30 results on '"Keim, Paul"'
Search Results
2. First draft genome sequence of a strain belonging to the Zoogloea genus and its gene expression in situ.
- Author
-
Muller, Emilie E. L., Narayanasamy, Shaman, Zeimes, Myriam, Laczny, Cédric C., Lebrun, Laura A., Herold, Malte, Hicks, Nathan D., Gillece, John D., Schupp, James M., Keim, Paul, and Wilmes, Paul
- Subjects
NUCLEOTIDE sequencing ,ZOOGLOEA ,GRAM-negative bacteria ,GENOMICS ,LIPID metabolism ,SEWAGE disposal plants - Abstract
The Gram-negative beta-proteobacterium Zoogloea sp. LCSB751 (LMG 29444) was newly isolated from foaming activated sludge of a municipal wastewater treatment plant. Here, we describe its draft genome sequence and annotation together with a general physiological and genomic analysis, as the first sequenced representative of the Zoogloea genus. Moreover, Zoogloea sp. gene expression in its environment is described using metatranscriptomic data obtained from the same treatment plant. The presented genomic and transcriptomic information demonstrate a pronounced capacity of this genus to synthesize poly-ß-hydroxyalkanoate within wastewater. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
3. Dominance of multidrug resistant CC271 clones in macrolide-resistant streptococcus pneumoniae in Arizona
- Author
-
Bowers, Jolene R., Driebe, Elizabeth M., Nibecker, Jennifer L., Wojack, Bette R., Sarovich, Derek S., Wong, Ada H., Brzoska, Pius M., Hubert, Nathaniel, Knadler, Andrew, Watson, Lindsey M., Wagner, David M., Furtado, Manohar R., Saubolle, Michael, Engelthaler, David M., Keim, Paul S., Bowers, Jolene R., Driebe, Elizabeth M., Nibecker, Jennifer L., Wojack, Bette R., Sarovich, Derek S., Wong, Ada H., Brzoska, Pius M., Hubert, Nathaniel, Knadler, Andrew, Watson, Lindsey M., Wagner, David M., Furtado, Manohar R., Saubolle, Michael, Engelthaler, David M., and Keim, Paul S.
- Abstract
BackgroundRates of resistance to macrolide antibiotics in Streptococcus pneumoniae are rising around the world due to the spread of mobile genetic elements harboring mef(E) and erm(B) genes and post-vaccine clonal expansion of strains that carry them. ResultsCharacterization of 592 clinical isolates collected in Arizona over a 10 year period shows 23.6% are macrolide resistant. The largest portion of the macrolide-resistant population, 52%, is dual mef(E)/erm(B)-positive. All dual-positive isolates are multidrug-resistant clonal lineages of Taiwan19F-14, mostly multilocus sequence type 320, carrying the recently described transposon Tn2010. The remainder of the macrolide resistant S. pneumoniae collection includes 31% mef(E)-positive, and 9% erm(B)-positive strains. ConclusionsThe dual-positive, multidrug-resistant S. pneumoniae clones have likely expanded by switching to non-vaccine serotypes after the heptavalent pneumococcal conjugate vaccine release, and their success limits therapy options. This upsurge could have a considerable clinical impact in Arizona.
- Published
- 2012
4. Meta-analysis to estimate the load of Leptospira excreted in urine: beyond rats as important sources of transmission in low-income rural communities.
- Author
-
Barragan, Veronica, Nieto, Nathan, Keim, Paul, and Pearson, Talima
- Subjects
LEPTOSPIRA ,LEPTOSPIROSIS ,VIRAL load ,SYSTEMATIC reviews ,META-analysis ,INFECTIOUS disease transmission - Abstract
Leptospirosis is a major zoonotic disease with widespread distribution and a large impact on human health. Carrier animals excrete pathogenic Leptospira primarily in their urine. Infection occurs when the pathogen enters a host through mucosa or small skin abrasions. Humans and other animals are exposed to the pathogen by direct contact with urine, contaminated soil or water. While many factors influence environmental cycling and the transmission of Leptospira to humans, the load of pathogenic Leptospira in the environment is likely to play a major role. Peridomestic rats are often implicated as a potential source of human disease; however exposure to other animals is a risk factor as well. The aim of this report is to highlight the importance of various carrier animals in terms of the quantity of Leptospira shed into the environment. For this, we performed a systematic literature review and a meta-analysis of the amount of pathogen that various animal species shed in their urine. Results: The quantity of pathogen has been reported for cows, deer, dogs, humans, mice, and rats, in a total of 14 research articles. We estimated the average Leptospira per unit volume shed by each animal species, and the daily environmental contribution by considering the total volume of urine excreted by each carrier animal. Rats excrete the highest quantity of Leptospira per millilitre of urine (median = 5.7 × 10
6 cells), but large mammals excrete much more urine and thus shed significantly more Leptospira per day (5.1 × 108 to 1.3 × 109 cells). Conclusions: Here we illustrate how, in a low-income rural Ecuadorian community, host population demographics, and prevalence of Leptospira infection can be integrated with estimates of shed Leptospira to suggest that peridomestic cattle may be more important than rats in environmental cycling and ultimately, transmission to humans. [ABSTRACT FROM AUTHOR]- Published
- 2017
- Full Text
- View/download PDF
5. Whole genome SNP typing to investigate methicillin-resistant Staphylococcus aureus carriage in a health-care provider as the source of multiple surgical site infections.
- Author
-
Roe, Chandler C., Horn, Kimberly S., Driebe, Elizabeth M., Bowers, Jolene, Terriquez, Joel A., Keim, Paul, and Engelthaler, David M.
- Subjects
SINGLE nucleotide polymorphisms ,METHICILLIN-resistant staphylococcus aureus ,SURGICAL site ,PHYLOGENY ,MEDICAL personnel ,DISEASES - Abstract
Background: Prevention of nosocomial transmission of infections is a central responsibility in the healthcare environment, and accurate identification of transmission events presents the first challenge. Phylogenetic analysis based on whole genome sequencing provides a high-resolution approach for accurately relating isolates to one another, allowing precise identification or exclusion of transmission events and sources for nearly all cases. We sequenced 24 methicillin-resistant Staphylococcus aureus (MRSA) genomes to retrospectively investigate a suspected point source of three surgical site infections (SSIs) that occurred over a one-year period. The source of transmission was believed to be a surgical team member colonized with MRSA, involved in all surgeries preceding the SSI cases, who was subsequently decolonized. Genetic relatedness among isolates was determined using whole genome single nucleotide polymorphism (SNP) data. Results: Whole genome SNP typing (WGST) revealed 283 informative SNPs between the surgical team member's isolate and the closest SSI isolate. The second isolate was 286 and the third was thousands of SNPs different, indicating the nasal carriage strain from the surgical team member was not the source of the SSIs. Given the mutation rates estimated for S. aureus, none of the SSI isolates share a common ancestor within the past 16 years, further discounting any common point source for these infections. The decolonization procedures and resources spent on the point source infection control could have been prevented if WGST was performed at the time of the suspected transmission, instead of retrospectively. Conclusions: Whole genome sequence analysis is an ideal method to exclude isolates involved in transmission events and nosocomial outbreaks, and coupling this method with epidemiological data can determine if a transmission event occurred. These methods promise to direct infection control resources more appropriately. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
