In vitro and in vivo influence of adjunct clarithromycin on the treatment of mucoid Pseudomonas aeruginosa

Khanh Q. Buia, Mary Anne Baneviciusa, Charles H. Nightingaleb, Richard Quintilianib and David P. Nicolaua,c,*

a Department of Pharmacy Research, b Office of Research Administration and c Division of Infectious Diseases, Hartford Hospital, 80 Seymour Street, Hartford, CT 06102, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recent in vitro and in vivo data have substantiated the beneficial effects of macrolides/ azalides for use against Pseudomonas aeruginosa. While macrolides/azalides are not very potent in vitro antimicrobial agents against this pathogen, they appear to have an adjunctive role by either altering the course of infection owing to their inhibition of biofilm production or modulation of the host anti-inflammatory response, or both. To determine the in vitro and in vivo effects of clarithromycin as adjunctive therapy with ceftazidime against a mucoidproducing strain of P. aeruginosa, we performed a standard time–kill experiment and a pneumonia model in mice, respectively. Time–kill studies were performed over a 24 h period with varying concentrations of clarithromycin and ceftazidime alone or in combination. Synergic activity was noted with the use of 0.5 x MIC of ceftazidime combined with either 0.5 or 2 x MIC of clarithromycin. Neutropenic mice were infected with 108 cfu of mucoid P. aeruginosa intranasally to produce pneumonia and subsequently treated with oral clarithromycin (100 mg/kg) and/or sc ceftazidime (1500 mg/kg) as monotherapy or in combination. The addition of 5 days of clarithromycin to the ceftazidime regimen significantly improved survival as compared with the ß-lactam alone (48% versus 32%, P = 0.04). While a statistically significant difference was not detected with the addition of 3 days of clarithromycin therapy, a trend towards improved survival was noted with this regimen (38% versus 32%). These data demonstrate the adjunctive potential of clarithromycin when administered in combination with an antipseudomonal agent for the treatment of mucoid-producing Pseudomonas in acute respiratory infection.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Despite the introduction of new antimicrobial therapies, Pseudomonas aeruginosa continues to be a pathogen responsible for considerable morbidity and mortality in respiratory tract infections. Patients suffering from chronic respiratory diseases such as cystic fibrosis and diffuse panbronchiolitis (DPB) often experience exacerbations from this bacterium, and it is not uncommon to isolate P. aerugin-osa from their sputum. Cystic fibrosis is widely recognized in the USA while DPB is more commonly diagnosed in the Japanese population. Although both non-mucoid- and mucoid-producing strains of P. aeruginosa are implicated in these infections, it is the mucoid strain that plays a significant role in the colonization and infection of these patients.1–3 Mucoid strains have the unique ability to produce exopolysaccharides or biofilms, notably alginate and glycocalyx. These tightly formed structures retard antibiotic penetration, promote bacterial adhesion to lung epithelia and prevent bacterial dehydration.4

Although the mainstay of treatment for Pseudomonas spp. continues to be ß-lactams, aminoglycosides and fluoroquinolones, recent evidence with erythromycin has shown that macrolides may also be beneficial in this infection.3,5–7 Macrolides have limited direct antibacterial activity against P. aeruginosa, but they have been shown to reduce the biofilm production in vitro.8,9 This attack on biofilms reduces a critical defence mechanism that shields the bacterium from antibiotics.1,4 Successful management of P. aeruginosa infection with macrolides is also related to their anti-inflammatory response. Modulating the host response during infection by inhibiting excessive neutrophil aggregation to the respiratory tract can be beneficial since it may be overreacting, leading to further tissue damage.7 With this evidence of macrolide activity against P. aeruginosa, we investigated the influence of adjunctive clarithromycin with ceftazidime using an in vitro time–kill model and a mouse pneumonia model against mucoid P. aeruginosa.


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

A mucoid P. aeruginosa (PSA13) isolated from a hospitalized patient was selected for study. The strain was maintained at –70°C in skimmed milk and subcultured out twice on to blood agar plates before use in vitro or in vivo.

