Displaying publications 21 - 40 of 319 in total

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  1. Electrotransformation of thermophilic bacterium Caldimonas manganoxidans
    Arai T, Aikawa S, Sudesh K, Kondo T, Kosugi A
    J Microbiol Methods, 2022 01;192:106375.
    PMID: 34793853 DOI: 10.1016/j.mimet.2021.106375
    Caldimonas manganoxidans is a Gram-negative, thermophilic, bioplastic-producing bacterium that is a promising strain to overcome the drawbacks of existing bioplastic manufacturing methods. However, genetic manipulation of this species has not previously been studied. Here, we developed an optimized electrotransformation protocol for C. manganoxidans by screening conditions, including the bacterial growth phase, electroporation buffer, pulse strength, and recovery time. The optimized transformation protocol obtained (3.1 ± 0.78) × 108 colony-forming units/μg DNA of plasmid pBBR1MCS-2. High transformation efficiency was observed when using plasmid DNA isolated from C. manganoxidans. The DNA methylases of Escherichia coli did not affect the transformation efficiency of C. manganoxidans. The electrotransformation technique proposed here will be beneficial for the genetic manipulation of thermophilic Caldimonas species.
    Matched MeSH terms: DNA, Bacterial/genetics
  2. Isolation and characterization of a phenol-degrading Rhodococcus sp. strain AQ5NOL 2 KCTC 11961BP
    Arif NM, Ahmad SA, Syed MA, Shukor MY
    J Basic Microbiol, 2013 Jan;53(1):9-19.
    PMID: 22581645 DOI: 10.1002/jobm.201100120
    In this work, we report on the isolation of a phenol-degrading Rhodococcus sp. with a high tolerance towards phenol. The isolate was identified as Rhodococcus sp. strain AQ5NOL 2, based on 16S rDNA analysis. The strain degraded phenol using the meta pathway, a trait shared by many phenol-degraders. In addition to phenol biodegradation, the strain was also capable of degrading diesel. Strain AQ5NOL 2 exhibited a broad optimum temperature for growth on phenol at between 20 °C and 35 °C. The best nitrogen sources were ammonium sulphate, glycine or phenylalanine, followed by proline, nitrate, leucine, and alanine (in decreasing efficiency). Strain AQ5NOL 2 showed a high tolerance and degradation capacity of phenol, for it was able to register growth in the presence of 2000 mg l(-1) phenol. The growth of this strain on phenol as sole carbon and energy source were modeled using Haldane kinetics with a maximal specific growth rate (μ(max)) of 0.1102 hr(-1), a half-saturation constant (K(s) ) of 99.03 mg l(-1) or 1.05 mmol l(-1), and a substrate inhibition constant (K(i)) of 354 mg l(-1) or 3.76 mmol l(-1). Aside from phenol, the strain could utilize diesel, 2,4-dinitrophenol and ρ-cresol as carbon sources for growth. Strain AQ5NOL 2 exhibited inhibition of phenol degradation by Zn(2+), Cu(2+), Cr(6+), Ag(+) and Hg(2+) at 1 mg l(-1).
    Matched MeSH terms: DNA, Bacterial/genetics
  3. An ultrasensitive hollow-silica-based biosensor for pathogenic Escherichia coli DNA detection
    Ariffin EY, Lee YH, Futra D, Tan LL, Karim NHA, Ibrahim NNN, et al.
    Anal Bioanal Chem, 2018 Mar;410(9):2363-2375.
