Comparative Studies on Ganoderma (Karst.) from Infected Oil Palm and Coconut Stumps with Special Reference to Their Morphological, Molecular and Isozyme Characteristics

The basal stem rot of oil palm caused by Ganoderma i s the most serious disease infecting oil palm in South-east Asia. It is believed that coconut stumps which are colonized by Ganoderma can act as sources of inoculum for i nfection to healthy palms through root contact. However, it is not known whether the Ganoderma infecting oil palm and those colonizing coconut stumps are the same species. Therefore, a comparative study was conducted to determine the similarities and differences between Ganoderma isolates from infected oil palm and coconut stumps, using a multidisciplinary approaches in which morphological. biochemical (intracellular and extracellular enzyme systems) and molecular characteristics (RAPD, RAMS, RFLP and direct sequencing of the ITS regions + 5 . 8 S gene of rDNA) were analysed. Based on the morphological characteristics of the basidiomata, Ganoderma from infected oil palm and coconut stumps conformed to the description of G. boninense in Steyaert's classification system for Ganoderma ( 1 967 and 1975 ). The growth on various media and at different temperatures, and the cultural characteristics of the isolates from infected oil palm and coconut stumps were similar with no significant difference observed between the two groups of Ganoderma. However, the isolates were somatically incompatible with one another, which indicated that they were genotypically distinct individuals and not clones of a genotypic individual. The isozyme profiles from intracellular and extracellular enzyme systems, and the DNA profiles from RAPD, RAMS and RFLP of the ITS regions + 5 . 8 S gene revealed that Ganoderma isolates from infected oil palm and coconut stumps were very variable. Nucleotide sequences of the ITS regions + 5 . 8 S gene of rDNA from a limited number of isolates also showed that the isolates from both groups of Ganoderma were very variable. The Southern hybridization of RAMS gel showed that labelled probes from oil palm and coconut hybridized to the common bands of 1 .2 kb by from primer (CGA)5 and 1 .4 kb band by primer (ACA)5 which indicated that the bands of the same molecular sizes are likely to be homologous. Cluster analysis based on data from biochemical and molecular characters, and phylogenetic analysis of the nucleotide sequence showed that the oil palm isolates and the coconut isolates did not cluster separately which indicated that isolates of both groups of Ganoderma are closely related

Morphological and Molecular Diversity of Aspergillus From Corn Grain Used as Livestock Feed

The study of Aspergillus from corn grains used as livestock feed is important to ensure the safety of the grains as the occurrence of Aspergillus in the corn grain can give an indication of mycotoxin being produced. Morphological and molecular identifications were applied to identify Aspergillus isolated from corn grains used as livestock feed. Morphologically, six species were tentatively identified, namely Aspergillus niger (Groups I and II), Aspergillus flavus, Aspergillus oryzae, Aspergillus fumigatus, Aspergillus clavatus and Aspergillus terreus. Isolates of A. niger was divided into two groups based on slight differences of their colony appearances. Molecular identification using internal transcribed spacer and β-tubulin sequences supported morphological identification except isolates for A. niger Group II isolates, which were molecularly identified as Aspergillus tubingensis. Neighbour-joining phylogenetic tree showed that isolates from the same species were grouped in the same clade. The present study showed that diverse species of Aspergillus are prevalent in corn grain used as livestock feed. It is also necessary to correctly identify the Aspergillus species to employ correct treatment of contaminated corn grains. The occurrence of well-known toxigenic species such as A. niger, A. flavus and A. fumigatus suggested the possible risk of mycotoxin contamination of the corn grain

Presence of vancomycin resistance among enterococcus isolates from stray cats in Universiti Putra Malaysia and selected neighbourhood in Sri Serdang, Selangor, Malaysia

The present study was undertaken to determine the distribution of Enterococcus species from stray cats in Universiti Putra Malaysia and Seri Serdang and the presence of vancomycin resistance among the isolates. Fifty-five rectal swabs were collected from stray cats found in UPM and around the area of Sri Serdang. The Enterococcus species isolated were inoculated onto vancomycin resistant enterococci (VRE) agar supplemented with 8 μg/mL of vancomycin. Biochemical tests such as catalase, bile-aesculin and 6.5% NaCI were conducted to further confirm VRE isolates. Multiplex polymerase chain reaction assay (PCR) were performed for Enterococcus genus and species identification and vancomycin-resistant gene detection. Presence of Enterococcus spp. were demonstrated in every rectal sample tested. Two species were identified: Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium). Among 55 isolates of Enterococcus tested, none was resistant to vancomycin at 8 μg/mL

Evaluation of the survival of vancomycin-resistant enterococci (VRE) isolated from chickens and possible inactivation by in-use concentration of Lindores®, Ecos Timsen® and Omnicide®

