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Daptomycin is a novel lipopeptide antibiotic with excellent activity against Gram-positive bacterial pathogens, but its therapeutic value for the treatment of invasive pneumococcal disease compared to that for the treatment of pneumococcal pneumonia is incompletely defined. We investigated the efficacy of daptomycin in two models of Streptococcus pneumoniae-induced lung infection, i.e., pneumococcal pneumonia and septic pneumococcal disease. Mice were infected with a bioluminescent, invasive serotype 2 S. pneumoniae strain or a less virulent serotype 19 S. pneumoniae strain and were then given semitherapeutic or therapeutic daptomycin or ceftriaxone. Readouts included survival; bacterial loads; and septic disease progression, as determined by biophotonic imaging. Semitherapeutic daptomycin treatment fully protected the mice against the progression of septic disease induced by serotype 2 S. pneumoniae, while therapeutic treatment of the mice with daptomycin or ceftriaxone led to ~70% or ~60% survival, respectively. In contrast, mice infected with serotype 19 S. pneumoniae developed severe pneumonia and lung leakage even in the presence of increased intra-alveolar daptomycin levels, resulting in only 40% survival, whereas the ceftriaxone-treated mice had 100% survival. Together, although daptomycin demonstrates little efficacy in the treatment of pneumococcal pneumonia, daptomycin is highly effective in preventing S. pneumoniae-induced septic death, thus possibly offering a therapeutic option for patients with life-threatening septic pneumococcal disease.
The Gram-positive bacterium Streptococcus pneumoniae is the most prevalent pathogen in community-acquired pneumonia (CAP). CAP is known to frequently progress to invasive pneumococcal disease, thereby causing significant morbidity and mortality worldwide. In developed countries, death from pneumococcal disease occurs primarily among elderly individuals, in whom bacteremic pneumonia is associated with case-fatality rates of 10 to 20% and in whom pneumococcal bacteremia is associated with a case-fatality rate of up to 60% (33). As such, S. pneumoniae causes more deaths from invasive infections than any other bacterium and is the fifth leading cause of death worldwide (12). The worldwide increase in the rates of resistance of S. pneumoniae to frequently used antibiotics such as beta-lactams and macrolides and the rapid global spread of multidrug-resistant clones require both the development of novel immunization strategies and the search for novel antibiotic substances that are active against S. pneumoniae strains resistant to other antimicrobial drugs (1, 9, 24). The clinical situation with S. pneumoniae is further complicated by the fact that pathogenicity profiles vary considerably between and among different serotypes of S. pneumoniae. For example, serotypes 19 and 23 of S. pneumoniae are considered less virulent in humans and mice, whereas serotypes 2 and 4 frequently cause invasive pneumococcal disease and multiorgan failure (MOF) (14). It is just the progression from local infection to sepsis and MOF that contributes to the unacceptably high mortality rates in intensive care units. In view of the distinct pathogenicity profiles of S. pneumoniae serotypes and strains that result in different clinical courses, novel antibiotic substances with the potential to cure infections with S. pneumoniae need to be rigorously validated in experimental model systems specifically representing the major clinical phenotypes of pneumococcal lung infections, i.e., localized pneumococcal pneumonia or invasive pneumococcal disease.
Daptomycin is a novel, transmembrane pore-forming 13-amino-acid compound that is produced by Streptomyces roseosporus (21). It belongs to the group of cyclic lipopeptides (for a review, see reference 25), which have particular efficacy against Gram-positive bacterial pathogens, whereas Gram-negative bacterial pathogens are not affected, probably due to their outer membrane (28). Daptomycin inserts into the bacterial cell membrane in a calcium-dependent manner and causes oligomerization of the daptomycin peptide into transmembrane pores, thereby triggering membrane leakage, the release of intracellular ions, and rapid cell death (28, 29). Daptomycin has potent bactericidal activity against a wide range of Gram-positive bacteria and antibiotic-resistant Gram-positive pathogens, such as vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus (MRSA), glycopeptide-intermediate S. aureus, and penicillin-resistant Streptococcus pneumoniae, for which there are few therapeutic alternatives (4, 27, 30). Importantly, the spontaneous bacterial acquisition of daptomycin resistance has rarely been observed (11, 27, 31). Until now, daptomycin has been approved for use for the treatment of complicated skin and soft tissue or skin structure infections caused by Gram-positive bacteria and for the treatment of endocarditis and staphylococcal sepsis (10). However, in a mouse model of S. pneumoniae or MRSA pneumonia, daptomycin failed to significantly purge bacterial loads at 24 h after infection, although the findings of time-response and survival studies were not reported (26). In contrast, daptomycin was found to be effective in a rat model of hematogenous pneumonia caused by S. aureus (26). Of note, in a phase III clinical trial for the treatment of patients with community-acquired pneumonia, daptomycin (applied at a dose of 4 mg/kg of body weight) failed to achieve superiority over ceftriaxone (efficacies, 79 and 87%, respectively) (23). Thus, the currently available experimental and clinical data for mice and humans suggest that daptomycin has little efficacy against Gram-positive bacterial infections of the lung, possibly due to interactions of the compound with pulmonary surfactant components (26), and is therefore currently not recommended for use for the monotherapy of pneumonia.
