Efficacy of sodium hypochlorite in overcoming antimicrobial resistance and eradicating biofilms in clinical pathogens from pressure ulcers

Abstract

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: Acinetobacter baumannii; Candida albicans; ESKAPE; Klebsiella pneumoniae; NaOCl; Staphylococcus aureus; biofilm; skin; sodium hypochlorite solution.

Figure 1

(A) The percentage of carbapenem-resistant Klebsiella pneumoniae (CRKP), multidrug-resistant Acinetobacter baumannii (MDRAB), tested non-susceptible or resistant to amikacin (AMK), ciprofloxacin (CIP), colistin (COL), gentamicin (GEN), imipenem (IMI), levofloxacin (LEV), meropenem (MER), tobramycin (TOB) and trimethoprim-sulfamethoxazole (TSM). (B) The percentage of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) tested non-susceptible or resistant to ceftaroline (CEF), daptomycin (DAP), erythromycin (ERY), fusidic acid (FUS), levofloxacin (LEV), linezolid (LIN), oxacillin (OXA), teicoplanin (TEI), tigecycline (TIG), and vancomycin (VAN). (C) Multiple antibiotic resistance (MAR) index box plots, (D) predictions of antibiotic resistance genes (ARG), and (E) disinfectant resistance genes (DRG) using CARD v3.2.8. (F) Linear regression analysis illustrates the correlation among the MAR index, ARGs, and DRGs. Significance was assessed by the Kruskal Wallis statistic test. ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001, ^****, p < 0.0001.

Figure 2

Comparative biofilm analysis of carbapenem-resistant Klebsiella pneumoniae (CRKP), multidrug-resistant Acinetobacter baumannii (MDRAB), methicillin-resistant Staphylococcus aureus (MRSA), and methicillin-susceptible S. aureus (MSSA) was quantitatively assessed by evaluating (A) the biomass using crystal violet (CV) staining, (B) the count of biofilm-forming cells (CFU/ml), (C) metabolic activity of biofilm cell (MAB) expressed as the area under the growth curve by the resazurin assay, and (D) the amount of extracellular DNA (eDNA) in the biofilm matrix. (E) Representative fluorescence microscopy images of biofilm formation for the indicated bacterial species stained with the Live/Dead BacLight kit after 24 h of incubation at 37°C. Computerized 3D reconstruction (X, Y, and Z axis) of the biofilm layers (in μm). Significance was assessed by the Kruskal Wallis statistic test. ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001, ^****, p < 0.0001.

Figure 3

(A) The antimicrobial and antibiofilm activity of an electrolytic sodium hypochlorite solution (NaOCl) against carbapenem-resistant Klebsiella pneumoniae (CRKP), multidrug-resistant Acinetobacter baumannii (MDRAB), methicillin-resistant Staphylococcus aureus (MRSA), and methicillin-susceptible S. aureus (MSSA) is displayed through survival cell fractions compared to untreated control strains at concentrations ranging from 0.125 to 0.275 mg/mL. (B) The minimum inhibitory concentration (MIC₉₀) is the lowest concentration (mg/ml) needed to inhibit 90% of planktonic bacterial growth relative to controls and the minimum biofilm eradication concentration (MBEC₉₀). The antimicrobial and antibiofilm activity of NaOCl is demonstrated through the survival of bacterial cell fractions, compared to untreated control strains, at concentrations ranging from 0.125 to 0.275 mg/mL. The minimum inhibitory concentration (MIC₉₀) and the minimum biofilm eradication concentration (MBEC₉₀) are defined as the lowest NaOCl concentrations required to inhibit 90% of planktonic and biofilm bacterial growth, respectively, compared to untreated controls. The MBEC₉₀/MIC₉₀ ratio was used to quantify the biofilm tolerance to NaOCl for all tested strains. Significance was assessed by the Kruskal Wallis statistic test. ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001, ^****, p < 0.0001.

Figure 4

(A) The percentage of C. albicans tested non-susceptible or resistant according to EUCAST breakpoints to amphotericin B, anidulafungin, fluconazole, itraconazole, micafungin, posaconazole, voriconazole. (B) The biofilm of 5 C. albicans clinical isolates was assessed by measuring the biomass with CV staining, (C) the count of biofilm-forming cells (CFU/ml), and (D) the metabolic activity of the biofilm cells with XTT. (E) Representative fluorescence microscopy image stained with the Live/Dead BacLight kit after 24 h of incubation at 37°C. The C. albicans ATCC 2091, classified as a strong biofilm producer, was included as a control strain. (F) The antimicrobial and antibiofilm activity of NaOCl against C. albicans isolates was demonstrated through survival cell fractions, compared to untreated control strains, at concentrations ranging from 0.125 to 0.275 mg/mL. (G) The minimum inhibitory concentration (MIC₉₀) and the minimum biofilm eradication concentration (MBEC₉₀) are defined as the lowest NaOCl concentrations (mg/ml) required to inhibit 90% of planktonic and biofilm bacterial growth, respectively, compared to untreated controls. Significance was assessed by the Kruskal Wallis statistic test. ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001, ^****, p < 0.0001.