    PMID: 29504083 DOI: 10.1007/s00216-018-0893-1
    A novel electrochemical DNA biosensor for ultrasensitive and selective quantitation of Escherichia coli DNA based on aminated hollow silica spheres (HSiSs) has been successfully developed. The HSiSs were synthesized with facile sonication and heating techniques. The HSiSs have an inner and an outer surface for DNA immobilization sites after they have been functionalized with 3-aminopropyltriethoxysilane. From field emission scanning electron microscopy images, the presence of pores was confirmed in the functionalized HSiSs. Furthermore, Brunauer-Emmett-Teller (BET) analysis indicated that the HSiSs have four times more surface area than silica spheres that have no pores. These aminated HSiSs were deposited onto a screen-printed carbon paste electrode containing a layer of gold nanoparticles (AuNPs) to form a AuNP/HSiS hybrid sensor membrane matrix. Aminated DNA probes were grafted onto the AuNP/HSiS-modified screen-printed electrode via imine covalent bonds with use of glutaraldehyde cross-linker. The DNA hybridization reaction was studied by differential pulse voltammetry using an anthraquinone redox intercalator as the electroactive DNA hybridization label. The DNA biosensor demonstrated a linear response over a wide target sequence concentration range of 1.0×10-12-1.0×10-2 μM, with a low detection limit of 8.17×10-14 μM (R2 = 0.99). The improved performance of the DNA biosensor appeared to be due to the hollow structure and rough surface morphology of the hollow silica particles, which greatly increased the total binding surface area for high DNA loading capacity. The HSiSs also facilitated molecule diffusion through the silica hollow structure, and substantially improved the overall DNA hybridization assay. Graphical abstract Step-by-step DNA biosensor fabrication based on aminated hollow silica spheres.
    Matched MeSH terms: DNA, Bacterial/genetics
  4. 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
  5. Fulltext Multi locus sequence typing of clinical Burkholderia pseudomallei isolates from Malaysia
    Arushothy R, Amran F, Samsuddin N, Ahmad N, Nathan S
    PLoS Negl Trop Dis, 2020 12;14(12):e0008979.
    PMID: 33370273 DOI: 10.1371/journal.pntd.0008979
    BACKGROUND: Melioidosis is a neglected tropical disease with rising global public health and clinical importance. Melioidosis is endemic in Southeast Asia and Northern Australia and is of increasing concern in Malaysia. Despite a number of reported studies from Malaysia, these reports are limited to certain parts of the country and do not provide a cohesive link between epidemiology of melioidosis cases and the nation-wide distribution of the causative agent Burkholderia pseudomallei.

    METHODOLOGY/PRINCIPLE FINDINGS: Here we report on the distribution of B. pseudomallei sequence types (STs) in Malaysia and how the STs are related to STs globally. We obtained 84 culture-confirmed B. pseudomallei from confirmed septicaemic melioidosis patients from all over Malaysia. Prior to performing Multi Locus Sequence Typing, the isolates were subjected to antimicrobial susceptibility testing and detection of the YLF/BTFC genes and BimA allele. Up to 90.5% of the isolates were sensitive to all antimicrobials tested while resistance was observed for antimicrobials typically administered during the eradication stage of treatment. YLF gene cluster and bimABp allele variant were detected in all the isolates. The epidemiological distribution patterns of the Malaysian B. pseudomallei isolates were analysed in silico using phylogenetic tools and compared to Southeast Asian and world-wide isolates. Genotyping of the 84 Malaysian B. pseudomallei isolates revealed 29 different STs of which 6 (7.1%) were novel. ST50 was identified as the group founder followed by subgroup founders ST376, ST211 and ST84. A low-level diversity is noted for the B. pseudomallei isolates described in this study while phylogenetic analysis associated the Malaysian STs to Southeast Asian isolates especially isolates from Thailand. Further analysis also showed a strong association that implicates agriculture and domestication activities as high-risk routes of infection.

    CONCLUSIONS/SIGNIFICANCE: In conclusion, MLST analysis of B. pseudomallei clinical isolates from all states in Malaysia revealed low diversity and a close association to Southeast Asian isolates.

    Matched MeSH terms: DNA, Bacterial/genetics
  6. Nocardiopsis deserti sp. nov., isolated from a high altitude Atacama Desert soil
    Asem MD, Salam N, Idris H, Zhang XT, Bull AT, Li WJ, et al.
    Int J Syst Evol Microbiol, 2020 May;70(5):3210-3218.