To the best of our knowledge, no detailed experimental data are available from investigations of the efficacy of daptomycin in defined mouse models of S. pneumoniae-induced lung infections either causing pneumonia in the absence of sepsis or causing fatal invasive pneumococcal disease. Therefore, in the current study, we made use of self-glowing, bioluminescent strains of S. pneumoniae with different virulence profiles to determine the efficacy of daptomycin compared to that of ceftriaxone in two models: a model of pneumococcal pneumonia caused by a less virulent serotype 19 S. pneumoniae strain and a model of septic pneumococcal disease caused by a highly invasive serotype 2 S. pneumoniae strain.
C57BL/6 mice were purchased from Charles River (Sulzfeld, Germany) and were kept under conventional conditions with free access to food and water. The mice were used in all experiments at 8 to 12 weeks of age, in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Hannover School of Medicine. Animal experiments were approved by local government authorities in Hannover, Germany.
In the current study, we employed two bioluminescent, self-glowing strains of S. pneumoniae, i.e., a highly virulent serotype 2 S. pneumoniae strain (strain D39 lux) and a capsular group 19 S. pneumoniae strain (strain EF3030 lux) with a low level of virulence. Both strains of S. pneumoniae contained a plasmid expressing the luxABCDE operon of Photorhabdus luminescens and an erythromycin resistance cassette for positive selection purposes and were generated as described recently (2, 5). Importantly, constitutive bioluminescence emission of the pneumococcal strains used did not require application of a substrate (self-glowing strains).
The serotype 2 S. pneumoniae strain was grown in Todd-Hewitt broth (Oxoid, Basingstoke, United Kingdom) supplemented with 0.5% yeast extract (Difco/BD Biosciences, Sparks, MD) in the presence of 0.2 μg/ml erythromycin (Sigma-Aldrich, Steinheim, Germany). The serotype 19 S. pneumoniae strain was grown in Todd-Hewitt broth supplemented with 10% fetal calf serum in the presence of 0.2 μg/ml erythromycin. Aliquots were snap-frozen in liquid nitrogen and were stored at −80°C. The S. pneumoniae stocks were quantified by plating serial dilutions of the bacteria on sheep blood agar plates (BD Biosciences, Heidelberg, Germany), followed by incubation of the plates at 37°C in 5% CO2 for 18 h and subsequent determination of the numbers of CFU.
Daptomycin was kindly provided by Novartis GmbH (Nürnberg, Germany). The comparator agent, ceftriaxone, was purchased from Ratiopharm GmbH (Ulm, Germany). Both daptomycin and ceftriaxone were freshly reconstituted in vehicle (sterile saline), according to the manufacturers' recommendations, immediately prior to each experiment. Mice received daily applications of either daptomycin, applied at a clinically relevant dose of 6 mg/kg body weight intraperitoneally (i.p.); ceftriaxone (20 mg/kg i.p.); or vehicle (12 ml/kg body weight i.p.). Determination of the MICs of daptomycin for the inhibition of S. pneumoniae growth was done by Etest (AB Biodisk, Solna, Sweden), as recommended by the manufacturer, and MICs of ≤0.094 μg/ml daptomycin were revealed for the serotype 2 and 19 S. pneumoniae strains. The MICs of the antibiotic ceftriaxone were tested in a semiautomated Micronaut system (Merlin, Bornheim-Hersel, Germany). Briefly, single colonies were inoculated in 0.9% NaCl to a McFarland standard of 0.5. Pneumococci (serotype 2 or 19) were mixed with M broth supplemented with phytagel and lysed horse blood, as recommended by the manufacturer, and inoculated in 96-well microtiter plates (E1-922-100 Micronaut-S. pneumococci and Haemophilus 2 plates). Following overnight incubation at 37°C in 5% CO2, the bacterial growth was determined photometrically at a wavelength of 690 nm. The MICs were interpreted by using an advanced expert system (AES; MCN-6 of the Merlin Micronaut system) and were found to be less than 0.125 μg/ml.
