Last data update: Jan 27, 2025. (Total: 48650 publications since 2009)
Records 1-30 (of 43 Records) |
Query Trace: Keim M[original query] |
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Strong population structure in Venezuelan populations of Coccidioides posadasii (preprint)
Teixeira MM , Alvarado P , Roe CC , Thompson GR 3rd , Patane JSL , Sahl JW , Keim P , Galgiani JN , Litvintseva AP , Matute DR , Barker BM . bioRxiv 2019 719328 Coccidioides posadasii is a pathogenic fungus that causes coccidioidomycosis in many arid regions of the Americas. One of these regions is bordered by the Caribbean Sea, and the surrounding landscape may play an important role in the dispersion of C. posadasii across South America through southeastern Mexico, Honduras, Guatemala and Venezuela. Comparative phylogenomic analyses of C. posadasii reveal that clinical strains from Venezuela are genetically distinct from the North American populations found in Arizona (AZ), Texas, Mexico, and the rest of South America (TX/MX/SA). We find evidence for admixture between the Venezuela and the North American populations of C. posadasii in Central America. As expected, the proportion of Venezuelan alleles in the admixed population decreases as latitude (and distance from Venezuela) increases. Our results indicate that the population in Venezuela may have been subjected to a recent bottleneck, and shows strong population structure. This analysis provides insight into potential for Coccidioides spp. to invade new regions. |
Burkholderia thailandensis Isolated from the Environment, United States.
Hall CM , Stone NE , Martz M , Hutton SM , Santana-Propper E , Versluis L , Guidry K , Ortiz M , Busch JD , Maness T , Stewart J , Sidwa T , Gee JE , Elrod MG , Petras JK , Ty MC , Gulvik C , Weiner ZP , Salzer JS , Hoffmaster AR , Rivera-Garcia S , Keim P , Kieffer A , Sahl JW , Soltero F , Wagner DM . Emerg Infect Dis 2023 29 (3) 618-621 ![]() ![]() Burkholderia thailandensis, an opportunistic pathogen found in the environment, is a bacterium closely related to B. pseudomallei, the cause of melioidosis. Human B. thailandensis infections are uncommon. We isolated B. thailandensis from water in Texas and Puerto Rico and soil in Mississippi in the United States, demonstrating a potential public health risk. |
Measuring the efficacy of a pilot public health intervention for engaging communities of Puerto Rico to rapidly write hurricane protection plans
Keim ME , Runnels LA , Lovallo AP , Pagan Medina M , Roman Rosa E , Ramery Santos M , Mahany M , Cruz MA . Prehosp Disaster Med 2020 36 (1) 1-10 OBJECTIVE: The efficacy is measured for a public health intervention related to community-based planning for population protection measures (PPMs; ie, shelter-in-place and evacuation). DESIGN: This is a mixed (qualitative and quantitative) prospective study of intervention efficacy, measured in terms of usability related to effectiveness, efficiency, satisfaction, and degree of community engagement. SETTING: Two municipalities in the Commonwealth of Puerto Rico are included. PARTICIPANTS: Community members consisting of individuals; traditional leaders; federal, territorial, and municipal emergency managers; municipal mayors; National Guard; territorial departments of education, health, housing, public works, and transportation; health care; police; Emergency Medical Services; faith-based organizations; nongovernmental organizations (NGOs); and the private sector. INTERVENTION: The intervention included four community convenings: one for risk communication; two for plan-writing; and one tabletop exercise (TTX). This study analyzed data collected from the project work plan; participant rosters; participant surveys; workshop outputs; and focus group interviews. MAIN OUTCOME MEASURES: Efficacy was measured in terms of ISO 9241-11, an international standard for usability that includes effectiveness, efficiency, user satisfaction, and "freedom from risk" among users. Degree of engagement was considered an indicator of "freedom from risk," measurable through workshop attendance. RESULTS: Two separate communities drafted and exercised ~60-page-long population protection plans, each within 14.5 hours. Plan-writing workshops completed 100% of plan objectives and activities. Efficiency rates were nearly the same in both communities. Interviews and surveys indicated high degrees of community satisfaction. Engagement was consistent among community members and variable among governmental officials. CONCLUSIONS: Frontline communities have successfully demonstrated the ability to understand the environmental health hazards in their own community; rapidly write consensus-based plans for PPMs; participate in an objective-based TTX; and perform these activities in a bi-lingual setting. This intervention appears to be efficacious for public use in the rapid development of community-based PPMs. |
Population Structure and Genetic Diversity among Isolates of Coccidioides posadasii in Venezuela and Surrounding Regions.
Teixeira MM , Alvarado P , Roe CC , Thompson GR 3rd , Patane JSL , Sahl JW , Keim P , Galgiani JN , Litvintseva AP , Matute DR , Barker BM . mBio 2019 10 (6) ![]() ![]() Coccidioides posadasii is a pathogenic fungus that causes coccidioidomycosis in many arid regions of the Americas. One of these regions is bordered by the Caribbean Sea, and the surrounding landscape may play an important role in the dispersion of C. posadasii across South America through southeastern Mexico, Honduras, Guatemala, and Venezuela. Comparative phylogenomic analyses of C. posadasii reveal that clinical strains from Venezuela are genetically distinct from the North American populations found in (i) Arizona and (ii) Texas, Mexico, and the rest of South America (TX/MX/SA). We find evidence for admixture between the Venezuela and the North American populations of C. posadasii in Central America. Additionally, the proportion of Venezuelan alleles in the admixed population decreases as latitude (and distance from Venezuela) increases. Our results indicate that the population in Venezuela may have been subjected to a recent bottleneck and shows a strong population structure. This analysis provides insight into potential for Coccidioides spp. to invade new regions.IMPORTANCE Valley Fever is a fungal disease caused by two species of fungi: Coccidioides immitis and C. posadasii These fungi are found throughout the arid regions of North and South America; however, our understanding of genetic diversity and disease in South America is limited. In this report, we analyze 10 new genomes of Coccidioides posadasii from regions bordering the Caribbean Sea. We show that these populations are distinct and that isolates from Venezuela are likely a result of a recent bottleneck. These data point to patterns that might be observed when investigating recently established populations. |
Burkholderia pseudomallei, the causative agent of melioidosis, is rare but ecologically established and widely dispersed in the environment in Puerto Rico.
