Giorgia Fabrizio1 , Francesca Sivori2 , Ilaria Cavallo2 ,

Mauro Truglio2 , Luigi Toma3 , Francesca Sperati4 ,

Massimo Francalancia2 , Francisco Obregon2 , Luisa Pamparau2 ,

Daniela Kovacs5 , Fulvia Pimpinelli2 * and Enea Gino Di Domenico1 *

¹Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Rome, Italy,

²Microbiology and Virology, San Gallicano Dermatological Institute, IRCCS, Rome, Italy, ³Medical

Directorate, IRCCS Regina Elena National Cancer Institute, Rome, Italy, ⁴UOSD Clinical Trial Center,

Biostatistics and Bioinformatics, IRCCS San Gallicano Dermatological Institute, Rome, Italy,

⁵Cutaneous Physiopathology, San Gallicano Dermatological Institute, IRCCS, Rome, Italy

Sodium hypochlorite (NaOCl) is widely recognized for its broad-spectrum antimicrobial efficacy in skin wound care. This study investigates the effectiveness of NaOCl against a range of bacterial and fungal isolates from pressure ulcer (PU) patients. We analyzed 20 bacterial isolates from PU patients, comprising carbapenem resistant Klebsiella pneumoniae (CRKP), multidrug-resistant Acinetobacter baumannii (MDRAB), methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), along with 5 Candida albicans isolates. Antibiotic resistance profiles were determined using standard susceptibility testing. Whole-genome sequencing (WGS) was employed to identify antimicrobial resistance genes (ARGs) and disinfectant resistance genes (DRGs). Genetic determinants of biofilm formation were also assessed. The antimicrobial activity of NaOCl was evaluated by determining the minimum inhibitory concentration (MIC) and the minimal biofilm eradication concentration (MBEC) for both planktonic and biofilm-associated cells. CRKP and MDRAB showed resistance to fluoroquinolones and carbapenems, while MRSA exhibited resistance to β-lactams and levofloxacin. MSSA displayed a comparatively lower resistance profile. WGS identified significant numbers of ARGs in CRKP and MDRAB, with fewer DRGs compared to MRSA and MSSA. All isolates possessed genes associated with fimbriae production and adhesion, correlating with pronounced biofilm biomass production. NaOCl demonstrated substantial antimicrobial activity against both planktonic cells and biofilms. The MIC90 for planktonic bacterial cells was 0.125  mg/mL, and the MBEC90 ranged from 0.225 to 0.5  mg/mL. For planktonic C. albicans, the MIC90 was 0.150  mg/ mL, and the MBEC90 was 0.250  mg/mL. These results highlight the challenge in treating biofilm-associated infections and underscore the potential of NaOCl as a robust antimicrobial agent against difficult-to-treat biofilm infections at concentrations lower than those typically found in commercial disinfectants.

KEYWORDS

NaOCl, biofilm, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Candida albicans, sodium hypochlorite solution, skin, ESKAPE