Antimicrobial agents and MIC determinations

Analytical grade clarithromycin (Abbott Laboratories, Chicago, IL, USA) and ceftazidime (Glaxo–Wellcome, Research Triangle Park, NC, USA) standard powders were obtained for use during the in vitro portion of this study. Ceftazidime (Ceptaz, Glaxo Pharmaceuticals, Research Triangle Park, NC, USA) for iv administration and clarithromycin suspension (Biaxin, Abbott Laboratories) were kindly provided by the manufacturers for use in the mouse pneumonia model. All drugs were reconstituted as directed before use. For in vivo administration, ceftazidime was administered by sc injection to the dorsal base of the neck while clarithromycin was administered as po suspension using a 20 gauge stainless steel feeding tube (Popper & Sons, Inc., New Hyde Park, NY, USA). The MIC of ceftazidime and clarithromycin for the test strain was determined in duplicate using an approved NCCLS methodology for broth microdilution.10 Cation-adjusted Mueller–Hinton broth (CAMHB, Becton Dickinson, Cockeysville, MD, USA) was used for all in vitro experiments. The MIC for PSA13 of ceftazidime was 2 mg/L, while that of clarithromycin was 64 mg/L.

Time–kill studies

Tests for synergy between clarithromycin and ceftazidime were performed using the time–kill methodology. Following an overnight growth of PSA13 in CAMHB at 37°C in ambient air, the inoculum was visually adjusted to a 0.5 McFarland standard (1 x 108 cfu/mL). A 0.1 mL aliquot of inoculum was removed and added to test tubes containing the desired antibiotic concentration and CAMHB to produce a starting inoculum of 1 x 106 cfu/mL. Both agents were tested alone and in combination at 0.25 x MIC, 0.5 x MIC, 1 x MIC, 2 x MIC, 8 x MIC and 16 x MIC to characterize the individual effects as well as the potential for synergy. A control tube containing PSA13 in CAMHB without the presence of antibiotic was also prepared with each run. Each tube was incubated at 37°C and aliquots were taken at times 0, 2, 4, 6, 12 and 24 h. During sampling, performed using aseptic techniques, 0.1 mL was removed and serially diluted 10-fold, with 10 µL plated on to blood agar plates. Viable colonies from the plates were counted after a 24 h incubation at 37°C. The lower limit of detection was 100 cfu/mL and antibiotic carryover was not observed. All time–kill experiments were run in duplicate. Synergy was defined as a >2 log10 decrease in the viable count with the combination at 24 h compared with that of the more active compound tested alone. A combination effect producing <2 log10 decrease in the viable counts was considered to be additive.

Production of bacterial pneumonia

This protocol was reviewed and approved by our Institutional Animal Care and Use Committee. All animals were cared for according to the guidelines provided by the United States Department of Health and Human Services.11 Female Swiss Webster mice (20–25 g, Taconic, Germantown, NY, USA) were given a 7 day period to acclimatize to the animal facility. Animals were provided with feed and water ad libitum and housed in climate- and light-controlled conditions. Mice were rendered transiently neutropenic by injecting cyclophosphamide (Bristol-Myers Squibb Co., Princeton, NJ, USA) ip at a dose of 150 mg/kg of body weight at –4, –1 and +1 days in relation to induction of pneumonia.

One day before inoculation, PSA13 was grown overnight in CAMHB. The overnight cultures were combined and centrifuged at 1700g for 25 min. After a pellet was formed, the supernatant was decanted. The pellet was reconstituted with sterile 0.9% saline to 1/100 of the original volume to produce a concentrated bacterial suspension (~1010 cfu/mL). All bacterial suspensions were pooled and a small aliquot was removed for serial dilution and subsequent plating on to agar to confirm bacterial concentrations. Each mouse was held in an upright position and infected intranasally with two 25 µL doses of the bacterial suspension separated by 5 min. Using a calibrated pipette, the bacterial suspension was placed on to the nares of the nose and this was allowed to be inhaled during normal respiration.