Hall CM , Jaramillo S , Jimenez R , Stone NE , Centner H , Busch JD , Bratsch N , Roe CC , Gee JE , Hoffmaster AR , Rivera-Garcia S , Soltero F , Ryff K , Perez-Padilla J , Keim P , Sahl JW , Wagner DM . PLoS Negl Trop Dis 2019 13 (9) e0007727 ![]() ![]() BACKGROUND: Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The global burden and distribution of melioidosis is poorly understood, including in the Caribbean. B. pseudomallei was previously isolated from humans and soil in eastern Puerto Rico but the abundance and distribution of B. pseudomallei in Puerto Rico as a whole has not been thoroughly investigated. METHODOLOGY/PRINCIPAL FINDINGS: We collected 600 environmental samples (500 soil and 100 water) from 60 sites around Puerto Rico. We identified B. pseudomallei by isolating it via culturing and/or using PCR to detect its DNA within complex DNA extracts. Only three adjacent soil samples from one site were positive for B. pseudomallei with PCR; we obtained 55 isolates from two of these samples. The 55 B. pseudomallei isolates exhibited fine-scale variation in the core genome and contained four novel genomic islands. Phylogenetic analyses grouped Puerto Rico B. pseudomallei isolates into a monophyletic clade containing other Caribbean isolates, which was nested inside a larger clade containing all isolates from Central/South America. Other Burkholderia species were commonly observed in Puerto Rico; we cultured 129 isolates from multiple soil and water samples collected at numerous sites around Puerto Rico, including representatives of B. anthina, B. cenocepacia, B. cepacia, B. contaminans, B. glumae, B. seminalis, B. stagnalis, B. ubonensis, and several unidentified novel Burkholderia spp. CONCLUSIONS/SIGNIFICANCE: B. pseudomallei was only detected in three soil samples collected at one site in north central Puerto Rico with only two of those samples yielding isolates. All previous human and environmental B. pseudomallei isolates were obtained from eastern Puerto Rico. These findings suggest B. pseudomallei is ecologically established and widely dispersed in the environment in Puerto Rico but rare. Phylogeographic patterns suggest the source of B. pseudomallei populations in Puerto Rico and elsewhere in the Caribbean may have been Central or South America. |
Performance standards for biological threat agent assays for Department of Defense applications
Beck L , Coates SG , Gee J , Hadfield T , Jackson P , Keim P , Lindler L , Olson V , Ostlund E , Roberto F , Samuel J , Sharma S , Tallent S , Wagner DM . J AOAC Int 2018 101 (6) 1665-1708 There has been a proliferation in the developmentof biological threat agent detection technologies foruse in the field by first responders and private-sectorend-users as well as in Department of Defense(DoD) applications in which active combat maybe occurring and in other parts of the world. Incontrast to the proliferation of detection methodology, there has been a lack of standards defining therequired performance of these technologies. Standardsare necessary to demonstrate the performanceand limitations of the tools, providing confidencein the data to allow appropriate response actions byend-users and responders. In the past, the Departmentof Homeland Security (DHS), Science andTechnology Directorate, funded AOAC to developstandards and perform conformity assessmentunder three efforts. The first effort began in 2003to evaluate the performance of lateral flow immunoassaydevices used by first responders to screensuspicious powders for Bacillus anthracis spores.These devices are colloquially known as "handheldassays" and are frequently referred to as"HHAs." AOAC formed the Task Force on Bacillusanthracis (TFBA), which created a specific set ofconsensus performance criteria and test protocols(i.e., standards). Five HHA manufacturers submittedtheir technologies to AOAC so that third-partylaboratories could evaluate the tools against the criteria. |
Improved Subtyping of Staphylococcus aureus Clonal Complex 8 Strains Based on Whole-Genome Phylogenetic Analysis.
Bowers JR , Driebe EM , Albrecht V , McDougal LK , Granade M , Roe CC , Lemmer D , Rasheed JK , Engelthaler DM , Keim P , Limbago BM . mSphere 2018 3 (3) ![]() Strains of Staphylococcus aureus in clonal complex 8 (CC8), including USA300, USA500, and the Iberian clone, are prevalent pathogens in the United States, both inside and outside health care settings. Methods for typing CC8 strains are becoming obsolete as the strains evolve and diversify, and whole-genome sequencing has shown that some strain types fall into multiple sublineages within CC8. In this study, we attempt to clarify the strain nomenclature of CC8, classifying the major strain types based on whole-genome sequence phylogenetics using both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) genomes. We show that isolates of the Archaic and Iberian clones from decades ago make up the most basal clade of the main CC8 lineages and that at least one successful lineage of CC8, made up mostly of MSSA, diverged before the other well-known strain types USA500 and USA300. We also show that the USA500 type includes two clades separated by the previously described "Canadian epidemic MRSA" strain CMRSA9, that one clade containing USA500 also contains the USA300 clade, and that the USA300-0114 strain type is not a monophyletic group. Additionally, we present a rapid, simple CC8 strain-typing scheme using real-time PCR assays that target single nucleotide polymorphisms (SNPs) derived from our CC8 phylogeny and show the significant benefit of using more stable genomic markers based on evolutionary lineages over traditional S. aureus typing techniques. This more accurate and accessible S. aureus typing system may improve surveillance and better inform the epidemiology of this very important pathogen.IMPORTANCEStaphylococcus aureus is a major human pathogen worldwide in both community and health care settings. Surveillance for S. aureus strains is important to our understanding of their spread and to informing infection prevention and control. Confusion surrounding the strain nomenclature of one of the most prevalent lineages of S. aureus, clonal complex 8 (CC8), and the imprecision of current tools for typing S. aureus make surveillance and source tracing difficult and sometimes misleading. In this study, we clarify the CC8 strain designations and propose a new typing scheme for CC8 isolates that is rapid and easy to use. This typing scheme is based on relatively stable genomic markers, and we demonstrate its superiority over traditional typing techniques. This scheme has the potential to greatly improve epidemiological investigations of S. aureus. |
African Lineage Brucella melitensis Isolates from Omani Livestock.
