Efficacy of azithromycin, clarithromycin and ß-lactam agents against experimentally induced bronchopneumonia caused by Haemophilus influenzae in mice

Shuichi Miyazaki*,, Toshihiko Fujikawa, Tetsuya Matsumoto, Kazuhiro Tateda and Keizo Yamaguchi

Department of Microbiology, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143-8540, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Azithromycin is an azalide with potent activity against Haemophilus influenzae including ampicillin-resistant strains. We evaluated the efficacy of azithromycin, clarithromycin and three ß -lactams when used for 1 day only and for 3 days for the treatment of a murine model of bronchopneumonia, using three strains of H. influenzae, two of which were ampicillin resistant. MICs of azithromycin (1–2 mg/L) and clarithromycin (4–8 mg/L) were similar for the three strains. The MICs of cefdinir and cefcapene for ß -lactamase-negative ampicillin-resistant (BLNAR) H. influenzae were 32 times higher than those for ß-lactamase-positive ampicillin-resistant and ampicillin-susceptible strains. The viable counts in the infected tissues of azithromycin-treated mice with bronchopneumonia caused by the susceptible strain TUM8, ß -lactamase-positive strain TUH36 and BLNAR strain TUH267 were less than the counts obtained with the other antibiotics used, irrespective of MIC. At a dose of 50 mg/kg, the area under the concentration curve and the half-life of azithromycin in the lungs were respectively three times higher and six times longer than those of clarithromycin. Our results indicate that azithromycin may be useful for both ampicillin-susceptible and ampicillin-resistant bronchopneumonial infections caused by H. influenzae.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Haemophilus influenzae is recognized as a frequent cause of a variety of infections, including acute otitis media, sinusitis, acute purulent exacerbation of chronic bronchitis and pneumonia. Among H. influenzae isolated during 1994 and 1995 in USA, 36.4% were found to produce ß-lactamase.1 The proportion of ß-lactamase-negative ampicillin-resistant (including intermediate resistance) (BLNAR) isolates was 2.5%. Resistance to ampicillin among H. influenzae has not changed since 1998 in the USA.2 Among H. influenzae isolated during 1996 and 1997 in Japan, 14.9% of isolates were ß-lactamase-positive while the proportion of BLNAR isolates was 44.4% of all ß-lactamase-negative isolates.3

It is well known that the activity of some oral cephalosporins against ß-lactamase-positive organisms and all oral cephalosporins against BLNAR organisms is lower than their activity against ß-lactamase-negative organisms. Thus, it is preferable to use other oral antibiotics with high in vitro and in vivo antibacterial activities against H. influenzae, including ampicillin-resistant organisms. The new macrolides (azalides), have been reported to reach high concentrations in lung tissues, thus improving their therapeutic potential against infections caused by obligatory or facultative pathogens, including H. influenzae and ampicillin-resistant strains.4

The purpose of our study was to determine whether the daily dose of various commercially available antimicrobial agents, including the frequency of administration, is effective in eliminating H. influenzae. The efficacies of various regimens for 1 day and for 3 days, as recommended by the suppliers, were evaluated by determination of viable bacterial counts in infected tissues, using the murine bronchopneumonia model.


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

ß-Lactamase-negative H. influenzae TUM8, ß-lactamase-positive H. influenzae TUH36 and BLNAR H. influenzae TUM267 were isolated from bronchopneumonia patients at our hospital, were non-typeable by the previously described method,5 and were stored at –80°C until use.

Antimicrobial agents

The following antimicrobial agents were used in this study: azithromycin (Pfizer Pharmaceuticals Inc., Tokyo, Japan); cefcapene, cefcapene pivoxil (Shionogi & Co., Osaka, Japan); clarithromycin (Taisho Pharmaceutical Co., Tokyo, Japan); cefdinir (Fujisawa Pharmaceutical Co., Osaka, Japan); and ampicillin (Meiji Seika Co., Tokyo, Japan).

In vitro susceptibility test

The MIC of each antimicrobial agent for the three strains was determined by the broth microdilution method in 0.1 mL volumes of Haemophilus test medium (HTM broth). Microdilution plates were inoculated with an automatic pin inoculator (MIC-2000; Dynatech Laboratories, Inc., Alexandria, VA, USA) to a final inoculum of c. 5 x 105 cfu/mL, and were incubated at 35°C for 20 h.

