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Fulltext Molecular characterization of putative virulence determinants in Burkholderia pseudomallei

Puah SM, Puthucheary SD, Wang JT, Pan YJ, Chua KH

ScientificWorldJournal, 2014;2014:590803.
PMID: 25215325 DOI: 10.1155/2014/590803

The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.

Matched MeSH terms: Virulence/genetics*
Comparison of Clinical Isolates of Aeromonas from Singapore and Malaysia with Regard to Molecular Identification, Virulence, and Antimicrobial Profiles

Khor WC, Puah SM, Koh TH, Tan JAMA, Puthucheary SD, Chua KH

Microb Drug Resist, 2018 May;24(4):469-478.
PMID: 29461928 DOI: 10.1089/mdr.2017.0083

OBJECTIVE: The objective of this study was to examine the species distribution, genetic relatedness, virulence gene profiles, antimicrobial sensitivities, and resistance gene distribution of clinical Aeromonas strains from Singapore and Malaysia.
METHODS: A total of 210 Aeromonas clinical isolates were investigated: 116 from Singapore General Hospital and 94 archived clinical isolates from University of Malaya Medical Center, Malaysia. The isolates were genetically identified based on the gcat gene screening and the partial sequences of the rpoD housekeeping gene. Genetic relatedness, distribution of 15 virulence genes and 4 beta-lactamase resistance genes, and susceptibility patterns to 11 antimicrobial agents were compared.
RESULTS: Of the 210 Aeromonas isolates, A. dhakensis-94 (45%) was the dominant species in Singapore and Malaysia. Species composition was similar and enterobacterial repetitive intergenic consensus-PCR did not show genetic relatedness between strains from the two countries. Of the 15 virulence genes, A. dhakensis and A. hydrophila harbored the most compared with other species. Different combinations of 9 virulence genes (exu, fla, lip, eno, alt, dam, hlyA, aexU, and ascV) were present in A. dhakensis, A. hydrophila, and A. veronii from both the countries. Distribution of virulence genes was species and anatomic site related. Majority (>80%) of the strains were susceptible to all antimicrobial agents tested, except amoxicillin and cephalothin. A. dhakensis strains from Malaysia significantly harbored the cphA gene compared with A. dhakensis from Singapore. Multidrug resistance was mostly detected in strains from peritoneal fluids of dialysis patients.
CONCLUSION: This study revealed A. dhakensis as the dominant species isolated in both geographic regions, and that it carried a high number of virulence genes. It also highlights the geographic-related differences of virulence gene distribution and antimicrobial resistance profiles of clinical Aeromonas strains from Singapore and Malaysia.

Matched MeSH terms: Virulence/genetics*
Fulltext Comparative genome sequencing and analyses of Mycobacterium cosmeticum reveal potential for biodesulfization of gasoline

Wee WY, Dutta A, Jayaraj J, Choo SW

PLoS One, 2019;14(4):e0214663.
PMID: 30964891 DOI: 10.1371/journal.pone.0214663

Mycobacterium cosmeticum is a nontuberculous Mycobacterium recovered from different water sources including household potable water and water collected at nail salon. Individual cases of this bacterium have been reported to be associated with gastrointestinal tract infections. Here we present the first whole-genome study and comparative analysis of two new clinically-derived Mycobacterium sp. UM_RHS (referred as UM_RHS after this) and Mycobacterium sp. UM_NYF (referred as UM_NYF after this) isolated from patients in Indonesia and Malaysia respectively to have a better understanding of the biological characteristic of these isolates. Both strains are likely Mycobacterium cosmeticum as supported by the evidence from molecular phylogenetic, comparative genomic and Average Nucleotide Identity (ANI) analyses. We found the presence of a considerably large number of putative virulence genes in the genomes of UM_RHS and UM_NYF. Interestingly, we also found a horizontally transferred genomic island carrying a putative dsz operon proposing that they may have potential to perform biodesulfization of dibenzothiophene (DBT) that may be effective in cost reduction and air pollution during fuel combustion. This comparative study may provide new insights into M. cosmeticum and serve as an important reference for future functional studies of this bacterial species.