The antibacterial efficacy of daptomycin compared to that of ceftriaxone was evaluated for the following treatment groups: (i) a group receiving semitherapeutic treatment, in which antibiotics (daptomycin or ceftriaxone versus vehicle) were administered intraperitoneally immediately prior to infection with either serotype 2 or serotype 19 S. pneumoniae (applied at either 5 × 106 CFU/mouse or 1.5 × 107 CFU/mouse), or (ii) a group receiving therapeutic treatment, in which the mice received antibiotics (daptomycin or ceftriaxone versus vehicle) 2 days after infection with serotype 2 S. pneumoniae (applied at 1.5 × 107 CFU/mouse). In those experiments in which the S. pneumoniae-infected mice were subjected to a time-response analysis of their bioluminescence emission, the antibiotics were always applied immediately after the respective biophotonic analysis.
Mice were anesthetized with tetrazoline hydrochloride (5 mg/kg) and ketamine (75 mg/kg). Subsequently, the mice were orotracheally intubated with a 29-gauge Abbocath catheter (Abbott, Wiesbaden, Germany), which was inserted into the trachea under visual control with transillumination of the neck region. Lung infection with S. pneumoniae was induced by orotracheal instillation of either 5 × 106 CFU (low-dose challenge) or 1.5 × 107 CFU (high-dose challenge) of serotype 2 or 19 S. pneumoniae in a volume of 50 μl Todd-Hewitt broth (THB). Vehicle-treated mice received intratracheal applications of THB only. Subsequent to infection, the mice were taken back to their cages, where they had free access to food and water, and were monitored daily for disease symptoms. The survival of the S. pneumoniae-infected mice was recorded daily over an observation period of 14 days.
At various time points postinfection, the S. pneumoniae-infected mice were killed with an overdose of isoflurane (Baxter, Unterschleissheim, Germany) and the bacterial loads in the blood, lung, liver, and spleen were determined. For the determination of bacteremia, peripheral blood was collected from the inferior vena cava, followed by the plating of serial dilutions on sheep blood agar plates. For determination of the numbers of CFU in the lungs, spleens, and livers, the organs were dissected and homogenized in Hanks' balanced salt solution without supplements by using a tissue homogenizer (IKA, Staufen, Germany). The resulting homogenates were filtered through a 100-μm-pore-size cell strainer (BD Falcon), and aliquots of each sample were then plated in 10-fold serial dilutions on sheep blood agar plates, followed by incubation at 37°C in 5% CO2 for determination of the bacterial loads, as recently described in detail (18, 34).
To evaluate the effect of semitherapeutic treatment (i.e., at the time of infection) or therapeutic treatment (2 days after infection) with daptomycin compared to that with ceftriaxone on the progression of S. pneumoniae-induced lung infection in individual mice over time, separate groups of serotype 2 S. pneumoniae (strain D39 lux)-infected or serotype 19 S. pneumoniae (strain EF3030 lux)-infected mice receiving daptomycin, ceftriaxone, or vehicle were subjected to bioluminescence analysis on days 1, 2, 4, and 7 of infection by using a bioluminescence analyzer (Xenogen, Alameda, CA). The imaging system employed measures the numbers of photons originating from the self-glowing pneumococci, which are collected by a charge-coupled-device camera, followed by conversion of the primary data into false color images by using IVIS Living Image (version 2.50.1) software (Xenogen). Areas of intense luminescence are depicted in red, areas of moderate luminescence are depicted in yellow and green, and areas of mild luminescence are depicted in blue. For bioluminescence analysis, lightly anesthetized mice were transferred in the supine position to an imaging chamber for an exposure time of 1 min. The images displayed in the current report are grey-scale photographs with an overlay image of bioluminescence obtained by using the aforementioned computer-generated false color scale. Images were taken with the widest lens opening (f-stop 1), the field of view was set equal to 19.2 cm, the subject height was set equal to 1.5 cm, and the binning was set to medium. Fatal septic disease progression is characterized by the emanation of high-intensity bioluminescence signals from the entire mouse (22).
