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Diaminobenzene schiff base, a novel class of DNA minor groove binder

Helal MH, Al-Mudaris ZA, Al-Douh MH, Osman H, Wahab HA, Alnajjar BO, et al.

Int J Oncol, 2012 Aug;41(2):504-10.
PMID: 22614449 DOI: 10.3892/ijo.2012.1491

Molecules that target the deoxyribonucleic acid (DNA) minor groove are relatively sequence specific and they can be excellent carrier structures for cytotoxic chemotherapeutic compounds which can help to minimize side effects. Two novel isomeric derivatives of diaminobenzene Schiff base [N,N'-bis (2-hydroxy-3-methoxybenzylidene)-1,2-diaminobenzene (2MJ) and N,N'-bis(2-hydroxy-3-methoxybenzylidene)-1,3-diaminobenzene (2MH)] were analyzed for their DNA minor groove binding (MGB) ability using viscometry, UV and fluorescence spectroscopy, computational modeling and clonogenic assay. The result shows that 2MJ and 2MH are strong DNA MGBs with the latter being more potent. 2MH can form interstrand hydrogen bond linkages at its oxygens with N3 of adenines. Changing the 2-hydroxy-3-methoxybenzylidene binding position to the 1,3 location on the diaminobenzene structure (2MJ) completely removed any viable hydrogen bond formation with the DNA and caused significant decrease in binding strength and minor groove binding potency. Neither compound showed any significant cytotoxicity towards human breast, colon or liver cancer cell lines.

Matched MeSH terms: DNA/chemistry*
Cloning of glyceraldehyde-3-phosphate dehydrogenase from an Antarctic psychrophilic bacterium by inverse and splinkerette PCR

Too WC, Liew YC, Few LL

J Basic Microbiol, 2008 Oct;48(5):430-5.
PMID: 18759222 DOI: 10.1002/jobm.200800008

Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterium codenamed pi9 was selected for the cloning of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme in the glycolytic pathway. Here, the cloning of an 1,113 bp fragment of GAPDH gene which covers the 1,002 bp open reading frame by using multiple PCR steps is described. The partial sequence of this gene was PCR amplified by using degenerate primers followed by the cloning of the flanking sequences by inverse and splinkerette PCR techniques. The success in cloning the GAPDH gene by PCR has bypassed the more time consuming genomic library construction and screening method. The full length GAPDH protein was subsequently expressed in E. coli, purified as His-tag protein and confirmed to be catalytically active. This work demonstrated the use of multiple PCR techniques to clone a gene based solely on sequence comparison. It also laid the foundation for further biochemical and structural characterizations of GAPDH from a psychrophilic bacterium by providing a highly purified recombinant protein sample.

Matched MeSH terms: DNA, Bacterial/genetics
The role of TGFbeta in human cancers

Wong SF, Lai LC

Pathology, 2001 Feb;33(1):85-92.
PMID: 11280615

Transforming growth factor beta (TGFbeta) is secreted as a large latent precursor from both normal and transformed cells which needs to be activated for biological activity. The active TGFbeta binds either directly to TbetaR-II or indirectly by binding to beta-glycan which then presents the TGFbeta to TbetaR-II. Formation of the TGFbeta-TbetaR-II complex rapidly leads to phosphorylation of TbetaR-I. TbetaR-I, in turn, phosphorylates receptor-specific Smads and induces their translocation into the nucleus. TGFbeta is able to act as a growth stimulator or inhibitor and elicits a broad spectrum of biological effects on various cell types. However, these cells may lose their sensitivity and responsiveness to TGFbeta. Down-regulation or loss of functional receptors, aberrant signal transduction pathways due to Smad mutations, loss of the cell's ability to activate latent TGFbeta, loss of the peptide itself or functional genes that control the transcription and translation of TGFbeta may contribute to development of cancer.