Vancomycin-resistant enterococci (VRE) are well-known ascendant nosocomial pathogens. The recent detection of epidemiologic strain carrying vanA gene in the community of people working with animals and in chickens has brought to the forefront the potential public health danger posed by these organism. The farm environment is a major source of VRE persistence in poultry farms. We carried out survival test to test the survival of the VRE isolates on dry condition and surface test to evaluate the inactivation of the isolates by in-use concentration of commonly used disinfectants. In the survival test, all isolates survived for at least 4 weeks in colony counts of (1.00 × 103 – 3.86 × 103 CFU/ml) under clean condition and (1.00 × 103 – 2.02 × 104) for soiled condition. Those that were suspended in 5% BSA solution to mimic organic matter load as obtainable on farms survived for at least 8 weeks at (1.54 × 102 – 1.34 × 103 CFU/ml). In the surface test, inactivation of VRE isolates by in-use concentration of Lindores, Omnicide and Ecos Timsen was tested using the European surface test (EST). All the tested disinfectants were active against the VRE isolates on both the standard test surface (stainless steel) and our test surface (wooden). The results shows microbiocidal effects (ME)for test disinfectants, i.e. the log10 CFU of micro-organisms compared between test biocide and control treated with distilled water, after 7 min of exposure as follows; Lindores® active on both surfaces 5.24 and 3.17, Ecos Timsen® active significantly on steel 4.90 than wood 2.98 and Omnicide® significantly less active on stainless steel 2.40 than on wood 3.50

ITS-PCR-RFLP analysis of Ganoderma sp. infecting industrial crops

Ganoderma is a disastrous pathogen that has been causing tremendous losses to economically important crops in many countries. Vast genetic variations have been observed among several Ganoderma species, even from the same host. In this study, genetic variation was assessed among 44 isolates of Ganoderma sp. isolated from the basidiocarps of four different hosts (oil palm, rubber, tea, and forest trees) collected from selected areas of Peninsular Malaysia. Restriction Fragment Length Polymorphism (RFLP) technique, using ITS1 and ITS4 primers, was used to amplify Internal Transcribed Spacer (ITS) regions. Amplified products were further digested using Bsu 151, Hind III and Taq I restriction enzymes. Cluster analysis with UPGMA using genetic distances clustered all the isolates studied into four main groups. Generally, Ganoderma isolates from the same host were clustered together. The isolates from tea and rubber were more closely related compared to oil palm and forest trees. Similarly, the Ganoderma isolates from the same host were also clustered together, and three species were identified, namely, G. boninense (from oil palm and coconut stumps), G. philippii (rubber) and G. australe (forest trees). The results obtained from the analysis showed that host preference was a possible factor in the differentiation of Ganoderma species

Species distribution and resistance phenotypes of vancomycin-resistant enterococcus isolated from pigs in Pulau Pinang, Malaysia

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens. The extensive use of avoparcin as a growth promoter in poultry and pigs is the hypothesized factor for the emergence of vancomycin resistance in enterococci in animals. As pork is one of the major protein sources for 30% of Malaysians, the present study was conducted to elucidate the role of pigs in the epidemiology of VRE. In this study, 220 rectal swabs were collected from pigs at 12 pig farms in Pulau Pinang. The study found 10 of 12 farms (83.3%) and 92 (41.8%) of the sampled pigs were positive for VRE. Of the 92 isolates examined by PCR, E. faecium (14%), E. casseliflavus (21.7%), E. gallinarum (1.1%) and other Enterococcus species (63.0%) were identified. VanA was detected in E. faecium and E. gallinarum. Questionnaire survey indicated that none of the sampled farms had used glycopeptides, either for growth promotion or for therapy. Tylosin, which has also been associated with vancomycin cross-resistance, was used in 41.8% of the sampled farms; however, there was no significant difference (P>0.05) between the proportion of VRE detected in the farms which used tylosin to those farms which did not. E-test on selected 49 isolates showed 16.0% of the isolates had MIC≤8 and 22.0% had MIC≥32. Single isolates of E. faecium and E. gallinarum, both possessed the resistance gene vanA, showed very high resistance (MIC>256). About 10.0% of the isolates, in which van genes was not detected, had MIC>32. In conclusion E. faecium and E. faecalis were found to be present at a low rate in the pigs sampled in this study. However, detection of vanA with high level of vancomycin resistance (MIC>256) highlights the potential public health threat associated with the pigs

Prevalence of methicillin-resistant Staphylococcus aureus in stray cats around colleges of Universiti Putra Malaysia and selected neighbourhoods of Seri Serdang, Selangor, Malaysia

The existence of Methicillin-resistant Staphylococcus aureus (MRSA) is due to the widespread utilization of antimicrobial agents. Besides humans, MRSA has not only been reported in livestock animals but also in pet animals such as the dogs and cats. This study was designed to estimate the prevalence of MRSA in stray cats around colleges of Universiti Putra Malaysia (UPM) and in selected neighbourhood of Sri Serdang. The samples for this study were taken from the nostrils of the stray cats using small sterile cotton swabs. The swabs were then inoculated onto blood agar (BA) and suspected colonies were gram stained. Gram positive cocci bacteria colonies were then inoculated onto mannitol salt agar (MSA) and yellowish colonies that grew were subjected to a series of biochemical tests such as the catalase, coagulase and Staphytect Plus latex agglutination test before they were inoculated onto the oxacillin resistance screening agar base (ORSAB). A total of seven (12.73%) MRSA from 55 samples were isolated from the stray cats. The results showed that the prevalence of MRSA in stray cats at colleges of Universiti Putra Malaysia and Seri Serdang are high (>10%). The stray cats may have contracted MRSA from contact with infected humans or via contaminated environments at the health care facilities such as the Student Medical Centre which is located in the college area of UPM and also from consuming contaminated raw meat from markets in Sri Serdang