Confirmation of bacterial pneumonia

To determine the appropriate initiation time for antibiotic therapy, lungs from neutropenic infected mice were harvested for bacterial density. At 0, 1, 2 and 3 days post-infection, groups of six to eight untreated mice were killed with carbon dioxide exposure followed by cervical dislocation. Under sterile conditions the thorax was opened and the right lung was dissected from the trachea and other structures, rinsed with saline, blotted dry, weighed, homogenized and quantitatively cultured. Bacterial growth was observed at 24 h post-infection and this was used as the time when all antibiotic treatments would begin.

Determination of the 50% protective dose (PD50)

Neutropenic mice were infected intranasally with 108 cfu of PSA13 and 24 h after infection the dose ranging studies were initiated. On days 1 and 2 post-infection a range of ceftazidime doses in 0.2 mL volumes were administered. Groups containing 10 mice were used for each dosing regimen. A control group of 10 mice received 0.2 mL of sterile 0.9% saline sc on days 1 and 2. The survival was recorded thrice daily for 7 days and plotted accordingly to determine the PD50 of ceftazidime. These preliminary studies revealed that a single dose of 1500 mg/kg sc on days 1 and 2 was required to produce a 50% survival rate.

Although death has historically been used as an end-point for studies of this type, it is no longer suitable to use this strict criterion in the current era of animal research. To comply with new standards set out by Hamm,12 every attempt was made to lessen the duration of pain and suffering by killing animals if they appeared to be in undue distress. Whether or not death resulted from this cause or natural progression of the disease process, the end-point data were treated identically with respect to statistical analysis.

Treatment regimens

Before antibiotic therapy, the neutropenic mice were randomized to one of four treatment regimens: ceftazidime alone sc at the PD50 dose; clarithromycin 100 mg/kg po q12h x six doses alone; ceftazidime at the PD50 plus clarithromycin 100 mg/kg po q12h x six doses; ceftazidime at the PD50 plus clarithromycin 100 mg/kg po q12h x 10 doses; or no treatment (control). In all regimens, all animals were manipulated equally so that when no antibiotic was administered, this was supplemented with 0.2 mL volume of saline sc or po. The dose of clarithromycin oral suspension was selected to simulate the human pharmacokinetic profile (expected Cmax = 2 mg/L and Cmin = 0.03 mg/L) observed during the administration of the 500 mg po q12h regimen.13

Data analysis

A sample size determination was performed using data from a previous study14 where ceftazidime was used alone or with azithromycin. To detect a difference with 80% power, 64 mice needed to be accrued in each treatment group. The logrank test for survival was used to compare differences in outcome at the end of the experiment. A P value < 0.05 was considered to be statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Time–kill studies

The time–kill kinetics of clarithromycin were studied with a range of 0.25–16 x MIC. Mean data for the duplicate runs are presented in Figure 1Go. While clarithromycin is primarily a bacteriostatic agent (<3 log10 reduction in bacteria), it was observed that the higher multiples of the MIC (8 and 16) produced bactericidal activity (>=3 log10 reduction in bacteria). Although the effect of clarithromycin against PSA13 was remarkable over the experimental concentration range studied, it must be noted that the physiologically achievable concentrations would best result in a killing profile that is appropriately characterized as bacteriostatic.



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Figure 1. Time–kill curves for a mucoid P. aeruginosa exposed to clarithromycin. Data points represent the mean of duplicate experiments. ({diamondsuit}), control; ({square}), 0.25 x MIC; ({triangleup}), 0.5 x MIC; ({blacksquare}), 1 x MIC; ({bigcirc}), 2 x MIC; (•), 8 x MIC; ({diamond}), 16 x MIC.