Matched MeSH terms: DNA-Binding Proteins/metabolism
Fulltext Population structure of Helicobacter pylori among ethnic groups in Malaysia: recent acquisition of the bacterium by the Malay population

Tay CY, Mitchell H, Dong Q, Goh KL, Dawes IW, Lan R

BMC Microbiol, 2009;9:126.
PMID: 19538757 DOI: 10.1186/1471-2180-9-126

Helicobacter pylori is a major gastric bacterial pathogen. This pathogen has been shown to follow the routes of human migration by their geographical origin and currently the global H. pylori population has been divided into six ancestral populations, three from Africa, two from Asia and one from Europe. Malaysia is made up of three major ethnic populations, Malay, Chinese and Indian, providing a good population for studying recent H. pylori migration and admixture.

Matched MeSH terms: DNA, Bacterial/genetics
Prevalence and molecular study of G6PD deficiency in Malaysian Orang Asli

Amini F, Ismail E, Zilfalil BA

Intern Med J, 2011 Apr;41(4):351-3.
PMID: 21507164 DOI: 10.1111/j.1445-5994.2011.02456.x

This study aims to define the prevalence and the molecular basis of G6PD deficiency in the Negrito tribe of the Malaysian Orang Asli. Four hundred and eighty seven consenting Negrito volunteers were screened for G6PD deficiency through the use of a fluorescent spot test. DNA from deficient individuals underwent PCR-RFLP analysis using thirteen recognized G6PD mutations. In the instances when the mutation could not be identified by PCR-RFLP, the entire coding region of the G6PD gene was subjected to DNA sequencing. In total, 9% (44/486) of the sample were found to be G6PD-deficient. However, only 25 samples were subjected to PCR-RFLP and DNA sequencing. Of these, three were found to carry Viangchan, one Coimbra and 16, a combination of C1311T in exon 11 and IVS11 T93C. Mutation(s) for the five remaining samples are unknown. The mean G6PD enzyme activity ranged 5.7 IU/gHb in deficient individuals. Our results demonstrate that the frequency of G6PD deficiency is higher among the Negrito Orang Asli than other Malaysian races. The dual presence of C1311T and IVS11 T93C in 64% of the deficient individuals (16/44) could well be a result of genetic drift within this isolated group.

Matched MeSH terms: Sequence Analysis, DNA/methods
Development and partial characterization of new marine cell line from brain of Asian sea bass Lates calcarifer for virus isolation

Hasoon MF, Daud HM, Abdullah AA, Arshad SS, Bejo HM

In Vitro Cell Dev Biol Anim, 2011 Jan;47(1):16-25.
PMID: 21082288 DOI: 10.1007/s11626-010-9348-5

A new cell line, Asian sea bass brain (ASBB), was derived from the brain tissue of Asian sea bass Lates calcarifer. This cell line was maintained in Leibovitz L-15 media supplemented with 10% fetal bovine serum (FBS). The ASBB cell line was subcultured more than 60 times over a period of 15 mo. The ASBB cell line consists predominantly of fibroblastic-like cells and was able to grow at temperatures between 20°C and 30°C with an optimum temperature of 25°C. The growth rate of these cells increased as the proportion of FBS increased from 2% to 20% at 25°C with optimum growth at the concentrations of 10% or 15% FBS. Polymerase chain reaction products were obtained from ASBB cells and tissues of sea bass with primer sets of microsatellite markers of sea bass. An isolate of piscine nodavirus from juveniles of marine fish species tested positive by IQ2000 kit for viral nervous necrosis detection and was examined for its infectivity to a fish cell line of ASBB. A marine fish betanodavirus was tested to determine the susceptibility of this new cell line in comparison with commercial highly permissive SSN-1 cells. The ASBB cell line was found to be susceptible to nodavirus (RGNNV genotype), and the infection was confirmed by comparison cytopathic effect (CPE) with commercial SSN-1 and reverse transcriptase-polymerase chain reaction. A nodavirus was further elucidated by electron microscopy, and the virus tested was shown to induce CPE on ASBB cells with significant high titer. This suggests that the ASBB cell line has good potential for the isolation of fish viruses.

