There have been a number of studies conducted in order to compare the efficiencies of recovery rates, utilizing different protocols, for the isolation of L. monocytogenes. However, the severity of multiple cell injury has not been included in these studies. In the current study, L. monocytogenes ATCC 19112 was injured by exposure to extreme temperatures (60°C and -20°C) for a one-step injury, and for a two-step injury the cells were transferred directly from a heat treatment to frozen state to induce a severe cell injury (up to 100% injury). The injured cells were then subjected to the US Food and Drug Administration (FDA), the ISO-11290, and the modified United States Department of Agriculture (mUSDA) protocols, and plated on TSAyeast (0.6% yeast), PALCAM agar, and CHROMAgar Listeria for 24 h or 48 h. The evaluation of the total recovery of injured cells was also calculated based on the costs involved in the preparation of media for each protocol. Results indicate that the mUSDA method is best able to aid the recovery of heat-injured, freeze-injured, and heat-freeze-injured cells and was shown to be the most cost effective for heat-freeze-injured cells.
Enzymatic saccharification of corn stover using Phanerochaete chrysosporium and Gloeophyllum trabeum and subsequent fermentation of the saccharification products to ethanol by Saccharomyces cerevisiae and Escherichia coli K011 were achieved. Prior to simultaneous saccharification and fermentation (SSF) for ethanol production, solid-state fermentation was performed for four days on ground corn stover using either P. chrysosporium or G. trabeum to induce in situ cellulase production. During SSF with S. cerevisiae or E. coli, ethanol production was the highest on day 4 for all samples. For corn stover treated with P. chrysosporium, the conversion to ethanol was 2.29 g/100 g corn stover with S. cerevisiae as the fermenting organism, whereas for the sample inoculated with E. coli K011, the ethanol production was 4.14 g/100 g corn stover. Corn stover treated with G. trabeum showed a conversion 1.90 and 4.79 g/100 g corn stover with S. cerevisiae and E. coli K011 as the fermenting organisms, respectively. Other fermentation co-products, such as acetic acid and lactic acid, were also monitored. Acetic acid production ranged between 0.45 and 0.78 g/100 g corn stover, while no lactic acid production was detected throughout the 5 days of SSF. The results of our experiment suggest that it is possible to perform SSF of corn stover using P. chrysosporium, G. trabeum, S. cerevisiae and E. coli K011 for the production of fuel ethanol.
Phenanthrene degradation by Polyporus sp. S133, a new phenanthrene-degrading strain, was investigated in this work. The analysis of degradation was performed by calculation of the remaining phenanthrene by gas chromatography-mass spectrometry. When cells were grown in phenanthrene culture after 92 h, all but 200 and 250 mg/l of the phenanthrene had been degraded. New metabolic pathways of phenanthrene and a better understanding of the phenoloxidases and dioxygenase mechanism involved in degradation of phenanthrene were explored in this research. The mechanism of degradation was determined through identification of the several metabolites; 9,10-phenanthrenequinone, 2,2'-diphenic acid, salicylic acid, and catechol. 9,10-Oxidation and ring cleavage to give 9,10-phenanthrenequinone is the major fate of phenanthrene in ligninolytic Polyporus sp. S133. The identification of 2,2'-diphenic acid in culture extracts indicates that phenanthrene was initially attacked through dioxigenation at C9 and C10 to give cis-9,10-dihydrodiol. Dehydrogenation of phenanthrene-cis-9,10-dihydrodiol to produce the corresponding diol, followed by ortho-cleavage of the oxygenated ring, produced 2,2'-diphenic acid. Several enzymes (manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase, and 2,3-dioxygenase) produced by Polyporus sp. S133 was detected during the incubation. The highest level of activity was shown at 92 h of culture.
Isozyme and protein electrophoresis data from mycelial extracts of 27 isolates of Trichoderma harzianum, 10 isolates of T. aureoviride and 10 isolates of T. longibrachiatum from Southern Peninsular Malaysia were investigated. The eight enzyme and a single protein pattern systems were analyzed. Three isozyme and total protein patterns were shown to be useful for the detection of three Trichoderma species. The isozyme and protein data were analyzed using the Nei and Li Dice similarity coefficient for pairwise comparison between individual isolates, species isolate group, and for generating a distance matrix. The UPGMA cluster analysis showed a higher degree of relationship between T. harzianum and T. aureoviride than to T. longibrachiatum. These results suggested that the T. harzianum isolates had high levels of genetic variation compared to the other isolates of Trichoderma species.
