Disinfectant Resistance and Biofilm Formation among Multidrug-Resistant Bacteria Isolated from Hospital Surfaces

Abstract: Background: Routine cleaning of high-touch surfaces in the hospital may leave healthcare-associated bacteria. Resistance might be increased if antimicrobial resistance is combined with the ability to form biofilms and withstand killing by standard disinfectants. In this study, the association of multidrug resistance, quantitative biofilm formation and disinfectant tolerance was assessed in the bacteria isolated from the hospital surfaces. Study design: Analytical cross sectional environmental study with controlled laboratory study was carried out in Imam Al-Sadiq Teaching Hospital and Al-Hilla Surgical Teaching Hospital of Hilla, Iraq between January and June 2026. Six categories of clinical units were sampled for a total of 360 high-touch surface swabs, using a stratified approach. Conventional and Vitek 2 Compact were used for identification of bacteria. Explanation of antimicrobial susceptibility was done according to the CLSI M100 36th edition. The crystal-violet microtiter-plate assay was used to quantify the biomass of biofilm. MIC and MBC testing, neutralizer-validated time-kill testing and a mature-biofilm challenge were used to evaluate formulations of sodium hypochlorite, benzalkonium chloride and hydrogen peroxide. Results: Culture was positive in 200/360 samples (55.6%; 95% CI 50.4-60.6), yielding 246 isolates; 171 (69.5%) were Gram-negative. MDR and XDR phenotypes occurred in 158/246 (64.2%) and 22/246 (8.9%), respectively. Biofilm production was detected in 193/246 (78.5%), including 148/246 (60.2%) moderate or strong producers. The median log10 reductions at the 5-minute contact time under dirty conditions were 5.28, 4.55 and 5.50 for sodium hypochlorite, benzalkonium chloride and hydrogen peroxide, respectively. A total of 301/738 (40.8%) pairs of isolates and products were operationally tolerant. Each 0.10 increase in biofilm OD was associated with higher adjusted odds of tolerance (aOR 1.39, 95% CI 1.27-1.51), as was MDR (aOR 2.50, 95% CI 1.49-4.20). Conclusion: Surface contamination was still prevalent and had a strong correlation with the pre-cleaning sampling. Operational killing was affected by MDR, biofilm biomass, organism group and disinfectant formulation. Routine environmental-cleaning audits should be followed by product verification at validated in-use conditions, correct dilution, removal of organic soil and maintenance of full wet contact time.