Jin J, Zhou H, Cui Z C, Wang L, Luo P F, Ji S Z, Hu X Y, Ma B, Wang G Y, Zhu S H, Xia Z F
Burn Institute of PLA, Department of Burn Surgery, the First Affiliated Hospital, Naval Military Medical University, Shanghai 200433, China.
Zhonghua Shao Shang Za Zhi. 2018 Apr 20;34(4):225-232. doi: 10.3760/cma.j.issn.1009-2587.2018.04.007.
To study the antiseptic effect of compound lysostaphin disinfectant and its preventive effect on infection of artificial dermis after graft on full-thickness skin defect wound in rats. (1) Each one standard strain of and were selected. Each 20 clinical strains of and were collected from those isolated from wound exudates of burn patients hospitalized in our wards from January 2014 to December 2016 according to the random number table. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of compound lysostaphin disinfectant to above-mentioned strains were detected. The experiment was repeated 3 times. Compared with the corresponding standard strain, the clinical strain with higher MIC and/or MBC was considered as having decreased sensitivity to the disinfectant. The percentage of strains of each of the three kinds of bacteria with decreased sensitivity was calculated. (2) Artificial dermis pieces were soaked in compound lysostaphin disinfectant for 5 min, 1 h, 2 h, and 4 h, respectively, with 21 pieces at each time point. After standing for 0 (immediately), 12, 24, 36, 48, 60, 72 h (with 3 pieces at each time point), respectively, the diameters of their inhibition zones to standard strains of and were measured. The experiment was repeated 3 times. The shortest soaking time corresponding to the longest standing time, after which the disinfectant-soaked artificial dermis could form an effective inhibition zone (with diameter more than 7 mm), was the sufficient soaking time of the disinfectant to the artificial dermis. (3) Forty Sprague-Dawley rats were divided into post injury day (PID) 3, 7, 14, and 21 sampling groups according to the random number table, with 10 rats in each group. A full-thickness skin defect wound with a diameter of 20 mm was made on both sides of the spine on the back of each rat. Immediately after injury, the artificial dermis without any treatment was grafted on the wound on left side of the spine (hereinafter referred to as control wound), while the sufficiently soaked artificial dermis with compound lysostaphin disinfectant was grafted on the wound on right side of the spine (hereinafter referred to as disinfectant wound). On PID 3, 7, 14, and 21, the gross condition of wounds of all the surviving rats was observed, and the new infection rates of control wounds and disinfectant wounds were calculated. Then, the rats in the sampling group with corresponding time were killed, and the full-thickness wound tissue containing artificial dermis was collected for quantitative analysis of bacteria. Bacteria content of the uninfected control wounds and that of the uninfected disinfectant wounds were compared. Data were processed with chi-square test and Wilcoxon rank sum test. (1) The MIC of compound lysostaphin disinfectant to standard strains of , and were 1/32, 1/32, and 1/512 of the original concentration of the disinfectant, respectively, and the MBC were 1/32, 1/16, and 1/512 of the original concentration of the disinfectant, respectively. The percentages of clinical strains of and with decreased sensitivity to compound lysostaphin disinfectant were 15% (3/20), 20% (4/20), and 10% (2/20), respectively. (2) After being soaked in compound lysostaphin disinfectant for 2 and 4 h, the longest standing time, after which the artificial dermis could form an effective inhibition zone against and were 24, 36, and 48 h respectively, longer than 12, 24, and 24 h of soaking for 5 min and 24, 24, and 36 h of soaking for 1 h. The sufficient soaking time of compound lysostaphin disinfectant to artificial dermis was 2 h. (3) On PID 3, no infection symptom was observed in all the wounds, and so both the new infection rate of control wounds and that of disinfectant wounds were 0. The artificial dermis was transparent but not well connected with the wound. On PID 7, the new infection rate of control wounds was 20.00% (6/30), which was obviously higher than 3.33% (1/30) of disinfectant wounds, (2)=4.043, <0.05. On the infected wound, a large amount of purulent exudates were observed, and the artificial dermis was not connected with the wound and degraded partially. On the uninfected wound, artificial dermis was transparent and had a partial connection with the wound. On PID 14 and 21, no new infected wound was observed, and so both the new infection rate of control wounds and that of disinfectant wounds were 0. There was no obvious improvement on the infected wounds. The collagen layers of artificial dermis in the uninfected wound established a good connection with the wound and were separating from the silica gel layer gradually. Infection occurred in 2, 3, 1 control wound (s) in PID 7, 14, and 21 sampling groups, respectively, and in 1 disinfectant wound in PID 14 sampling group. The bacteria content of the infected wounds tissue was 0.79×10(6) to 7.22×10(9) colony-forming unit (CFU)/g. The bacteria content of uninfected control wounds tissue in PID 3, 7, and 14 sampling groups were (3.43±1.88)×10(2,) (2.37±0.43)×10(3,) and (8.40±1.03)×10(3) CFU/g, respectively, which were significantly higher than (0.33±0.12)×10(2,) (0.43±0.17)×10(3,) (2.16±0.52)×10(3) CFU/g of uninfected disinfectant wounds tissue (=-3.780, -3.554, -3.334, <0.05). The bacteria content of uninfected control wounds tissue and that of uninfected disinfectant wounds tissue in PID 21 sampling group were similar (=-0.490, >0.05). Compound lysostaphin disinfectant has quite strong antibacterial ability against and . Clinical strains of the three kinds of bacteria were highly sensitive to compound lysostaphin disinfectant. Saturation of absorption of compound lysostaphin disinfectant achieves in artificial dermis after 2 hours' soaking. After 24, 36, and 48 hours' standing, the soaked artificial dermis still has the antibacterial effect on and respectively. The infection rate and the bacteria content of full-thickness skin defect wound in rats are all decreased when grafted with soaked artificial dermis.
