Characterization of biofilm formation by Myroides odoratimimus isolated from post-traumatic calcaneal recurrent ulcer in diabetic patient

Introduction: Myroides odoratimimus is an aerobic, non-motile, non-fermenting, yellow-pigmented Gram-negative rod-shaped bacterium, known for its opportunistic pathogenicity in humans. Widely distributed in nature, it causes infections in immunocompromised patients due to its multi-drug resistance. Selecting the optimal antibiotic therapy for the treatment of M. odoratimimus infections is challenging due to limited clinical experience with this micro-organism and its reported multidrugresistance. In this study, a clinical strain of M. odoratimimus isolated from a recurrent calcaneal ulcer of a 65-year-old male diabetic patient was characterized for its ability to form biofilm. Materials and Methods: The strain was identified as M. odoratimimus by both MALDI-TOF and 16SrDNA sequencing. Biofilm formation was evaluated, under different pH (5.5, 7.2, and 8.5) and glucose concentrations (5, 12.5, and 25 mM), on polystyrene (cristal violet) and in a “skin‑like” model (viable count). MIC and MBC of levofloxacin (LVX), meropenem (MPM), and tigecycline (TGC) were determined, both on planktonic and 7-day biofilms cells. Activity against 7-day biofilm was assessed both by viable cell count, and confocal (CLSM) and electronic (FIB-SEM) microscopy. The expression of antibiotic resistance-related genes (MUS-1; gyrA; AcrB) during biofilm formation was evaluated by RT-PCR. Results: M. odoratimimus produced relevant amount of biofilm biomass, in a time- dependent manner, but regardless pH and glucose concentration. MBC values between planktonic and sessile cells were different for LVX only. None of the tested antibiotics was able to completely eradicate preformed biofilm. Particularly, MPM and LVX caused a significant, although dose‑independent, reduction of cell viability, as also confirmed by microscopy. RT-PCR showed that MUS-1, gyrA and AcrB are over-expressed during the transition from planktonic to sessile (biofilm) lifestyle. Conclusions: Our results showed, for the first time, that M. odoratimimus is able to form relevant amount of inherently antibiotic-resistant biofilm that might play a role in the pathogenesis of chronic ulcer infections. The present study, along with others reported in literature, clearly suggested that significance of M. odoratimimus isolation in clinical specimens should always be discussed between the clinical microbiologist and the clinician, and that antimicrobial assay is necessary to guide therapeutic decisions.