Antibacterial activities of gemifloxacin, levofloxacin, gatifloxacin, moxifloxacin and erythromycin against intracellular Legionella pneumophila and Legionella micdadei in human monocytes

Aldona L. Baltch1,2,*, Lawrence H. Bopp1, Raymond P. Smith1,2, Phyllis B. Michelsen1,2 and William J. Ritz1,2

1 Infectious Disease Section, Stratton VA Medical Center, Albany, NY, USA; 2 Albany Medical College, Albany, NY, USA


* Correspondence address. Infectious Disease Research, 111D, Stratton VA Medical Center, 113 Holland Avenue, Albany, NY 12208, USA. Tel: +1-518-626-6416; Fax: +1-518-626-6564; E-mail: aldona.baltch{at}med.va.gov

Received 10 December 2004; returned 22 February 2005; revised 10 March 2005; accepted 9 May 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The antibacterial activity of a new fluoroquinolone, gemifloxacin, was tested against intracellular Legionella pneumophila and Legionella micdadei and was compared with the activities of levofloxacin, gatifloxacin, moxifloxacin and erythromycin.

Methods: For intracellular assays, bacteria were used to infect human monocyte-derived macrophages prepared from heparinized blood of healthy volunteers. Antibiotics were added following phagocytosis. Numbers of viable bacteria were determined at 0, 24, 48, 72 and 96 h.

Results: The intracellular antibacterial activity of gemifloxacin was concentration- and time-dependent. All of the quinolones had similar activities against L. pneumophila and L. micdadei at 10 x MIC, but there were minor differences: at 24 h moxifloxacin was significantly more active than the other quinolones against L. pneumophila, while gemifloxacin was more active against L. micdadei (P < 0.01). All of the quinolones were markedly more active than erythromycin (P < 0.01). The antibacterial effect of gemifloxacin against L. pneumophila following drug removal at 24 h persisted for 72 h at 20 x MIC but not at 10 x MIC, while for L. micdadei the antibacterial effect persisted for 24 h at 10 x MIC.

Conclusions: All of the quinolones had similar activities against intracellular L. pneumophila and L. micdadei and were markedly more effective than erythromycin.

Keywords: Legionella spp. , in vitro models , fluoroquinolones , macrolides , phagocytes


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although the most frequent cause of Legionnaires' disease is Legionella pneumophila serogroup 1, it is well known that Legionella micdadei can also be a cause of community-acquired and nosocomial pneumonia.13 The mortality rate associated with these infections continues to be high, especially in immunocompromised and intubated patients in intensive care units.1,4,5 Legionella spp. are intracellular pathogens that survive and multiply in human macrophages;68 therefore, antimicrobial agents must have intracellular activity in order to be useful for therapy of legionella infections.813 Until recently, infections caused by Legionella spp. were treated with erythromycin and, in patients failing therapy, with added rifampicin.4,9,14 Quinolones are able to enter phagocytic cells and in vitro studies have demonstrated that they are highly active against Legionella spp.9,11,13,14 Therefore, they may be preferred for treatment of infections caused by these bacteria.

Gemifloxacin (SB-265805, LB20304a) is a new pyrrolidine-type fluoroquinolone antibacterial agent with enhanced affinity for topoisomerase IV and broad-spectrum activity against Gram-positive and Gram-negative pathogens, including anaerobes and organisms causing respiratory tract infections.1519 Furthermore, gemifloxacin is active against Legionella spp., Mycoplasma pneumoniae and Chlamydia pneumoniae.16,2023

In this report, the effect of increasing concentrations of gemifloxacin on intracellular L. pneumophila and L. micdadei in human monocytes is described. The intracellular activity of gemifloxacin was compared with the activities of erythromycin and three other quinolones (levofloxacin, gatifloxacin and moxifloxacin). The activities of gemifloxacin and erythromycin were also compared following removal of these antimicrobials from the assay system.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

