- Email: [email protected]
Low-dose aerosol model of pneumococcal pneumonia in the mouse: utility for evaluation of antimicrobial efficacy
International Journal of Antimicrobial Agents 26 (2005) 497–503
Low-dose aerosol model of pneumococcal pneumonia in the mouse: utility for evaluation of antimicrobial efficacy夽 Eric Nuermberger a,∗ , Kris Helke b , William R. Bishai a b
a Division of Infectious Diseases, Department of Medicine, 1503 E. Jefferson St., Baltimore, MD 21231, USA Departments of Comparative Medicine and Pathology, 811 Broadway Research Building, Baltimore, MD 21205, USA
Received 20 January 2005; accepted 31 August 2005
Abstract Current mouse models of pneumococcal infection have two disadvantages: (1) those that are not based on lung infections do not take into account the tissue pharmacokinetics of drugs in the lung parenchyma; and (2) those that are pneumonia models typically use large infectious doses to produce fulminant infections. The objective of this study was to determine the utility of a low-dose aerosol pneumonia model for evaluation of antimicrobial efficacy. Mice infected with penicillin-susceptible or non-susceptible pneumococci were left untreated or treated for 2.5 days with ertapenem in a range of doses. Efficacy was determined by the change in log10 colony-forming unit (CFU) counts and survival. Low-dose aerosol infection with the penicillin-susceptible strain 6303 produced an indolent pneumonia that was reliably lethal 1–2 weeks after infection. Ertapenem demonstrated bactericidal activity and prevented mortality over a range of doses after infection with strain 6303, but demonstrated only bacteriostatic activity at the highest doses used against the more resistant 1980 strain. A beneficial effect on survival was seen at doses approaching bioequivalence with the standard human dosage. The low-dose aerosol model of pneumococcal pneumonia in the mouse is a viable alternative model for the evaluation of antimicrobial efficacy. It may be particularly useful in the evaluation of drugs that concentrate in the alveolar epithelial lining fluid or lung parenchyma. © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Streptococcus pneumoniae; Carbapenem; Ertapenem; Antibiotic
1. Introduction Streptococcus pneumoniae is the most common aetiological agent of community-acquired pneumonia (CAP), a disease that causes 2–3 million infections each year in the USA alone [1]. The dramatic increase in the prevalence of antimicrobial resistance among pneumococcal isolates is therefore cause for alarm. Today, up to one-third of US isolates have reduced susceptibility to penicillin [2,3]. There is also an increasing prevalence of resistance to agents currently recommended for empirical treatment of CAP. Nearly 15% of isolates are now resistant to ceftriaxone, 25% are resistant to 夽 A portion of these results were presented at the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy, September, 2002, San Diego, CA, USA. ∗ Corresponding author. Tel.: +1 410 502 0580; fax: +1 410 614 8173. E-mail address: [email protected] (E. Nuermberger).
macrolides [3] and 1.5–2% of adult isolates are resistant to levofloxacin [4]. It is difficult to assess accurately the impact of in vitro antimicrobial resistance on clinical outcomes for CAP caused by drug-resistant S. pneumoniae (DRSP) through epidemiological studies. When properly conducted, studies in animal models of DRSP infection can provide critical information on antimicrobial efficacy that is directly relevant to the treatment of human infections [5]. Murine models of pneumococcal infection currently used to evaluate antimicrobial efficacy include the neutropenic thigh infection model [6,7], the intraperitoneal infection model [8] and intranasal or intratracheal instillation [9–13]. Each model has its disadvantages. The two former models are not based on lung infections and therefore do not take into account achievable concentrations of drugs in the lung parenchyma that are expected to contribute to efficacy against pneumonia, including pneumonia caused by the extracellular pathogen
0924-8579/$ – see front matter © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2005.08.022
498
E. Nuermberger et al. / International Journal of Antimicrobial Agents 26 (2005) 497–503
S. pneumoniae. Although it is a model of pneumonia, the intratracheal instillation model has typically involved a large infectious dose (5 × 106 to 5 × 108 colony-forming units (CFUs)) and resulted in a rapidly progressive infection with the onset of bacteraemia within 24–48 h and death within 72 h [9,10,13,14]. Under the conditions of such a fulminant infection, it is possible that the outcome of therapy is determined primarily by attainable drug concentrations in serum rather than concentrations achieved at the site of primary infection in the lung parenchyma. Intranasal instillation of a smaller infectious dose (105 –106 CFUs) has been used successfully to create a more indolent infection, although a number of pneumococcal strains tested still resulted in mortality rates of 50% or greater within 96 h [15,16]. All of the above models are also disadvantageous in that each animal must be infected individually by skilled personnel, making the infection step especially labour intensive. Drawing on our experience with aerosol infection for murine models of tuberculosis, we hypothesised that aerosol infection of mice with S. pneumoniae could deliver a smaller infectious dose to the lungs and produce a more indolent form of pneumonia than the intratracheal instillation model. Indolent, but ultimately lethal, pneumococcal infection of mice via the aerosol route has been described previously [17]; however, the model was not well characterised at the time and, to our knowledge, has never been used for evaluation of antimicrobial efficacy. Aerosol infection could also be a labour-saving model, since up to 125 mice can be infected simultaneously using our apparatus. Based on these hypotheses, we set out to create a low-dose aerosol infection model of pneumococcal pneumonia in the mouse as an alternative to existing models. We sought to demonstrate the utility of the model by using it to evaluate the efficacy of a newly available antibiotic, ertapenem. Ertapenem is a new once-daily carbapenem recently approved for the treatment of CAP caused by penicillin-susceptible S. pneumoniae (PSSP). Like ceftriaxone, its pharmacokinetic profile allows once-daily administration. Efficacy and safety similar to ceftriaxone have been demonstrated in two randomised, double-blind multicentre trials [18,19]. We have evaluated the efficacy of ertapenem both against PSSP and penicillin-resistant S. pneumoniae (PRSP) infections in our model.
