Richard Okinaka | Northern Arizona University (original) (raw)

Papers by Richard Okinaka

PLoS ONE, 2014

Sequence analyses and subtyping of Bacillus anthracis strains from Georgia reveal a single distin... more Sequence analyses and subtyping of Bacillus anthracis strains from Georgia reveal a single distinct lineage (Aust94) that is ecologically established. Phylogeographic analysis and comparisons to a global collection reveals a clade that is mostly restricted to Georgia. Within this clade, many groups are found around the country, however at least one subclade is only found in the eastern part. This pattern suggests that dispersal into and out of Georgia has been rare and despite historical dispersion within the country, for at least for one lineage, current spread is limited.

Biochemistry, 1977

Nuclei isolated from cultured Chinese hamster cells were treated with micrococcal nuclease and ly... more Nuclei isolated from cultured Chinese hamster cells were treated with micrococcal nuclease and lysed, and the resulting chromatin subunit classes (nucleosomes) were purified by sedimentation and resedimentation through isokinetic sucrose gradients. Nucleosomes isolated from [3H]thymidine-labeled cells were analyzed for DNA size using both polyacrylamide gel and electron microscopic techniques. Nucleosomes isolated from [14C]lysine-labeled cells were analyzed for protein content using a sodium dodecyl sulfate-polyacrylamide gel system. The results from monitoring the [14c]lysine in each protein indicate that, in the nucleosome classes (monomer through tetramer), the molar ratios of histones H2A, H2B, H3, and H4 are equivalent. Furthermore, in each population of the nucleosome classes monomer through tetramer, it was possible to demonstrate that this histone unit (H2A + H2B + H3 + H4) is present, on the average, in the amount of two for monomers, four for dimers, six for trimers, and eight for tetramers. This is direct experimental confirmation of the prediction of R.D. Kornberg [(1974) Science 184, 868] concerning the substructure of chromatin.

Analyst, 2000

A very fast and ultrasensitive method has been developed for the detection and quantitation of sp... more A very fast and ultrasensitive method has been developed for the detection and quantitation of specific nucleic and sequences of bacterial origin in solution. The method is based on a two-color, single fluorescent molecule detection technique developed in our laboratory. The technique was applied to the detection of Bacillus anthracis DNA in solution.

PLOS One, 2009

Disease introduction into the New World during colonial expansion is well documented and had a ma... more Disease introduction into the New World during colonial expansion is well documented and had a major impact on indigenous populations; however, few diseases have been associated with early human migrations into North America. During the late Pleistocene epoch, Asia and North America were joined by the Beringian Steppe ecosystem which allowed animals and humans to freely cross what would become a water barrier in the Holocene. Anthrax has clearly been shown to be dispersed by human commerce and trade in animal products contaminated with Bacillus anthracis spores. Humans appear to have brought B. anthracis to this area from Asia and then moved it further south as an ice-free corridor opened in central Canada ,13,000 ybp. In this study, we have defined the evolutionary history of Western North American (WNA) anthrax using 2,850 single nucleotide polymorphisms (SNPs) and 285 geographically diverse B. anthracis isolates. Phylogeography of the major WNA B. anthracis clone reveals ancestral populations in northern Canada with progressively derived populations to the south; the most recent ancestor of this clonal lineage is in Eurasia. Our phylogeographic patterns are consistent with B. anthracis arriving with humans via the Bering Land Bridge. This northern-origin hypothesis is highly consistent with our phylogeographic patterns and rates of SNP accumulation observed in current day B. anthracis isolates. Continent-wide dispersal of WNA B. anthracis likely required movement by later European colonizers, but the continent's first inhabitants may have seeded the initial North American populations.

Analytical Chemistry, 2008

Genomics, 1996

man autoantigen , where the Ku70 DNA-dependent protein kinase (DNA-PK) consists of and Ku80 subun... more man autoantigen , where the Ku70 DNA-dependent protein kinase (DNA-PK) consists of and Ku80 subunits were found to form heterodimers three polypeptide subunits: Ku70, Ku80, and the DNA- ; Fran-PK catalytic subunit (DNA-PKcs). Mammalian mutants coeur et al., 1986). The Ku70/80 heterodimer binds with deficient in either Ku80 or DNA-PKcs function have high affinity to the end of double-stranded DNA and to been shown to be lacking in DNA double-strand break single-stranded DNA transitions (Mimori and Hardin, repair and V(D)J recombination, respectively. The 1986). The DNA binding activity of the complex has precise role of the Ku70 gene in this process has not been shown to be suppressed by IgG in patients with yet been determined, in part because no cell lines, anithe disease (Mimori and Hardin, 1986).