6. Comparative genomic analyses reveal broad diversity in botulinum-toxinproducing Clostridia.
- Author
-
Williamson, Charles H. D., Sahl, Jason W., Smith, Theresa J., Xie, Gary, Foley, Brian T., Smith, Leonard A., Fernández, Rafael A., Lindström, Miia, Korkeala, Hannu, Keim, Paul, Foster, Jeffrey, and Hill, Karen
- Subjects
COMPARATIVE genomics ,BOTULINUM toxin ,CLOSTRIDIA ,NEUROTOXIC agents ,NUCLEOTIDE sequencing - Abstract
Background: Clostridium botulinum is a diverse group of bacteria characterized by the production of botulinum neurotoxin. Botulinum neurotoxins are classified into serotypes (BoNT/A-G), which are produced by six species/ Groups of Clostridia, but the genetic background of the bacteria remains poorly understood. The purpose of this study was to use comparative genomics to provide insights into the genetic diversity and evolutionary history of bacteria that produce the potent botulinum neurotoxin. Results: Comparative genomic analyses of over 170 Clostridia genomes, including our draft genome assemblies for 59 newly sequenced Clostridia strains from six continents and publicly available genomic data, provided in-depth insights into the diversity and distribution of BoNT-producing bacteria. These newly sequenced strains included Group I and II strains that express BoNT/A,/B,/E, or/F as well as bivalent strains. BoNT-producing Clostridia and closely related Clostridia species were delineated with a variety of methods including 16S rRNA gene, concatenated marker genes, core genome and concatenated multi-locus sequencing typing (MLST) gene phylogenies that related whole genome sequenced strains to publicly available strains and sequence types. These analyses illustrated the phylogenetic diversity in each Group and the diversity of genomic backgrounds that express the same toxin type or subtype. Comparisons of the botulinum neurotoxin genes did not identify novel toxin types or variants. Conclusions: This study represents one of the most comprehensive analyses of whole genome sequence data for Group I and II BoNT-producing strains. Read data and draft genome assemblies generated for 59 isolates will be a resource to the research community. Core genome phylogenies proved to be a powerful tool for differentiating BoNT-producing strains and can provide a framework for the study of these bacteria. Comparative genomic analyses of Clostridia species illustrate the diversity of botulinum-neurotoxin-producing strains and the plasticity of the genomic backgrounds in which bont genes are found. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
7. Estimated herd prevalence and sequence types of Coxiella burnetii in bulk tank milk samples from commercial dairies in Indiana.
- Author
-
Bauer, Amy E., Olivas, Sonora, Cooper, Maria, Hornstra, Heidie, Keim, Paul, Pearson, Talima, and Johnson, April J.
- Subjects
COXIELLA burnetii ,DISEASE prevalence ,MILK analysis ,DAIRY farms ,SINGLE nucleotide polymorphisms ,DIAGNOSTIC use of polymerase chain reaction - Abstract
Background: Coxiella burnetii is the etiologic agent of Q fever, a zoonotic disease causing influenza-like illness, pregnancy loss, cardiovascular disease and chronic fatigue syndrome in people. C. burnetii is considered to be enzootic in ruminants, but clinical signs of infection do not always manifest. National studies have documented the presence of C. burnetii in dairy herds in Indiana. This represents an opportunity to better characterize the distribution and prevalence of C. burnetii infection at the state scale, allowing evaluation of the need for surveillance and response planning to occur at this level. A cross-sectional study was conducted to estimate the herd prevalence of C. burnetii in commercial cattle dairies in Indiana and characterize the strains of C. burnetii within these dairies. Results: Bulk tank milk samples were collected between June and August of 2011 by the Indiana State Board of Animal Health (ISBOAH). A total of 316 of these samples were tested for the IS1111 transposon of C. burnetii using quantitative real time polymerase chain reaction (PCR). Single nucleotide polymorphism (SNP) genotyping was used to identify the multispacer sequence genotypes (ST) present in samples where the IS1111 transposon was identified. The geographic distribution of dairies testing positive for C. burnetii DNA and the identified STs were also evaluated. The estimated overall herd prevalence for C. burnetii DNA was 61.1 % (95 % CI 55.6-66.3 %). The highest estimated regional prevalence was 70.2 % in the Central region of Indiana. An ST was identifiable in 74 of the positive 178 samples (41.6 %) and none of the 10 negative samples tested. Of these samples, 71 (95.9 %) were identified as ST20, 2 (2.7 %) as ST8 and a combination of ST20 and ST8 was identified in a single sample. Conclusions: C. burnetii is present in dairy herds throughout Indiana. Indiana follows national trends with ST20 most commonly identified. The presence of multiple STs in a single bulk tank sample indicates thatmultiple strains of C. burnetii can circulate within a herd. This supports potential transmission of C. burnetii between goats and cattle, presenting the potential for a switch in the dominant genotype found in a given species. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
8. Phylogenetically typing bacterial strains from partial SNP genotypes observed from direct sequencing of clinical specimen metagenomic data.
- Author
-
Sahl, Jason W., Schupp, James M., Rasko, David A., Colman, Rebecca E., Foster, Jeffrey T., and Keim, Paul
- Subjects
BACTERIA phylogeny ,SINGLE nucleotide polymorphisms ,NUCLEOTIDE sequence ,METAGENOMICS ,MICROBIAL genomics - Abstract
We describe an approach for genotyping bacterial strains from low coverage genome datasets, including metagenomic data from complex samples. Sequence reads from unknown samples are aligned to a reference genome where the allele states of known SNPs are determined. The Whole Genome Focused Array SNP Typing (WG-FAST) pipeline can identify unknown strains with much less read data than is needed for genome assembly. To test WG-FAST, we resampled SNPs from real samples to understand the relationship between low coverage metagenomic data and accurate phylogenetic placement. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