The total numbers of bronchoalveolar lavage (BAL) fluid leukocytes were determined from whole-lung washes of S. pneumoniae-infected mice, as recently described in detail (19, 20). In brief, mice were euthanized with an overdose of isoflurane (Forene; Abbott Laboratories), and the tracheas of the mice were exposed and cannulated with a shortened 20-gauge needle that was firmly fixed to the trachea. Subsequently, 300-μl aliquots of ice-cold sterile phosphate-buffered saline were instilled, followed by careful aspiration, until a BAL fluid volume of 1.5 ml was collected. Bronchoalveolar lavage was then continued until an additional BAL fluid volume of 4.5 ml was collected. The whole-lung washes were subjected to centrifugation at 1,400 rpm (4°C, 10 min), and the cell pellets were pooled to determine the total numbers of BAL fluid leukocytes. The quantification of the BAL fluid leukocyte subsets was done on the differential cell counts of Pappenheim-stained cytocentrifuge preparations by using the overall morphological criteria, including the cell size and the shape of the nuclei, and the subsequent multiplication of those values with the respective absolute BAL fluid cell counts.
In selected experiments, we determined the effect of daptomycin treatment on the lung permeability induced in mice after infection with different serotypes of S. pneumoniae. Lung permeability was determined essentially as described recently (8, 16, 17, 35). In brief, mice received an intravenous injection of fluorescein isothiocyanate-labeled human albumin (1 mg/mouse in 100 μl saline; Sigma, Deisenhofen, Germany) 1 h before they were killed. Subsequently, undiluted BAL fluid samples and serum samples (diluted 1/10 in saline) were placed in a 96-well microtiter plate, and the fluorescence intensities were measured with a fluorescence spectrometer (FL 880 microplate fluorescence reader; Bio-Tek, Bad Friedrichshall, Germany) operating at an absorbance of 488 nm and an emission wavelength of 525 ± 20 nm. The lung permeability index is defined as the ratio of the fluorescence signals of undiluted BAL fluid samples relative to the fluorescence signals of 1/100-diluted serum samples.
Mouse serum daptomycin concentrations were determined by a previously published (15) high-performance liquid chromatography (HPLC) method, with some modifications. In short, proteins of 50 μl serum were precipitated by the addition of 100 μl methanol, and after centrifugation, 50 μl of the clear supernatant was injected into the HPLC system. The separation of daptomycin was achieved by gradient elution on a C8 reversed-phase column with an acetonitrile buffer (20 mM trifluoroacetic acid, 15 mM triethylamine) gradient. The calibration range was 3.5 to 350 μg/ml, and the accuracy of the assay was better than 1.5%. The determination of daptomycin in BAL fluids (which is anticipated to contain low concentrations) was found to require sample cleanup and concentration by solid-phase extraction (SPE). After conditioning of Oasis HLB SPE columns (Waters, Milford, CA) with 1 ml methanol and 1 ml water, 1 ml of BAL fluid was passed through, and the columns were subsequently rinsed with 1 ml water and sucked dry for 5 min under vacuum. Daptomycin was eluted from the columns with 1 ml methanol, and the extract was evaporated under vacuum. The residue was reconstituted with 25 μl methanol and 75 μl water, and 50 μl of this mixture was injected into the HPLC system by using the same conditions described for the determination of daptomycin in serum samples. The calibration range for BAL samples was 5 to 800 ng/ml, and the precision was better than 2.5%.
All data are given as means ± standard errors of the means (SEMs). The differences between the controls and the respective treatment groups over time were analyzed by one-way analysis of variance, followed by the post hoc Tukey test for paired comparisons, by using the SPSS for Windows software package. Survival rates and survival curves were compared by the Pearson chi-square test and log-rank test, respectively. The differences between the groups were assumed to be statistically significant when P values were at least ≤0.05.
In initial experiments in which a semitherapeutic treatment protocol was used and in which the mice received the antibiotic at the time of infection, we evaluated the efficacy of daptomycin at protecting mice against low-dose infection with a highly virulent serotype 2 S. pneumoniae strain (5 × 106 CFU/mouse). As shown in Fig. Fig.1,1, semitherapeutic daptomycin treatment fully protected the mice against a low-dose S. pneumoniae infection during the entire observation period of 14 days, whereas vehicle-treated mice showed a survival rate of only ~60% (Fig. (Fig.1;1; P < 0.05). The results of the analysis of the bacterial loads in the lungs, spleens, and livers, as well as the bacterial loads and levels of bacteremia in the blood of mice receiving daptomycin semitherapeutically are given in Table Table1.1. Interestingly, the loads of the highly virulent serotype 2 S. pneumoniae strain were detectable in the lungs of daptomycin-treated mice up until day 10 of infection, albeit at significantly lower levels than in the vehicle-treated mice. At the same time, the early bacteremia that developed in the blood, as well as the spleens and livers, of mice in either treatment group was rapidly purged from the daptomycin-treated mice within 24 h of treatment. These data illustrate that although daptomycin was not highly effective in purging pneumococcal loads from the lung itself, the antibiotic provided full protection against serotype 2 S. pneumoniae-induced sepsis in mice.