Experimental bronchopneumonia caused by H. influenzae

The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Toho University School of Medicine. The effects of azithromycin and other antibiotics on bronchopneumonia caused by H. influenzae were determined using the previously reported mouse model.6 For airway injury, 40 µL of 1% formalin was instilled intranasally into 4-week-old male ICR mice (Nihon Charles River Co., Kanagawa) under ketamine– xyline anaesthesia. An overnight culture of H. influenzae was inoculated into brain–heart infusion broth (Difco, Detroit, MI, USA) supplemented with haemin and NAD at a final concentration of 5%, and the culture was incubated at 35°C for 3 h. Bacterial cells were harvested by centrifugation then suspended in Eagle's minimal essential medium (MEM) to a volume similar to that of the original culture. This bacterial suspension was added to MFL cell monolayers, and the monolayers were incubated at 35°C for 1 h with gentle shaking. Free-floating bacteria were removed and discarded. The monolayer was then washed three times with saline and cell-bound organisms were removed from the flask and suspended in MEM. Three days after treatment of mice with formalin as described above, 50 µL of a cell-bound organism suspension (1.9–4.2 x 104 cfu/ animal) was instilled intranasally into anaesthetized mice. At 48 h after infection, the antibiotics were administered to each group (n = 14) for 1 or 3 days. The dose and frequency of administration of each antibiotic for human dosing as recommended by the suppliers were as follows: azithromycin 500 mg od; clarithromycin 200 mg bd; cefdinir 100 mg tds; cefcapene pivoxil 100 mg tds; ampicillin 250– 500 mg four to six times per day. Based on this schedule, we determined that the dose ratios of azithromycin, clarithromycin, cefdinir, cefcapene pivoxil and ampicillin were 5, 2, 1, 1 and 5, respectively. The frequency and dose of oral administration in the mice of azithromycin, clarithromycin, cefdinir, cefcapene pivoxil and ampicillin were: 100 mg/kg od, 40 mg/kg bd, 20 mg/kg tds, 20 mg/kg tds and 100 mg/kg tds, respectively. Tissues from mice (n = 7) treated for 3 days were obtained 18 h after the last administration of antibiotics while those (n = 7) treated with the 1 day regimen were obtained at 24 h or 48 h post-administration of antibiotics.

The lungs and trachea were removed and homogenized in 2 mL of sterile saline, and 0.1 mL aliquots of serial 10-fold dilutions of the homogenate were sub-cultured onto chocolate agar for determination of viable counts. Since the original tissue homogenate suspension inhibited growth of H. influenzae, but a 10-fold dilution of it did not, the detectable limit of bacteria in the lower respiratory organs was 2 x102 cfu/set of lower respiratory tract organs (tissues). The results are presented as the mean ± s.d. of log cfu/tissue (total organ counts). Statistical analysis was performed by the Mann–Whitney U-test. The bacterial number in samples with counts under the detectable limit was set at 1.9 x 102 cfu/animal for statistical analysis.

Pharmacokinetics in mice with pneumonia

Pharmacokinetic studies were conducted in 4-week-old ICR male mice (body weight, c. 20 g). Bronchopneumonia was experimentally induced using TUM8 as described above. Two days after infection, mice were treated orally with 0.2 mL of azithromycin or clarithromycin at a dose of 50 mg/kg. Blood samples were collected post-mortem at 0, 15 and 30 min, and at 1, 2, 4, 6, 8, 10, 12, 16, 20 and 24 h after administration of azithromycin and clarithromycin. The lungs were harvested, weighed and stored at –80°C until used for analysis. The blood samples were centrifuged to separate the serum. The serum and tissue samples were analysed by the paper disc bio-method, using Micrococcus luteus ATCC 9471 as the assay organism. The detection limits of clarithromycin and azithromycin were 0.1 and 0.0125 mg/L, respectively.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Efficacy of various drugs administered for 1 day in mice infected with H. influenzae

The antibacterial activity of azithromycin against strain TUM8 was four times greater than that of clarithromycin, but was at least four times less than for the three ß-lactams (Table 1Go). In the TUM8 infection model (Table 1Go), the number of bacteria in the lung tissue of the mice treated with azithromycin decreased markedly below the detection limit, with the exception of one set of lung tissues from one mouse where the organism count was 4.28 log cfu. The viable counts in lung tissue from mice treated with cefcapene pivoxil decreased significantly to less than the control and that of the cefdinir-treated mice, while in the mice treated with ampicillin, the viable count was significantly less than the control and the clarithromycin- and the cefdinir-treated mice at 24 h post-administration of antibiotic.