Forty-seven Salmonella Typhimurium (33 zoonotic, 14 clinical) strains were tested for antimicrobial resistance using the standard disk diffusion method. Presence of relevant resistance genes and class 1 integrons were investigated by using PCR. Pulsed-field gel electrophoresis (PFGE) and plasmid profiling were carried out to determine the genomic diversity of Salmonella Typhimurium. Approximately 57.4% of S. Typhimurium were multidrug resistant (MDR) and showed high resistance rates to tetracycline (70.2%), sulphonamides (57.4%), streptomycin (53.1%), ampicillin (29.7%), nalidixic acid (27.6%), kanamycin (23.4%), chloramphenicol (21.2%) and trimethoprim (19.1%). Resistance towards cephalosporins was noted for cephalothin (27.6%), cephradine (21.2%), amoxicillin clavulanic acid (17.0%) and cephalexin (17.0%). Resistance genes, blaTEM, strA, aadA, sul1, sul2, tet(A), tet(B) and tet(C) were detected among the drug resistant strains. Thirty-three strains (70.2%) carried class 1 integrons, which were grouped in 9 different profiles. DNA sequencing identified sat, aadA, pse-1 and dfrA genes in variable regions on class 1 integrons. Thirty-five strains (74.4%) were subtyped to 22 different plasmid profiles, each with 1 - 6 plasmids (2.0 to 95 kb). PFGE subtyped the 47 strains into 39 profiles. In conclusion, high rates of multidrug-resistance were found among the Malaysian Salmonella Typhimurium strains. The emergence of multidrug-resistant Salmonella Typhimurium to cephalosporin antibiotics was also observed. The strains were very diverse and no persistent clone was observed. The emergence of MDR Salmonella Typhimurium is a worldwide problem and this report provides information for the better understanding of the prevalence and epidemiology of MDR S. Typhimurium in Malaysia.
Garcinia is commonly found in Malaysia, but limited information is available regarding endophytic fungi associated with this plant. In this study, 24 endophytic fungi were successfully recovered from different parts of two Garcinia species. Characterization of endophytic fungi was performed based on the conserved internal transcribed spacer (ITS) region sequence analysis and the antimicrobial properties. Results revealed that fruits of the plant appeared to be the highest inhabitation site (38 %) as compared with others. Glomerella sp., Guignardia sp., and Phomopsis sp. appeared to be the predominant endophytic fungi group in Garcinia mangostana and Garcinia parvifolia. Phylogenetic relationships of the isolated endophytic fungi were estimated from the sequences of the ITS region. On the other hand, antibacterial screening showed 11 of the isolates possessed positive response towards pathogenic and nonpathogenic bacteria. However, there was no direct association between certain antibacterial properties with the specific genus observed.
The purpose of this study was to investigate the biosafety of Vibrio parahaemolyticus in raw salad vegetables at wet market and supermarket in Malaysia. A combination of Most Probable Number - Polymerase Chain Reaction (MPN-PCR) method was applied to detect the presence of V. parahaemolyticus and to enumerate their density in the food samples. The study analyzed 276 samples of common vegetables eaten raw in Malaysia (Wild cosmos = 8; Japanese parsley = 21; Cabbage = 30; Lettuce = 16; Indian pennywort = 17; Carrot = 31; Sweet potato = 29; Tomato = 38; Cucumber = 28; Four winged bean = 26; Long bean = 32). The samples were purchased from two supermarkets (A and B) and two wet markets (C and D). The occurrence of V. parahaemolyticus detected was 20.65%, with higher frequency of V. parahaemolyticus in vegetables obtained from wet markets (Wet market C = 27.27%Wet Market D = 32.05%) compared to supermarkets (Supermarket A = 1.64%; Supermarket B = 16.67%). V. parahaemolyticus was most prevalent in Indian pennywort (41.18%). The density of V. parahaemolyticus in all the samples ranged from <3 up to >2400 MPN/g, mostly <3 MPN/g concentration. Raw vegetables from wet markets contained higher levels of V. parahaemolyticus compared to supermarkets. V. parahaemolyticus were present in raw vegetables although in low numbers. The results suggest that raw vegetables act as a transmission route for V. parahaemolyticus. This study will be the first biosafety assessment of V. parahaemolyticus in raw vegetables in Malaysia.
Recently, many cases related to viral gastroenteritis outbreaks have been reported all over the world. Noroviruses are found to be leading as the major cause of outbreaks of acute gastroenteritis. Patients with the acute gastroenteritis normally found to be positive with norovirus when stools and vomit were analyzed. This paper reviews various activities and previous reports that describe norovirus contaminated in various food matrixes and relationship between food handlers. Lately, a numbers of norovirus outbreaks have been reported which are involved fresh produce (such as vegetables, fruits), shellfish and prepared food. Food produces by infected food handlers may therefore easily contaminated. In addition, food that required much handling and have been eaten without heat treatment gave the high risk for getting foodborne illnesses. The standard method for detection of norovirus has already been available for stool samples. However, only few methods for detection of norovirus in food samples have been developed until now.