    PMID: 32320378 DOI: 10.1099/ijsem.0.004158
    The taxonomic status of a Nocardiopsis strain, designated H13T, isolated from a high altitude Atacama Desert soil, was established by using a polyphasic approach. The strain was found to have chemotaxonomic, cultural and morphological characteristics consistent with its classification within the genus Nocardiopsis and formed a well-supported clade in the Nocardiopsis phylogenomic tree together with the type strains of Nocardiopsis alborubida, Nocardiopsis dassonvillei and Nocardiopsis synnematoformans. Strain H13T was distinguished from its closest relatives by low average nucleotide identity (93.2-94.9 %) and in silico DNA-DNA hybridization (52.5-62.4 %) values calculated from draft genome assemblies and by a range of phenotypic properties. On the basis of these results, it is proposed that the isolate be assigned to the genus Nocardiopsis as Nocardiopsis deserti sp. nov. with isolate H13T (=CGMCC 4.7585T=KCTC 49249T) as the type strain.
    Matched MeSH terms: DNA, Bacterial/genetics
  7. Detection of Bartonella sp. in ticks and their small mammal hosts in mangrove forests of Peninsular Malaysia
    Asyikha R, Sulaiman N, Mohd-Taib FS
    Trop Biomed, 2020 Dec 01;37(4):919-931.
    PMID: 33612746 DOI: 10.47665/tb.37.4.919
    Bacteria of the genus Bartonella have been known as emerging zoonotic pathogens for several human diseases including cat scratch disease, Carrion's disease and trench fever. Numerous species of small mammals have been reported to play a role as a suitable reservoir to many pathogenic Bartonella. These infections are thought to be transmitted through blood-feeding arthropod vectors such as ticks, fleas and lice. The purpose of this study is to detect the presence of Bartonella species from tick samples collected from small mammals in mangrove forests of Peninsular Malaysia. Herein, 38 individual ticks and their small mammals host were evaluated for the presence of Bartonella DNA by conventional PCR targeting the 16S rRNA intergenic spacer region (ITS) and partial sequencing of 460 bp from this locususing Bartonella genus-specific primers. Two tick individuals from Dermacentor auratus and Haemaphysalis hystricis collected from Rattus tiomanicus (host), were PCR-positive for Bartonella DNA amplification. No Bartonella amplification was possible in other tick species (Amblyomma sp.). Phylogenetic analysis of ITS fragments demonstrated that the sequences from ticks were closely related to Bartonella phoceensis, a species that has been reported from black rats (Rattus rattus) in Australia. This is the first report of a Bartonella bacteria detected in ticks from small mammals in Malaysia. Further research should be warranted to investigate the transmission of Bartonella and the potential impact of this zoonotic pathogen in animals and humans as this mangrove ecosystem is significant for local economy and tourism.
    Matched MeSH terms: DNA, Bacterial/genetics
  8. Fulltext First report of cervicofacial lymphadenitis due to Mycobacterium haemophilum in an immunocompromised adult patient
    Atiya N, Sulaiman H, Chong J, Ng KP
    J Infect Dev Ctries, 2015 Mar;9(3):313-6.
    PMID: 25771471 DOI: 10.3855/jidc.5208
    We report the first case of an immunocompromised adult patient presenting with cervicofacial lymphadenitis due to Mycobacterium haemophilum, confirmed using hsp65 gene sequencing and line-probe assays. In resource-limited settings, especially in developing countries, appropriate culture methods and rapid molecular diagnostic tools such as hsp65 gene sequencing for identification of this organism may not be readily available. This may cause M. haemophilum infections to go unrecognised or lead to delays in diagnosis. Lack of heightened awareness about the potential for this mycobacterial species to cause infections may also contribute to possible underestimation of M. haemophilum cases in the developing world.
    Matched MeSH terms: DNA, Bacterial/genetics
  9. Fulltext Psychrotolerant Mesorhizobium sp. Isolated from Temperate and Cold Desert Regions Solubilizes Potassium and Produces Multiple Plant Growth Promoting Metabolites
    Baba ZA, Hamid B, Sheikh TA, Alotaibi SH, El Enshasy HA, Ansari MJ, et al.
    Molecules, 2021 Sep 23;26(19).
