Fréhel C, Offredo C, de Chastellier C
INSERM U411, Laboratoire de Microbiologie, UFR de Médecine Necker Enfants Malades, Paris, France.
Infect Immun. 1997 Jul;65(7):2792-802. doi: 10.1128/iai.65.7.2792-2802.1997.
Murine bone marrow-derived macrophages (Mphis) infected with virulent strains of Mycobacterium avium (TMC 724 and a human clinical isolate) or with an avirulent opaque variant that spontaneously dissociates from the virulent human clinical isolate were subjected to a prolonged and continuous treatment with clarithromycin added at the MIC. The efficiency of this antibiotic in terms of inhibition of bacterial growth and bacterial degradation was evaluated during a 21-day treatment period. Growth was assessed by determination of CFU of intracellular bacteria and by a quantitative ultrastructural analysis which allowed us also to determine the extent of bacterial degradation. A similar treatment was applied to the same strains growing in liquid medium. Our data show that in liquid medium, clarithromycin caused a 90% decrease in CFU within 7 days of treatment. When applied to Mphis infected with virulent M. avium, clarithromycin immediately arrested bacterial growth but was unable to fully kill and degrade intracellularly growing virulent bacteria. After 21 days of treatment, 25% of intracellular bacteria were still morphologically intact. These bacteria resumed growth upon removal of the antibiotic, with a normal replication rate. These bacteria had not become more resistant to the drug, since the MIC was unchanged as compared to the one determined for the initial stock used to infect Mphis. Our data therefore suggest that the intraphagosomal environment protects bacteria from degradation. We propose that the inability of the drug to completely destroy bacteria is the result of a limited accessibility of the drug due to prevention of fusions between the immature phagosomes in which virulent bacteria reside and lysosomes in which clarithromycin accumulates. In accord with our proposal, we show that the avirulent opaque variant, which does not prevent phagosome-lysosome fusions (unpublished data), is finally destroyed by clarithromycin even within the phagosomal environment.
用鸟分枝杆菌的强毒株（TMC 724和一株人临床分离株）或用从强毒力人临床分离株自发解离的无毒力不透明变体感染的小鼠骨髓来源巨噬细胞（Mphis），接受添加了最低抑菌浓度（MIC）的克拉霉素的长期连续处理。在21天的处理期内评估了这种抗生素在抑制细菌生长和细菌降解方面的效率。通过测定细胞内细菌的菌落形成单位（CFU）以及通过定量超微结构分析来评估生长情况，定量超微结构分析还使我们能够确定细菌降解的程度。对在液体培养基中生长的相同菌株进行了类似处理。我们的数据表明，在液体培养基中，克拉霉素在处理7天内使CFU减少了90%。当应用于感染强毒力鸟分枝杆菌的Mphis时，克拉霉素立即阻止了细菌生长，但无法完全杀死和降解细胞内生长的强毒力细菌。处理21天后，25%的细胞内细菌在形态上仍然完整。去除抗生素后，这些细菌以正常的复制速率恢复生长。这些细菌对该药物并没有变得更具抗性，因为与用于感染Mphis的初始菌株所测定的MIC相比没有变化。因此，我们的数据表明吞噬体内环境保护细菌不被降解。我们提出，药物无法完全破坏细菌是由于毒力细菌所在的未成熟吞噬体与克拉霉素积累的溶酶体之间的融合受到阻止，导致药物可及性有限。与我们的提议一致，我们表明无毒力不透明变体（未发表数据）不会阻止吞噬体 - 溶酶体融合，即使在吞噬体内环境中最终也会被克拉霉素破坏。
