The organism designated the SF agent was originally isolated in Japan in 1962 from Stellantchasmus falcatus metacercaria parasitic on gray mullet fish. The SF agent resembles members of the genus Ehrlichia morphologically and exhibits weak antigenic cross-reactivity with Ehrlichia sennetsu. This organism causes mild clinical signs in dogs, but severe splenomegaly and lymphadenopathy in mice. This suggests that the SF agent may be similar to either Neorickettsia helminthoeca, an intracellular parasite of a fluke and the cause of salmon poisoning disease in dogs, or E. sennetsu, the causative agent of human sennetsu ehrlichiosis in Japan and Malaysia. In order to determine the phylogenetic relationship between the SF agent and other ehrlichial species, the 16S rRNA gene was amplified by the PCR and sequenced. The SF agent sequence was most closely related to the sequences of Ehrlichia risticii (level of sequence similarity, 99.1%), the causative agent of Potomac horse fever, and E. sennetsu (level of sequence similarity, 98.7%). The next most similar sequence was that of N. helminthoeca, but the level of sequence similarity was only 93.7%. E. sennetsu, E. risticii, the SF agent, and N. helminthoeca formed a distinct cluster that was separated from all other ehrlichial species. As determined by immunofluorescence labeling, antiserum against the SF agent cross-reacted strongly with E. sennetsu, E. risticii, and N. helminthoeca. When three genetically distinct ehrlichial isolates obtained from horses with Potomac horse fever were compared with the SF agent, we found that the SF agent was most closely related to Ohio isolate 081, followed by IllinoisT (T = type strain) and a Kentucky isolate. We observed strong antigenic cross-reactivities and similarities in Western blot (immunoblot) reaction profiles when we compared the SF agent, E. risticii, and E. sennetsu; however, weaker antigenic cross-reactivity was observed when the SF agent and N. helminthoeca were compared. Our results indicate that the SF agent is antigenically more closely related to E. risticii and E. sennetsu than to N. helminthoeca. The biological and antigenic characteristics and the 16S rRNA sequence data suggest that the SF agent is a new species that belongs to the genus Ehrlichia.
Phytoplasmas (mycoplasmalike organisms, MLOs) associated with mitsuba (Japanese hone-wort) witches'-broom (JHW), garland chrysanthemum witches'-broom (GCW), eggplant dwarf (ED), tomato yellows (TY), marguerite yellows (MY), gentian witches'-broom (GW), and tsu-wabuki witches'-broom (TW) in Japan were investigated by polymerase chain reaction (PCR) amplification of DNA and restriction enzyme analysis of PCR products. The phytoplasmas could be separated into two groups, one containing strains JHW, GCW, ED, TY, and MY, and the other containing strains GW and TW, corresponding to two groups previously recognized on the basis of transmission by Macrosteles striifrons and Scleroracus flavopictus, respectively. The strains transmitted by M. striifrons were classified in 16S rRNA gene group 16SrI, which contains aster yellows and related phytoplasma strains. Strains GW and TW were classified in group 16SrIII, which contains phytoplasmas associated with peach X-disease, clover yellow edge, and related phytoplasmas. Digestion of amplified 16S rDNA with HpaII indicated that strains GW and TW were affiliated with subgroup 16SrIII-B, which contains clover yellow edge phytoplasma. All seven strains were distinguished from other phytoplasmas, including those associated with clover proliferation, ash yellows, elm yellows, and beet leafhopper-transmitted virescence in North America, and Malaysian periwinkle yellows and sweet potato witches'-broom in Asia.
We have previously reported the phenotypic characterization of methicillin-resistant Staphylococcus aureus (MRSA) clinical strains isolated in Malaya University Hospital in the period 1987 to 1989 using antibiogram, coagulase typing, plasmid profiles, and phage typing. Here, we report the analysis of the same strains with three genotyping methods; ribotyping, pulsed-field gel electrophoresis (PFGE) typing, and IS431 typing (a restriction enzyme fragment length polymorphism analysis using an IS431 probe). Ribotyping could discriminate 46 clinical MRSA strains into 5 ribotypes, PFGE typing into 22 types, and IS431 typing into 15 types. Since the differences of the three genotyping patterns from strain to strain were quite independent from one another, the combined use of the three genotyping methods could discriminate 46 strains into 39 genotypes. Thus, the powerful discriminatory ability of the combination was demonstrated.
