Caenorhabditis elegans (C. elegans) have been widely used as an infection model for mammalian related pathogens with promising results. The bacterial factors required for virulence in non-mammalian host C. elegans play a role in mammalian systems. Previous reported that Salmonella found in vegetable and poultry meat could be potential health hazards to human. This study evaluated the pathogenicity of various serovars of Salmonella enterica (S. enterica) that recovered from local indigenous vegetables and poultry meat using C. elegans as a simple host model. Almost all S. enterica isolates were capable of colonizing the intestine of C. elegans, causing a significant reduction in the survival of nematodes. The colonization of Salmonella in C. elegans revealed that the ability of S. enterica in killing C. elegans correlates with its accumulation in the intestine to achieve full pathogenicity. Using this model, the virulence mechanisms of opportunistic pathogenic S. enterica were found to be not only relevant for the interactions of the bacteria with C. elegans but also with mammalian hosts including humans. Hence, C. elegans model could provide valuable insight into preliminary factors from the host that contributes to the environmental bacterial pathogenesis scenario.
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.
Little is known on the biosafety level of Vibrio spp. in freshwater fish in Malaysia. The purpose of this study was to investigate the prevalence and concentration of Vibrio spp. and V. parahaemolyticus in
freshwater fish using the Most Probable Number-Polymerase Chain Reaction (MPN-PCR) method. The study was conducted on 150 samples from two types of freshwater fish commonly sold at hypermarkets, i.e. Pangasius hypophthalmus (catfish) and Oreochromis sp. (red tilapia). Sampling was done on the flesh, intestinal tract and gills of each fish. The prevalence of Vibrio spp. and V. parahaemolyticus was found to be 98.67% and 24% respectively with higher percentages detected in samples from the gills followed by the intestinal tract and flesh. Vibrio spp. was detected in almost all red tilapia and catfish samples. V. parahaemolyticus was detected in 25% of the catfish samples compared to 22.6% of red tilapia fish. The density of Vibrio spp. and V. parahaemolyticus in the samples ranged from 0 to 1.1x107 MPN/g. Although the maximum value was 1.1x107 MPN/g, most samples had microbial loads ranging from 0 to >104 MPN/g. The outcome on the biosafety assessment of Vibrio spp. and V. parahaemolyticus in freshwater fish indicates another potential source of food safety issues to consumers.
Nineteen strains of vancomycin-resistant Enterococcus faecium isolated from 10 of 75 (13.3%) tenderloin beef samples were examined for resistance to selected antibiotics, presence of plasmids, and genetic diversity by random amplification of polymorphic DNA analysis. All strains showed multiple resistant to the antibiotics tested. Multiple antibiotic indexing of the vancomycin-resistant E. faecium strains showed that all (100%) originated from high risk contamination environments where antibiotics were often used. Plasmids ranging in size from 1.5 to 36 megadalton were detected in 15 of 19 (79%) strains. Thus, three plasmid profiles and eight antibiotypes were observed among the E. faecium strains. A high degree of polymorphism was obtained by combining the results of the two primers used; with the 19 E. faecium strains being differentiated into 19 RAPD-types. These preliminary results suggest that RAPD-PCR has application for epidemiologic studies and that resistance patterns and plasmid profiling could be used as an adjunct to RAPD for the typing of E. faecium in the study area.
Salmonellosis is an important public health problem and causes large economic losses in the poultry industry. The emergence of molecular technology has opened various possibilities for constructing tailor-made proteins, particularly protein E from bacteriophage PhiX174 for the
production of bacterial ghosts (BGs) applied in vaccines purposes. In the present study, the plamdaPRcI-Elysis plasmid carrying the PhiX174 lysis gene E and thermo-sensitive lamda PR-cl857 regulatory system was constructed. Two Salmonella Enteritidis (SE-2 and SE- 4) and one Salmonella Typhimurium (ST-4) isolates were able to uptake the lysis plasmid via electrotransformation. Generation of ghosts was enhanced by increasing the incubation temperature up to 42˚C. Cell viability of SE-2, SE-4 and ST-4 decreased ranging in log 2.7 to log 4.1 cycles after lysis induction. Moreover, SE-2 and SE-4 exhibited the earliest reduction of CFU after 3 h of incubation. Our results may provide a promising avenue for the development of Salmonella BGs vaccines.
