Chlamydia suis is a swine pathogen that also causes zoonotic infections. Many strains contain a tet(C) cassette conferring tetracycline resistance originally acquired by horizontal gene transfer from a Gram-negative donor. As we and others have described the capacity for Chlamydiaceae to exchange DNA by homologous recombination, tet(C)-containing C. suis strains represent a potential source for antibiotic-resistance spread within and between Chlamydia species. Here, we examine the genetics of

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... t(C)-transfer among C. suis strains. Tetracycline-sensitive C. suis strain S45 was simultaneously or sequentially co-infected with tetracycline-resistant C. suis strains in McCoy cells. Potential recombinants were clonally purified by plaque assay. C. suis strain Rogers132, which lacks IS200/IS605 transposases, was the most efficient donor, producing three confirmed recombinants of 56 (5.4%) clones with a minimal inhibitory concentration (MIC) of ~8µg/mL. Resistance was stable when recombinants were grown initially in tetracycline at twice the MIC of S45 (0.032 µg/mL). Genomic analysis revealed that tet(C) had integrated into the S45 genome by homologous recombination at two sites in different recombinants: a 55kb exchange between nrqF and pckG, and a 175kb exchange between kdsA and cysQ. Neither site was associated with repeats or motifs associated with recombination hotspots. Our findings show that cassette transfer into S45 has low frequency, does not require IS200/IS605 transposases, 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 by Chlamydia species that infect humans, a matter of public health importance.