AbstractTo enumerate bacteria adherent to medical devices, Vortex-Sonication-Vortex Method (VSVM) and BioTimer Assay (BTA) have been applied. VSVM counts detached microorganisms whereas BTA enumerates adherent microorganisms through microbial metabolism. However, the limitation of VSVM consists in incomplete detachment of adherent microorganisms while BTA is unable to identify microbial genera and species. Herein, the combined use of VSVM and BTA for the diagnosis and enumeration of adherent microorganisms causing implant-associated-infections (IAIs) is reported. Over 2016–2018, 46 patients with IAIs were enrolled and their 82 explanted devices were submitted firstly to VSVM and then to BTA. VSVM plus BTA detected microorganisms in 39/46 patients (84.7%) compared with 32/46 (69.5%) and 31/46 (67.3%) by VSVM and BTA alone, respectively. Likely, combined methods led to microorganism detection in 54/82 devices (65.9%) compared with each method alone [43/82 (52.4%), 44/82 (53.6%) for VSVM and BTA, respectively]. The combination of both methods (concordance 75.6%) raised the sensitivity of microbial analysis in IAIs compared with either VSVM or BTA alone, thus representing a simple and accurate way for the identification and enumeration of microorganisms adherent on devices. Moreover, BTA reagent applied in a new apparatus allowed also the enumeration of the microorganisms adherent on different segments of cardiac electrodes, thus contributing to define IAIs pathogenesis.