Matched MeSH terms: DNA Primers/genetics
HIV type 1 subtypes in Malaysia include B, C, and E

Brown TM, Robbins KE, Sinniah M, Saraswathy TS, Lee V, Hooi LS, et al.

AIDS Res Hum Retroviruses, 1996 Nov 20;12(17):1655-7.
PMID: 8947304
Matched MeSH terms: DNA, Viral/analysis
Fulltext Whole-genome sequencing to establish relapse or re-infection with Mycobacterium tuberculosis: a retrospective observational study

Bryant JM, Harris SR, Parkhill J, Dawson R, Diacon AH, van Helden P, et al.

Lancet Respir Med, 2013 Dec;1(10):786-92.
PMID: 24461758 DOI: 10.1016/S2213-2600(13)70231-5

BACKGROUND: Recurrence of tuberculosis after treatment makes management difficult and is a key factor for determining treatment efficacy. Two processes can cause recurrence: relapse of the primary infection or re-infection with an exogenous strain. Although re-infection can and does occur, its importance to tuberculosis epidemiology and its biological basis is still debated. We used whole-genome sequencing-which is more accurate than conventional typing used to date-to assess the frequency of recurrence and to gain insight into the biological basis of re-infection.
METHODS: We assessed patients from the REMoxTB trial-a randomised controlled trial of tuberculosis treatment that enrolled previously untreated participants with Mycobacterium tuberculosis infection from Malaysia, South Africa, and Thailand. We did whole-genome sequencing and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing of pairs of isolates taken by sputum sampling: one from before treatment and another from either the end of failed treatment at 17 weeks or later or from a recurrent infection. We compared the number and location of SNPs between isolates collected at baseline and recurrence.
FINDINGS: We assessed 47 pairs of isolates. Whole-genome sequencing identified 33 cases with little genetic distance (0-6 SNPs) between strains, deemed relapses, and three cases for which the genetic distance ranged from 1306 to 1419 SNPs, deemed re-infections. Six cases of relapse and six cases of mixed infection were classified differently by whole-genome sequencing and MIRU-VNTR. We detected five single positive isolates (positive culture followed by at least two negative cultures) without clinical evidence of disease.
INTERPRETATION: Whole-genome sequencing enables the differentiation of relapse and re-infection cases with greater resolution than do genotyping methods used at present, such as MIRU-VNTR, and provides insights into the biology of recurrence. The additional clarity provided by whole-genome sequencing might have a role in defining endpoints for clinical trials.
FUNDING: Wellcome Trust, European Union, Medical Research Council, Global Alliance for TB Drug Development, European and Developing Country Clinical Trials Partnership.

Matched MeSH terms: DNA, Bacterial/genetics*
Fulltext Differences in the bacteriome of swab, saliva, and tissue biopsies in oral cancer

Gopinath D, Menon RK, Wie CC, Banerjee M, Panda S, Mandal D, et al.

Sci Rep, 2021 01 13;11(1):1181.
PMID: 33441939 DOI: 10.1038/s41598-020-80859-0

Microbial dysbiosis has been implicated in the pathogenesis of oral cancer. We analyzed the compositional and metabolic profile of the bacteriome in three specific niches in oral cancer patients along with controls using 16SrRNA sequencing (Illumina Miseq) and DADA2 software. We found major differences between patients and control subjects. Bacterial communities associated with the tumor surface and deep paired tumor tissue differed significantly. Tumor surfaces carried elevated abundances of taxa belonging to genera Porphyromonas, Enterobacteriae, Neisseria, Streptococcus and Fusobacteria, whereas Prevotella, Treponema, Sphingomonas, Meiothermus and Mycoplasma genera were significantly more abundant in deep tissue. The most abundant microbial metabolic pathways were those related to fatty-acid biosynthesis, carbon metabolism and amino-acid metabolism on the tumor surface: carbohydrate metabolism and organic polymer degradation were elevated in tumor tissues. The bacteriome of saliva from patients with oral cancer differed significantly from paired tumor tissue in terms of community structure, however remained similar at taxonomic and metabolic levels except for elevated abundances of Streptococcus, Lactobacillus and Bacteroides, and acetoin-biosynthesis, respectively. These shifts to a pro-inflammatory profile are consistent with other studies suggesting oncogenic properties. Importantly, selection of the principal source of microbial DNA is key to ensure reliable, reproducible and comparable results in microbiome studies.

