This study aimed to study the antimicrobial resistance, genetic characterization, and molecular epidemiology of Ureaplasma species in order to provide clinicians sufficient data to select optimal strategies of treatment for genitourinary tract infections of infertile male patients. Firstly, a total of 817 clinical semen specimens were detected for Ureaplasma species by molecular detection. Secondly, culture and identification of Ureaplasma species were achieved by using Mycoplasma ICS Test, and the antimicrobial susceptibility tests were determined by using broth microdilution assay. Then, the tetracycline resistance genetic determinants in Ureaplasma species were identified by PCR, and the fluoroquinolone and macrolide resistance genetic determinants were identified by DNA sequencing. Finally, the molecular epidemiology of Ureaplasma species was studied by both multilocus sequence typing (MLST) and expanded MLST (eMLST) schemes. Among the 817 semen specimens, 320 (39.17%) specimens were positive for Ureaplasma species. The percentages of resistance in 320 isolates against LEV, MXF, TET, and ERY were 47.5%, 39.38%, 19.69%, and 3.75%, respectively. The tet(M) and int-Tn genes were detected positive in all the tetracycline-resistant isolates. One macrolide-resistant UU isolate had a novel amino acid alteration (R66T) in L4 ribosomal protein and another UU isolate harbored a novel alteration (S109T) in L22. In fluoroquinolone-resistant isolates, S83L substitution in the ParC was predominant. In this area, ST22 and eST16 were the most prevalent ST and eST, respectively. One ST and 3 eSTs were newly identified in this study. This study has demonstrated that ERY can be first-line therapy for Ureaplasma species infections.