9. Genotyping of Coxiella burnetii from domestic ruminants and human in Hungary: indication of various genotypes.
- Author
-
Sulyok, Kinga M., Kreizinger, Zsuzsa, Hornstra, Heidie M., Pearson, Talima, Szigeti, Alexandra, Dán, Ádám, Balla, Eszter, Keim, Paul S., and Gyuranecz, Miklós
- Subjects
COXIELLA burnetii ,GENETIC polymorphisms ,BIOLOGICAL adaptation ,CATTLE genome mapping - Abstract
Background Information about the genotypic characteristic of Coxiella burnetii from Hungary is lacking. The aim of this study is to describe the genetic diversity of C. burnetii in Hungary and compare genotypes with those found elsewhere. A total of 12 samples: (cattle, n = 6, sheep, n = 5 and human, n = 1) collected from across Hungary were studied by a 10-loci multispacer sequence typing (MST) and 6-loci multiple-locus variable-number of tandem repeat analysis (MLVA). Phylogenetic relationships among MST genotypes show how these Hungarian samples are related to others collected around the world. Results Three MST genotypes were identified: sequence type (ST) 20 has also been identified in ruminants from other European countries and the USA, ST28 was previously identified in Kazakhstan, and the proposed ST37 is novel. All MST genotypes yielded different MLVA genotypes and three different MLVA genotypes were identified within ST20 samples alone. Two novel MLVA types 0-9-5-5-6-2 (AG) and 0-8-4-5-6-2 (AF) (Ms23-Ms24-Ms27-Ms28- Ms33-Ms34) were defined in the ovine materials correlated with ST28 and ST37. Samples from different parts of the phylogenetic tree were associated with different hosts, suggesting host-specific adaptations. Conclusions Even with the limited number of samples analysed, this study revealed high genetic diversity among C. burnetii in Hungary. Understanding the background genetic diversity will be essential in identifying and controlling outbreaks. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
10. Real-time PCR assays for genotyping of Cryptococcus gattii in North America.
- Author
-
Kelley, Erin J., Driebe, Elizabeth M., Etienne, Kizee, Brandt, Mary E., Schupp, James M., Gillece, John D., Trujillo, Jesse S., Lockhart, Shawn R., Deak, Eszter, Keim, Paul S., and Engelthaler, David M.
- Subjects
POLYMERASE chain reaction ,CRYPTOCOCCUS ,NUCLEOTIDE sequencing ,EPIDEMIOLOGY - Abstract
Background Cryptococcus gattii has been the cause of an ongoing outbreak starting in 1999 on Vancouver Island, British Columbia and spreading to mainland Canada and the US Pacific Northwest. In the course of the outbreak, C. gattii has been identified outside of its previously documented climate, habitat, and host disease. Genotyping of C. gattii is essential to understand the ecological and geographical expansion of this emerging pathogen. Methods We developed and validated a mismatch amplification mutation assay (MAMA) real-time PCR panel for genotyping C. gattii molecular types VGI-VGIV and VGII subtypes a,b,c. Subtype assays were designed based on whole-genome sequence of 20 C. gattii strains. Publically available multilocus sequence typing (MLST) data from a study of 202 strains was used for the molecular type (VGI-VGIV) assay design. All assays were validated across DNA from 112 strains of diverse international origin and sample types, including animal, environmental and human. Results Validation revealed each assay on the panel is 100% sensitive, specific and concordant with MLST. The assay panel can detect down to 0.5 picograms of template DNA. Conclusions The (MAMA) real-time PCR panel for C. gattii accurately typed a collection of 112 diverse strains and demonstrated high sensitivity. This is a time and cost efficient method of genotyping C. gattii best suited for application in large-scale epidemiological studies. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
11. High prevalence and two dominant host-specific genotypes of Coxiella burnetii in U.S. milk.
- Author
-
Pearson, Talima, Hornstra, Heidie M., Hilsabeck, Remy, Gates, Lauren T., Olivas, Sonora M., Birdsell, Dawn M., Hall, Carina M., German, Sabrina, Cook, James M., Seymour, Meagan L., Priestley, Rachael A., Kondas, Ashley V., Friedman, Christine L. Clark, Price, Erin P., Schupp, James M., Liu, Cindy M., Price, Lance B., Massung, Robert F., Kersh, Gilbert J., and Keim, Paul
- Subjects
COXIELLA burnetii ,HOST specificity (Biology) ,MILK ,Q fever ,SINGLE nucleotide polymorphisms ,DAIRY processing ,PUBLIC health - Abstract
Background Coxiella burnetii causes Q fever in humans and Coxiellosis in animals; symptoms range from general malaise to fever, pneumonia, endocarditis and death. Livestock are a significant source of human infection as they shed C. burnetii cells in birth tissues, milk, urine and feces. Although prevalence of C. burnetii is high, few Q fever cases are reported in the U.S. and we have a limited understanding of their connectedness due to difficulties in genotyping. Here, we develop canonical SNP genotyping assays to evaluate spatial and temporal relationships among C. burnetii environmental samples and compare them across studies. Given the genotypic diversity of historical collections, we hypothesized that the current enzootic of Coxiellosis is caused by multiple circulating genotypes. We collected A) 23 milk samples from a single bovine herd, B) 134 commercial bovine and caprine milk samples from across the U.S., and C) 400 bovine and caprine samples from six milk processing plants over three years. Results We detected C. burnetii DNA in 96% of samples with no variance over time. We genotyped 88.5% of positive samples; bovine milk contained only a single genotype (ST20) and caprine milk was dominated by a second type (mostly ST8). Conclusions The high prevalence and lack of genotypic diversity is consistent with a model of rapid spread and persistence. The segregation of genotypes between host species is indicative of species-specific adaptations or dissemination barriers and may offer insights into the relative lack of human cases and characterizing genotypes. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
12. Rapid and robust phylotyping of spa t003, a dominant MRSA clone in Luxembourg and other European countries.
- Author
-
Engelthaler, David M., Kelley, Erin, Driebe, Elizabeth M., Bowers, Jolene, Eberhard, Carl F., Trujillo, Jesse, Decruyenaere, Frederic, Schupp, James M., Mossong, Joel, Keim, Paul, and Even, Jos
- Subjects
METHICILLIN-resistant staphylococcus aureus ,CLONING ,GENOMES ,POLYMERASE chain reaction - Abstract
Background: spa typing is a common genotyping tool for methicillin-resistant Staphylococcus aureus (MRSA) in Europe. Given the high prevalence of dominant clones, spa-typing is proving to be limited in its ability to distinguish outbreak isolates from background isolates. New molecular tools need to be employed to improve subtyping of dominant local MRSA strains (e.g., spa type t003). Methods: Phylogenetically critical, or canonical, SNPs (can-SNPs) were identified as subtyping targets through sequence analysis of 40 MRSA whole genomes from Luxembourg. Real-time PCR assays were designed around target SNPs and validated using a repository of 240 previously sub-typed and epidemiologically characterized Luxembourg MRSA isolates, including 153 community and hospital isolates, 69 isolates from long term care (LTC) facilities, and 21 prospectively analyzed MRSA isolates. Selected isolates were also analyzed by whole genome SNP typing (WGST) for comparison to the SNP assays and other subtyping techniques. Results: Fourteen real-time PCR assays were developed and validated, including two assays to determine presence of spa t003 or t008. The other twelve assays successfully provided a high degree of resolution within the t003 subtype. WGST analysis of the LTC facility isolates provided greater resolution than other subtyping tools, identifying clusters indicative of ongoing transmission within LTC facilities. Conclusions: canSNP-based PCR assays are useful for local level MRSA phylotyping, especially in the presence of one or more dominant clones. The assays designed here can be easily adapted for investigating t003 MRSA strains in other regions in Western Europe. WGST provides substantially better resolution than other typing methods. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
13. Within-host evolution of Brucella canis during a canine brucellosis outbreak in a kennel.
- Author
-
Gyuranecz, Miklós, Rannals, Brandy D., Allen, Christina A., Jánosi, Szilárd, Keim, Paul S., and Foster, Jeffrey T.