Foster JT , Walker FM , Rannals BD , Hussain MH , Drees KP , Tiller RV , Hoffmaster AR , Al-Rawahi A , Keim P , Saqib M . Front Microbiol 2017 8 2702 ![]() Brucellosis is a common livestock disease in the Middle East and North Africa, but remains poorly described in the region both genetically and epidemiologically. Traditionally found in goats and sheep, Brucella melitensis is increasingly recognized as infecting camels. Most studies of brucellosis in camels to date have focused on serological surveys, providing only limited understanding of the molecular epidemiology of circulating strains. We genotyped B. melitensis isolates from Omani camels using whole genome SNP assays and VNTRs to provide context for regional brucellosis cases. We identified a lineage of B. melitensis circulating in camels as well as in goats, sheep, and cattle in Oman. This lineage is genetically distinct from most genotypes from the Arabian Peninsula and from isolates from much of the rest of the Middle East. We then developed diagnostic assays that rapidly identify strains from this lineage. In analyses of genotypes from throughout the region, Omani isolates were genetically most closely related to strains from brucellosis cases in humans and livestock in North Africa. Our findings suggest an African origin for B. melitensis in Oman that has likely occurred through the trade of infected livestock. Moreover, African lineages of B. melitensis appear to be undersampled and consequently are underrepresented in genetic databases for Brucella. As we begin to more fully understand global genomic diversity of B. melitensis, finding and characterizing these unique but widespread lineages is essential. We predict that increased sampling of humans and livestock in Africa will reveal little known diversity in this important zoonotic pathogen. |
AOAC SMPR 2016.010: Standard Method Performance Requirements (SMPRs) for DNA-based methods of detecting Burkholderia pseudomallei in field-deployable, Department of Defense aerosol collection devices
Gee J , Arce J , Beck LC , Blank TR , Blyn L , Cahall R , Clark AJ , Currie B , Damer K , Davenport M , DeShazer D , Johns M , Keim PS , Kiss K , Lesho M , Lin N , Morse SA , Naraghi-Arani P , Ozanich R , Roberto F , Rozak D , Sahl J , Schaefer F , Schutzer S , Schweizer HP , Sozhamannan S , Tuanyok A , Coates S . J AOAC Int 2017 100 (1) 261-265 Intended Use: Field-deployed use for analysis of | aerosol collection filters and/or liquids | 1 Applicability | Detection of Burkholderia pseudomallei in collection buffers | from aerosol collection devices. Field-deployable assays are | preferred. | 2 Analytical Technique | Molecular detection of nucleic acid. | 3 Definitions | Acceptable minimum detection level (AMDL).— | Predetermined minimum level of an analyte, as specified by | an expert committee which must be detected by the candidate | method at a specified probability of detection (POD). | Exclusivity.—Study involving pure nontarget strains, which | are potentially cross-reactive, that shall not be detected or | enumerated by the candidate method. | Inclusivity.—Study involving pure target strains that shall be | detected or enumerated by the candidate method. | Maximum time-to-result.—Maximum time to complete an | analysis starting from the collection buffer to assay result. | Probability of detection (POD).—Proportion of positive | analytical outcomes for a qualitative method for a given matrix | at a specified analyte level or concentration with a ≥0.95 | confidence interval. | System false-negative rate.—Proportion of test results that | are negative contained within a population of known positives. | System false-positive rate.—Proportion of test results that are | positive contained within a population of known negatives. | 4 Method Performance Requirements | See Table 1. | 5 System Suitability Tests and/or Analytical Quality Control | The controls listed in Table 2 shall be embedded in assays as | appropriate. Manufacturer must provide written justification if | controls are not embedded in the assay. |
Burkholderia humptydooensis sp. nov., a New Species Related to Burkholderia thailandensis and the Fifth Member of the Burkholderia pseudomallei Complex.