BMC Genomics, 2002

Background: Complete sequencing and annotation of the 96.2 kb Bacillus anthracis plasmid, pXO2, p... more Background: Complete sequencing and annotation of the 96.2 kb Bacillus anthracis plasmid, pXO2, predicted 85 open reading frames (ORFs). Bacillus cereus and Bacillus thuringiensis isolates that ranged in genomic similarity to B. anthracis, as determined by amplified fragment length polymorphism (AFLP) analysis, were examined by PCR for the presence of sequences similar to 47 pXO2 ORFs.

PLOS One, 2009

Disease introduction into the New World during colonial expansion is well documented and had a ma... more Disease introduction into the New World during colonial expansion is well documented and had a major impact on indigenous populations; however, few diseases have been associated with early human migrations into North America. During the late Pleistocene epoch, Asia and North America were joined by the Beringian Steppe ecosystem which allowed animals and humans to freely cross what would become a water barrier in the Holocene. Anthrax has clearly been shown to be dispersed by human commerce and trade in animal products contaminated with Bacillus anthracis spores. Humans appear to have brought B. anthracis to this area from Asia and then moved it further south as an ice-free corridor opened in central Canada ,13,000 ybp. In this study, we have defined the evolutionary history of Western North American (WNA) anthrax using 2,850 single nucleotide polymorphisms (SNPs) and 285 geographically diverse B. anthracis isolates. Phylogeography of the major WNA B. anthracis clone reveals ancestral populations in northern Canada with progressively derived populations to the south; the most recent ancestor of this clonal lineage is in Eurasia. Our phylogeographic patterns are consistent with B. anthracis arriving with humans via the Bering Land Bridge. This northern-origin hypothesis is highly consistent with our phylogeographic patterns and rates of SNP accumulation observed in current day B. anthracis isolates. Continent-wide dispersal of WNA B. anthracis likely required movement by later European colonizers, but the continent's first inhabitants may have seeded the initial North American populations.

Infection Genetics and Evolution, 2009

Biosensors & Bioelectronics, 2004

Rapid, accurate, and sensitive detection of biothreat agents requires a broad-spectrum assay capa... more Rapid, accurate, and sensitive detection of biothreat agents requires a broad-spectrum assay capable of discriminating between closely related microbial or viral pathogens. Moreover, in cases where a biological agent release has been identified, forensic analysis demands detailed genetic signature data for accurate strain identification and attribution. To date, nucleic acid sequences have provided the most robust and phylogentically illuminating signature information. Nucleic acid signature sequences are not often linked to genomic or extrachromosomal determinants of virulence, a link that would further facilitate discrimination between pathogens and closely related species. Inextricably coupling genetic determinants of virulence with highly informative nucleic acid signatures would provide a robust means of identifying human, livestock, and agricultural pathogens. By means of example, we present here an overview of two general applications of microarray-based methods for: (1) the identification of candidate virulence factors; and (2) the analysis of genetic polymorphisms that are coupled to Bacillus anthracis virulence factors using an accurate, low cost solid-phase mini-sequencing assay. We show that microarray-based analysis of gene expression can identify potential virulence associated genes for use as candidate signature targets, and, further, that microarray-based single nucleotide polymorphism assays provide a robust platform for the detection and identification of signature sequences in a manner independent of the genetic background in which the signature is embedded. We discuss the strategy as a general approach or pipeline for the discovery of virulence-linked nucleic acid signatures for biothreat agents.