2.2. Bacterial isolates Two S. pneumoniae strains were used in this study: ATCC strain 6303 (a type III strain) and strain 1980, a clinical isolate obtained from the clinical microbiology laboratory at Johns Hopkins Hospital that was not typed. Ertapenem susceptibility was determined by Etest (AB Biodisk, Solna, Sweden) and penicillin susceptibility was determined according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines. Both tests were performed in the clinical microbiology laboratory at Johns Hopkins Hospital. 2.3. Animals Six-to-eight-week-old female CBA/J mice, weighing 17–19 g, were purchased from Jackson Laboratories (Bar Harbor, ME). The mice were allowed to acclimate to laboratory conditions for least 4 days before use. Neutropenia was induced by intraperitoneal administration of cyclophosphamide (Mead Johnson, Princeton, NJ) at a dose of 150 mg/kg daily for 3 days beginning 3 days prior to infection, a regimen that results in neutropenia for 3–5 days after infection. All animal care and experimental manipulations were approved by the institutional Animal Care and Use Committee. 2.4. Low-dose aerosol infection Broth cultures of the test organism were grown overnight from frozen stocks in brain–heart infusion (BHI) broth (Difco, Detroit, MI) supplemented with 10% equine serum (HyClone, Logan, UT). On the morning of infection, a 1 mL aliquot was transferred to fresh BHI broth. When the culture reached log phase with an optical density at 600 nm of 0.3–0.4, a volume of 100 mL was centrifuged (3500 rpm for 20 min at 37 ◦ C) and the pellet was re-suspended in 10 mL of phosphate-buffered saline (PBS). The titre of this suspension (estimated at 1010 CFUs/mL) was determined by plating serial dilutions on Columbia blood agar plates (Fisher, Pittsburgh, PA). The suspension was then used to infect mice with 103.5 –104 CFUs using the Inhalation Exposure System (Glas-Col, Terre Haute, IN), a self-contained programmable apparatus that uses a nebuliser–Venturi unit to generate a uniform infectious aerosol from liquid cultures. 2.5. Natural history of infection in untreated animals
2. Materials and methods 2.1. Antimicrobial agents Ertapenem powder (lot no. 002C089) was kindly provided by Merck Research Laboratories (Rahway, NJ). Drug solutions were prepared on the day of infection in sterile distilled water, frozen in aliquots at −70 ◦ C and thawed immediately before use.
Neutropenic and non-neutropenic mice were infected with strain 6303 as described above. Two mice were sacrificed immediately before infection to serve as pre-infection controls. Four mice were sacrificed within 15 min of infection and then four mice daily thereafter. Two of the four mice were used for lung and spleen cultures. The two remaining mice were used for histopathological assessment. For quantitative lung CFU counts, the lungs were removed aseptically, mixed with 5 mL of sterile PBS and homogenised
E. Nuermberger et al. / International Journal of Antimicrobial Agents 26 (2005) 497–503
using the Stomacher 80 lab system (Seward, London, UK). Serial dilutions of the homogenates were plated on Columbia blood agar plates (Fisher) and incubated at 37 ◦ C with 5% CO2 . CFU counts were determined the following day. The lower limit of detection was 50 CFU per organ. The spleens from the same mice were removed aseptically and homogenised in 5 mL PBS before plating on blood agar. The lower limit of detection was 25 CFU per organ. For histopathological assessment, lungs were removed en bloc and perfused with neutral buffered formalin (NBF) solution (10%). After fixation in NBF for a minimum of 48 h, lungs were sectioned and embedded in paraffin. Sections were cut to 5 m thickness and placed on negatively-charged glass slides. The sections were then stained with haematoxylin and eosin (H&E) and a tissue Gram stain. The slides were then reviewed in a blinded, systematic fashion by a veterinary pathologist (K.H.), with specific attention to the degree of alveolar haemorrhage and oedema, inflammatory cell infiltration and pleural reaction. 2.6. Therapeutic efficacy of ertapenem as determined by lung CFU counts Mice were randomised immediately after aerosol infection (Day 0) into treatment groups to receive no treatment or ertapenem doses ranging from 2–200 mg/kg administered every 12 h (q12h) or every 6 h (q6h). Treatment commenced on Day 3 post infection for mice infected with the 6303 strain and on Day 1 post infection for mice infected with the 1980 strain. Antibiotics were administered by intraperitoneal injection for 2.5 days to achieve a total of five or ten doses depending on the dosing frequency. Five mice in the untreated control group were sacrificed prior to the initiation of treatment for baseline CFU counts from lung homogenates as described above. Five mice from each group were sacrificed daily during treatment for CFU counts from lung homogenates to determine the effect of antimicrobial therapy.
499
2.8. Statistical analysis All CFU counts were log-transformed before analysis. Multiple comparisons between group mean log10 CFU counts were made by one-way analysis of variance (ANOVA) with Bonferroni’s post-test. Survival curves were generated using the Kaplan–Meier method. Individual curves were compared using the Mantel–Haenszel test and, for each pneumococcal isolate, the survival trend associated with increasing doses of ertapenem was analysed by the log-rank test for trend. All analyses were performed using GraphPad Prism v.4 (GraphPad Software, San Diego, CA). 3. Results 3.1. Minimum inhibitory concentrations (MICs) for S. pneumoniae strains MICs of penicillin against strains 6303 and 1980 were ≤0.03 g/mL and 3 g/mL, respectively. MICs of ertapenem were 0.008 g/mL and 1 g/mL, respectively. 3.2. Natural history of infection in untreated mice In three separate time course studies, aerosol infection of neutropenic mice with strain 6303 produced an indolent, though ultimately lethal, pneumonia, whereas normal mice routinely cleared the infection. The log10 CFU counts from a representative time course experiment are presented in Fig. 1. After infection with 3.9 log10 CFUs, lung counts increased in surviving neutropenic mice over ensuing days, reaching 6.4 log10 CFUs by 3 days and 7.9 logs by 6 days post infection (Fig. 1). Cultures of spleen homogenates were performed as a measure of disseminated infection. As seen in Fig. 1, spleen cultures were negative for the first 3 days. Dissemination
2.7. Therapeutic efficacy of ertapenem as determined by survival Seven to nine mice per treatment group were randomly chosen at Day 0 for survival analysis. These mice were caged separately from mice to be sacrificed for CFU counts and monitored for 21 days after infection to measure the effect of antibiotic treatment on survival. To reduce the potential for prolonged suffering, mice were euthanized if they demonstrated signs of irreversibly fatal infection: reduced activity, hunched posture, piloerection and/or tachypnoea. Mice were assessed every 6 h during the treatment phase and once daily thereafter. Deaths by natural infection and those following euthanasia were considered as the same endpoint for survival analysis.
Fig. 1. Natural history of aerosol pneumococcal infection. Change in lung log10 colony-forming unit (CFU) counts (solid lines) and proportion of positive spleen cultures (dotted line) over time after aerosol infection with Streptococcus pneumoniae strain 6303 in neutropenic and normal CBA/J mice. Lower limit of detection for lung counts was 1.7 log10 CFUs. Error bars denote standard deviations.