The aim of the present study was to examine the prevalence of thermophilic Campylobacter spp. (Campylobacter jejuni and Campylobacter coli) in soil, poultry manure, irrigation water, and freshly harvested vegetables from vegetable farms in Malaysia. C. jejuni was detected in 30.4% and 2.7% of the soil samples, 57.1% and 0% of the manure samples, and 18.8% and 3% of the vegetable samples from farm A and farm B, respectively, when using the MPNPCR method. Campylobacter spp. was not found in any of the irrigation water samples tested. Therefore, the present results indicate that the aged manure used by farm A was more contaminated than the composted manure used by farm B. Mostly, the leafy and root vegetables were contaminated. C. coli was not detected in any of the samples tested in the current study. Both farms tested in this study were found to be contaminated by campylobacters, thereby posing a potential risk for raw vegetable consumption in Malaysia. The present results also provide baseline data on Campylobacter contamination at the farm level.
The objectives of this study were to determine the antibiotypes, SCCmec subtypes, PVL carriage, and genetic diversity of MRSA strains from a tertiary hospital. Sixtysix MRSA strains were selected randomly (2003, 2004, and 2007) and tested for the Panton-Valentine leukocidin gene, mecA gene, and SCCmec type via a PCR. The antibiograms were determined using a standard disc diffusion method, and the genetic diversity of the isolates was determined by PFGE. Thirty-four antibiograms were obtained, with 55% of the 66 strains exhibiting resistance to more than 4 antimicrobials. All the isolates remained susceptible to vancomycin, and low resistance rates were noted for fusidic acid (11%), rifampicin (11%), and clindamycin acid (19%). The MRSA isolates that were multisensitive (n=12) were SCCmec type IV, whereas the rest (multiresistant) were SCCmec type III. Only two isolates (SCCmec type IV) tested positive for PVL, whereas all the isolates were mecA-positive. The PFGE was very discriminative and subtyped the 66 isolates into 55 pulsotypes (F=0.31-1.0). The multisensitive isolates were distinctly different from the multidrug-resistant MRSA. In conclusion, no vancomycin-resistant isolate was observed. The Malaysian MDR MRSA isolates were mostly SCCmec type III and negative for PVL. These strains were genetically distinct from the SCCmec type IV strains, which were sensitive to SXT, tetracycline, and erythromycin. Only two strains were SCCmec IV and PVL-positive. The infections in the hospital concerned were probably caused by multiple subtypes of MRSA.
Hepatitis B core antigen (HBcAg) is an important serological marker used in the diagnosis of hepatitis B virus (HBV) infections. In the current study, a fast and efficient preparative purification protocol for truncated HBcAg from Escherichia coli disruptate was developed. The recombinant HBcAg was first captured by anion exchange expanded bed adsorption chromatography integrated with a cell disruption process. This online capture process has shortened the process time and eliminated the "hold-up" period that may be detrimental to the quality of target protein. The eluted product from the expanded bed adsorption chromatography was subsequently purified using size-exclusion chromatography. The results showed that this novel purification protocol achieved a recovery yield of 45.1% with a product purity of 88.2%, which corresponds to a purification factor of 4.5. The recovered HBcAg is still biologically active as shown by ELISA test.
Two culture-independent methods, namely ribosomal DNA libraries and denaturing gradient gel electrophoresis (DGGE), were adopted to examine the microbial community of a Malaysian light crude oil. In this study, both 16S and 18S rDNAs were PCR-amplified from bulk DNA of crude oil samples, cloned, and sequenced. Analyses of restriction fragment length polymorphism (RFLP) and phylogenetics clustered the 16S and 18S rDNA sequences into seven and six groups, respectively. The ribosomal DNA sequences obtained showed sequence similarity between 90 to 100% to those available in the GenBank database. The closest relatives documented for the 16S rDNAs include member species of Thermoincola and Rhodopseudomonas, whereas the closest fungal relatives include Acremonium, Ceriporiopsis, Xeromyces, Lecythophora, and Candida. Others were affiliated to uncultured bacteria and uncultured ascomycete. The 16S rDNA library demonstrated predomination by a single uncultured bacterial type by >80% relative abundance. The predomination was confirmed by DGGE analysis.