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.
Salmonella remains to be a major foodborne pathogen for animals and humans and is the
leading cause of foodborne infections and outbreaks in various countries. Salmonella Enteritidis
is one of the most frequently isolated serotypes in poultry and poultry products from human
food poisoning cases. It can cause mild to acute gastroenterititis as well as other common
food poisoning symptoms when infection takes place in human. Nucleic acid amplification
technologies such as Polymerase Chain Reaction (PCR) is a tool that is rapid and sensitive
for detection of bacterial pathogen. We report the successful detection of S. Enteritidis by
PCR in raw chicken meat artificially-contaminated with serial concentration of S. Enteritidis
using crude DNA extracts as DNA template. PCR primers, ENT-F and ENT-R targeted on sdfI
gene were used to amplify DNA region unique to S. Enteritidis with crude DNA extract of the
samples, yielded product with the size of 303 bp. These primers were specific to S. Enteritidis
when tested by in-silico simulation against genome database of targeted bacterial species and
confirmed in PCR as amplification bands were observed with S. Typhimurium, S. Polarum and
S. Gallinarum. The established PCR can detect as few as 9.4 X 101
CFU/ml of inoculated S.
Enteritidis concentration and proved that pre-enrichment effect have significant effect on PCR
detection by increasing 1000-fold of the sensitivity limit compared to the non pre-enriched
samples. The PCR technique indicated that it can be successfully coupled with pre-enrichment
step to offer advantage in routine screening and surveillance of bacterial contamination in food
samples.
Broiler part samples (80 fresh and 80 chilled) were examined for the prevalence and numbers of C. jejuni and C. coli by employing most-probable-number (MPN) and polymerase chain reaction (PCR) techniques. The prevalence of the bacteria was high where C. jejuni was detected in 92.5% fresh and 53.8% chilled samples while C. coli in 80.0% fresh and 56.3% chilled. The number of these bacteria in the positive fresh and chilled samples was from 3 to more than 2400 MPN/g and from 3 to 290 MPN/g, respectively. Antibiotic resistance test (using Kirby-Bauer disc diffusion method) on 10 C. jejuni and 13 C. coli isolates toward ampicillin, tobramycin, enrofloxacin, ciprofloxacin, tetracycline, cephalothin, gentamicin and norfloxacin revealed high resistance toward all antibiotics (20.0% - 100.0%). All isolates were resistant to at least two antibiotics. This study highlights the potential of multidrug-resistant C. jejuni and C. coli transmission to humans through fresh and chilled broiler parts. Consecutive studies with bigger sample sizes and covering all over Malaysia are warranted in future.
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.
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.
The introduction of new agricultural commodities and products derived from modernbiotechnology may have an impact on human and animal health, the environment and economiesof countries. As more Genetically Modified Organisms (GMO) enter markets worldwide, themonitoring of GMOs is being preferred for obvious reasons such as determination of seed purity,verification of non-GMO status of agricultural crops and fulfilling GMO labeling provisions, tomention a few. Numerous GMO analytical methods which include screening, identification andquantification have been developed to reliably determine the presence and/or amount of GMOin agricultural commodities, in raw agricultural materials and in processed and refined ingredients.The detection of GMOs relies on the detection of transgenic DNA or protein material. For routineanalysis, a good sample preparation technique should reproducibly generate DNA/protein ofsufficient quality, purity and yield while minimizing the effects of inhibition andcontamination.
The key sample preparation steps include homogenization, pretreatment, extraction andpurification. Due to the fact that analytical laboratories receive samples that are often processedand refined, the quality and quantity of transgenic target analyte (e.g. protein and DNA) frequentlychallenge the sensitivity of any detection method. With the development of GMO analysistechniques, the Polymerase Chain Reaction (PCR) technique has been the mainstay for GMOdetection, and the real-time PCR is the most effective and important method for GMOquantification. The choice of target sequence; for example a promoter, a terminator, a gene, or ajunction between two of these elements, is the single most important factor controlling the specificity of the PCR method. Recent developments include event-specific methods, particularlyuseful for identification and quantification of GM content. Although PCR technology has obvious
limitations, the potentially high degree of sensitivity and specificity explains why PCR in its various
formats, is currently the leading analytical technology employed in GMO analysis. Comparatively, immunoassays are becoming attractive tools for rapid field monitoring for the integrity of agricultural commodities in identity preservation systems, whereby non-specialised personnel can employ them in cost-effective manner. This review discusses various popular extraction methodologies and summarises the current status of the most widely used and easily applicable GMO analysis technologies in laboratories, namely the PCR and immunoassay technologies.