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 testedby 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 multidrugresistant (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.
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.
Peanuts are widely consumed as the main ingredient in many local dishes in Malaysia. However, the tropical climate in Malaysia (high temperature and humidity) favours the growth of fungi from Aspergillus section Flavi, especially during storage. Most of the species from this section, such as A. flavus, A. parasiticus and A. nomius, are natural producers of aflatoxins. Precise identification of local isolates and information regarding their ability to produce aflatoxins are very important to evaluate the safety of food marketed in Malaysia. Therefore, this study aimed to identify and characterize the aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi in peanuts and peanut-based products. A polyphasic approach, consisting of morphological and chemical characterizations was applied to 128 isolates originating from raw peanuts and peanut-based products. On the basis of morphological characters, 127 positively identified as Aspergillus flavus, and the other as A. nomius. Chemical characterization revealed six chemotype profiles which indicates diversity of toxigenic potential. About 58.6%, 68.5%, and 100% of the isolates are positive for aflatoxins, cyclopiazonic acid and aspergillic acid productions respectively. The majority of the isolates originating from raw peanut samples (64.8%) were aflatoxigenic, while those from peanut-based products were less toxigenic (39.1%). The precise identification of these species may help in developing control strategies for aflatoxigenic fungi and aflatoxin contamination in peanuts, especially during storage. These findings also highlight the possibility of the co-occurrence of other toxins, which could increase the potential toxic effects of peanuts.
Bacteriophages are ubiquitous in our world, mainly in the oceans, soil, the water and food
we consume. They can be used efficiently in modern biotechnology, as well as alternatives
to antibiotics for many antibiotic resistant bacterial strains. Phages can be used as vehicles
for vaccines both DNA and protein, for the detection of pathogenic bacterial strain, as biocontrol
agents in agriculture and food industry. This review outlines the properties as well
as the influence of different external physical and chemical factors like temperature and
acidity on phage persistence. A better understanding of the complex problem of phage
sensitivity to external factors may be useful for other researchers working with phages.
Furthermore, the applications of bacteriophages were described in this paper as well.
Strains of Aeromonas hydrophila isolates from skin lesions of the common freshwater fish, Telapia mossambica, were screened for the presence of plasmid DNA by agarose gel electrophoresis and tested for susceptibility to 10 antimicrobial agents. Of the 21 fish isolates examined, all were resistant to ampicillin and sensitive to gentamycin. Most isolates were resistant to streptomycin (57%), tetracycline (48%) and erythromycin (43%). While seven of 21 isolates harboured plasmids, with sizes ranging from 3 to 63.4 kilobase pair (kb), it was only possible to associate the presence of a plasmid with antibiotic resistance (ampicillin and tetracycline) in strain AH11. Both the plasmid and the associated antimicrobial resistance could be transferred to an Escherichia coli recipient by single-step conjugation at a frequency of 4.3 x 10(-3) transconjugants per donor cell.
