Intraoperative Airborne Contamination in Operating Rooms: A Multiparametric Analysis Integrating Culture‐Based Microbiology, Metagenomic Sequencing, Particle Counts, and Procedural Determinants

Airborne microbial contamination and particulate matter in operating rooms (ORs) contribute to the risk of surgical site infections (SSIs). However, few studies have integrated culture-based approaches with MALDI-TOF MS identification and metagenomic NGS during real surgical activity. This study combines microbiological, molecular, particulate, and procedural data to provide a multifactorial view of intraoperative contamination dynamics. This study aimed at characterizing airborne microbiota, particulate matter, and key procedural determinants of contamination in ORs. Forty-two surgical procedures across three specialties were monitored. Airborne microorganisms were isolated using culture-based methods and identified with the MALDI Biotyper, whereas the OR airbiome was assessed using shotgun metagenomic sequencing. Airborne particle concentrations (≥ 0.5 and ≥5 μm), microclimatic parameters, staff presence, door openings, electrosurgical use, and procedural order were recorded. Statistical analyses included Wilcoxon and Kruskal–Wallis tests with Dunn′s post hoc comparisons, Kendall′s τ and Spearman′s ρ correlations, and rank-based effect sizes. Airborne microbial contamination was low in both ISO 5 and ISO 7 rooms (mean 14.02 ± 6.88 CFU/m3), with a predominance of skin-associated taxa. Culture-based methods detected viable, fast-growing species, whereas NGS revealed additional environmental, slow-growing, and anaerobic taxa, including clinically significant bacteria and yeasts such as Cutibacterium acnes (C. acnes), Finegoldia magna (F. magna), and Malassezia restricta (M. restricta). Cross-method concordance was weak to moderate (Spearman′s ρ = 0.35; Kendall′s τ = 0.24), indicating complementary detection. Median particle concentrations were significantly higher in ISO 7 than in ISO 5 ORs. In ISO 7 rooms, particle loads varied by specialty and tended to be higher during open procedures. Integrated analyses showed a decoupling of microbial and particulate contamination in ISO 5 rooms, whereas ISO 7 rooms exhibited more interconnected contamination dynamics, with significant associations between electrosurgical device use and airborne microbial contamination (ρ = 0.45, p = 0.025), between fine (≥ 0.5 μm) and coarse (≥ 5 μm) particle concentrations (ρ = 0.65, p < 0.001), and an inverse association between procedure order and staff count (ρ = −0.66, p < 0.001). Integrating culture-based and molecular techniques may provide a more comprehensive characterization of the airborne microbiota in ORs. The findings suggest that ISO 5 laminar-flow systems are associated with more stable airborne contamination patterns, whereas ISO 7 rooms exhibit greater interactions between airborne contamination and procedural variables. Incorporating molecular profiling and particle metrics into OR surveillance may support environmental monitoring strategies relevant to infection prevention and control.