- Subjects
BRUCELLA ,DOG diseases ,CELL culture ,TANDEM repeats ,ANIMAL genetics ,EPIDEMIOLOGY - Abstract
Background: Little is currently known about Brucella evolution within the host during infection. The current study is the first to employ fine-scale genotyping on an isolate collection derived from a Brucella canis outbreak. Eight isolates of B. canis, cultured from different tissues of three dogs (female, stud dog, puppy of another female) from a single kennel over three months were genetically characterized with a 15-marker multi-locus, variable-number tandem repeat (VNTR) analysis (MLVA) to assess the genetic relatedness of isolates and potential rapid mutational changes. Results: MLVA discriminated among the otherwise indistinguishable isolates from different animals and from isolates collected at different time points within each host, with different VNTR alleles being detected at multiple dates and tissue sites. We suspect that all isolates cultured from the female, puppy, and stud dogs originated from the same strain, with subsequent rapid in vivo mutations. However, high mutation rates and apparent in several of the loci prevented making definitive epidemiological relationships among isolates. Conclusions: This investigation highlights the rapid in vivo genetic mutations of several VNTRs of B. canis over a short time period in the host and the emergence of alternate alleles. However, this work also suggests the challenges of using highly mutable VNTRs to infer epidemiological relationships of strains within a short duration outbreak. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
14. FungiQuant: A broad-coverage fungal quantitative real-time PCR assay.
- Author
-
Liu, Cindy M., Kachur, Sergey, Dwan, Michael G., Abraham, Alison G., Aziz, Maliha, Hsueh, Po-Ren, Yu-Tsung Huang, Busch, Joseph D., Lamit, Louis J., Gehring, Catherine A., Keim, Paul, and Price, Lance B.
- Subjects
POLYMERASE chain reaction ,RIBOSOMAL RNA ,NUCLEOTIDE sequence ,ASPERGILLOSIS ,ERGOSTEROL ,ASPERGILLUS fumigatus - Abstract
Background: Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for new broad-coverage techniques. Methods: We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Results: We designed FungiQuant, a TaqMan® qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuant's is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r²-values of >0.99 against the 69 fungal species tested. Additionally, FungiQuant inter- and intra-run coefficients of variance ranged from <10% and <20%, respectively. We further showed that FungiQuant has a limit of quantification 25 copies and a limit of detection at 5 copies. Lastly, by comparing results from human-only background DNA with low-level fungal DNA, we showed that amplification in two or three of a FungiQuant performed in triplicate is statistically significant for true positive fungal detection. Conclusions: FungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
15. Detection of Burkholderia pseudomallei O-antigen serotypes in near-neighbor species.
- Author
-
Stone, Joshua K., Mayo, Mark, Grasso, Stephanie A., Ginther, Jennifer L., Warrington, Stephanie D., Allender, Christopher J., Doyle, Adina, Georgia, Shalamar, Kaestli, Mirjam, Broomall, Stacey M., Karavis, Mark A., Insalaco, Joseph M., Hubbard, Kyle S., McNew, Lauren A., Gibbons, Henry S., Currie, Bart J., Keim, Paul, and Tuanyok, Apichai
- Subjects
MELIOIDOSIS ,BURKHOLDERIA pseudomallei ,VACCINES ,IMMUNIZATION ,BIOSYNTHESIS - Abstract
Background: Burkholderia pseudomallei is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS) as a potential vaccine target because it is known as one of the most important antigenic epitopes in B. pseudomallei. Complicating this strategy are the four different B. pseudomallei LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of Burkholderia species. Here, we identified the presence of multiple B. pseudomallei O-antigen types and sero-crossreactivity in its near-neighbor species. Results: PCR screening of O-antigen biosynthesis genes, phenotypic characterization using SDS-PAGE, and immunoblot analysis showed that majority of B. mallei and B. thailandensis strains contained the typical O-antigen type A. In contrast, most of B. ubonensis and B. thailandensis-like strains expressed the atypical O-antigen types B and B2, respectively. Most B. oklahomensis strains expressed a distinct and non-seroreactive O-antigen type, except strain E0147 which expressed O-antigen type A. O-antigen type B2 was also detected in B. thailandensis 82172, B. ubonensis MSMB108, and Burkholderia sp. MSMB175. Interestingly, B. thailandensis-like MSMB43 contained a novel serotype B positive O-antigen. Conclusions: This study expands the number of species which express B. pseudomallei O-antigen types. Further work is required to elucidate the full structures and how closely these are to the B. pseudomallei O-antigens, which will ultimately determine the efficacy of the near-neighbor B serotypes for vaccine development. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
16. BactQuant: An enhanced broad-coverage bacterial quantitative real-time PCR assay.
- Author
-
Liu, Cindy M., Aziz, Maliha, Kachur, Sergey, Po-Ren Hsueh, Yu-Tsung Huang, Keim, Paul, and Price, Lance B.
- Subjects
FUNGUS-bacterium relationships ,MOBILE genetic elements ,FUNGAL ecophysiology ,MOLECULAR genetics ,DNA - Abstract
Background: Bacterial load quantification is a critical component of bacterial community analysis, but a culture-independent method capable of detecting and quantifying diverse bacteria is needed. Based on our analysis of a diverse collection of 16 S rRNA gene sequences, we designed a broad-coverage quantitative real-time PCR (qPCR) assay-BactQuant-for quantifying 16 S rRNA gene copy number and estimating bacterial load. We further utilized in silico evaluation to complement laboratory-based qPCR characterization to validate BactQuant. Methods: The aligned core set of 4,938 16 S rRNA gene sequences in the Greengenes database were analyzed for assay design. Cloned plasmid standards were generated and quantified using a qPCR-based approach. Coverage analysis was performed computationally using >670,000 sequences and further evaluated following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Results: A bacterial TaqMan&Reg; qPCR assay targeting a 466 bp region in V3-V4 was designed. Coverage analysis showed that 91% of the phyla, 96% of the genera, and >80% of the 89,537 species analyzed contained at least one perfect sequence match to the BactQuant assay. Of the 106 bacterial species evaluated, amplification efficiencies ranged from 81 to 120%, with r2-value of >0.99, including species with sequence mismatches. Inter- and intra-run coefficient of variance was <3% and <16% for Ct and copy number, respectively. Conclusions: The BactQuant assay offers significantly broader coverage than a previously reported universal bacterial quantification assay BactQuant in vitro performance was better than the in silico predictions. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
