The genus Streptomonospora is a group of extremely halophilic filamentous actinomycetes that form a distinct branch in the 16S rRNA gene phylogenetic tree adjacent to the genera Nocardiopsis and Thermobifida, family Norcadiopsaceae. To date, genus Streptomonospora only contain two validly described species which are Streptomonospora salina and Streptomonospora alba. During a biodiversity study on halophilic filamentous actinomycetes from 18 co-ordinates in Barrientos Island, Antarctic, numerous actinomycetes strains were isolated. To identify whether these isolates were members of the genus Streptomonospora, a genus specific primer that allow the rapid detection of the genus Streptomonospora by means of PCR amplification was used. Furthermore molecular cloning was performed to make identical and multiple copies of the target gene. In addition, morphological characteristic identification was performed to validate isolates with positive amplification during PCR.
Aflatoxin contamination in foods is a global concern as they are carcinogenic, teratogenic and mutagenic compounds. The aflatoxin-producing fungi, mainly from the Aspergillus section Flavi, are ubiquitous in nature and readily contaminate various food commodities, thereby affecting human's health. The incidence of aflatoxigenic Aspergillus spp. and aflatoxins in various types of food, especially raw peanuts and peanut-based products along the supply chain has been a concern particularly in countries having tropical and sub-tropical climate, including Malaysia. These climatic conditions naturally support the growth of Aspergillus section Flavi, especially A. flavus, particularly when raw peanuts and peanut-based products are stored under inappropriate conditions. Peanut supply chain generally consists of several major stakeholders which include the producers, collectors, exporters, importers, manufacturers, retailers and finally, the consumers. A thorough examination of the processes along the supply chain reveals that Aspergillus section Flavi and aflatoxins could occur at any step along the chain, from farm to table. Thus, this review aims to give an overview on the prevalence of Aspergillus section Flavi and the occurrence of aflatoxins in raw peanuts and peanut-based products, the impact of aflatoxins on global trade, and aflatoxin management in peanuts with a special focus on peanut supply chain in Malaysia. Furthermore, aflatoxin detection and quantification methods as well as the identification of Aspergillus section Flavi are also reviewed herein. This review could help to shed light to the researchers, peanut stakeholders and consumers on the risk of aflatoxin contamination in peanuts along the supply chain.
In this study, Listeria (L.) monocytogenes isolated from milk and milk products in Kaduna, Nigeria, were subjected to a multiplex PCR assay to identify virulence-associated genes (such as prf A, inl A, hly A, act A, and iap). Of the 36 isolates, 9 (25%) were positive for one or two virulence-associated genes. Based on the sample type, 6 (16.9%) of the isolates that possessed virulence-associated genes were obtained from raw milk, 2 (3.2%) from "Manshanu," and 1 (2.8%) from "Kindrimo." Sequence and phylogenetic analysis based on the 16S rRNA revealed that Nigerian L. monocytogenes isolates (NGA 34A, NGA 35A, NGA 41A, and NGA 38A), when compared with reference L. monocytogenes, were grouped into two distinct clusters, A and B, with sequence (NGA 34A, NGA 35A, and NGA 41A) phylogenetically closer to J1776; N1-011A; R2-502; J1816; and J2-031, whereas L. monocytogenes isolate (NGA 38A) clustered with EDG; J1-220; J1926; J1817; and J2-1091. The separation of the Nigerian L. monocytogenes isolates into linage A (responsible for epidemic listeriosis) and lineage B (responsible for sporadic cases of listeriosis) is of public health concern and that local isolates might have potentials for human food borne listeriosis based on the virulence factors so far identified.
Oxalic acid was evaluated as a treatment for reducing populations of naturally occurring microorganisms on raw chicken. Raw chicken breasts were dipped in solutions of oxalic acid (0, 0.5, 1.0, 1.5, and 2.0%, wt/vol) for 10, 20, and 30 min, individually packed in oxygen-permeable polyethylene bags, and stored at 4 degrees C. Total plate counts of aerobic bacteria and populations of Pseudomonas spp. and Enterobacteriaceae on breasts were determined before treatment and after storage for 1, 3, 7, 10, and 14 days. The pH and Hunter L, a, and b values of the breast surface were measured. Total plate counts were ca. 1.5 and 4.0 log CFU/g higher on untreated chicken breasts after storage for 7 and 14 days, respectively, than on breasts treated with 0.5% oxalic acid, regardless of dip time. Differences in counts on chicken breasts treated with water and 1.0 to 2.0% of oxalic acid were greater. Populations of Pseudomonas spp. on chicken breasts treated with 0.5 to 2.0% oxalic acid and stored at 4 degrees C for 1 day were less than 2 log CFU/g (detection limit), compared with 5.14 log CFU/g on untreated breasts. Pseudomonas grew on chicken breasts treated with 0.5% oxalic acid to reach counts not exceeding 3.88 log CFU/g after storage for 14 days. Counts on untreated chicken exceeded 8.83 log CFU/g at 14 days. Treatment with oxalic acid caused similar reductions in Enterobacteriaceae counts. Kocuria rhizophila was the predominant bacterium isolated from treated chicken. Other common bacteria included Escherichia coli and Empedobacter brevis. Treatment with oxalic acid caused a slight darkening in color (decreased Hunter L value), retention of redness (increased Hunter a value), and increase in yellowness (increased Hunter b value). Oxalic acid has potential for use as a sanitizer to reduce populations of spoilage microorganisms naturally occurring on raw chicken, thereby extending chicken shelf life.