Tuanyok A , Mayo M , Scholz H , Hall CM , Allender CJ , Kaestli M , Ginther J , Spring-Pearson S , Bollig MC , Stone JK , Settles EW , Busch JD , Sidak-Loftis L , Sahl JW , Thomas A , Kreutzer L , Georgi E , Gee JE , Bowen RA , Ladner JT , Lovett S , Koroleva G , Palacios G , Wagner DM , Currie BJ , Keim P . Appl Environ Microbiol 2016 83 (5) ![]() During routine screening for endemic Burkholderia pseudomallei from water wells in northern Australia, Gram-staining-negative bacteria (strains MSMB43T, MSMB121, and MSMB122) with a similar morphology and biochemical pattern to B. pseudomallei and B. thailandensis were co-isolated with B. pseudomallei on Ashdown's selective agar. To determine the exact taxonomic position of these strains and to distinguish them from B. pseudomallei and B. thailandensis, they were subjected to a series of phenotypic and molecular analyses. Biochemical and fatty acid methyl esters analysis was unable to distinguish B. humptydooensis sp. nov. from closely related species. In MALDI-TOF analysis, all isolates grouped together in a cluster separate from other Burkholderia spp. 16S rRNA and recA sequence analysis demonstrated phylogenetic placement for B. humptydooensis sp. nov. in a novel clade within the B. pseudomallei group. MLST analysis of the three isolates in comparison with MLST data from 3,340 B. pseudomallei strains and related taxa revealed a new sequence type (ST318). Genome to genome distance calculations and average nucleotide identity of all isolates to both B. thailandensis and B. pseudomallei, based on whole genome sequences, also confirmed B. humptydooensis sp. nov. as a novel Burkholderia species within the pseudomallei complex. Molecular analyses clearly demonstrate that strains MSMB43T, MSMB121, and MSMB122 belong to a novel Burkholderia species for which the name Burkholderia humptydooensis sp. nov. is proposed with the type strain MSMB43T (=BAA-2767; LMG 29471, CP013380-CP013382). IMPORTANCE: Burkholderia pseudomallei is a soil dwelling bacteria and the causative agent of melioidosis. The genus Burkholderia consists of a diverse group of species with the closest relatives of B. pseudomallei referred to as the B. pseudomallei complex. A proposed novel species, B. humptydooensis sp. nov, was isolated from a bore water sample from the Northern Territory in Australia. B. humptydooensis sp. nov., is phylogenetically distinct from B. pseudomallei and other members of the pseudomallei complex making it the fifth member of this important group of bacteria. |
Massive dispersal of coxiella burnetii among cattle across the United States
Olivas S , Hornstra H , Priestley RA , Kaufman E , Hepp C , Sonderegger DL , Handady K , Massung RF , Keim P , Kersh GJ , Pearson T . Microb Genom 2016 2 (8) e000068 ![]() Q-fever is an underreported disease caused by the bacterium Coxiella burnetii, which is highly infectious and has the ability to disperse great distances. It is a completely clonal pathogen with low genetic diversity and requires whole-genome analysis to identify discriminating features among closely related isolates. C. burnetii, and in particular one genotype (ST20), is commonly found in cow's milk across the entire dairy industry of the USA. This single genotype dominance is suggestive of host-specific adaptation, rapid dispersal and persistence within cattle. We used a comparative genomic approach to identify SNPs for high-resolution and high-throughput genotyping assays to better describe the dispersal of ST20 across the USA. We genotyped 507 ST20 cow milk samples and discovered three subgenotypes, all of which were present across the entire country and over the complete time period studied. Only one of these sub-genotypes was observed in a single dairy herd. The temporal and geographic distribution of these sub-genotypes is consistent with a model of large-scale, rapid, frequent and continuous dissemination on a continental scale. The distribution of subgenotypes is not consistent with wind-based dispersal alone, and it is likely that animal husbandry and transportation practices, including pooling of milk from multiple herds, have also shaped the patterns. On the scale of an entire country, there appear to be few barriers to rapid, frequent and large-scale dissemination of the ST20 subgenotypes. |
A Bacillus anthracis Genome Sequence from the Sverdlovsk 1979 Autopsy Specimens.
Sahl JW , Pearson T , Okinaka R , Schupp JM , Gillece JD , Heaton H , Birdsell D , Hepp C , Fofanov V , Noseda R , Fasanella A , Hoffmaster A , Wagner DM , Keim P . mBio 2016 7 (5) ![]() Anthrax is a zoonotic disease that occurs naturally in wild and domestic animals but has been used by both state-sponsored programs and terrorists as a biological weapon. A Soviet industrial production facility in Sverdlovsk, USSR, proved deficient in 1979 when a plume of spores was accidentally released and resulted in one of the largest known human anthrax outbreaks. In order to understand this outbreak and others, we generated a Bacillus anthracis population genetic database based upon whole-genome analysis to identify all single-nucleotide polymorphisms (SNPs) across a reference genome. Phylogenetic analysis has defined three major clades (A, B, and C), B and C being relatively rare compared to A. The A clade has numerous subclades, including a major polytomy named the trans-Eurasian (TEA) group. The TEA radiation is a dominant evolutionary feature of B. anthracis, with many contemporary populations having resulted from a large spatial dispersal of spores from a single source. Two autopsy specimens from the Sverdlovsk outbreak were deep sequenced to produce draft B. anthracis genomes. This allowed the phylogenetic placement of the Sverdlovsk strain into a clade with two Asian live vaccine strains, including the Russian Tsiankovskii strain. The genome was examined for evidence of drug resistance manipulation or other genetic engineering, but none was found. The Soviet Sverdlovsk strain genome is consistent with a wild-type strain from Russia that had no evidence of genetic manipulation during its industrial production. This work provides insights into the world's largest biological weapons program and provides an extensive B. anthracis phylogenetic reference. IMPORTANCE: The 1979 Russian anthrax outbreak resulted from an industrial accident at the Soviet anthrax spore production facility in the city of Sverdlovsk. Deep genomic sequencing of two autopsy specimens generated a draft genome and phylogenetic placement of the Soviet Sverdlovsk anthrax strain. While it is known that Soviet scientists had genetically manipulated Bacillus anthracis with the potential to evade vaccine prophylaxis and antibiotic therapeutics, there was no genomic evidence of this from the Sverdlovsk production strain genome. The whole-genome SNP genotype of the Sverdlovsk strain was used to precisely identify it and its close relatives in the context of an extensive global B. anthracis strain collection. This genomic identity can now be used for forensic tracking of this weapons material on a global scale and for future anthrax investigations. |
Genotyping and Axenic Growth of Coxiella burnetii Isolates Found in the United States Environment.