Applied and Environmental Microbiology, 2008

Ten variable-number tandem-repeat (VNTR) regions identified within the complete genomic sequence ... more Ten variable-number tandem-repeat (VNTR) regions identified within the complete genomic sequence of Clostridium botulinum strain ATCC 3502 were used to characterize 59 C. botulinum strains of the botulism neurotoxin A1 (BoNT/A1) to BoNT/A4 (BoNT/A1-A4) subtypes to determine their ability to discriminate among the serotype A strains. Two strains representing each of the C. botulinum serotypes B to G, including five bivalent strains, and two strains of the closely related species Clostridium sporogenes were also tested. Amplified fragment length polymorphism analyses revealed the genetic diversity among the serotypes and the high degree of similarity among many of the BoNT/A1 strains. The 10 VNTR markers amplified fragments within all of the serotype A strains but were less successful with strains of other serotypes. The composite multiple-locus VNTR analysis of the 59 BoNT/A1-A4 strains and 3 bivalent B strains identified 38 different genotypes. Thirty genotypes were identified among the 53 BoNT/A1 and BoNT/A1(B) strains, demonstrating discrimination below the subtype level. Contaminating DNA within crude toxin preparations of three BoNT/A subtypes (BoNT/A1 to BoNT/A3) also supported amplification of all of the VNTR regions. These markers provide clinical and forensics laboratories with a rapid, highly discriminatory tool to distinguish among C. botulinum BoNT/A1 strains for investigations of botulism outbreaks.

Journal of Bacteriology, 2000

Bacillus anthracis is one of the most genetically homogeneous pathogens described, making strain ... more Bacillus anthracis is one of the most genetically homogeneous pathogens described, making strain discrimination particularly difficult. In this paper, we present a novel molecular typing system based on rapidly evolving variable-number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) uses the combined power of multiple alleles at several marker loci. In our system, fluorescently labeled PCR primers are used to produce PCR amplification products from eight VNTR regions in the B. anthracis genome. These are detected and their sizes are determined using an ABI377 automated DNA sequencer. Five of these eight loci were discovered by sequence characterization of molecular markers (vrrC 1 , vrrC 2 , vrrB 1 , vrrB 2 , and CG3), two were discovered by searching complete plasmid nucleotide sequences (pXO1-aat and pXO2-at), and one was known previously (vrrA). MLVA characterization of 426 B. anthracis isolates identified 89 distinct genotypes. VNTR markers frequently identified multiple alleles (from two to nine), with Nei's diversity values between 0.3 and 0.8. Unweighted pair-group method arithmetic average cluster analysis identified six genetically distinct groups that appear to be derived from clones. Some of these clones show worldwide distribution, while others are restricted to particular geographic regions. Human commerce doubtlessly has contributed to the dispersal of particular clones in ancient and modern times.

Bacillus cereus is ubiquitous in nature, and while most isolates appear to be harmless, some are ... more Bacillus cereus is ubiquitous in nature, and while most isolates appear to be harmless, some are associated with food-borne illnesses, periodontal diseases, and other more serious infections. In one such infection, B. cereus G9241 was identified as the causative agent of a severe pneumonia in a Louisiana welder in 1994. This isolate was found to harbor most of the B. anthracis virulence plasmid pXO1 (13). Here we report the characterization of two clinical and one environmental B. cereus isolate collected during an investigation of two fatal pneumonia cases in Texas metal workers. Molecular subtyping revealed that the two cases were not caused by the same strain. However, one of the three isolates was indistinguishable from B. cereus G9241. PCR analysis demonstrated that both clinical isolates contained B. anthracis pXO1 toxin genes. One clinical isolate and the environmental isolate collected from that victim's worksite contained the cap A, B, and C genes required for capsule biosynthesis in B. anthracis. Both clinical isolates expressed a capsule; however, neither was composed of poly-D-glutamic acid. Although most B. cereus isolates are not opportunistic pathogens and only a limited number cause food-borne illnesses, these results demonstrate that some B. cereus strains can cause severe and even fatal infections in patients who appear to be otherwise healthy.

