Isolation and Characterisation of Bacteria and Fungus from the Intestine of Sea Cucumber Acaudina molpadioides

Acaudina molpadioidesor locally known as ‘beronok’ is a high valued sea cucumber that is widely distributed in the muddy shores in the west coast Peninsular Malaysia and being consumed by the local people as traditional healthy delicacies. They are made into dishes and are usually consumed raw as ‘kerabu’. The aim of this study was to isolate and characterise the bacteria and fungus from the intestine of A. molpadioidesusing standard method for biochemical tests, safety assessments and molecular identification. A total of 100samples were collectedrandomlyfrom Pulau Langkawi, Malaysia and 1642 isolates were obtained from the intestine. Biochemical tests, safety evaluation and molecular identification were performed. Six strains (AM8h, AM47e, AM59a, AM67d, AM80d, and AM84d1) were selected for characterisation as they showed distinct morphology and from the biochemical tests. Further molecular identification showed the strains were identified as Priestia megaterium, Carnobacterium maltaromaticum, Bacillus tropicus, Staphylococcus saprophyticus, Bacillus cereus, and Yarrowia lipolytica(GenBank accession number: MZ947169, MZ934727, MZ947170, MZ934728, MZ934726, and MZ956769). The results indicated that both bacteria and fungus were presence in the intestine of A. molpadioides, hence there is a need for adequate measures in consuming this sea cucumber ra

Distribution of bacteriophages in food and environment samples

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

Isolation and characterization of six vibrio parahaemolyticus lytic bacteriophages from seafood samples

Vibrio parahaemolyticus is a foodborne pathogen that is frequently isolated from a variety of seafood. To control this pathogenic Vibrio spp., the implementation of bacteriophages in aquaculture and food industries have shown a promising alternative to antibiotics. In this study, six bacteriophages isolated from the seafood samples demonstrated a narrow host range specificity that infecting only the V. parahaemolyticus strains. Morphological analysis revealed that bacteriophages Vp33, Vp22, Vp21, and Vp02 belong to the Podoviridae family, while bacteriophages Vp08 and Vp11 were categorized into the Siphoviridae family. All bacteriophages were composed of DNA genome and showed distinctive restriction fragment length polymorphism. The optimal MOI for bacteriophage propagation was determined to be 0.001 to 1. One-step growth curve revealed that the latent period ranged from 10 to 20 min, and the burst size of bacteriophage was approximately 17 to 51 PFU/cell. The influence of temperature and pH levels on the stability of bacteriophages showed that all bacteriophages were optimally stable over a wide range of temperatures and pH levels. In vitro lytic activity of all bacteriophages demonstrated to have a significant effect against V. parahaemolyticus. Besides, the application of a bacteriophage cocktail instead of a single bacteriophage suspension was observed to have a better efficiency to control the growth of V. parahaemolyticus. Results from this study provided a basic understanding of the physiological and biological properties of the isolated bacteriophages before it can be readily used as a biocontrol agent against the growth of V. parahaemolyticus

Quantification and antibiotic susceptibility of Salmonella spp., Salmonella enteritidis and Salmonella typhimurium in raw vegetables (ulam)

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

Isolation of food-borne pathogen bacteriophages from retail food and environmental sewage

Bacteriophages are the viruses of bacteria and are widely distributed in the biosphere, exhibiting dramatic manifestations both in liquid cultures and on solid media. In this study, bacteriophages were isolated from different types of food (beef, chicken meats, cucumber, lettuce, clam, cockles and shrimp) and sewage samples using 6 reference pathogen strains (Salmonella Enteritidis, Salmonella Typhimurium, Campylobacter jejuni, Vibrio parahaemolyticus, Listeria monocytogenes and Escherichia coli). A total of 29 bacteriophage isolates were obtained and further examined for titer via agar overlay assay. The titers were determined within the range of 108 to 1011 PFU/mL. Our results showed that diverse of bacteriophages are naturally present in a variety of foods

Occurrence of Escherichia coli harbouring stx genes in popiah, a Malaysian street food

Irrespective of its health effects, street foods are very popular with the consumers. The main purpose of this research was to study the biosafety of Escherichia coli in popiah, a Malaysian street food sold at a roadside food stall and a restaurant in Sri Serdang, Selangor, Malaysia, using the combination of the most probable number (MPN)-Polymerase Chain Reaction (PCR) assay-plating on Eosin Methylene Blue (EMB) agar methods. Using these biomolecular methods, E. coli was detected in 12/15 (80%) and 11/15 (73%) of the collected samples from the roadside food stall and the restaurant respectively. The incidence of stx virulence-associated genes was detected in 1/15 (7%) among the E. coli isolated from samples taken from the roadside food stall while the E. coli isolated from the restaurant was 3/15 (20%). The density of E. coli ranged from 1100 MPN/g and the density of E. coli positive with stx genes was <3 to 53 MPN /g in samples from both the roadside food stall and the restaurant. The presence of the stx-positive E. coli in popiah are significant to risk assessments of food and epidemiological studies. Therefore, from the information obtained in this study, it is obvious that the screening for STEC markers in food samples would be useful for food safety survey

Antimicrobial resistance of Listeria monocytogenes and Salmonella Enteritidis isolated from vegetable farms and retail markets in Malaysia

