Department of Microbiology, St Marianna University School of Medicine, 2-16-1 Sugao Miyamae-ku, Kawasaki 216-8511, Japan
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Abstract |
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Introduction |
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THP-1 is a human monocytic cell line,9 which matures into macrophage-like adherent cells following stimulation with phorbol 12-myristate 13-acetate10 or 1, 25-dihydroxy vitamin D3.11 Cirillo et al.12 reported that L. pneumophila could invade and multiply within THP-1 cells in a manner similar to that in human monocytes and macrophages. In this study, we established a simple method using THP-1 to assess the intracellular activity of antimicrobial agents. We used this system to examine the activity of several drugs against L. pneumophila, including grepafloxacin, a recently developed fluoroquinolone, which is reported to have good intracellular permeability.4,13
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Materials and methods |
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A clinical isolate of L. pneumophila serogroup 1, SMUM-353, from the sputum of a Japanese patient with pneumonia, was used in all experiments. The susceptibility of this strain to antimicrobial agents was compared with that of L. pneumophila serogroup 1 type strain, SMUM-352 (Philadelphia 1, ATCC 33152). L. pneumophila was stored in sterile skimmed milk at 80°C until use. A sample of each strain was cultured on to buffered charcoal yeast extract agar supplemented with -ketoglutarate (BCYE-
; Difco Laboratories, Detroit, MI, USA) and incubated for 48 h at 35°C. Pure colonies of L. pneumophila were inoculated into a buffered yeast extract broth supplemented with
-ketoglutarate (BYE-
; Difco Laboratories) and incubated for 24 h at 35°C on a shaker. BYE-
was prepared with 10 g/L ACES (Research Organics Inc., Cleveland, OH, USA), 10 g/L yeast extract (Difco Laboratories), 0.25 g/L ferric pyrophosphate, 0.4 g/L l-cysteine hydrochloride and 1 g/L
-ketoglutarate (Wako Ltd, Osaka, Japan), and BCYE-
contained, in addition, 3 g/L charcoal and 15 g/L agar. For the inoculum for all experimental procedures, the bacteria were washed and suspended in sterile distilled water and then prepared to 1 x 108 cfu/mL on the basis of previously prepared standards by optical density measurement at 420 nm.
Antimicrobial agents
The antimicrobial agents used in this study were kindly provided by the following suppliers: ampicillin (Meiji Seika Kaisha, Tokyo, Japan); azithromycin (Pfizer Pharmaceutical Inc., Tokyo, Japan); cefotiam (Takeda Chemical Industries, Osaka, Japan); ciprofloxacin (Bayer Yakuhin, Osaka, Japan); clarithromycin (Taisho Pharmaceutical Co., Tokyo, Japan); clindamycin (Pharmacia and Upjohn, Tokyo, Japan); erythromycin (Dainippon Pharmaceutical Co., Osaka, Japan); grepafloxacin (Otsuka Pharmaceutical Co., Osaka, Japan); imipenem (Banyu Pharmaceutical Co., Tokyo, Japan); minocycline (Lederle, Tokyo, Japan) and rifampicin (Daiichi Pharmaceutical Co., Tokyo, Japan).
Determination of MIC
MICs were determined using a broth microdilution method with BYE- broth.14 For this purpose, 5 µL of a BYE-
broth containing L. pneumophila (1 x 107/mL) was added to 0.1 mL of BYE-
broth containing standard antibiotic concentrations (final concentration, 5 x 105 cfu/mL) in microtitre wells, and incubated for 48 h at 35°C. The MIC represented the lowest antibiotic concentration that exhibited no visible bacterial growth. Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as the control strains. We also determined the minimum bactericidal activity (MBC) using 0.01 mL of the bacterial suspension from wells that demonstrated no visible growth. The bacterial suspension was inoculated on to the surface of BCYE-
agar and incubated for 72 h at 35°C.14 The MBC represented the lowest antibiotic concentration resulting in 99.9% killing of the bacterial inoculum.
Cell line and culture conditions
THP-1 (kindly provided by Dr Shigeru Tsuchiya, Tohoku University, Sendai, Japan), a human monocytic cell line,9 was maintained in continuous culture in RPMI 1640 medium (Iwaki Ltd, Funabashi, Japan) containing 10% fetal calf serum (BioWhittaker, Inc., Walkersville, MD, USA) (RPMI 164010% FCS) in 5% CO2 at 37°C.10,11
Infection of THP-1 cells
In each experiment, THP-1 cells were washed with RPMI 1640, counted and seeded in 24-well tissue culture dishes at a density of 5 x 105 cells/well with RPMI 164010% FCS. Cell viability was >95% as determined by the trypan blue dye exclusion method. THP-1 cells were pretreated with 16 nM phorbol 12-myristate 13-acetate for 24 h in 5% CO2 at 37°C to induce maturation of the monocytes into macrophage-like adherent cells.10 L. pneumophila were added at a bacteria:cell ratio of 1:20 or 1:2 and spun on to monolayers at 600g for 10 min and then incubated for 50 min. Cells were washed twice with RPMI 1640 to remove extracellular bacteria. In experiments that evaluated the intracellular activity of antimicrobial agents, each antimicrobial agent was added in various concentrations at this point. In control samples (0 h after inoculation), the culture supernatant in each well (0.5 mL) was harvested and centrifuged at 2000g for 10 min to pellet extracellular bacteria. The pellet was resuspended in 0.5 mL of distilled water and cells adherent to the wells were disrupted by adding 0.5 mL distilled water over 15 min to harvest intracellular bacteria. This procedure did not alter the viability of bacteria.2 The number of viable bacteria in solutions was determined by colony counting on BCYE- agar after serial dilutions in distilled water. After incubation for 24 and 48 h in 5% CO2 at 37°C, colony numbers were counted in a similar manner.
