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.
The effect of 18 different chemicals, which included acidic compounds (sulfuric acid, chloridric acid, phosphoric acid, benzoic acid, citric acid, acetic acid), alkaline compounds (ammonia, sodium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide), salts (acetate ammonium, sodium bisulfite, sodium hydrosulfite, sodium chloride, sodium sulfate) and oxidising agents (hydrogen peroxide, sodium hypochlorite), on the reduction of aflatoxins B(1), B(2), G(1) and G(2) and ochratoxin A (OTA) was investigated in black and white pepper. OTA and aflatoxins were determined using HPLC after immunoaffinity column clean-up. Almost all of the applied chemicals showed a significant degree of reduction on mycotoxins (p < 0.05). The lowest and highest reduction of aflatoxin B(1), which is the most dangerous aflatoxin, was 20.5% ± 2.7% using benzoic acid and 54.5% ± 2.7% using sodium hydroxide. There was no significant difference between black and white peppers (p < 0.05).
Thirty-five veterinary isolates of Salmonella enteritidis were characterized by their susceptibility to 10 antimicrobial agents and by their plasmid profiles on agarose gel electrophoresis. All were susceptible to carbenicillin, chloramphenicol and nalidixic acid but 89% were resistant to tetracycline. When examined, 91% of the isolates harboured plasmids, with sizes ranging from 9.8 to 60 MDa. However, it was only possible to associate the presence of plasmids with tetracycline resistance; plasmids occurring in 90% of the tetracycline-resistant isolates. In conjugation experiments, with Escherichia coli K12 Nal(r) as recipient, the tetracycline resistance in three selected S. enteritidis isolates was observed to transfer at frequencies of 3.0×10(-3) to 1.0×10(-2)/donor cell. The concomitant transfer of a 56-MDa or 60-MDa plasmid in these three S. enteritidis isolates was also detected.
Vibrio parahaemolyticus is a gram negative bacterium and causes gastrointestinal illness in humans. In this study, twenty five out of fifty cockle samples from Padang, Indonesia produced purple colonies when they were grown on selective medium, CHROMagarTM Vibrio. Specific–PCR for toxR gene detection gave positive results in which a band with 368 base pairs size appeared on the gel for all the isolates that confirmed the presence of V. parahaemolyticus. In the virulence properties test, all the isolates showed negative results for tdh and trh genes detection. The results indicate that the isolates under this study do not contain virulence properties that correlate to the ability of infection and diseases, which means that they are nonpathogenic.
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.
An awareness of Escherichia coli as a foodborne pathogen and illness causing bacterium has been increased among consumers. Moreover, there is demand for natural product in order to reduce synthetic product that can cause toxic to the human. In this study, antibacterial activity, in term of MIC, MBC and killing-time curve of methanolic extract of Boesenbergia rotunda have been tested against a standard E. coli ATCC 25922 and two E. coli isolated from milk products using Clinical and Laboratory Standard Institute (CLSI) methods. The results show that B. rotunda extract was susceptible to all E. coli strains. The MIC and MBC values of B. rotunda extract against E. coli ranged 0.019 mg/mL 2.5 mg/mL and 0.039 mg/mL – 5.0 µg/mL, respectively. Killing-time curves were constructed at concentrations of 0x MIC, 1/2x MIC, 1x MIC, and 2x MIC. All E. coli strains can be killed with concentration of 2x MIC after 2 hours. The results show that B. rotunda extract has potential antibacterial activity against E. coli.
Molecular typing methods have been widely applied for many purposes. In this study, such methods were adopted as DNA fingerprinting tools to determine the origin and divergence of virulent Vibrio parahaemolyticus strains found in local seafood. Although not all strain carry virulent tdh and trh gene, increasing prevalence demands an effective fingerprinting scheme which can constantly monitor and trace the sources of such emerging food pathogens. By using ERIC-, RAPD-, and BOX-PCR methods, 33 Vibrio parahaemolyticus isolates from local Malaysia bloody clam (Anadara granosa) and Lala (Orbicularia orbiculata) with confirmed presence of tdh and trh gene were characterised, followed by determination of clonal relatedness among virulent strains using cluster analysis and discriminatory index. This study also involved application of Immunomagnetic Separation (IMS) Method which significantly improved the specificity of strain isolation. Cluster analysis using Unweighted Pair Group Mathematical Averaging (UPGMA) and Dice Coefficient shown clustering according to isolation food source, IMS level and haemolysin gene possessed. Nevertheless, different DNA fingerprinting methods generated different clustering at different similarity cut-off percentage, regardless as individual or as composite dendrograms. ERIC- and RAPD-PCR composite fingerprinting relatively shown the highest discriminatory index at following similarity cutoff percentage: 0.68 at 50%; 0.83 at 65%; and 0.93 at 75%. Discriminatory power increased with similarity cut-off percentage. However, result also suggested that BOX-PCR might be an effective fingerprinting tool, as it generated three clusters with no single-colony isolate at 70% similarity cut-off. This study not only achieved its objective to determine clonal relatedness among virulent strains from local seafood via characterisation, but also speculated the best possible combination of molecular typing methods to effectively do so.