500
E. Nuermberger et al. / International Journal of Antimicrobial Agents 26 (2005) 497–503
of infection from the lung occurred in one-half of the neutropenic animals on Days 4 and 5, and in all of the animals by Day 6. Normal mice had culture-negative spleens at each time point. Microscopic examination of lung samples from each time course experiment demonstrated similar results. Lung tissue taken just before and within 1 h after aerosol infection (Day 0) revealed normal lung architecture (Fig. 2A). Initial foci of alveolitis were noted by Day 1 and were characterised by leakage of red blood cells, mild interstitial oedema and increased cellularity in the interstitium. Modest progression was seen on Day 2, with increasing haemorrhage and interstitial oedema (Fig. 2B). Peribronchiolar oedema was evident, as was proliferation of Type II pneumocytes and intra-alveolar macrophages in the alveoli. Rare diplococci adherent to the alveolar epithelium were visible on Gramstained specimens (Fig. 2B, inset). Infiltration of neutrophils began between Day 3 and Day 5, giving rise to patchy but widespread foci of inflammation that were both peripheral (with occasional pleural reaction) and peribronchiolar in distribution (Fig. 2C). Gram-stained specimens demonstrated numerous Gram-positive diplococci in these areas (Fig. 2C, inset). With the increasing influx of neutrophils, these lesions progressed over Days 4–6, with increasing haemorrhage, consolidation and necrosis (Fig. 2D). In general, the bacterial burden was highest in the most severe lesions. Lobar involvement was common and translocation of infection to the pleural space occurred less commonly during this period. By Day
6, perivascular infiltrates were apparent in most samples, concomitant with the presence of disseminated infection. 3.3. Efficacy of ertapenem against pneumonia caused by PSSP The efficacy of ertapenem over a range of doses (2, 10 and 50 mg/kg q12h) was determined against the PSSP strain 6303. Two outcome measures were used: reduction in lung log10 CFU counts and survival. The bactericidal activity of each antimicrobial regimen was demonstrated by a ≥3 log10 reduction in mean log10 CFU counts relative to pre-treatment controls after 24 h of treatment (P < 0.01 vs. pre-treatment controls for the 2 mg/kg and 50 mg/kg doses; P < 0.05 for the 10 mg/kg dose) (Fig. 3). The bactericidal activity of ertapenem increased with the size of the dose in that all mice treated with 50 mg/kg were culture-negative within 24 h of treatment, whilst two and one of the five mice from the 2 mg/kg and 10 mg/kg groups, respectively, were still culturepositive after 48 h. Mortality occurred in 88% of untreated mice between 7 and 13 days post infection, after which point no further deaths occurred (median time to death, 10.5 days). One mouse in the group receiving ertapenem at 50 mg/kg died shortly after the first dose and was censored. Treatment with any dose of ertapenem resulted in improved survival (P = 0.0002) (Fig. 4). No significant differences were noted between the antibiotic regimens.
Fig. 2. Histopathological appearance of mouse lung following induction of transient neutropenia and infection with a mouse-passaged isolate of Streptococcus pneumoniae strain 6303. (A) Day 0, following sham infection (H&E, 100×). (B) Day 2 after aerosol infection with S. pneumoniae (H&E, 100×). (C) Day 3 after aerosol infection (H&E, 100×). (D) Day 4 after aerosol infection (H&E, 100×). Size bars are 100 m in length. Insets are representative tissue Gram stains at higher magnification (400×), showing Gram-positive diplococci (arrowheads).
E. Nuermberger et al. / International Journal of Antimicrobial Agents 26 (2005) 497–503
Fig. 3. Log10 colony-forming unit (CFU) counts in lungs of neutropenic mice after aerosol infection with Streptococcus pneumoniae strain 6303 and either no treatment () or treatment with ertapenem at 2 mg/kg (), 10 mg/kg () or 50 mg/kg () every 12 h. Treatment with the indicated regimen was administered intraperitoneally for a total of five doses beginning on Day 3. Error bars denote standard deviations. Lower limit of detection was 1.7 log10 CFUs.
3.4. Efficacy of ertapenem against pneumonia caused by PRSP The efficacy of ertapenem over a wider range of doses (2–200 mg/kg given q12h or q6h) was determined against the PRSP strain 1980. The activity of ertapenem was dose dependent (Fig. 5). When given only twice daily (q12h), ertapenem dosages up to 100 mg/kg failed to prevent increases in the log10 CFU count over the first 24 h of treatment. Increasing the frequency of administration to give ertapenem at a dosage of 100 mg/kg q6h produced bacteriostatic activity, but was not statistically significantly better than no treatment, although the number of mice in each group was small. An ertapenem dosage of 200 mg/kg q6h resulted in a 0.7 log10 reduction in CFU counts in the first 24 h and was significantly different from no treatment (P < 0.05).
501
Fig. 4. Survival of mice following aerosol infection with Streptococcus pneumoniae strain 6303 followed by either no treatment () or by treatment with ertapenem at 2 mg/kg (), 10 mg/kg () or 50 mg/kg () every 12 h. Ertapenem was administered intraperitoneally for a total of five doses beginning on Day 3. The mouse counted dead on Day 4 died shortly after the first dose and was not considered a treatment failure in the survival analysis.
The mortality rate was 88% in untreated animals (median time to death, 3 days). Ertapenem had a more significant dosedependent effect on survival (P < 0.0001 for the trend), even when significant reductions in log10 CFU counts at 24 h were not apparent (Fig. 6). Mice receiving ertapenem dosages less than 100 mg/kg q12h died at rates that were not significantly different from those of untreated animals (median time to death, 3–4 days). At 100 mg/kg q12h, ertapenem resulted in a statistically significant improvement in survival over doses of 50 mg/kg or less (P < 0.005), but only dosages of 100 mg/kg or 200 mg/kg q6h completely prevented mortality.
4. Discussion Surveillance studies have shown that at any given time, 5–10% of immunocompetent adults harbour viable S. pneumoniae in the nasopharynx [20]. Pneumonia is believed to result from microaspiration of these colonising organisms into the distal airspaces. Proliferation of bacteria incites an inflammatory response to produce an initial alveolitis, then
Fig. 5. Change in log10 colony-forming units (CFUs) in lungs of neutropenic mice after aerosol infection with penicillin-susceptible Streptococcus pneumoniae strain 6303 and penicillin-resistant strain 1980 following 24 h of treatment with ertapenem (E) at the indicated dosage (in mg/kg). Error bars denote standard deviations.
