In Vitro Inhibitory Activity of Three Hospital Biocides Against Multidrug-Resistant Clinical Isolates: A Pilot Study From Morocco

Background Biocides are central to infection prevention and control, but incorrect preparation or dilution may reduce antimicrobial activity. This exploratory pilot study evaluated visible-growth inhibition produced by three hospital biocide working solutions against selected multidrug-resistant clinical isolates from a Moroccan tertiary-care hospital. Methodology A prospective laboratory-based study was conducted from March 1 to April 10, 2026. A total of 33 nonduplicate isolates were tested: Klebsiella pneumoniae (n = 15), Escherichia coli (n = 7), Pseudomonas aeruginosa (n = 5), Acinetobacter baumannii (n = 3), and methicillin-resistant Staphylococcus aureus (n = 3). Sodium hypochlorite 0.5%, hydrogen peroxide 3%, and ethanol 70% working solutions were serially diluted twofold with sterile water before inoculation. Subsequently, 50 µL of each prepared dilution was mixed 1:1 with 50 µL of bacterial suspension. Consequently, the highest final in-well biocide concentrations were 0.25% sodium hypochlorite, 1.5% hydrogen peroxide, and 35% ethanol. The estimated final bacterial inoculum was 5 × 105 CFU/mL. After continuous broth exposure at 37°C for 18-24 hours, growth was assessed visually by turbidity and/or a bacterial pellet. Results A total of 30 isolates (90.9%) were recovered from hospitalized patients. No visible growth was observed for any isolate at the highest test condition (prepared working solutions of 0.5% sodium hypochlorite, 3% hydrogen peroxide, and 70% ethanol; final in-well concentrations 0.25%, 1.5%, and 35%, respectively). With sodium hypochlorite, growth occurred in 10/33 (30.3%) isolates at the 1/2 pre-inoculation dilution (0.125% final in-well), and all isolates grew at 1/8 (0.03125% final). Hydrogen peroxide suppressed visible growth through 1/16 (0.09375% final); growth occurred in 10/33 isolates at 1/32 (0.046875% final), and all isolates grew at 1/128. Ethanol suppressed visible growth through 1/8 (4.375% final); 27/33 isolates (81.8%) grew at 1/16 (2.1875% final), and all isolates grew at 1/32. Conclusions Under this prolonged broth model, the three biocides inhibited visible growth at the highest test conditions, while serial dilution produced a product-specific loss of inhibitory activity. Because equal volumes of biocide solution and inoculum were mixed, final in-well concentrations were half the prepared product concentrations. These findings support strict adherence to validated preparation and use procedures but do not define operational disinfectant concentrations, bactericidal surface efficacy, or clinical biocide resistance.