Journal of Bacteriology, 2002

The complete sequencing and annotation of the 181.7-kb Bacillus anthracis virulence plasmid pXO1 ... more The complete sequencing and annotation of the 181.7-kb Bacillus anthracis virulence plasmid pXO1 predicted 143 genes but could only assign putative functions to 45. Hybridization assays, PCR amplification, and DNA sequencing were used to determine whether pXO1 open reading frame (ORF) sequences were present in other bacilli and more distantly related bacterial genera. Eighteen Bacillus species isolates and four other bacterial species were tested for the presence of 106 pXO1 ORFs. Three ORFs were conserved in most of the bacteria tested. Many of the pXO1 ORFs were detected in closely related Bacillus species, and some were detected only in B. anthracis isolates. Three isolates, Bacillus cereus D-17, B. cereus 43881, and Bacillus thuringiensis 33679, contained sequences that were similar to more than one-half of the pXO1 ORF sequences examined. The majority of the DNA fragments that were amplified by PCR from these organisms had DNA sequences between 80 and 98% similar to that of pXO1. Pulsed-field gel electrophoresis revealed large potential plasmids present in both B. cereus 43881 (341 kb) and B. thuringiensis ATCC 33679 (327 kb) that hybridized with a DNA probe composed of six pXO1 ORFs.

Applied and Environmental Microbiology, 2005

Following detection of putative Francisella species in aerosol samples from Houston, Texas, we su... more Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities.

Emerging Infectious Diseases, 2008

A small number of conserved canonical single nucleotide polymorphisms (canSNP) that defi ne major... more A small number of conserved canonical single nucleotide polymorphisms (canSNP) that defi ne major phylogenetic branches for Bacillus anthracis were used to place a Sverdlovsk patient's B. anthracis genotype into 1 of 12 subgroups. Reconstruction of the pagA gene also showed a unique SNP that defi nes a new lineage for B. anthracis.

Members of the genus Brucella are worldwide pathogens of wildlife and livestock and are the most ... more Members of the genus Brucella are worldwide pathogens of wildlife and livestock and are the most common zoonotic infection in humans. In general, Brucella exhibit a range of hostspecificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella are highly conserved, which makes differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main Brucella species or clades and thus, enabled us to develop Real-Time PCR assays around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate into its previously determined Brucella clades. Six of the seven clade-specific assays detected DNA concentrations of less than 10 femtograms, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons among lineages of clonal bacteria and provides a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major Brucella groups that will be valuable for clinical and forensic applications.

A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism... more A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype Burkholderia pseudomallei and Burkholderia mallei isolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neighbors, such as Burkholderia thailandensis and Burkholderia cepacia.

PLoS ONE, 2014

Sequence analyses and subtyping of Bacillus anthracis strains from Georgia reveal a single distin... more Sequence analyses and subtyping of Bacillus anthracis strains from Georgia reveal a single distinct lineage (Aust94) that is ecologically established. Phylogeographic analysis and comparisons to a global collection reveals a clade that is mostly restricted to Georgia. Within this clade, many groups are found around the country, however at least one subclade is only found in the eastern part. This pattern suggests that dispersal into and out of Georgia has been rare and despite historical dispersion within the country, for at least for one lineage, current spread is limited.

Biochemistry, 1977

Nuclei isolated from cultured Chinese hamster cells were treated with micrococcal nuclease and ly... more Nuclei isolated from cultured Chinese hamster cells were treated with micrococcal nuclease and lysed, and the resulting chromatin subunit classes (nucleosomes) were purified by sedimentation and resedimentation through isokinetic sucrose gradients. Nucleosomes isolated from [3H]thymidine-labeled cells were analyzed for DNA size using both polyacrylamide gel and electron microscopic techniques. Nucleosomes isolated from [14C]lysine-labeled cells were analyzed for protein content using a sodium dodecyl sulfate-polyacrylamide gel system. The results from monitoring the [14c]lysine in each protein indicate that, in the nucleosome classes (monomer through tetramer), the molar ratios of histones H2A, H2B, H3, and H4 are equivalent. Furthermore, in each population of the nucleosome classes monomer through tetramer, it was possible to demonstrate that this histone unit (H2A + H2B + H3 + H4) is present, on the average, in the amount of two for monomers, four for dimers, six for trimers, and eight for tetramers. This is direct experimental confirmation of the prediction of R.D. Kornberg [(1974) Science 184, 868] concerning the substructure of chromatin.

Analyst, 2000

A very fast and ultrasensitive method has been developed for the detection and quantitation of sp... more A very fast and ultrasensitive method has been developed for the detection and quantitation of specific nucleic and sequences of bacterial origin in solution. The method is based on a two-color, single fluorescent molecule detection technique developed in our laboratory. The technique was applied to the detection of Bacillus anthracis DNA in solution.