Matched MeSH terms: Virulence/genetics
Fulltext Evolutionary study of Yersinia genomes deciphers emergence of human pathogenic species

Tan SY, Tan IK, Tan MF, Dutta A, Choo SW

Sci Rep, 2016 10 31;6:36116.
PMID: 27796355 DOI: 10.1038/srep36116

On record, there are 17 species in the Yersinia genus, of which three are known to be pathogenic to human. While the chromosomal and pYV (or pCD1) plasmid-borne virulence genes as well as pathogenesis of these three species are well studied, their genomic evolution is poorly understood. Our study aims to predict the key evolutionary events that led to the emergence of pathogenic Yersinia species by analyzing gene gain-and-loss, virulence genes, and "Clustered regularly-interspaced short palindromic repeats". Our results suggest that the most recent ancestor shared by the human pathogenic Yersinia was most probably an environmental species that had adapted to the human body. This might have led to ecological specialization that diverged Yersinia into ecotypes and distinct lineages based on differential gene gain-and-loss in different niches. Our data also suggest that Y. pseudotuberculosis group might be the donor of the ail virulence gene to Y. enterocolitica. Hence, we postulate that evolution of human pathogenic Yersinia might not be totally in parallel, but instead, there were lateral gene transfer events. Furthermore, the presence of virulence genes seems to be important for the positive selection of virulence plasmid. Our studies provide better insights into the evolutionary biology of these bacteria.

Matched MeSH terms: Virulence/genetics
Fulltext YersiniaBase: a genomic resource and analysis platform for comparative analysis of Yersinia

Tan SY, Dutta A, Jakubovics NS, Ang MY, Siow CC, Mutha NV, et al.

BMC Bioinformatics, 2015;16:9.
PMID: 25591325 DOI: 10.1186/s12859-014-0422-y

Yersinia is a Gram-negative bacteria that includes serious pathogens such as the Yersinia pestis, which causes plague, Yersinia pseudotuberculosis, Yersinia enterocolitica. The remaining species are generally considered non-pathogenic to humans, although there is evidence that at least some of these species can cause occasional infections using distinct mechanisms from the more pathogenic species. With the advances in sequencing technologies, many genomes of Yersinia have been sequenced. However, there is currently no specialized platform to hold the rapidly-growing Yersinia genomic data and to provide analysis tools particularly for comparative analyses, which are required to provide improved insights into their biology, evolution and pathogenicity.

Matched MeSH terms: Virulence/genetics*
Possible virulence factors involved in bacteraemia caused by Aeromonas hydrophila

Vadivelu J, Puthucheary SD, Phipps M, Chee YW

J Med Microbiol, 1995 Mar;42(3):171-4.
PMID: 7884797

Eighteen strains of Aeromonas hydrophila from patients with bacteraemia were investigated for possible virulence factors. Cytotoxin and haemolysin were produced by all strains, whereas cholera toxin-like factor was produced by 33% of strains only. Enterotoxin production was not detected. Haemagglutination of guinea-pig, fowl and rabbit erythrocytes was demonstrated by 83%, 67% and 61% of strains, respectively. Fucose- and mannose-sensitive haemagglutinins were predominant. None of the strains agglutinated sheep erythrocytes. Extrachromosomal DNA was detected in 17 strains, 16 of which had a plasmid (3.6-5.1 MDa), the majority being between 4.6 and 5.1 MDa.