17. Genotyping of Brucella species using clade specific SNPs.
- Author
-
Foster, Jeffrey T., Price, Lance B., Beckstrom-Sternberg, Stephen M., Pearson, Talima, Brown, William D, Kiesling, Danika M., Allen, Christina A., Liu, Cindy M., Beckstrom-Sternberg, James, Roberto, Frank F., and Keim, Paul
- Subjects
BRUCELLOSIS ,GENOMES ,SINGLE nucleotide polymorphisms ,BIOLOGICAL assay ,PHYLOGENY - Abstract
Background: Brucellosis is a worldwide disease of mammals caused by Alphaproteobacteria in the genus Brucella. The genus is genetically monomorphic, requiring extensive genotyping to differentiate isolates. We utilized two different genotyping strategies to characterize isolates. First, we developed a microarray-based assay based on 1000 single nucleotide polymorphisms (SNPs) that were identified from whole genome comparisons of two B. abortus isolates, one B. melitensis, and one B. suis. We then genotyped a diverse collection of 85 Brucella strains at these SNP loci and generated a phylogenetic tree of relationships. Second, we developed a selective primer-extension assay system using capillary electrophoresis that targeted 17 high value SNPs across 8 major branches of the phylogeny and determined their genotypes in a large collection (n = 340) of diverse isolates. Results: Our 1000 SNP microarray readily distinguished B. abortus, B. melitensis, and B. suis, differentiating B. melitensis and B. suis into two clades each. Brucella abortus was divided into four major clades. Our capillary-based SNP genotyping confirmed all major branches from the microarray assay and assigned all samples to defined lineages. Isolates from these lineages and closely related isolates, among the most commonly encountered lineages worldwide, can now be quickly and easily identified and genetically characterized. Conclusions: We have identified clade-specific SNPs in Brucella that can be used for rapid assignment into major groups below the species level in the three main Brucella species. Our assays represent SNP genotyping approaches that can reliably determine the evolutionary relationships of bacterial isolates without the need for whole genome sequencing of all isolates. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
18. Single nucleotide polymorphisms for assessing genetic diversity in castor bean (Ricinus communis).
- Author
-
Foster, Jeffrey T., Allan, Gerard J., Chan, Agnes P., Rabinowicz, Pablo D., Ravel, Jacques, Jackson, Paul J., and Keim, Paul
- Subjects
CASTOR beans ,OILSEEDS ,GENETIC polymorphisms ,POPULATION genetics ,BOTANY - Abstract
Background: Castor bean (Ricinus communis) is an agricultural crop and garden ornamental that is widely cultivated and has been introduced worldwide. Understanding population structure and the distribution of castor bean cultivars has been challenging because of limited genetic variability. We analyzed the population genetics of R. communis in a worldwide collection of plants from germplasm and from naturalized populations in Florida, U.S. To assess genetic diversity we conducted survey sequencing of the genomes of seven diverse cultivars and compared the data to a reference genome assembly of a widespread cultivar (Hale). We determined the population genetic structure of 676 samples using single nucleotide polymorphisms (SNPs) at 48 loci. Results: Bayesian clustering indicated five main groups worldwide and a repeated pattern of mixed genotypes in most countries. High levels of population differentiation occurred between most populations but this structure was not geographically based. Most molecular variance occurred within populations (74%) followed by 22% among populations, and 4% among continents. Samples from naturalized populations in Florida indicated significant population structuring consistent with local demes. There was significant population differentiation for 56 of 78 comparisons in Florida (pairwise population ϕPT values, p < 0.01). Conclusion: Low levels of genetic diversity and mixing of genotypes have led to minimal geographic structuring of castor bean populations worldwide. Relatively few lineages occur and these are widely distributed. Our approach of determining population genetic structure using SNPs from genome-wide comparisons constitutes a framework for high-throughput analyses of genetic diversity in plants, particularly in species with limited genetic diversity. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
19. Accurate, rapid and high-throughput detection of strain-specific polymorphisms in Bacillus anthracis and Yersinia pestis by next-generation sequencing.
- Author
-
Cummings, Craig A., Chung, Christina A. Bormann, Fang, Rixun, Barker, Melissa, Brzoska, Pius, Williamson, Phillip C., Beaudry, Jodi, Matthews, Molly, Schupp, James, Wagner, David M, Birdsell, Dawn, Vogler, Amy J., Furtado, Manohar R., Keim, Paul, and Budowle, Bruce
- Subjects
GENETIC polymorphisms ,POPULATION genetics ,BACILLUS anthracis ,BACILLUS (Bacteria) ,GENOMICS - Abstract
Background: In the event of biocrimes or infectious disease outbreaks, high-resolution genetic characterization for identifying the agent and attributing it to a specific source can be crucial for an effective response. Until recently, in-depth genetic characterization required expensive and time-consuming Sanger sequencing of a few strains, followed by genotyping of a small number of marker loci in a panel of isolates at or by gel-based approaches such as pulsed field gel electrophoresis, which by necessity ignores most of the genome. Next-generation, massively parallel sequencing (MPS) technology (specifically the Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD™) system) is a powerful investigative tool for rapid, cost-effective and parallel microbial whole-genome characterization. Results: To demonstrate the utility of MPS for whole-genome typing of monomorphic pathogens, four Bacillus anthracis and four Yersinia pestis strains were sequenced in parallel. Reads were aligned to complete reference genomes, and genomic variations were identified. Resequencing of the B. anthracis Ames ancestor strain detected no false-positive single-nucleotide polymorphisms (SNPs), and mapping of reads to the Sterne strain correctly identified 98% of the 133 SNPs that are not clustered or associated with repeats. Three geographically distinct B. anthracis strains from the A branch lineage were found to have between 352 and 471 SNPs each, relative to the Ames genome, and one strain harbored a genomic amplification. Sequencing of four Y. pestis strains from the Orientalis lineage identified between 20 and 54 SNPs per strain relative to the CO92 genome, with the single Bolivian isolate having approximately twice as many SNPs as the three more closely related North American strains. Coverage plotting also revealed a common deletion in two strains and an amplification in the Bolivian strain that appear to be due to insertion element-mediated recombination events. Most private SNPs (that is, a, variant found in only one strain in this set) selected for validation by Sanger sequencing were confirmed, although rare falsepositive SNPs were associated with variable nucleotide tandem repeats. Conclusions: The high-throughput, multiplexing capability, and accuracy of this system make it suitable for rapid whole-genome typing of microbial pathogens during a forensic or epidemiological investigation. By interrogating nearly every base of the genome, rare polymorphisms can be reliably discovered, thus facilitating high-resolution strain tracking and strengthening forensic attribution. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
20. Identifying experimental surrogates for Bacillus anthracis spores: a review.
- Author
-
Greenberg, David L., Busch, Joseph D., Keim, Paul, and Wagner, David M.