PLOS One, 2009

Disease introduction into the New World during colonial expansion is well documented and had a ma... more Disease introduction into the New World during colonial expansion is well documented and had a major impact on indigenous populations; however, few diseases have been associated with early human migrations into North America. During the late Pleistocene epoch, Asia and North America were joined by the Beringian Steppe ecosystem which allowed animals and humans to freely cross what would become a water barrier in the Holocene. Anthrax has clearly been shown to be dispersed by human commerce and trade in animal products contaminated with Bacillus anthracis spores. Humans appear to have brought B. anthracis to this area from Asia and then moved it further south as an ice-free corridor opened in central Canada ,13,000 ybp. In this study, we have defined the evolutionary history of Western North American (WNA) anthrax using 2,850 single nucleotide polymorphisms (SNPs) and 285 geographically diverse B. anthracis isolates. Phylogeography of the major WNA B. anthracis clone reveals ancestral populations in northern Canada with progressively derived populations to the south; the most recent ancestor of this clonal lineage is in Eurasia. Our phylogeographic patterns are consistent with B. anthracis arriving with humans via the Bering Land Bridge. This northern-origin hypothesis is highly consistent with our phylogeographic patterns and rates of SNP accumulation observed in current day B. anthracis isolates. Continent-wide dispersal of WNA B. anthracis likely required movement by later European colonizers, but the continent's first inhabitants may have seeded the initial North American populations.

Analytical Chemistry, 2008

Genomics, 1996

man autoantigen , where the Ku70 DNA-dependent protein kinase (DNA-PK) consists of and Ku80 subun... more man autoantigen , where the Ku70 DNA-dependent protein kinase (DNA-PK) consists of and Ku80 subunits were found to form heterodimers three polypeptide subunits: Ku70, Ku80, and the DNA- ; Fran-PK catalytic subunit (DNA-PKcs). Mammalian mutants coeur et al., 1986). The Ku70/80 heterodimer binds with deficient in either Ku80 or DNA-PKcs function have high affinity to the end of double-stranded DNA and to been shown to be lacking in DNA double-strand break single-stranded DNA transitions (Mimori and Hardin, repair and V(D)J recombination, respectively. The 1986). The DNA binding activity of the complex has precise role of the Ku70 gene in this process has not been shown to be suppressed by IgG in patients with yet been determined, in part because no cell lines, anithe disease (Mimori and Hardin, 1986).

BMC Genomics, 2002

Background: Complete sequencing and annotation of the 96.2 kb Bacillus anthracis plasmid, pXO2, p... more Background: Complete sequencing and annotation of the 96.2 kb Bacillus anthracis plasmid, pXO2, predicted 85 open reading frames (ORFs). Bacillus cereus and Bacillus thuringiensis isolates that ranged in genomic similarity to B. anthracis, as determined by amplified fragment length polymorphism (AFLP) analysis, were examined by PCR for the presence of sequences similar to 47 pXO2 ORFs.

PLOS One, 2009

Disease introduction into the New World during colonial expansion is well documented and had a ma... more Disease introduction into the New World during colonial expansion is well documented and had a major impact on indigenous populations; however, few diseases have been associated with early human migrations into North America. During the late Pleistocene epoch, Asia and North America were joined by the Beringian Steppe ecosystem which allowed animals and humans to freely cross what would become a water barrier in the Holocene. Anthrax has clearly been shown to be dispersed by human commerce and trade in animal products contaminated with Bacillus anthracis spores. Humans appear to have brought B. anthracis to this area from Asia and then moved it further south as an ice-free corridor opened in central Canada ,13,000 ybp. In this study, we have defined the evolutionary history of Western North American (WNA) anthrax using 2,850 single nucleotide polymorphisms (SNPs) and 285 geographically diverse B. anthracis isolates. Phylogeography of the major WNA B. anthracis clone reveals ancestral populations in northern Canada with progressively derived populations to the south; the most recent ancestor of this clonal lineage is in Eurasia. Our phylogeographic patterns are consistent with B. anthracis arriving with humans via the Bering Land Bridge. This northern-origin hypothesis is highly consistent with our phylogeographic patterns and rates of SNP accumulation observed in current day B. anthracis isolates. Continent-wide dispersal of WNA B. anthracis likely required movement by later European colonizers, but the continent's first inhabitants may have seeded the initial North American populations.