    PMID: 34641302 DOI: 10.3390/molecules26195758
    Soil potassium (K) supplement depends intensively on the application of chemical fertilizers, which have substantial harmful environmental effects. However, some bacteria can act as inoculants by converting unavailable and insoluble K forms into plant-accessible forms. Such bacteria are an eco-friendly approach for enhancing plant K absorption and consequently reducing utilization of chemical fertilization. Therefore, the present research was undertaken to isolate, screen, and characterize the K solubilizing bacteria (KSB) from the rhizosphere soils of northern India. Overall, 110 strains were isolated, but only 13 isolates showed significant K solubilizing ability by forming a halo zone on solid media. They were further screened for K solubilizing activity at 0 °C, 1 °C, 3 °C, 5 °C, 7 °C, 15 °C, and 20 °C for 5, 10, and 20 days. All the bacterial isolates showed mineral K solubilization activity at these different temperatures. However, the content of K solubilization increased with the upsurge in temperature and period of incubation. The isolate KSB (Grz) showed the highest K solubilization index of 462.28% after 48 h of incubation at 20 °C. The maximum of 23.38 µg K/mL broth was solubilized by the isolate KSB (Grz) at 20 °C after 20 days of incubation. Based on morphological, biochemical, and molecular characterization (through the 16S rDNA approach), the isolate KSB (Grz) was identified as Mesorhizobium sp. The majority of the strains produced HCN and ammonia. The maximum indole acetic acid (IAA) (31.54 µM/mL) and cellulase (390 µM/mL) were produced by the isolate KSB (Grz). In contrast, the highest protease (525.12 µM/mL) and chitinase (5.20 µM/mL) activities were shown by standard strain Bacillus mucilaginosus and KSB (Gmr) isolate, respectively.
    Matched MeSH terms: DNA, Bacterial/genetics
  10. Fulltext Antimicrobial activities of Bacillus velezensis strains isolated from stingless bee products against methicillin-resistant Staphylococcus aureus
    Baharudin MMA, Ngalimat MS, Mohd Shariff F, Balia Yusof ZN, Karim M, Baharum SN, et al.
    PLoS One, 2021;16(5):e0251514.
    PMID: 33974665 DOI: 10.1371/journal.pone.0251514
    Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have reached epidemic proportions globally. Therefore, there is an urgent need for a continuous supply of antibiotics to combat the problem. In this study, bacteria initially identified as species belonging to the Bacillus amyloliquefaciens operational group were re-identified based on the housekeeping gene, gyrB. Cell-free supernatants (CFS) from the strains were used for antimicrobial tests using the agar well diffusion assay against MRSA and various types of pathogenic bacteria. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and physicochemical characteristics of the CFS were determined. Based on gyrB sequence analysis, five strains (PD9, B7, PU1, BP1 and L9) were identified as Bacillus velezensis. The CFS of all B. velezensis strains showed broad inhibitory activities against Gram-negative and -positive as well as MRSA strains. Strain PD9 against MRSA ATCC 33742 was chosen for further analysis as it showed the biggest zone of inhibition (21.0 ± 0.4 mm). The MIC and MBC values obtained were 125 μl/ml. The crude antimicrobial extract showed bactericidal activity and was stable at various temperatures (40-80°C), pH (4-12), surfactants (Tween 20, Tween 80, SDS and Triton X-100) and metal ions (MgCI2, NaCI2, ZnNO3 and CuSO4) when tested. However, the crude extract was not stable when treated with proteinase K. All these properties resembled the characteristics of peptides. The antimicrobial compound from the selected strain was purified by using solvent extraction method and silica gel column chromatography. The purified compound was subjected to High Performance Liquid Chromatography which resulted in a single peak of the anti-MRSA compound being detected. The molecular weight of the anti-MRSA compound was determined by using SDS-PAGE and zymogram. The size of the purified antimicrobial peptide was approximately ~ 5 kDa. The antimicrobial peptide produced from B. velezensis strain PD9 is a promising alternative to combat the spread of MRSA infections in the future.
    Matched MeSH terms: DNA, Bacterial/genetics
  11. Decolourisation of Acid Orange 7 recalcitrant auto-oxidation coloured by-products using an acclimatised mixed bacterial culture
    Bay HH, Lim CK, Kee TC, Ware I, Chan GF, Shahir S, et al.
    Environ Sci Pollut Res Int, 2014 Mar;21(5):3891-906.