- Subjects
BACILLUS anthracis ,BACILLUS (Bacteria) ,ANTHRAX ,BACTERIAL diseases ,BACTERIAL spores - Abstract
Bacillus anthracis, the causative agent of anthrax, is a proven biological weapon. In order to study this threat, a number of experimental surrogates have been used over the past 70 years. However, not all surrogates are appropriate for B. anthracis, especially when investigating transport, fate and survival. Although B. atrophaeus has been widely used as a B. anthracis surrogate, the two species do not always behave identically in transport and survival models. Therefore, we devised a scheme to identify a more appropriate surrogate for B. anthracis. Our selection criteria included risk of use (pathogenicity), phylogenetic relationship, morphology and comparative survivability when challenged with biocides. Although our knowledge of certain parameters remains incomplete, especially with regards to comparisons of spore longevity under natural conditions, we found that B. thuringiensis provided the best overall fit as a non-pathogenic surrogate for B. anthracis. Thus, we suggest focusing on this surrogate in future experiments of spore fate and transport modelling. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
21. Bacillus anthracis in China and its relationship to worldwide lineages.
- Author
-
Simonson, Tatum S., Okinaka, Richard T., Bingxiang Wang, Easterday, W. Ryan, Huynh, Lynn, U'Ren, Jana M., Dukerich, Meghan, Zanecki, Shaylan R., Kenefic, Leo J., Beaudry, Jodi, Schupp, James M., Pearson, Talima, Wagner, David M., Hoffmaster, Alex, Ravel, Jacques, and Keim, Paul
- Subjects
BACILLUS anthracis ,BACILLUS (Bacteria) ,NUCLEOTIDES ,GENETIC polymorphisms - Abstract
Background: The global pattern of distribution of 1033 B. anthracis isolates has previously been defined by a set of 12 conserved canonical single nucleotide polymorphisms (canSNP). These studies reinforced the presence of three major lineages and 12 sub-lineages and sub-groups of this anthrax-causing pathogen. Isolates that form the A lineage (unlike the B and C lineages) have become widely dispersed throughout the world and form the basis for the geographical disposition of "modern" anthrax. An archival collection of 191 different B. anthracis isolates from China provides a glimpse into the possible role of Chinese trade and commerce in the spread of certain sub-lineages of this pathogen. Canonical single nucleotide polymorphism (canSNP) and multiple locus VNTR analysis (MLVA) typing has been used to examine this archival collection of isolates. Results: The canSNP study indicates that there are 5 different sub-lineages/sub-groups in China out of 12 previously described world-wide canSNP genotypes. Three of these canSNP genotypes were only found in the western-most province of China, Xinjiang. These genotypes were A.Br.008/ 009, a sub-group that is spread across most of Europe and Asia; A.Br.Aust 94, a sub-lineage that is present in Europe and India, and A.Br.Vollum, a lineage that is also present in Europe. The remaining two canSNP genotypes are spread across the whole of China and belong to sub-group A.Br.001/002 and the A.Br.Ames sub-lineage, two closely related genotypes. MLVA typing adds resolution to the isolates in each canSNP genotype and diversity indices for the A.Br.008/009 and A.Br.001/002 sub-groups suggest that these represent older and established clades in China. Conclusion: B. anthracis isolates were recovered from three canSNP sub-groups (A.Br.008/009, A.Br.Aust94, and A.Br.Vollum) in the western most portion of the large Chinese province of Xinjiang. The city of Kashi in this province appears to have served as a crossroads for not only trade but the movement of diseases such as anthrax along the ancient "silk road". Phylogenetic inference also suggests that the A.Br.Ames sub-lineage, first identified in the original Ames strain isolated from Jim Hogg County, TX, is descended from the A.Br.001/002 sub-group that has a major presence in most of China. These results suggest a genetic discontinuity between the younger Ames sub-lineage in Texas and the large Western North American sub-lineage spread across central Canada and the Dakotas. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
22. Microarrays for global expression constructed with a low redundancy set of 27,500 sequenced cDNAs representing an array of developmental stages and physiological conditions of the soybean plant.
- Author
-
Vodkin, Lila O., Khanna, Anupama, Shealy, Robin, Clough, Steven J., Gonzalez, Delkin Orlando, Philip, Reena, Zabala, Gracia, Thibaud-Nissen, Françoise, Sidarous, Mark, Strömvik, Martina V., Shoop, Elizabeth, Schmidt, Christina, Retzel, Ernest, Erpelding, John, Shoemaker, Randy C., Rodriguez-Huete, Alicia M., Polacco, Joseph C., Coryell, Virginia, Keim, Paul, and Gong, George
- Subjects
DNA microarrays ,ANTISENSE DNA ,SOYBEAN ,GENE expression ,MESSENGER RNA ,POLYMERASE chain reaction - Abstract
Background: Microarrays are an important tool with which to examine coordinated gene expression. Soybean (Glycine max) is one of the most economically valuable crop species in the world food supply. In order to accelerate both gene discovery as well as hypothesis-driven research in soybean, global expression resources needed to be developed. The applications of microarray for determining patterns of expression in different tissues or during conditional treatments by dual labeling of the mRNAs are unlimited. In addition, discovery of the molecular basis of traits through examination of naturally occurring variation in hundreds of mutant lines could be enhanced by the construction and use of soybean cDNA microarrays. Results: We report the construction and analysis of a low redundancy 'unigene' set of 27,513 clones that represent a variety of soybean cDNA libraries made from a wide array of source tissue and organ systems, developmental stages, and stress or pathogen-challenged plants. The set was assembled from the 5′ sequence data of the cDNA clones using cluster analysis programs. The selected clones were then physically reracked and sequenced at the 3′ end. In order to increase gene discovery from immature cotyledon libraries that contain abundant mRNAs representing storage protein gene families, we utilized a high density filter normalization approach to preferentially select more weakly expressed cDNAs. All 27,513 cDNA inserts were amplified by polymerase chain reaction. The amplified products, along with some repetitively spotted control or 'choice' clones, were used to produce three 9,728-element microarrays that have been used to examine tissue specific gene expression and global expression in mutant isolines. Conclusions: Global expression studies will be greatly aided by the availability of the sequence-validated and low redundancy cDNA sets described in this report. These cDNAs and ESTs represent a wide array of developmental stages and physiological conditions of the soybean plant. We also demonstrate that the quality of the data from the soybean cDNA microarrays is sufficiently reliable to examine isogenic lines that differ with respect to a mutant phenotype and thereby to define a small list of candidate genes potentially encoding or modulated by the mutant phenotype. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
23. Dominance of multidrug resistant CC271 clones in macrolide-resistant streptococcus pneumoniae in Arizona.
- Author
-
Bowers JR, Driebe EM, Nibecker JL, Wojack BR, Sarovich DS, Wong AH, Brzoska PM, Hubert N, Knadler A, Watson LM, Wagner DM, Furtado MR, Saubolle M, Engelthaler DM, and Keim PS
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Arizona epidemiology, Child, Child, Preschool, Cluster Analysis, DNA Transposable Elements, Female, Genes, Bacterial, Genotype, Humans, Infant, Male, Middle Aged, Molecular Epidemiology, Multilocus Sequence Typing, Pneumococcal Infections microbiology, Polymerase Chain Reaction, Streptococcus pneumoniae genetics, Streptococcus pneumoniae isolation & purification, Young Adult, Anti-Bacterial Agents pharmacology, Drug Resistance, Multiple, Bacterial, Macrolides pharmacology, Pneumococcal Infections epidemiology, Streptococcus pneumoniae classification, Streptococcus pneumoniae drug effects
- Abstract
Background: Rates of resistance to macrolide antibiotics in Streptococcus pneumoniae are rising around the world due to the spread of mobile genetic elements harboring mef(E) and erm(B) genes and post-vaccine clonal expansion of strains that carry them., Results: Characterization of 592 clinical isolates collected in Arizona over a 10 year period shows 23.6% are macrolide resistant. The largest portion of the macrolide-resistant population, 52%, is dual mef(E)/erm(B)-positive. All dual-positive isolates are multidrug-resistant clonal lineages of Taiwan19F-14, mostly multilocus sequence type 320, carrying the recently described transposon Tn2010. The remainder of the macrolide resistant S. pneumoniae collection includes 31% mef(E)-positive, and 9% erm(B)-positive strains., Conclusions: The dual-positive, multidrug-resistant S. pneumoniae clones have likely expanded by switching to non-vaccine serotypes after the heptavalent pneumococcal conjugate vaccine release, and their success limits therapy options. This upsurge could have a considerable clinical impact in Arizona.
