Microbial Risk Assessment of Thermophilic Campylobacter SPP. in Raw Vegetables from Farm to Table

The first aim of this study was to determine the prevalence and number of thermophilic Campylobacter spp. (Campylobacter jejuni, Campylobacter coli and Campylobacter fetus) in raw vegetables (ulam) at pre-harvest and retail level, soil and animal manure in an organic and a traditional vegetable farm. The biosafety of Campylobacter jejuni was assessed by phenotypic (antibiotic resistance) and genotypic (presence of virulent and toxin genes) as well as RAPD-PCR characteristics of the strains isolated from vegetables. A kitchen simulation study was conducted to provide decontamination and crosscontamination data and information for estimation of the risk of acquiring campylobacteriosis from consumption of ulam using a step-wise risk assessment. The prevalence of thermophilic Campylobacter spp. in 309 (number of samples) raw vegetables purchased from two supermarkets and a wet market was relatively high, 29% to 68%. Campylobacter jejuni (25.5% to 67.7%) and C. coli (21.6% to 65.7%) were predominant species isolated; while C. fetus was only detected in two samples (1.9%) from one of the supermarkets. Only 18.3% of Campylobacter-MPN-PCR positive samples were recovered by enumeration-plating method indicating that routine enumeration-plating methods has very low recovery rate for Campylobacter spp. from vegetables. The study was extended to investigate the level of contamination with Campylobacter spp. in vegetables farms. A total of 172 samples of animal manure (n=18), soil (n=60), irrigation water (n=45) and vegetables (n=49) samples were collected from both an organic and a conventional vegetable farm. The organic vegetable farm (20.5%) was found to have a higher prevalence of Campylobacter spp. compared to the vegetable farm practicing conventional farming (2%). The low contamination level in the conventional farm was most probably due to the bed-burning practice and the use of composted manure in the farm. Campylobacter coli was not detected in all the samples from both farms. Soil (30.4%) and animal manure (57.1%) sampled from the organic vegetable farm were found to harbor Campylobacter spp. and C. jejuni. However, none of the irrigation water samples examined from both farms were positive for Campylobacter spp. RAPD-PCR fingerprinting and antibiotic resistance profiling indicated that multi-resistant Campylobacter spp. might be wide-spread in the study area. Clustering of C. jejuni isolates based on RAPD-PCR profiles suggested that some isolates from different sources and locations were genotypically closely related. Clusters A2, A3, A5 and A6 comprised C. jejuni strains isolated from raw vegetables in the supermarkets and a wet market. All clusters including B1 and B3, which comprised strains only from supermarkets, were actually consisted of isolates from different sources. The isolates showed multi-resistance to as many as 10 antibiotics tested. All the isolates were detected to carry the virulent genes, cadF, ceuE and flaA. However, toxin genes detection indicated only 16.1% and 10.7% of the isolates carry cdtB and cdtC toxin genes, respectively; while none of the isolates carry cdtA gene. The potential of raw salad vegetables as a vehicle in C. jejuni transmission was demonstrated by a step-wise risk assessment. Based on the assumptions used in the step-wise risk assessment, the annual number of cases of campylobacteriosis acquired from the consumption of ulam is estimated to be 4992/100,000 of Malaysian population, assuming that 10% of Campylobacter spp. infection translates into illness. However, the risk estimate was predicted to reduce to 175/100,000 if an extra blanching step was incorporated into the model. In conclusion, there is an immediate need for further investigation to look into the wide-spread problem of Campylobacter spp. in ready-to-eat foods, such as salad and ulam, in Malaysia

Performance of air-cathode microbial fuel cell with wood charcoal as electrodes

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 fnely 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 modifcation is required to increase the performance of the fuel cell

Optimization of multiplex PCR conditions for rapid detection of Escherichia coli O157:H7 virulence genes

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, stx 1, and stx 2) 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 (fliC h7 gene) and virulence genes (eaeA, rfbE, hly, stx 1, and stx 2) 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

Antibiotic susceptibility and genotyping by RAPD of Campylobacter jejuni isolated from retailed ready-to-eat sushi

A study to determine the antibiotic sensitivity pattern and genotyping using RAPD-PCR was performed on 50 C. jejuni isolated from sushi retailed in different supermarkets. With less than half of the isolates susceptible to the antibiotics tested, resistant to two or more antibiotics were observed in most of the isolates. The banding patterns obtained from RAPD-PCR revealed that no predominant clone exists and the bacterial population is rather diverse. Hence, the resistance of the C. jejuni to different classes of antibiotic as well as their diverse genotypes suggests that these C. jejuni isolates were generated from different sources in the contaminated supermarkets where sushi were retailed. Our data showed that C. jejuni can be an important reservoir for resistance genes and that study with comprehensive collections of samples are urgently required to establish better measures to reduce or eliminate the risk from antibiotic resistant and pathogenic bacteria originating from minimally processed ready-to-eat food

Assessment of knowledge, attitude and practices concerning food safety among restaurant workers in Putrajaya, Malaysia