Kersh GJ , Priestley RA , Hornstra HM , Self JS , Fitzpatrick KA , Biggerstaff BJ , Keim P , Pearson T , Massung RF . Vector Borne Zoonotic Dis 2016 16 (9) 588-94 ![]() Coxiella burnetii is a gram-negative bacterium that is the etiologic agent of the zoonotic disease Q fever. Common reservoirs of C. burnetii include sheep, goats, and cattle. These animals shed C. burnetii into the environment, and humans are infected by inhalation of aerosols. A survey of 1622 environmental samples taken across the United States in 2006-2008 found that 23.8% of the samples contained C. burnetii DNA. To identify the strains circulating in the U.S. environment, DNA from these environmental samples was genotyped using an SNP-based approach to derive sequence types (ST) that are also compatible with multispacer sequence typing methods. Three different sequence types were observed in 31 samples taken from 19 locations. ST8 was associated with goats and ST20 with dairy cattle. ST16/26 was detected in locations with exposure to various animals and also in locations with no direct animal contact. Viable isolates were obtained for all three sequence types, but only the ST20 and ST16/26 isolates grew in acidified citrate cysteine medium (ACCM)-2 axenic media. Examination of a variety of isolates with different sequence types showed that ST8 and closely related isolates did not grow in ACCM-2. These results suggest that a limited number of C. burnetii sequence types are circulating in the U.S. environment and these strains have close associations with specific reservoir species. Growth in ACCM-2 may not be suitable for isolation of many C. burnetii strains. |
Local Population Structure and Patterns of Western Hemisphere Dispersal for Coccidioides spp., the Fungal Cause of Valley Fever.
Engelthaler DM , Roe CC , Hepp CM , Teixeira M , Driebe EM , Schupp JM , Gade L , Waddell V , Komatsu K , Arathoon E , Logemann H , Thompson GR 3rd , Chiller T , Barker B , Keim P , Litvintseva AP . mBio 2016 7 (2) e00550-16 ![]() ![]() ![]() Coccidioidomycosis (or valley fever) is a fungal disease with high morbidity and mortality that affects tens of thousands of people each year. This infection is caused by two sibling species, Coccidioides immitis and C. posadasii, which are endemic to specific arid locales throughout the Western Hemisphere, particularly the desert southwest of the United States. Recent epidemiological and population genetic data suggest that the geographic range of coccidioidomycosis is expanding, as new endemic clusters have been identified in the state of Washington, well outside the established endemic range. The genetic mechanisms and epidemiological consequences of this expansion are unknown and require better understanding of the population structure and evolutionary history of these pathogens. Here we performed multiple phylogenetic inference and population genomics analyses of 68 new and 18 previously published genomes. The results provide evidence of substantial population structure in C. posadasii and demonstrate the presence of distinct geographic clades in central and southern Arizona as well as dispersed populations in Texas, Mexico, South America, and Central America. Although a smaller number of C. immitis strains were included in the analyses, some evidence of phylogeographic structure was also detected in this species, which has been historically limited to California and Baja, Mexico. Bayesian analyses indicated that C. posadasii is the more ancient of the two species and that Arizona contains the most diverse subpopulations. We propose a southern Arizona-northern Mexico origin for C. posadasii and describe a pathway for dispersal and distribution out of this region. IMPORTANCE: Coccidioidomycosis, or valley fever, is caused by the pathogenic fungi Coccidioides posadasii and C. immitis The fungal species and disease are primarily found in the American desert southwest, with spotted distribution throughout the Western Hemisphere. Initial molecular studies suggested a likely anthropogenic movement of C. posadasii from North America to South America. Here we comparatively analyze eighty-six genomes of the two Coccidioides species and establish local and species-wide population structures to not only clarify the earlier dispersal hypothesis but also provide evidence of likely ancestral populations and patterns of dispersal for the known subpopulations of C. posadasii. |
Dietary intake of polyunsaturated fatty acids and fish among US children 12-60 months of age
Keim SA , Branum AM . Matern Child Nutr 2015 11 (4) 987-98 This study aimed to estimate intake of individual polyunsaturated fatty acids (PUFAs), identify major dietary sources of PUFAs and estimate the proportion of individuals consuming fish among US children 12-60 months of age, by age and race and ethnicity. The study employed a cross-sectional design using US National Health and Nutrition Examination Survey data. Representative sample of US population based on selected counties. SUBJECTS: 2496 US children aged 12-60 months. Mean daily intake of n-6 PUFAs and eicosapentaenoic acid (EPA) varied by age, with children 12-24 months of age having lower average intakes (mg or g day(-1) ) than children 49-60 months of age and the lowest n6 : n3 ratio, upon adjustment for energy intake. Docosahexaenoic acid (DHA) intake was low (20 mg day(-1) ) compared to typical infant intake and did not change with age. Compared to non-Hispanic white children, Mexican American children had higher DHA and arachidonic acid (AA) intake. In the previous 30 days, 53.7% of children ever consumed fish. Non-Hispanic black children were more likely than non-Hispanic white children to have consumed fish (64.0% vs. 53.0%). Results indicate low prevalence of fish intake and key n-3 PUFAs, relative to n-6 fatty acids, which suggests room for improvement in the diets of US children. More research is needed to determine how increasing dietary intakes of n-3 PUFAs like DHA could benefit child health. |
Genomic Analysis of the Emergence and Rapid Global Dissemination of the Clonal Group 258 Klebsiella pneumoniae Pandemic.
Bowers JR , Kitchel B , Driebe EM , MacCannell DR , Roe C , Lemmer D , de Man T , Rasheed JK , Engelthaler DM , Keim P , Limbago BM . PLoS One 2015 10 (7) e0133727 ![]() Multidrug-resistant Klebsiella pneumoniae producing the KPC carbapenemase have rapidly spread throughout the world, causing severe healthcare-associated infections with limited antimicrobial treatment options. Dissemination of KPC-producing K. pneumoniae is largely attributed to expansion of a single dominant strain, ST258. In this study, we explore phylogenetic relationships and evolution within ST258 and its clonal group, CG258, using whole genome sequence analysis of 167 isolates from 20 countries collected over 17 years. Our results show a common ST258 ancestor emerged from its diverse parental clonal group around 1995 and likely acquired blaKPC prior to dissemination. Over the past two decades, ST258 has remained highly clonal despite diversity in accessory elements and divergence in the capsule polysaccharide synthesis locus. Apart from the large recombination event that gave rise to ST258, few mutations set it apart from its clonal group. However, one mutation occurs in a global transcription regulator. Characterization of outer membrane protein sequences revealed a profile in ST258 that includes a truncated OmpK35 and modified OmpK37. Our work illuminates potential genomic contributors to the pathogenic success of ST258, helps us better understand the global dissemination of this strain, and identifies genetic markers unique to ST258. |
Comparative analysis of subtyping methods against a whole-genome-sequencing standard for Salmonella enterica serotype Enteritidis.
