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Safa Boujemaa,1 Béhija Mlik,1 Helmi Mardassi,2 Boutheina Ben Abdelmoumen Mardassi1 1Group of Mycoplasmas, Laboratory of Molecular Microbiology, Vaccinology, and Biotechnology Development. Institut Pasteur De Tunis, Université De Tunis El Manar, Tunis, Tunisia; 2Unit of Typing & Genetics of Mycobacteria, Laboratory of Molecular Microbiology, Vaccinology, and Biotechnology Development. Institut Pasteur De Tunis, Université De Tunis El Manar, Tunis, TunisiaCorrespondence: Boutheina Ben Abdelmoumen MardassiGroup of Mycoplasmas, Laboratory of Molecular Microbiology, Vaccinology, and Biotechnology Development. Institut Pasteur De Tunis, Université De Tunis El Manar, Tunis, TunisiaTel +216 71 847 609Fax +216 71 791 833Email boutheina.mardassi@pasteur.tnAbstract: Antimicrobial resistance in a number of bacterial pathogens has been shown to spread clonally. To our knowledge, data about the phylodistribution of drug resistance in Mycoplasma hominis are very scarce. The aims of this study were to assess the antimicrobial susceptibility of Mycoplasma hominis clinical strains in Tunisia, to identify the molecular basis of antibiotic resistance, and to investigate the phylogenetic relationships of resistant strains. This study included 65 molecularly typed Mycoplasma hominis clinical strains recovered from Tunisian patients over 18 years (2000– 2018). The antimicrobial susceptibility was tested against nine antibacterial agents using the broth microdilution method. Minimum spanning tree was constructed to establish the phylogenetic relationships among resistant isolates. Fluoroquinolones, doxycycline, and josamycine were found to be the most effective antibacterial agents. However, 22 strains belonging to 11 expanded multilocus sequence types (eSTs) proved resistant to tetracycline. The majority of these eSTs were genetically related, indicative of clonal expansion of tetracycline resistance. The present study provides relevant information on the antibiotic susceptibility of Tunisian M. hominis clinical strains, lending support to a clonal transmission of tetracycline resistance. This is likely to have an important implication in monitoring the spread of drug resistance among M. hominis.Keywords: Mycoplasma hominis, antibiotic resistance, tetracycline, clonal transmission, expanded multilocus sequence type