Matched MeSH terms: DNA, Bacterial/genetics
Molecular detection of pathogens from ectoparasites recovered from peri-domestic animals, and the first description of a Candidatus Midichloria sp. from Haemaphysalis wellingtoni from rural communities in Malaysia

Khoo JJ, Husin NA, Lim FS, Oslan SNH, Mohd Azami SNI, To SW, et al.

Parasitol Int, 2021 Feb;80:102202.
PMID: 33038482 DOI: 10.1016/j.parint.2020.102202

Rural communities in Malaysia have been shown to be exposed to Coxiella, Borrelia and rickettsial infections in previous seroprevalence studies. Further research is necessary to identify the actual causative agents and the potential vectors of these infections. The arthropods parasitizing peri-domestic animals in these communities may serve as the vector in transmitting arthropod-borne and zoonotic agents to the humans. Molecular screening of bacterial and zoonotic pathogens from ticks and fleas collected from dogs, cats and chickens from six rural communities in Malaysia was undertaken. These communities were made up of mainly the indigenous people of Malaysia, known as the Orang Asli, as well as settlers in oil palm plantations. The presence of Coxiella burnetii, Borrelia, and rickettsial agents, including Rickettsia and Anaplasma, was investigated by performing polymerase chain reaction (PCR) and DNA sequencing. Candidatus Rickettsia senegalensis was detected in one out of eight pools of Ctenocephalides felis fleas. A relapsing fever group Borrelia sp. was identified from one of seven Haemaphysalis hystricis ticks tested. The results from the PCR screening for Anaplasma unexpectedly revealed the presence of Candidatus Midichloria sp., a potential tick endosymbiont, in two out of fourteen Haemaphysalis wellingtoni ticks tested. C. burnetii was not detected in any of the samples tested. The findings here provide evidence for the presence of potentially novel strains of rickettsial and borrelial agents in which their impact on public health risks among the rural communities in Malaysia merit further investigation. The detection of a potential endosymbiont of ticks also suggest that the presence of tick endosymbionts in the region is not fully explored.

Matched MeSH terms: Sequence Analysis, DNA/veterinary
Fulltext Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny

Cappellini E, Welker F, Pandolfi L, Ramos-Madrigal J, Samodova D, Rüther PL, et al.

Nature, 2019 10;574(7776):103-107.
PMID: 31511700 DOI: 10.1038/s41586-019-1555-y

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.

Matched MeSH terms: DNA, Ancient/analysis*
Fulltext A Sensitive, Colorimetric, High-Throughput Loop-Mediated Isothermal Amplification Assay for the Detection of Plasmodium knowlesi

Britton S, Cheng Q, Grigg MJ, William T, Anstey NM, McCarthy JS

Am J Trop Med Hyg, 2016 07 06;95(1):120-2.
PMID: 27162264 DOI: 10.4269/ajtmh.15-0670

The simian parasite Plasmodium knowlesi is now the commonest cause of malaria in Malaysia and can rapidly cause severe and fatal malaria. However, microscopic misdiagnosis of Plasmodium species is common, rapid antigen detection tests remain insufficiently sensitive and confirmation of P. knowlesi requires polymerase chain reaction (PCR). Thus available point-of-care diagnostic tests are inadequate. This study reports the development of a simple, sensitive, colorimetric, high-throughput loop-mediated isothermal amplification assay (HtLAMP) diagnostic test using novel primers for the detection of P. knowlesi. This assay is able to detect 0.2 parasites/μL, and compared with PCR has a sensitivity of 96% for the detection of P. knowlesi, making it a potentially field-applicable point-of-care diagnostic tool.

