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Background: Contamination of healthcare surfaces contributes to Healthcare-associated infections (HAIs) transmission, representing a global concern. Conventional chemicals-based sanitation shows limitations in controlling surface contamination and related HAIs onset, and can select multi-resistant pathogen species. Recently, a sanitizing procedure involving probiotic-based detergents was shown to decrease surface pathogens up to 90% more than conventional disinfectants, without selecting resistant species. This study aims to analyse the influence of a probiotic-based sanitizing intervention on HAIs incidence and typology. Design: Seven Italian hospitals representative of all Italian geographical areas will be recruited for participation in a multicentre, prospective, randomised, pre-post interventional study, analysing simultaneously for a consecutive 18- months period both microbial surface contamination and HAIs occurrence. The intervention will consist in the substitution of the conventional cleaning procedure (chlorine-based) with a probiotic-based one. In the preintervention phase, hospitals will maintain conventional sanitizing procedures; in the post-intervention phase, the probiotic-based sanitation will be applied, allowing a buffer period for the stabilization of the new method. The participating hospitals will be randomly allocated in the following groups: no-intervention (one hospital), intervention-1st group (three hospitals) and intervention-2nd group (three hospitals), entering the study with a 5- months delay. During the whole study period, all patients admitted to the recruited hospital wards will be continuously surveyed for HAI occurrence. Meanwhile, surface bioburden will be monitored monthly by both biological and molecular assays. Discussion: This study will be the first to provide robust data on the impact of sanitation procedures on healthcare associated infections onset and typology, as no previously reported studies evaluates simultaneously and continuously for a period of 18 months both the environmental surface bioburden, its resistome and the healthcare associated infections onset. Results: The results generated from this study might be important for the development of future guidelines to modulate environmental microbiota and to ameliorate hospital environmental cleaning, implementing prevention strategies to reduce HAIs onset.