The aim of this study is to compare the occurrence of thermophilic Campylobacter spp. in chicken retail at wet markets and hypermarkets. Campylobacter contaminations in chicken samples from wet market (70.7%) were comparatively lower than chicken samples sold in hypermarket (91.4%). Of the 77 Campylobacter isolates, 59 (76.6%) were identified as Campylobacter jejuni and 18 (23.4%) isolates were identified as C. coli. All Campylobacterisolates are multi-resistant to the antimicrobial agents. Most of the isolates were resistant to tetracycline (92.2%) and erythromycin (98.7%). This study concluded that chicken samples from both wet market and hypermarket were contaminated with Campylobacter, most of which are antimicrobial-resistant strains.
Pathogenic Vibrio parahaemolyticus is one of the leading causes of bacterial gastroenteritis in many countries. Among the strains examined, 36 RAPD-types were found when amplified with primers OPA8 and OPA10. The analysis shows the majority of V. parahaemolyticus isolates originated from seafood were branched into four major clusters at 18.2%, 20.7% 34% and 3.4% similarity levels. This suggests that there is potential for a single strain to be distributed widely within a population and there also potential for multiple contaminating strains of different clonal lineages to be present within the same population. Optimum temperature (37ºC) was the highest and stable formation of biofilm. The total percentage of biofilm formation at 37ºC was 33.33% for each of weak, moderate and strong biofilm producers. Room temperature produces 61.1% of weak biofilm producer, while 13. 89% for moderate biofilm producers and produce 25% of strong biofilm. While a total of 91.67% weak biofilm producers at 4ºC and 8:33% for room temperature and no growth of strong biofilm. Upon analysis, strong biofilm was tracked from the largest group at 37°C and room temperature which produce 27.27% of strong biofilm producer respectively. Interestingly, they are derived from cockles.
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.
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.
Listeria monocytogenes (L. monocytogenes) is a gram positive food-borne pathogen that is able to form biofilm on food factory surfaces. Formation of biofilm makes the bacteria much more resistance to environmental stresses such as disinfectant. The extracellular polymeric matrix (biofilm structure) which is mostly comprised of sticky extracellular polysaccharides (EPS) and proteins can protect bacteria in a harsh condition. The efficiency of four disinfectants on removing L. monocytogenes biofilm was investigated. Five concentration levels (100, 50, 25, 12.5, and 6.25%) of disinfectants were tested. In the microtitre assay, the optical density at 595 nm CV-OD595 value, was used to measure the amount of remained biofilm after 24 h. Results showed that disinfectants did not have significant effect on removing L. monocytogenes biofilm. Formation of L. monocytogenes biofilm significantly decreased the efficiency of disinfectants. Biofilm produced by strain number 9 showed higher resistance to disinfectant. Low concentrations (
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.
In this study, RAPD-PCR and ERIC-PCR were used to study the epidemiology of V. parahaemolyticus isolated from cockles in Padang, Indonesia. The Gold Oligo OPAR3 primer produced bands ranged from 1-8 with sizes from 0.2 – 5.0 kb and the Gold Oligo OPAR8 primer produced 1-7 bands with sizes 0.7 – 1.5 kb. Both primers produced twenty five RAPD patterns with a few isolates failed to produce any products. Based on phylogenetic dendrogram, all the isolates can be divided into 6 major clusters with similarity between 0 to 52%. For the ERIC primer, it produced bands ranged from 3-15 with sizes from 0.1 – 5.0 kb and twenty seven different ERIC patterns. Construction of the phylogenetic dendogram showed the isolates can be divided into 4 major clusters with similarity between 56 to 86%. The high diversity of both processes may be due to the multiple contamination sources of V. parahaemolyticus.
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.