L. pneumophila strain L-1033 (serogroup 1) and L. micdadei strain 99-4164, both low-passage clinical isolates, were obtained from the Wadsworth Center, New York State Department of Health, Albany, NY, USA. Both strains are positive for the mip gene by PCR.24 From stock cultures frozen at –70°C in 20% glycerol, subcultures were made on buffered charcoal yeast extract agar supplemented with 5% {alpha}-ketoglutarate (BCYE{alpha}; BBL Microbiology Systems, Cockeysville, MD, USA) and incubated at 35°C. Before each experiment, several colonies from a 48 h culture were subcultured from BCYE{alpha} agar to buffered yeast extract (BYE) broth, which was prepared with 10 g/L yeast extract, 10 g/L ACES, 1 g/L {alpha}-ketoglutarate, 0.25 g/L ferric pyrophosphate and 0.4 g/L L-cysteine hydrochloride (all from Sigma Chemical Co., St Louis, MO, USA), adjusted to pH 6.90, and filter sterilized. BYE broth was supplemented with 0.1% (w/v) bovine serum albumin (BSA) (Sigma) for growth of L. micdadei, since growth of this strain in broth was minimal without the addition of BSA. Cultures were then incubated at 35°C for 18 h in a shaking water bath. Cells were diluted to 1 x 107 cfu/mL (L. pneumophila) and 1 x 108 cfu/mL (L. micdadei) in RPMI 1640 medium (Sigma) containing 10% fetal bovine serum (RPMI+). Final counts of the diluted inocula were determined in duplicate using the standard plate count method and BCYE{alpha} agar. Plates were incubated for from 48 to 72 h at 35°C before colonies were counted.

Antimicrobial agents

Standard powders of the antimicrobial agents were obtained from the following sources: gemifloxacin, GlaxoSmithKline (Harlow, UK); levofloxacin, RW Johnson Pharmaceutical Research Institute (Raritan, NJ, USA); gatifloxacin, Bristol-Myers Squibb (Princeton, NJ, USA); moxifloxacin, Bayer AG (West Haven, CT, USA); erythromycin, Sigma. Antibiotic solutions were made fresh for each experiment as directed by the supplier, filter-sterilized (0.45 µm pore size) and used on the same day.

Antimicrobial susceptibility testing

MIC determinations were made in BYE broth using the macrotube dilution method.25,26 For L. micdadei, broth was supplemented with 0.1% BSA and the inoculum was carefully adjusted so that the starting cell density was 5 x 105 cfu/mL.27 Growth was evaluated at 24 and 48 h (L. pneumophila) and 48 and 72 h (L. micdadei), and each MIC determination was repeated three to five times. Controls were Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212. The MICs (mg/L) for L. pneumophila strain L-1033 were as follows: gemifloxacin, 0.016; levofloxacin, 0.016; gatifloxacin, 0.016; moxifloxacin, 0.03; and erythromycin, 0.5. For L. micdadei strain 99-4164 the MICs (mg/L) were: gemifloxacin, 0.008; levofloxacin, 0.016; gatifloxacin, 0.016; moxifloxacin, 0.016; and erythromycin, 0.5.

Preparation of human monocytes

Monocytes were prepared from heparinized blood of healthy human donors who had signed an informed consent form approved by the Institutional Review Board of the Stratton VA Medical Center, Albany, NY, USA. Mononuclear cells were separated from whole blood using Histopaque 1077 (Sigma), yielding a ≥98% pure mononuclear cell preparation. The separated mononuclear cells were resuspended in RPMI 1640+ to a concentration of 2 x 106 cells/mL. Cell viability was >98% as determined by the Trypan Blue dye exclusion test.