 
In contrast, ceftazidime was clearly bactericidal starting at 2 x MIC (Figure 2Go). It also showed concentrationindependent activity over the dosing range that was not as noticeable with clarithromycin. To evaluate the potential for synergic effects, a low concentration of ceftazidime was combined with varying concentrations of clarithromycin. Higher concentrations of ceftazidime were not used for synergy experiments because as monotherapy, they were already close to the limit of detection and any synergic effect would have been unobservable.



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Figure 2. Time–kill curves for a mucoid P. aeruginosa exposed to ceftazidime. Data points represent the mean of duplicate experiments. ({diamondsuit}), control; ({square}), 0.25 x MIC; ({triangleup}), 0.5 x MIC; ({blacksquare}), 1 x MIC; ({bigcirc}), 2 x MIC; (•), 8 x MIC; ({diamond}), 16 x MIC.

 
Figure 3Go represents the data for the low concentrations of ceftazidime in combination with a range of clarithromycin concentrations. When ceftazidime 0.5 x MIC was combined with either clarithromycin 0.5 or 2 x MIC, a synergic effect was noted when compared with results obtained when ceftazidime was tested at this concentration alone. With increasing concentration of clarithromycin, an additive effect was seen.



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Figure 3. Time–kill curves for a mucoid P. aeruginosa exposed to clarithromycin (CLR) and ceftazidime (CAZ) combinations. Data points represent the mean of duplicate experiments. (—{diamondsuit}—), control; (—{blacksquare}—), CLR 0.5 x MIC; (—{blacktriangleup}—), CAZ 0.5 x MIC; (—{diamond}—), CLR 0.5 x MIC, CAZ 0.5 x MIC; (—{triangleup}—), CLR 2 x MIC, CAZ 0.5 x MIC; (—•—), CLR 8 x MIC, CAZ 0.5 x MIC; (—x—), CLR 16 x MIC, CAZ 0.5 x MIC; (—{triangleup}—), CLR 2 x MIC; (—{bigcirc}—), CLR 8 x MIC; (—{diamondsuit}—), CLR 16 x MIC.

 
Mouse pneumonia model

Neutropenic mice were infected with PSA13 and treatment was begun 24 h later. Only groups receiving ceftazidime alone or in combination achieved the required number of animals for statistical analysis. This was done intentionally because the benefit of adjunctive clarithromycin therapy would only be compared with the most active agent: ceftazidime. The use of clarithromycin alone was used to confirm the prediction that the macrolide has very little effect as monotherapy in vivo even though the in vitro data were promising. While the final survival rate of 30% (range of 20–50% over three experimental study periods) observed for animals receiving ceftazidime was less than the PD50, this did not affect the final conclusions since the prime objective was to determine the adjunctive effects with addition of clarithromycin. From these data (Figure 4Go), a multiple comparison using the logrank test was made. When the survival of mice treated with ceftazidime plus 10 doses (5 days) of clarithromycin was compared with those treated with ceftazidime alone, a statistically significant difference was found (48% versus 32%, P = 0.0409). While a statistically significant difference was not detected during the comparison between ceftazidime plus six doses (3 days) of clarithromycin and ceftazidime alone, a trend towards improved survival was noted with the addition of clarithromycin (38% versus 32%). No difference was revealed between the two ceftazidime plus clarithromycin groups (P = 0.19).



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Figure 4. Survival data for murine pneumonia model owing to a strain of mucoid P. aeruginosa. Infection occurred at 0 h and treatment began at 24 h. CAZ, ceftazidime; CLR, clarithromycin (n = total number of mice accrued in each group). ({diamond}), control (n = 24); ({blacksquare}), CLR q12h x 6 (n = 25); ({blacktriangleup}), CAZ q24h x 2 (n = 65); ({bigcirc}), CAZ q24h x 2 + CLR q12h x 6 (n = 64); (•), CAZ q24h x 2 + CLR q12h x 10 (n = 65).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Traditional antibiotics such as ß-lactams, aminoglycosides and fluoroquinolones for the treatment of P. aeruginosa pneumonia have continued to provide the best clinical outcomes even for a disease with significant mortality. With increasing resistance to these antibiotics, it will be necessary to explore additional treatments that will produce enhanced activity. In vitro experiments have shown that some of the expression factors related to virulence and pathogenicity of this organism can be diminished in the presence of macrolide/azalide antibiotics.15–18 In addition, data from our group using a P. aeruginosa (PSA 8, non-mucoid strain) peritonitis model has shown a slower rate of mortality when animals were treated with azithromycin.14,18 These data are further supported by the beneficial effect observed with the addition of the macrolides to the treatment regimen for diffuse panbronchiolitis, a clinical setting where mucoid strains of P. aeruginosa often produce a chronic infection.1–3

