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Microbiol."],"abstract":"<jats:p>The<jats:italic>Chlamydiaceae<\/jats:italic>are a family of obligate intracellular, gram-negative bacteria known to readily exchange DNA by homologous recombination upon co-culture<jats:italic>in vitro<\/jats:italic>, allowing the transfer of antibiotic resistance residing on the chlamydial chromosome. Among all the obligate intracellular bacteria, only<jats:italic>Chlamydia<\/jats:italic>(<jats:italic>C<\/jats:italic>.)<jats:italic>suis<\/jats:italic>naturally integrated a tetracycline resistance gene into its chromosome. Therefore, in order to further investigate the readiness of<jats:italic>Chlamydia<\/jats:italic>to exchange DNA and especially antibiotic resistance,<jats:italic>C. suis<\/jats:italic>is an excellent model to advance existing co-culture protocols allowing the identification of factors crucial to promote homologous recombination<jats:italic>in vitro<\/jats:italic>. With this strategy, we co-cultured tetracycline-resistant with rifamycin group-resistant<jats:italic>C. suis<\/jats:italic>, which resulted in an allover recombination efficiency of 28%. We found that simultaneous selection is crucial to increase the number of recombinants, that sub-inhibitory concentrations of tetracycline inhibit rather than promote the selection of double-resistant recombinants, and identified a recombination-deficient<jats:italic>C. suis<\/jats:italic>field isolate, strain SWA-110 (1-28b). While tetracycline resistance was detected in field isolates, rifampicin\/rifamycin resistance (RifR) had to be induced<jats:italic>in vitro<\/jats:italic>. Here, we describe the protocol with which RifR<jats:italic>C. suis<\/jats:italic>strains were generated and confirmed. Subsequent whole-genome sequencing then revealed that G530E and D461A mutations in<jats:italic>rpoB<\/jats:italic>, a gene encoding for the \u03b2-subunit of the bacterial RNA polymerase (RNAP), was likely responsible for rifampicin and rifamycin resistance, respectively. Finally, whole-genome sequencing of recombinants obtained by co-culture revealed that recombinants picked from the same plate may be sibling clones and confirmed<jats:italic>C. suis<\/jats:italic>genome plasticity by revealing variable, apparently non-specific areas of recombination.<\/jats:p>","DOI":"10.3389\/fmicb.2021.630293","type":"journal-article","created":{"date-parts":[[2021,6,30]],"date-time":"2021-06-30T06:03:37Z","timestamp":1625033017000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":11,"title":["Generation of Tetracycline and Rifamycin Resistant Chlamydia Suis Recombinants"],"prefix":"10.3389","volume":"12","author":[{"given":"Hanna","family":"Marti","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sankhya","family":"Bommana","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Timothy D.","family":"Read","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Theresa","family":"Pesch","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Barbara","family":"Pr\u00e4hauser","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Deborah","family":"Dean","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicole","family":"Borel","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2021,6,30]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"2590","DOI":"10.1128\/AAC.42.10.2590","article-title":"Characterization of mutations in the rpoB gene that confer rifampin resistance in Staphylococcus aureus","volume":"42","author":"Aubry-Damon","year":"1998","journal-title":"Antimicrob. 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