Department of Microbiology and Immunology, Shimane Medical University, Izumo, Shimane 693-8501, Japan
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Abstract |
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Introduction |
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It has been reported that Mycobacterium tuberculosis and M. avium invade and replicate within A-549 cells (a human type II alveolar epithelial cell line), that the degree of invasiveness is in the order of virulence of the organisms11 and that intracellular multiplication of M. tuberculosis is much more vigorous in A-549 cells than in human monocytes.12 McDonough & Kress 13 also suggested the possibility that M. tuberculosis gains access to the host lymphatic system and circulatory system by directly penetrating the alveolar epithelial lining of infected lung, and that non-professional phagocytes play important roles as sites of infection and multiplication of these mycobacteria. In this context, we have attempted to compare the behaviour of MAC in lung epithelial cells with that in macrophages, and have determined the efficacies with which KRM-1648 and clarithromycin kill the organisms within these cells. In this study, we examined the profiles of the growth of MAC within A-549 cells, the THP-1 human macrophage cell line, which has immature macrophage function, 14 and murine peritoneal macrophages when cultured in medium in the presence or absence of either KRM-1648 or clarithromycin.
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Materials and methods |
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M. avium strain N-444 (which shows low virulence in mice) 15 and Mycobacterium intracellulare strain N-260 (highly virulent in mice),15 both of which we isolated from patients with MAC infection, were cultured in 7H9 broth (Difco Laboratories, Detroit, MI, USA). Bacterial suspensions were prepared in phosphate-buffered saline (PBS) containing 0.1% (w/v) bovine serum albumin (BSA) and frozen at 80°C until use.
Mice
Female BALB/c mice were purchased from Japan Clea Co., Osaka, Japan.
Antimicrobial agents
KRM-1648 (Kaneka Corporation, Hyogo, Japan) and clarithromycin (Taisho Pharmaceutical Co., Tokyo, Japan), which were initially dissolved in dimethylsuphoxide, were diluted in prescribed media before use. The maximum concentration of compound detected in lungs (Cmax(lung)) in mice administered the drugs at oral doses equivalent to the clinical dosage was as follows: 0.8 mg/L for KRM-1648 when given at 2 mg/kg (personal communication; Dr T. Hidaka, Kaneka Corporation), and 7.0 mg/L for clarithromycin when given at 10 mg/kg.16
MIC and MBC determination
MICs of test drugs were determined as reported previously,3,10 by either an agar dilution method using Middlebrook 7H11 medium or a broth dilution method using 7HSF medium (a broth medium with the same composition as 7H11 agar but without malachite green), as described by Yajuko et al.17 MBCs were determined as described previously.10 Briefly, after MIC determinations using 7HSF medium, MBCs were determined by inoculating 10 µL samples from wells in which test agents allowed no visible growth of the organisms, on to a 7H11 agar plate, followed by 14 day cultivation. MBCs were read as minimum concentrations of drugs causing >99.9% killing of the inoculated organisms.
Intracellular growth of organisms
Macrophage monolayer cultures were prepared by seeding 1 x 106 of Zymosan A-induced peritoneal exudate cells of 8- to 12-week-old BALB/c mice on 16 mm culture wells (Becton Dickinson & Co., Lincoln Park, NJ, USA). Monolayer cultures of A-549 cells (American Type Culture Collection, Rockville, MD, USA) and THP-1 cells (American Type Culture Collection) were prepared by seeding 1 x 105 or 2 x 10 5 and 3 x 105 or 4 x 105 of cultured cells, respectively, on 16 mm culture wells and the latter cells were pretreated with 20 ng/mL phorbol myristate acetate for 18 h before use to arrest their growth. These cells were then infected with 4 x 106 or 5 x 106 cfu/mL of test organisms in a 0.5 mL portion of Ham's F-12K medium supplemented with 5% fetal bovine serum (FBS) (Bio Whittaker Co., Walkersville, MD, USA) at 37°C in a CO2 incubator (5% CO295% humidified air) for 2 h. After washing with 2% (v/v) FBS- Hanks' balanced salt solution (HBSS) to remove extracellular organisms, the MAC-infected cells were cultured in 1.0 mL of 1% (v/v) FBS- Ham's F-12K medium (5% FBS for peritoneal macrophages) in the presence or absence of test antimicrobials for up to 7 days. At intervals, the cells were lysed with 0.07% (w/v) sodium dodecylsulphate followed by subsequent neutralization with 6% (w/v) BSA-PBS, and were then washed with distilled water by centrifugation (2000g for 30 min). The recovered organisms were resuspended in 1.0 mL of distilled water, and 10 µL portions of serial 10-fold dilutions were spotted on to 7H11 agar plates. The number of residual bacterial cfu was estimated by counting microcolonies, formed after cultivation for 57 days at 37°C in a CO2 incubator, microscopically at x 15 magnification.