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Table 1. Mean (s.d.) viable total organ counts (log10 cfu/tissue) in lungs of mice infected with H. influenzae and treated with antimicrobial agents for 1 day
 
The antibacterial activity of azithromycin against strain TUH36 was four or more times greater than clarithromycin and ampicillin, but was 16 or more times less than cefdinir and cefcapene. In murine infection with TUH36 organisms, the viable counts in the two azithromycin treatment groups and the cefdinir treatment group at 24 h post-administration decreased significantly to less than that of the control, the clarithromycin and the ampicillin treatment groups. The viable counts in lung tissue of the cefcapene pivoxil treatment group at 24 h post-administration decreased significantly to less than that of the control and the azithromycin-, clarithromycin-, cefdinir- and ampicillin-treated mice.

The antibacterial activity of azithromycin against strain TUH267 was two to eight times greater than the other antibiotics. In murine infection with TUH267 organisms, the number of bacteria in the lung tissue decreased only in azithromycin-treated mice, which was less than the detection limit and significantly lower than in the other groups.

Efficacy of various drugs administered for 3 days in mice infected with strain TUH8

When the antibiotics were used for 1 day in H. influenzae-infected mice, azithromycin caused the largest reduction in lung tissue bacterial counts, excluding infections caused by ampicillin-resistant strains. When we evaluated the efficacies of various drugs when the period of administration was 3 days, the viable counts from lung tissue in the azithromycin and the cefcapene pivoxil treatment groups were significantly lower than in the control and the clarithromycin, cefdinir and ampicillin treatment groups (Table 2Go). The viable counts in the cefdinir and ampicillin treatment groups were significantly lower than in the clarithromycin treatment and the control groups.


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Table 2. Mean (S.D.) viable total organ counts (log10 cfu/tissue) in lungs of mice infected with H. influenzae TUM8 and treated with antimicrobial agents for 3 days
 
Pharmacokinetic studies

A single oral dose of azithromycin or clarithromycin at 50 mg/kg body weight produced a maximum concentration of the drug in the lung (Cmax) of 8.89 and 12.49 µg/g, respectively, and a half-life (tH) of 9.99 and 1.64 h, respectively. The areas under the concentration–time curves (AUC) for the same drugs were 147.58 and 47.73 µg•h/mL.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The prevalence of ß-lactamase-positive strains of H. influenzae has progressively increased in the UK, USA and Japan.13,7 In 1980, the problem of drug-resistant H. influenzae was complicated further by the description of ß- lactamase-negative strains that were resistant to ampicillin by unknown mechanisms, but possibly by elaboration of altered penicillin-binding proteins.810 These strains, referred to as BLNAR, have remained relatively uncommon in the USA (MIC >= 2 mg/L; 4%) and UK (MIC >= 1 mg/L; 5.8%).2,7 In contrast, BLNAR, defined as an MIC of >=1 mg/L, was noted in 40.5% of H. influenzae isolates, and BLNAR, defined as an MIC of >=2 mg/L was noted in 20.6% of H. influenzae isolates in Japan.3 Interestingly, the MIC90 of cefditoren, cefteram, cefdinir, cefpodoxime and minocycline for BLNAR isolates were two or more times higher than for ß-lactamase-producing isolates.3 In addition, the MIC90s of other test drugs except cefditoren and cefteram for ß-lactamase-producing isolates were four or more times higher than for ß-lactamase-negative isolates. Similar results were obtained in the present study. MICs of azithromycin and clarithromycin for ampicillin-resistant organisms were similar to those for ampicillin-susceptible organisms. The MICs of cefdinir and cefcapene for BLNAR, however, were higher than for ß-lactamase-positive ampicillin-resistant and ampicillin-susceptible strains.