Matched MeSH terms: Virulence/genetics
Fulltext The Effects of Chinese Herbal Medicines on the Quorum Sensing-Regulated Virulence in Pseudomonas aeruginosa PAO1

Chong YM, How KY, Yin WF, Chan KG

Molecules, 2018 04 21;23(4).
PMID: 29690523 DOI: 10.3390/molecules23040972

The quorum sensing (QS) system has been used by many opportunistic pathogenic bacteria to coordinate their virulence determinants in relation to cell-population density. As antibiotic-resistant bacteria are on the rise, interference with QS has been regarded as a novel way to control bacterial infections. As such, many plant-based natural products have been widely explored for their therapeutic roles. These natural products may contain anti-QS compounds that could block QS signals generation or transmission to combat QS pathogens. In this study, we report the anti-QS activities of four different Chinese herbal plant extracts: Poria cum Radix pini, Angelica dahurica, Rhizoma cibotii and Schizonepeta tenuifolia, on Pseudomonas aeruginosa PAO1. All the plants extracted using hexane, chloroform and methanol were tested and found to impair swarming motility and pyocyanin production in P.aeruginosa PAO1, particularly by Poria cum Radix pini. In addition, all the plant extracts also inhibited violacein production in C.violaceum CV026 up to 50% while bioluminescence activities were reduced in lux-based E. coli biosensors, pSB401 and pSB1075, up to about 57%. These anti-QS properties of the four medicinal plants are the first documentation that demonstrates a potential approach to attenuate pathogens’ virulence determinants.

Matched MeSH terms: Virulence/genetics
Fulltext Whole-genome sequence of multi-drug resistant Pseudomonas aeruginosa strains UY1PSABAL and UY1PSABAL2 isolated from human broncho-alveolar lavage, Yaoundé, Cameroon

Madaha EL, Mienie C, Gonsu HK, Bughe RN, Fonkoua MC, Mbacham WF, et al.

PLoS One, 2020;15(9):e0238390.
PMID: 32886694 DOI: 10.1371/journal.pone.0238390

Pseudomonas aeruginosa has been implicated in a wide range of post-operation wound and lung infections. A wide range of acquired resistance and virulence markers indicate surviving strategy of P. aeruginosa. Complete-genome analysis has been identified as efficient approach towards understanding the pathogenicity of this organism. This study was designed to sequence the entire genome of P. aeruginosa UY1PSABAL and UY1PSABAL2; determine drug-resistance profiles and virulence factors of the isolates; assess factors that contribute toward stability of the genomes; and thereafter determine evolutionary relationships between the strains and other isolates from similar sources. The genomes of the MDR P. aeruginosa UY1PSABAL and UY1PSABAL2 were sequenced on the Illumina Miseq platform. The raw sequenced reads were assessed for quality using FastQC v.0.11.5 and filtered for low quality reads and adapter regions using Trimmomatic v.0.36. The de novo genome assembly was made with SPAdes v.3.13 and annotated using Prokka v.2.1.1 annotation pipeline; Rapid Annotation using Subsytems Technology (RAST) server v.2.0; and PATRIC annotation tool v.3.6.2. Antimicrobial resistance genes and virulence determinants were searched through the functional annotation data generated from Prokka, RAST and PATRIC annotation pipelines; In addition to ResFinder and Comprehensive Antibiotic Resistance Database (CARD) which were employed to determine resistance genes. The PHAge Search Tool Enhanced Release (PHASTER) web server was used for the rapid identification and annotation of prophage sequences within bacterial genome. Predictive secondary metabolites were identified with AntiSMASH v.5.0. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and cas genes regions were also investigated with the CRISPRone and CRISPRFinder server. The genome sizes of 7.0 and 6.4 Mb were determined for UY1PSABAL and UY1PSABAL2 strains with G+C contents of 66.1% and 66.48% respectively. β-lactamines resistance genes blaPAO, aminoglycoside phosphorylating enzymes genes aph(3')-IIb, fosfomycine resistance gene fosA, vancomycin vanW and tetracycline tetA were among identified resistance genes harboured in both isolates. UY1PSABAL bore additional aph(6)-Id, aph(3'')-Ib, ciprofloxacin-modifying enzyme crpP and ribosomal methylation enzyme rmtB. Both isolates were found harbouring virulence markers such as flagella and type IV pili; and also present various type III secretion systems such as exoA, exoS, exoU, exoT. Secondary metabolites such as pyochelin and pyoverdine with iron uptake activity were found within the genomes as well as quorum-sensing systems, and various fragments for prophages and insertion sequences. Only the UY1PSABAL2 contains CRISPR-Cas system. The phylogeny revealed a very close evolutionary relationship between UY1PSABAL and the similar strain isolated from Malaysia; the same trend was observed between UY1PSABAL2 and the strain from Chinese origin. Complete analyses of the entire genomes provide a wide range of information towards understanding pathogenicity of the pathogens in question.