Intracellular assay

Aliquots (1 mL) of a human mononuclear cell suspension at a density of 2 x 106 cell/mL were delivered to the wells of 24-well plates (Corning/Costar Corp., Cambridge, MA, USA) and allowed to adhere for 2 h. Monocytes adhered to the wells in contiguous monolayers. Non-adherent cells, including lymphocytes, were then removed. From this point, the adhered cells were considered to be monocyte-derived macrophages (MDM). The adherent and contiguous monolayer was gently washed once with RPMI 1640+. Bacteria suspended in RPMI+ (1 mL at 1 x 107 cfu/mL for L. pneumophila and 1 mL at 1 x 108 cfu/mL for L. micdadei) were then added to the wells. A higher multiplicity of infection was used for L. micdadei (which is phagocytosed less efficiently than L. pneumophila in our in vitro model) in order to obtain an adequate number of intracellular bacteria following phagocytosis and washing. After allowing 1 h for phagocytosis, the medium was removed and the monolayer was gently washed once with RPMI+. Additional washing (up to three times) did not further decrease the number of bacteria in the well. Following phagocytosis and washing, 5 x 104 to 1 x 105 intracellular bacteria/well remained. Antimicrobials were then added at desired concentrations to duplicate wells. The plates were then incubated at 37°C in a 5% CO2 atmosphere. At each time point, (0, 24, 48, 72 and 96 h) the supernatants were removed by aspiration. In order to lyse the MDM, 1 mL of sterile distilled water was added to each well and the plate was incubated for 5 min at room temperature. Adherent material was then removed from the monolayers by scraping with a sterile plastic transfer pipette, after which the contents of the wells were transferred to test tubes and the lysis procedure described above was repeated. Lysates were then mixed vigorously by vortexing for 15 s. Complete lysis of the MDM was verified microscopically. Viable bacteria in the lysates were enumerated in duplicate on BCYE{alpha} agar using the standard plate count method. The limit of detection was 20 cfu/mL. A subset of experiments involved removal of the antibiotics 24 h after their addition. In these experiments, drug retention and drug removal wells were run in parallel. For the drug removal wells, supernatants were removed by aspiration from all wells (whether or not they contained antibiotics) at 24 h and replaced with fresh RPMI+ containing no antibiotics. The wells in these experiments were then processed at 48, 72 and 96 h after infection as described above. Experiments were performed three times for each assay condition.

Statistical analyses

Statistical analysis utilized analysis of variance methodology.28 Analyses were performed using the bacterial counts (log10 cfu/mL) of lysed MDM at 24, 48, 72 or 96 h minus the bacterial counts of lysed MDM at 0 h (immediately following phagocytosis, washing and addition of antibiotics). Results are expressed as percentages of the geometric mean counts at 0 h (geometric mean numbers of cfu/mL at 0, 24, 48, 72 and 96 h divided by the corresponding values at 0 h, multiplied by 100). The level of significance was 0.01.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Figure 1 shows the intracellular activity of gemifloxacin at 0.1, 0.25, 0.5, 1, 5 and 10 x MIC against L. pneumophila L-1033 (Figure 1a) and L. micdadei 99-4164 (Figure 1b). Increasing the gemifloxacin concentration from 0.1 to 10 x MIC increased its intracellular activity against L. pneumophila and L. micdadei (P < 0.01). At 0.1 x MIC, gemifloxacin had minimal but significant activity against L. pneumophila (P < 0.01). However, at gemifloxacin concentrations from 0.25 to 10 x MIC, the numbers of viable intracellular bacteria were markedly reduced. At 10 x MIC, there were >3 log10 units fewer viable bacteria than at 0.1 x MIC. The number of viable intracellular bacteria decreased gradually from 0 to 72 h. In contrast to the activity against L. pneumophila, the activity of gemifloxacin against L. micdadei increased quite uniformly as the concentration of the antibiotic increased from 0.1 to 10 x MIC. These results differ from those observed for L. pneumophila, where intracellular killing increased dramatically when the antibiotic concentration was increased from 0.1 to 0.25 x MIC.



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Figure 1. Time–kill curves demonstrating in vitro activity of gemifloxacin against (a) intracellular L. pneumophila L-1033 and (b) intracellular L. micdadei 99-4164. No antibiotic (filled circles); 0.1 x MIC (open circles); 0.25 x MIC (filled inverted triangles); 0.5 x MIC (open inverted triangles); 1 x MIC (filled squares); 5 x MIC (open squares); 10 x MIC (filled diamonds). Coefficients of variation (SEM divided by the geometric mean) ranged from 0.1 to 0.27.

