Evaluation of a human monocytic cell line THP-1 model for assay of the intracellular activities of antimicrobial agents against Legionella pneumophila

Hiromu Takemura*, Hiroyuki Yamamoto, Hiroyuki Kunishima, Hideaki Ikejima, Takashi Hara, Keiji Kanemitsu, Shigemi Terakubo, Yoko Shoji, Mitsuo Kaku and Jingoro Shimada

Department of Microbiology, St Marianna University School of Medicine, 2-16-1 Sugao Miyamae-ku, Kawasaki 216-8511, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We examined the intracellular activities of 11 antimicrobial agents against Legionella pneumophila using a human monocyte-derived cell line, THP-1. Colony counting and microscopic examination of L. pneumophila co-incubated with THP-1 cells (5 x 105 cells/well) were performed. Both extra- and intra-cellular multiplication of L. pneumophila were observed and were dependent on the inoculum of L. pneumophila in the culture; L. pneumophila did not grow in the cell culture medium alone. Light microscopic examination confirmed that extracellular L. pneumophila originated from THP-1 cells disrupted by bacterial multiplication. L. pneumophila multiplied by 3–4 logs after 24 h incubation with THP-1 cells and their number remained stable at 106–107 cfu/mL until 72 h. The results of viability studies using four antimicrobial agents—ciprofloxacin, erythromycin, minocycline and rifampicin—demonstrated that our system was suitable for the intracellular activity assay. We used a concept of ‘minimum extracellular concentration inhibiting intracellular multiplication’ (MIEC) to evaluate the intracellular activity of antimicrobial agents. The MIECs of three ß-lactams were markedly higher than their conventional MICs while those of macrolides, quinolones, rifampicin and minocycline were similar to their MICs. Our results suggest that evaluation of the clinical efficacy of drugs against L. pneumophila should include determination of their intracellular activity against the bacteria, which could be measured using our assay system in THP-1 cells.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Legionella pneumophila, the causative organism of legionnaires' disease, multiplies intracellularly within the phagosomes of human monocytes and macrophages.13 The activity of certain antimicrobial agents against intracellular L. pneumophila is minimal, for example ß-lactams, because of poor penetration into the phagosome.24 Furthermore, clinical observations,5 case reports6 and the results of in vivo studies2,7 suggest that effective treatment of Legionnaires' disease requires the use of drugs that are effective against both extra- and intra-cellular bacteria. While tests are available to evaluate the intracellular activity of drugs against Legionella spp., the application of these tests may be difficult in many clinical laboratories, since they require the collection of cells from humans or animals, for example human peripheral monocytes,2,3 or guinea pig peritoneal4 and alveolar7,8 macrophages.

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{alpha}, 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


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria

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 {alpha}-ketoglutarate (BCYE-{alpha}; 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 {alpha}-ketoglutarate (BYE-{alpha}; Difco Laboratories) and incubated for 24 h at 35°C on a shaker. BYE-{alpha} 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 {alpha}-ketoglutarate (Wako Ltd, Osaka, Japan), and BCYE-{alpha} 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-{alpha} broth.14 For this purpose, 5 µL of a BYE-{alpha} broth containing L. pneumophila (1 x 107/mL) was added to 0.1 mL of BYE-{alpha} 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-{alpha} 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 1640–10% 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 1640–10% 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-{alpha} 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MICs and MBCs of antimicrobial agents for L. pneumophila

The MICs and MBCs estimated using the broth microdilution method for L. pneumophila are shown in Table IGo. 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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Table I. MICs, MBCs and MIECs of antimicrobial agents for Legionella pneumophila serogroup 1
 
Growth of L. pneumophila associated with THP-1 cells

The growth curves of L. pneumophila SMUM-353 in RPMI 1640–10% FCS medium with and without THP-1 cells at two bacteria:cell ratios are shown in the FigureGo. 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)Go, 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 (106–107 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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Figure. Growth of Legionella pneumophila SMUM-353 in RPMI 1640–10% FCS alone ({blacktriangleup}) and in THP-1 cells at a bacteria:cell ratio of 1:2 ({circ}), 1:20 (•). (a) Extracellular L. pneumophila; number of L. pneumophila in the culture supernatant. (b) Intracellular L. pneumophila; number of L. pneumophila in adherent THP-1 cells. The detection limit in this assay system is 50 cfu/mL. Data represent the mean ± s.d. of three independent experiments.