Matched MeSH terms: DNA Primers/genetics
Fulltext Genome-Wide Novel Genic Microsatellite Marker Resource Development and Validation for Genetic Diversity and Population Structure Analysis of Banana

Biswas MK, Bagchi M, Biswas D, Harikrishna JA, Liu Y, Li C, et al.

Genes (Basel), 2020 12 09;11(12).
PMID: 33317074 DOI: 10.3390/genes11121479

Trait tagging through molecular markers is an important molecular breeding tool for crop improvement. SSR markers encoded by functionally relevant parts of a genome are well suited for this task because they may be directly related to traits. However, a limited number of these markers are known for Musa spp. Here, we report 35136 novel functionally relevant SSR markers (FRSMs). Among these, 17,561, 15,373 and 16,286 FRSMs were mapped in-silico to the genomes of Musa acuminata, M. balbisiana and M. schizocarpa, respectively. A set of 273 markers was validated using eight accessions of Musa spp., from which 259 markers (95%) produced a PCR product of the expected size and 203 (74%) were polymorphic. In-silico comparative mapping of FRSMs onto Musa and related species indicated sequence-based orthology and synteny relationships among the chromosomes of Musa and other plant species. Fifteen FRSMs were used to estimate the phylogenetic relationships among 50 banana accessions, and the results revealed that all banana accessions group into two major clusters according to their genomic background. Here, we report the first large-scale development and characterization of functionally relevant Musa SSR markers. We demonstrate their utility for germplasm characterization, genetic diversity studies, and comparative mapping in Musa spp. and other monocot species. The sequences for these novel markers are freely available via a searchable web interface called Musa Marker Database.

Matched MeSH terms: DNA, Plant/genetics
Fulltext Can telomere length predict bone health? A review of current evidence

Wong SK, Ima-Nirwana S, Chin KY

Bosn J Basic Med Sci, 2020 Nov 02;20(4):423-429.
PMID: 32156247 DOI: 10.17305/bjbms.2020.4664

Telomeres are repetitive DNA sequences located at the end of chromosomes that serve as a protective barrier against chromosomal deterioration during cell division. Approximately 50-200 base pairs of nucleotides are lost per cell division, and new repetitive nucleotides are added by the enzyme telomerase, allowing telomere maintenance. Telomere shortening has been proposed as an indicator for biological aging, but its relationship with age-related osteoporosis is ambiguous. We summarize the current evidence on the relationship between telomere length and bone health in experimental and epidemiological studies, which serve as a scientific reference for the development of novel diagnostic markers of osteoporosis or novel therapeutics targeting telomere and telomerase of bone cells to treat osteoporosis.

Matched MeSH terms: DNA/analysis
Fulltext Evaluation of BOX-PCR and ERIC-PCR as Molecular Typing Tools for Pathogenic Leptospira