 
Figure 2 shows the activities of gemifloxacin, levofloxacin, gatifloxacin, moxifloxacin and erythromycin at 10 x MIC against intracellular L. pneumophila (Figure 2a) and intracellular L. micdadei (Figure 2b). The activities of the quinolones were similar to one another with two exceptions. First, at 24 h moxifloxacin was significantly more active against L. pneumophila than the other quinolones (P < 0.01). Otherwise the activities of the quinolones against L. pneumophila varied by <0.5%. Secondly, at 24 h gemifloxacin was significantly more active than the other quinolones against L. micdadei (P < 0.01). For both L. pneumophila and L. micdadei, there were so few surviving bacteria at 48 and 72 h that the differences in activity between the quinolones were no longer meaningful. For both L. pneumophila and L. micdadei, all of the quinolones were significantly more active than erythromycin at 24, 48 and 72 h (P < 0.01).



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Figure 2. Time–kill curves comparing the in vitro activities of gemifloxacin, other quinolones and erythromycin at 10 x MIC against (a) intracellular L. pneumophila strain L-1033 and (b) intracellular L. micdadei strain 99-4164. No antibiotic (filled circles); gemifloxacin (open circles); levofloxacin (filled inverted triangles); gatifloxacin (open inverted triangles); moxifloxacin (filled squares); erythromycin (open squares). Coefficients of variation ranged from 0.08 to 0.69.

 
Figure 3 shows the activities of gemifloxacin and erythromycin against intracellular L. pneumophila and L. micdadei following antibiotic removal 24 h after infection and addition of antibiotic. The effects of antibiotic removal (10 x MIC) were similar for both L. pneumophila and L. micdadei (Figure 3a and b). Removal of either antibiotic resulted in a decrease in the rate of bacterial death and, in some cases, resumption of growth (P < 0.01). In contrast, the rates of bacterial death were relatively constant throughout the course of the experiments if the antibiotics were not removed. Consequently, there were significantly more viable bacteria at 96 h post-infection when antibiotics were removed than when they were not (P < 0.01). Antibiotic removal experiments at 20 x MIC were conducted only for L. pneumophila. In contrast to the results obtained at 10 x MIC, removal of 20 x MIC of gemifloxacin from the assay did not decrease the rate of bacterial killing, while removal of erythromycin under the same conditions resulted in regrowth of the organism (Figure 3c; P < 0.01).



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Figure 3. Time–kill curves comparing the in vitro activities of gemifloxacin and erythromycin, with or without antibiotic removal 24 h post-infection/antibiotic addition, against intracellular Legionella spp. No antibiotic (filled circles); no antibiotic, mock drug removal (open circles); erythromycin (filled inverted triangles); erythromycin with drug removal (open inverted triangles); gemifloxacin (filled squares); gemifloxacin with drug removal (open squares). (a) L. pneumophila strain L-1033: antibiotics at 10 x MIC; (b) L. micdadei strain 99-4164: antibiotics at 10 x MIC; (c) L. pneumophila strain L-1033: antibiotics at 20 x MIC. Coefficients of variation ranged from 0.08 to 0.1

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The extracellular activity of gemifloxacin against Legionella spp. has been studied,16 and there is one report of its intracellular activity against L. pneumophila in guinea pig alveolar macrophages.29 However, to our knowledge ours is the first study comparing the activities of gemifloxacin, other quinolones and erythromycin against intracellular L. pneumophila and L. micdadei in freshly prepared human MDM.

