Nozawa R T, Kato H, Yokota T
Antimicrob Agents Chemother. 1984 Dec;26(6):841-4. doi: 10.1128/AAC.26.6.841.
We developed a rapid, quantitative culture method to estimate the replication of Mycobacterium avium-intracellulare complex (MAIC) in human peripheral blood mononuclear cells. Mononuclear cells were plated in a 96-well tray, infected with clinically isolated strains of MAIC in the presence of autologous plasma, and further cultivated for 1 to 2 weeks in a tissue culture medium. No MAIC cells proliferated extracellularly, since human plasma inhibited extracellular growth of the mycobacteria. The mononuclear cells were lysed through a brief treatment with alkali, and surviving intracellular mycobacteria were diluted and plated with tissue culture medium in a 96-well tray. Mycobacterial colonies were counted under a microscope after a 5-day incubation. The number of viable MAIC cells continuously increased, reaching 10 times the number of inoculated cells in a week. Thus, mononuclear phagocytes were the permissive site for the replication of MAIC. Intra- and extracellular susceptibilities of seven MAIC strains to four aminoglycoside antibiotics were then studied. The mycobacteria were most susceptible in vitro to dibekacin (MICs, 3.13 to 12.5 micrograms/ml). Dibekacin at 12.5 micrograms/ml was bacteriostatic to five of seven strains in the monocytes. Also, intracellular replication of the other two strains was greatly suppressed by that concentration of dibekacin.
我们开发了一种快速定量培养方法,用于评估鸟分枝杆菌-胞内分枝杆菌复合体(MAIC)在人外周血单个核细胞中的复制情况。将单个核细胞接种于96孔板中,在自体血浆存在的情况下,用临床分离的MAIC菌株进行感染,并在组织培养基中进一步培养1至2周。由于人血浆抑制分枝杆菌的细胞外生长,因此没有MAIC细胞在细胞外增殖。通过短暂的碱处理使单个核细胞裂解,将存活的细胞内分枝杆菌稀释后接种于96孔板中的组织培养基中。孵育5天后,在显微镜下计数分枝杆菌菌落。存活的MAIC细胞数量持续增加,一周内达到接种细胞数量的10倍。因此,单核吞噬细胞是MAIC复制的允许位点。随后研究了7株MAIC菌株对4种氨基糖苷类抗生素的细胞内和细胞外敏感性。分枝杆菌在体外对双去氧卡那霉素最敏感(最低抑菌浓度为3.13至12.5微克/毫升)。12.5微克/毫升的双去氧卡那霉素对单核细胞中7株菌株中的5株具有抑菌作用。此外,该浓度的双去氧卡那霉素可极大地抑制另外2株菌株的细胞内复制。