502
E. Nuermberger et al. / International Journal of Antimicrobial Agents 26 (2005) 497–503
Fig. 6. Survival of mice following aerosol infection with Streptococcus pneumoniae strain 1980 followed by either no treatment (), or treatment with 2 mg/kg (), 10 mg/kg (䊉), 50 mg/kg () or 100 mg/kg () ertapenem every 12 h, or treatment with 100 mg/kg () or 200 mg/kg () ertapenem every 6 h. Ertapenem was administered for a total of 2.5 days beginning on Day 1.
local consolidation. At the time of clinical presentation, bacteraemia is present in 20% of patients [21], but the frequency in outpatients with CAP is certainly lower. The low-dose aerosol mouse model of pneumococcal pneumonia offers close parallels to its human counterpart. Mice infected by low-dose aerosol with S. pneumoniae strain 6303 developed an indolent, though ultimately fatal, pneumonia with no evidence of dissemination before the fourth day after infection. This model of primary pneumonia with delayed dissemination of infection allows for the institution of antimicrobial therapy before the animals become bacteraemic. As a model of isolated, non-bacteraemic pneumonia, it may be particularly appropriate for the evaluation of antimicrobials that are believed to concentrate in the alveolar epithelial lining fluid. For such drugs, evaluation in models that do not take intrapulmonary concentrations into account may be misleading [22]. A second advantage of our model is its convenience with regard to the infection of mice. Up to 125 mice may be infected simultaneously, rather than requiring individual handling by skilled personnel to create infection. We demonstrated the utility of the low-dose aerosol pneumonia model by evaluating the efficacy of ertapenem against infections caused by PSSP and PRSP strains. Ertapenem is highly active against PSSP in vitro [23]. Although PRSP isolates typically have reduced susceptibility to ertapenem, most isolates have MICs of 1–2 g/mL [23]. According to current NCCLS guidelines, pneumococci are considered susceptible to ertapenem when MICs are ≤1 g/mL and intermediately susceptible when the MIC is 2 g/mL. Although it was not our goal to validate these susceptibility breakpoints, our results are consistent with them. At a dosage of 200 mg/kg q6h, ertapenem had bacteriostatic activity against the PRSP isolate with an ertapenem MIC of 1 g/mL. These results are similar to those of other investigators who observed consistent bactericidal activity in a neutropenic thigh infection model against isolates for which MICs were <2 g/mL, but
only bacteriostatic activity against most isolates for which MICs were ≥2 g/mL [7]. These investigators administered an ertapenem dose of 50 mg/kg q6h to mice with renal impairment induced by uranyl nitrate and reduced clearance of the drug. This dosage best simulated ertapenem pharmacokinetics in humans for their model. Since the elimination half-life of ertapenem is expected to increase at least two- to three-fold in the setting of renal impairment induced by uranyl nitrate [7,24,25], our dosage of 200 mg/kg q6h is not excessive. Furthermore, both studies demonstrated that even doses without evident bactericidal activity reduced mortality in neutropenic mice. There are several legitimate criticisms of our model. First, pneumococcal pneumonia in humans follows aspiration of bacteria from the upper airways, not aerosol infection. Nevertheless, these aspiration events involve relatively small numbers of pneumococci that are expected to descend to the distal airways to incite infection. Our data show that the selective deposition of limited numbers of bacteria in the distal airways is more likely to result in a more indolent infection than intratracheal instillation, giving rise to an infection that is more representative of isolated, non-bacteraemic pneumonia. A second potential limitation of our model is the need to use mice that are neutropenic at the time of infection to obtain reliably lethal infections. Whilst a neutropenic mouse is not necessarily representative of most human hosts with pneumococcal pneumonia, it may afford the most accurate assessment of antimicrobial activity. Several groups of investigators have described enhancement of antimicrobial activity in non-neutropenic mice versus neutropenic mice [7,26,27]. The presence of neutrophils may, in fact, obscure significant differences in antimicrobial regimens. For example, in one of these studies, the use of non-neutropenic CBA/J mice did not allow differentiation of the effect of two different ertapenem doses, whilst the use of neutropenic mice did [7]. Furthermore, many drug-resistant strains come from pneumococcal serotypes such as 6, 14, 19 and 23 that are typically avirulent in mice [28]. Neutropenia provides a more level playing field for the evaluation of drug efficacy against such strains without necessarily facilitating dissemination of infection [29]. We originally hoped to avoid the use of immunosuppression and so chose to use inbred CBA/J mice, which are susceptible to infection with S. pneumoniae in the absence of neutropenia. However, many pneumococcal strains, especially drug-resistant isolates, will not cause a lethal infection in non-neutropenic mice via the aerosol route. In this report, we have demonstrated that effective antimicrobial drugs reduce lung CFU counts and improve survival in aerosol-infected mice. Differential efficacy was observed against infections with PSSP and PRSP strains. We therefore believe that the low-dose aerosol model of pneumococcal pneumonia in the mouse is a viable and time-saving alternative model for the evaluation of antimicrobial efficacy. It may be particularly useful in the evaluation of drugs that concentrate in the alveolar epithelial lining fluid or lung parenchyma.
E. Nuermberger et al. / International Journal of Antimicrobial Agents 26 (2005) 497–503
Acknowledgments Financial support was provided by Merck and Co., Inc. and NRSA# RR07002. Jim Dick, PhD, provided strain 1980 and supervised drug susceptibility testing. M. Chris Zink, DVM, PhD, gave valuable guidance on preparation of the manuscript.