Pulsed field gel electrophoresis analysis of genomic DNA was used to investigate genetic diversity among Dichelobacter nodosus from footrot in sheep in Malaysia. Twelve Dichelobacter nodosus strains isolated from lesion materials from infected sheep were confirmed as Dichelobacter nodosus by polymerase chain reaction technique using the species-specific Dichelobacter nodosus 16S RNA sequence Ac and C as primers. Pulsed field gel electrophoresis banding profiles using restriction enzymes ApaI (5'GGGCCC3'), SfiI (5'GGCCNNNNNGGCC3') and SmaI ('5CCCGGG3') enabled the 12 Dichelobacter nodosus strains to be differentiated into eight different PFGE patterns and thus genome-types, with F (coefficient of similarity) values ranging from 0.17 to 1.0 (ApaI), 0.14 to 1.0 (SfiI) and 0.22 to 1.0 (SmaI). Strains with origin in different farms were shown to have different PFGE patterns (two strains, M7 and M8 were the only exception). On the basis of their PFGE, all field strains used in the study differed from the reference strains. Our data revealed that there are several clonal types of Dichelobacter nodosus isolates and indicated that there is probably more than one source of this pathogen on the farms studied. The study showed that strains of D. nodosus exhibited considerable genetic diversity using this method and that genomic analysis by pulsed field gel electrophoresis was useful in discriminating the D. nodosus strains.
Ninety-two strains of lactic acid bacteria (LAB) were isolated from a Malaysian food ingredient, chili bo, stored for up to 25 days at 28 degreesC with no benzoic acid (product A) or with 7,000 mg of benzoic acid kg-1 (product B). The strains were divided into eight groups by traditional phenotypic tests. A total of 43 strains were selected for comparison of their sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) whole-cell protein patterns with a SDS-PAGE database of LAB. Isolates from product A were identified as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus farciminis, Pediococcus acidilactici, Enterococcus faecalis, and Weissella confusa. Five strains belonging to clusters which could not be allocated to existing species by SDS-PAGE were further identified by 16S rRNA sequence comparison. One strain was distantly related to the Lactobacillus casei/Pediococcus group. Two strains were related to Weissella at the genus or species level. Two other strains did not belong to any previously described 16S rRNA group of LAB and occupied an intermediate position between the L. casei/Pediococcus group and the Weissella group and species of Carnobacterium. The latter two strains belong to the cluster of LAB that predominated in product B. The incidence of new species and subspecies of LAB in chili bo indicate the high probability of isolation of new LAB from certain Southeast Asian foods. None of the isolates exhibited bacteriocin activity against L. plantarum ATCC 14917 and LMG 17682.
Paralactobacillus selangorensis gen. nov., sp. nov. is described. This organism, isolated from a Malaysian food ingredient called chili bo, is an obligatory homofermentative, rod-shaped lactic acid bacterium. The G+C content is 46.1-46.2+/-0.3 mol%. Earlier 16S rRNA studies showed that this organism constitutes a new taxon distantly related to the Lactobacillus casei-Pediococcus group. A phenotypic description that distinguishes Paralactobacillus selangorensis from other genera of lactic acid bacteria is presented. The type strain of Paralactobacillus selangorensis is LMG 17710T.
A taxonomic study was conducted to clarify the relationships of two bacterial populations belonging to the genus Weissella. A total of 39 strains originating mainly from Malaysian foods (22 strains) and clinical samples from humans (9 strains) and animals (6 strains) were analysed using a polyphasic taxonomic approach. The methods included classical phenotyping, whole-cell protein electrophoresis, 16S and 23S rDNA RFLP (ribotyping), determination of 16S rDNA sequence homologies and DNA-DNA reassociation levels. Based on the results, the strains were considered to represent two different species, Weissella confusa and a novel Weissella species, for which the name Weissella cibaria sp. nov. is proposed. Weisella confusa possessed the highest 16S rDNA sequence similarity to Weisella cibaria, but the DNA-DNA reassociation experiment showed hybridization levels below 49% between the strains studied. The numerical analyses of Weisella confusa and Weisella cibaria strains did not reveal any specific clustering with respect to the origin of the strains. Based on whole-cell protein electrophoresis, and ClaI and HindIII ribotyping patterns, food and clinical isolates were randomly located in the two species-specific clusters obtained.