In the next set of experiments, we questioned whether semitherapeutic daptomycin treatment would also protect the mice from lethal pneumococcal sepsis in a high-dose serotype 2 S. pneumoniae infection model. As shown in Fig. Fig.2,2, the challenge of vehicle-treated mice with a high dose of the serotype 2 S. pneumoniae strain (1.5 × 107 CFU/mouse) resulted in an overall mortality rate of ~70% by day 7 postinfection. In contrast, semitherapeutic treatment of mice with daptomycin or the comparator antibiotic, ceftriaxone, fully protected the mice against the invasive pneumococcal disease induced by serotype 2 S. pneumoniae (Fig. (Fig.2;2; P < 0.01 for both treatments compared to the results obtained for the vehicle-treated mice). Analysis of the bacterial loads in serotype 2 S. pneumoniae-infected mice receiving semitherapeutic daptomycin or ceftriaxone treatment showed that under high-dose infection conditions, S. pneumoniae was also detectable in the lungs of mice up until day 5 of infection, whereas strong bacterial outgrowth was demonstrated in the distal lung airspaces of the vehicle-treated mice (Table (Table2).2). In contrast, ceftriaxone treatment of the mice resulted in the rapid clearance of the bacteria from the lungs by 24 h of infection. Again, however, daptomycin was as effective as ceftriaxone in preventing serotype 2 S. pneumoniae-induced bacteremia and sepsis in mice, as determined by analysis of the peripheral blood, spleens, and livers (Table (Table2).2). Importantly, the residual bacterial counts observed in the lungs of daptomycin-treated mice had no influence on the overall rate of survival, again supporting the conclusion that the prevention of septic disease progression is more important in view of survival rather than the complete removal of bacteria from distal airspaces, at least in this model of serotype 2 S. pneumoniae-induced lung infection.
We next determined the efficacy of daptomycin compared to that of ceftriaxone on the survival of mice under therapeutic treatment conditions, i.e., when the mice were administered antibiotics at day 2 of the high-dose infection with the serotype 2 S. pneumoniae strain (1.5 × 107 CFU/mouse). As shown in Fig. Fig.3A,3A, high-dose challenge of the mice with the serotype 2 S. pneumoniae strain resulted in an overall rate of survival of the vehicle-treated mice of 20% by day 5 postinfection. In contrast, the therapeutic application of daptomycin resulted in a significantly improved rate of survival of 70% (P < 0.05), with a 60% survival rate being observed in the ceftriaxone treatment group by day 3 postinfection. Biophotonic imaging of mice therapeutically treated with daptomycin or ceftriaxone (Fig. (Fig.3B,3B, right) demonstrated the complete loss of bioluminescence emission over time, reflecting the strong antibacterial activities of the substances in mice after challenge with a high dose of the serotype 2 S. pneumoniae strain, while, at the same time, mice receiving the vehicle only demonstrated a continuous increase in bioluminescence emission over time until death (Fig. (Fig.3B,3B, left).
Having determined the survival in mice therapeutically treated with daptomycin subsequent to infection with a high dose of the serotype 2 S. pneumoniae strain, we next analyzed the bacterial loads and levels of bacteremia in peripheral blood and lung tissue of the mice in the respective treatment groups. As shown in Table Table3,3, the bacterial loads in the lungs and blood of the mice at day 2 postinfection with serotype 2 S. pneumoniae determined immediately prior to therapeutic application of the respective antibiotics ranged from ~107 CFU in the lungs to ~108 CFU in the blood of the mice. Analysis of the bacterial loads in the lungs and blood and determination of the level of bacteremia on day 1 posttreatment (i.e., on day 3 postinfection) with either daptomycin or ceftriaxone showed a much higher level of reduction in the bacterial loads in the blood than in the lungs for both antibiotic substances tested. Again, while ceftriaxone led to the complete elimination of S. pneumoniae from the lungs by day 2 of therapeutic treatment (day 4 postinfection), low bacterial counts were still detectable in the lungs of daptomycin-treated mice by day 3 of therapeutic treatment (day 5 postinfection). Together with the currently chosen therapeutic treatment regimen, these data strongly support the conclusion that daptomycin appears to be as effective as the comparator substance, ceftriaxone, for the treatment of invasive pneumococcal disease.