Deng X , Shariat N , Driebe EM , Roe CC , Tolar B , Trees E , Keim P , Zhang W , Dudley EG , Fields PI , Engelthaler DM . J Clin Microbiol 2014 53 (1) 212-8 ![]() A retrospective investigation was performed to evaluate whole genome sequencing as a benchmark for comparing molecular subtyping methods for Salmonella enterica serotype Enteritidis (SE) and survey the population structure of commonly encountered SE outbreak isolates in the United States. A total of 52 SE isolates representing 16 major outbreaks and three sporadic cases between 2001 and 2012 were sequenced and subjected to subtyping by four different methods: 1) whole genome single nucleotide polymorphism typing (WGST), 2) multiple loci VNTR (variable-number tandem repeat) analysis (MLVA), 3) clustered regularly interspaced short palindromic repeats combined with multi-virulence-locus sequence typing (CRISPR-MVLST) and 4) pulsed-field gel electrophoresis (PFGE). WGST resolved all outbreak clusters and provided useful robust phylogenetic inference with high epidemiological correlation. While both MLVA and CRISPR-MVLST yielded higher discriminatory power than PFGE, MLVA outperformed in delineating outbreak clusters whereas CRISPR-MVLST showed the potential to trace major lineages and ecological origins of SE. Our results suggested that whole genome sequencing makes a viable platform for the evaluation and benchmarking molecular subtyping methods. |
Transmission of methicillin-resistant Staphylococcus aureus infection through solid organ transplantation: confirmation via whole genome sequencing.
Wendt JM , Kaul D , Limbago BM , Ramesh M , Cohle S , Denison AM , Driebe EM , Rasheed JK , Zaki SR , Blau DM , Paddock CD , McDougal LK , Engelthaler DM , Keim PS , Roe CC , Akselrod H , Kuehnert MJ , Basavaraju SV . Am J Transplant 2014 14 (11) 2633-9 ![]() We describe two cases of donor-derived methicillin-resistant Staphylococcus aureus (MRSA) bacteremia that developed after transplantation of organs from a common donor who died from acute MRSA endocarditis. Both recipients developed recurrent MRSA infection despite appropriate antibiotic therapy, and required prolonged hospitalization and hospital readmission. Comparison of S. aureus whole genome sequence of DNA extracted from fixed donor tissue and recipients' isolates confirmed donor-derived transmission. Current guidelines emphasize the risk posed by donors with bacteremia from multidrug-resistant organisms. This investigation suggests that, particularly in the setting of donor endocarditis, even a standard course of prophylactic antibiotics may not be sufficient to prevent donor-derived infection. |
Valley fever: finding new places for an old disease: Coccidioides immitis found in Washington State soil associated with recent human infection
Litvintseva AP , Marsden-Haug N , Hurst S , Hill H , Gade L , Driebe EM , Ralston C , Roe C , Barker BM , Goldoft M , Keim P , Wohrle R , Thompson GR 3rd , Engelthaler DM , Brandt ME , Chiller T . Clin Infect Dis 2014 60 (1) e1-3 We used real-time PCR and culture to demonstrate persistent colonization of soils by Coccidioides immitis, an agent of Valley Fever, in Washington State linked to recent human infections and located outside of the endemic range. Whole genome sequencing confirmed genetic identity between isolates from soil and one of the case-patients. |
Cryptococcus gattii in North American Pacific Northwest: whole-population genome analysis provides insights into species evolution and dispersal.
Engelthaler DM , Hicks ND , Gillece JD , Roe CC , Schupp JM , Driebe EM , Gilgado F , Carriconde F , Trilles L , Firacative C , Ngamskulrungroj P , Castaneda E , Lazera Mdos S , Melhem MS , Perez-Bercoff A , Huttley G , Sorrell TC , Voelz K , May RC , Fisher MC , Thompson GR 3rd , Lockhart SR , Keim P , Meyer W . mBio 2014 5 (4) e01464-14 ![]() The emergence of distinct populations of Cryptococcus gattii in the temperate North American Pacific Northwest (PNW) was surprising, as this species was previously thought to be confined to tropical and semitropical regions. Beyond a new habitat niche, the dominant emergent population displayed increased virulence and caused primary pulmonary disease, as opposed to the predominantly neurologic disease seen previously elsewhere. Whole-genome sequencing was performed on 118 C. gattii isolates, including the PNW subtypes and the global diversity of molecular type VGII, to better ascertain the natural source and genomic adaptations leading to the emergence of infection in the PNW. Overall, the VGII population was highly diverse, demonstrating large numbers of mutational and recombinational events; however, the three dominant subtypes from the PNW were of low diversity and were completely clonal. Although strains of VGII were found on at least five continents, all genetic subpopulations were represented or were most closely related to strains from South America. The phylogenetic data are consistent with multiple dispersal events from South America to North America and elsewhere. Numerous gene content differences were identified between the emergent clones and other VGII lineages, including genes potentially related to habitat adaptation, virulence, and pathology. Evidence was also found for possible gene introgression from Cryptococcus neoformans var. grubii that is rarely seen in global C. gattii but that was present in all PNW populations. These findings provide greater understanding of C. gattii evolution in North America and support extensive evolution in, and dispersal from, South America. IMPORTANCE: Cryptococcus gattii emerged in the temperate North American Pacific Northwest (PNW) in the late 1990s. Beyond a new environmental niche, these emergent populations displayed increased virulence and resulted in a different pattern of clinical disease. In particular, severe pulmonary infections predominated in contrast to presentation with neurologic disease as seen previously elsewhere. We employed population-level whole-genome sequencing and analysis to explore the genetic relationships and gene content of the PNW C. gattii populations. We provide evidence that the PNW strains originated from South America and identified numerous genes potentially related to habitat adaptation, virulence expression, and clinical presentation. Characterization of these genetic features may lead to improved diagnostics and therapies for such fungal infections. The data indicate that there were multiple recent introductions of C. gattii into the PNW. Public health vigilance is warranted for emergence in regions where C. gattii is not thought to be endemic. |
Whole-genome analysis of Exserohilum rostratum from an outbreak of fungal meningitis and other infections.