E. coli O157:H7 is associated with life threatening diseases such as hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Raw milk is considered a high risk food as it is highly nutritious and serves as an ideal medium for bacterial growth. The aim of this study was to investigate the prevalence of E. coli O157:H7 in raw cow, goat and buffalo milk samples. MPN-PCR method targeting the major virulence rfbE gene and fliCH7gene of E. coli O157:H7 was used. Total of 177 raw milk samples were collected from local dairy farms in the state of Selangor, Malaysia. The highest prevalence of E. coli O157:H7 was found in raw cow milk (18.75%). E. coli O157:H7 was detected in 7.32% and 3.57% of raw goat and buffalo milk, respectively. The estimated quantity of E. coli O157:H7 in raw cow, goat and buffalo milk ranged from
The organic foods’ market is becoming one of the rapidly growing sections in agricultural economies in the world. During the last two decades, food-borne outbreaks associated with fresh produce have rapidly increased. E. coli O57:H7, the caustic agent of acute hemorrhagic diarrhea and abdominal cramps, is mainly associated with meat and poultry product outbreaks but frequent outbreaks linked to the consumption of vegetables have been reported. The aim of this study was to investigate prevalence of E. coli O157:H7 in some organic foods. A total of 230 organic food samples including four-winged bean, tomato, white radish, red cabbage, chinese cabbage, lettuce, cucumber and chicken form retailed groceries and supermarkets in Malaysia were investigated. Low prevalence of E. coli O157:H7 was detected in organic vegetables and chickens. The estimated quantity of E. coli O157:H7 in all samples ranged from 2400 MPN/g. The overall MPN/g estimate of E. coli O157:H7 in the samples from organic groceries was higher than supermarket with the maximum of >2400 MPN/g. Most of the samples from supermarket showed a minimum of
This study aims to determine the presence of extended-spectrum (ESBL) in Klebsiella pneumoniae isolated from raw vegetables by genotypic and phenotypic method. Fifty-three K. pneumoniae isolates that were obtained by plating method were confirmed by PCR. Isolates obtained were screened for their resistance to selected antibiotics. Phenotypic tests for ESBL detection is basically to confirm production of ESBL, in this study two types of antibiotics used which were amoxycillin/clavulanic Acid (AMC, 30 µg) and ceftazidime (CAZ, 30 µg), The resistance were 5/53 (9.4%) and 1/53 (1.9%), respectively. However, it was interesting to observe that none of the K. pneumoniae isolates demonstrated the presence of any of the bla genes by using genotypic method except blaTEM gene has been detected in two isolates out of 53 isolates of K. pneumoniae in this research.
Foodborne diseases are mainly caused by bacterial contamination which can lead to severe diarrhea. This study aimed to detect the presence of Shiga toxin-Producing Escherichia coli O157, Escherichia coli non-O157 and virulence gene in raw vegetables. The samples were purchased from wet market and hypermarket in Selangor. The detections were carried out by using the combination methods of Most Probable Number-Polymerase Chain Reaction (MPNPCR). A total of 37(18.5%) samples were found to be contaminated by STEC. Out of these 37 isolates, four (10.8%) of the isolates were E. coli O157 while 33(89.2%) were E. coli nonO157. However, there was no E. coli O157:H7 detected in all the samples. The occurrence of Shiga toxin-Producing E. coli in edible raw vegetables samples suggests the importance of this pathogen in vegetables. Therefore, more studies are required to remove this pathogen from vegetables.
Presence of Norovirus in food can cause viral gasteroenteritis. Recently, lots of reports relating to Norovirus in food have been published. Special attention must be paid to the raw foods as they are not subjected to further heat treatment. In this study, pegaga, kesum, tauge and ulam raja (popular salad vegetables in Malaysia) were investigated for Norovirus. A total of 32 samples from each type of salad vegetables were purchased from local market and analyzed using One-step RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) for both genogroups namely Norovirus Genogroup I and Genogroup II. Results showed that tauge had the highest contamination with Norovirus Genogroup I (15.6%) comparing to pegaga (9.4%), kesum (12.5%)
and ulam raja (0%). Samples were free from Norovirus Genogroup II. The study showed that raw vegetables are high-risk foods and can be contaminated with Norovirus.
Four Vibrio cholerae O139 Bengal strains isolated from surface water were characterized by antibiotic resistance, plasmid profile, presence of cholera toxin gene and random amplification of polymorphic DNA (RAPD) analysis. All four strains exhibit multiple resistance towards the antibiotics tested with a multiple antibiotic resistance index of 0.5-0.66, and harboured a 2.0 MDa non-conjugative plasmid. The Vibrio cholerae O139 Bengal were positive for the cholera toxin gene. Antibiotyping and random amplification of polymorphic DNA analysis with four primers proved to be useful in discriminating the isolates. RAPD proved to be more sensitive. These results reveal that there is significant genetic diversity among the Vibrio cholerae O139 Bengal strains studied.
