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      First Draft Genome Sequence of a Human Coxiella burnetii Isolate, Originating from the Largest Q Fever Outbreak Ever Reported, the Netherlands, 2007 to 2010

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          Abstract

          In 2009, Coxiella burnetii caused a large regional outbreak of Q fever in South Limburg, the Netherlands. Here, we announce the genome draft sequence of a human C. burnetii isolate, strain NL-Limburg, originating from this outbreak, including a brief summary of the genome’s general features.

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          Q fever.

          Q fever is a zoonosis with many manifestations. The most common clinical presentation is an influenza-like illness with varying degrees of pneumonia and hepatitis. Although acute disease is usually self-limiting, people do occasionally die from this condition. Endocarditis is the most frequent chronic presentation. Although Q fever is widespread, practitioner awareness and clinical manifestations vary from region to region. Geographically limited studies suggest that chronic fatigue syndrome and cardiovascular disease are long-term sequelae. An effective whole-cell vaccine is licensed in Australia. Live and acellular vaccines have also been studied, but are not currently licensed.
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            Comparative genomics reveal extensive transposon-mediated genomic plasticity and diversity among potential effector proteins within the genus Coxiella.

            Genetically distinct isolates of Coxiella burnetii, the cause of human Q fever, display different phenotypes with respect to in vitro infectivity/cytopathology and pathogenicity for laboratory animals. Moreover, correlations between C. burnetii genomic groups and human disease presentation (acute versus chronic) have been described, suggesting that isolates have distinct virulence characteristics. To provide a more-complete understanding of C. burnetii's genetic diversity, evolution, and pathogenic potential, we deciphered the whole-genome sequences of the K (Q154) and G (Q212) human chronic endocarditis isolates and the naturally attenuated Dugway (5J108-111) rodent isolate. Cross-genome comparisons that included the previously sequenced Nine Mile (NM) reference isolate (RSA493) revealed both novel gene content and disparate collections of pseudogenes that may contribute to isolate virulence and other phenotypes. While C. burnetii genomes are highly syntenous, recombination between abundant insertion sequence (IS) elements has resulted in genome plasticity manifested as chromosomal rearrangement of syntenic blocks and DNA insertions/deletions. The numerous IS elements, genomic rearrangements, and pseudogenes of C. burnetii isolates are consistent with genome structures of other bacterial pathogens that have recently emerged from nonpathogens with expanded niches. The observation that the attenuated Dugway isolate has the largest genome with the fewest pseudogenes and IS elements suggests that this isolate's lineage is at an earlier stage of pathoadaptation than the NM, K, and G lineages.
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              Isolation from animal tissue and genetic transformation of Coxiella burnetii are facilitated by an improved axenic growth medium.

              We recently described acidified citrate cysteine medium (ACCM), which supports host cell-free (axenic) growth of Coxiella burnetii. After 6 days of incubation, greater than 3 logs of growth was achieved with the avirulent Nine Mile phase II (NMII) strain. Here, we describe modified ACCM and culture conditions that support improved growth of C. burnetii and their use in genetic transformation and pathogen isolation from tissue samples. ACCM was modified by replacing fetal bovine serum with methyl-β-cyclodextrin to generate ACCM-2. Cultivation of NMII in ACCM-2 with moderate shaking and in 2.5% oxygen yielded 4 to 5 logs of growth over 7 days. Similar growth was achieved with the virulent Nine Mile phase I and G isolates of C. burnetii. Colonies that developed after 6 days of growth in ACCM-2 agarose were approximately 0.5 mm in diameter, roughly 5-fold larger than those formed in ACCM agarose. By electron microscopy, colonies consisted primarily of the C. burnetii small cell variant morphological form. NMII was successfully cultured in ACCM-2 when medium was inoculated with as little as 10 genome equivalents contained in tissue homogenates from infected SCID mice. A completely axenic C. burnetii genetic transformation system was developed using ACCM-2 that allowed isolation of transformants in about 2 1/2 weeks. Transformation experiments demonstrated clonal populations in colonies and a transformation frequency of approximately 5 × 10(-5). Cultivation in ACCM-2 will accelerate development of C. burnetii genetic tools and provide a sensitive means of primary isolation of the pathogen from Q fever patients.
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                Author and article information

                Journal
                Genome Announc
                Genome Announc
                ga
                ga
                GA
                Genome Announcements
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                2169-8287
                7 May 2015
                May-Jun 2015
                : 3
                : 3
                : e00445-15
                Affiliations
                [a ]Department of Biological Safety, Federal Institute for Risk Assessment, Berlin, Germany
                [b ]Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute for Bacterial Infections and Zoonoses, National Reference Laboratory for Fever, Jena, Germany
                [c ]Public Health Service South Limburg, Department of Sexual Health, Infectious Diseases, and Environmental Health, Sittard-Geleen, the Netherlands
                [d ]Department of Internal Medicine, Atrium Medical Center, Heerlen, the Netherlands
                [e ]School of Public Health and Primary Care (CAPHRI), Maastricht University Medical Centre (MUMC+), Maastricht, the Netherlands
                [f ]Department of Internal Medicine III, RWTH Aachen University, Aachen, Germany
                Author notes
                Address correspondence to J. A. Hammerl, jens.hammerl@ 123456web.de .
                Article
                genomeA00445-15
                10.1128/genomeA.00445-15
                4424315
                25953164
                a3c2aae3-318a-4839-8e33-c8458e504711
                Copyright © 2015 Hammerl et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.

                History
                : 30 March 2015
                : 3 April 2015
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 12, Pages: 2, Words: 1288
                Categories
                Prokaryotes
                Custom metadata
                May/June 2015
                free

                Genetics
                Genetics

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