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1.
Int J Syst Evol Microbiol ; 66(8): 2803-2812, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27045762

ABSTRACT

Strains of members of the genus Corynebacterium derived from ophthalmologic patients in Japan, Belgium and Switzerland and found to be closely related to-, but distinguishable from Corynebacterium mastitidis by 16S rRNA gene sequencing, were characterized using biochemical, chemotaxonomic, MALDI-TOF mass spectrometry and antimicrobial susceptibility methods and DNA-DNA hybridization as well as by whole-genome sequencing (WGS). Based on this investigation, we describe Corynebacterium lowii sp. nov. and Corynebacterium oculi sp. nov., derived from human ocular specimens, as well as emend the description of Corynebacterium mastitidis. Type strains for these species are: C. lowii R-50085T (=LMG 28276T =CCUG 65815T) and C. oculi R-50187T (=LMG 28277T =CCUG 65816T). DNA G+C content was found to be 62.2 % (by HPLC) and 62.8 % (by WGS) for C. lowii R-50085T, 64.1 % (HPLC) and 64.8 % (WGS) for C. oculi R-50187T and 67.8 % (HPLC) for C. mastitidis LMG 19040T [=S-8T =CCUG 38654T =CECT 4843T =CIP 105509T =DSM 44356T =IFO (NBRC)16160T =JCM 12269T].


Subject(s)
Corynebacterium/classification , Eye/microbiology , Phylogeny , Bacterial Typing Techniques , Base Composition , Belgium , Corynebacterium/genetics , Corynebacterium/isolation & purification , DNA, Bacterial/genetics , Fatty Acids/chemistry , Humans , Japan , Nucleic Acid Hybridization , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Switzerland
2.
BMC Genomics ; 14: 895, 2013 Dec 17.
Article in English | MEDLINE | ID: mdl-24341328

ABSTRACT

BACKGROUND: The Streptococcus Anginosus Group (SAG) represents three closely related species of the viridans group streptococci recognized as commensal bacteria of the oral, gastrointestinal and urogenital tracts. The SAG also cause severe invasive infections, and are pathogens during cystic fibrosis (CF) pulmonary exacerbation. Little genomic information or description of virulence mechanisms is currently available for SAG. We conducted intra and inter species whole-genome comparative analyses with 59 publically available Streptococcus genomes and seven in-house closed high quality finished SAG genomes; S. constellatus (3), S. intermedius (2), and S. anginosus (2). For each SAG species, we sequenced at least one numerically dominant strain from CF airways recovered during acute exacerbation and an invasive, non-lung isolate. We also evaluated microevolution that occurred within two isolates that were cultured from one individual one year apart. RESULTS: The SAG genomes were most closely related to S. gordonii and S. sanguinis, based on shared orthologs and harbor a similar number of proteins within each COG category as other Streptococcus species. Numerous characterized streptococcus virulence factor homologs were identified within the SAG genomes including; adherence, invasion, spreading factors, LPxTG cell wall proteins, and two component histidine kinases known to be involved in virulence gene regulation. Mobile elements, primarily integrative conjugative elements and bacteriophage, account for greater than 10% of the SAG genomes. S. anginosus was the most variable species sequenced in this study, yielding both the smallest and the largest SAG genomes containing multiple genomic rearrangements, insertions and deletions. In contrast, within the S. constellatus and S. intermedius species, there was extensive continuous synteny, with only slight differences in genome size between strains. Within S. constellatus we were able to determine important SNPs and changes in VNTR numbers that occurred over the course of one year. CONCLUSIONS: The comparative genomic analysis of the SAG clarifies the phylogenetics of these bacteria and supports the distinct species classification. Numerous potential virulence determinants were identified and provide a foundation for further studies into SAG pathogenesis. Furthermore, the data may be used to enable the development of rapid diagnostic assays and therapeutics for these pathogens.