Infection Genetics and Evolution, 2009

Biosensors & Bioelectronics, 2004

Rapid, accurate, and sensitive detection of biothreat agents requires a broad-spectrum assay capa... more Rapid, accurate, and sensitive detection of biothreat agents requires a broad-spectrum assay capable of discriminating between closely related microbial or viral pathogens. Moreover, in cases where a biological agent release has been identified, forensic analysis demands detailed genetic signature data for accurate strain identification and attribution. To date, nucleic acid sequences have provided the most robust and phylogentically illuminating signature information. Nucleic acid signature sequences are not often linked to genomic or extrachromosomal determinants of virulence, a link that would further facilitate discrimination between pathogens and closely related species. Inextricably coupling genetic determinants of virulence with highly informative nucleic acid signatures would provide a robust means of identifying human, livestock, and agricultural pathogens. By means of example, we present here an overview of two general applications of microarray-based methods for: (1) the identification of candidate virulence factors; and (2) the analysis of genetic polymorphisms that are coupled to Bacillus anthracis virulence factors using an accurate, low cost solid-phase mini-sequencing assay. We show that microarray-based analysis of gene expression can identify potential virulence associated genes for use as candidate signature targets, and, further, that microarray-based single nucleotide polymorphism assays provide a robust platform for the detection and identification of signature sequences in a manner independent of the genetic background in which the signature is embedded. We discuss the strategy as a general approach or pipeline for the discovery of virulence-linked nucleic acid signatures for biothreat agents.

Applied and Environmental Microbiology, 2008

Ten variable-number tandem-repeat (VNTR) regions identified within the complete genomic sequence ... more Ten variable-number tandem-repeat (VNTR) regions identified within the complete genomic sequence of Clostridium botulinum strain ATCC 3502 were used to characterize 59 C. botulinum strains of the botulism neurotoxin A1 (BoNT/A1) to BoNT/A4 (BoNT/A1-A4) subtypes to determine their ability to discriminate among the serotype A strains. Two strains representing each of the C. botulinum serotypes B to G, including five bivalent strains, and two strains of the closely related species Clostridium sporogenes were also tested. Amplified fragment length polymorphism analyses revealed the genetic diversity among the serotypes and the high degree of similarity among many of the BoNT/A1 strains. The 10 VNTR markers amplified fragments within all of the serotype A strains but were less successful with strains of other serotypes. The composite multiple-locus VNTR analysis of the 59 BoNT/A1-A4 strains and 3 bivalent B strains identified 38 different genotypes. Thirty genotypes were identified among the 53 BoNT/A1 and BoNT/A1(B) strains, demonstrating discrimination below the subtype level. Contaminating DNA within crude toxin preparations of three BoNT/A subtypes (BoNT/A1 to BoNT/A3) also supported amplification of all of the VNTR regions. These markers provide clinical and forensics laboratories with a rapid, highly discriminatory tool to distinguish among C. botulinum BoNT/A1 strains for investigations of botulism outbreaks.

Journal of Bacteriology, 2000

Bacillus anthracis is one of the most genetically homogeneous pathogens described, making strain ... more Bacillus anthracis is one of the most genetically homogeneous pathogens described, making strain discrimination particularly difficult. In this paper, we present a novel molecular typing system based on rapidly evolving variable-number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) uses the combined power of multiple alleles at several marker loci. In our system, fluorescently labeled PCR primers are used to produce PCR amplification products from eight VNTR regions in the B. anthracis genome. These are detected and their sizes are determined using an ABI377 automated DNA sequencer. Five of these eight loci were discovered by sequence characterization of molecular markers (vrrC 1 , vrrC 2 , vrrB 1 , vrrB 2 , and CG3), two were discovered by searching complete plasmid nucleotide sequences (pXO1-aat and pXO2-at), and one was known previously (vrrA). MLVA characterization of 426 B. anthracis isolates identified 89 distinct genotypes. VNTR markers frequently identified multiple alleles (from two to nine), with Nei's diversity values between 0.3 and 0.8. Unweighted pair-group method arithmetic average cluster analysis identified six genetically distinct groups that appear to be derived from clones. Some of these clones show worldwide distribution, while others are restricted to particular geographic regions. Human commerce doubtlessly has contributed to the dispersal of particular clones in ancient and modern times.