Litvintseva AP , Hurst S , Gade L , Frace MA , Hilsabeck R , Schupp JM , Gillece JD , Roe C , Smith D , Keim P , Lockhart SR , Changayil S , Weil MR , MacCannell DR , Brandt ME , Engelthaler DM . J Clin Microbiol 2014 52 (9) 3216-22 ![]() Exserohilum rostratum was the cause of most cases of fungal meningitis and other infections associated with the injection of contaminated methylprednisolone acetate produced by the New England Compounding Center (NECC). Until this outbreak, very few human cases of Exserohilum had been reported and very little was known about this dematiaceous fungus, which usually infects plants. Here we report using whole genome sequencing (WGS) for detection of single nucleotide polymorphisms (SNP) and phylogenetic analysis to investigate molecular origin of the outbreak using 22 isolates of E. rostratum isolated from 19 case-patients with meningitis or epidural/spinal abscesses, six isolates isolated from contaminated NECC vials, and seven isolates that are unrelated to the outbreak. Our analysis indicates that all 28 isolates associated with the outbreak had nearly identical genomes of 33.8 Mb. A total of eight SNPs were detected among the outbreak genomes, with no more than two SNPs separating any two of the 28 genomes. The outbreak genomes were separated from the next most closely related control strain by approximately 136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest a possibility of cryptic speciation in E. rostratum. |
Real-time PCR assays for genotyping of Cryptococcus gattii in North America
Kelley EJ , Driebe EM , Etienne K , Brandt ME , Schupp JM , Gillece JD , Trujillo JS , Lockhart SR , Deak E , Keim PS , Engelthaler DM . BMC Microbiol 2014 14 (1) 125 ![]() 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. |
Francisella tularensis subsp. tularensis group A.I, United States
Birdsell DN , Johansson A , Ohrman C , Kaufman E , Molins C , Pearson T , Gyuranecz M , Naumann A , Vogler AJ , Myrtennas K , Larsson P , Forsman M , Sjodin A , Gillece JD , Schupp J , Petersen JM , Keim P , Wagner DM . Emerg Infect Dis 2014 20 (5) 861-5 We used whole-genome analysis and subsequent characterization of geographically diverse strains using new genetic signatures to identify distinct subgroups within Francisella tularensis subsp. tularensis group A.I: A.I.3, A.I.8, and A.I.12. These subgroups exhibit complex phylogeographic patterns within North America. The widest distribution was observed for A.I.12, which suggests an adaptive advantage. |
High prevalence and two dominant host-specific genotypes of Coxiella burnetii in U.S. milk.
Pearson T , Hornstra HM , Hilsabeck R , Gates LT , Olivas SM , Birdsell DM , Hall CM , German S , Cook JM , Seymour ML , Priestley RA , Kondas AV , Clark Friedman CL , Price EP , Schupp JM , Liu CM , Price LB , Massung RF , Kersh GJ , Keim P . BMC Microbiol 2014 14 (1) 41 ![]() 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. |
Whole genome sequence typing to investigate the Apophysomyces outbreak following a tornado in Joplin, Missouri, 2011.
Etienne KA , Gillece J , Hilsabeck R , Schupp JM , Colman R , Lockhart SR , Gade L , Thompson EH , Sutton DA , Neblett-Fanfair R , Park BJ , Turabelidze G , Keim P , Brandt ME , Deak E , Engelthaler DM . PLoS One 2012 7 (11) e49989 ![]() Case reports of Apophysomyces spp. in immunocompetent hosts have been a result of traumatic deep implantation of Apophysomyces spp. spore-contaminated soil or debris. On May 22, 2011 a tornado occurred in Joplin, MO, leaving 13 tornado victims with Apophysomyces trapeziformis infections as a result of lacerations from airborne material. We used whole genome sequence typing (WGST) for high-resolution phylogenetic SNP analysis of 17 outbreak Apophysomyces isolates and five additional temporally and spatially diverse Apophysomyces control isolates (three A. trapeziformis and two A. variabilis isolates). Whole genome SNP phylogenetic analysis revealed three clusters of genotypically related or identical A. trapeziformis isolates and multiple distinct isolates among the Joplin group; this indicated multiple genotypes from a single or multiple sources. Though no linkage between genotype and location of exposure was observed, WGST analysis determined that the Joplin isolates were more closely related to each other than to the control isolates, suggesting local population structure. Additionally, species delineation based on WGST demonstrated the need to reassess currently accepted taxonomic classifications of phylogenetic species within the genus Apophysomyces. |
Public health needs assessments of Tutuila Island, American Samoa, after the 2009 tsunami
Choudhary E , Chen TH , Martin C , Vagi S , Roth J Jr , Keim M , Noe R , Ponausuia SE , Lemusu S , Bayleyegn T , Wolkin A . Disaster Med Public Health Prep 2012 6 (3) 209-16 OBJECTIVE: An 8.3 magnitude earthquake followed by tsunami waves devastated American Samoa on September 29, 2009, resulting in widespread loss of property and public services. An initial and a follow-up Community Needs Assessment for Public Health Emergency Response (CASPER) objectively quantified disaster-affected population needs. METHODS: Using a 2-stage cluster sampling method of CASPER, a household questionnaire eliciting information about medical and basic needs, illnesses, and injuries was administered. To assess response efforts, percent changes in basic and medical needs, illnesses, and injuries between the initial and follow-up CASPER were calculated. RESULTS: During the initial CASPER (N = 212 households), 47.6% and 51.6% of households reported needing a tarpaulin and having no electricity, respectively. The self-reported greatest needs were water (27.8%) and financial help with cleanup (25.5%). The follow-up CASPER (N = 207 households) identified increased vector problems compared to pre-tsunami, and food (26%) was identified as the self-reported greatest need. As compared to the initial CASPER, the follow-up CASPER observed decreases in electricity (-78.3%), drinking water (-44.4%), and clothing (-26.6%). CONCLUSION: This study highlights the use of CASPER during the response and recovery phases following a disaster. The initial CASPER identified basic needs immediately after the earthquake, whereas the follow-up CASPER assessed effectiveness of relief efforts and identified ongoing community needs. |