References [1] Centers for Disease Control and Prevention. Premature deaths, monthly mortality and monthly physician contacts—United States. MMWR Morb Mortal Wkly Rep 1997;46:556–61. [2] Thornsberry C, Sahm DF, Kelly LJ, et al. Regional trends in antimicrobial resistance among clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in the United States: results from the TRUST Surveillance Program, 1999–2000. Clin Infect Dis 2002;34(Suppl. 1):S4–16. [3] Doern GV, Heilmann KP, Huynh HK, Rhomberg PR, Coffman SL, Brueggemann AB. Antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in the United States during 1999–2000, including a comparison of resistance rates since 1994–1995. Antimicrob Agents Chemother 2001;45:1721–9. [4] Karlowsky JA, Thornsberry C, Critchley IA, et al. Susceptibilities to levofloxacin in Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis clinical isolates from children: results from 2000–2001 and 2001–2002 TRUST studies in the United States. Antimicrob Agents Chemother 2003;47:1790–7. [5] Craig WA. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin Infect Dis 1998;26:1–10. [6] Andes D, Craig WA. In vivo activities of amoxicillin and amoxicillin–clavulanate against Streptococcus pneumoniae: application to breakpoint determinations. Antimicrob Agents Chemother 1998;42:2375–9. [7] Xuan D, Banevicius M, Capitano B, Kim MK, Nightingale C, Nicolau D. Pharmacodynamic assessment of ertapenem (MK-0826) against Streptococcus pneumoniae in a murine neutropenic thigh infection model. Antimicrob Agents Chemother 2002;46:2990–5. [8] Knudsen JD, Frimodt-Moller N, Espersen F. Experimental Streptococcus pneumoniae infection in mice for studying correlation of in vitro and in vivo activities of penicillin against pneumococci with various susceptibilities to penicillin. Antimicrob Agents Chemother 1995;39:1253–8. [9] Azoulay-Dupuis E, Vallee E, Veber B, Bedos JP, Bauchet J, Pocidalo JJ. In vivo efficacy of a new fluoroquinolone, sparfloxacin, against penicillin-susceptible and -resistant and multiresistant strains of Streptococcus pneumoniae in a mouse model of pneumonia. Antimicrob Agents Chemother 1992;36:2698–703. [10] Kim MK, Zhou W, Tessier PR, et al. Bactericidal effect and pharmacodynamics of cethromycin (ABT-773) in a murine pneumococcal pneumonia model. Antimicrob Agents Chemother 2002;46:3185–92. [11] Moine P, Vallee E, Azoulay-Dupuis E, et al. In vivo efficacy of a broad-spectrum cephalosporin, ceftriaxone, against penicillinsusceptible and -resistant strains of Streptococcus pneumoniae in a mouse pneumonia model. Antimicrob Agents Chemother 1994;38:1953–8. [12] Takashima K, Tateda K, Matsumoto T, et al. Establishment of a model of penicillin-resistant Streptococcus pneumoniae pneumonia in healthy CBA/J mice. J Med Microbiol 1996;45:319–22.
503
[13] Hoffman HL, Klepser ME, Ernst EJ, Petzold CR, Sa’adah LM, Doern GV. Influence of macrolide susceptibility on efficacies of clarithromycin and azithromycin against Streptococcus pneumoniae in a murine lung infection model. Antimicrob Agents Chemother 2003;47:739–46. [14] Bergeron Y, Ouellet N, Deslauriers AM, Simard M, Olivier M, Bergeron MG. Cytokine kinetics and other host factors in response to pneumococcal pulmonary infection in mice. Infect Immun 1998;66:912–22. [15] Dallaire F, Ouellet N, Bergeron Y, et al. Microbiological and inflammatory factors associated with the development of pneumococcal pneumonia. J Infect Dis 2001;184:292–300. [16] Tateda K, Takashima K, Miyazaki H, Matsumoto T, Hatori T, Yamaguchi K. Noncompromised penicillin-resistant pneumococcal pneumonia CBA/J mouse model and comparative efficacies of antibiotics in this model. Antimicrob Agents Chemother 1996;40:1520–5. [17] Coil JA, Dickerman JD, Boulton E. Increased susceptibility of splenectomized mice to infection after exposure to an aerosolized suspension of type III Streptococcus pneumoniae. Infect Immun 1978;21:412–6. [18] Ortiz-Ruiz G, Caballero-Lopez J, Friedland IR, Woods GL, Carides A. A study evaluating the efficacy, safety, and tolerability of ertapenem versus ceftriaxone for the treatment of communityacquired pneumonia in adults. Clin Infect Dis 2002;34:1076–83. [19] Vetter N, Cambronero-Hernandez E, Rohlf J, et al. A prospective, randomized, double-blind multicenter comparison of parenteral ertapenem and ceftriaxone for the treatment of hospitalized adults with community-acquired pneumonia. Clin Ther 2002;24: 1770–85. [20] Musher DM. Streptococcus pneumoniae. In: Mandell GL, Bennett JE, Dolin R, editors. Principles and practice of infectious diseases. Philadelphia, PA: Churchill Livingstone; 2000. p. 2128–47. [21] Musher DM, Alexandraki I, Graviss EA, et al. Bacteremic and nonbacteremic pneumococcal pneumonia. A prospective study. Medicine (Baltimore) 2000;79:210–21. [22] Maglio D, Capitano B, Banevicius MA, Geng Q, Nightingale CH, Nicolau DP. Differential efficacy of clarithromycin in lung versus thigh infection models. Chemotherapy 2004;50:63–6. [23] Livermore DM, Sefton AM, Scott GM. Properties and potential of ertapenem. J Antimicrob Chemother 2003;52:331–44. [24] Van Ogtrop ML, Andes D, Craig WA. In vivo antimicrobial activity of MK-0826, a new carbapenem, against various penicillin-resistant pneumococci. In: Program and Abstracts of the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. F-48. [25] Gill CJ, Jackson JJ, Gerckens LS, et al. In vivo activity and pharmacokinetic evaluation of a novel long-acting carbapenem antibiotic, MK-826 (L-749,345). Antimicrob Agents Chemother 1998;42:1996–2001. [26] Mattoes HM, Banevicius M, Li D, et al. Pharmacodynamic assessment of gatifloxacin against Streptococcus pneumoniae. Antimicrob Agents Chemother 2001;45:2092–7. [27] Craig WA. Post-antibiotic effects in experimental infection models: relationship to in-vitro phenomena and to treatment of infections in man. J Antimicrob Chemother 1993;31(Suppl. D):149–58. [28] Briles DE, Crain MJ, Gray BM, Forman C, Yother J. Strong association between capsular type and virulence for mice among human isolates of Streptococcus pneumoniae. Infect Immun 1992;60: 111–6. [29] Wang E, Simard M, Ouellet N, Bergeron Y, Beauchamp D, Bergeron MG. Pathogenesis of pneumococcal pneumonia in cyclophosphamide-induced leukopenia in mice. Infect Immun 2002;70:4226–38.
