Generation of Tetracycline and Rifamycin Resistant Chlamydia Suis Recombinants.

Hanna Marti,Sankhya Bommana,Deborah Dean,Timothy D Read,Nicole Borel,Barbara Prähauser,Theresa Pesch

doi:10.3389/fmicb.2021.630293

Abstract

The Chlamydiaceae are a family of obligate intracellular, gram-negative bacteria known to readily exchange DNA by homologous recombination upon co-culture in vitro, allowing the transfer of antibiotic resistance residing on the chlamydial chromosome. Among all the obligate intracellular bacteria, only Chlamydia (C.) suis naturally integrated a tetracycline resistance gene into its chromosome. Therefore, in order to further investigate the readiness of Chlamydia to exchange DNA and especially antibiotic resistance, C. suis is an excellent model to advance existing co-culture protocols allowing the identification of factors crucial to promote homologous recombination in vitro. With this strategy, we co-cultured tetracycline-resistant with rifamycin group-resistant C. suis, 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 C. suis field isolate, strain SWA-110 (1-28b). While tetracycline resistance was detected in field isolates, rifampicin/rifamycin resistance (RifR) had to be induced in vitro. Here, we describe the protocol with which RifR C. suis strains were generated and confirmed. Subsequent whole-genome sequencing then revealed that G530E and D461A mutations in rpoB, a gene encoding for the β-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 C. suis genome plasticity by revealing variable, apparently non-specific areas of recombination.

Highlights

The obligate intracellular bacterial family Chlamydiaceae comprises a single genus with a wide range of species that infect human and animal hosts (Sachse and Borel, 2020)
The updated structure of the treated monolayers (Tet)-island for all three strains is shown in Figure 3 and the Tet-island sequences with annotations are listed in Supplementary Table 2
The minimal inhibitory concentration (MIC) of all three strains was determined in order to establish a selection concentration for tetracycline

Summary

Introduction

The obligate intracellular bacterial family Chlamydiaceae comprises a single genus with a wide range of species that infect human and animal hosts (Sachse and Borel, 2020). The most common cause of bacterial sexually transmitted infections (STI) and infectious blindness worldwide is Chlamydia (C.) trachomatis (Jordan et al, 2020). It is the most widelystudied Chlamydia species. The closest phylogenetic relatives to C. trachomatis are the murine species C. muridarum and the porcine species C. suis. The latter is commonly detected in domesticated pigs and wild boar (Hotzel et al, 2004; Schautteet and Vanrompay, 2011; Sachse and Borel, 2020). C. suis is a zoonotic pathogen, causing asymptomatic or very mild ocular infection among pig farm workers as well as pharyngeal and rectal infections among abattoir employees (De Puysseleyr et al, 2014, 2017)

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