Appropriate and safe antibacterial agents able to decontaminate meat surfaces have long been big concern of meat industry. In an attempt to manage beef carcass contamination, spray wash treatments utilizing three concentrations (1, 1.5 and 2%) of acetic, lactic, propionic and formic acids were performed to evaluate their efficacy in reducing numbers of Escherichia coli O157:H7 and Staphylococcus aureus on meat tissues. The procured beef pieces of freshly slaughtered animals were decontaminated with hot water and then inoculated with E. coli O157:H7 and S. aureus individually which then were spray washed with organic acids separately. The total plate count of the treated samples showed that the populations of bacteria decreased after being exposed to organic acids. Spray wash of formic acid resulted in the highest reduction of both bacterial species on meat surface. Significantly, higher log reductions were obtained for S. aureus than E. coli O157:H7. It was concluded that organic acids are highly effective in decontaminating meat surfaces and organic acids are shown to be safe, simple, efficient, and cheap modality of meat decontamination which can be highly recommended for industrial scales.
The aims of this communication were to study characterization of serogroups among Salmonella isolates and the relationship of antimicrobial resistance to serogroups. Multiple antimicrobial resistance (MAR) was performed on 189 Salmonella enterica isolates associated with 38 different serovars that were recovered from poultry and four types of indigenous vegetables.
In vitro experiments were undertaken to study the adhesion and colonization to tracheal mucosa, lung and aorta explants from freshly killed rabbits of two different strains of Pasteurella multocida. Serotype A:3 (capsulated, fimbriae +, haemagglutination -, dermonecrotic toxin -) isolated from a rabbit with rhinitis, and serotype D:1 (non-capsulated, fimbriae +, haemagglutination +, dermonecrotic toxin +) isolated from a dead rabbit with septicaemia, were used. When the explants were observed under the scanning electron microscope, the type D strain was highly adherent to trachea and aorta explants compared to the type A strain. Adhesion to lung explants was best achieved by the type A strain after 45 min incubation, but after 2 h incubation no significant difference was observed between the strains. Our data indicate that the presence of fimbriae and the absence of capsule seem to enhance the adherence of P. multocida type D strain to tracheal tissue. The capsular material of P. multocida type A strain and the toxin of the type D strain seem to influence the adherence to lung tissue in rabbit. Adhesion of strain D to aorta may indicate the expression of receptors on the endothelium to that strain and may also explain the ability of certain strains to cause septicaemia.
Sixteen 8- to 9-week-old Pasteurella multocida-free rabbits were divided into two equal groups. Eight rabbits in one group were inoculated intranasally with P. multoida type A:3. The other eight were inoculated intranasally with phosphate-buffered saline and used as controls. Nasal swabs taken before and after inoculation were cultured for bacterial isolation. Post-mortem nasal swabs and lung samples were cultured for bacteriological isolation. Nasal mucosa and lung samples were collected and processed for transmission electron microscopy. Pasteurella multocida was isolated from the nasal cavity of all infected rabbits and from the lungs of four infected rabbits. Degenerative ultrastructural changes in epithelial cells and endothelial cells were seen in the infected rabbits. Deciliation of the ciliated epithelium and hyperplasia of the goblet cells in the nasal mucosa were noted. Thickening of the alveolar septa due to hyperplasia of type II pneumocytes, swelling of the endothelial lining of capillaries and infiltration of inflammatory cells were also observed. Intracellular invasion of the nasal epithelial cells and of type II pneumocytes by the organism was observed. Coccobacilli were observed in membrane-bound vacuoles in the cytoplasm of these cells. The vacuoles were adjacent to the host-cell mitochondria and some of these vacuoles appeared to be fused to the mitochondrial membrane. Some type I pneumocytes with intracellular membrane-bound vacuoles containing bacterial cells showed protrusions, which appeared to detach into the alveolar lumina. These results indicated that P. multocida serotype A:3 in rabbits can invade the epithelial cell and cause structural changes in the interstitium, epithelium and endothelium. Heterophils and macrophages appear to play important roles in tissue injury.