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  • Title: [Effect of hypochloric acid on Escherichia coli biofilm and the clinical efficacy of hypochloric acid for wounds with Escherichia coli infection].
    Author: Liu J, Wu BL, Zhu WZ, Liu J, Wang T, Geng MM, Bai L, Liu Y.
    Journal: Zhonghua Shao Shang Yu Chuang Mian Xiu Fu Za Zhi; 2022 Mar 20; 38(3):242-250. PubMed ID: 35325969.
    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 940th 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 940th 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 10th 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 3rd, 7th, and 10th 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 10th 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 10th 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 10th 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 7th and 10th 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 7th and 10th 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 10th 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 7th and 10th 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 7th and 10th 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. 目的： 探讨次氯酸对大肠埃希菌生物膜的作用及其对大肠埃希菌感染创面的临床疗效。 方法： 收集从解放军联勤保障部队第940医院5个临床科室2019年9—12月25例患者（男16例、女9例，年龄32~67岁）送检标本分离出的大肠埃希菌菌株中细菌生物膜形成能力最强的1株菌进行实验研究。将大肠埃希菌分别与162.96、81.48、40.74、20.37、10.18、5.09、2.55、1.27、0.64、0.32 μg/mL的次氯酸共培养，筛选次氯酸最低杀菌浓度（MBC）；将大肠埃希菌与筛选的MBC次氯酸分别作用2、5、10、20、30、60 min，筛选次氯酸的最短杀菌时间。分别于培养6、12、24、48、72、96 h，采用扫描电子显微镜观察大肠埃希菌生物膜形成情况。大肠埃希菌培养72 h后，分别加入1、2、4、8、16倍MBC的次氯酸，筛选次氯酸对大肠埃希菌的最低生物膜清除浓度（MBEC）。于大肠埃希菌中分别加入1、2、4、8倍MBEC的次氯酸及无菌生理盐水，作用10 min后，采用活/死细菌染色试剂盒检测活、死细胞数，并计算死菌率（样本数为5）。2020年1—12月，解放军联勤保障部队第940医院烧伤整形外科收治41例符合入选标准的感染创面患者，对其进行前瞻性随机对照试验。采用随机数字表法将患者分为次氯酸组21例［男13例、女8例，年龄（46±14）岁］和聚维酮碘组20例［男14例、女6例，年龄（45±19）岁］。2组患者分别用100 μg/mL次氯酸、50 mg/mL聚维酮碘溶液浸透的无菌纱布湿敷，每天换药1次。首次换药前、第10天换药时，取创面及创缘组织，采用琼脂培养法培养细菌并定量分析组织细菌量。首次换药前和第3、7、10天换药时，肉眼观察创面渗出量和肉芽组织生长情况并评分。对数据行单因素方差分析、Dunnett-t检验、独立样本t检验、Mann-Whitney U检验、Wilcoxon符号秩检验、χ2检验或Fisher确切概率法检验。 结果： 次氯酸对大肠埃希菌的MBC为10.18 μg/mL，MBC的次氯酸对大肠埃希菌的最短杀菌时间为2 min。培养6、12 h，大肠埃希菌处于完全游离状态；随着培养时间的延长，大肠埃希菌逐渐聚集、黏附，至培养72 h形成成熟的生物膜。次氯酸对大肠埃希菌的MBEC为20.36 μg/mL。与1、2、4、8倍MBEC的次氯酸作用10 min后，大肠埃希菌死菌率均明显高于与无菌生理盐水作用10 min后（t值分别为6.11、25.04、28.90、40.74，P<0.01）。第10天换药时，次氯酸组患者创面组织细菌量为2.61（2.20，3.30）×104集落形成单位（CFU）/g，明显少于聚维酮碘组的4.77（2.18，12.48）×104 CFU/g（Z=2.06，P<0.05）；次氯酸组和聚维酮碘组患者创面组织细菌量均明显少于首次换药前的2.97（2.90，3.04）×106、2.97（1.90，7.95）×106 CFU/g（Z值分别为4.02、3.92，P<0.01）。第10天换药时，次氯酸组患者创面渗出量评分明显低于聚维酮碘组（Z=2.07，P<0.05）。与首次换药前比较，次氯酸组患者第7、10天换药时创面渗出量评分均明显降低（Z值分别为-3.99、-4.12，P<0.01），聚维酮碘组患者第7、10天换药时创面渗出量评分均明显降低（Z值分别为-3.54、-3.93，P<0.01）。第10天换药时，次氯酸组患者创面肉芽组织生长评分明显高于聚维酮碘组（Z=2.02，P<0.05）。与首次换药前比较，次氯酸组患者第7、10天换药时创面肉芽组织生长评分均明显升高（Z值分别为-3.13、-3.67，P<0.01），聚维酮碘组患者第7、10天换药时创面肉芽组织生长评分均明显升高（Z值分别为-3.12、-3.50，P<0.01）。 结论： 次氯酸对游离状态和生物膜状态的大肠埃希菌均有杀灭作用，低浓度的次氯酸对成熟的大肠埃希菌生物膜可起到快速杀菌作用，且次氯酸浓度越高，杀菌效果越好。100 μg/mL次氯酸能有效减少患者大肠埃希菌感染创面的细菌负荷，表现为创面渗出的减少、间接促进肉芽组织生长，较传统外用抗菌剂聚维酮碘疗效更好。.
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