| Summary: | <b>Background</b>: Bacterial biofilm on the surface of tracheostomy tubes (TTs) is a potential reservoir of potentially pathogenic bacteria, including <i>S. aureus</i>. For this reason, our study aimed to investigate biofilm production in vitro and the presence of <i>ica</i>AD and MSCRAMM genes in clinical <i>S. aureus</i> strains derived from TTs, with respect to antibiotic resistance and genetic variability. <b>Methods:</b> The clonality of the <i>S. aureus</i> strains was analyzed by the PFGE method. The assessment of drug resistance was based on the EUCAST recommendations. The isolates were evaluated for biofilm production by the microtiter plate method and the slime-forming ability was tested on Congo red agar (CRA). The presence of <i>ica</i>AD genes was investigated by PCR and MSCRAMM genes were detected by multiplex PCR. <b>Results:</b> A total of 60 patients were enrolled in the study. One TT was obtained from each patient (n = 60). Twenty-one TTs (35%) were colonized with S. aureus. A total of 24 strains were isolated as 3 patients showed colonization with 2 SA clones (as confirmed by PFGE). PFGE showed twenty-two unique molecular profiles. Two isolates (8%) turned out to be MRSA, but 50% were resistant to chloramphenicol, 25% to erythromycin and 8% to clindamycin (two cMLS<sub>B</sub> and four iMLS<sub>B</sub> phenotypes were detected). The microtiter plate method with crystal violet confirmed that 96% of the strains were biofilm formers. Representative strains were visualized by SEM. All isolates had <i>clf</i>AB, <i>fnb</i>A, <i>ebp</i>S and <i>ica</i>AD. Different MSCRAMM gene combinations were observed. <b>Conclusions:</b> the present study showed that the <i>S. aureus</i> isolated from the TTs has a high diversity of genotypes, a high level of antibiotic resistance and ability to produce biofilm.
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