Matched MeSH terms: Virulence/genetics
Nucleotide sequence and phylogenetic analysis of a segment of a highly virulent strain of infectious bursal disease virus

Chong LK, Omar AR, Yusoff K, Hair-Bejo M, Aini I

Acta Virol., 2001;45(4):217-26.
PMID: 11885928

The complete nucleotide sequences encoding precursor polyprotein (VP2-VP3-VP4) and VP5 of a highly virulent (hv) infectious bursal disease virus (IBDV), UPM97/61 was determined. Comparison of the deduced amino acid sequences with the published ones revealed 8 common amino acid substitutions, which were found only in the hv IBDV including the UPM97/61 strain. Three of the amino acid substitutions (222 Ala, 256 Ile and 294 Ile) were used as a marker for determining hv IBDV strains. The other five substitutions (685 Asn, 715 Ser, 751 Asp, 990 Val and 1005 Ala) were also conserved in hv IBDV strains isolated in various countries. UPM97/61 strain demonstrated also 8 unique amino acid substitutions of which 3 were in VP2, 4 in VP3 and 1 in VP4. There was 1 unique amino acid substitution in VP5 at position 19 (Asp-->Gly) not found in other strains. However, all the strains have a conserved 49 Arg. The amino acid sequence of UPM97/61 strain differed by 1.09% from the Japanese (OKYM) and Hong Kong (HK46) strains, and by 1.48% from the Israeli (IBDVKS) and European (UK661) strains. Hence, UPM97/61 is more closely related to the hv strains from Asia. However, phylogenetic analysis indicated that the origin of UPM97/61 might be the same as that of other hv strains isolated from other parts of the world.

Matched MeSH terms: Virulence/genetics
Sequence and phylogenetic analysis of the VP2 gene of very virulent infectious bursal disease virus isolates

Hoque MM, Omar AR, Hair-Bejo M, Aini I

J. Biochem. Mol. Biol. Biophys., 2002 Apr;6(2):93-9.
PMID: 12186763

Previously we have shown that very virulent infectious bursal disease viruses (vvIBDV) that are SspI, TaqI and StyI positive (92/04, 97/61 and 94/B551) but not SspI and TaqI positive and StyI negative (94/273) cause high mortality, up to 80% in specific-pathogen-free chickens with significant damage of the bursal as well as nonbursal tissues. In this study, we sequenced the VP2 gene (1351 bp) of the 92/04, 94/273 and 94/B551 and compared them with other IBDV strains. All the isolates have the unique amino acid residues at positions 222A, 256I, 294I and 299S found in other vvIBDV strains. The deduced VP2 amino acids encoded by 92/04 is identical to the vvIBDV strains from Israel (IBDVKS), Japan (OKYM) and Europe (UK661), whereas the 94/273 and 94/B551 isolates have one to three amino acid substitutions. The 94/273 has two amino acid substitutions at positions 254 G to S and at 270 A to E that have not been reported before from vvIBDV strains. The 94/B551 also has one amino acid substitution at position 300 E to S, which is uncommon among other vvIBDV strains. However, phylogenetic analysis suggested that the isolates are very close to each other and all of them may have derived from the same origin as vvIBDV strains isolated from China, Japan and Europe. Even though antigenic index analysis of the 94/273 and 94/B551 indicated that the isolates are unique compared to other IBDV strains, their antigenic variation remain to be determined by monoclonal antibody study.