Bacillus cereus is ubiquitous in nature, and while most isolates appear to be harmless, some are ... more Bacillus cereus is ubiquitous in nature, and while most isolates appear to be harmless, some are associated with food-borne illnesses, periodontal diseases, and other more serious infections. In one such infection, B. cereus G9241 was identified as the causative agent of a severe pneumonia in a Louisiana welder in 1994. This isolate was found to harbor most of the B. anthracis virulence plasmid pXO1 (13). Here we report the characterization of two clinical and one environmental B. cereus isolate collected during an investigation of two fatal pneumonia cases in Texas metal workers. Molecular subtyping revealed that the two cases were not caused by the same strain. However, one of the three isolates was indistinguishable from B. cereus G9241. PCR analysis demonstrated that both clinical isolates contained B. anthracis pXO1 toxin genes. One clinical isolate and the environmental isolate collected from that victim's worksite contained the cap A, B, and C genes required for capsule biosynthesis in B. anthracis. Both clinical isolates expressed a capsule; however, neither was composed of poly-D-glutamic acid. Although most B. cereus isolates are not opportunistic pathogens and only a limited number cause food-borne illnesses, these results demonstrate that some B. cereus strains can cause severe and even fatal infections in patients who appear to be otherwise healthy.

Journal of Bacteriology, 2002

The complete sequencing and annotation of the 181.7-kb Bacillus anthracis virulence plasmid pXO1 ... more The complete sequencing and annotation of the 181.7-kb Bacillus anthracis virulence plasmid pXO1 predicted 143 genes but could only assign putative functions to 45. Hybridization assays, PCR amplification, and DNA sequencing were used to determine whether pXO1 open reading frame (ORF) sequences were present in other bacilli and more distantly related bacterial genera. Eighteen Bacillus species isolates and four other bacterial species were tested for the presence of 106 pXO1 ORFs. Three ORFs were conserved in most of the bacteria tested. Many of the pXO1 ORFs were detected in closely related Bacillus species, and some were detected only in B. anthracis isolates. Three isolates, Bacillus cereus D-17, B. cereus 43881, and Bacillus thuringiensis 33679, contained sequences that were similar to more than one-half of the pXO1 ORF sequences examined. The majority of the DNA fragments that were amplified by PCR from these organisms had DNA sequences between 80 and 98% similar to that of pXO1. Pulsed-field gel electrophoresis revealed large potential plasmids present in both B. cereus 43881 (341 kb) and B. thuringiensis ATCC 33679 (327 kb) that hybridized with a DNA probe composed of six pXO1 ORFs.

Applied and Environmental Microbiology, 2005

Following detection of putative Francisella species in aerosol samples from Houston, Texas, we su... more Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities.

Emerging Infectious Diseases, 2008

A small number of conserved canonical single nucleotide polymorphisms (canSNP) that defi ne major... more A small number of conserved canonical single nucleotide polymorphisms (canSNP) that defi ne major phylogenetic branches for Bacillus anthracis were used to place a Sverdlovsk patient's B. anthracis genotype into 1 of 12 subgroups. Reconstruction of the pagA gene also showed a unique SNP that defi nes a new lineage for B. anthracis.

Members of the genus Brucella are worldwide pathogens of wildlife and livestock and are the most ... more Members of the genus Brucella are worldwide pathogens of wildlife and livestock and are the most common zoonotic infection in humans. In general, Brucella exhibit a range of hostspecificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella are highly conserved, which makes differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main Brucella species or clades and thus, enabled us to develop Real-Time PCR assays around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate into its previously determined Brucella clades. Six of the seven clade-specific assays detected DNA concentrations of less than 10 femtograms, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons among lineages of clonal bacteria and provides a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major Brucella groups that will be valuable for clinical and forensic applications.

A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism... more A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype Burkholderia pseudomallei and Burkholderia mallei isolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neighbors, such as Burkholderia thailandensis and Burkholderia cepacia.