In Vitro Antibacterial and Anti-Biofilm Activities of Rifampicin-Based Combinations Against Clinical Stenotrophomonas maltophilia Isolates: High Synergy with Rifampicin–Colistin

Background/Objectives: Stenotrophomonas maltophilia is an important opportunistic pathogen with intrinsic multidrug resistance (MDR) and strong biofilm-forming ability. This study evaluated the in vitro antibacterial and anti-biofilm activities of rifampicin (RIF)-based combinations against 50 clinical S. maltophilia isolates, with a particular focus on those exhibiting colistin (COL) resistance. Methods: Antimicrobial susceptibility, biofilm formation, and RIF-based combinations were evaluated using broth microdilution, crystal violet, and checkerboard assays, respectively. RIF/COL synergy was further assessed by time-kill assays and scanning electron microscopy (SEM) of mature biofilms. Results: The isolates showed susceptibility to levofloxacin (74.0%) and tigecycline (66.0%), whereas high resistance rates were observed for RIF (98.0%), COL (92.0%), and fosfomycin (84.0%). Overall, 98.0% of the isolates were phenotypically classified as MDR, and 92.0% were biofilm producers, of which 93.5% were classified as strong biofilm producers. Among the RIF-based combinations, RIF/COL exhibited the highest synergistic activity (90.0%), followed by RIF/tigecycline (20.0%). Time-kill kinetics demonstrated potent and sustained RIF/COL synergy against three representative isolates, achieving ≥2 log10 CFU/mL reductions starting at 4–8 h post-incubation. SEM revealed more pronounced structural alterations in established biofilms and more evident cellular damage following RIF/COL treatment than following either monotherapy. Conclusions: These in vitro findings support the further investigation of RIF/COL as a potential therapeutic combination against MDR S. maltophilia, particularly for isolates exhibiting high-level COL resistance and in the treatment of established biofilms.