Low-dose aerosol model of pneumococcal pneumonia in the mouse: utility for evaluation of antimicrobial efficacy夽 Eric Nuermberger a,∗ , Kris Helke b , William R. Bishai a b
a Division of Infectious Diseases, Department of Medicine, 1503 E. Jefferson St., Baltimore, MD 21231, USA Departments of Comparative Medicine and Pathology, 811 Broadway Research Building, Baltimore, MD 21205, USA
Received 20 January 2005; accepted 31 August 2005
Abstract Current mouse models of pneumococcal infection have two disadvantages: (1) those that are not based on lung infections do not take into account the tissue pharmacokinetics of drugs in the lung parenchyma; and (2) those that are pneumonia models typically use large infectious doses to produce fulminant infections. The objective of this study was to determine the utility of a low-dose aerosol pneumonia model for evaluation of antimicrobial efficacy. Mice infected with penicillin-susceptible or non-susceptible pneumococci were left untreated or treated for 2.5 days with ertapenem in a range of doses. Efficacy was determined by the change in log10 colony-forming unit (CFU) counts and survival. Low-dose aerosol infection with the penicillin-susceptible strain 6303 produced an indolent pneumonia that was reliably lethal 1–2 weeks after infection. Ertapenem demonstrated bactericidal activity and prevented mortality over a range of doses after infection with strain 6303, but demonstrated only bacteriostatic activity at the highest doses used against the more resistant 1980 strain. A beneficial effect on survival was seen at doses approaching bioequivalence with the standard human dosage. The low-dose aerosol model of pneumococcal pneumonia in the mouse is a viable alternative model for the evaluation of antimicrobial efficacy. It may be particularly useful in the evaluation of drugs that concentrate in the alveolar epithelial lining fluid or lung parenchyma. © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Streptococcus pneumoniae; Carbapenem; Ertapenem; Antibiotic
1. Introduction Streptococcus pneumoniae is the most common aetiological agent of community-acquired pneumonia (CAP), a disease that causes 2–3 million infections each year in the USA alone [1]. The dramatic increase in the prevalence of antimicrobial resistance among pneumococcal isolates is therefore cause for alarm. Today, up to one-third of US isolates have reduced susceptibility to penicillin [2,3]. There is also an increasing prevalence of resistance to agents currently recommended for empirical treatment of CAP. Nearly 15% of isolates are now resistant to ceftriaxone, 25% are resistant to 夽 A portion of these results were presented at the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy, September, 2002, San Diego, CA, USA. ∗ Corresponding author. Tel.: +1 410 502 0580; fax: +1 410 614 8173. E-mail address: [email protected] (E. Nuermberger).
macrolides [3] and 1.5–2% of adult isolates are resistant to levofloxacin [4]. It is difficult to assess accurately the impact of in vitro antimicrobial resistance on clinical outcomes for CAP caused by drug-resistant S. pneumoniae (DRSP) through epidemiological studies. When properly conducted, studies in animal models of DRSP infection can provide critical information on antimicrobial efficacy that is directly relevant to the treatment of human infections [5]. Murine models of pneumococcal infection currently used to evaluate antimicrobial efficacy include the neutropenic thigh infection model [6,7], the intraperitoneal infection model [8] and intranasal or intratracheal instillation [9–13]. Each model has its disadvantages. The two former models are not based on lung infections and therefore do not take into account achievable concentrations of drugs in the lung parenchyma that are expected to contribute to efficacy against pneumonia, including pneumonia caused by the extracellular pathogen
0924-8579/$ – see front matter © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2005.08.022
498
E. Nuermberger et al. / International Journal of Antimicrobial Agents 26 (2005) 497–503
S. pneumoniae. Although it is a model of pneumonia, the intratracheal instillation model has typically involved a large infectious dose (5 × 106 to 5 × 108 colony-forming units (CFUs)) and resulted in a rapidly progressive infection with the onset of bacteraemia within 24–48 h and death within 72 h [9,10,13,14]. Under the conditions of such a fulminant infection, it is possible that the outcome of therapy is determined primarily by attainable drug concentrations in serum rather than concentrations achieved at the site of primary infection in the lung parenchyma. Intranasal instillation of a smaller infectious dose (105 –106 CFUs) has been used successfully to create a more indolent infection, although a number of pneumococcal strains tested still resulted in mortality rates of 50% or greater within 96 h [15,16]. All of the above models are also disadvantageous in that each animal must be infected individually by skilled personnel, making the infection step especially labour intensive. Drawing on our experience with aerosol infection for murine models of tuberculosis, we hypothesised that aerosol infection of mice with S. pneumoniae could deliver a smaller infectious dose to the lungs and produce a more indolent form of pneumonia than the intratracheal instillation model. Indolent, but ultimately lethal, pneumococcal infection of mice via the aerosol route has been described previously [17]; however, the model was not well characterised at the time and, to our knowledge, has never been used for evaluation of antimicrobial efficacy. Aerosol infection could also be a labour-saving model, since up to 125 mice can be infected simultaneously using our apparatus. Based on these hypotheses, we set out to create a low-dose aerosol infection model of pneumococcal pneumonia in the mouse as an alternative to existing models. We sought to demonstrate the utility of the model by using it to evaluate the efficacy of a newly available antibiotic, ertapenem. Ertapenem is a new once-daily carbapenem recently approved for the treatment of CAP caused by penicillin-susceptible S. pneumoniae (PSSP). Like ceftriaxone, its pharmacokinetic profile allows once-daily administration. Efficacy and safety similar to ceftriaxone have been demonstrated in two randomised, double-blind multicentre trials [18,19]. We have evaluated the efficacy of ertapenem both against PSSP and penicillin-resistant S. pneumoniae (PRSP) infections in our model.
2.2. Bacterial isolates Two S. pneumoniae strains were used in this study: ATCC strain 6303 (a type III strain) and strain 1980, a clinical isolate obtained from the clinical microbiology laboratory at Johns Hopkins Hospital that was not typed. Ertapenem susceptibility was determined by Etest (AB Biodisk, Solna, Sweden) and penicillin susceptibility was determined according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines. Both tests were performed in the clinical microbiology laboratory at Johns Hopkins Hospital. 2.3. Animals Six-to-eight-week-old female CBA/J mice, weighing 17–19 g, were purchased from Jackson Laboratories (Bar Harbor, ME). The mice were allowed to acclimate to laboratory conditions for least 4 days before use. Neutropenia was induced by intraperitoneal administration of cyclophosphamide (Mead Johnson, Princeton, NJ) at a dose of 150 mg/kg daily for 3 days beginning 3 days prior to infection, a regimen that results in neutropenia for 3–5 days after infection. All animal care and experimental manipulations were approved by the institutional Animal Care and Use Committee. 2.4. Low-dose aerosol infection Broth cultures of the test organism were grown overnight from frozen stocks in brain–heart infusion (BHI) broth (Difco, Detroit, MI) supplemented with 10% equine serum (HyClone, Logan, UT). On the morning of infection, a 1 mL aliquot was transferred to fresh BHI broth. When the culture reached log phase with an optical density at 600 nm of 0.3–0.4, a volume of 100 mL was centrifuged (3500 rpm for 20 min at 37 ◦ C) and the pellet was re-suspended in 10 mL of phosphate-buffered saline (PBS). The titre of this suspension (estimated at 1010 CFUs/mL) was determined by plating serial dilutions on Columbia blood agar plates (Fisher, Pittsburgh, PA). The suspension was then used to infect mice with 103.5 –104 CFUs using the Inhalation Exposure System (Glas-Col, Terre Haute, IN), a self-contained programmable apparatus that uses a nebuliser–Venturi unit to generate a uniform infectious aerosol from liquid cultures. 2.5. Natural history of infection in untreated animals
2. Materials and methods 2.1. Antimicrobial agents Ertapenem powder (lot no. 002C089) was kindly provided by Merck Research Laboratories (Rahway, NJ). Drug solutions were prepared on the day of infection in sterile distilled water, frozen in aliquots at −70 ◦ C and thawed immediately before use.