Bilung LM, Pui CF, Su'ut L, Apun K

Dis Markers, 2018;2018:1351634.
PMID: 30154937 DOI: 10.1155/2018/1351634

In the last decades, leptospirosis had gained public health concern due to morbidity and mortality rates caused by pathogenic Leptospira. The need for rapid and robust molecular typing methods to differentiate this zoonotic pathogen is of utmost importance. Various studies had been conducted to determine the genetic relatedness of Leptospira isolates using molecular typing methods. In this study, 29 pathogenic Leptospira isolates from rat, soil, and water samples in Sarawak, Malaysia, were characterized using BOX-PCR and ERIC-PCR. The effectiveness of these two methods with regard to the ease of interpretation, reproducibility, typeability, and discriminatory power was also being evaluated. Using BOX-PCR, six clusters and 3 single isolates were defined at a genetic distance percentage of 11.2%. ERIC-PCR clustered the isolates into 6 clusters and 2 single isolates at a genetic distance percentage of 6.8%. Both BOX-PCR and ERIC-PCR produced comparable results though the discriminatory index for ERIC-PCR (0.826) was higher than that for BOX-PCR (0.809). From the constructed dendrogram, it could be summarized that the isolates in this study were highly heterogeneous and genetically diverse. The findings from this study indicated that there is no genetic relatedness among the pathogenic Leptospira isolates in relation to the locality, source, and identity, with some exceptions. Out of the 29 pathogenic Leptospira isolates studied, BOX-PCR and ERIC-PCR successfully discriminated 4 isolates (2 isolates each) into the same cluster in relation to sample sources, as well as 2 isolates into the same cluster in association with the sample locality. Future studies shall incorporate the use of other molecular typing methods to make a more thorough comparison on the genetic relatedness of pathogenic Leptospira.

Matched MeSH terms: DNA, Bacterial/genetics*
Development of a species-specific PCR-RFLP targeting rpoD gene fragment for discrimination of Aeromonas species

Puah SM, Khor WC, Kee BP, Tan JAMA, Puthucheary SD, Chua KH

J Med Microbiol, 2018 Sep;67(9):1271-1278.
PMID: 30024365 DOI: 10.1099/jmm.0.000796

PURPOSE: The taxonomy of Aeromonas keeps expanding and their identification remains problematic due to their phenotypic and genotypic heterogeneity. In this study, we aimed to develop a rapid and reliable polymerase chain reaction-restriction fragment length polymorphism assay targeting the rpoD gene to enable the differentiation of aeromonads into 27 distinct species using microfluidic capillary electrophoresis.
METHODOLOGY: A pair of degenerate primers (Aero F: 5'-YGARATCGAYATCGCCAARCGB-3' and Aero R: 5'-GRCCDATGCTCATRCGRCGGTT-3') was designed that amplified the rpoD gene of 27 Aeromonas species. Subsequently, in silico analysis enabled the differentiation of 25 species using the single restriction endonuclease AluI, while 2 species, A. sanarelli and A. taiwanensis, required an additional restriction endonuclease, HpyCH4IV. Twelve type strains (A. hydrophila ATCC7966T, A. caviae ATCC15468T, A. veronii ATCC9071T, A. media DSM4881T, A. allosaccharophila DSM11576T, A. dhakensis DSM17689T, A. enteropelogens DSM7312T, A. jandaei DSM7311T, A. rivuli DSM22539T, A. salmonicida ATCC33658T, A. taiwanensis DSM24096T and A. sanarelli DSM24094T) were randomly selected from the 27 Aeromonas species for experimental validation.Results/key findings. The twelve type strains demonstrated distinctive RFLP patterns and supported the in silico digestion. Subsequently, 60 clinical and environmental strains from our collection, comprising nine Aeromonas species, were used for screening examinations, and the results were in agreement.
CONCLUSION: This method provides an alternative method for laboratory identification, surveillance and epidemiological investigations of clinical and environmental specimens.

Matched MeSH terms: DNA Primers/genetics
Fulltext Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme

Hancock SJ, Phan MD, Peters KM, Forde BM, Chong TM, Yin WF, et al.

Antimicrob Agents Chemother, 2017 02;61(2).
PMID: 27872077 DOI: 10.1128/AAC.01740-16

Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including blaCMY and blaNDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a blaNDM-1-positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of blaNDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this blaNDM-containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies.

