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Berlin Brandenburg

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  • 1
    Language: English
    In: Microbiology (Reading, England), October 2013, Vol.159(Pt 10), pp.2109-17
    Description: A culture-independent genome sequencing approach was developed and used to examine genomic variability in Chlamydia trachomatis-positive specimens that were collected from patients in the Seattle, WA, USA, area. The procedure is based on an immunomagnetic separation approach with chlamydial LPS-specific mAbs, followed by DNA purification and total DNA amplification, and subsequent Illumina-based sequence analysis. Quality of genome sequencing was independent of the total number of inclusion-forming units determined for the sample and the amount of non-chlamydial DNA in the Illumina libraries. A geographically and temporally linked clade of isolates was identified with evidence of several different regions of recombination and variable ompA sequence types, suggesting that recombination is common within outbreaks. Culture-independent sequence analysis revealed a linkage pattern at two nucleotide positions that was unique to the genomes of isolates from patients, but not in C. trachomatis recombinants generated in vitro. These data demonstrated that culture-independent sequence analysis can be used to rapidly and inexpensively collect genome data from patients infected by C. trachomatis, and that this approach can be used to examine genomic variation within this species.
    Keywords: Genetic Variation ; Recombination, Genetic ; Bacteriological Techniques -- Methods ; Chlamydia Infections -- Microbiology ; Chlamydia Trachomatis -- Genetics ; Genitalia -- Microbiology
    ISSN: 13500872
    E-ISSN: 1465-2080
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  • 2
    In: The Journal of Infectious Diseases, 2017, Vol. 215(11), pp.1657-1665
    Description: Genome sequence analysis of clinical samples demonstrated that identical or nearly identical Chlamydia trachomatis strains can be isolated from individual patients for up to 5 years. These data provide evidence for chlamydial persistence, even when patients are treated with antibiotics.
    Keywords: 〈Kwd〉 〈Italic Toggle="Yes"〉Chlamydia Trachomatis〈/Italic〉 〈/Kwd〉 ; Genomics ; Persistent Infection ; Sexually Transmitted Infection.
    ISSN: 0022-1899
    E-ISSN: 1537-6613
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  • 3
    Language: English
    In: Journal of bacteriology, 01 August 2016, Vol.198(15), pp.2131-9
    Description: Intracellular bacterial pathogens in the family Chlamydiaceae are causes of human blindness, sexually transmitted disease, and pneumonia. Genetic dissection of the mechanisms of chlamydial pathogenicity has been hindered by multiple limitations, including the inability to inactivate genes that would prevent the production of elementary bodies. Many genes are also Chlamydia-specific genes, and chlamydial genomes have undergone extensive reductive evolution, so functions often cannot be inferred from homologs in other organisms. Conditional mutants have been used to study essential genes of many microorganisms, so we screened a library of 4,184 ethyl methanesulfonate-mutagenized Chlamydia trachomatis isolates for temperature-sensitive (TS) mutants that developed normally at physiological temperature (37°C) but not at nonphysiological temperatures. Heat-sensitive TS mutants were identified at a high frequency, while cold-sensitive mutants were less common. Twelve TS mutants were mapped using a novel markerless recombination approach, PCR, and genome sequencing. TS alleles of genes that play essential roles in other bacteria and chlamydia-specific open reading frames (ORFs) of unknown function were identified. Temperature-shift assays determined that phenotypes of the mutants manifested at distinct points in the developmental cycle. Genome sequencing of a larger population of TS mutants also revealed that the screen had not reached saturation. In summary, we describe the first approach for studying essential chlamydial genes and broadly applicable strategies for genetic mapping in Chlamydia spp. and mutants that both define checkpoints and provide insights into the biology of the chlamydial developmental cycle. Study of the pathogenesis of Chlamydia spp. has historically been hampered by a lack of genetic tools. Although there has been recent progress in chlamydial genetics, the existing approaches have limitations for the study of the genes that mediate growth of these organisms in cell culture. We used a genetic screen to identify conditional Chlamydia mutants and then mapped these alleles using a broadly applicable recombination strategy. Phenotypes of the mutants provide fundamental insights into unexplored areas of chlamydial pathogenesis and intracellular biology. Finally, the reagents and approaches we describe are powerful resources for the investigation of these organisms.
    Keywords: Recombination, Genetic ; Temperature ; Chlamydia Trachomatis -- Physiology
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 4
    In: Infection and Immunity, 2010, Vol. 78(9), p.3678
    Description: Chlamydia trachomatis is the leading cause of infectious blindness worldwide and is the most commonly reported pathogen causing sexually transmitted infections. Tarp (translocated actin recruiting phosphoprotein), a type III secreted effector that mediates actin nucleation, is central to C. trachomatis infection. The phylogenetic analysis of tarP from reference strains as well as ocular, genital, and lymphogranuloma venereum (LGV) clinical isolates demonstrated an evolutionary relationship with disease phenotype, with LGV and ocular isolates branched into clades that were separate from the urogenital isolates. The sequence analysis of Tarp indicated a high degree of variability and identified trends within clinical groupings. Tarps from LGV strains contained the highest number of tyrosine-rich repeat regions (up to nine) and the fewest (two) predicted actin binding domains. The converse was noted for Tarp proteins from ocular isolates that contained up to four actin binding domains and as few as one tyrosine-rich repeat region. The results suggest that Tarp is among the few known genes to play a role in C. trachomatis adaptations to specific niches within the host.