Loop-mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. Following its first discovery by Notomi et al. ( Nucleic Acids Res 28: E63), LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro-organisms in humans, livestock and plants. In this review, available types of LAMP techniques will be discussed together with their applications in detection of various micro-organisms. Up to date, there are varieties of LAMP detection methods available including colorimetric and fluorescent detection, real-time monitoring using turbidity metre and detection using lateral flow device which will also be highlighted in this review. Apart from that, commercialization of LAMP technique had also been reported such as lyophilized form of LAMP reagents kit and LAMP primer sets for detection of pathogenic micro-organisms. On top of that, advantages and limitations of this molecular detection method are also described together with its future potential as a diagnostic method for infectious disease.
The peanut supply chain in Malaysia is dominated by three main stakeholders (importers, manufacturers, retailers). The present study aimed to determine the levels and critical points of aflatoxin and fungal contamination in peanuts along the supply chain. Specifically, two types of raw peanuts and six types of peanut-based products were collected (N = 178). Samples were analysed for aflatoxins by using high-performance liquid chromatography. Results revealed that the aflatoxin contamination was significantly higher (P ≤ 0.05) in raw peanuts and peanut-based products from the retailers. However, there was no significant difference (P ≥ 0.05) in fungal contamination for both types of peanuts except for the total fungal count in raw peanuts from the retailers. Furthermore, raw peanut kernels from the retailers were the most contaminated ones ranged from
Isolation of bacteriocinogenic lactic acid bacteria (LAB) from the Malaysian mould-fermented product tempeh and characterization of the produced bacteriocin(s).
A total of 31 strains of Vibrio cholerae O1 (10 from outbreak cases and 7 from surface water) and non-O1 (4 from clinical and 10 from surface water sources) isolated between 1993 and 1997 were examined with respect to presence of cholera enterotoxin (CT) gene by PCR-based assays, resistance to antibiotics, plasmid profiles and random amplified polymorphic DNA (RAPD) analysis. All were resistant to 9 or more of the 17 antibiotics tested. Identical antibiotic resistance patterns of the isolates may indicate that they share a common mode of developing antibiotic resistance. Furthermore, the multiple antibiotic resistance indexing showed that all strains tested originated from high risk contamination. Plasmid profile analysis by agarose gel electrophoresis showed the presence of small plasmids in 12 (7 non-O1 and 5 O1 serotypes) with sizes ranging 1.3-4.6 MDa. The CT gene was detected in all clinical isolates but was present in only 14 (6 O1 serotype and 8 non-O1 serotype) isolates from environmental waters. The genetic relatedness of the clinical and environmental Vibrio cholerae O1 and non-O1 strains was investigated by RAPD fingerprinting with four primers. The four primers generated polymorphisms in all 31 strains of Vibrio cholerae tested, producing bands ranging from < 250 to 4500 bp. The RAPD profiles revealed a wide variability and no correlation with the source of isolation. This study provides evidence that Vibrio cholerae O1 and non-O1 have significant public health implications.
Twenty-one Vibrio parahaemolyticus isolates representing 21 samples of coastal seawater from three beaches in peninsular Malaysia were found to be sensitive to streptomycin, norfloxacin and chloramphenicol. Resistance was observed to penicillin (100%), ampicillin (95.2%), carbenicilin (95.2%), erythromycin (95.2%), bacitracin (71.4%), cephalothin (28.6%), moxalactam (28.6%), kanamycin (19.1%), tetracycline (14.3%), nalidixic acid (9.5%) and gentamicin (9.5%). Plasmids of 2.6 to 35.8 mDa were detected among plasmid-containing isolates. All isolates carried the Vp-toxR gene specific to V. parahaemolyticus and were negative for the tdh gene, but only one isolate was positive for the trh gene. DNA fingerprinting of the isolates using ERIC-PCR and PFGE showed that the isolates belong to two major clonal groups, with several isolates from different locations in the same group, indicating the presence of similar strains in the different locations.