 
Antimicrobial activity of agents against intracellular L. pneumophila

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 IIGo). 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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Table II. Effects of antimicrobial agents on extra- and intra-cellular multiplication of Legionella pneumophilaa
 
Evaluation of intracellular activity of antimicrobial agents by MIECs

The results in Table IIGo 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 IGo). MIECs of three ß-lactams—ampicillin, cefotiam and imipenem—were 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.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we established a THP-1 Legionella infection model for evaluation of intracellular activities of antimicrobial agents against L. pneumophila multiplying intracellularly. Similar studies have been reported previously by other investigators using human peripheral monocytes,2 macrophages,3 or guinea pig peritoneal4 or alveolar macrophages.7,8 Although monocytes or macrophages can be collected from humans or animals, we believe that an assay system using an established cell line is more suitable to simplify and standardize laboratory testing. Previous studies1720 have also used certain cell lines of the monocyte –macrophage lineage, such as U93717 and HL-6018 to evaluate the intracellular activities of drugs for L. pneumophila. Sato & Tomioka21 have examined the intracellular antimicrobial activities of drugs for Mycobacterium avium–intracellulare complex using THP-1 cells,21 although we are not aware of any similar published studies with L. pneumophila. In this study, the results of viability determinations using four drugs—ciprofloxacin, erythromycin, mino-cycline and rifampicin—for L. pneumophila co-incubated with THP-1, were in agreement with those reported previously.2,3,20

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 IIGo) 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.


    Notes
 
* Corresponding author. Tel: +81-44-977-8111; Fax: +81-44-977-7818; E-mail: takeh{at}marianna-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Horwitz, M. A. & Silverstein, S. C. (1980). Legionnaires' disease bacterium (Legionella pneumophila) multiplies intracellularly in human monocytes. Journal of Clinical Investigation 66, 441–50.[ISI][Medline]

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11 . Fujii, T., Kadota, J., Morikawa, T., Matsubara, Y., Kawakami, K., Iida, K. et al. (1996). Inhibitory effect of erythromycin on interleukin 8 production by 1-alpha, 25-dihydroxyvitamin D3-stimulated THP-1 cells. Antimicrobial Agents and Chemotherapy 40, 1548–51.[Abstract]

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13 . Ridgway, G. L., Salman, H., Robbins, M. J., Dencer, C. & Felmingham, D. (1997). The in-vitro activity of grepafloxacin against Chlamydia spp., Mycoplasma spp., Ureaplasma urealyticum and Legionella spp. Journal of Antimicrobial Chemotherapy 40, Suppl. A, 31–4.[Abstract/Free Full Text]

14 . Liebers, D. M., Baltch, A. L., Smith, R. P., Hammer, M. C. & Conroy, J. V. (1989). Susceptibility of Legionella pneumophila to eight antimicrobial agents including four macrolides under different assay conditions. Journal of Antimicrobial Chemotherapy 23, 37–41.[Abstract]

15 . Nakagawara, A. & Nathan, C. F. (1983). A simple method for counting adherent cells: application to cultured human monocytes, macrophages and multinucleated giant cells. Journal of Immunological Methods 56, 261–8.[ISI][Medline]

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17 . Ramirez, J. A., Summersgill, J. T., Miller, R. D., Meyers, T. L. & Raff, M. J. (1993). Comparative study of the bactericidal activity of ampicillin/sulbactam and erythromycin against intracellular Legionella pneumophila. Journal of Antimicrobial Chemotherapy 32, 93–9.[Abstract]

18 . Stout, J. E., Arnold, B. & Yu, V. L. (1998). Activity of azithromycin, clarithromycin, roxithromycin, dirithromycin, quinupristin/ dalfopristin and erythromycin against Legionella species by intracellular susceptibility testing in HL-60 cells. Journal of Antimicrobial Chemotherapy 41, 289–91.[Abstract]

19 . Stokes, D. H., Wilkinson, M. J., Tyler, J., Slocombe, B. & Sutherland, R. (1989). Bactericidal effects of amoxycillin/clavulanic acid against intracellular Legionella pneumophila in tissue culture studies. Journal of Antimicrobial Chemotherapy 23, 547–56.[Abstract]

20 . 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, 1392–8.[Abstract/Free Full Text]

21 . Sato, K. & Tomioka, H. (1999). Antimicrobial activities of benzoxazinorifamycin (KRM-1648) and clarithromycin against Mycobacterium avium–intracellulare complex within murine peritoneal macrophages, human macrophage-like cells and human alveolar epithelial cells. Journal of Antimicrobial Chemotherapy 43, 351–7.[Abstract/Free Full Text]

Received 31 January 2000; returned 4 May 2000; revised 25 May 2000; accepted 28 June 2000