    Keywords: Clinical Isolates ; Nucleation ; Phylogeny ; Adaptations ; Phosphoproteins ; Niches ; Lymphogranuloma Venereum ; Actin ; Blindness ; Pathogens ; Infection ; Evolution ; Chlamydia Trachomatis ; Microorganisms & Parasites ; Genetics & Taxonomy;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 5
    In: Infection and Immunity, 2010, Vol. 78(6), p.2544
    Description: The human pathogen Chlamydia trachomatis exists as multiple serovariants that have distinct organotropisms for different tissue sites. Culture and epidemiologic data have demonstrated that serovar G is more prevalent, while serovar E is less prevalent, for rectal isolates from men having sex with men (MSM). The relative prevalence of these serovars is the opposite for isolates from female cervical infections. In contrast, the prevalence of serovar J isolates is approximately the same at the different tissue sites, and these isolates are the only C-class strains that are routinely cultured from MSM populations. These correlations led us to hypothesize that polymorphisms in open reading frame (ORF) sequences correlate with the different tissue tropisms of these serovars. To explore this possibility, we sequenced and compared the genomes of clinical anorectal and cervical isolates belonging to serovars E, G, and J and compared these genomes with each other, as well as with a set of previously sequenced genomes. We then used PCR- and restriction digestion-based genotyping assays performed with a large collection of recent clinical isolates to show that polymorphisms in ORFs CT144, CT154, and CT326 were highly associated with rectal tropism in serovar G isolates and that polymorphisms in CT869 and CT870 were associated with tissue tropism across all serovars tested. The genome sequences collected were also used to identify regions of likely recombination in recent clinical strains. This work demonstrated that whole-genome sequencing along with comparative genomics is an effective approach for discovering variable loci in Chlamydia spp. that are associated with clinical presentation.
    Keywords: Clinical Isolates ; Genomes ; Rectum ; Data Processing ; Genotyping ; Tropism ; Anorectal ; Pathogens ; Infection ; Recombination ; Organotropism ; Genomics ; Open Reading Frames ; Sex ; Chlamydia Trachomatis ; Microorganisms & Parasites ; Protein-Nucleic Acids Association ; Human Diseases;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 6
    Language: English
    In: The Journal of infectious diseases, 01 October 2005, Vol.192(7), pp.1229-36
    Description: We compared growth rate and host-cell transcriptional responses of a Chlamydia trachomatis variant strain and a prototype strain. Growth dynamics were estimated by 16S rRNA level and by inclusion-forming units (IFUs) at different times after infection in HeLa cells. When inoculated at the same multiplicity of infection and observed 24-48 h after infection, the variant 16S rRNA transcriptional level was 3%-4% that of the prototype, and the IFUs of the variant strain were 0.1%-1% those of the prototype. Specific host-cell transcriptional responses to the variant were identified in a global-expression microarray in which variant strain-infected cells were compared with mock-infected and prototype strain-infected cells. In variant strain-infected cells, 47% (16/34) of specifically induced host genes were related to immunity and 32% (8/25) of specifically suppressed genes were related to lipid metabolism. The variant strain grew significantly more slowly and induced a modified host-cell transcriptional response, compared with the prototype strain.
    Keywords: Genetic Variation ; Chlamydia Trachomatis -- Growth & Development ; Inclusion Bodies -- Metabolism ; Proteins -- Metabolism
    ISSN: 0022-1899
    E-ISSN: 15376613
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  • 7
    Language: English
    In: The Journal of Infectious Diseases, 15 November 2001, Vol.184(10), pp.1350-1354
    Description: The relationship of Chlamydia trachomatis inclusion-forming units in quantitative culture to clinical manifestations and inflammation in urogenital disease was assessed in 1179 patients attending a sexually transmitted diseases clinic. In women, greater inclusion-forming unit counts were associated with cervical mucopus (3000 vs. 450 ifu), amount and character of cervical discharge, 〉30 polymorphonuclear cells (PMNL) per high-power field (hpf) on Gram stain (2050 vs. 320 ifu), and diagnoses of mucopurulent cervicitis (MPC; 2550 vs. 300 ifu) and pelvic inflammatory disease (PID; 3000 vs. 578 ifu). In men, greater inclusion-forming unit counts were associated with urethral discharge (85 vs. 44 ifu), amount and character of discharge, and 〉10 PMNL/hpf (95 vs. 50 ifu). These associations persisted on multivariate analysis. Thus, chlamydial replication is associated with MPC and PID in women, urethritis in men, and inflammation in both. Since infections with high inclusion counts may be the most transmissible, identification and treatment of patients with these chlamydia-associated syndromes is important in control programs.