A total of 78 samples comprising different types of street foods, sold in different locations in Malaysia, were examined for the presence of Enterobacter cloacae. E. cloacae contamination was recorded in 9% of the samples examined. Tests for susceptibility to 12 different antibiotics showed that all were resistant to six or more antibiotics, but susceptible to chloramphenicol and gentamicin. Plasmids of four different sizes were detected from the three plasmid positive isolates. RAPD analysis using four primers yielded completely different banding patterns for all E. cloacae studied. In Malaysia, no published information on street foods in the epidemiological investigation of E.cloacae related disease is available. However, their occurrences have provided compelling evidence that the risk of disease transmission caused by E. cloacae through street foods is moderate.
Since the introduction of the molecularly imprinting technology (MIT) in 1970s, it becomes an emerging technology with the potential for wide-ranging applications in food manufacturing, processing, analysis and quality control. It has been successfully applied in food microbiology, removal of undesirable components
from food matrices, detection of hazardous residues or pollutants and sensors. Molecularly imprinted solid-phase extraction (MISPE) is the most common application so far. The review describes the methods of making the molecularly imprinted polymer systems, the application of the technology in food safety issues and the remaining challenges.
Studies indicate that bacterial cross-contamination occurs during food preparation where bacteria can retent on the food contact surfaces and cause illness. The study evaluated the adherence of Campylobacter spp. to cutting boards, blades of knives and hands after cutting chilled, raw broiler parts (thighs + drumsticks, wings and livers). The adherence to cucumber cuts that were cut using the unwashed boards and knives was also analyzed. Generally, utensils have higher mean of Campylobacter spp. retained to them (1.4-223.3 MPN/ml rinse) than hands (0.7-43.4 MPN/ml rinse); however, Mann-Whitney U test showed no significant differences in the bacterial numbers found among the different surfaces. The transfer rates of Campylobacter spp. from utensils to cucumber cuts varied from 0% to more than 100%. The bacteria detected could be from the utensils and cucumber contamination before purchase or due to other factors where further investigation is required. The possibility is there for Campylobacter to spread to contact surfaces during chilled broiler handling; therefore, utensils and hands involved should be washed thoroughly especially before ready-to-eat food preparation.
Application of surface plasmon resonance (SPR) biosensor in detection of genetically modified organism (GMO) is demonstrated. A total of four biotinylated probes namely Tnosb, P35Sb, LECb and TSQb were successfully immobilized onto the SA chip. Results analysis indicated that the SPR system with the sensor chip immobilized with the Tnosb, P35Sb, LECb and TSQb biotinylated probes potentially detect complementary standard fragments as low as 1 nM. Biospecific interaction analysis (BIA), employing surface plasmon resonance (SPR) and biosensor technologies provide easy, rapid and automatable approach in detection of GMOs. Short assay times, label free DNA hybridization reaction and no toxic compounds are required, i.e. ethidium bromide, and the reusability of the sensor surface are some of the factors that contribute to the general advantages of the surface plasmon resonance (SPR) biosensor system in detection of GMOs.
Local wood charcoal was used as the main component of the electrodes of an air-cathode microbial
fuel cell (air-cathode MFC) in current study. The air cathode was build with finely milled charcoal powder and cement plaster as binder; while anode was made up of a packed bed of charcoal granules. Mangrove estuary brackish water was inoculated in the anodic chamber as the fuel and a source of exoelectrogens. The constructed fuel cell was monitored by measuring the potential over time. The MFC generated a stable power density at 33mW/m2 (0.5V) under a load of 200Ω after 72 hours of operation. An open circuit voltage (OCV) of 0.7mV was obtained after 15 hours operating under open circuit. The result of power generation by the constructed fuel cell indicating that wood charcoal could be used as electrode in an MFC and that brackish water contained potential exoelectrogens. However, further investigation and modification is required to increase the performance of the fuel cell.
A total of 32 clinical strains of Vibrio cholerae, including members of the 01 and 0139 serogroup
were collected from Klang, Selangor; Penang Island; Samarahan, Sarawak and Miri, Sarawak in Malaysia. In general, all the isolates except the 0139 serotype expressed low resistance to all the antibiotics tested with their Multiple Antibiotic Resistance (MAR) indices ranged from 0.10 to 0.48. The presence of ctx gene that encoded the cholera toxin was confirmed in all these clinical isolates by polymerase chain reaction. The results from the RAPD-PCR were analyzed using the RAPDistance software (Version 1.04). From the dendrogram generated, two main groups were observed which were subdivided into two clusters each. The Selangor’s isolates and the 0139 Penang’s isolates formed one group whereas the Samarahan, Sarawak isolates and the Miri, Sarawak isolates made up the other group, thus delineating their different sources of origin based on their geographical location.