The pathogenicity profiles differ considerably between and among different serotypes of S. pneumoniae (14). As opposed to serotype 2 S. pneumoniae, which rapidly triggers sepsis in mice, serotype 19 S. pneumoniae is known to primarily cause pneumonia in mice without septic disease progression (6, 35, 36). Therefore, we also determined the efficacy of daptomycin in a model of S. pneumoniae lobar pneumonia induced by infection with a low dose of serotype 19 S. pneumoniae (5 × 106 CFU/mouse). As shown in Table Table4,4, the semitherapeutic treatment of mice with daptomycin did not the improve the elimination of the bacterial pathogen from the distal airspaces over that achieved in the vehicle-treated mice during the up to 10-day observation period. In contrast to the bacteremia observed in mice after challenge with a low dose of a highly virulent serotype 2 S. pneumoniae strain (Table (Table1),1), infection of the mice with a low dose of a serotype 19 S. pneumoniae strain did not cause any bacteremia in mice in either treatment group.
Because approximately 95% of the vehicle-treated mice were found to survive the current low-dose infection with serotype 19 S. pneumoniae (data not shown), we further evaluated the efficacy of daptomycin in a mouse model of a high-dose challenge with a serotype 19 S. pneumoniae strain (1.5 × 107 CFU/mouse), using semitherapeutic treatment conditions. As shown in Fig. Fig.4,4, the vehicle-treated mice demonstrated 20% survival by day 7 postinfection. Importantly, the semitherapeutic treatment of mice with daptomycin had no significant impact on the survival of mice infected with 1.5 × 107 CFU serotype 19 S. pneumoniae, with an overall rate of survival of approximately 40% by day 6 postinfection (Fig. (Fig.4).4). In sharp contrast, treatment of the mice with the comparator antibiotic, ceftriaxone, resulted in 100% survival of the mice after infection with serotype 19 S. pneumoniae (P < 0.01). Determination of the bacterial loads in the lungs and blood of vehicle-, daptomycin-, or ceftriaxone-treated mice infected with serotype 19 S. pneumoniae revealed the superior antibacterial efficacy of ceftriaxone in purging the serotype 19 S. pneumoniae infection, resulting in the complete clearance of bacteria from lung parenchymal tissue by day 1 postinfection (Table (Table5).5). In contrast, treatment of the mice with daptomycin did not reduce the bacterial loads in their lungs during the first 3 days of infection, while lower CFU counts were observed by days 4 and 5 postinfection (Table (Table5).5). As expected in the currently employed pneumonia model, weak and transient bacteremia was detectable only in vehicle-treated mice on day 4 postinfection, whereas daptomycin- and ceftriaxone-treated mice did not develop bacteremia during the observation period of 5 days. These data suggest that in contrast to pneumococcal sepsis, in pneumococcal pneumonia, the outcome appears to be critically dependent on the rapid elimination of the pathogen from the alveolar compartment to limit the degree of acute lung injury. Thus, unlike ceftriaxone, daptomycin demonstrated only weak and nonsignificant efficacy in improving survival in a mouse model of severe pneumococcal pneumonia induced by a serotype 19 S. pneumoniae strain.
So far, we have demonstrated that daptomycin is highly effective in preventing death induced by highly virulent serotype 2 S. pneumoniae but not serotype 19 S. pneumoniae with a low level of virulence. We next tested whether the highly invasive serotype 2 S. pneumoniae strain would trigger an increased lung barrier dysfunction in mice, thereby possibly improving the bioavailability of the antibiotic within lung parenchymal tissue. Because not all vehicle-treated mice challenged with 1.5 × 107 CFU/mouse developed septic disease progression, we used our biophotonic imaging approach to specifically identify those mice demonstrating progression from pneumonia to sepsis after infection with serotype 2 S. pneumoniae (Fig. (Fig.5A,5A, vehicle) and to identify those mice developing only pneumococcal pneumonia from day 2 to day 3 of infection with serotype 19 S. pneumoniae (Fig. (Fig.5B,5B, vehicle). Unexpectedly, we found that mice analyzed at day 3 of infection (day 1 after the beginning of daptomycin treatment) with serotype 19 S. pneumoniae with a low level of virulence responded with five- to sixfold higher levels of lung permeability compared with that for mice infected with highly virulent serotype 2 S. pneumoniae, as exemplified in Fig. 5A and B and as summarized in Fig. Fig.5C.5C. Interestingly, such differences in lung permeability were not due to the differences in neutrophil counts observed in the BAL fluids of serotype 2 or 19 S. pneumoniae-infected mice (Fig. (Fig.5D).5D). Importantly however, serotype 19 S. pneumoniae-infected mice but not serotype 2 S. pneumoniae-infected mice demonstrated increased daptomycin levels in their BAL fluids over time when they were analyzed at 0.5 h, 2 h, and 4 h immediately after the second application of daptomycin on day 2 of treatment (i.e., day 3 postinfection) (Fig. (Fig.5E),5E), while at the same time, no overt differences in serum daptomycin levels were noted between the groups (Fig. (Fig.5F).5F). These data suggest that the increased lung permeability induced by serotype 19 S. pneumoniae actually leads to increased daptomycin access to the bronchoalveolar compartment.
