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.
We describe a convenient, versatile and safe method for preparing bacterial DNA for ribotyping analysis. In this method, extraction of bacterial DNA from Salmnonella typhi and Burkholderia pseudomallei. and subsequent restriction endonuclease digestion, was performed in agarose blocks/plugs thus minimizing shearing and loss of DNA, problems commonly associated with liquid phase phenol extraction. Digested DNA in the plugs was then electrophoresed directly, transferred to nylon membranes and hybridized with labeled rDNA probes in the usual manner to provide reproducible restriction patterns. This method is particularly useful for bacterial species where standard DNA extraction in the liquid phase using phenol has been problematic (e.g. B. pseudomallei) but can be used for any bacterial species. The DNA extracted within the agarose plugs can be stored for long periods and can be used in other, widely-used typing methods such as pulsed-field gel electrophoresis (PFGE) and PCR-based techniques. Embedding live cells directly in agarose plugs also minimizes the risk of exposure to these virulent human pathogens among laboratory workers.
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.
Eighty-four strains of Vibrio cholerae O1, O139 and non-O1/non-O139 from clinical and environmental sources were investigated for the presence of the toxin co-regulated pilus gene, tcpA, the virulence cassette genes ctxA, zot, ace and cep and also for their ability to elaborate haemolysin and protease. The ctxA and zot genes were detected using DNA-DNA hybridization while the ace, cep and tcpA genes were detected using PCR. Production of haemolysin and protease was detected using mammalian erythrocytes and an agar diffusion assay respectively. Analysis of their virulence profiles showed six different groups designated Type I to Type VI and the major distinguishing factor among these profiles was in the in vitro production of haemolysin and/or protease. Clinical O1, O139 and environmental O1 strains were similar with regard to presence of the virulence cassette genes. All environmental O1 strains with the exception of one were found to possess ctxA, zot and ace giving rise to the probability that these strains may actually be of clinical origin. One strain which had only cep but none of the toxin genes may be a true environmental isolate. The virulence cassette and colonization factor genes were absent in all non-O1/non-O139 environmental strains but production of both the haemolysin and protease was present, indicating that these may be putative virulence factors. These findings suggest that with regard to its pathogenic potential, only strains of the O1 and O139 serogroup that possess the tcpA gene which encodes the phage receptor, have the potential to acquire the CTX genetic element and become choleragenic.
Ten strains of Salmonella weltevreden isolated from poultry sources were examined and found to contain plasmid DNA ranging in size from 1.8 to 68.5 MD. All isolates were susceptible to carbenicillin, cephalothin, ceftriazone, gentamicin, kanamycin and nalidixic acid, but resistance to bacitracin (100%), penicillin G (100%), rifampicin (100%), sulphamethoxazole (100%), cefuroxime (80%) and tetracycline (60%) was recorded. The 55 MD plasmid of strain SW5 determined resistance to penicillin G and tetracycline, which was transmissible to the E. coli K12 recipient at a frequency of 3.52 x 10(-5) transconjugants per input donor cell. The results of arbitrarily primed polymerase chain reaction (AP-PCR), using two 10-mer oligonucleotides and PCR-ribotyping to differentiate between the ten strains of S. weltevreden were compared. The strains were separated into ten different genome types by AP-PCR but were indistinguishable by PCR-ribotyping. These results suggest that poultry may constitute a reservoir for disseminating antibiotic resistance and that AP-PCR may be a valuable tool for epidemiological studies.
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.
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.
In many parts of Asia, the inaccessibility and high cost of diagnostic tests have hampered the study of community-acquired pneumonia (CAP) caused by atypical respiratory pathogens.
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)).