Previous studies found that H. influenzae is an intracellular organism.6,11,12 In fact, H. influenzae colonizes and multiplies in both intracellular and extracellular locations.13 It is well known that azithromycin shows a potent activity against this species in vitro. Moreover, elimination half-lives of many of the new macrolides (including azithromycin) are longer than that of erythromycin, e.g. 4.2 h for clarithromycin14 and about 53 h for azithromycin in humans.15 One of the other characteristics of these antibiotics is the high penetration into lung tissues, especially azithromycin, which attains a high intracellular concentration after oral administration.16 Taken together, these data indicate that new macrolides may be useful antimicrobial agents for community-acquired infections caused by H. influenzae.

The aim of the present study was to determine whether a single administration of azithromycin per day was sufficient to cure bronchopneumonia infections caused by H. influenzae. The murine model of H. influenzae bronchopneumonia is a reasonable simulation of clinical infection. The infection is maintained for >7 days, and there is usually no death in untreated controls. Azithromycin was the most potent of the antibiotics tested under the present experimental conditions. Local concentration of the antibiotic at the site of infection is a key factor in determining its therapeutic efficacy. As described previously, azithromycin and clarithromycin can achieve high tissue and plasma concentration ratios and have a long half-life.17 Cmax, tH and AUC of cefcapene pivoxil and cefinir when administered in mice at 50 mg/kg were 4.1 and 0.7 µg/g, 0.78 and 1.90 h, and 6.6 and 2.2 µg•h/g, respectively.18 Those of ampicillin in mice were 1.7 µg/g, 1.34 h and 3.81 µg•h/g, respectively, at a dose of 40 mg/kg.19 These parameters were apparently lower than those of azithromycin at a dose of 50 mg/kg in the present study. In this regard, Craig20,21 reported that the efficacy of ß-lactams and macrolides, except azithromycin, is mainly dependent upon time above MIC and that azithromycin efficacy is mainly dependent upon the AUC/ MIC ratio. MICs of azithromycin for organisms used in this study were four times lower than those of clarithromycin. Musher et al.22 reported that bactericidal activity, which correlates with opsonizing activity of normal serum, might protect the host against non-typeable H. influenzae infection. The post-antibiotic effect, post-antibiotic sub-MIC effect and sub-MIC effect of azithromycin were greater than for clarithromycin for H. influenzae.23 These data support the superior efficacy of azithromycin compared with the other antibiotics studied. In this regard, although the experimental conditions were different, the present results are consistent with those of Piper et al.24 Our results showed that the efficacy of the comparative test antibiotics except clarithromycin, when administered for 3 days was significantly better than the control. This finding is coincident with the administration period of those drugs being longer than azithromycin. On the other hand, this model may underestimate clarithromycin efficacy against H. influenzae infection because rodents do not produce the active 14-hydroxy metabolite of clarithromycin, which is twice as potent as the parent compound against H. influenzae.25 Further studies are necessary to identify the factor(s) responsible for the difference in the response of the different infecting strains.


    Notes
 
* Corresponding author. Tel: +81-3-3762-4151; Fax: +81-3-5493-5415; E-mail: shuichi{at}med.toho-u.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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19 . Ishida, Y., Kurosaka, Y., Murakami, Y., Otani, T. & Yamaguchi, K. (1999). Therapeutic effect of oral levofloxacin, ciprofloxacin, and ampicillin on experimental murine pneumonia caused by penicillin intermediate Streptococcus pneumoniae for which the minimum inhibitory concentrations of the quinolones are similar. Chemotherapy 45, 183–91.[ISI][Medline]

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23 . Odenholt-Tornqvist, I., Lowdin, E. & Cars, O. (1995). Postantibiotic effects and postantibiotic sub-MIC effects of roxithromycin, clarithromycin, and azithromycin on respiratory tract pathogens. Antimicrobial Agents and Chemotherapy 39, 221–6.[Abstract]

24 . Piper, K. E., Rouse, M. S., Steckelberg, J. M., Wilson, W. R. & Patel, R. (1999). Ketolide treatment of Haemophilus influenzae experimental pneumonia. Antimicrobial Agents and Chemotherapy 43, 708–10.[Abstract/Free Full Text]

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Received 8 February 2001; returned 26 April 2001; revised 18 May 2001; accepted 8 June 2001