Bacteriophage effectiveness in hard surfaces decontamination

Objectives: Persistently contaminated hospital surfaces represent a reservoir of infectious pathogens, hence contributing to the development of healthcare associated infections (HAI), which are often sustained by antibiotic-resistant pathogens. So far, decontamination of hospital surfaces was performed by conventional disinfectants, that cannot prevent recontamination phenomena, which occur in about 30 minutes. Previous studies suggested that specific bacteriophage mixtures could be used to reduce bacterial viability on food, thus the present study was aimed to determine the effect of phage treatment as decontaminating agents on hard surfaces, specifically targeting hospital pathogens. Methods: Both Gram positive and Gram negative bacteria were included in the study, focusing on strains most frequently detected on hospital surfaces, based on previous observations [Caselli E. et al., PLoS One 2016]. Bacterial strains were applied on sterile ceramic tiles and plastic surfaces, at a concentration similar to what detected on hospital surfaces. Namely, cultures of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Proteus mirabilis (ATCC 29906) were separately seeded on surfaces at 102 or 103 CFU per 24 cm2 (corresponding to 4,000 or 40,000 CFU/m2), and dried. Phage mixtures (Staphylococcal phage and Pyophage; Eliava Institute) were then applied uniformly on surfaces after dilution in saline at 1, 10, 100 and 1000 multiplicity of infection (m.o.i.; phage PFU/bacterial CFU ratio), and incubated at room temperature for 0.5, 1, 3, 6 and 24 hours. Bacterial survival was determined by standard CFU count after applying surfaces contact Rodac plates containing the selective media for the analyzed bacterial strain. All assays were also repeated by diluting phage preparations in conventional detergents at work dilution, to ascertain their usability during the usual sanitation procedures. Results: Both used phage mixtures reduced bacterial CFU on artificially contaminated surfaces 70-90%, depending on the used m.o.i., after 1 hour of incubation, compared to what detected on control surfaces (treated with phage diluent only). After 6 hours, almost no survivors were detected, and the results were maintained in the subsequent 24 hours. Phages activity was dose-dependent, but no statistically significant differences were observed between 100 and 1,000 m.o.i.. The results were similar for all the tested bacteria. Notably, phages retained their 100% activity when diluted in detergents commonly used for hospital surface sanitation. Conclusions: Our data indicate that bacteriophages are active in decontaminating in vitro dry hard surfaces, acting against pathogens levels similar to those detected on field on hospital surfaces. They are active within 1 hour at room temperature, and maintain their full activity when suspended in conventional detergents at work dilution. These features render phage mixtures suitable for use as sanitizing agents, and, especially in consideration of the high proportion of antibiotic-resistant isolates on hospital surfaces, open the way to the development of innovative products for the effective elimination of nosocomial pathogens on field.