Molecular relatedness of methicillin-resistant s. aureus isolates from staff, environment and pets at university veterinary hospital in Malaysia

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a problem in veterinary medicine and is no longer considered as a mere nosocomial pathogen. We studied the occurrence of MRSA in veterinary personnel, cats and dogs and the environmental premises in University Veterinary Hospital (UVH). We found the prevalence of MRSA as follows: UVH 2/28 (7.1%) staff, 8/100 (8%) of the pets [5/50 (10%) of the dogs and 3/50 (6%) of the cats)], and 9/28 (4.5%) of the environmental samples. Antibiotic sensitivity tests (AST) show multi-resistance characteristics of the MRSA and the minimum inhibitory concentration (MIC) values for the isolates ranged from 1.5 µg to >256 µg/ml. Molecular typing by using multi-locus sequence typing (MLST), staphylococcal protein A typing (spa typing) and pulsed-field gel electrophoresis (PFGE) was conducted and the results from MLST indicated that an isolate from a veterinary personnel (PG21), typed as ST1241 belonged to the same clonal complex (CC) as the two isolates from two dogs (DG16 and DG20), both being typed as ST59. The PFGE results revealed that the two isolates from two veterinary personnel, PG21 and PG16 belonged to closely related MRSA strains with isolates from dog (DG36) and from environmental surface (EV100) respectively. The fact that PFGE revealed close similarity between isolates from humans, a dog and environmental surfaces indicates the possibility for either of them to be the source of MRSA and the potential routes and risks of spread

Genetic variability of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis isolates from humans, chickens and pigs in Malaysia

Vancomycin-resistant enterococci (VRE) have been reported to be present in humans, chickens, and pigs in Malaysia. In the present study, representative samples of VRE isolated from these populations were examined for similarities and differences by using the multilocus sequence typing (MLST) method. Housekeeping genes of Enterococcus faecium (n = 14) and Enterococcus faecalis (n = 11) isolates were sequenced and analyzed using the MLST databases eBURST and goeBURST. We found five sequence types (STs) of E. faecium and six STs of E. faecalis existing in Malaysia. Enterococcus faecium isolates belonging to ST203, ST17, ST55, ST79, and ST29 were identified, and E. faecium ST203 was the most common among humans. The MLST profiles of E. faecium from humans in this study were similar to the globally reported nosocomial-related strain lineage belonging to clonal complex 17 (CC17). Isolates from chickens and pigs have few similarities to those from humans, except for one isolate from a chicken, which was identified as ST203. E. faecalis isolates were more diverse and were identified as ST4, ST6, ST87, ST108, ST274, and ST244, which were grouped as specific to the three hosts. E. faecalis, belonging to the high-risk CC2 and CC87, were detected among isolates from humans. In conclusion, even though one isolate from a chicken was found clonal to that of humans, the MLST analysis of E. faecium and E. faecalis supports the findings of others who suggest VRE to be predominantly host specific and that clinically important strains are found mainly among humans. The infrequent detection of a human VRE clone in a chicken may in fact suggest a reverse transmission of VRE from humans to animals

Molecular identification and ochratoxigenic potential of black Aspergillus from various substrates and indoor environment

Aspergillus section Nigri or black Aspergillus is characterized by dark brown to black colonies. Several species of this group showcase the ability to produce ochratoxin A (OTA), contaminating various types of food and feed. This study was conducted to identify black Aspergillus isolated from rice, groundnuts, spices, corn grains, soil, and indoor environment and determine the ability of the isolates to produce OTA. Based on ITS, β-tubulin and calmodulin sequences, 177 isolates of black Aspergillus from various substrates and indoor environment were identified as A. niger, A. aculeatus, and A. tubingensis. By using two sets of primers to amplify two ochratoxin biosynthesis genes (PKS15KS and PKS15C-MeT genes), the OTA genes were detected in only six A. niger isolates from rice and indoor environment. These six A. niger isolates produced 8 to 308 μg/g of OTA as quantified using UPLC analysis. No OTA gene was detected in any of the A. tubingensis and A. aculeatus isolates. In conclusion, three black Aspergillus species, A. niger, A. aculeatus, and A. tubingensis, were identified from rice, groundnuts, spices, corn grains, soil, and indoor environment. Only six isolates of A. niger produced OTA indicating most of the A. niger isolates were non-OTA producers. These results could thus portray the occurrence of OTA in the field. Since both the number of isolates producing OTA and the levels of OTA production were low, it could be possibly assumed that the occurrence as well as the levels of OTA in the field are also low