Reactive nitrogen intermediates production by MAC-infected cells
Culture fluids from MAC-infected cells were harvested and the NO2 concentration in them was measured using Griess reagent, as an indicator of the extent of production of reactive nitrogen intermediates (RNIs) during cultivation, as described previously.18
Statistical calculation
Statistical analysis was performed using Student's t-test.
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Results |
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Figure 1 shows the rate of bacterial growth of M. avium
N-444 () 15 and M. intracellulare
N-260 () 15 in peritoneal macrophages, THP-1
cells and A-549 cells. The growth rate of M. intracellulare N-260 was consistently
higher than that of M. avium N-444 regardless of the type of cell in which the organisms
were (P < 0.01). The growth of both MAC organisms was much more vigorous in
THP-1 and A-549 cells than in peritoneal macrophages.
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Figure 3 shows the effects of KRM-1648 0.8 mg/L against the intracellular growth of M. intracellulare N-260 residing in peritoneal macrophages, THP-1 cells and A-549 cells. The organisms in peritoneal macrophages were progressively killed by KRM-1648 during a 7 day cultivation (Figure 3a), but KRM-1648-mediated bacterial elimination was incomplete for M. intracellulare N-260 within THP-1 and A-549 cells (Figure 3b and c). In THP-1 and A-549 cells, rapid bacterial killing was observed during the first 24 h of incubation, and progressive regrowth of the organisms occurred thereafter. This closely resembles the mode of behaviour of MAC organisms in the visceral organs (lungs and spleen) of infected mice treated with KRM-1648.8,19
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Figure 4 shows the effects of clarithromycin at the C max (7 mg/L) against M. intracellulare in peritoneal macrophages, THP-1 cells or A-549 cells. Less dramatic effects were seen than those observed for the case of peritoneal macrophages with KRM-1648, and differences between the three cell lines were less marked. In this experiment, we did not observe the generation of clarithromycin-resistant mutants from M. intracellulare populations in THP-1 or A-549 cells during cultivation of these cells in the presence of clarithromycin at 7 mg/L. That is, when ten colonies of the organism were isolated from M. intracellulare-infected THP-1 cells or A-549 cells after 7 days' cultivation, the MIC of clarithromycin for all of these colonies was 6.25 mg/L. This value was identical to the MIC for the parent M. intracellulare N-260 strain.
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Discussion |
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Another interesting finding of the present study is that M. intracellulare organisms within A-549 and THP-1 cells were more resistant to KRM-1648-mediated bactericidal effects than were those in peritoneal macrophages (Figure 3). This is consistent with the finding by Mehta et al.12 of amikacin-mediated killing of M. tuberculosis in A-549 cells and in macrophages. Notably, M. intracellulare in A-549 and THP-1 cells was not completely inhibited even by extracellular concentrations (0.8- 1 mg/L) of KRM-1648 that were more than ten-fold the MIC value (0.06 mg/L). This profile is very different from that of KRM-1648- mediated killing of M. intracellulare within peritoneal macrophages. As proposed by Frehel et al.,23 the ineffectiveness of KRM-1648 against M. intracellulare in A-549 and THP-1 cells may have been partly caused by limited access of the drug to organisms as immature phagosomes in which virulent MAC pathogens reside and lysosomes in which KRM-1648 may accumulate, may not fuse with each other. Although the results obtained in this study pertain to a particular M. intracellulare N-260 strain, they indicate that it is important to consider the roles of alveolar epithelial cells as sites of bacterial invasion and multiplication in the lungs of MAC patients who are receiving chemotherapeutic regimens that include KRM-1648 treatment.
On the other hand, clarithromycin displayed moderate levels of antimicrobial activity against M. intracellulare residing in peritoneal macrophages, THP-1 cells or A-549 cells. Profiles of clarithromycin-mediated killing and inhibition of M. intracellulare within these three types of cells did not differ from each other markedly (Figure 4). Since clarithromycin was effective against M. intracellulare in A-549 cells as well as the organisms in peritoneal macrophages, it appears that the efficacy of clarithromycin delivery to M. intracellulare growing in A-549 cells are nearly the same as those in macrophages.
Recently, Bermudez et al.24 have reported that MAC organisms which are adapted to the intracellular environment of macrophages invade macrophages by complement receptor- or mannose receptor-independent pathways, unlike MAC growing extracellularly, and that such organisms resist tumour necrosis factor-a-mediated microbicidal activity of host macrophages. Further studies of the efficacies of these drugs against MAC in macrophages and A-549 cells are currently under way using organisms growing intracellularly in macrophages as an inoculum for infection.
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Acknowledgments |
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Notes |
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References |
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Received 29 May 1998; returned 15 August 1998; revised 12 September 1998; accepted 26 October 1998