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Abstract：
BACKGROUND: The Mycobacterium avium complex includes the commonest non-tuberculous mycobacteria associated with human infections. These infections have been associated with the production of biofilms in many cases, but there are only a few studies about biofilms produced by the species included in this group.
METHODS: Three collection strains (M. avium ATCC25291, M. intracellulare ATCC13950, and M. chimaera DSM756), three clinically significant strains (647, 657, and 655), and three clinically non-significant ones (717, 505, and 575) of each species were included. The clinical significance of the clinical isolates was established according to the internationally accepted criteria. The biofilm ultrastructure was studied by Confocal-Laser Scanning Microscopy by using BacLight Live-Dead and Nile Red stains. The viability, covered surface, height, and relative autofluorescence were measured in several images/strain. The effect of clarithromycin was studied by using the technique described by Muñoz-Egea et al. with modifications regarding incubation time. The study included clarithromycin in the culture medium at a concentration achievable in the lungs (11.3 mg/L), using one row of wells as the control without antibiotics. The bacterial viability inside the biofilm is expressed as a percentage of viable cells. The differences between the different parameters of the biofilm ultrastructure were analyzed by using the Kruskal-Wallis test. The correlation between bacterial viability in the biofilm and treatment time was evaluated by using Spearman\'s rank correlation coefficient (ρ).
RESULTS: The strains showed differences between them with all the studied parameters, but neither a species-specific pattern nor a clinical-significance-specific pattern were detected. For the effect of clarithromycin, the viability of the bacteria contained in the biofilm was inversely proportional to the exposure time of the biofilm (ρ > -0.3; p-value < 0.05), excluding two M. chimaera strains (M. chimaera DSM756 and 575), which showed a weak positive correlation with treatment time (0.2 < ρ < 0.39; p-value < 0.05). Curiously, despite a clarithromycin treatment of 216 h, the percentage of the biofilm viability of the strains evaluated here was not less than 40% at best (M. avium 717).
CONCLUSIONS: All the M. avium complex strains studied can form biofilm in vitro, but the ultrastructural characteristics between them suggest that these are strain-specific characteristics unrelated to the species or the clinical significance. The clarithromycin effect on MAC species is biofilm-age/time-of-treatment-dependent and appears to be strain-specific while being independent of the clinical significance of the strain.
METHODS: Three collection strains (M. avium ATCC25291, M. intracellulare ATCC13950, and M. chimaera DSM756), three clinically significant strains (647, 657, and 655), and three clinically non-significant ones (717, 505, and 575) of each species were included. The clinical significance of the clinical isolates was established according to the internationally accepted criteria. The biofilm ultrastructure was studied by Confocal-Laser Scanning Microscopy by using BacLight Live-Dead and Nile Red stains. The viability, covered surface, height, and relative autofluorescence were measured in several images/strain. The effect of clarithromycin was studied by using the technique described by Muñoz-Egea et al. with modifications regarding incubation time. The study included clarithromycin in the culture medium at a concentration achievable in the lungs (11.3 mg/L), using one row of wells as the control without antibiotics. The bacterial viability inside the biofilm is expressed as a percentage of viable cells. The differences between the different parameters of the biofilm ultrastructure were analyzed by using the Kruskal-Wallis test. The correlation between bacterial viability in the biofilm and treatment time was evaluated by using Spearman\'s rank correlation coefficient (ρ).
RESULTS: The strains showed differences between them with all the studied parameters, but neither a species-specific pattern nor a clinical-significance-specific pattern were detected. For the effect of clarithromycin, the viability of the bacteria contained in the biofilm was inversely proportional to the exposure time of the biofilm (ρ > -0.3; p-value < 0.05), excluding two M. chimaera strains (M. chimaera DSM756 and 575), which showed a weak positive correlation with treatment time (0.2 < ρ < 0.39; p-value < 0.05). Curiously, despite a clarithromycin treatment of 216 h, the percentage of the biofilm viability of the strains evaluated here was not less than 40% at best (M. avium 717).
CONCLUSIONS: All the M. avium complex strains studied can form biofilm in vitro, but the ultrastructural characteristics between them suggest that these are strain-specific characteristics unrelated to the species or the clinical significance. The clarithromycin effect on MAC species is biofilm-age/time-of-treatment-dependent and appears to be strain-specific while being independent of the clinical significance of the strain.