The in vitro time–kill studies in this investigation revealed that clarithromycin, in combination with ceftazidime, has the ability to produce synergic bactericidal effects. It is unlikely that clarithromycin's contribution to this result was directly related to its antibacterial property since very little inhibition was noted when this antibiotic was used alone. Therefore, the likely explanation for the observed synergic effect is the ability of macrolide sub-MIC concentrations to inhibit formation of glycocalyx and/or alginate in P. aeruginosa.5,6,15,17 This mechanism is supported by a similar study with non-mucoid-producing P. aeruginosa where our group found no survival benefit with the addition of azithromycin.19 By reducing the secretion of these exogenous polysaccharides, the penetration of ceftazidime into the immediate space surrounding the bacteria may have been enhanced, resulting in substantially improved bactericidal activity as compared with the ß-lactam alone.

Similar to the beneficial effects of clarithromycin in the in vitro testing conditions, the in-vivo data also support the adjunctive role of clarithromycin in the management of mucoid P. aeruginosa strains. The exposure to cyclophosphamide provided 5 days of transient neutropenia, diminishing the role of neutrophils even as they recover.20–22 This current study highlights two important facts: (i) clarithromycin does have beneficial effects against mucoid-producing Pseudomonas sp. during acute infection when used as an adjuvant, and (ii) the duration of treatment is vital in determining overall benefit of this adjunctive therapy. This latter point is emphasized by the investigation of Tateda and colleagues,6 which suggests that macrolide sub-MIC concentrations require a longer exposure time to produce bacterial killing. In addition to a direct influence on the microorganism via the inhibition of bacterial exopolysaccharides, adjunctive clarithromycin may also result in an anti-inflammatory effect. Data with the macrolides have shown that they reduce the number of neutrophils and neutrophil-derived elastolytic-like activity in the lower respiratory tract,5 inhibit neutrophil chemoattractant,23 and elevate the CD4+/CD8+ ratio by reducing the number of CD8+ lymphocytes.7

Our study did not set out to determine which factor was most important in determining outcome but it is likely that at any one period of time, a combination of effects was required to keep the bacteria in check. During the first 5 days of the study when the mice were neutropenic, the anti-inflammatory response was blunted and had a minimal effect on survival, whereas inhibition of exopolysaccharides promoted the penetration of ceftazidime or reduced the adhesion of Pseudomonas sp. to the respiratory tract. As the neutrophil counts began to recover, the benefits of clarithromycin may be more a result of its anti-inflammatory effects, with minimal effects on direct bacterial clearance. The dual role of clarithromycin underscores the fact that its activity against Pseudomonas sp. is not mutually exclusive but rather working in concert to alleviate an infection. The positive clinical outcomes with chronic infection and data from our study for the treatment of acute infections may warrant additional clinical studies to determine the usefulness of this agent against pseudomonal infections.


    Acknowledgments
 
The authors would like to thank Christina Turley for her technical assistance and Jeff Mather for his assistance with the data analysis. This work was supported by a grant from Abbott Laboratories.


    Notes
 
* Corresponding author. Tel: +1-860-545-3941; Fax: +1-860-545-5112; E-mail: dinicola{at}harthosp.org Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 23 March 1999; returned 12 July 1999; revised 22 July 1999; accepted 21 September 1999