A collection of 207 historically relevant Burkholderia pseudomallei isolates was analyzed by multilocus sequence typing (MLST). The strain collection contains environmental isolates obtained from a geographical distribution survey of B. pseudomallei isolates in Thailand (1964 to 1967), as well as stock cultures and colony variants from the U.S. Army Medical Research Unit (Malaysia), the Walter Reed Army Institute for Research, and the Pasteur Institute (Vietnam). The 207 isolates of the collection were resolved into 80 sequence types (STs); 56 of these were novel. eBURST diagrams predict that the historical-collection STs segregate into three complexes when analyzed separately. When added to the 760 isolates and 365 STs of the B. pseudomallei MLST database, the historical-collection STs cluster significantly within the main complex of the eBURST diagram in an ancestral pattern and alter the B. pseudomallei "population snapshot." Differences in colony morphology among reference isolates were found not to affect the STs assigned, which were consistent with the original isolates. Australian ST84 is likely characteristic of B. pseudomallei isolates of Southeast Asia rather than Australia, since multiple environmental isolates from Thailand and Malaysia share this ST with the single Australian clinical isolate in the MLST database. Phylogenetic evidence is also provided suggesting that Australian isolates may not be distinct from those of Thailand, since ST60 is common to environmental isolates from both countries. MLST and eBURST are useful tools for the study of population biology and epidemiology, since they provide methods to elucidate new genetic relationships among bacterial isolates.
Nine Escherichia coli O157: H7/- strains isolated primarily from non-clinical sources in Thailand and Japan carried the stx(2) gene but did not produce Stx2 toxin in a reversed passive latex agglutination (RPLA) assay. A strain (EDL933) bearing a stx(2) phage (933W) was compared to a strain (Thai-12) that was Stx2-negative but contained the stx(2) gene. To study the lack of Stx2 production, the Thai-12 stx(2) gene and its upstream nucleotide sequence were analyzed. The Thai-12 stx(2) coding region was intact and Stx2 was expressed from a cloned stx(2) gene using a plasmid vector and detected using RPLA. A lacZ fusion analysis found the Thai-12 stx(2) promoter non-functional. Because the stx(2) gene is downstream of the late promoter in the stx(2) phage genome, the antitermination activity of Q protein is essential for strong stx(2) transcription. Thai-12 had the q gene highly homologous to that of Phi21 phage but not to the 933W phage. High-level expression of exogenous q genes demonstrated Q antitermination activity was weak in Thai-12. Replication of stx(2) phage was not observed in Stx2-negative strains. The q-stx(2) gene sequence of Thai-12 was well conserved in all Stx2-negative strains. A PCR assay to detect the Thai-12 q-stx(2) sequence demonstrated that 30% of O157 strains from marketed Malaysian beef carried this sequence and they produced little or no Stx2. These results suggest that stx(2)-positive O157 strains that produce little or no Stx2 may be widely distributed in the Asian environment.
In this study we describe a triplex real-time PCR assay that enables the identification of S. aureus and detection of two important antibiotic resistant genes simultaneously using real-time PCR technology in a single assay. In this triplex real-time PCR assay, the mecA (methicillin resistant), femA (species specific S. aureus) and aacA-aphD (aminoglycoside resistant) genes were detected in a single test using dual-labeled Taqman probes. The assay gives simultaneous information for the identification of S. aureus and detection of methicillin and aminoglycoside resistance in staphylococcal isolates. 152 clinical isolates were subjected to this triplex real-time PCR assay. The results of the triplex real-time PCR assay correlated with the results of the phenotypic antibiotic susceptibility testing. The results obtained from triplex real-time PCR assay shows that the primer and probe sets were specific for the identification of S. aureus and were able to detect methicillin- and aminoglycoside-resistant genes. The entire assay can be performed within 3 h which is a very rapid method that can give simultaneous information for the identification of S. aureus and antibiotic resistance pattern of a staphylococcal isolate. The application of this rapid method in microbiology laboratories would be a valuable tool for the rapid identification of the S. aureus isolates and determination of their antibiotic resistance pattern with regards to methicillin and aminoglycosides.
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.
