Comparison of the antibacterial activities of ampicillin, ciprofloxacin, clarithromycin, telithromycin and quinupristin/dalfopristin against intracellular non-typeable Haemophilus influenzae

Irini Lazou Ahrén, Eva Karlsson, Arne Forsgren and Kristian Riesbeck*

Department of Medical Microbiology, University Hospital Malmö, Lund University, S-205 02 Malmö, Sweden

Received 25 March 2002; returned 28 May 2002; revised 16 August 2002; accepted 27 August 2002


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Non-typeable Haemophilus influenzae, which is a cause of disease in the upper and lower respiratory tract, can survive intracellularly in human epithelial cells and macrophages. We studied the in vitro activity of five antibiotics against intracellular non-typeable H. influenzae in human type II alveolar epithelial cells. The eukaryotic cells were loaded with bacteria, and extracellular bacteria were killed by gentamicin. After the cells were washed, antibiotics were added at concentrations of 0.12–64 mg/L for 18 h before the numbers of viable intracellular bacteria were determined. Of the antibiotics tested, ciprofloxacin and quinupristin/dalfopristin were the most potent agents, followed by clarithromycin and telithromycin. Ampicillin was not active against intracellularly localized, non-typeable H. influenzae.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In recent years, it has been shown that non-typeable Haemophilus influenzae can invade epithelial cells and macrophages and, in addition, survive intracellularly.1,2 It has also been demonstrated experimentally that H. influenzae, localized in epithelial cell layers, is shielded from antibiotic activity.3 Furthermore, the intracellular localization of non-typeable H. influenzae might serve as a reservoir, promoting recurrent infections in otitis-prone children and in patients with chronic obstructive pulmonary disease (COPD).4,5 Therefore, it is of interest to elucidate the intracellular antibacterial activities of recently developed antibiotics.

Penicillins have repeatedly been shown to poorly penetrate eukaryotic cells. In contrast, ciprofloxacin penetrates and is bactericidal intracellularly. In spite of the low in vitro antibacterial activity of clarithromycin and telithromycin against non-typeable H. influenzae, as measured by bactericidal (MBC) and bacteriostatic (MIC) concentrations, good in vivo activities have been reported for these drugs in the treatment of community-acquired pneumonia caused by non-typeable H. influenzae.6,7 Intracellular accumulation is a hallmark of these drugs, and consequently intracellular antibacterial activity has been proposed as a major factor for bacterial eradication. Treatment with quinupristin/dalfopristin is approved for use in patients with nosocomial pneumonia, skin and soft tissue infections, and clinically significant infections due to vancomycin-resistant Enterococcus faecium.8 In vitro, quinupristin/dalfopristin has a moderate effect on non-typeable H. influenzae, partly in parallel with clarithromycin and telithromycin, but is accumulated intracellularly.8 The goal of the present investigation was to study the intracellular effect against non-typeable H. influenzae of clarithromycin, telithromycin and quinupristin/dalfopristin in comparison with ciprofloxacin and ampicillin in vitro.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibacterial agents

Ciprofloxacin (Bayer, Wuppertal, Germany), clarithromycin (Abbot, Queensborough, UK), quinupristin/dalfopristin and telithromycin (Aventis Pharma,Vitry sur Seine, France) were studied. Ampicillin (Astra, Södertälje, Sweden) was used as control, and gentamicin (Schering-Plough, Stockholm, Sweden) was applied for the killing of all the extracellular bacteria in the experimental procedure. All antibiotics supplied as powders, except for gentamicin (which was obtained as a soluble preparation), were solubilized according to the manufacturers’ instructions and further diluted in RPMI 1640 medium (Gibco-BRL, Paisley, UK) supplemented with HEPES buffer and 5% fetal calf serum (FCS). MICs were determined using a broth dilution method. Each antibiotic was serially diluted in 2 mL of RPMI 1640 supplemented with HEPES buffer and FCS. Bacteria (105 cfu) were resuspended in 1 mL of phosphate-buffered saline (PBS), and 100 µL was added to each tube containing antibiotics followed by incubation at 37°C and 5% CO2 for 18 h. MBCs were determined by culturing 100 µL of each dilution on chocolate agar plates. All drugs were tested at final concentrations ranging from 0.12 to 64 mg/L.