Neutropenic and non-neutropenic mice were infected with strain 6303 as described above. Two mice were sacrificed immediately before infection to serve as pre-infection controls. Four mice were sacrificed within 15 min of infection and then four mice daily thereafter. Two of the four mice were used for lung and spleen cultures. The two remaining mice were used for histopathological assessment. For quantitative lung CFU counts, the lungs were removed aseptically, mixed with 5 mL of sterile PBS and homogenised
E. Nuermberger et al. / International Journal of Antimicrobial Agents 26 (2005) 497–503
using the Stomacher 80 lab system (Seward, London, UK). Serial dilutions of the homogenates were plated on Columbia blood agar plates (Fisher) and incubated at 37 ◦ C with 5% CO2 . CFU counts were determined the following day. The lower limit of detection was 50 CFU per organ. The spleens from the same mice were removed aseptically and homogenised in 5 mL PBS before plating on blood agar. The lower limit of detection was 25 CFU per organ. For histopathological assessment, lungs were removed en bloc and perfused with neutral buffered formalin (NBF) solution (10%). After fixation in NBF for a minimum of 48 h, lungs were sectioned and embedded in paraffin. Sections were cut to 5 m thickness and placed on negatively-charged glass slides. The sections were then stained with haematoxylin and eosin (H&E) and a tissue Gram stain. The slides were then reviewed in a blinded, systematic fashion by a veterinary pathologist (K.H.), with specific attention to the degree of alveolar haemorrhage and oedema, inflammatory cell infiltration and pleural reaction. 2.6. Therapeutic efficacy of ertapenem as determined by lung CFU counts Mice were randomised immediately after aerosol infection (Day 0) into treatment groups to receive no treatment or ertapenem doses ranging from 2–200 mg/kg administered every 12 h (q12h) or every 6 h (q6h). Treatment commenced on Day 3 post infection for mice infected with the 6303 strain and on Day 1 post infection for mice infected with the 1980 strain. Antibiotics were administered by intraperitoneal injection for 2.5 days to achieve a total of five or ten doses depending on the dosing frequency. Five mice in the untreated control group were sacrificed prior to the initiation of treatment for baseline CFU counts from lung homogenates as described above. Five mice from each group were sacrificed daily during treatment for CFU counts from lung homogenates to determine the effect of antimicrobial therapy.
499
2.8. Statistical analysis All CFU counts were log-transformed before analysis. Multiple comparisons between group mean log10 CFU counts were made by one-way analysis of variance (ANOVA) with Bonferroni’s post-test. Survival curves were generated using the Kaplan–Meier method. Individual curves were compared using the Mantel–Haenszel test and, for each pneumococcal isolate, the survival trend associated with increasing doses of ertapenem was analysed by the log-rank test for trend. All analyses were performed using GraphPad Prism v.4 (GraphPad Software, San Diego, CA). 3. Results 3.1. Minimum inhibitory concentrations (MICs) for S. pneumoniae strains MICs of penicillin against strains 6303 and 1980 were ≤0.03 g/mL and 3 g/mL, respectively. MICs of ertapenem were 0.008 g/mL and 1 g/mL, respectively. 3.2. Natural history of infection in untreated mice In three separate time course studies, aerosol infection of neutropenic mice with strain 6303 produced an indolent, though ultimately lethal, pneumonia, whereas normal mice routinely cleared the infection. The log10 CFU counts from a representative time course experiment are presented in Fig. 1. After infection with 3.9 log10 CFUs, lung counts increased in surviving neutropenic mice over ensuing days, reaching 6.4 log10 CFUs by 3 days and 7.9 logs by 6 days post infection (Fig. 1). Cultures of spleen homogenates were performed as a measure of disseminated infection. As seen in Fig. 1, spleen cultures were negative for the first 3 days. Dissemination
2.7. Therapeutic efficacy of ertapenem as determined by survival Seven to nine mice per treatment group were randomly chosen at Day 0 for survival analysis. These mice were caged separately from mice to be sacrificed for CFU counts and monitored for 21 days after infection to measure the effect of antibiotic treatment on survival. To reduce the potential for prolonged suffering, mice were euthanized if they demonstrated signs of irreversibly fatal infection: reduced activity, hunched posture, piloerection and/or tachypnoea. Mice were assessed every 6 h during the treatment phase and once daily thereafter. Deaths by natural infection and those following euthanasia were considered as the same endpoint for survival analysis.
Fig. 1. Natural history of aerosol pneumococcal infection. Change in lung log10 colony-forming unit (CFU) counts (solid lines) and proportion of positive spleen cultures (dotted line) over time after aerosol infection with Streptococcus pneumoniae strain 6303 in neutropenic and normal CBA/J mice. Lower limit of detection for lung counts was 1.7 log10 CFUs. Error bars denote standard deviations.
500
E. Nuermberger et al. / International Journal of Antimicrobial Agents 26 (2005) 497–503
of infection from the lung occurred in one-half of the neutropenic animals on Days 4 and 5, and in all of the animals by Day 6. Normal mice had culture-negative spleens at each time point. Microscopic examination of lung samples from each time course experiment demonstrated similar results. Lung tissue taken just before and within 1 h after aerosol infection (Day 0) revealed normal lung architecture (Fig. 2A). Initial foci of alveolitis were noted by Day 1 and were characterised by leakage of red blood cells, mild interstitial oedema and increased cellularity in the interstitium. Modest progression was seen on Day 2, with increasing haemorrhage and interstitial oedema (Fig. 2B). Peribronchiolar oedema was evident, as was proliferation of Type II pneumocytes and intra-alveolar macrophages in the alveoli. Rare diplococci adherent to the alveolar epithelium were visible on Gramstained specimens (Fig. 2B, inset). Infiltration of neutrophils began between Day 3 and Day 5, giving rise to patchy but widespread foci of inflammation that were both peripheral (with occasional pleural reaction) and peribronchiolar in distribution (Fig. 2C). Gram-stained specimens demonstrated numerous Gram-positive diplococci in these areas (Fig. 2C, inset). With the increasing influx of neutrophils, these lesions progressed over Days 4–6, with increasing haemorrhage, consolidation and necrosis (Fig. 2D). In general, the bacterial burden was highest in the most severe lesions. Lobar involvement was common and translocation of infection to the pleural space occurred less commonly during this period. By Day
6, perivascular infiltrates were apparent in most samples, concomitant with the presence of disseminated infection. 3.3. Efficacy of ertapenem against pneumonia caused by PSSP The efficacy of ertapenem over a range of doses (2, 10 and 50 mg/kg q12h) was determined against the PSSP strain 6303. Two outcome measures were used: reduction in lung log10 CFU counts and survival. The bactericidal activity of each antimicrobial regimen was demonstrated by a ≥3 log10 reduction in mean log10 CFU counts relative to pre-treatment controls after 24 h of treatment (P < 0.01 vs. pre-treatment controls for the 2 mg/kg and 50 mg/kg doses; P < 0.05 for the 10 mg/kg dose) (Fig. 3). The bactericidal activity of ertapenem increased with the size of the dose in that all mice treated with 50 mg/kg were culture-negative within 24 h of treatment, whilst two and one of the five mice from the 2 mg/kg and 10 mg/kg groups, respectively, were still culturepositive after 48 h. Mortality occurred in 88% of untreated mice between 7 and 13 days post infection, after which point no further deaths occurred (median time to death, 10.5 days). One mouse in the group receiving ertapenem at 50 mg/kg died shortly after the first dose and was censored. Treatment with any dose of ertapenem resulted in improved survival (P = 0.0002) (Fig. 4). No significant differences were noted between the antibiotic regimens.