Five out of the nine benzene-toulene-ethylbenzene-xylene (BTEX) tolerant bacteria that demonstrated high protease activity on skim milk agar were isolated. Among them, isolate 115b identified as Bacillus pumilus exhibited the highest protease production. The protease produced was stable in 25% (v/v) benzene and toluene and it was activated 1.7 and 2.5- fold by n-dodecane and n-tetradecane, respectively. The gene encoding the organic solvent tolerant protease was cloned and its nucleotide sequence determined. Sequence analysis revealed an open reading frame (ORF) of 1,149 bp that encoded a polypeptide of 383 amino acid residues. The polypeptide composed of 29 residues of signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids with a calculated molecular mass of 27,846 Da. This is the only report available to date on organic solvent tolerant protease from B. pumilus.
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)).
A Na(+)/H(+) antiporter gene was isolated from alkaliphilic Bacillus sp. G1. The full-length sequence of the Na(+)/H(+) antiporter gene was obtained using a genome walking method, and designated as g1-nhaC. An ORF preceded by a promoter-like sequence and a Shine-Dalgarno sequence, and followed by a terminator-like sequence was identified. The deduced amino acid sequence consists of 535 amino acids, and a calculated molecular mass of 57 776 Da. g1-nhaC was subsequently cloned into pET22b(+) and expressed in Escherichia coli BL21 (DE3). Recombinant E. coli harboring the g1-nhaC gene was able to grow in modified L medium at various concentrations of NaCl (0.2-2.0 M) at different pH values. The recombinant bacteria grew well in the medium with concentrations of NaCl as high as 1.75 M at pH 8.0-9.0. Minimal growth was observed at 2.0 M NaCl, pH 8.0-9.0. At pH 10, the recombinant bacteria grew well in a medium with a low concentration of NaCl (0.2 M). These results suggested that the g1-NhaC antiporter from Bacillus sp. G1 plays a role in Na(+) extrusion at lower pH values and in pH homeostasis at pH 10 under Na(+)-limiting conditions.
Genes encoding the quinolones resistance determining regions (QRDRs) in Streptococcus pneumoniae were detected by PCR and the sequence analysis was carried out to identify point mutations within these regions. The study was carried out to observe mutation patterns among S. pneumoniae strains in Malaysia. Antimicrobial susceptibility testing of 100 isolates was determined against various antibiotics, out of which 56 strains were categorised to have reduced susceptibility to ciprofloxacin (>or=2 microg/mL). These strains were subjected to PCR amplification for presence of the gyrA, parC , gyrB and parE genes. Eight representative strains with various susceptibilities to fluoroquinolones were sequenced. Two out of the eight isolates that were sequenced were shown to have a point mutation in the gyrA gene at position Ser81. The detection of mutation at codon Ser81 of the gyrA gene suggested the potential of developing fluoroquinolone resistance among S. pneumoniae isolates in Malaysia. However, further experimental work is required to confirm the involvement of this mutation in the development of fluoroquinolone resistance in Malaysia.
Efflux-mediated resistance has been recognized as an important contributor of antibiotic resistance in bacteria, especially in methicillin-resistant Staphylococcus aureus (MRSA) isolates. This study was carried out to detect and analyze efflux genes (norA and mdeA) and active efflux activity in a collection of Malaysian MRSA and methicillin-sensitive S. aureus (MSSA) clinical isolates. Nineteen isolates including three ATCC S. aureus reference strains were subjected to PCR detection and DNA sequence analysis for norA and mdeA and active efflux detection using modified minimum inhibitory concentration (MIC) assay. From the 19 isolates, 18 isolates harboured the mdeA gene while 16 isolates contained norA gene. DNA sequence analysis reveals 98-100% correlation between the PCR product and the published DNA sequences in GenBank. In addition, 16 isolates exhibited active efflux activity using the ethidium bromide (EtBr)-reserpine combination MIC assay. To our knowledge, this is the first report on the detection of efflux genes and active efflux activity amongst Malaysian clinical isolates of MRSA/MSSA. Detection of active efflux activity may explain the previous report on efflux-mediated drug resistance profile amongst the local clinical isolates.