Listeriosis and salmonellosis are the major foodborne illnesses worldwide. Over the last decade, increasing reports about the antibiotic resistance of Listeria monocytogenes and Salmonella from diverse sources have prompted public health concerns, especially in developing countries with over reliance or misuse of antibiotic drugs in the treatment of humans and animals. In this study, antibiotic susceptibility profiles of 58 L. monocytogenes and 12 Salmonella Enteritidis strains from vegetable farms and retail markets in Malaysia were tested by the standard disk diffusion method. Listeria monocytogenes isolates were found to exhibit 100% resistance to penicillin G. Also, high resistance patterns were observed for meropenem (70.7%) and rifampicin (41.4%). The multiple antibiotic resistance (MAR) index of L. monocytogenes isolates ranged from 0.11 to 0.56. Besides, the antibiogram results revealed that multidrug-resistant (MDR) S. Enteritidis were detected and all the S. Enteritidis isolates demonstrated resistance to at least four antibiotics. Ampicillin, amoxicillin, and trimethoprim failed to inhibit all the S. Enteritidis strains. Salmonella Enteritidis isolates also displayed high resistance to nalidixic acid (75.0%), trimethoprim-sulfamethoxazole (75.0%), and chloramphenicol (66.7%). Findings in this study indicated that vegetables could be potential sources of multidrug resistance of L. monocytogenes and S. Enteritidis, which can be a serious issue and a major concern for public health. Thus, there is a great need for surveillance programs in Malaysia to continuously monitor the antibiotic resistance profiles of important pathogens

Use of a lytic bacteriophage to control Salmonella Enteritidis in retail food

A Salmonella Enteritidis lytic bacteriophage designated as SE07 was isolated from retail meat samples. Electron micrograph revealed that phage SE07 belonged to family Podoviridae. Phage SE07 was relatively stable at the temperature ranging from 28 to 65 °C. Furthermore, it exhibited remarkable pH stability (between pH 4.0–11.0). The effectiveness of phage SE07 was determined in different food matrices (fruit juice, fresh eggs, beef and chicken meat) experimentally contaminated with S. Enteritidis. A significant reduction of S. Enteritidis population (about 2 log cycles) was obtained in fruit juice and fresh eggs after incubation at 4 °C for 48 h. Furthermore, within the same period, bacterial population was reduced by 2.1 and 2.0 log cycles on the bacteriophage treated beef and chicken meat samples, respectively. In this study, the obtained data suggested that phage SE07 might have potential uses against Salmonella food-borne infections

Simulation of decontamination and transmission of Escherichia coli O157:H7, Salmonella Enteritidis, and Listeria monocytogenes during handling of raw vegetables in domestic kitchens

Epidemiological data indicates that a large number of foodborne illnesses are attributed to cross-contamination during food preparation in the domestic kitchen. The objectives of this study were to evaluate the efficiency of household washing practices in removing Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Enteritidis on artificially contaminated lettuce and to determine the transfer rate of these three foodborne pathogens from contaminated lettuce to wash water, tomato, cabbage, and cutting boards during washing and cutting processes. Washing under the running tap water with scrubbing for 60 s was the most effective method in reducing pathogen populations by 1.86 to 2.60 log10 CFU/g. Also, final rinsing and scrubbing practices were found to enhance the efficiency of washing treatment. In this study, the transfer rates of S. Enteritidis, E. coli O157:H7, and L. monocytogenes from cutting board to cabbage and tomato via cutting process (17.5 to 31.7%) were higher (P<0.05) than from wash water to cabbage and tomato (0.8 to 23.0%) during washing treatment. Overall, our findings suggest that wash water and cutting board can be potential vehicles in the dissemination of foodborne pathogens. Therefore, there is a need to promote consumer awareness for proper handling practices in the kitchen to minimise the risk of foodborne infection

Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from short mackerels (Rastrelliger brachysoma) in Malaysia

Numerous prevalence studies and outbreaks of Vibrio parahaemolyticus infection have been extensively reported in shellfish and crustaceans. Information on the quantitative detection of V. parahaemolyticus in finfish species is limited. In this study, short mackerels (Rastrelliger brachysoma) obtained from different retail marketplaces were monitored with the presence of total and pathogenic strains of V. parahaemolyticus. Out of 130 short mackerel samples, 116 (89.2%) were detected with the presence of total V. parahaemolyticus and microbial loads of total V. parahaemolyticus ranging from 105 MPN/g. Prevalence of total V. parahaemolyticus was found highest in wet markets (95.2%) followed by minimarkets (89.1%) and hypermarkets (83.3%). Pathogenic V. parahaemolyticus strains (tdh+ and/or trh+) were detected in 16.2% (21 of 130) of short mackerel samples. The density of tdh+ V. parahaemolyticus strains were examined ranging from 3.6 to >105 MPN/g and microbial loads of V. parahaemolyticus strains positive for both tdh and trh were found ranging from 300 to 740 MPN/g. On the other hand, antibiotic susceptibility profiles of V. parahaemolyticus strains isolated from short mackerels were determined through disc diffusion method in this study. Assessment of antimicrobial susceptibility profile of V. parahaemolyticus revealed majority of the isolates were highly susceptible to ampicillin sulbactam, meropenem, ceftazidime, and imipenem, but resistant to penicillin G and ampicillin. Two isolates (2.99%) exhibited the highest multiple antibiotic resistance (MAR) index value of 0.41 which shown resistance to 7 antibiotics. Results of the present study demonstrated that the occurrence of pathogenic V. parahaemolyticus strains in short mackerels and multidrug resistance of V. parahaemolyticus isolates could be a potential public health concerns to the consumer. Furthermore, prevalence data attained from the current study can be further used to develop a microbial risk assessment model to estimate health risks associated with the consumption of short mackerels contaminated with pathogenic V. parahaemolyticus