Evaluation of antimicrobial and sporicidal activity of hypochlorous acid against clinical isolates from diabetic foot gangrene patients

  • M. A. Mahdi Al-Iraqia University
  • M. A. Abbas Middle Technical University
  • M. M. Khlaif Al-Iraqia University
Keywords: disinfectant, diabetic foot ulcer, sporicidal activity, Clostridium perfringens.

Abstract

Diabetic foot ulcers and gangrene, often infected with multidrug-resistant pathogens and resilient bacterial spores, require effective topical antisepsis to prevent systemic infection and promote healing. This study compared the concentration- and time-dependent bactericidal and sporicidal activity of four common disinfectants against clinical isolates from diabetic foot infections. Forty-five clinical swab samples were collected from DFU patients at Baghdad Teaching Hospi t al. Pathogens were isolated and identified using the Vitek 2 Compact system. Quantitative time-kill assays and dilution-neutralization methods were employed to determine the log 10 reduction of vegetative bacteria against different type s of disinfectant and exposure times (1 to 30 minutes). S taphylococcus aureus was the most frequent isolate (40.8%), next to P seudomonas aeruginosa (24.5%) and Acinetobacter baumannii (20.4%). The time-kill study revealed that HOCl (200 ppm) was the most effective and acted the fastest to kill S. aureus , achieving a 6.94 ± 0.71 log 10 reduction of bacteria within 1 minute Moreover, HOCl also proved high ly effective in kill ing bacteria spores, achieving a 7.87 ± 0.76 log 10 reduction in C lostridium perfringens spores in 20 minutes. NaOCl (0.05%) showed high bactericidal activity with a 7.60 ± 1.19 log 10 reduction against S. aureus but with a long exposure time. HOCl 200 ppm showed rapid bactericidal and sporicidal activity against diabetic foot pathogens, which makes it an effective yet safe clinical-grade wound disinfectant.

References

Anagnostopoulos, A. G., Rong, A., Miller, D., Tran, A. Q., Head, T., Lee, M. C., & Lee, W. W. (2018). 0.01% hypochlorous acid as an alternative skin antiseptic: An in vitro comparison. Dermatologic Surgery, 44(12), 1489–1493.

Anwar, S., Alrumaihi, F., Sarwar, T., Babiker, A. Y., Khan, A. A., Prabhu, S. V., & Rahmani, A. H. (2024). Exploring therapeutic potential of catalase: Strategies in disease prevention and management. Biomolecules, 14(6), 697.

Bezsenyi, A., Sági, G., Makó, M., Wojnárovits, L., & Takács, E. (2021). The effect of hydrogen peroxide on the biochemical oxygen demand (BOD) values measured during ionizing radiation treatment of wastewater. Radiation Physics and Chemistry, 189, 109773.

Boecker, D., Zhang, Z., Breves, R., Herth, F., Kramer, A., & Bulitta, C. (2023). Antimicrobial efficacy, mode of action and in vivo use of hypochlorous acid (HOCl) for prevention or therapeutic support of infections. GMS Hygiene and Infection Control, 18, Doc07.

British Standards Institution (2019). BS EN 1276:2019. Chemical disinfectants and antiseptics. Quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics. Test method and requirements (Phase 1).

Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2010). Jawetz, Melnick & Adelberg’s Medical Microbiology. McGraw-Hill, New York.

Byun, B. Y., Cho, H.-Y., Hwang, H.-J., Mah, J.-H., Liu, Y., Tang, J., & Kang, D.-H. (2011). Optimization and evaluation of heat-shock condition for spore enumeration being used in thermal-process verification: Differential responses of spores and vegetative cells of Clostridium sporogenes to heat shock. Food Science and Biotechnology, 20(3), 751–757.

Chung, I., Ryu, H., Yoon, S.-Y., & Ha, J. C. (2022). Health effects of sodium hypochlorite: Review of published case reports. Environmental Analysis Health and Toxicology, 37(1), e2022006.

Coaguila-Llerena, H., Ferraz, E. R., Mendes, B. R., da Silva, L. R., Rossa Jr., C., Cerri, P. S., & Faria, G. (2025). Biological advantages of calcium hypochlorite solution over sodium hypochlorite for regenerative endodontic procedures: An ex vivo and in vitro study on human apical papilla. International Endodontic Journal, 59(1), 153–162.

da Cruz Nizer, W. S., Inkovskiy, V., & Overhage, J. (2020). Surviving reactive chlorine stress: Responses of gram-negative bacteria to hypochlorous acid. Microorganisms, 8(8), 1220.