    Keywords: Health sciences -- Medical conditions -- Infections -- Oral contraceptives ; Social sciences -- Population studies -- Human populations -- Oral contraceptives ; Biological sciences -- Biology -- Microbiology -- Oral contraceptives ; Health sciences -- Medical conditions -- Symptoms -- Oral contraceptives ; Health sciences -- Medical conditions -- Diseases -- Oral contraceptives ; Health sciences -- Medical sciences -- Immunology -- Oral contraceptives ; Health sciences -- Medical conditions -- Symptoms -- Oral contraceptives ; Health sciences -- Medical conditions -- Diseases -- Oral contraceptives ; Health sciences -- Medical conditions -- Diseases -- Oral contraceptives ; Health sciences -- Health care industry -- Health care services -- Oral contraceptives
    ISSN: 00221899
    E-ISSN: 15376613
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  • 8
    Language: English
    In: The Journal of Infectious Diseases, 1 October 2001, Vol.184(7), pp.879-884
    Description: Unique Chlamydia trachomatis strains characterized by multiple nonfusing inclusions were recently described. These strains lack evidence of the protein IncA in the inclusion membrane and have mutations in the incA gene. This study evaluated the epidemiology and clinical manifestations of patients infected with nonfusing mutant strains (case patients) and compared them with patients infected with wild-type fusing strains (control subjects). Both male and female case patients had fewer signs of infection than did control subjects (P = .016 and P = .019, respectively). Female case patients also had fewer symptoms of infection (P = .02). Median inclusion-forming unit (ifu) counts were lower in male and female case patients (P = .045 and P = .135, respectively). Thus, nonfusing strains of C trachomatis more often produce subclinical infections than do normal fusing strains and have lower median ifu counts. From a prevention perspective, the data underscore the importance of screening programs to detect and treat inapparent C. trachomatis infection.
    Keywords: Health sciences -- Medical conditions -- Infections ; Health sciences -- Medical conditions -- Symptoms ; Biological sciences -- Biology -- Microbiology ; Biological sciences -- Biology -- Anatomy ; Health sciences -- Health and wellness -- Public health ; Health sciences -- Medical sciences -- Immunology ; Biological sciences -- Biology -- Genetics ; Information science -- Data products -- Databases ; Health sciences -- Medical conditions -- Symptoms ; Behavioral sciences -- Anthropology -- Ethnology
    ISSN: 00221899
    E-ISSN: 15376613
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  • 9
    Language: English
    In: BMC Microbiology, June 20, 2013, Vol.13(1)
    Description: Background Pre-genomic and post-genomic studies demonstrate that chlamydiae actively recombine in vitro and in vivo, although the molecular and cellular biology of this process is not well understood. In this study, we determined the genome sequence of twelve Chlamydia trachomatis recombinants that were generated in vitro under antibiotic selection. These strains were used to explore the process of recombination in Chlamydia spp., including analysis of candidate recombination hotspots, and to correlate known C. trachomatis in vitro phenotypes with parental phenotypes and genotypes. Results Each of the 190 examined recombination events was the product of homologous recombination, and no candidate targeting motifs were identified at recombination sites. There was a single deletion event in one recombinant progeny that resulted in the removal of 17.1 kilobases between two rRNA operons. There was no evidence for preference for any specific region of the chromosome for recombination, and analyses of a total of over 200 individual recombination events do not provide any support for recombination hotspots in vitro. Two measurable phenotypes were analyzed in these studies. First, the efficiency of attachment to host cells in the absence of centrifugation was examined, and this property segregated to regions of the chromosome that carry the polymorphic membrane protein (Pmp) genes. Second, the formation of secondary inclusions within cells varied among recombinant progeny, but this did not cleanly segregate to specific regions of the chromosome. Conclusions These experiments examined the process of recombination in C. trachomatis and identified tools that can be used to associate phenotype with genotype in recombinant progeny. There were no data supporting the hypothesis that particular nucleotide sequences are preferentially used for recombination in vitro. Selected phenotypes can be segregated by analysis of recombination, and this technology may be useful in preliminary analysis of the relationship of genetic variation to phenotypic variation in the chlamydiae. Keywords: Chlamydia, Recombination, Hotspot, Attachment, Secondary inclusions
    Keywords: Chromosomes ; Chlamydia ; Phenotypes
    ISSN: 1471-2180
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  • 10
    In: Antimicrobial Agents and Chemotherapy, 2008, Vol. 52(5), p.1855
    ISSN: 0066-4804
    ISSN: 00664804
    Source: American Society of Microbiology
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