorphism of codon 86 in pfmdr1 (encoding P. falciparum multidrug resistance protein 1) was investigated among 75 samples of P. falciparum and 2 samples of P. knowlesi. A pfmdr1 N86Y mutation was detected in 1 out of 29 samples and 45 out of 46 samples obtained from Ranong and Yala Provinces, respectively. It is interesting that pfmdr1 was detected in two P. knowlesi DNA samples obtained previously from Ranong Province which was 99% homologous to pfmdr1 obtained from falciparum parasites in the same area but the mutation was not observed. The difference in multidrug resistance protein in Plasmodium obtained from those two border areas of Thailand will be of use in monitoring drug resistance in these border regions of the country.
A total of 57 Vibrio vulnificus isolates from coastal water were characterized for their antimicrobial resistance, plasmid profiles and were typed by the PCR-based techniques: a random amplification of polymorphic DNA (RAPD) method and the enterobacterial repetitive intergenic consensus sequence (ERIC) method. All isolates were susceptible to chloramphenicol, nalidixic acid, tetracycline and trimethoprim-sulfamethoxazole. Fifty-one isolates were resistant to one or more of the other antibiotics tested. Plasmid analysis indicated that only 18 isolates carried small plasmids of 1.6 to 16 megadaltons. Analysis of the RAPD and ERIC DNA fingerprints of the V. vulnificus isolates with Gel Compare and cluster analysis software revealed significant genetic heterogeneity among these isolates. The combination of RAPD and ERIC analysis allowed us to distinguish all isolates. Thus, the combination of the two techniques is recommended for epidemiological investigation.
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.
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.
Little is known on the biosafety level of Vibrio spp. in freshwater fish in Malaysia. The purpose of this study was to investigate the prevalence and concentration of Vibrio spp. and V. parahaemolyticus in
freshwater fish using the Most Probable Number-Polymerase Chain Reaction (MPN-PCR) method. The study was conducted on 150 samples from two types of freshwater fish commonly sold at hypermarkets, i.e. Pangasius hypophthalmus (catfish) and Oreochromis sp. (red tilapia). Sampling was done on the flesh, intestinal tract and gills of each fish. The prevalence of Vibrio spp. and V. parahaemolyticus was found to be 98.67% and 24% respectively with higher percentages detected in samples from the gills followed by the intestinal tract and flesh. Vibrio spp. was detected in almost all red tilapia and catfish samples. V. parahaemolyticus was detected in 25% of the catfish samples compared to 22.6% of red tilapia fish. The density of Vibrio spp. and V. parahaemolyticus in the samples ranged from 0 to 1.1x107 MPN/g. Although the maximum value was 1.1x107 MPN/g, most samples had microbial loads ranging from 0 to >104 MPN/g. The outcome on the biosafety assessment of Vibrio spp. and V. parahaemolyticus in freshwater fish indicates another potential source of food safety issues to consumers.
The development of the biological synthesis of nanoparticles using microorganisms or plant extracts plays an important role in the field of nanotechnology as it is environmentally friendly and does not involve any harmful chemicals. In this study, the synthesis of silver nanoparticles using the leaves extract of Chinese tea from Camellia sinensis is reported. The synthesized nanoparticles were characterized using UV-vis spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy. The XRD analysis shows that the synthesized silver nanoparticles are of face-centered cubic structure. Well-dispersed silver nanoparticles with an approximate size of 4 nm were observed in the TEM image. The application of the green synthesized nanoparticles can be used in many fields such as cosmetics, foods, and medicine.
Twenty-five and three strains of Escherichia coli O157:H7 were identified from 25 tenderloin beef and three chicken meat burger samples, respectively. The bacteria were recovered using the immunomagnetic separation procedure followed by selective plating on sorbitol MacConkey agar and were identified as E. coli serotype O157:H7 with three primer pairs that amplified fragments of the SLT-I, SLT-II and H7 genes in PCR assays. Susceptibility testing to 14 antibiotics showed that all were resistant to two or more antibiotics tested. Although all 28 strains contained plasmid, there was very little variation in the plasmid sizes observed. The most common plasmid of 60 MDa was detected in all strains. We used DNA fingerprinting by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) to compare the 28 E. coli O157:H7 strains. At a similarity level of 90%, the results of PFGE after restriction with XbaI separated the E. coli O157:H7 strains into 28 single isolates, whereas RAPD using a single 10-mer oligonucleotides separated the E. coli O157:H7 strains into two clusters and 22 single isolates. These typing methods should aid in the epidemiological clarification of the E. coli O157:H7 in the study area.
Four Vibrio cholerae O139 Bengal strains isolated from surface water were characterized by antibiotic resistance, plasmid profile, presence of cholera toxin gene and random amplification of polymorphic DNA (RAPD) analysis. All four strains exhibit multiple resistance towards the antibiotics tested with a multiple antibiotic resistance index of 0.5-0.66, and harboured a 2.0 MDa non-conjugative plasmid. The Vibrio cholerae O139 Bengal were positive for the cholera toxin gene. Antibiotyping and random amplification of polymorphic DNA analysis with four primers proved to be useful in discriminating the isolates. RAPD proved to be more sensitive. These results reveal that there is significant genetic diversity among the Vibrio cholerae O139 Bengal strains studied.
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.
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.
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.
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