Subject(s)
Genome, Bacterial , Phylogeny , Streptococcus anginosus/classification , Streptococcus anginosus/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Order , Gene Transfer, Horizontal , Genes, Bacterial , Genetic Loci , Genomics , Histidine Kinase , Minisatellite Repeats , Molecular Sequence Data , Polymorphism, Single Nucleotide , Protein Kinases/genetics , Repetitive Sequences, Nucleic Acid , Streptococcus anginosus/pathogenicity , Virulence/genetics , Virulence Factors/genetics
3.
J Mol Diagn ; 12(4): 469-75, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20466837

ABSTRACT

Molecular diagnostic tools capable of identifying Shiga toxin-specific genetic determinants in stool specimens permit an unbiased approach to detect Shiga toxin-producing Escherichia coli (STEC) in clinical samples and can indicate when culture-based isolation methods are required. It is increasingly recognized that clinically relevant STEC are not limited to the singular O157 serotypes, and therefore diagnostic assays targeting toxin-encoding determinants must be able to account for any genetic variation that exists between serotypes. In this study conventional PCR and four real-time PCR assays (HybProbe, TaqMan, SYBR Green, and LUX) targeting the stx1 and stx2 Shiga toxin coding sequences were used to identify STEC in enriched stool samples (n = 36) and a panel of O157 and non-O157 strains (n = 64). PCR assays targeting stx1 and stx2 had variable specificity and sensitivity values with enriched stool samples. Molecular assays using DNA from pure cultures revealed that some primers were not sensitive to all stx2 variants. This evaluation concluded that the TaqMan-based probes were most appropriate in high throughput clinical diagnostic laboratories in consideration of cost, turn around time, and assay performance.


Subject(s)
Feces/microbiology , Polymerase Chain Reaction/methods , Shiga-Toxigenic Escherichia coli/genetics , Shiga-Toxigenic Escherichia coli/isolation & purification , Biological Assay , Humans , Polymerase Chain Reaction/economics , Sensitivity and Specificity , Shiga Toxin 1/genetics , Shiga Toxin 2/genetics , Time Factors
4.
BMC Microbiol ; 8: 89, 2008 Jun 06.
Article in English | MEDLINE | ID: mdl-18538029

ABSTRACT

BACKGROUND: Salmonella enterica serovar Heidelberg ranks amongst the most prevalent causes of human salmonellosis in Canada and an increase in resistance to extended spectrum cephalosporins (ESC) has been observed by the Canadian Integrated Program for Antimicrobial Resistance Surveillance. This study examined the genetic relationship between S. Heidelberg isolates from livestock, abattoir, retail meat, and clinical human specimens to determine whether there was a link between the emergence of MDR S. Heidelberg in chicken agri-food sources and the simultaneous increase of MDR S. Heidelberg in human clinical samples. RESULTS: Chromosomal genetic homogeneity was observed by pulsed-field gel electrophoresis (PFGE), DNA sequence-based typing (SBT) and DNA microarray-based comparative genomic hybridization (CGH). Sixty one percent of isolates were indistinguishable by PFGE conducted using XbaI and BlnI restriction enzymes. An additional 15% of isolates had PFGE patterns that were closely related to the main cluster. SBT did not identify DNA polymorphisms and CGH revealed only genetic differences between the reference S. Typhimurium strain and S. Heidelberg isolates. Genetic variation observed by CGH between S. Heidelberg isolates could be attributed to experimental variation. Alternatively, plasmid content was responsible for differences in antimicrobial susceptibility, and restriction fragment length polymorphism (RFLP) analyses followed by replicon typing identified two divergent plasmid types responsible for ESC resistance. CONCLUSION: Due to the overall limited genetic diversity among the isolates, it was not possible to identify variable traits that would be suitable for source tracking between human and agri-food isolates of S. Heidelberg in Canada.


Subject(s)
Animals, Domestic/microbiology , Drug Resistance, Multiple, Bacterial , Meat/microbiology , Salmonella Infections/microbiology , Salmonella enterica/classification , Salmonella enterica/genetics , Animals , Bacterial Typing Techniques , Canada , Cattle , Cephalosporins/pharmacology , Chickens , Chromosomes, Bacterial/genetics , Genetic Variation , Humans , Microbial Sensitivity Tests , Plasmids/chemistry , Plasmids/genetics , Polymorphism, Restriction Fragment Length , Salmonella enterica/drug effects , Salmonella enterica/isolation & purification , Swine , Turkeys
5.
BMC Microbiol ; 7: 87, 2007 Oct 01.
Article in English | MEDLINE | ID: mdl-17908316