研究复方溶葡萄球菌消毒剂的抗菌效果及其对大鼠全层皮肤缺损创面植皮后人工真皮感染的预防作用。(1)选取金黄色葡萄球菌、大肠杆菌的标准菌株各1株。按照随机数字表法,从2014年1月至2016年12月在我科住院的烧伤患者创面分泌物中分离的金黄色葡萄球菌、大肠杆菌临床菌株各20株。检测复方溶葡萄球菌消毒剂对上述菌株的最低抑菌浓度(MIC)和最低杀菌浓度(MBC)。实验重复3次。与相应标准菌株比较,MIC和/或MBC较高的临床菌株视为对消毒剂敏感性降低。计算3种细菌中敏感性降低菌株的百分比。(2)将人工真皮片分别浸泡于复方溶葡萄球菌消毒剂中5分钟、1小时、2小时和4小时,每个时间点21片。分别于浸泡后0(即刻)、12、24、36、48、60、72小时(每个时间点3片)静置后,测量其对金黄色葡萄球菌、大肠杆菌标准菌株的抑菌圈直径。实验重复3次。浸泡时间最短且静置时间最长,浸泡后的人工真皮能形成有效抑菌圈(直径大于7mm)的时间,即为消毒剂对人工真皮的充足浸泡时间。(3)将40只Sprague-Dawley大鼠按照随机数字表法分为伤后3天、7天、14天和21天取样组,每组10只。在每只大鼠背部脊柱两侧制作直径为20mm的全层皮肤缺损创面。伤后即刻,将未作任何处理的人工真皮移植于脊柱左侧创面(以下简称对照创面),将充分浸泡复方溶葡萄球菌消毒剂的人工真皮移植于脊柱右侧创面(以下简称消毒剂创面)。于伤后3天、7天、14天和21天,观察所有存活大鼠创面的大体情况,计算对照创面和消毒剂创面的新感染率。然后,处死相应时间取样组的大鼠,采集含有人工真皮的全层创面组织进行细菌定量分析。比较未感染对照创面和未感染消毒剂创面的细菌含量。数据采用卡方检验和Wilcoxon秩和检验进行处理。(1)复方溶葡萄球菌消毒剂对金黄色葡萄球菌、大肠杆菌标准菌株的MIC分别为消毒剂原液浓度的1/32、1/32和1/512,MBC分别为消毒剂原液浓度的1/32、1/16和1/512。金黄色葡萄球菌、大肠杆菌临床菌株对复方溶葡萄球菌消毒剂敏感性降低的百分比分别为15%(3/20)、20%(4/20)和10%(2/20)。(2)人工真皮浸泡复方溶葡萄球菌消毒剂2小时和4小时后,对金黄色葡萄球菌、大肠杆菌能形成有效抑菌圈的最长静置时间分别为24、36和48小时,长于浸泡5分钟的12、24和24小时以及浸泡1小时的24、24和36小时。复方溶葡萄球菌消毒剂对人工真皮的充足浸泡时间为2小时。(3)伤后3天,所有创面均未观察到感染症状,对照创面和消毒剂创面的新感染率均为0。人工真皮呈透明状,但与创面连接不佳。伤后7天,对照创面的新感染率为20.00%(6/30),明显高于消毒剂创面的3.33%(1/30),(χ2)=4.043,P<0.05。感染创面上可见大量脓性分泌物,人工真皮与创面分离且部分降解。未感染创面上,人工真皮呈透明状,与创面部分连接。伤后14天和21天,未观察到新的感染创面,对照创面和消毒剂创面的新感染率均为0。感染创面无明显改善。未感染创面人工真皮的胶原层与创面建立了良好连接,并逐渐与硅胶层分离。伤后7天、14天和21天取样组对照创面分别有2、3、1个发生感染,伤后14天取样组消毒剂创面有1个发生感染。感染创面组织的细菌含量为0.79×10(6)至7.22×10(9)菌落形成单位(CFU)/克。伤后3天、7天和14天取样组未感染对照创面组织的细菌含量分别为(3.43±1.88)×10(2)、(2.37±0.43)×10(3)和(8.40±1.03)×10(3)CFU/克,明显高于未感染消毒剂创面组织的(0.33±0.12)×10(2)、(0.43±0.17)×10(3)、(2.16±0.52)×10(3)CFU/克(Z=-3.780,-3.554,-3.334,P<0.05)。伤后21天取样组未感染对照创面组织与未感染消毒剂创面组织的细菌含量相近(Z=-0.490,P>0.05)。复方溶葡萄球菌消毒剂对金黄色葡萄球菌、大肠杆菌有较强的抗菌能力。3种细菌的临床菌株对复方溶葡萄球菌消毒剂高度敏感。复方溶葡萄球菌消毒剂浸泡人工真皮2小时后吸收达到饱和。浸泡后的人工真皮分别于静置24、36和48小时后,对金黄色葡萄球菌、大肠杆菌仍有抗菌作用。大鼠全层皮肤缺损创面移植浸泡后的人工真皮后,感染率和细菌含量均降低。