Matched MeSH terms: Virulence/genetics
Fulltext Virulence gene profiles and antimicrobial susceptibility of Salmonella Brancaster from chicken

Khoo E, Roslee R, Zakaria Z, Ahmad NI

J Vet Sci, 2023 Nov;24(6):e82.
PMID: 38031519 DOI: 10.4142/jvs.23053

BACKGROUND: The current conventional serotyping based on antigen-antisera agglutination could not provide a better understanding of the potential pathogenicity of Salmonella enterica subsp. enterica serovar Brancaster. Surveillance data from Malaysian poultry farms indicated an increase in its presence over the years.
OBJECTIVE: This study aims to investigate the virulence determinants and antimicrobial resistance in S. Brancaster isolated from chickens in Malaysia.
METHODS: One hundred strains of archived S. Brancaster isolated from chicken cloacal swabs and raw chicken meat from 2017 to 2022 were studied. Two sets of multiplex polymerase chain reaction (PCR) were conducted to identify eight virulence genes associated with pathogenicity in Salmonella (invasion protein gene [invA], Salmonella invasion protein gene [sipB], Salmonella-induced filament gene [sifA], cytolethal-distending toxin B gene [cdtB], Salmonella iron transporter gene [sitC], Salmonella pathogenicity islands gene [spiA], Salmonella plasmid virulence gene [spvB], and inositol phosphate phosphatase gene [sopB]). Antimicrobial susceptibility assessment was conducted by disc diffusion method on nine selected antibiotics for the S. Brancaster isolates. S. Brancaster, with the phenotypic ACSSuT-resistance pattern (ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracycline), was subjected to PCR to detect the corresponding resistance gene(s).
RESULTS: Virulence genes detected in S. Brancaster in this study were invA, sitC, spiA, sipB, sopB, sifA, cdtB, and spvB. A total of 36 antibiogram patterns of S. Brancaster with a high level of multidrug resistance were observed, with ampicillin exhibiting the highest resistance. Over a third of the isolates displayed ACSSuT-resistance, and seven resistance genes (β-lactamase temoneira [blaTEM], florfenicol/chloramphenicol resistance gene [floR], streptomycin resistance gene [strA], aminoglycoside nucleotidyltransferase gene [ant(3″)-Ia], sulfonamides resistance gene [sul-1, sul-2], and tetracycline resistance gene [tetA]) were detected.
CONCLUSION: Multidrug-resistant S. Brancaster from chickens harbored an array of virulence-associated genes similar to other clinically significant and invasive non-typhoidal Salmonella serovars, placing it as another significant foodborne zoonosis.

Matched MeSH terms: Virulence/genetics
Fulltext Occurrence of virulent genes among environmental isolates of Legionella pneumophila serogroup 1 strains from various parts of peninsular Malaysia

Arushothy R, Ahmad N

Trop Biomed, 2008 Dec;25(3):259-61.
PMID: 19287368

Legionella pneumophila are intracellular pathogens, associated with human disease, attributed to the presence and absence of certain virulent genes. In this study, virulent gene loci (lvh and rtxA regions) associated with human disease were determined. Thirty-three cooling tower water isolates, isolated between 2004 to 2006, were analyzed for the presence of these genes by PCR method. Results showed that 19 of 33 (57.5%) of the L. pneumophila serogroup 1 isolates have both the genes. Six (18.2%) of the isolates have only the lvh gene and 2 (6.1%) of the isolates have only the rtxA gene. However, both genes were absent in 6 (18.2%) of the L. pneumophila isolates. The result of our study provides some insight into the presence of the disease causing L. pneumophila serogroup 1 in the environment. Molecular epidemiological studies will provide better understanding of the prevalence of the disease in Malaysia.

Matched MeSH terms: Virulence/genetics

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