The present work investigated the profile and biodiversity of lactic acid bacteria (LAB) isolated from selected manufactured and homemade fermented foods in Malaysia. A total of 55 LAB were isolated from 20 samples, and identified based on the sequencing of 16S rRNA gene. The LAB isolates were identified as Lacticaseibacillus rhamnosus (34.5%), Lactiplantibacillus plantarum (20%), Limosilactobacillus fermentum (20%), Lacticaseibacillus paracasei (12.7%), Lacticaseibacillus casei (3.6%), Lactobacillus sp. (1.8%), Enterococcus faecalis (3.6%), Enterococcus faecium (1.8%), and Enterococcus durans (1.8%). Majority (94%) of the LAB isolates exhibited broad-spectrum antimicrobial activity against selected foodborne pathogens, and four isolates (L. fermentum SC1001, L. paracasei K2003, and L. rhamnosus KF1002 and MK2003) could produce bacteriocin-like inhibitory substance (BLIS). Lacticaseibacillus paracasei M1001 (homemade mozzarella) exhibited high-temperature tolerance and acid resistance, was homofermentative, and generated good antimicrobial activity, which strongly implied its potential for industrial applications. The present work results would potentially widen our knowledge of LAB diversity in Malaysian fermented foods and provide a potential for their applications in the food industry or other purposes.
Filth flies at wet markets can be a vector harbouring multiple antimicrobial-resistant (MAR) nontyphoidal Salmonella (NTS), and such strains are a significant threat to public health as they may cause severe infections in humans. This study aims to investigate the prevalence of antimicrobial-resistant NTS, especially Salmonella Enteritidis and S. Typhimurium harboured by filth flies at wet markets, and investigate their survival in the simulated gastric fluid (SGF). Filth flies (n = 90) were captured from wet markets in Klang, Malaysia, and processed to isolate Salmonella spp. The isolates (n = 16) were identified using the multiplex-touchdown PCR and assessed their antimicrobial susceptibility against 11 antimicrobial agents. Finally, three isolates with the highest MAR index were subjected to SGF survival tests. It was observed that 17.8 % of flies (n = 16/90) harbouring Salmonella, out of which 10 % (n = 9/90) was S. Enteritidis, 2.2 % (n = 2/90) was S. Typhimurium, and 5.6 % was unidentified serotypes of Salmonella enterica subsp. I. 43.8 % (n = 7/16) were confirmed as MAR, and they were observed to be resistant against ampicillin, chloramphenicol, kanamycin, streptomycin, and nalidixic acid. Three strains, F35, F75, and F85 demonstrated the highest MAR index and were able to survive (>6-log10) in the SGF (180 min), indicating their potential virulence and invasiveness. This study provides significant insights into the prevalence and severity of MAR nontyphoidal Salmonella harboured by filth flies in wet markets, which may help inform strategies for controlling the spread and outbreak of foodborne disease.
An earlier electrochemical mechanism of DNA detection was adapted and specified for the detection of Vibrio parahaemolyticus in real samples. The reader, based on a screen printed carbon electrode, was modified with polylactide-stabilized gold nanoparticles and methylene blue was employed as the redox indicator. Detection was assessed using a microprocessor to measure current response under controlled potential. The fabricated sensor was able to specifically distinguish complementary, non-complementary and mismatched oligonucleotides. DNA was measured in the range of 2.0 × 10(-8)-2.0 × 10(-13) M with a detection limit of 2.16 pM. The relative standard deviation for 6 replications of differential pulse voltammetry (DPV) measurement of 0.2 µM complementary DNA was 4.33%. Additionally, cross-reactivity studies against various other food-borne pathogens showed a reliably sensitive detection of the target pathogen. Successful identification of Vibrio parahaemolyticus (spiked and unspiked) in fresh cockles, combined with its simplicity and portability demonstrate the potential of the device as a practical screening tool.
A simple but promising electrochemical DNA nanosensor was designed, constructed and applied to differentiate a few food-borne pathogens. The DNA probe was initially designed to have a complementary region in Vibrio parahaemolyticus (VP) genome and to make different hybridization patterns with other selected pathogens. The sensor was based on a screen printed carbon electrode (SPCE) modified with polylactide-stabilized gold nanoparticles (PLA-AuNPs) and methylene blue (MB) was employed as the redox indicator binding better to single-stranded DNA. The immobilization and hybridization events were assessed using differential pulse voltammetry (DPV). The fabricated biosensor was able to specifically distinguish complementary, non-complementary and mismatched oligonucleotides. DNA was measured in the range of 2.0×10(-9)-2.0×10(-13)M with a detection limit of 5.3×10(-12)M. The relative standard deviation for 6 replications of DPV measurement of 0.2µM complementary DNA was 4.88%. The fabricated DNA biosensor was considered stable and portable as indicated by a recovery of more than 80% after a storage period of 6 months at 4-45°C. Cross-reactivity studies against various food-borne pathogens showed a reliably sensitive detection of VP.
Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.
Fresh pennywort (Centella asiatica) is usually eaten raw as 'ulam' or salad-like lettuce. Unfortunately, the fresh pennywort has the potential to cause foodborne outbreaks due to pathogens present on the surface and between the leaves, as washing the pennywort using tap water alone cannot guarantee that the pathogens are eliminated. Thus, the efficacies of several sanitizing solutions, i.e., sodium chloride, sodium hypochlorite, acetic acid, acidic electrolyzed water (acidic EW), alkaline electrolyzed water (alkaline EW), and a combination of acidic EW and alkaline EW (acidic-alkaline EW), were evaluated for their potential applications as washing solutions for pennywort. Washing using acidic EW alone or in combination with alkaline EW (two-step washing) reduced the microbial count. In sensory evaluation, all sanitizer solutions were accepted by the panellists with a score greater than 5, except those washing with acetic acid. Overall, the use of acidic EW, either alone or in combination with alkaline EW, was the best treatment to decontaminate microbes while maintaining the physicochemical and sensory properties of pennywort leaves.
The screening of antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, yeast and fungi was carried out on isopropanol extracts prepared from 121 isolates of endophytic fungi isolated from medicinal plants in Malaysia. Sensitivity was found to vary among the microorganisms. Bacillus subtilis, Saccharomyces cerevisiae and Alternaria sp. were susceptible to extracts from three, two and two isolates of endophytic fungi, respectively. None were found effective against Salmonella typhimurium. Sixteen endophytic fungal isolates tested were also found to exhibit antitumor activity in the yeast cell-based assay.
The main aim of this study was to combine the techniques of most probable number (MPN) and polymerase chain reaction (PCR) for quantifying the prevalence and numbers of Campylobacter spp. in ulam, a popular Malaysian salad dish, from a traditional wet market and two modern supermarkets in Selangor, Malaysia. A total of 309 samples of raw vegetables which are used in ulam were examined in the study. The prevalences of campylobacters in raw vegetables were, for supermarket I, Campylobacter spp., 51.9%; Campylobacter jejuni, 40.7%; and Campylobacter coli, 35.2%: for supermarket II, Campylobacter spp., 67.7%; C. jejuni, 67.7%; and C. coli, 65.7%: and for the wet market, Campylobacter spp., 29.4%; C. jejuni, 25.5%; and C. coli, 22.6%. In addition Campylobacter fetus was detected in 1.9% of raw vegetables from supermarket I. The maximum numbers of Campylobacter spp. in raw vegetables from supermarkets and the wet market were >2400 and 460 MPN/g, respectively.
Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhea among infants in developing countries. A total of 38 EPEC isolates, obtained from diarrhea patients of Hospital Miri, Sarawak, were investigated through plasmid profile, antibiotic resistance and randomly amplified polymorphic DNA (RAPD) analysis. From the 8 types of antibiotics used, all isolates were 100% resistant to furoxime, cephalothin and sulphamethoxazole and showed high multiple antibiotic resistant (MAR) indexes, ranging from 0.5 to 1.0. In plasmid profiling, 22 isolates (58%) showed the presence of one or more plasmids in the range 1.0 to 30.9 mDa. The dendrogram obtained from the results of the RAPD-PCR discriminated the isolates into 30 single isolates and 3 clusters at the level of 40% similarity. The EPEC isolates were highly diverse, as shown by their differing plasmid profiles, antibiotic resistance patterns and RAPD profiles.
This study has evaluated the use of a commercially available Rainbow agar O157 and polymerase chain reaction (PCR) assays for the detection of Shiga-like toxin producing Escherichia coli and to serotype E. coli O157:H7 from raw meat. The Rainbow agar O157 was found to be selective and sensitive for the screening of the E. coli O157 from artificially and naturally contaminated meat samples. Shiga-like toxin producing E. coli were identified with two primer pairs that amplified fragments of the SLT-I (384 bp) and SLT-II (584 bp). E. coli O157:H7 was serotyped with a primer pair specified for the H7 flagellar gene, which amplify specific DNA fragments (625 bp) from all E. coli O157:H7 strains. The use of Rainbow agar O157 described allows for the presumptive isolation of E. coli O157 in 24 hours. Identification and confirmation of the presumptive isolates as E. coli O157:H7 by PCR assays require additional 6-8 hours. The above-mentioned screening and identification procedures should prove to be a very useful method since it allows for the specific detection of E. coli O157:H7.