The aim of this study is to evaluate the knowledge, attitudes and practices on food safety held by food handlers working in restaurants in Putrajaya, Malaysia. Inappropriate food handling is the main factor contributing to food borne disease outbreaks. A total of 127 food handlers were randomly selected from 23 restaurants and the data collected in September 2013 through self-administered questionnaires were analyzed using the SPSS version 16. Generally knowledge of food safety was good with the mean score 90.3 ± 7.787. However, respondents lacked knowledge about the hazards of reheating cooked food (75.1 ± 25.662) and the safe temperature of cooked food (71.9 ± 33.548). Knowledge differed significantly by age groups (F=2.530; p=0.044). Respondents had positive attitudes about food safety, with a mean score of 93.9 ± 6.813, although there were significant differences between trained and untrained workers (t=2.406; p=0.018); Malaysian ethnic groups (F=2.502; p=0.034); Malaysians and non-Malaysians (t=3.273; p=0.001) and due to differences in education levels (F=6.057; p=0.003). The mean score for practice was 92.9 ± 7.647 and again there were significant differences related to education levels (F=1.345; p=0.003), gender (t=-2.120; p=0.036) and ethnicity of Malaysian workers (F=2.502; p=0.034). A strong relationship was found between knowledge and attitudes about food safety (r=0.266; p=0.002) and between knowledge and practice (r=0.203; p=0.022). In conclusion, this study suggests that food handlers in Putrajaya restaurants displayed good knowledge, a positive attitude and an excellent practices regarding food safety. Yet results showed the food workers still lack basic knowledge of food safety, particularly related to reheated food and safe temperatures for cooked food

Assessment of Knowledge, Attitude and Practices Concerning Food Safety among Restaurant Workers in Putrajaya, Malaysia

The aim of this study is to evaluate the knowledge, attitudes and practices on food safety held by food handlers working in restaurants in Putrajaya, Malaysia. Inappropriate food handling is the main factor contributing to food borne disease outbreaks. A total of 127 food handlers were randomly selected from 23 restaurants and the data collected in September 2013 through self-administered questionnaires were analyzed using the SPSS version 16. Generally knowledge of food safety was good with the mean score 90.3 ± 7.787. However, respondents lacked knowledge about the hazards of reheating cooked food (75.1 ± 25.662) and the safe temperature of cooked food (71.9 ± 33.548). Knowledge differed significantly by age groups (F=2.530; p=0.044). Respondents had positive attitudes about food safety, with a mean score of 93.9 ± 6.813, although there were significant differences between trained and untrained workers (t=2.406; p=0.018); Malaysian ethnic groups (F=2.502; p=0.034); Malaysians and non-Malaysians (t=3.273; p=0.001) and due to differences in education levels (F=6.057; p=0.003). The mean score for practice was 92.9 ± 7.647 and again there were significant differences related to education levels (F=1.345; p=0.003), gender (t=-2.120; p=0.036) and ethnicity of Malaysian workers (F=2.502; p=0.034). A strong relationship was found between knowledge and attitudes about food safety (r=0.266; p=0.002) and between knowledge and practice (r=0.203; p=0.022). In conclusion, this study suggests that food handlers in Putrajaya restaurants displayed good knowledge, a positive attitude and an excellent practices regarding food safety. Yet results showed the food workers still lack basic knowledge of food safety, particularly related to reheated food and safe temperatures for cooked food. Keywords: Knowledge, Attitude, Practices, food safety, Putrajaya, Malaysia

Prevalence of Campylobacter spp. in retailed ready-to-eat sushi

The prevalence of Campylobacter spp. in retailed sushi were examined using the techniques of polymerase chain reaction (PCR) in combination with most probable number (MPN) to quantify the bacteria in 150 samples obtained from three supermarkets. The average prevalence of Campylobacter spp. in retailed sushi was 26.6% with 32%, 16% and 32% from supermarket I, II and III, respectively. Campylobacter jejuni was found to be the predominant species in retailed sushi with 82.49% of all Campylobacter spp. positive samples. Campylobacter coli was not detected in all samples. The maximum MPN number of Campylobacter spp. in retailed sushi purchased from supermarket I, II and III ranged from 3.6-11.0 MPN/g, 9.4->1100 MPN/g and 27-1100 MPN/g, respectively. The isolation of C. jejuni from a variety of ready-to-eat retail sushi may indicate that these products can act as possible vehicles for the dissemination of food-borne campylobacteriosis

Food court hygiene assessment and food safety knowledge, attitudes and practices of food handlers in Putrajaya

This cross sectional study aimed to explored the pattern of socio-demographic distribution, to assess the level of KAP of food safety; and the relationship with the level of premise cleanliness in the food courts at Putrajaya. Distribution of food handlers socio-demographic profile was Malaysian (62.0%), male (70.4%), working experienced in food industry (82.0%) and attended food handler training (85.0%). The mean age was 28.7 years and 85.4% having income not less than RM 1,500 monthly. 78.5% of the food handlers at educational level were found as primary/secondary school. 15.0% of the respondents had not attended the food sanitation training. The findings reveal that food handlers' KAP were high with a mean percentage score more than 79.0%.The majority of the food courts in Putrajaya had consistently moderate level of cleanliness (63.5%) with the mean of 83.03%. Only 27.4% of the food courts were in the level of clean situation (>89% of premise cleanliness score) and 9.1% were not in the clean condition (<70% of premise cleanliness score)

Characterization of Enterobacter cloacae isolated from street foods

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

Detection of virulence genes and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) analysis among raw vegetables isolates of Campylobacter jejuni

A total of 20 (n=20) raw vegetable isolates of Campylobacter jejuni were examined for their virulence genes and genetic diversity by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) analysis. All raw vegetable isolates of C. jejuni were encoded cadF and ceuE genes. None of the C. jejuni isolates was cdtA positive. Whereas, 12 (12/20) isolates were positive for cdtB and 6 (6/20) were positive for cdtC. All the 20 isolates of C. jejuni were subtyped and they produced 18 ERIC-PCR profiles namely E1 to E18. Dendrogram performed from cluster analysis showed that the 20 isolates were grouped into 2 major clusters, cluster I and II indicating the high level of local geographical genetic variation. The detection of virulence genes among C. jejuni strains isolated from raw vegetables raised concern on the threat of campylobacteriosis