Development and validation of Burkholderia pseudomallei-specific real-time PCR assays for clinical, environmental or forensic detection applications
Price EP , Dale JL , Cook JM , Sarovich DS , Seymour ML , Ginther JL , Kaufman EL , Beckstrom-Sternberg SM , Mayo M , Kaestli M , Glass MB , Gee JE , Wuthiekanun V , Warner JM , Baker A , Foster JT , Tan P , Tuanyok A , Limmathurotsakul D , Peacock SJ , Currie BJ , Wagner DM , Keim P , Pearson T . PLoS One 2012 7 (5) e37723 ![]() The bacterium Burkholderia pseudomallei causes melioidosis, a rare but serious illness that can be fatal if untreated or misdiagnosed. Species-specific PCR assays provide a technically simple method for differentiating B. pseudomallei from near-neighbor species. However, substantial genetic diversity and high levels of recombination within this species reduce the likelihood that molecular signatures will differentiate all B. pseudomallei from other Burkholderiaceae. Currently available molecular assays for B. pseudomallei detection lack rigorous validation across large in silico datasets and isolate collections to test for specificity, and none have been subjected to stringent quality control criteria (accuracy, precision, selectivity, limit of quantitation (LoQ), limit of detection (LoD), linearity, ruggedness and robustness) to determine their suitability for environmental, clinical or forensic investigations. In this study, we developed two novel B. pseudomallei specific assays, 122018 and 266152, using a dual-probe approach to differentiate B. pseudomallei from B. thailandensis, B. oklahomensis and B. thailandensis-like species; other species failed to amplify. Species specificity was validated across a large DNA panel (>2,300 samples) comprising Burkholderia spp. and non-Burkholderia bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published B. pseudomallei-specific assays, BurkDiff and TTS1, demonstrated comparable performance of all assays, providing between 99.7 and 100% specificity against our isolate panel. Last, we subjected 122018 and 266152 to rigorous quality control analyses, thus providing quantitative limits of assay performance. Using B. pseudomallei as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated B. pseudomallei assays for the scientific research community. |
The public health impact of industrial disasters
Keim ME . Am J Disaster Med 2011 6 (5) 265-72 The recent Deepwater Horizon oil spill and Japanese earthquake/tsunami radiation disaster have increased public concerns regarding the public health impact of industrial disasters. Industrial disasters are known to impose a unique set of challenges for public health emergency response. There are critical gaps in scientific knowledge regarding assessment and control of public health disasters related to industrial releases of hazardous materials. There is also a fundamental lack of familiarity regarding industrial disasters among the public health and medical communities, in general. There are few sources in the current public health literature that review this disaster phenomenon in a comprehensive manner. This article offers a review of the public health impact and unique considerations related to industrial disasters. |
Diversity of 16S-23S rDNA internal transcribed spacer (ITS) reveals phylogenetic relationships in Burkholderia pseudomallei and its near-neighbors.
Liguori AP , Warrington SD , Ginther JL , Pearson T , Bowers J , Glass MB , Mayo M , Wuthiekanun V , Engelthaler D , Peacock SJ , Currie BJ , Wagner DM , Keim P , Tuanyok A . PLoS One 2011 6 (12) e29323 ![]() Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species. |
Molecular epidemiology of anthrax cases associated with recreational use of animal hides and yarn in the United States.
Marston CK , Allen CA , Beaudry J , Price EP , Wolken SR , Pearson T , Keim P , Hoffmaster AR . PLoS One 2011 6 (12) e28274 ![]() To determine potential links between the clinical isolate to animal products and their geographic origin, we genotyped (MLVA-8, MVLA-15, and canSNP analysis) 80 environmental and 12 clinical isolates and 2 clinical specimens from five cases of anthrax (California in 1976 [n = 1], New York in 2006 [n = 1], Connecticut in 2007 [n = 2], and New Hampshire in 2009[n = 1]) resulting from recreational handling of animal products. For the California case, four clinical isolates were identified as MLVA-8 genotype (GT) 76 and in the canSNP A.Br.Vollum lineage, which is consistent with the Pakistani origin of the yarn. Twenty eight of the California isolates were in the A.Br.Vollum canSNP lineage and one isolate was in the A.Br. 003/004 canSNP sub-group. All 52 isolates and both clinical specimens related to the New York and Connecticut cases were MLVA-8 GT 1. The animal products associated with the NY and CT cases were believed to originate from West Africa, but no isolates from this region are available to be genotyped for comparison. All isolates associated with the New Hampshire case were identical and had a new genotype (GT 149). Isolates from the NY, CT and NH cases diverge from the established canSNP phylogeny near the base of the A.Br.011/009. This report illustrates the power of the current genotyping methods and the dramatically different epidemiological conditions that can lead to infections (i.e., contamination by a single genotype versus widespread contamination of numerous genotypes). These cases illustrate the need to acquire and genotype global isolates so that accurate assignments can be made about isolate origins. |
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