Comprehensive evaluation of the bactericidal and anti-pathogenic efficacy of chemical disinfectants against antibiotic resistant ESKAPE clinical strains

The persistence of nosocomial pathogens in healthcare settings represents a challenge for cleaning and disinfection. Few studies have addressed the efficacy of chemical disinfectants against emergent and multidrug (MDR) resistant ESKAPE bacteria. The aim of this study was to quantify the in-use and off-label efficacy of three commercially available disinfectants: hydrogen-peroxide-, alcohol- and chlorine-based formulations — against twelve genomically characterized ESKAPE pathogens(ST101/258 Klebsiella pneumoniae, ST2 Acinetobacter baumannii, ST131/62 Escherichia coli, ST357 Pseudomonas aeruginosa, ST1 MRSA and ST612/203 vanA-positive Enterococcus faecium) plus four reference strains, and to determine how sub-lethal exposures influence key virulence traits. Disinfectant activity (log₁₀-reduction, LR) was measured by a quantitative suspension test (EN14885) with and without 3% bovine serum albumin. Additional assays simulated real-life use (contaminated nitrile/latex gloves), early biofilm formation (crystal-violet microplate assay) and evaluated the secretion of proteases, phospholipases, lipases and haemolysins. Statistical significance was assessed by one-way ANOVA or unpaired t-tests (P ≤ 0.05). All disinfectants achieved ≥ 5 LR at the manufacturer-specified concentration and contact time; however, efficacy dropped markedly when contact time was halved or concentration reduced. The hydrogen-peroxide-based disinfectant demonstrated minor differences in efficacy. Specifically, the clinical E. faecium 16 VRE strain exhibited higher MBCs compared to the reference strain (3-fold increase) (P < 0.05). A similar behavior was noticed for both clinical Pseudomonas strains that had 4-fold higher MBCs, compared to the P. aeruginosa reference strain (P < 0.05) at a 5-minute contact time. Organic soiling significantly impaired hydrogen peroxide- and chlorine-based disinfectants' activity. On artificially contaminated gloves, the label-strength alcohol-based disinfectant eradicated all strains after 60 s, whereas 30 s exposure or ≥ 25% dilution permitted recovery of up to 10⁵ CFU cm⁻². Sub-inhibitory chlorine or peroxide concentrations reduced early biofilm biomass by 40–70% (P < 0.05) and suppressed extracellular protease/phospholipase activity, while equivalent alcohol exposure had no effect. Manufacturer-recommended concentration and contact time are critical for reliable killing of MDR ESKAPE pathogens. Shortened contact or dilution—common in clinical practice—creates a survival window and may differentially select tolerant species. Chlorine and peroxide formulations additionally attenuate biofilm initiation and soluble virulence factors, suggesting a dual antimicrobial/anti-pathogenic benefit. These findings support strict compliance with label instructions and encourage formulation optimization that couples rapid killing with anti-virulence activity.