ABSTRACT

BACKGROUND: Salmonella enterica serovar Enteritidis has emerged as a significant foodborne pathogen throughout the world and is commonly characterized by phage typing. In Canada phage types (PT) 4, 8 and 13 predominate and in 2005 a large foodborne PT13 outbreak occurred in the province of Ontario. The ability to link strains during this outbreak was difficult due to the apparent clonality of PT13 isolates in Canada, as there was a single dominant pulsed-field gel electrophoresis (PFGE) profile amongst epidemiologically linked human and food isolates as well as concurrent sporadic strains. The aim of this study was to perform comparative genomic hybridization (CGH), DNA sequence-based typing (SBT) genomic analyses, plasmid analyses, and automated repetitive sequence-based PCR (rep-PCR) to identify epidemiologically significant traits capable of subtyping S. Enteritidis PT13. RESULTS: CGH using an oligonucleotide array based upon chromosomal coding sequences of S. enterica serovar Typhimurium strain LT2 and the Salmonella genomic island 1 successfully determined major genetic differences between S. Typhimurium and S. Enteritidis PT13, but no significant strain-to-strain differences were observed between S. Enteritidis PT13 isolates. Individual loci (safA and fliC) that were identified as potentially divergent in the CGH data set were sequenced in a panel of S. Enteritidis strains, and no differences were detected between the PT13 strains. Additional sequence-based typing was performed at the fimA, mdh, manB, cyaA, citT, caiC, dmsA, ratA and STM0660 loci. Similarly, no diversity was observed amongst PT13 strains. Variation in plasmid content between PT13 strains was observed, but macrorestriction with BglII did not identify further differences. Automated rep-PCR patterns were variable between serovars, but S. Enteritidis PT13 strains could not be differentiated. CONCLUSION: None of the methods identified any significant variation between PT13 strains. Greater than 11,300 base pairs of sequence for each of seven S. Enteritidis PT13 strains were analyzed without detecting a single polymorphic site, although diversity between different phage types of S. Enteritidis was observed. These data suggest that Canadian S. Enteritidis PT13 strains are highly related genetically.


Subject(s)
Disease Outbreaks , Food Microbiology , Salmonella Food Poisoning/microbiology , Salmonella Infections/microbiology , Salmonella enteritidis/genetics , Animals , Canada/epidemiology , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field , Genetic Variation , Humans , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Poultry , Poultry Diseases/microbiology , Retrospective Studies , Salmonella Food Poisoning/epidemiology , Salmonella Infections/epidemiology , Salmonella enteritidis/classification
6.
J Clin Microbiol ; 45(11): 3771-3, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17804662

ABSTRACT

A coinfection of O177:NM and O55:H7 Shiga toxin-producing Escherichia coli (STEC) was identified for a child with acute bloody diarrhea and hemolytic uremic syndrome by using culture and serotype-specific molecular reagents. The profile of O157-related genetic islands revealed that the O55:H7 isolate was highly similar to O157 STEC whereas the O177:NM isolate lacked several fimbrial O islands and non-locus-of-enterocyte-effacement effector determinants. However, both STEC serotypes are known to cause serious disease, and the significant repertoire of virulence determinants in both strains made it impossible to determine their individual contributions to the clinical symptoms.


Subject(s)
Shiga-Toxigenic Escherichia coli/isolation & purification , Base Sequence , Child, Preschool , Hemolytic-Uremic Syndrome/microbiology , Humans , Molecular Sequence Data , Serotyping , Shiga-Toxigenic Escherichia coli/classification , Shiga-Toxigenic Escherichia coli/genetics
7.
J Med Microbiol ; 56(Pt 5): 620-628, 2007 May.
Article in English | MEDLINE | ID: mdl-17446284