- Published
- 2012
- Full Text
- View/download PDF
24. Tularemia in Alaska, 1938 - 2010.
- Author
-
Hansen CM, Vogler AJ, Keim P, Wagner DM, and Hueffer K
- Subjects
- Alaska epidemiology, Animals, Francisella tularensis genetics, History, 20th Century, History, 21st Century, Humans, Tularemia epidemiology, Animals, Wild microbiology, Disease Reservoirs microbiology, Francisella tularensis isolation & purification, Population Surveillance, Tularemia history
- Abstract
Tularemia is a serious, potentially life threatening zoonotic disease. The causative agent, Francisella tularensis, is ubiquitous in the Northern hemisphere, including Alaska, where it was first isolated from a rabbit tick (Haemophysalis leporis-palustris) in 1938. Since then, F. tularensis has been isolated from wildlife and humans throughout the state. Serologic surveys have found measurable antibodies with prevalence ranging from < 1% to 50% and 4% to 18% for selected populations of wildlife species and humans, respectively. We reviewed and summarized known literature on tularemia surveillance in Alaska and summarized the epidemiological information on human cases reported to public health officials. Additionally, available F. tularensis isolates from Alaska were analyzed using canonical SNPs and a multi-locus variable-number tandem repeats (VNTR) analysis (MLVA) system. The results show that both F. t. tularensis and F. t. holarctica are present in Alaska and that subtype A.I, the most virulent type, is responsible for most recently reported human clinical cases in the state.
- Published
- 2011
- Full Text
- View/download PDF
25. An attenuated strain of Bacillus anthracis (CDC 684) has a large chromosomal inversion and altered growth kinetics.
- Author
-
Okinaka RT, Price EP, Wolken SR, Gruendike JM, Chung WK, Pearson T, Xie G, Munk C, Hill KK, Challacombe J, Ivins BE, Schupp JM, Beckstrom-Sternberg SM, Friedlander A, and Keim P
- Subjects
- Bacillus anthracis classification, Base Sequence, Genome, Bacterial, Molecular Sequence Data, Phylogeny, Polymorphism, Single Nucleotide, Bacillus anthracis genetics, Bacillus anthracis growth & development, Chromosome Inversion
- Abstract
Background: An isolate originally labeled Bacillus megaterium CDC 684 was found to contain both pXO1 and pXO2, was non-hemolytic, sensitive to gamma-phage, and produced both the protective antigen and the poly-D-glutamic acid capsule. These phenotypes prompted Ezzell et al., (J. Clin. Microbiol. 28:223) to reclassify this isolate to Bacillus anthracis in 1990., Results: We demonstrate that despite these B. anthracis features, the isolate is severely attenuated in a guinea pig model. This prompted whole genome sequencing and closure. The comparative analysis of CDC 684 to other sequenced B. anthracis isolates and further analysis reveals: a) CDC 684 is a close relative of a virulent strain, Vollum A0488; b) CDC 684 defines a new B. anthracis lineage (at least 51 SNPs) that includes 15 other isolates; c) the genome of CDC 684 contains a large chromosomal inversion that spans 3.3 Mbp; d) this inversion has caused a displacement of the usual spatial orientation of the origin of replication (ori) to the termination of replication (ter) from 180° in wild-type B. anthracis to 120° in CDC 684 and e) this isolate also has altered growth kinetics in liquid media., Conclusions: We propose two alternative hypotheses explaining the attenuated phenotype of this isolate. Hypothesis 1 suggests that the skewed ori/ter relationship in CDC 684 has altered its DNA replication and/or transcriptome processes resulting in altered growth kinetics and virulence capacity. Hypothesis 2 suggests that one or more of the single nucleotide polymorphisms in CDC 684 has altered the expression of a regulatory element or other genes necessary for virulence.
- Published
- 2011
- Full Text
- View/download PDF
26. Phylogeography of Francisella tularensis subspecies holarctica from the country of Georgia.
- Author
-
Chanturia G, Birdsell DN, Kekelidze M, Zhgenti E, Babuadze G, Tsertsvadze N, Tsanava S, Imnadze P, Beckstrom-Sternberg SM, Beckstrom-Sternberg JS, Champion MD, Sinari S, Gyuranecz M, Farlow J, Pettus AH, Kaufman EL, Busch JD, Pearson T, Foster JT, Vogler AJ, Wagner DM, and Keim P
- Subjects
- Cluster Analysis, DNA, Bacterial chemistry, DNA, Bacterial genetics, Francisella tularensis isolation & purification, Georgia (Republic), Molecular Sequence Data, Sequence Analysis, DNA, Francisella tularensis classification, Francisella tularensis genetics, Phylogeography, Tularemia microbiology
- Abstract
Background: Francisella tularensis, the causative agent of tularemia, displays subspecies-specific differences in virulence, geographic distribution, and genetic diversity. F. tularensis subsp. holarctica is widely distributed throughout the Northern Hemisphere. In Europe, F. tularensis subsp. holarctica isolates have largely been assigned to two phylogenetic groups that have specific geographic distributions. Most isolates from Western Europe are assigned to the B.Br.FTNF002-00 group, whereas most isolates from Eastern Europe are assigned to numerous lineages within the B.Br.013 group. The eastern geographic extent of the B.Br.013 group is currently unknown due to a lack of phylogenetic knowledge about populations at the European/Asian juncture and in Asia. In this study, we address this knowledge gap by describing the phylogenetic structure of F. tularensis subsp. holarctica isolates from the country of Georgia, and by placing these isolates into a global phylogeographic context., Results: We identified a new genetic lineage of F. tularensis subsp. holarctica from Georgia that belongs to the B.Br.013 group. This new lineage is genetically and geographically distinct from lineages previously described from the B.Br.013 group from Central-Eastern Europe. Importantly, this new lineage is basal within the B.Br.013 group, indicating the Georgian lineage diverged before the diversification of the other known B.Br.013 lineages. Although two isolates from the Georgian lineage were collected nearby in the Ukrainian region of Crimea, all other global isolates assigned to this lineage were collected in Georgia. This restricted geographic distribution, as well as the high levels of genetic diversity within the lineage, is consistent with a relatively older origin and localized differentiation., Conclusions: We identified a new lineage of F. tularensis subsp. holarctica from Georgia that appears to have an older origin than any other diversified lineages previously described from the B.Br.013 group. This finding suggests that additional phylogenetic studies of F. tularensis subsp. holarctica populations in Eastern Europe and Asia have the potential to yield important new insights into the evolutionary history and phylogeography of this broadly dispersed F. tularensis subspecies.
