次氯酸对大肠埃希菌生物膜的作用及大肠埃希菌感染创面的临床疗效

刘江; 吴宝林; 朱万招; 刘洁; 王彤; 耿毛毛; 白莉; 刘毅

doi:10.3760/cma.j.cn501120-20201112-00471

次氯酸对大肠埃希菌生物膜的作用及大肠埃希菌感染创面的临床疗效

doi: 10.3760/cma.j.cn501120-20201112-00471

1.
榆林市第一医院烧伤整形外科，榆林　　719000
2.
宁夏医科大学临床医学院，银川　　750000
3.
解放军联勤保障部队第940医院烧伤整形外科，兰州　　730050
4.
兰州大学第二医院烧伤整形与创面修复外科，兰州　　730030
5.
榆林市中医医院重症医学科，榆林　　719000

详细信息

通讯作者:
刘毅，Email：liuyi196402@163.com

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出版历程
- 收稿日期: 2020-11-12

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

1.
Department of Burns and Plastic Surgery, the First Hospital of Yulin, Yulin 719000, China
2.
Clinical Medical College, Ningxia Medical University, Yinchuan 750000, China
3.
Department of Burns and Plastic Surgery, the 940th Hospital of the Joint Logistic Support Force of PLA, Lanzhou 730050, China
4.
Department of Burns and Plastic Surgery ＆ Wound Repair Surgery, Lanzhou University Second Hospital, Lanzhou 730030, China
5.
Intensive Care Unit, Traditional Chinese Medicine Hospital of Yulin, Yulin 719000, China

More Information

Corresponding author: Liu Yi, Email: liuyi196402@163.com

摘要

摘要: 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）×10⁴CFU/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）×10⁶CFU/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。
- 次氯酸 /
- 大肠杆菌 /
- 生物膜 /
- 感染性创面
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.
- Hypochloric acid /
- Escherichia coli /
- Biofilm /
- Infectious wounds
刘江：直接参与，文章撰写；吴宝林、朱万招、刘洁、王彤、耿毛毛：实施研究、采集数据；白莉：统计分析，分析/解释数据；刘毅：获取研究经费，技术或材料支持、指导，对文章的知识性内容做批评性审阅

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严重烧伤过程中,由血管渗漏诱发的休克是导致患者死亡的主要原因之一，但血管渗漏的病理生理机制尚不清楚。南京医科大学附属苏州医院整形外科孙炳伟教授团队近期在《Burns & Trauma》发文《Neutrophil-derived heparin binding protein triggers vascular leakage and synergizes with myeloperoxidase at the early stage of severe burns (with video)》，该研究通过ELISA的检测患者血浆中中性粒细胞相关颗粒蛋白[肝素结合蛋白（HBP）、髓过氧化物酶（MPO）、中性粒细胞弹性蛋白酶及基质金属蛋白酶9以及糖萼分解产物(透明质酸、硫酸乙酰肝素及多配体聚糖1)含量，观察到烧伤患者中性粒细胞相关颗粒蛋白和糖萼分解产物的含量均明显升高。采用体外人微血管内皮细胞-1细胞、小鼠烧烫伤模型及重组HBP注射模型的研究观察到，HBP可导致比烧伤更严重的血管渗漏。通过瞬时热刺激模型观察到人来源外周血中性粒细胞分泌HBP、MPO明显增多，而MPO并不能单独诱发血管渗漏，但其可以促进HBP诱导血管通透性增加的程度。进一步分析结果显示，MPO的催化产物——次氯酸能诱发血管内皮细胞CD44胞外结构域脱落，进而损伤糖萼。糖萼受损后，则上调血管内皮细胞内细胞间黏附分子1及血管细胞黏附分子1的表达水平，促使中性粒细胞更加牢固黏附，加重血管渗漏。该研究阐明了中性粒细胞来源的HBP和MPO分别通过不同途径调控血管内皮细胞和糖萼的新机制，从而为重度烧伤的治疗提供新靶点。