Matched MeSH terms: DNA Transposable Elements; DNA Replication
Fulltext Development and evaluation of a Multiplex Polymerase Chain Reaction for the detection of Salmonella species

Thong KL, Teh CS, Chua KH

Trop Biomed, 2014 Dec;31(4):689-97.
PMID: 25776594 MyJurnal

The present study aims to develop a system which consists of four pairs of primers that specifically detects Salmonella spp., Salmonella serovar Typhi and Salmonella serovar Paratyphi A with an internal amplification control. The system, when applied in Polymerase Chain Reaction (PCR) under specific conditions, reaction mixture and cycling temperatures produced four bands; 784 bp, 496 bp, 332 bp and 187 bp. The DNA band 784 bp is present in all Salmonella spp., while the bands of 496 bp and 332 bp are only present in S. Paratyphi A and S. Typhi, respectively. An internal amplification control as indicated by the 187 bp shows the system is working in optimum condition in all the tests. This multiplex PCR was evaluated on 241 bacterial cultures and 691 naturally contaminated samples. Overall, this multiplex PCR detection system provides a single step for simultaneous detection of DNAs of Salmonella spp., S. Typhi and S. Paratyphi A.

Matched MeSH terms: DNA Primers/genetics
Fulltext DNA fingerprinting of human isolates of Burkholderia pseudomallei from different geographical regions of Malaysia

Chua KH, See KH, Thong KL, Puthucheary SD

Trop Biomed, 2010 Dec;27(3):517-24.
PMID: 21399594 MyJurnal

Melioidosis is an infectious disease caused by Burkholderia pseudomallei and endemic in Southeast Asia. One hundred and forty six clinical isolates of B. pseudomallei from different states in Malaysia were obtained and molecular typing was carried out using pulsed-field gel electrophoresis (PFGE). Overall, nine clusters were successfully identified. Burkholderia pseudomallei isolates used in this study were found to be genetically diverse and there were differences in the clusters of isolates from peninsular and east Malaysia. BS9 cluster was the most common cluster and found in all the states while BS2 cluster only existed in a particular state. Based on the PFGE analysis, the distribution of different B. pseudomallei clinical isolates in Malaysia was mapped.

Matched MeSH terms: DNA Fingerprinting*
Fulltext Capsular serotyping of Pasteurella multocida from various animal hosts - a comparison of phenotypic and genotypic methods

Arumugam ND, Ajam N, Blackall PJ, Asiah NM, Ramlan M, Maria J, et al.

Trop Biomed, 2011 Apr;28(1):55-63.
PMID: 21602769

One hundred and fourteen strains of Pasteurella multocida were isolated from different domestic animals species (cattle, buffalo, sheep, goat, pig, rabbit, dog, cat), avian species (chicken, duck, turkey) and wild animals (deer, tiger, orang utan, marmoset). The serogroups of P. multocida were determined by both conventional capsular serotyping and a multiplex PCR assay targeting specific capsular genes. Based on the conventional serotyping method, the 114 strains of P. multocida were subtyped into 55 species-specific (untypeable strains) P. multocida, 15 serogroup A, 23 serogroup B and 21 serogroup D. Based on the multiplex PCR assay on the specific capsular genes associated with each serogroup, the 114 strains were further divided to 22 species-specific P. multocida (KMT1 - 460 bp), 53 serogroup A (A - 1,044 bp), 33 serogroup B (B - 760 bp) and 6 serogroup D (D - 657 bp). No serogroup E (511 bp) or F (851 bp) was detected among the Malaysian P. multocida. PCR-based typing was more discriminative and could further subtype the previously untypeable strains. Overall, there was a significant and positive correlation between both methods in serogrouping P. multocida (r = 0.7935; p<0.4893). Various serogroups of P. multocida were present among the livestock with 75% of the strains belonging to serogroups A or B. PCR serotyping was therefore a highly species-specific, sensitive and robust method for detection and differentiation of P. multocida serogroups compared to conventional serotyping. To the best of our knowledge, this is the first report from Malaysia of the application of a PCR to rapidly define the species-specific P. multocida and its serogroups as an important zoonotic pathogen in Malaysia.

Matched MeSH terms: DNA, Bacterial/genetics

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