Bacteria, epithelial cells and culture conditions

The non-typeable H. influenzae strain 6-9547 was a nasopharyngeal isolate from our department as described previously.1 Bacteria were grown in brain–heart infusion broth (Difco, Detroit, MI, USA) supplemented with nicotinamide adenine dinucleotide and haemin (both at 10 mg/L). In all experiments, bacteria were grown to stationary phase overnight at 37°C with shaking, followed by 1/100 dilution in fresh pre-warmed medium. The bacteria were further incubated at 37°C until OD600 reached 0.4. The human lung carcinoma epithelial cell line A-549 (CCL-185) was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in RPMI 1640 supplemented with HEPES buffer, 5% FCS, 2 mM L-glutamine and gentamicin at a concentration of 12 mg/L (referred to as ‘culture medium’) at 37°C and 5% CO2. Cells were added to 24-well culture plates (Nunc, Roskilde, Denmark) the day before the experiment (0.5 mL with 1 x 105 cells was added per well). On the day of the experiments, cells (now expanded to 2 x 105 per well) were washed twice in gentamicin-free RPMI 1640.

Internalization assay

To examine bacterial invasion, a modified method that we described recently was used.1 Bacteria (10 µL of 1–5 x 109 cfu/mL) were added to each well containing 2 x 105 cells in gentamicin-free culture medium followed by incubation at 37°C and 5% CO2 for 3 h. To kill the extracellular bacteria, gentamicin was added at a final concentration of 100 mg/L. After 2 h of gentamicin treatment, cells containing 80 x 103 bacteria were washed twice with PBS and either harvested or incubated with the indicated antibiotics at concentrations ranging from 0.12 to 64 mg/L for an additional 18 h. The addition of the antibacterial agents did not change the pH (7.2–7.4) in the cell cultures. To harvest cells and bacteria, cells were washed twice with PBS, detached by 0.25% trypsin-versene, resuspended in 1 mL of PBS and transferred to a glass tube containing eight glass pearls. Trypsin-versene did not interfere with bacterial survival. All cells were mechanically lysed by vigorous vortexing for 1 min. To determine the number of viable intracellular bacteria, 10 and 100 µL of the lysates were plated on chocolate agar plates. Colony forming units (cfu) were counted after 24 h of incubation at 37°C. The lower limit of detection was 30 cfu per 2 x 105 cells.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MICs and MBCs of the antibacterial agents

The non-typeable H. influenzae strain 6-9547 was susceptible to all antibiotics tested including ampicillin with MBCs 4- to 16-fold higher than the MICs (Table 1). For comparison, peak serum concentrations and cellular and extracellular (C/E) ratios for the various antibiotics have been included in Table 1.913 Peak serum concentrations were significantly above the estimated MBCs for ampicillin and ciprofloxacin, whereas Cmax for clarithromycin, quinupristin/dalfopristin and telithromycin was below the MBC.


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Table 1.  Extracellular MICs and MBCs, peak serum concentrations, intracellular/extracellular (C/E) ratios and concentrations required for intracellular killing of bacteria for antibiotics against H. influenzae 6-9547
 
Antibacterial activity against intracellularly residing non-typeable H. influenzae

To determine the intracellular activities of the selected antibacterial agents, A-549 epithelial cells (2 x 105) were loaded with non-typeable H. influenzae. A multiplicity of infection (MOI) of 100 bacteria per mammalian cell was used and resulted in an invasive capacity of 1.6% of the inoculum.1 Extracellular bacteria were killed by gentamicin, and the remaining intracellular bacteria were incubated in the absence or presence of antibiotic. After 18 h, control epithelial cells incubated without any additional antimicrobial agent contained ~3500 viable bacteria.