ABSTRACT

Serogroup classifications based upon the O-somatic antigen of Shiga toxin-producing Escherichia coli (STEC) provide significant epidemiological information on clinical isolates. Each O-antigen determinant is encoded by a unique cluster of genes present between the gnd and galF chromosomal genes. Alternatively, serogroup-specific polymorphisms might be encoded in loci that are encoded outside of the O-antigen gene cluster. Segments of the core bacterial loci mdh, gnd, gcl, ppk, metA, ftsZ, relA and metG for 30 O26 STEC strains have previously been sequenced, and comparative analyses to O157 distinguished these two serogroups. To screen these loci for serogroup-specific traits within a broader range of clinically significant serogroups, DNA sequences were obtained for 19 strains of 10 additional STEC serogroups. Unique alleles were observed at the gnd locus for each examined STEC serogroup, and this correlation persisted when comparative analyses were extended to 144 gnd sequences from 26 O-serogroups (comprising 42 O : H-serotypes). These included O157, O121, O103, O26, O5 : non-motile (NM), O145 : NM, O113 : H21, O111 : NM and O117 : H7 STEC; and furthermore, non-toxin encoding O157, O26, O55, O6 and O117 strains encoded distinct gnd alleles compared to STEC strains of the same serogroup. DNA sequencing of a 643 bp region of gnd was, therefore, sufficient to minimally determine the O-antigen of STEC through molecular means, and the location of gnd next to the O-antigen gene cluster offered additional support for the co-inheritance of these determinants. The gnd DNA sequence-based serogrouping method could improve the typing capabilities for STEC in clinical laboratories, and was used successfully to characterize O121 : H19, O26 : H11 and O177 : NM clinical isolates prior to serological confirmation during outbreak investigations.


Subject(s)
DNA, Bacterial/genetics , Escherichia coli Proteins/genetics , Escherichia coli/classification , Escherichia coli/genetics , O Antigens/genetics , Phosphogluconate Dehydrogenase/genetics , Shiga Toxins/biosynthesis , Bacterial Typing Techniques/methods , Base Sequence , DNA, Bacterial/chemistry , Escherichia coli/isolation & purification , Escherichia coli/metabolism , Escherichia coli Infections/microbiology , Genes, Bacterial , Genetic Markers , Humans , Molecular Epidemiology/methods , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , Sequence Homology
8.
J Med Microbiol ; 56(Pt 3): 336-341, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17314363

ABSTRACT

An outbreak of Legionnaires' disease at a long-term care facility in Ontario, Canada from September to October 2005 resulted in the death of 23 residents and the illness of 112 other people. In response, molecular methods were developed to detect Legionella pneumophila in clinical lung samples and to subtype isolates from clinical and environmental samples. The targeted genetic loci included Legionella-specific virulence determinants (mip, icmO, sidA and lidA) and core bacterial determinants (ftsZ, trpS and dnaX). An established amplified fragment length polymorphism typing method provided the first indication of genetic relatedness between strains recovered from clinical samples and strains cultured from environmental samples taken from the outbreak site. These associations were verified using the European Working Group for Legionella Infections sequence-based typing protocol targeting the flaA, pilE, asd, mip, mompS and proA loci. These molecular typing methods confirmed the outbreak source as a contaminated air conditioning cooling tower.


Subject(s)
Bacterial Typing Techniques , Disease Outbreaks , Legionella pneumophila/classification , Legionella pneumophila/genetics , Legionellosis/epidemiology , Legionellosis/microbiology , Bacterial Proteins/genetics , Cluster Analysis , DNA Fingerprinting/methods , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Humans , Legionella pneumophila/isolation & purification , Lung/microbiology , Molecular Epidemiology , Molecular Sequence Data , Nucleic Acid Amplification Techniques , Ontario/epidemiology , Sequence Analysis, DNA , Skilled Nursing Facilities , Virulence Factors/genetics
9.
Microbes Infect ; 9(1): 21-7, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17194608

ABSTRACT

A Salmonella genomic island 1 (SGI1) isogenic strain pair was constructed using Salmonella enterica serovar Typhimurium LT2 (ST LT2). Real-time quantitative reverse transcriptase PCR revealed detectable mRNA transcripts for all 44 putative ORFs encoded within the SGI1. The highest levels of transcripts observed in SGI1 encoded ORFs were found in genes conferring antibiotic resistance to ampicillin, streptomycin/spectinomycin, and sulphonamides. Abundant mRNA transcripts, relative to gapA, were also noted for one putative regulatory ORF and seven ORFs of unknown function encoded within SGI1, whose products could represent factors contributing to increases in virulence and/or fitness of the organism. DNA microarray analysis revealed the differential expression of known factors that contribute to virulence in many pathogens. Twenty-two chromosomal genes were significantly upregulated in ST LT2 harboring SGI1, which included increased expression of iron and sialic acid utilization genes. Decreased expression was noted for 15 genes in ST LT2 harboring SGI1, including genes involved in chemotaxis and motility. This is the first report examining gene expression within the SGI1, as well as its potential effect on global gene expression, and sets the foundation for future studies involving the effect of SGI1 in other Salmonella spp.


