人体肘窝部皮肤菌群培养方法的建立及其在特应性皮炎疾病辅助诊断中的应用探索

Establishment of a culture method for skin flora of human antecubital fossa and its application in assistance in diagnosis of atopic dermatitis

目的建立一种肘窝部皮肤菌群的分离培养方法, 探讨其在特应性皮炎(atopic dermatitis, AD)疾病辅助诊断中的应用价值。方法采用前瞻性试验性研究方法, 选取符合Williams诊断标准的AD患者8例和健康者8例, 采集其肘窝部皮肤刷洗标本进行皮肤菌群的分离培养, 探讨生长温度、气体环境、pH值、盐浓度和培养基等条件对皮肤细菌生长的影响, 建立人体皮肤菌群培养的方法。基于该方法, 选取2019年9月至2020年3月就诊于广东省中医院皮肤科的AD患者34例[年龄(14.43±8.03)岁, 男21例, 女13例]和健康志愿者者26例[年龄(29.38±7.47)岁, 男12例, 女14例], 对其肘窝部皮肤菌群进行定量培养, 采用MALDI-TOF质谱技术和16S rRNA基因测序技术进行菌种鉴定和菌落计数。采用Kruskal-WallisH检验比较AD患者和健康者肘窝部皮肤菌群结构差异, 以及AD患者中肘部皮损者与肘部非皮损者的皮肤菌群结构差异。最终, 结合AD患者的菌群培养结果及其局部皮肤损伤严重程度, 探讨金黄色葡萄球菌定量计数在AD诊断中的应用价值。结果人体肘窝部皮肤菌群培养的最适宜生长温度为28~32 ℃, 最适pH值为6~7, 最适盐浓度范围为0.5%~5.0%, 其中以血平板和巧克力平板检出细菌种类和数量最多。定量细菌培养结果比较发现:健康对照组肘部皮肤菌群在种属上的多样性比AD组(AD皮损组和AD非皮损组)高, 而AD患者中AD皮损组肘窝部皮肤细菌的总体分布密度明显高于AD非皮损组和健康对照组(H=24.25, P<0.05;H=13.41, P<0.05), 且从AD皮损组中检出的金黄色葡萄球菌的占比与AD的严重程度呈显著正相关(r=0.411, P<0.05)。结论基于上述培养方法, 采用定量培养获得的皮肤菌群多样性数据, 尤其是血平板上金黄色葡萄球菌的占比, 可作为评估AD患者皮损严重程度的微生物学指标, 对指导AD疾病的临床治疗及预后判断有重要意义。

Objective To establish a method for the isolation and culture of antecubital fossa skin flora,and to investigate its application value in the assistance in diagnosis of atopic dermatitis(AD).Methods In this prospective experimental study,a total of 8 AD patients who met the Williams diagnostic criteria and 8 healthy volunteers were recruited.The skin specimens of the antecubital fossa were collected for the isolation and culture of skin flora.The effects of growth temperature,gas environment,pH value,salt concentration,and culture medium on the growth of skin flora were investigated to establish a culture method for human skin flora.Based on the established skin flora culture method,34 AD patients who were(14.43±8.03)years old,including 21 males and 13 females,and 26 healthy volunteers who were(29.38±7.47)years old,including 12 males and 14 females,admitted to Department of Dermatology,Guangdong Provincial Hospital of Traditional Chinese Medicine from September 2019 to March 2020 were selected,and the skin specimens of the antecubital fossa were subjected to culture.MALDI-TOF mass spectrometry and 16S rRNA gene sequencing were used to identify and count all the bacteria growing in the fixed unit area on the medium.The differences in skin microbiome between the AD patients and the healthy volunteers and between the AD patients with lesional and non-lesional skin were compared by Kruskal-Wallis H test.Combined with the severity of local skin damage in the AD patients,the application value of quantitative Staphylococcus aureus count in the diagnosis of atopic dermatitis was discussed.Results The optimum growth temperature for the culture of human antecubital fossa skin flora was 28-32℃,the optimum pH was 6-7,and the optimum salt concentration range was 0.5%-5.0%.More bacteria species and quantities were detected in Columbia blood and Haemophilus chocolate agars.Comparison of quantitative bacterial culture results showed that the species diversity of antecubital skin bacteria in the healthy control group was generally higher than that in the AD patients(the AD skin lesions and non-skin lesions groups);the total distribution density of skin bacteria in antecubital fossa of the AD skin lesions group was significantly higher than those of the AD non-skin lesions group and the healthy control group(H=24.25,P<0.05;H=13.41,P<0.05).The proportion of Staphylococcus aureus detected in the patients with skin lesions was significantly positively correlated with the severity of AD(r=0.411,P<0.05).Conclusions Based on the above quantitative microbial culture method,the skin flora diversity data,especially the proportion of Staphylococcus aureus quantitatively cultured on Columbia blood agar,can be used as a microbiological index to evaluate the severity of skin lesions in AD patients,which is of great significance for guiding the clinical treatment and prognosis evaluation of AD.