Edwards-Jones, V. (2025). Hypochlorous acid in healthcare: A comprehensive review of applications, production and safety. International Journal of Biomedical and Clinical Analysis, 5(2), 61–70.

Fabrizio, G., Sivori, F., Cavallo, I., Truglio, M., Toma, L., Sperati, F., Francalancia, M., Obregon, F., Pamparau, L., Kovacs, D., Pimpinelli, F., & Di Domenico, E. G. (2024). Efficacy of sodium hypochlorite in overcoming antimicrobial resistance and eradicating biofilms in clinical pathogens from pressure ulcers. Frontiers in Microbiology, 15, 1432883.

Fukuzaki, S. (2006). Mechanisms of actions of sodium hypochlorite in cleaning and disinfection processes. Biocontrol Science, 11(4), 147–157.

Goda, H., Yamaoka, H., Nakayama-Imaohji, H., Kawata, H., Horiuchi, I., Fujita, Y., Nagao, T., Tada, A., Terada, A., & Kuwahara, T. (2017). Microbicidal effects of weakly acidified chlorous acid water against feline calicivirus and Clostridium difficile spores under protein-rich conditions. PLoS One, 12(5), e0176718.

Habeeb, T. A., Shaebth, L. J., & Abdulameer, N. A. (2021). Isolation of bacteria from diabetic foot patients in Hospital of Al-Dewaniyah City, Iraq. Annals of the Romanian Society for Cell Biology, 25(5), 187–192.

Hidalgo, E., & Dominguez, C. (2000). Growth-altering effects of sodium hypochlorite in cultured human dermal fibroblasts. Life Sciences, 67(11), 1331–1344.

Irawan, D., Lesmana, R., & Sahiratmadja, E. (2024). Hypochlorous acid for wound healing in diabetic rats: Effect on MMP-9 and histology. Clinical, Cosmetic and Investigational Dermatology, 17, 1853–1861.

Kaymaz, B., Gölge, U. H., Ozyalvaclı, G., Kömürcü, E., Goksel, F., Mermerkaya, M. U., & Doral, M. N. (2015). Effects of boric acid on the healing of Achilles tendons of rats. Knee Surgery, Sports Traumatology, Arthroscopy, 24(12), 3738–3744.

Lewandowski, R. B., Stępińska, M., Osuchowski, Ł., Kasprzycka, W., Dobrzyńska, M., Mierczyk, Z., & Trafny, E. A. (2024). The HOCl dry fog – is it safe for human cells? PLoS One, 19(5), e0304602.

Linley, E., Denyer, S. P., McDonnell, G., Simons, C., & Maillard, J.-Y. (2012). Use of hydrogen peroxide as a biocide: new consideration of its mechanisms of biocidal action. Journal of Antimicrobial Chemotherapy, 67(7), 1589–1596.

MacFaddin, J. F. (2000). Biochemical tests for identification of medical bacteria. Williams & Wilkins, Baltimore.

Matar, T. A., & Saleh, N. I. (2024). Detection of multidrug resistant bacterial infections isolated from patients with diabetic foot ulcers in Iraq. Central Asian Journal of Medical and Natural Sciences, 5(4), 410–418.

Mercado, H. R., Sosa, S. A. M., Higuera, J. A. G., & González, F. J. O. (2007). Microorganismos bacteriológicos más frecuentes y resistencia en las infecciones de pie del diabético. Revista Mexicana de Angiología, 35(4), 177–184.

Mung’ong’o, S. G., & Mugoyela, V. (2007). Quality of chlorine-based antiseptics and disinfectants circulating in Dar es Salaam, Tanzania. Tanzania Medical Journal, 22(1), 17–19.

Newsom, S. W. B., & Ridgway, G. L. (2014). The history of decontamination in hospitals. In: Walker, J. T. (Ed.). Decontamination in hospitals and healthcare. Elsevier, Amsterdam. Pp. 20–41.