Gemifloxacin had concentration-dependent antibacterial activity intracellularly against both L. pneumophila and L. micdadei. However, some important differences were observed. First, although antibacterial activity against L. pneumophila was clearly concentration-dependent, the relationship between increased inhibition and/or killing and concentration was not linear. There was a substantial decrease in the number of viable intracellular bacteria when the gemifloxacin concentration was increased from 0.1 to 0.25 x MIC (~2 log10 units), compared with a further decrease of only 1.5 log10 units as the gemifloxacin concentration was increased from 0.25 to 10 x MIC. Similar results were seen for L. micdadei as the gemifloxacin concentration was raised from 0.25 to 0.5 x MIC. However, for L. micdadei the magnitude of the difference between 0.25 and 0.5 x MIC was much smaller (~1 log10 unit) than that observed for L. pneumophila between 0.1 and 0.25 x MIC. These results suggest that caution should be taken in making assumptions about the linearity of the antibacterial response to increasing concentrations of antibiotic, especially in complex situations where bacterial growth is primarily or exclusively intracellular. Secondly, L. pneumophila grew intracellularly in untreated MDM, generally increasing in number by 1–2 log10 units during the first 48 h of the assay. This rate of growth is slower than that reported in some cell types29,30 and greater than that reported in others.31 We have found that the intracellular growth rate of L. pneumophila is not only host cell-dependent, but within MDM is L. pneumophila strain-dependent. It is also important to remember that L. pneumophila L-1033 does not grow extracellularly in the RPMI+ medium used to sustain the MDM, so all increases in numbers of viable bacteria are the result of intracellular growth in the MDM. In contrast to L. pneumophila, the number of viable intracellular L. micdadei decreased intracellularly in untreated MDM (P < 0.01). This was unexpected, since monocytes/MDM are not reported to be able to kill L. micdadei. However, we have tested by pulsed-field gel electrophoresis DNA fingerprinting three genetically distinct low-passage clinical isolates of L. micdadei, including the strain used for this study, and have found that for each there is a gradual decline in the number of viable intracellular bacteria for at least 72 h after phagocytosis. In contrast, for a high-passage, laboratory-adapted strain (ATCC 33204), the rate of intracellular die-off is substantially greater (L.H. Bopp, A.L. Baltch and R.P. Smith, unpublished results). For each of the four L. micdadei strains examined, the death rate in MDM in the presence of RPMI+ medium was greater than the corresponding death rate in RPMI+ medium alone.

At 10 x MIC, the antibacterial activities of the quinolones against L. pneumophila were similar, although there were some minor differences at 24 h, and significantly greater than that of erythromycin (P < 0.01). Similar results were seen for L. micdadei at 10 x MIC. It is important to remember that at these concentrations, where the quinolones were clearly superior to erythromycin, the quinolone concentrations were still only 0.08–0.3 mg/L, while the erythromycin concentration was 5 mg/L. Since quinolone concentrations ≥100 x MIC for these organisms are readily attainable in human serum, further investigation of this phenomenon at higher quinolone concentrations seems warranted.

Antibiotic removal assays at 10 x MIC for both L. pneumophila and L. micdadei showed that upon removal of the antibiotic, the intracellular bacteria recovered. Growth or death rates following removal of both erythromycin and gemifloxacin resembled those of the corresponding untreated controls. For L. pneumophila, when the antibiotic concentration was increased to 20 x MIC the bacteria recovered after removal of erythromycin. However, following removal of gemifloxacin at 20 x MIC, the rate of bacterial death continued to be the same as when the drug was not removed for an additional 72 h. These results suggest that, even though drug concentrations in excess of 10 x MIC may not be necessary to achieve satisfactory intracellular killing of L. pneumophila, they may be useful for prolonging the post-antibiotic effect and allowing alteration of the dosing schedule in treatment of legionella infections.16,32

In conclusion, killing of intracellular L. pneumophila and L. micdadei in MDM was dependent upon drug concentration. All of the quinolones tested had similar activities (with minor differences) and were more active than erythromycin.


    Acknowledgements
 
This work was supported by GlaxoSmithKline Pharmaceuticals and, in part, by the US Department of Veterans Affairs.


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 Materials and methods
 Results
 Discussion
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