    PMID: 24293297 DOI: 10.1007/s11356-013-2331-4
    This study focuses on the biodegradation of recalcitrant, coloured compounds resulting from auto-oxidation of Acid Orange 7 (AO7) in a sequential facultative anaerobic-aerobic treatment system. A novel mixed bacterial culture, BAC-ZS, consisting of Brevibacillus panacihumi strain ZB1, Lysinibacillus fusiformis strain ZB2, and Enterococcus faecalis strain ZL bacteria were isolated from environmental samples. The acclimatisation of the mixed culture was carried out in an AO7 decolourised solution. The acclimatised mixed culture showed 98 % decolourisation within 2 h of facultative anaerobic treatment using yeast extract and glucose as co-substrate. Subsequent aerobic post treatment caused auto-oxidation reaction forming dark coloured compounds that reduced the percentage decolourisation to 73 %. Interestingly, further agitations of the mixed culture in the solution over a period of 48 h significantly decolourise the coloured compounds and increased the decolourisation percentage to 90 %. Analyses of the degradation compounds using UV-visible spectrophotometer, Fourier transform infrared spectroscopy (FTIR) and high performance liquid chromatography (HPLC) showed complete degradation of recalcitrant AO7 by the novel BAC-ZS. Phytotoxicity tests using Cucumis sativus confirmed the dye solution after post aerobic treatment were less toxic compared to the parent dye. The quantitative real-time PCR revealed that E. faecalis strain ZL was the dominant strain in the acclimatised mix culture.
    Matched MeSH terms: DNA, Bacterial/genetics
  12. Fulltext Molecular analysis of asymptomatic bacteriuria Escherichia coli strain VR50 reveals adaptation to the urinary tract by gene acquisition
    Beatson SA, Ben Zakour NL, Totsika M, Forde BM, Watts RE, Mabbett AN, et al.
    Infect Immun, 2015 May;83(5):1749-64.
    PMID: 25667270 DOI: 10.1128/IAI.02810-14
    Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.
    Matched MeSH terms: DNA, Bacterial/genetics
  13. Fulltext Isolation and molecular characterization of Leptospira interrogans and Leptospira borgpetersenii isolates from the urban rat populations of Kuala Lumpur, Malaysia
    Benacer D, Mohd Zain SN, Amran F, Galloway RL, Thong KL
    Am J Trop Med Hyg, 2013 Apr;88(4):704-9.
    PMID: 23358635 DOI: 10.4269/ajtmh.12-0662
    Rats are considered the principal maintenance hosts of Leptospira. The objectives of this study were isolation and identification of Leptospira serovars circulating among urban rat populations in Kuala Lumpur. Three hundred urban rats (73% Rattus rattus and 27% R. norvegicus) from three different sites were trapped. Twenty cultures were positive for Leptospira using dark-field microscopy. R. rattus was the dominant carrier (70%). Polymerase chain reaction (PCR) confirmed that all isolates were pathogenic Leptospira species. Two Leptospira serogroups, Javanica and Bataviae, were identified using microscopic agglutination test (MAT). Pulsed-field gel electrophoresis (PFGE) identified two serovars in the urban rat populations: L. borgpetersenii serovar Javanica (85%) and L. interrogans serovar Bataviae (15%). We conclude that these two serovars are the major serovars circulating among the urban rat populations in Kuala Lumpur. Despite the low infection rate reported, the high pathogenicity of these serovars raises concern of public health risks caused by rodent transmission of leptospirosis.
    Matched MeSH terms: DNA, Bacterial/genetics
  14. Pathogenic and saprophytic Leptospira species in water and soils from selected urban sites in peninsular Malaysia
    Benacer D, Woh PY, Mohd Zain SN, Amran F, Thong KL
    Microbes Environ, 2013;28(1):135-40.
