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Title: [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].
Author: Jin J, Zhou H, Cui ZC, Wang L, Luo PF, Ji SZ, Hu XY, Ma B, Wang GY, Zhu SH, Xia ZF.
Journal: Zhonghua Shao Shang Za Zhi; 2018 Apr 20; 34(4):225-232. PubMed ID: 29690741.
Abstract:
Objective: 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. Methods: (1) Each one standard strain of Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus were selected. Each 20 clinical strains of Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus 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 Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus 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. Results: (1) The MIC of compound lysostaphin disinfectant to standard strains of Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii 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 Klebsiella pneumoniae, Acinetobacter baumannii and Staphylococcus aureus 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 Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus, 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, χ(2)=4.043, P<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 (Z=-3.780, -3.554, -3.334, P<0.05). The bacteria content of uninfected control wounds tissue and that of uninfected disinfectant wounds tissue in PID 21 sampling group were similar (Z=-0.490, P>0.05). Conclusions: Compound lysostaphin disinfectant has quite strong antibacterial ability against Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus. 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 Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus, 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. 目的： 研究复合溶葡萄球菌酶消毒剂的抗菌作用及其对全层皮肤缺损大鼠移植人工真皮后感染的预防作用。 方法： (1)取肺炎克雷伯菌、鲍氏不动杆菌、金黄色葡萄球菌标准菌株各1株，采用随机数字表法选取从笔者单位2014年1月—2016年12月收治烧伤患者创面分泌物中分离出的肺炎克雷伯菌、鲍氏不动杆菌、金黄色葡萄球菌各20株，测定复合溶葡萄球菌酶消毒剂对上述菌株的最低抑菌浓度(MIC)及最低杀菌浓度(MBC)，实验重复3次。临床菌株MIC和/或MBC高于对应标准菌株者判定为对该消毒剂敏感性降低，计算3种细菌各自的敏感性降低菌株百分比。(2)将人工真皮于复合溶葡萄球菌酶消毒剂中分别浸泡5 min、1 h、2 h、4 h(每个时间点21块)，另分别于静置0(即刻)、12、24、36、48、60、72 h(每个时间点3块)后测量其对金黄色葡萄球菌、肺炎克雷伯菌、鲍氏不动杆菌标准菌株抑菌圈直径，实验重复3次。以浸泡消毒剂的人工真皮可形成有效抑菌圈(直径>7 mm)的最长静置时间所对应的最短浸泡时间为该消毒剂对人工真皮的充分浸泡时间。(3)取40只SD大鼠，采用随机数字表法分为伤后3、7、14、21 d取材组，每组10只。于每只大鼠背部脊柱两侧各做1个直径20 mm的全层皮肤缺损创面，伤后即刻脊柱左侧创面移植未进行任何处理的人工真皮(下称对照创面)，脊柱右侧创面移植充分浸泡复合溶葡萄球菌酶消毒剂的人工真皮(下称消毒剂创面)。伤后3、7、14、21 d，观察所有存活大鼠创面大体情况，计算对照创面和消毒剂创面的新发感染率，随后处死对应时间取材组大鼠，取所有包含人工真皮的全层创面组织行细菌定量，对比未感染对照创面和未感染消毒剂创面的细菌含量。对数据进行χ(2)检验和Wilcoxon秩和检验。 结果： (1)复合溶葡萄球菌酶消毒剂对金黄色葡萄球菌、肺炎克雷伯菌、鲍氏不动杆菌标准菌株的MIC分别为该消毒剂原始浓度的1/32、1/32、1/512，MBC分别为该消毒剂原始浓度的1/32、1/16、1/512。肺炎克雷伯菌、鲍氏不动杆菌、金黄色葡萄球菌临床菌株对复合溶葡萄球菌酶消毒剂敏感性降低菌株百分比分别为15%(3/20)、20%(4/20)、10%(2/20)。(2)浸泡复合溶葡萄球菌酶消毒剂2、4 h后人工真皮对肺炎克雷伯菌、鲍氏不动杆菌、金黄色葡萄球菌标准菌株可形成有效抑菌圈的最长静置时间均分别为24、36、48 h，久于浸泡5 min的12、24、24 h和浸泡1 h的24、24、36 h。2 h为该消毒剂对人工真皮的充分浸泡时间。(3)伤后3 d，大鼠所有创面均未见感染症状，对照创面及消毒剂创面新发感染率均为0。人工真皮透亮，但与创面尚未建立良好连接。伤后7 d，大鼠对照创面新发感染率为20.00%(6/30)，明显高于消毒剂创面的3.33%(1/30)，χ(2)＝4.043，P<0.05。感染创面可见大量脓性分泌物，人工真皮与创面未形成连接且部分降解；未感染创面人工真皮透亮，并与创面建立部分连接。伤后14、21 d均未观察到新的感染创面，大鼠对照创面及消毒剂创面新发感染率均为0。感染创面未见明显好转；未感染创面人工真皮胶原层与创面已建立良好连接，硅胶层和胶原层逐渐分离。伤后7、14、21 d取材组大鼠分别有2、3、1个对照创面出现感染，且伤后14 d取材组大鼠1个消毒剂创面出现感染，创面组织细菌含量为0.79×10(6)～7.22×10(9)集落形成单位(CFU)/g。伤后3、7、14 d取材组大鼠未感染对照创面组织细菌含量分别为(3.43±1.88)×10(2)、(2.37±0.43)×10(3)、(8.40±1.03)×10(3) CFU/g，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/g(Z＝－3.780、－3.554、－3.334，P<0.05)；伤后21 d取材组未感染对照创面与未感染消毒剂创面组织细菌含量相近(Z＝－0.490，P>0.05)。 结论： 复合溶葡萄球菌酶消毒剂对肺炎克雷伯菌、鲍氏不动杆菌、金黄色葡萄球菌具有较强的抗菌能力，这3个菌种的临床菌株对该消毒剂的敏感性均较高；人工真皮浸泡复合溶葡萄球菌酶消毒剂2 h后吸收饱和，静置24、36、48 h后仍分别对肺炎克雷伯菌、鲍氏不动杆菌、金黄色葡萄球菌具有抑菌作用，移植于大鼠全层皮肤缺损创面后感染率降低，并减少了创面组织细菌含量。.

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