In the current study, we investigated the efficacy of the cyclic lipopeptide daptomycin in two mouse models of either pneumococcal pneumonia induced by a serotype 19 S. pneumoniae strain with a low level of virulence or pneumococcal sepsis induced by a highly virulent serotype 2 S. pneumoniae strain under both semitherapeutic and therapeutic treatment conditions. Semitherapeutic treatment with daptomycin was highly effective in inhibiting the septic disease progression in mice induced by serotype 2 S. pneumoniae, leading to 100% survival of the mice after a low-dose or even a high-dose bacterial challenge. Under therapeutic treatment conditions, daptomycin was as effective as the comparator substance, ceftriaxone, at inhibiting septic disease progression in mice after challenge with a high dose of a serotype 2 S. pneumoniae strain. Importantly, although daptomycin was not as effective as ceftriaxone in rapidly purging the bacterial loads from the lung parenchymal tissue of serotype 2 S. pneumoniae-infected mice, such residual bacterial loads had no influence on the overall survival of the daptomycin-treated mice. In sharp contrast, daptomycin was not found to be effective for the treatment of pneumococcal pneumonia induced by a serotype 19 S. pneumoniae strain with a low level of virulence. Even the semitherapeutic application of daptomycin failed to purge the bacterial loads from the lungs of mice in response to a low-dose or a high-dose challenge with serotype 19 S. pneumoniae. Consequently, daptomycin treatment of mice infected with serotype 19 S. pneumoniae resulted in a survival rate of 40%, whereas 100% survival was observed in the ceftriaxone-treated mice. These data show that the novel cyclic lipopeptide daptomycin demonstrates little efficacy for the treatment of low-virulence serotype 19 S. pneumoniae-induced pneumococcal pneumonia but proves to be highly effective for the treatment of life-threatening septic pneumococcal disease progression subsequent to pulmonary infection with highly virulent serotype 2 S. pneumoniae.
To the best of our knowledge, the current study is the first to provide detailed information about the efficacy of the antibiotic substance daptomycin for the treatment of S. pneumoniae-induced lung infections with distinct clinical courses. The pathogenicity profiles of S. pneumoniae differ significantly between and among serotypes. This fact is evidenced by the clinical observation that some pneumococcal lung infections cause focal pneumonia, sinusitis, or otitis media in the absence of a septic component, while others trigger severe septic complications (33). Just the septic component of S. pneumoniae-induced lung infection causes significant morbidity and mortality rates worldwide and is the most frequent severe complication observed in patients with CAP. The results of studies investigating the efficacy of daptomycin in pneumococcal infection models currently available are limited, in our view, because in those studies the efficacies of daptomycin were evaluated only in animal models (or the clinical condition) of S. pneumoniae-induced CAP and not in animal models of septic pneumococcal disease progression (23, 26). Thus, in view of the various pathogenicity profiles of different pneumococcal strains, novel antibiotic substances with activities against S. pneumoniae should be evaluated with defined models representing the major clinical phenotypes of lung infections with S. pneumoniae, i.e., pneumococcal pneumonia and septic pneumococcal disease.