All obligate bacterial endosymbionts of free-living amoebae currently described are affiliated with the alpha-Proteobacteria, the Chlamydiales or the phylum Cytophaga-Flavobacterium-Bacteroides. Here, six rod-shaped gram-negative obligate bacterial endosymbionts of clinical and environmental isolates of Acanthamoeba spp. from the USA and Malaysia are reported. Comparative 16S rDNA sequence analysis demonstrated that these endosymbionts form a novel, monophyletic lineage within the beta-Proteobacteria, showing less than 90% sequence similarity to all other recognized members of this subclass. 23S rDNA sequence analysis of two symbionts confirmed this affiliation and revealed the presence of uncommon putative intervening sequences of 146 bp within helix-25 that shared no sequence homology to any other bacterial rDNA. In addition, the 23S rRNA of these endosymbionts displayed one polymorphism at the target site of oligonucleotide probe BET42a that is conserved in all other sequenced beta-Proteobacteria. Intra-cytoplasmatic localization of the endosymbionts within the amoebal host cells was confirmed by electron microscopy and fluorescence in situ hybridization with a specific 16S rRNA-targeted oligonucleotide probe. Based on these findings, the provisional name 'Candidatus Procabacter acanthamoebae' is proposed for classification of a representative of the six endosymbionts of Acanthamoeba spp. studied in this report. Comparative 18S rDNA sequence analysis of the Acanthamoeba host cells revealed their membership with either Acanthamoeba 18S rDNA sequence type T5 (Acanthamoeba lenticulata) or sequence type T4, which comprises the majority of all Acanthamoeba isolates.
Lactic acid bacteria (LAB) are the predominant micro-organisms in tempoyak, a Malaysian acid-fermented condiment. In a study on the diversity of LAB in this product, three isolates could not be identified using SDS-PAGE of whole-cell proteins or API 50 CH. The taxonomic position of the three isolates was clarified in the present study. 16S rDNA sequencing classified a representative strain in the genus Lactobacillus, clearly separated from all known species, and most closely related to the Lactobacillus reuteri phylogenetic group. DNA-DNA hybridization experiments and an extensive phenotypic description confirm that the strains represent a single and separate novel species among the obligately heterofermentative lactobacilli. The three isolates are distinguished at the intra-species level by plasmid profiling, pulsed-field gel electrophoresis of macro-restriction fragments and biochemical features. The name Lactobacillus durianis sp. nov. is proposed for the novel taxon and the type strain is LMG 19193T (= CCUG 45405T).
Five strains of phytase-producing, gram-negative, non-spore-forming, non-motile, small, stout, rod-shaped, strictly anaerobic, fermentative bacteria were isolated from the rumens of cattle in Malaysia. All five strains had morphological, physiological and biochemical features in common. Although these strains had many physiological and biochemical characteristics that were identical to those of the Mitsuokella multacida type strain (ATCC 27723T), they could be distinguished from this species by means of the following characteristics: a smaller cell size (1.2-2.4 microm long and 0.6-0.8 microm wide); a lower final pH value (3.8-4.0) in peptone/yeast extract/glucose broth; inhibition by 0.001% brilliant green; insensitivity to kanamycin (100 microg ml(-1)) and penicillin (10 microg ml(-1)); a higher optimum growth temperature (approx. 42 degrees C); the ability to grow at 45 and 47 degrees C; the ability to ferment glycerol, sorbitol and amidon; and the inability to ferment mannitol, rhamnose, D-tagatose and melezitose. The G+C content of the type strain (M 9T) of these five strains was 56.9 mol%. Analysis of the 16S rRNA gene sequence of type strain M 9T indicated that the strain falls within the genus Mitsuokella. The sequence similarity between type strain M 9T and Mitsuokella multacida was 98.7%. The DNA-DNA relatedness between type strain M 9T and Mitsuokella multacida type strain DSM 20544T (= ATCC 27723T) was 63.8%, indicating that, in spite of a high level of similarity for the 16S rRNA gene sequence, type strain M 9T is independent of Mitsuokella multacida at the species level. On the basis of these results, a new species, Mitsuokella jalaludinii sp. nov., is proposed for these strains. The type strain is M 9T (= DSM 13811T = ATCC BAA-307T).