Microscopic examination
To confirm intracellular survival and multiplication of L. pneumophila in THP-1 cells, THP-1 cells adherent to the plastic and/or floating in the culture supernatant were examined by light microscopy and transmission electron microscopy. For light microscopy, THP-1 cells were seeded in 24-well dishes as described above except that a plastic coverslip (Ø 13.5 mm; Celldesk R1, Sumitomo Bakelite Co., Tokyo, Japan) was placed on the bottom of the well.15 After incubation for 24 or 48 h, the coverslip was fixed with 4% paraformaldehyde in PBS and stained using the technique of Giménez.16 For electron microscopy, THP-1 cells infected with L. pneumophila were removed from the coverslips by a rubber cell scraper, fixed in 2.5% glutaraldehyde and treated with 1% OsO4 for 2 h.
Expression of results of intracellular activity assay of antimicrobial agents
The minimum extracellular concentration inhibiting intracellular multiplication (MIEC), the lowest concentration of the agent that reduced the total colony count (extra- plus intra-cellular) of L. pneumophila to <10% of the agent-free control at 24 h, is a concept reported previously3 for the evaluation of the intracellular activities of antimicrobial agents. To confirm that MIEC was applicable to our assay system using THP-1, we first examined the antimicrobial activities of four representative drugs, ciprofloxacin, erythromycin, minocycline and rifampicin, all of which are active against intracellular L. pneumophila. Results were expressed as the inhibition ratio (colony count of L. pneumophila with agent/colony count without agent x 100%) of extracellular, intracellular and total colony count of L. pneumophila. We also attempted to evaluate the intracellular activity of some other antimicrobial agents against L. pneumophila using MIEC.
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Results |
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The MICs and MBCs estimated using the broth microdilution method for L. pneumophila are shown in Table I. The most potent drug against both strains was rifampicin, followed by ciprofloxacin, grepafloxacin, clarithromycin and imipenem. Clindamycin was less potent than any other drug tested. The MBCs of rifampicin, ciprofloxacin, grepafloxacin and azithromycin were equal to or one dilution higher than their MICs, while those of the other drugs were much higher than their MICs.
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The growth curves of L. pneumophila SMUM-353 in RPMI 164010% FCS medium with and without THP-1 cells at two bacteria:cell ratios are shown in the Figure. The mean colony counts of L. pneumophila in the THP-1 culture supernatant (extracellular) and in the adherent cells (intracellular) are shown in the Figure in (a) and (b)
, respectively. The number of extracellular L. pneumophila at 0 h represented the number of bacteria remaining after double washing. L. pneumophila multiplied both intra- and extracellularly in the presence of THP-1 cells, increasing between 2 and 4 logs within 24 h. Although the extra-and intra-cellular L. pneumophila counts at 0 h were influenced by the bacteria:cell ratio, the final colony counts at 24 h were almost equal, c.106 cfu/mL at both ratios, and the counts remained stable at 48 and 72 h (106107 cfu/mL). Conversely, in the absence of THP-1 cells, L. pneumophila did not grow and gradually died in the medium RPMI 1640 10% FCS. Thus, the extracellular L. pneumophila were suspected to have originated from cells disrupted by multiplication of the bacteria. To examine this phenomenon, light microscopic examination using Giménez staining was performed.12,16 At the time of inoculation (0 h), several adherent THP-1 cells containing one or two bacteria were detected in the microscopic field, but no extracellular bacteria were seen. At 24 h after inoculation, intracellular growth of bacteria was seen in several adherent THP-1 cells. The number of THP-1 cells adherent on the plastic coverslip surface decreased and excessive numbers of floating disrupted cells appeared in the culture supernatant. These cells were disrupted by multiplication of bacteria and contained many bacteria. The number of disrupted THP-1 cells in the culture supernatant increased further at 48 h. These findings confirmed that extracellular L. pneumophila originated from disrupted THP-1 cells. Intracellular multiplication and destruction of THP-1 by L. pneumophila at 24 h were also confirmed by transmission electron microscopy.1,12
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Colony counts of L. pneumophila in the THP-1 culture supernatant (extracellular) and in the adherent cells (intracellular) were examined after 24 and 48 h treatment with ciprofloxacin, erythromycin, minocycline and rifampicin. Each drug was added after removing the extracellular bacteria (0 h), and all tests were performed at a bacteria:cell ratio of 1:20. All four drugs caused inhibition of the extra-, intra-cellular and total (extra- plus intra-cellular) growth of L. pneumophila when used at concentrations close to their MICs determined by the broth microdilution method (Table II). Furthermore, the activities of all four drugs were time- and concentration-dependent. In our assay system, at the concentrations at which the total inhibition ratio were <10% at 24 h, the extra-, intra-cellular and total inhibition ratios at 48 h with all four drugs were only c.1%, and at higher concentrations the ratios at 48 h were <1%. Thus the values lower than 10% at 24 h were considered as sufficient growth inhibition of L. pneumophila.