刘双庆，编译自《Burns Trauma》,2021,9：tkab030；姚咏明，审校

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1 培养各时间点临床来源大肠埃希菌生物膜形成情况扫描电子显微镜×20 000，图中标尺为10 μm。1A、1B.分别为培养6、12 h，大肠埃希菌处于游离状态，呈散在分布并黏附在培养皿表面，未形成胞外聚合物（EPS），图1B黏附数较图1A增多；1C.培养24 h，大肠埃希菌开始聚集、黏附，有少量的EPS连接菌体；1D.培养48 h，大肠埃希菌产生的EPS进一步增多，呈膜状结构；1E.培养72 h，大肠埃希菌产生的大量EPS呈大片膜状并将自身完全包裹；1F.培养96 h，一部分大肠埃希菌从EPS中播散、脱落

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2 与无菌生理盐水或4种浓度的次氯酸作用10 min后临床来源大肠埃希菌生物膜活性激光扫描共聚焦显微镜×60，图中标尺为10 μm。2A.与MBEC的次氯酸作用后，死菌率较低；2B.与2倍MBEC次氯酸作用后，死菌率较图2A升高；2C.与4倍MBEC次氯酸作用后，死菌率较图2B升高；2D.与8倍MBEC次氯酸作用后，死菌率较图2C升高；2E.与无菌生理盐水作用后，死菌率明显低于图2A

注：红色荧光代表死菌，绿色荧光代表活菌，黄色代表活菌和死菌重叠；最低生物膜清除浓度（MBEC）为20.36 μg/mL

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表1 2组感染性创面患者一般资料比较

组别	例数	性别（例）		年龄（岁，x¯±s）	创面面积（cm²，x¯±s）	创面类型（例）
组别	例数	男	女	年龄（岁，x¯±s）	创面面积（cm²，x¯±s）	动静脉溃疡	糖尿病足	压力性溃疡	外伤创面	热力烧伤创面
次氯酸组	21	13	8	46±14	18±5	2	4	8	2	5
聚维酮碘组	20	14	6	45±19	20±5	1	5	6	4	4
统计量值		χ²=0.30		t=0.20	t=1.61	—
P值		0.585		0.844	0.116	1.000
注：“—”表示无此统计量值

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表2 2组感染性创面患者各时间点创面组织细菌量比较

表2. [M（Q1，Q3）]

组别	例数	首次换药前（×10⁶CFU/g）	第10天换药时（×10⁴CFU/g）	Z值	P值
次氯酸组	21	2.97（2.90，3.04）	2.61（2.20，3.30）	4.02	<0.001
聚维酮碘组	20	2.97（1.90，7.95）	4.77（2.18，12.48）	3.92	<0.001
Z值		0.03	2.06
P值		0.979	0.039
注：CFU为集落形成单位

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表3 2组感染性创面患者创面渗出量评分比较[分，M（Q₁，Q₃）]

组别	例数	首次换药前	第3天换药时	第7天换药时	第10天换药时
次氯酸组	21	2（2，3）	2（2，3）	1（1，2）^a	1（0，1）^a
聚维酮碘组	20	2（2，3）	2（2，3）	1（1，2）^a	1（1，1）^a
Z值		0.14	0.30	0.55	2.07
P值		0.891	0.762	0.584	0.038
注：与组内首次换药前比较，^aP<0.01

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表4 2组感染性创面患者换药前后创面肉芽组织生长情况评分比较[分，M（Q₁，Q₃）]

组别	例数	首次换药前	第3天换药时	第7天换药时	第10天换药时
次氯酸组	21	1（0，1）	1（0，1）	1（1，2）^a	2（1，2）^a
聚维酮碘组	20	1（0，1）	1（0，1）	1（1，2）^a	1（1，2）^a
Z值		0.12	0.45	0.47	2.02
P值		0.902	0.654	0.639	0.043
注：与组内首次换药前比较，^aP<0.01

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