Subject(s)
Gene Expression Regulation, Bacterial/genetics , Genomic Islands , Salmonella typhi/genetics , Conjugation, Genetic , DNA, Bacterial/genetics , Oligonucleotide Array Sequence Analysis , Open Reading Frames , Phenotype , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction
10.
J Med Microbiol ; 56(Pt 1): 56-65, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17172518

ABSTRACT

The development of rapid and sensitive molecular techniques for the detection of Vibrio species would be useful for the surveillance of sporadic infections and management of major outbreaks. Comparative sequence analysis of the ftsZ gene in the predominant Vibrio species that cause human disease revealed distinct alleles for each examined species, including Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Light Upon eXtension (LUX) real-time PCR assays were developed to target these species-specific polymorphisms, and were successful in rapidly differentiating the major pathogenic Vibrio species. Luminex liquid microsphere array technology was used to develop a comprehensive assay capable of simultaneously detecting V. cholerae, V. parahaemolyticus and V. vulnificus. These assays permitted the identification of a presumptive V. parahaemolyticus isolate as Vibrio alginolyticus, which was verified using additional molecular characterization.


Subject(s)
Bacterial Proteins/genetics , Bacterial Typing Techniques/methods , Cytoskeletal Proteins/genetics , Polymerase Chain Reaction/methods , Vibrio/genetics , Bacterial Typing Techniques/instrumentation , Base Sequence , DNA, Bacterial/chemistry , DNA, Bacterial/isolation & purification , Genetic Variation , Microspheres , Molecular Sequence Data , Phylogeny , Polymorphism, Genetic , Reproducibility of Results , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Species Specificity , Vibrio/classification , Vibrio cholerae/genetics , Vibrio parahaemolyticus/genetics , Vibrio vulnificus/genetics
11.
Microbes Infect ; 8(7): 1915-22, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16713724

ABSTRACT

Multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104, resistant to ampicillin, chloramphenicol/florfenicol, streptomycin, sulfonamides, and tetracycline, has disseminated worldwide. The resistance genes reside on the 43-kb Salmonella genomic island 1 (SGI1), which is transferable. Drug-resistant variants of SGI1 have been identified in numerous serotypes. Strains harboring SGI1 may be more virulent and have a tendency to rapidly disseminate.


Subject(s)
Drug Resistance, Multiple, Bacterial/genetics , Genomic Islands/genetics , Salmonella typhimurium/genetics , Animals , Humans , Salmonella Infections/epidemiology , Salmonella Infections/microbiology , Salmonella Infections, Animal/epidemiology , Salmonella Infections, Animal/microbiology , Salmonella typhimurium/pathogenicity , Virulence
12.
Antimicrob Agents Chemother ; 49(5): 2112-5, 2005 May.
Article in English | MEDLINE | ID: mdl-15855541

ABSTRACT

Chromosomal beta-lactamase genes (bla(KLUY)) from six Kluyvera georgiana strains isolated in Guyana were cloned and expressed in Escherichia coli. KLUY-1 exhibited 100% amino acid identity with the extended-spectrum beta-lactamase CTX-M-14. We also show that a 2.7-kb Kluyvera chromosomal region exhibits 99% nucleotide identity to a portion of In60 that includes bla(CTX-M-9).


Subject(s)
Escherichia coli Proteins/genetics , Kluyvera/enzymology , Kluyvera/genetics , beta-Lactamases/genetics , Amino Acid Sequence , Anti-Bacterial Agents/pharmacology , Chromosomes, Bacterial/genetics , Cloning, Molecular , Deoxyribonuclease EcoRI/chemistry , Escherichia coli/genetics , Guyana , Humans , Kluyvera/drug effects , Microbial Sensitivity Tests , Molecular Sequence Data
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