    PMID: 23363618
    Leptospira species were studied in water and soils from selected urban sites in Malaysia. A total of 151 water (n=121) and soil (n=30) samples were collected from 12 recreational lakes and wet markets. All samples were filtered and inoculated into semi-solid Ellinghausen and McCullough modified by Johnson and Harris (EMJH) media supplemented with additional 5-fluorouracil. The cultures were then incubated at 30°C and observed under a dark field microscope with intervals of 10 days. A PCR assay targeting the rrs gene was used to confirm the genus Leptospira among the isolates. Subsequently, the pathogenic status of the isolates was determined using primer sets G1/G2 and Sapro1/Sapro2, which target the secY and rrs genes, respectively. The isolates were identified at serogroup level using the microscopic agglutination test (MAT) while their genetic diversity was assessed by pulsed field gel electrophoresis (PFGE). Based on dark field microscopy, 23.1% (28/121) water and 23.3% (7/30) soil cultures were positive for Leptospira spp. Of the 35 positive cultures, only 8 were pure and confirmed as Leptospira genus by PCR assay. Two out of 8 isolates were confirmed as pathogenic, 5 were saprophytic and one was intermediate. These 8 isolates were negative for the 25 reference hyperimmune rabbit sera tested in the MAT. PFGE showed that all 8 of these environmental Leptospira spp. were genetically diverse. In conclusion, the presence of pathogenic Leptospira spp. in the urban Malaysian environment may indicate and highlight the importance of water screening, especially in recreational lakes, in order to minimize any chance of Leptospira infection.
    Matched MeSH terms: DNA, Bacterial/genetics
  15. Molecular characterization of carbapenemase and cephalosporinase genes among clinical isolates of Acinetobacter baumannii in a tertiary medical centre in Malaysia
    Biglari S, Alfizah H, Ramliza R, Rahman MM
    J Med Microbiol, 2015 Jan;64(Pt 1):53-8.
    PMID: 25381148 DOI: 10.1099/jmm.0.082263-0
    Antimicrobial resistance in Acinetobacter baumannii is a growing public health concern and an important pathogen in nosocomial infections. We investigated the genes involved in resistance to carbapenems and cephalosporins in clinical A. baumannii isolates from a tertiary medical centre in Malaysia. A. baumannii was isolated from 167 clinical specimens and identified by sequencing of the 16S rRNA and rpoB genes. The MIC for imipenem, meropenem, ceftazidime and cefepime were determined by the E-test method. The presence of carbapenemase and cephalosporinase genes was investigated by PCR. The isolates were predominantly nonsusceptible to carbapenems and cephalosporins (>70 %) with high MIC values. ISAba1 was detected in all carbapenem-nonsusceptible A. baumannii harbouring the blaOXA-23-like gene. The presence of blaOXA-51-like and ISAba1 upstream of blaOXA-51 was not associated with nonsusceptibility to carbapenems. A. baumannii isolates harbouring ISAba1-blaADC (85.8 %) were significantly associated with nonsusceptibility to cephalosporins (P<0.0001). However, ISAba1-blaADC was not detected in a minority (<10 %) of the isolates which were nonsusceptible to cephalosporins. The acquired OXA-23 enzymes were responsible for nonsusceptibility to carbapenems in our clinical A. baumannii isolates and warrant continuous surveillance to prevent further dissemination of this antibiotic resistance gene. The presence of ISAba1 upstream of the blaADC was a determinant for cephalosporin resistance. However, the absence of this ISAba1-blaADC in some of the isolates may suggest other resistance mechanisms and need further investigation.
    Matched MeSH terms: DNA, Bacterial/genetics
  16. 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*
  17. Taxonomic study of Weissella confusa and description of Weissella cibaria sp. nov., detected in food and clinical samples
    Björkroth KJ, Schillinger U, Geisen R, Weiss N, Hoste B, Holzapfel WH, et al.
    Int J Syst Evol Microbiol, 2002 Jan;52(Pt 1):141-148.
    PMID: 11837296 DOI: 10.1099/00207713-52-1-141
    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.
    Matched MeSH terms: DNA, Bacterial/genetics
  18. 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*
  19. Classification of thermophilic actinobacteria isolated from arid desert soils, including the description of Amycolatopsis deserti sp. nov
    Busarakam K, Brown R, Bull AT, Tan GY, Zucchi TD, da Silva LJ, et al.
    Antonie Van Leeuwenhoek, 2016 Feb;109(2):319-34.
