[Effect of hypochloric acid on Escherichia coli biofilm and the clinical efficacy of hypochloric acid for wounds with Escherichia coli infection]

Abstract

Objective: To investigate the effect of hypochloric acid on Escherichia coli biofilm and the clinical efficacy of hypochloric acid for wounds with Escherichia coli infection. Methods: One strain of Escherichia coli with the strongest bacterial biofilm forming ability among the strains isolated from specimens in 25 patients (16 males and 9 females, aged 32-67 years) from five clinical departments of the 940^th Hospital of the Joint Logistic Support Force was collected for the experimental study from September to December 2019. The Escherichia coli was cultured with hypochloric acid at 162.96, 81.48, 40.74, 20.37, 10.18, 5.09, 2.55, 1.27, 0.64, and 0.32 μg/mL respectively to screen the minimum bactericidal concentration (MBC) of hypochloric acid. The Escherichia coli was cultured with hypochloric acid at the screened MBC for 2, 5, 10, 20, 30, and 60 min respectively to screen the shortest bactericidal time of hypochloric acid. The biofilm formation of Escherichia coli was observed by scanning electron microscopy at 6, 12, 24, 48, 72, and 96 h of incubation, respectively. After 72 h of culture, hypochloric acid at 1, 2, 4, 8, and 16 times of MBC was respectively added to Escherichia coli to screen the minimum biofilm eradicate concentration (MBEC) of hypochloric acid against Escherichia coli. After hypochloric acid at 1, 2, 4, and 8 times of MBEC and sterile saline were respectively added to Escherichia coli for 10 min, the live/dead bacterial staining kit was used to detect the number of live and dead cells, with the rate of dead bacteria calculated (the number of samples was 5). From January to December 2020, 41 patients with infectious wounds meeting the inclusion criteria and admitted to the Department of Burns and Plastic Surgery of the 940^th Hospital of Joint Logistic Support Force of PLA were included into the prospective randomized controlled trial. The patients were divided into hypochloric acid group with 21 patients (13 males and 8 females, aged (46±14) years) and povidone iodine group with 20 patients (14 males and 6 females, aged (45±19) years) according to the random number table. Patients in the 2 groups were respectively dressed with sterile gauze soaked with hypochloric acid of 100 μg/mL and povidone iodine solution of 50 mg/mL with the dressings changed daily. Before the first dressing change and on the 10^th day of dressing change, tissue was taken from the wound and margin of the wound for culturing bacteria by agar culture method and quantifying the number of bacteria. The amount of wound exudate and granulation tissue growth were observed visually and scored before the first dressing change and on the 3^rd, 7^th, and 10^th days of dressing change. Data were statistically analyzed with one-way analysis of variance, Dunnett-t test, independent sample t test, Mann-Whitney U test, Wilcoxon signed-rank test, chi-square test, or Fisher's exact probability test. Results: The MBC of hypochloric acid against Escherichia coli was 10.18 μg/mL, and the shortest bactericidal time of hypochloric acid with MBC against Escherichia coli was 2 min. Escherichia coli was in a completely free state after 6 and 12 h of culture and gradually aggregated and adhered with the extension of culture time, forming a mature biofilm at 72 h of culture. The MBEC of hypochloric acid against Escherichia coli was 20.36 μg/mL. The Escherichia coli mortality rates after incubation with hypochloric acid at 1, 2, 4, and 8 times of MBEC for 10 min were significantly higher than that after incubation with sterile saline (with t values of 6.11, 25.04, 28.90, and 40.74, respectively, P<0.01). The amount of bacteria in the wound tissue of patients in hypochloric acid group on the 10^th day of dressing change was 2.61 (2.20, 3.30)×10⁴ colony forming unit (CFU)/g, significantly less than 4.77 (2.18, 12.48)×10⁴ CFU/g in povidone iodine group (Z=2.06, P<0.05). The amounts of bacteria in the wound tissue of patients in hypochloric acid group and povidone iodine group on the 10^th day of dressing change were significantly less than 2.97 (2.90, 3.04)×10⁶ and 2.97 (1.90, 7.95)×10⁶ CFU/g before the first dressing change (with Z values of 4.02 and 3.92, respectively, P<0.01). The score of wound exudate amount of patients in hypochloric acid group on the 10^th day of dressing change was significantly lower than that in povidone iodine group (Z=2.07, P<0.05). Compared with those before the first dressing change, the scores of wound exudate amount of patients in hypochloric acid group on the 7^th and 10^th days of dressing change were significantly decreased (with Z values of -3.99 and -4.12, respectively, P<0.01), and the scores of wound exudate amount of patients in povidone iodine group on the 7^th and 10^th days of dressing change were significantly decreased (with Z values of -3.54 and -3.93, respectively, P<0.01). The score of wound granulation tissue growth of patients in hypochloric acid group on the 10^th day of dressing change was significantly higher than that in povidone iodine group (Z=2.02, P<0.05). Compared with those before the first dressing change, the scores of wound granulation tissue growth of patients in hypochloric acid group on the 7^th and 10^th days of dressing change were significantly increased (with Z values of -3.13 and -3.67, respectively, P<0.01), and the scores of wound granulation tissue growth of patients in povidone iodine group on the 7^th and 10^th days of dressing change were significantly increased (with Z values of -3.12 and -3.50, respectively, P<0.01). Conclusions: Hypochloric acid can kill Escherichia coli both in free and biofilm status. Hypochloric acid at a low concentration shows a rapid bactericidal effect on mature Escherichia coli biofilm, and the higher the concentration of hypochloric acid, the better the bactericidal effect. The hypochloric acid of 100 μg/mL is effective in reducing the bacterial load on wounds with Escherichia coli infection in patients, as evidenced by a reduction in wound exudate and indirect promotion of granulation tissue growth, which is more effective than povidone iodine, the traditional topical antimicrobial agent.