- Published
- 2011
- Full Text
- View/download PDF
27. Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer.
- Author
-
Pearson T, Giffard P, Beckstrom-Sternberg S, Auerbach R, Hornstra H, Tuanyok A, Price EP, Glass MB, Leadem B, Beckstrom-Sternberg JS, Allan GJ, Foster JT, Wagner DM, Okinaka RT, Sim SH, Pearson O, Wu Z, Chang J, Kaul R, Hoffmaster AR, Brettin TS, Robison RA, Mayo M, Gee JE, Tan P, Currie BJ, and Keim P
- Subjects
- Australia, DNA, Bacterial chemistry, DNA, Bacterial genetics, Genome, Bacterial, Humans, Molecular Epidemiology, Phylogeny, Polymorphism, Single Nucleotide, Sequence Analysis, DNA, Sequence Homology, Burkholderia pseudomallei genetics, Gene Transfer, Horizontal physiology, Genes, Bacterial, Genetics, Population
- Abstract
Background: Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction., Results: Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia., Conclusion: We describe an Australian origin for B. pseudomallei, characterized by a single introduction event into Southeast Asia during a recent glacial period, and variable levels of lateral gene transfer within populations. These patterns provide insights into mechanisms of genetic diversification in B. pseudomallei and its closest relatives, and provide a framework for integrating the traditionally separate fields of population genetics and phylogenetics for other bacterial species with high levels of lateral gene transfer.
- Published
- 2009
- Full Text
- View/download PDF
28. Francisella tularensis subsp. novicida isolated from a human in Arizona.
- Author
-
Birdsell DN, Stewart T, Vogler AJ, Lawaczeck E, Diggs A, Sylvester TL, Buchhagen JL, Auerbach RK, Keim P, and Wagner DM
- Abstract
Background: Francisella tularensis is the etiologic agent of tularemia and is classified as a select agent by the Centers for Disease Control and Prevention. Currently four known subspecies of F. tularensis that differ in virulence and geographical distribution are recognized:tularensis (type A), holarctica (type B), mediasiatica, and novicida. Because of the Select Agent status and differences in virulence and geographical location, the molecular analysis of any clinical case of tularemia is of particular interest. We analyzed an unusual Francisella clinical isolate from a human infection in Arizona using multiple DNA-based approaches., Findings: We report that the isolate is F. tularensis subsp. novicida, a subspecies that is rarely isolated., Conclusion: The rarity of this novicida subspecies in clinical settings makes each case study important for our understanding of its role in disease and its genetic relationship with other F. tularensis subspecies.
- Published
- 2009
- Full Text
- View/download PDF
29. Genomic islands from five strains of Burkholderia pseudomallei.
- Author
-
Tuanyok A, Leadem BR, Auerbach RK, Beckstrom-Sternberg SM, Beckstrom-Sternberg JS, Mayo M, Wuthiekanun V, Brettin TS, Nierman WC, Peacock SJ, Currie BJ, Wagner DM, and Keim P
- Subjects
- Gene Transfer, Horizontal, RNA, Transfer genetics, Terminology as Topic, Burkholderia mallei genetics, Genetic Variation, Genomic Islands
- Abstract
Background: Burkholderia pseudomallei is the etiologic agent of melioidosis, a significant cause of morbidity and mortality where this infection is endemic. Genomic differences among strains of B. pseudomallei are predicted to be one of the major causes of the diverse clinical manifestations observed among patients with melioidosis. The purpose of this study was to examine the role of genomic islands (GIs) as sources of genomic diversity in this species., Results: We found that genomic islands (GIs) vary greatly among B. pseudomallei strains. We identified 71 distinct GIs from the genome sequences of five reference strains of B. pseudomallei: K96243, 1710b, 1106a, MSHR668, and MSHR305. The genomic positions of these GIs are not random, as many of them are associated with tRNA gene loci. In particular, the 3' end sequences of tRNA genes are predicted to be involved in the integration of GIs. We propose the term "tRNA-mediated site-specific recombination" (tRNA-SSR) for this mechanism. In addition, we provide a GI nomenclature that is based upon integration hotspots identified here or previously described., Conclusion: Our data suggest that acquisition of GIs is one of the major sources of genomic diversity within B. pseudomallei and the molecular mechanisms that facilitate horizontally-acquired GIs are common across multiple strains of B. pseudomallei. The differential presence of the 71 GIs across multiple strains demonstrates the importance of these mobile elements for shaping the genetic composition of individual strains and populations within this bacterial species.
- Published
- 2008
- Full Text
- View/download PDF
30. Tandem repeat regions within the Burkholderia pseudomallei genome and their application for high resolution genotyping.
- Author
-
U'Ren JM, Schupp JM, Pearson T, Hornstra H, Friedman CL, Smith KL, Daugherty RR, Rhoton SD, Leadem B, Georgia S, Cardon M, Huynh LY, DeShazer D, Harvey SP, Robison R, Gal D, Mayo MJ, Wagner D, Currie BJ, and Keim P
- Subjects
- DNA, Bacterial chemistry, DNA, Bacterial genetics, Genotype, Polymerase Chain Reaction, Sequence Analysis, DNA, Burkholderia pseudomallei genetics, Genome, Bacterial, Tandem Repeat Sequences genetics
- Abstract
Background: The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across a diverse panel of 31 isolates including B. pseudomallei, B. mallei and B. thailandensis in order to identify loci with varying degrees of polymorphism. A subset of these tandem repeat arrays were subsequently developed into a multiple-locus VNTR analysis to examine 66 B. pseudomallei and 21 B. mallei isolates from around the world, as well as 95 lineages from a serial transfer experiment encompassing ~18,000 generations., Results: B. pseudomallei contains a preponderance of tandem repeat loci throughout its genome, many of which are duplicated elsewhere in the genome. The majority of these loci are composed of repeat motif lengths of 6 to 9 bp with 4 to 10 repeat units and are predominately located in intergenic regions of the genome. Across geographically diverse B. pseudomallei and B.mallei isolates, the 32 VNTR loci displayed between 7 and 28 alleles, with Nei's diversity values ranging from 0.47 and 0.94. Mutation rates for these loci are comparable (>10-5 per locus per generation) to that of the most diverse tandemly repeated regions found in other less diverse bacteria., Conclusion: The frequency, location and duplicate nature of tandemly repeated regions within the B. pseudomallei genome indicate that these tandem repeat regions may play a role in generating and maintaining adaptive genomic variation. Multiple-locus VNTR analysis revealed extensive diversity within the global isolate set containing B. pseudomallei and B. mallei, and it detected genotypic differences within clonal lineages of both species that were identical using previous typing methods. Given the health threat to humans and livestock and the potential for B. pseudomallei to be released intentionally, MLVA could prove to be an important tool for fine-scale epidemiological or forensic tracking of this increasingly important environmental pathogen.
- Published
- 2007
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.