Rai, S., Gupta, T. P., Shaki, O., & Kale, A. (2021). Hydrogen peroxide: Its use in an extensive acute wound to promote wound granulation and infection control – is it better than normal saline? The International Journal of Lower Extremity Wounds, 22(3), 563–577.

Ricci, A., Zara, S., di Giacomo, V., Gallorini, M., Rapino, M., Di Pietro, N., Cipollina, A., Piattelli, A., & Cataldi, A. (2025). SOD-1/2 involvement in the antioxidant molecular events occurring upon complex magnetic fields application in an in vitro H2O2 oxidative stress-induced endothelial cell model. International Journal of Molecular Sciences, 26(17), 8600.

Ríos-Castillo, A. G., González-Rivas, F., & Rodríguez-Jerez, J. J. (2017). Bactericidal efficacy of hydrogen peroxide-based disinfectants against Gram-positive and Gram-negative bacteria on stainless steel surfaces. Journal of Food Science, 82(10), 2351–2356.

Romanowski, E. G., Stella, N. A., Yates, K. A., Brothers, K. M., Kowalski, R. P., & Shanks, R. M. Q. (2018). In vitro evaluation of a hypochlorous acid hygiene solution on established biofilms. Eye and Contact Lens: Science and Clinical Practice, 44(2), S187–S191.

Sadeghpour Heravi, F., Zakrzewski, M., Vickery, K., G. Armstrong, D., & Hu, H. (2019). Bacterial diversity of diabetic foot ulcers: Current status and future prospectives. Journal of Clinical Medicine, 8(11), 1935.

Severing, A.-L., Rembe, J.-D., Koester, V., & Stuermer, E. K. (2018). Safety and efficacy profiles of different commercial sodium hypochlorite / hypochlorous acid solutions (NaClO/HClO): Antimicrobial efficacy, cytotoxic impact and physicochemical parameters in vitro. Journal of Antimicrobial Chemotherapy, 74(2), 365–372.

Shah, A. B., Maharjan, R., Shrestha, B. P., & Chaudhary, P. (2017). A randomized controlled trial comparing EUSOL versus antibiotic loaded collagen granules as dressing agents in the management of traumatic infected wounds. International Journal of Orthopaedics Sciences, 3(2c), 157–162.

Slaughter, R. J., Watts, M., Vale, J. A., Grieve, J. R., & Schep, L. J. (2019). The clinical toxicology of sodium hypochlorite. Clinical Toxicology, 57(5), 303–311.

Son, S. T., Han, S.-K., Lee, T. Y., Namgoong, S., & Dhong, E.-S. (2017). The microbiology of diabetic foot infections in Korea. Journal of Wound Management and Research, 13(1), 8–12.

Stańkowska, M., Garbacz, K., Korzon-Burakowska, A., Bronk, M., Skotarczak, M., & Szymańska-Dubowik, A. (2022). Microbiological, clinical and radiological aspects of diabetic foot ulcers infected with methicillin-resistant and -sensitive Staphylococcus aureus. Pathogens, 11(6), 701.

Ulfig, A., & Leichert, L. I. (2020). The effects of neutrophil-generated hypochlorous acid and other hypohalous acids on host and pathogens. Cellular and Molecular Life Sciences, 78(2), 385–414.

Vargová, M., Veszelits Laktičová, K., Hromada, R., Cimboláková, I., Uher, I., Papajová, I., & Peter, K. (2020). Sanitation and the environment. In: Uher, I. (Ed.). Environmental factors affecting human health. IntechOpen, London.

Yung, L., Leung, F., Yao, X., Chen, Z.-Y., & Huang, Y. (2006). Reactive oxygen species in vascular wall. Cardiovascular and Hematological Disorders-Drug Targets, 6(1), 1–19.

Zhu, G., Wang, Q., Lu, S., & Niu, Y. (2017). Hydrogen peroxide: A potential wound therapeutic target. Medical Principles and Practice, 26(4), 301–308.

Published
2026-05-04
How to Cite
Mahdi, M. A., Abbas, M. A., & Khlaif, M. M. (2026). Evaluation of antimicrobial and sporicidal activity of hypochlorous acid against clinical isolates from diabetic foot gangrene patients. Regulatory Mechanisms in Biosystems, 17(3), e26068. https://doi.org/10.15421/0226068