Silverman and colleagues recently reported a lack of efficacy of daptomycin in a mouse model of bronchoalveolar pneumonia, although no results of time-response analyses of bacterial clearance or survival were reported (26). The data from the current study extend the findings presented in that report by demonstrating that daptomycin is not effective in eliminating serotype 19 S. pneumoniae from the lungs of mice during an observation period of more than 5 days. In addition, we found that only weak but nonsignificant protection from S. pneumoniae-induced death triggered by a high-dose challenge of mice with serotype 19 S. pneumoniae could be achieved by daptomycin treatment. One possible reason for the reduced ability of daptomycin to kill pneumococci within the lungs has been related to its interference with surfactant components (26). Another aspect might be the bioavailability of daptomycin in the lungs of mice, which, similar to its bioavailability in the brain, appears to be limited, possibly due to the tight alveolocapillary barrier (7). Importantly, we found for the first time that daptomycin levels are, indeed, very low in the BAL fluids of uninfected control mice, despite high serum daptomycin levels. These data taken alone strongly support the argument that the access of daptomycin to the bronchoalveolar space is limited under baseline conditions. Importantly, we observed that it was not the highly virulent serotype 2 S. pneumoniae strain but, rather, was the less virulent serotype 19 S. pneumoniae strain that significantly increased the lung permeability on day 3 postinfection, leading to the increased access of daptomycin to the bronchoalveolar space as early as 0.5 h, 2 h, and 4 h after the application of daptomycin on day 3 of infection. Taken together, these observations allow three important conclusions to be made. (i) Under baseline conditions, daptomycin access to the bronchoalveolar space appears to be limited. (ii) Different S. pneumoniae serotypes causing different degrees of lung permeability in mice differentially promoted the bioavailability of daptomycin in the distal lung airspaces, which may be of particular importance for broad-spectrum lipopeptide preparations exhibiting less surfactant interference (3). (iii) The lack of efficacy of daptomycin in curing serotype 19 S. pneumoniae-induced pneumonia in mice was not due to the principal lack of the bioavailability of the compound in distal lung airspaces but, rather, was most probably due to its inactivation by the lung surfactant system.
Serotype 2 S. pneumoniae is known to rapidly trigger invasive pneumococcal disease and death in mice, which appears to be correlated with the initial infection dose (18). In the current study, we found that a high-dose challenge of mice with highly virulent serotype 2 S. pneumoniae caused only a weak increase in lung permeability in mice, which was surprising in view of the severe bacteremia that developed in these mice, as also evidenced by biophotonic imaging. Although daptomycin was highly efficacious in improving the survival of mice challenged with serotype 2 S. pneumoniae under both semitherapeutic and therapeutic treatment conditions, daptomycin did not prove as efficacious as the comparator agent, ceftriaxone, in purging serotype 2 pneumococcal loads within the lung itself. However, such residual bacterial colonization of distal airspaces with serotype 2 S. pneumoniae did not affect the overall rate of survival of the mice, and the termination of daptomycin treatment after day 5 postinfection with serotype 2 S. pneumoniae did not again trigger invasive disease in these mice during an additional observation period of 14 days (data not shown), most probably due to the infection-induced protective immune responses of the lung.
The data from the current study provide three clinically important conclusions. First, in those patients with pneumococcal lung infections in whom, according to clinical criteria, progression from pneumonia to invasive pneumococcal disease occurs, daptomycin should significantly reduce the rate of sepsis-related mortality when it is given as part of combination therapy with a lung-active antibiotic. Importantly, contrary to cell wall-active beta-lactam antibiotics (13), daptomycin, due to its mode of action, has no or little immunomodulatory activity that might further aggravate the proinflammatory cytokine storms that are part of the systemic inflammatory response to invasive pneumococcal infections, which is an important aspect in the management of sepsis therapy (32). Second, when it is considered that daptomycin therapy was more effective against serotype 2 pneumococcal infections than serotype 19 pneumococcal infections of the lung, information about the serotype of the causative pathogen might possibly affect the future design of antibiotic strategies in patients, although one must keep in mind that the invasive capacity of a given serotype is also influenced by noncapsular virulence factors. As such, the data from the present study strongly suggest that daptomycin is very likely to be of clinical relevance for the treatment of pneumococcal infections induced by highly virulent serotypes/clones and, possibly, for the treatment of immunocompromised patients known to frequently develop severe complications in response to relatively harmless bacterial infections (34). Third, the observation that the less invasive S. pneumoniae serotypes causing lobar pneumonia in mice significantly increased the low baseline bioavailability of daptomycin within the lung distal air spaces might favor the efficacy profile of broad-spectrum lipopeptides showing decreased or even no surfactant interference for the treatment of lobar pneumonias in humans.
Collectively, the study described here provides a detailed investigation of the efficacy profile of the antibiotic substance daptomycin for the treatment of pneumococcal pneumonia compared to that for the treatment of invasive pneumococcal disease. We provide experimental evidence that in response to low-dose and high-dose infections with S. pneumoniae and under both semitherapeutic and therapeutic treatment conditions, daptomycin is particularly efficacious for the treatment of pneumococcal sepsis but not pneumococcal pneumonia. Thus, we believe that daptomycin may have the potential to be used for combination therapy with a lung-active antibiotic, particularly for patients with life-threatening septic complications of community-acquired Gram-positive bacterial infections of the lung.
This study was financially supported by Novartis Pharma.
Published ahead of print on 16 November 2009.