The taxonomic position of three actinomycete strains isolated from Malaysian soil was established by using a polyphasic approach. The isolates formed chains composed of four spores on the tip of sporophores branching from the aerial mycelium, and their chemotaxonomic properties were common to those of members of the family Streptosporangiaceae. These phenotypic properties as well as a phylogenetic analysis based on 16S rRNA gene sequences indicated that they should be classified in the genus Microtetraspora. The three isolates showed a unique pattern of cultural, physiological and biochemical properties that distinguished them from previously described species of the genus Microtetraspora. The isolates showed more than 72% DNA relatedness to each other, but only 58% or less relatedness to any previously described species. On the basis of the data presented, a new species of the genus Microtetraspora, Microtetraspora malaysiensis, is proposed. The type strain of the new species is strain H47-7(T) (=JCM 11278(T)=DSM 44579(T)).
An obligate intracellular bacterium was isolated from urine samples from 7 (3.5%) of 202 fruit bats (Eonycteris spelaea) in peninsular Malaysia. The bacterium produced large membrane-bound inclusions in human, simian, and rodent cell lines, including epithelial, fibroblastlike, and lymphoid cells. Thin-section electron microscopy showed reticulate bodies dividing by binary fission and elementary bodies in the inclusions; mitochondria surrounded the inclusions. The inclusions were positive for periodic acid-Schiff stain but could not be stained by fluorescein-labeled anti-Chlamydia trachomatis major outer membrane protein monoclonal antibody. The bacterium was resistant to penicillin and streptomycin (MICs > 256 mg/L) but susceptible to tetracycline (MIC = 0.25 mg/L) and chloramphenicol (MIC = 0.5 mg/L). Sequence analysis of the 16SrRNA gene indicated that it was most closely related to 2 isolates of Waddlia chondrophila (94% and 96% identity). The 16S and 23S rRNA gene signatures were only 91% identical. We propose this novel bacterium be called W. malaysiensis.
Three lactic acid bacterial (LAB) strains obtained from a Malaysian acid-fermented condiment, tempoyak (made from pulp of the durian fruit), showed analogous but distinct patterns after screening by SDS-PAGE of whole-cell proteins and comparison with profiles of all recognized LAB species. 16S rRNA gene sequencing of one representative strain showed that the taxon belongs phylogenetically to the genus Leuconostoc, with its nearest neighbour being Leuconostoc fructosum (98 % sequence similarity). Biochemical characteristics and DNA-DNA hybridization experiments demonstrated that the strains differ from Leuconostoc fructosum and represent a single, novel Leuconostoc species for which the name Leuconostoc durionis sp. nov. is proposed. The type strain is LMG 22556(T) (= LAB 1679(T) = D-24(T) = CCUG 49949(T)).
This study aims to identify Acinetobacter of clinical isolates from the University of Malaya Medical Centre (UMMC), Kuala Lumpur, to the species level by 16S rDNA sequencing.
In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.
Coptotermes gestroi, the Asian subterranean termite (AST), is an economically important structural and agricultural pest that has become established in many areas of the world. For the first time, phylogeography was used to illuminate the origins of new found C. gestroi in the US Commonwealth of Puerto Rico; Ohio, USA; Florida, USA; and Brisbane, Australia. Phylogenetic relationships of C. gestroi collected in indigenous locations within Malaysia, Thailand, and Singapore as well as from the four areas of introduction were investigated using three genes (16S rRNA, COII, and ITS) under three optimality criteria encompassing phenetic and cladistic assumptions (maximum parsimony, maximum likelihood, and neighbor-joining). All three genes showed consistent support for a close genetic relationship between C. gestroi samples from Singapore and Ohio, whereas termite samples from Australia, Puerto Rico, and Key West, FL were more closely related to those from Malaysia. Shipping records further substantiated that Singapore and Malaysia were the likely origin of the Ohio and Australia C. gestroi, respectively. These data provide support for using phylogeography to understand the dispersal history of exotic termites. Serendipitously, we also gained insights into concerted evolution in an ITS cluster from rhinotermitid species in two genera.
