title: Tet(C) Gene Transfer betweenChlamydia suisStrains Occurs by Homologous Recombination after Co-infection: Implications for Spread of Tetracycline-Resistance amongChlamydiaceae.
abstract: Chlamydia suisis a swine pathogen that has also recently been found to cause zoonotic infections of the human eye, pharynx, and gastrointestinal tract. Many strains contain a tetracycline class C gene [tet(C)] cassette that confers tetracycline resistance. The cassette was likely originally acquired by horizontal gene transfer from a Gram-negative donor after the introduction of tetracycline into animal feed in the 1950s. Various research groups have described the capacity for differentChlamydiaspecies to exchange DNA by homologous recombination. Since over 90% ofC. suisstrains are tetracycline resistant, they represent a potential source for antibiotic-resistance spread within and betweenChlamydiaceaespecies. Here, we examined the genetics oftet(C)-transfer amongC. suisstrains. Tetracycline-sensitiveC. suisstrain S45 was simultaneously or sequentially co-infected with tetracycline-resistantC. suisstrains in McCoy cells. Potential recombinants were clonally purified by a harvest assay derived from the classic plaque assay.C. suisstrain Rogers132, lacking transposases IS200and IS605, was the most efficient donor, producing two unique recombinants detected in three of the 56 (5.4%) clones screened. Recombinants were found to have a minimal inhibitory concentration (MIC) of 8-16 μg/mL for tetracycline. Resistance remained stable over 10 passages as long as recombinants were initially grown in tetracycline at twice the MIC of S45 (0.032 μg/mL). Genomic analysis revealed thattet(C) had integrated into the S45 genome by homologous recombination at two unique sites depending on the recombinant: a 55 kb exchange betweennrqF andpckG, and a 175 kb exchange betweenkdsA andcysQ. Neither site was associated with inverted repeats or motifs associated with recombination hotspots. Our findings show that cassette transfer into S45 has low frequency, does not require IS200/IS605transposases, is stable if initially grown in tetracycline, and results in multiple genomic configurations. We provide a model for stable cassette transfer to better understand the capability for cassette acquisition byChlamydiaceaespecies that infect humans, a matter of public health importance.
authors.affiliation: Center for Immunobiology and Vaccine Development, University of California at San Francisco/Benioff Children's Hospital Oakland Research Institute, Oakland CA, USA.
authors.forename: Hanna
authors.initials: H
authors.lastname: Marti
authors.affiliation: Center for Immunobiology and Vaccine Development, University of California at San Francisco/Benioff Children's Hospital Oakland Research Institute, Oakland CA, USA.
authors.forename: Hoyon
authors.initials: H
authors.lastname: Kim
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, AtlantaGA, USA; Department of Human Genetics, Emory University School of Medicine, AtlantaGA, USA.
authors.forename: Sandeep J
authors.initials: SJ
authors.lastname: Joseph
authors.affiliation: Center for Immunobiology and Vaccine Development, University of California at San Francisco/Benioff Children's Hospital Oakland Research Institute, Oakland CA, USA.
authors.forename: Stacey
authors.initials: S
authors.lastname: Dojiri
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, AtlantaGA, USA; Department of Human Genetics, Emory University School of Medicine, AtlantaGA, USA.
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Read
authors.affiliation: Center for Immunobiology and Vaccine Development, University of California at San Francisco/Benioff Children's Hospital Oakland Research Institute, OaklandCA, USA; Joint Graduate Program in Bioengineering, University of California, San Francisco, San FranciscoCA, USA; Joint Graduate Program in Bioengineering, University of California, Berkeley, BerkeleyCA, USA; Departments of Medicine and Pediatrics, University of California, San Francisco, San FranciscoCA, USA.
authors.forename: Deborah
authors.initials: D
authors.lastname: Dean
keywords: Chlamydia; Chlamydia suis; Chlamydia trachomatis; genomic island; homologous recombination; tetracycline resistance; transposon