    PMID: 26809280 DOI: 10.1007/s10482-015-0635-8
    The taxonomic position of 26 filamentous actinobacteria isolated from a hyper-arid Atacama Desert soil and 2 from an arid Australian composite soil was established using a polyphasic approach. All of the isolates gave the diagnostic amplification product using 16S rRNA oligonucleotide primers specific for the genus Amycolatopsis. Representative isolates had chemotaxonomic and morphological properties typical of members of the genus Amycolatopsis. 16S rRNA gene analyses showed that all of the isolates belong to the Amycolatopsis methanolica 16S rRNA gene clade. The Atacama Desert isolates were assigned to one or other of two recognised species, namely Amycolatopsis ruanii and Amycolatopsis thermalba, based on 16S rRNA gene sequence, DNA:DNA relatedness and phenotypic data; emended descriptions are given for these species. In contrast, the two strains from the arid Australian composite soil, isolates GY024(T) and GY142, formed a distinct branch at the periphery of the A. methanolica 16S rRNA phyletic line, a taxon that was supported by all of the tree-making algorithms and by a 100 % bootstrap value. These strains shared a high degree of DNA:DNA relatedness and have many phenotypic properties in common, some of which distinguished them from all of the constituent species classified in the A. methanolica 16S rRNA clade. Isolates GY024(T) and GY142 merit recognition as a new species within the A. methanolica group of thermophilic strains. The name proposed for the new species is Amycolatopsis deserti sp. nov.; the type strain is GY024(T) (=NCIMB 14972(T) = NRRL B-65266(T)).
    Matched MeSH terms: DNA, Bacterial/genetics
  20. Effect of dietary fiber on the methanogen community in the hindgut of Lantang gilts
    Cao Z, Liang JB, Liao XD, Wright AD, Wu YB, Yu B
    Animal, 2016 Oct;10(10):1666-76.
    PMID: 27052363 DOI: 10.1017/S1751731116000525
    The primary objective of this study was to investigate the effect of dietary fiber on methanogenic diversity and community composition in the hindgut of indigenous Chinese Lantang gilts to explain the unexpected findings reported earlier that Lantang gilts fed low-fiber diet (LFD) produced more methane than those fed high-fiber diet (HFD). In total, 12 Lantang gilts (58.7±0.37 kg) were randomly divided into two dietary groups (six replicates (pigs) per group) and fed either LFD (NDF=201.46 g/kg) or HFD (NDF=329.70 g/kg). Wheat bran was the main source of fiber for the LFD, whereas ground rice hull (mixture of rice hull and rice bran) was used for the HFD. Results showed that the methanogens in the hindgut of Lantang gilts belonged to four known species (Methanobrevibacter ruminantium, Methanobrevibacter wolinii, Methanosphaera stadtmanae and Methanobrevibacter smithii), with about 89% of the methanogens belonging to the genus Methanobrevibacter. The 16S ribosomal RNA (rRNA) gene copies of Methanobrevibacter were more than three times higher (P0.05) was observed in 16S rRNA gene copies of Fibrobacter succinogenes between the two dietary groups, and 18S rRNA gene copies of anaerobic fungi in gilts fed LFD were lower than (P<0.05) those fed HFD. To better explain the effect of different fiber source on the methanogen community, a follow-up in vitro fermentation using a factorial design comprised of two inocula (prepared from hindgut content of gilts fed two diets differing in their dietary fiber)×four substrates (LFD, HFD, wheat bran, ground rice hull) was conducted. Results of the in vitro fermentation confirmed that the predominant methanogens belonged to the genus of Methanobrevibacter, and about 23% methanogens was found to be distantly related (90%) to Thermogymnomonas acidicola. In vitro fermentation also seems to suggest that fiber source did change the methanogens community. Although the density of Methanobrevibacter species was positively correlated with CH4 production in both in vivo (P<0.01, r=0.737) and in vitro trials (P<0.05, r=0.854), which could partly explain the higher methane production from gilts fed LFD compared with those in the HFD group. Further investigation is needed to explain how the rice hull affected the methanogens and inhibited CH4 emission from gilts fed HFD.
    Matched MeSH terms: DNA, Bacterial/genetics
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