The taxonomic status of two actinomycetes isolated from the wall of a hypogean Roman catacomb was established based on a polyphasic investigation. The organisms were found to have chemical and morphological markers typical of members of the genus Amycolatopsis. They also shared a range of chemical, molecular and phenotypic markers which served to separate them from representatives of recognized Amycolatopsis species. The new isolates formed a branch in the Amycolatopsis 16S rRNA gene sequence tree with Amycolatopsis minnesotensis NRRL B-24435(T), but this association was not supported by a particularly high bootstrap value or by the product of the maximum-parsimony tree-making algorithm. The organisms were distinguished readily from closely related Amycolatopsis species based on a combination of phenotypic properties and from all Amycolatopsis strains by their characteristic menaquinone profiles, in which tetra-hydrogenated menaquinones with 11 isoprene units predominated. The combined genotypic and phenotypic data indicate that the isolates merit recognition as representing a novel species of the genus Amycolatopsis. The name proposed for this novel species is Amycolatopsis nigrescens sp. nov., with type strain CSC17Ta-90(T) (=HKI 0330(T)=DSM 44992(T)=NRRL B-24473(T)).
The phylogenetic relationship of Coptotermes gestroi (Wasmann) and Coptotermes vastator Light (Isoptera: Rhinotermitidae) was determined using DNA sequence comparisons of mitochondrial genes. Partial sequences of the ribosomal RNA small subunit 12S, ribosomal RNA large subunit 16S, and mitochondrial COII were obtained from nine populations of C. gestroi from South East Asia (Malaysia, Singapore, Thailand, and Indonesia) and four populations of C. vastator from the Philippines and Hawaii. In addition, four populations of Coptotermes formosanus Shiraki and Globitermes sulphureus (Haviland) were used as the outgroups. Consensus sequences were obtained and aligned. C. vastator and C. gestroi are synonymous, based on high sequence homology across the 12S, 16S, and COII genes. The interspecific pairwise sequence divergence, based on Kimura 2-parameter model between C. gestroi and C. vastator, varied only up to 0.80%. Morphometric measurements of 16 characteristics revealed numerous overlaps between the examined individuals of both species. Based on the molecular phylogenetics and morphometric data, it is proposed that C. vastator is a junior synonym of C. gestroi.
Burkholderia pseudomallei is a Gram-negative saprophytic soil bacterium, which is the etiologic agent of melioidosis, a severe and fatal infectious disease occurring in human and animals. Distinct clinical and animal isolates have been shown to exhibit differences in phenotypic trait such as growth rate, colony morphology, antimicrobial resistance, and virulence. This study was carried out to gain insight into the intrinsic differences between 4 clinical and 6 animal B. pseudomallei isolates from Malaysia. The 16S rRNA-encoding genes from these 10 isolates of B. pseudomallei were sequenced to confirm the identity of these isolates along with the avirulent Burkholderia thailandensis. The nucleotide sequences indicated that the 16S rRNA-encoding genes among the 10 B. pseudomallei isolates were identical to each other. However, the nucleotide sequence differences in the 16S rRNA-encoding genes appeared to be B. pseudomallei and B. thailandensis specific. The growth rate of all B. pseudomallei isolates was determined by generating growth curves at 37 degrees C for 72 h. The isolates were found to differ in growth rates with doubling time varying from 1.5 to 2.3 h. In addition, the B. pseudomallei isolates exhibited considerable variation in colony morphology when grown on Ashdown media, brain-heart infusion agar, and Luria-Bertani agar over 9 days of observation. Antimicrobial susceptibility tests indicated that 80% of the isolates examined were Amp(R) Cb(R) Kn(R) Gm(R) Chl(S) Te(S). Virulence of the B. pseudomallei clinical and animal isolates was evaluated in B. pseudomallei-susceptible BALB/c mice. Most of the clinical isolates were highly virulent. However, virulence did not correlate with isolate origin since 2 of the animal isolates were also highly virulent.
Three actinomycetes isolated from the surfaces of rocks in a medieval slate mine were examined in a polyphasic taxonomic study. Chemotaxonomic and morphological characteristics of the isolates were typical of strains of the genus Amycolatopsis. The isolates had identical 16S rRNA gene sequences and formed a distinct phyletic line towards the periphery of the Amycolatopsis mediterranei clade, being most closely related to Amycolatopsis rifamycinica. The organisms shared a wide range of genotypic and phenotypic markers that distinguished them from their closest phylogenetic neighbours. On the basis of these results, a novel species, Amycolatopsis saalfeldensis sp. nov., is proposed. The type strain is HKI 0457(T) (=DSM 44993(T)=NRRL B-24474(T)).