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The results in Table II show first, that the concept of MIEC, described above, was valid in our assay system using THP-1 cells, and secondly that each antimicrobial agent, at a concentration equal to its MIEC, inhibited intracellular growth of L. pneumophila at 24 and 48 h. Thus, we determined the MIEC of several other agents and compared them with their broth dilution MIC (Table I
). MIECs of three ß-lactamsampicillin, cefotiam and imipenemwere markedly higher than their MICs. The MIECs of other agents that are considered to be clinically effective, were almost the same as their MICs, although the MIEC of minocycline was relatively lower and that of rifampicin was higher than their respective MICs.
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Discussion |
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Vildé et al.3 defined the concept of the MIEC to evaluate the intracellular activities of antimicrobial agents against L. pneumophila using human monocyte-derived macrophages. The MIEC represented the lowest concentration of antimicrobial agent that caused 10% reduction in the total (extra- plus intra-cellular bacteria) number of L. pneumophila, compared with agent-free controls at 24 h. In their report they have shown that the definition of the MIEC was reasonable using statistical analysis. To simplify the assay, we determined whether the MIEC was applicable to our assay system using THP-1 cells. Since microscopic examination demonstrated that extracellular L. pneumophila originated from THP-1 cells disrupted by multiplication of bacteria, the total number of bacteria is available for determination of MIEC in this assay system. Furthermore, viability studies using four drugs indicated that each drug inhibited extra- and intra-cellular growth of L. pneumophila at 48 h (Table II) and even at 72 h (data not shown) at a higher concentration than the MIEC. Thus, 24 h incubation was a valid time-point for estimation of both intracellular activity of drugs and MIEC. Higa et al.20 used another definition of MIEC to evaluate the intracellular activities of drugs for L. pneumophila. In their report, the MIEC was defined as the minimum concentration of drugs resulting in 50% inhibition of the bacterial cytopathic effect. MIEC values of drugs in their report were similar to our results although they used a different definition of MIEC from ours. Consequently, the MIECs in this report are probably a reasonable and reliable indicator of the intracellular activity of drugs.
As the results of our study and those of previous reports indicate, the MIECs of the antimicrobial agents tested were not consistent with their conventional broth dilution MICs.24,20 The MIECs of ß-lactams were markedly higher than their MICs, while those of agents that are considered clinically effective were almost the same as their MICs. Moreover, even the MIECs of agents known to have good cell permeability were not always equivalent to their MIC values. MIECs are influenced by the drug permeability of the phagosome, the environment in the phagosome, for example pH, and the native antimicrobial mechanisms present in cells; we suggest that these may all contribute to discrepancies between MIEC and MIC values. Thus, to assess the effectiveness of drugs against pathogens, especially intracellular bacteria, assays of intracellular drug activity such as MIEC may be necessary in addition to conventional MIC determinations. Furthermore, assay systems using human-derived cells such as THP-1 are likely to be more relevant in the evaluation and prediction of antibiotic efficacy in humans than systems using cells derived from other species.20
Grepafloxacin is a new orally active and injectable fluoroquinolone with good intracellular permeability.4,13 In a previous report, grepafloxacin was found to inhibit the intracellular growth of L. pneumophila at a concentration twice its conventional MIC.4 In this study, we found that the MIEC of grepafloxacin was lower (25% of that of ciprofloxacin) although their broth dilution MICs were similar. These results were consistent with our finding that the intracellular concentration of grepafloxacin was two- to three-fold that of ciprofloxacin in this assay system (data not shown).
In conclusion, our assay system represents an excellent and useful method for the evaluation of antimicrobial activities of drugs against intracellular pathogens such as Legionella spp. However, further studies are required to refine the method of MIEC against intracellular pathogens as intracellular growth rates may vary.
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Notes |
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References |
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Higa, F., Kusano, N., Tateyama, M., Shinzato, T., Arakaki, N., Kawakami, K. et al. (1998). Simplified quantitative assay system for measuring activities of drugs against intracellular Legionella pneumophila. Journal of Clinical Microbiology 36, 13928.
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Sato, K. & Tomioka, H. (1999). Antimicrobial activities of benzoxazinorifamycin (KRM-1648) and clarithromycin against Mycobacterium aviumintracellulare complex within murine peritoneal macrophages, human macrophage-like cells and human alveolar epithelial cells. Journal of Antimicrobial Chemotherapy 43, 3517.
Received 31 January 2000; returned 4 May 2000; revised 25 May 2000; accepted 28 June 2000