As shown in Figure 1(a), ampicillin at concentrations of 0.12–64 mg/L killed only ~50% of the initially remaining bacteria. In contrast, ciprofloxacin and quinupristin/dalfopristin at concentrations of 1 mg/L killed 95% of the intracellular non-typeable H. influenzae (Figure 1, Table 1). Considerably higher concentrations of these two drugs were required (32 and 8 mg/L, respectively) to reach 99% killing of non-typeable H. influenzae. Clarithromycin and telithromycin were less potent in killing intracellular non-typeable H. influenzae. Concentrations of 8–16 mg/L of clarithromycin and telithromycin were required to kill 95% of the intracellular bacteria, whereas 64 and 16 mg/L, respectively, killed 99%.



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Figure 1. Intracellular survival of non-typeable H. influenzae in the presence of (a) ampicillin, ciprofloxacin and clarithromycin and (b) quinupristin/dalfopristin and telithromycin. Mean values ± S.D. are shown for two separate experiments. After 18 h, control cells without any antibacterial agents harboured 3552 ± 70 bacteria. Duplicates were included at all concentrations. All drugs were tested in parallel in the same experiments.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Non-typeable H. influenzae is a pathogen associated with diseases such as otitis media, cystic fibrosis and COPD. Together with Moraxella catarrhalis and Streptococcus pneumoniae, H. influenzae accounts for up to 50% of episodes of acute exacerbations of chronic bronchitis.14 Interestingly, in a study by Murphy and collaborators,15 26.3% of COPD patients were shown to have multiple H. influenzae strains simultaneously in their sputum. Different antimicrobial susceptibility patterns characterized the various strains. Notwithstanding the previous reports on the in vivo and in vitro activity of various antibacterial agents against H. influenzae, the susceptibility of intracellularly localized bacteria has not been investigated.

The present investigation shows that the intracellular bactericidal capacity of ciprofloxacin and quinupristin/dalfopristin, as analysed against non-typeable H. influenzae, is significantly higher than that of clarithromycin and telithromycin. Penicillins (here represented by ampicillin) have in general been shown to have limited access to the intracellular space, with C/E ratios <1 (Table 1).16 In contrast, the fluoroquinolone ciprofloxacin,17 the macrolide clarithromycin,18 the streptogramin quinupristin/dalfopristin19 and the ketolide telithromycin20 demonstrate C/E ratios of 10:1 to 100:1 or higher depending on the particular experimental setting. The intracellular distribution of antibiotics will influence their ultimate biological activity. A prerequisite for a beneficial intracellular antibacterial effect is the localization of the antibiotic and the pathogen in the same cellular compartment.21,22 Thus, intracellular bioactivity is not a common property among antibacterial agents, even though they are accumulated intracellularly.

The present study was performed in vitro, which does not take into account biologically active metabolites. For example, clarithromycin is dependent on its active metabolite 14-hydroxyclarithromycin for optimal Gram-negative killing.23 The concentration of the metabolite can vary in some individuals and depends on their particular liver function in addition to concomitantly administered drugs. Thus, the antibacterial effect of clarithromycin plus its metabolite might be higher in an in vivo clinical setting.

In order to keep the epithelial cells in good shape, an incubator with 5% CO2-enriched milieu was used in the present study. These might not be optimal conditions for some antibiotics such as clarithromycin.24 However, we measured the pH in all cell cultures and found that neither the various antibacterial agents nor incubation overnight with epithelial cells changed the pH below 7.2–7.4. Thus, our experimental pH conditions were most likely optimal.


    Acknowledgements
 
This work was supported by grants from the Alfred Österlund, the Anna and Edwin Berger, the Crafoord, the Greta and Johan Kock, the Magnus Bergvall, the T. H. C. Bergh and the Åke Wiberg Foundations, the Swedish Medical Research Council, the Swedish Society of Medicine and the Cancer Foundation at the University Hospital in Malmö.


    Footnotes
 
* Corresponding author. Tel: +46-40-331340; Fax: +46-40-336234; E-mail: kristian.riesbeck{at}mikrobiol.mas.lu.se Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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