Quantification of Listeria monocytogenes in salad vegetables by MPN-PCR

The aim of this study was to assess the most probable number-polymerase chain reaction (MPN-PCR) 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 artifcial and naturally contaminated samples. Based on recovery of L. monocytogenes from artifcially 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

Detection and quantification of Vibrio parahaemolyticus in vegetables and environmental samples at farm level

The purpose of this study was to detect and quantify total and pathogenic Vibrio parahaemolyticus from vegetables and environmental samples at the farm level in Cameron Highlands, Pahang, Malaysia. Most Probable Number (MPN) – Polymerase Chain Reaction (PCR) method was used to detect toxR, tdh and trh genes and to quantify their concentration in samples. Samples obtained were cabbage (20), carrot (10), cucumber (10), lettuce (31), tomato (18), manure (10), soil (12), surface swab (21) and water (14), with a total of 146 samples. Sampling locations involved were three vegetable farms, two packing houses and one loading bay. Based on the results, overall, 13.7% of samples were present with V. parahaemolyticus toxR (maximum concentration 1100 MPN/g), with the highest detection in cabbage (6%). Vibrio parahaemolyticus tdh was detected in 1.4% samples (maximum concentration 7.3 MPN/g), and V. parahaemolyticus trh could not be detected in any samples. No tdh and trh genes could be detected from the recovered isolates. This finding highlighted that vegetables and environmental samples could potentially be contaminated with V. parahaemolyticus which poses risk to consumers. This study could be useful in future food safety risk communication and management programmes

Biosafety of Vibrio parahaemolyticus from vegetables based on antimicrobial sensitivity and RAPD profiling

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 strain

Detection of Beta-Agonist Residues in Meat Using Enzyme Linked Immunosorbent Assay and Gas Chromatography Mass Spectrometry

The prevalence, type and concentration of beta-agonist residues in the liver and meat of three types of livestock animals i.e. goats, cattle and swine were studied using Gas Chromatography-Mass Spectrometry. Beta-agonist residues were only detected in swine with a prevalence of 16.6% in meat and 20% in liver sampled. The concentration of beta-agonist residues in the positive samples ranged between 1ng/g to 9ng/g. The performance of the multi-residue analysis method used was assessed through recovery studies and found to be varied among the beta-agonists wherein terbutaline showed the highest recovery values (78-83%) whereas salbutamol showed the lowest recovery values (22% -31%). The coefficient of variation (C.V.) had values between 1-12% which indicate acceptable variation for the method. In the second phase of this study, three ELISA test-kits, i.e. Randox ELISA beta-agonist test kit, Euro-Diagnostica beta-agonist test kit and Ridascreen beta-agonist test kit were evaluated for screening of meat and liver for beta-agonist residues in fortified and field incurred tissue samples. It was found that the Randox beta-agonist test kit was more suitable as a screening tool due to its accuracy, ease of use and lower cost. The test-kit was able to detect beta-agonists at the minimum level of detection as claimed by the suppliers. The performance of the method as assessed through recovery rates of the beta-agonists in fortified samples was satisfactory with a low coefficient of variation (1-3%). Reproducibility, as measured through the coefficient of correlation was also satisfactory. For field-incurred positive samples, the test kit showed a sensitivity of 100% and a low rate of false positives (less than 10%) for goat and cow tissues. However a high rates of apparent false positives (50%-65%) was obtained for tissues of swine. The third phase of the study evaluated the cross-reactivities of the antibodies within the three test kits to other veterinary drugs normally administered to swine. It was found that sulfachlorpyridazine; sulfamethaxine; penicillin G and amantidine cross-reacted with all three ELISA test kits to give a positive response

Microbiological Risk Assessment of Listeria Monocytogenes in Minimally Processed Vegetables

The purpose of this study was to carry out a microbiological risk assessment on Listeria. monocytogenes in vegetables that are consumed in the minimally processed state in Malaysia. The prevalence and number of Listeria spp. and L. monocytogenes were determined in raw vegetables from pre-harvest and retail level. Environmental samples of soil, water and animal manure were also obtained from vegetable farms. Characterisation of strains isolated from vegetables was carried out by phenotypic (antibiotic resistance) and genotypic (Random Amplification of Polymorphic DNA) methods. A kitchen simulation study was conducted to provide decontamination and cross-contamination data and information for estimation of the risk of acquiring listeriosis from consumption of minimally processed vegetables using a step-wise risk assessment as well as a stochastic approach using simulation software. At the retail level, 306 vegetable samples were examined over a one-year period (February 2008 to January 2009). The prevalence of Listeria spp. was 33.3% while the prevalence for L. monocytogenes was 22.5 %. L. monocytogenes was frequently found in yardlong bean (n=32) at 31.3% and Japanese parsley (n=33) at 27.2%. At the farm level, 50 vegetable samples were taken. Both Listeria spp and L. monocytogenes were detected in 16% and 6 % of the samples respectively. Among the environmental samples (n=94), Listeria spp. and L. monocytogenes were detected in 47.6% and 38.1 (n=21) of soil samples; 77.8% and 61.1% of manure samples (n=18); 25% and 12.5% of environmental swabs (n=40). It was not however detected in samples of the irrigation water (n=15). From the kitchen simulation study, it was found that the mean percent transfer rate from vegetables (n=45) to the wash water ranged from 32.4% to 60.2%, from wash water to cucumber was 24.9% to 66.3%; from vegetables to chopping board was 18.9 to 32.2%; from chopping board to cucumber was 5.4 to 75.3%. Washing of the vegetables in tap water caused a 0.3-log reduction of L. monocytogenes attached to the vegetables. Characterization of 71 strains isolated from the 306 samples of vegetables was done by Random Amplification of Polymorphic DNA (RAPD). It was found that the strains could be grouped into 6 clusters and 1 solitary isolate. This shows that the strains that have been isolated demonstrate genetic variability and is of importance to the Microbial Risk Assessment as the different strains would have variations in virulence and pathogenicity. In terms of antimicrobial susceptibility, the Multiple Antibiotic Resistance Index (MARI) of the strains ranged from 0.06 to 0.63. Only 14 % of the strains had MARI values higher than 0.2. MARI values less than 0.2 indicate strains from origins where antibiotics are seldom or never used. The importance of the antimicrobial study to the Microbial Risk Assessment would be that some of the strains exhibit multi-resistance to drugs used in the treatment of listeriosis. The estimation of risk of acquiring listeriosis from consuming minimally processed vegetables was done using a deterministic and stochastic approach. An exponential dose-response model was used to estimate the probability of illness in low risk and high risk group of consumers. The estimated mean risk per serving for salads was 1.42 x 10-5 per 100 000 population for the healthy low-risk population. For the high risk group, the risk estimate was 1.23 x 10-2 per 100 000 population for AIDS patients, 3.55 x 10-4 per 100 000 population for diabetics and 1.09 x 10-4 per 100 000 population for the elderly population respectively. The exposure assessment model was most sensitive to the input distribution describing the serving size. This implies that the serving size was the input parameter that most strongly influenced the risk and would be the primary control option in trying to reduce risk. In conclusion, a risk assessment has been conducted that can lay the foundation for more comprehensive studies as well as alert health authorities to be more vigilant about listeriosis especially among the vulnerable population