摘要：
背景：鸟分枝杆菌复合物包括与人类感染相关的最常见的非结核分枝杆菌。在许多情况下，这些感染与生物膜的产生有关，但是关于该组中包含的物种产生的生物膜的研究很少。
方法：三种收集菌株(M.aiumATCC25291,M.胞内ATCC13950,andM.chimaeraDSM756),三个临床显著菌株(647、657和655)，每个物种中包括3个临床上不显著的物种(717,505和575)。根据国际公认的标准确定临床分离株的临床意义。通过使用BacLight活死菌和尼罗河红染色，通过共聚焦激光扫描显微镜研究了生物膜的超微结构。生存能力,覆盖表面,高度,和相对自发荧光在几个图像/菌株中测量。使用Muñoz-Egea等人描述的技术研究了克拉霉素的作用。关于孵化时间的修改。该研究包括在培养基中的克拉霉素在肺中可达到的浓度（11.3mg/L），使用一排没有抗生素的井作为对照。生物膜内的细菌活力表示为活细胞的百分比。采用Kruskal-Wallis试验分析生物膜超微结构不同参数之间的差异。通过使用Spearman等级相关系数(ρ)评估生物膜中细菌活力与处理时间之间的相关性。
结果：菌株在所有研究参数下显示出它们之间的差异，但未检测到物种特异性模式和临床意义特异性模式。对于克拉霉素的作用，生物膜中包含的细菌的活力与生物膜的暴露时间成反比（ρ>-0.3；p值<0.05），不包括两种嵌合体菌株(M.嵌合体DSM756和575)，与治疗时间呈微弱正相关(0.2<ρ<0.39；p值<0.05)。奇怪的是,尽管克拉霉素治疗了216小时，此处评估的菌株的生物膜活力百分比最多不低于40%(M.avium717).
结论：研究的所有鸟分枝杆菌复合菌株均可在体外形成生物膜，但是它们之间的超微结构特征表明，这些是与物种或临床意义无关的菌株特异性特征。克拉霉素对MAC物种的作用是生物膜年龄/治疗时间依赖性的，并且似乎是菌株特异性的，而与菌株的临床意义无关。
方法：三种收集菌株(M.aiumATCC25291,M.胞内ATCC13950,andM.chimaeraDSM756),三个临床显著菌株(647、657和655)，每个物种中包括3个临床上不显著的物种(717,505和575)。根据国际公认的标准确定临床分离株的临床意义。通过使用BacLight活死菌和尼罗河红染色，通过共聚焦激光扫描显微镜研究了生物膜的超微结构。生存能力,覆盖表面,高度,和相对自发荧光在几个图像/菌株中测量。使用Muñoz-Egea等人描述的技术研究了克拉霉素的作用。关于孵化时间的修改。该研究包括在培养基中的克拉霉素在肺中可达到的浓度（11.3mg/L），使用一排没有抗生素的井作为对照。生物膜内的细菌活力表示为活细胞的百分比。采用Kruskal-Wallis试验分析生物膜超微结构不同参数之间的差异。通过使用Spearman等级相关系数(ρ)评估生物膜中细菌活力与处理时间之间的相关性。
结果：菌株在所有研究参数下显示出它们之间的差异，但未检测到物种特异性模式和临床意义特异性模式。对于克拉霉素的作用，生物膜中包含的细菌的活力与生物膜的暴露时间成反比（ρ>-0.3；p值<0.05），不包括两种嵌合体菌株(M.嵌合体DSM756和575)，与治疗时间呈微弱正相关(0.2<ρ<0.39；p值<0.05)。奇怪的是,尽管克拉霉素治疗了216小时，此处评估的菌株的生物膜活力百分比最多不低于40%(M.avium717).
结论：研究的所有鸟分枝杆菌复合菌株均可在体外形成生物膜，但是它们之间的超微结构特征表明，这些是与物种或临床意义无关的菌株特异性特征。克拉霉素对MAC物种的作用是生物膜年龄/治疗时间依赖性的，并且似乎是菌株特异性的，而与菌株的临床意义无关。