Fig. 2. Histopathological appearance of mouse lung following induction of transient neutropenia and infection with a mouse-passaged isolate of Streptococcus pneumoniae strain 6303. (A) Day 0, following sham infection (H&E, 100×). (B) Day 2 after aerosol infection with S. pneumoniae (H&E, 100×). (C) Day 3 after aerosol infection (H&E, 100×). (D) Day 4 after aerosol infection (H&E, 100×). Size bars are 100 m in length. Insets are representative tissue Gram stains at higher magnification (400×), showing Gram-positive diplococci (arrowheads).
E. Nuermberger et al. / International Journal of Antimicrobial Agents 26 (2005) 497–503
Fig. 3. Log10 colony-forming unit (CFU) counts in lungs of neutropenic mice after aerosol infection with Streptococcus pneumoniae strain 6303 and either no treatment () or treatment with ertapenem at 2 mg/kg (), 10 mg/kg () or 50 mg/kg () every 12 h. Treatment with the indicated regimen was administered intraperitoneally for a total of five doses beginning on Day 3. Error bars denote standard deviations. Lower limit of detection was 1.7 log10 CFUs.
3.4. Efficacy of ertapenem against pneumonia caused by PRSP The efficacy of ertapenem over a wider range of doses (2–200 mg/kg given q12h or q6h) was determined against the PRSP strain 1980. The activity of ertapenem was dose dependent (Fig. 5). When given only twice daily (q12h), ertapenem dosages up to 100 mg/kg failed to prevent increases in the log10 CFU count over the first 24 h of treatment. Increasing the frequency of administration to give ertapenem at a dosage of 100 mg/kg q6h produced bacteriostatic activity, but was not statistically significantly better than no treatment, although the number of mice in each group was small. An ertapenem dosage of 200 mg/kg q6h resulted in a 0.7 log10 reduction in CFU counts in the first 24 h and was significantly different from no treatment (P < 0.05).
501
Fig. 4. Survival of mice following aerosol infection with Streptococcus pneumoniae strain 6303 followed by either no treatment () or by treatment with ertapenem at 2 mg/kg (), 10 mg/kg () or 50 mg/kg () every 12 h. Ertapenem was administered intraperitoneally for a total of five doses beginning on Day 3. The mouse counted dead on Day 4 died shortly after the first dose and was not considered a treatment failure in the survival analysis.
The mortality rate was 88% in untreated animals (median time to death, 3 days). Ertapenem had a more significant dosedependent effect on survival (P < 0.0001 for the trend), even when significant reductions in log10 CFU counts at 24 h were not apparent (Fig. 6). Mice receiving ertapenem dosages less than 100 mg/kg q12h died at rates that were not significantly different from those of untreated animals (median time to death, 3–4 days). At 100 mg/kg q12h, ertapenem resulted in a statistically significant improvement in survival over doses of 50 mg/kg or less (P < 0.005), but only dosages of 100 mg/kg or 200 mg/kg q6h completely prevented mortality.
4. Discussion Surveillance studies have shown that at any given time, 5–10% of immunocompetent adults harbour viable S. pneumoniae in the nasopharynx [20]. Pneumonia is believed to result from microaspiration of these colonising organisms into the distal airspaces. Proliferation of bacteria incites an inflammatory response to produce an initial alveolitis, then
Fig. 5. Change in log10 colony-forming units (CFUs) in lungs of neutropenic mice after aerosol infection with penicillin-susceptible Streptococcus pneumoniae strain 6303 and penicillin-resistant strain 1980 following 24 h of treatment with ertapenem (E) at the indicated dosage (in mg/kg). Error bars denote standard deviations.
502
E. Nuermberger et al. / International Journal of Antimicrobial Agents 26 (2005) 497–503
Fig. 6. Survival of mice following aerosol infection with Streptococcus pneumoniae strain 1980 followed by either no treatment (), or treatment with 2 mg/kg (), 10 mg/kg (䊉), 50 mg/kg () or 100 mg/kg () ertapenem every 12 h, or treatment with 100 mg/kg () or 200 mg/kg () ertapenem every 6 h. Ertapenem was administered for a total of 2.5 days beginning on Day 1.
local consolidation. At the time of clinical presentation, bacteraemia is present in 20% of patients [21], but the frequency in outpatients with CAP is certainly lower. The low-dose aerosol mouse model of pneumococcal pneumonia offers close parallels to its human counterpart. Mice infected by low-dose aerosol with S. pneumoniae strain 6303 developed an indolent, though ultimately fatal, pneumonia with no evidence of dissemination before the fourth day after infection. This model of primary pneumonia with delayed dissemination of infection allows for the institution of antimicrobial therapy before the animals become bacteraemic. As a model of isolated, non-bacteraemic pneumonia, it may be particularly appropriate for the evaluation of antimicrobials that are believed to concentrate in the alveolar epithelial lining fluid. For such drugs, evaluation in models that do not take intrapulmonary concentrations into account may be misleading [22]. A second advantage of our model is its convenience with regard to the infection of mice. Up to 125 mice may be infected simultaneously, rather than requiring individual handling by skilled personnel to create infection. We demonstrated the utility of the low-dose aerosol pneumonia model by evaluating the efficacy of ertapenem against infections caused by PSSP and PRSP strains. Ertapenem is highly active against PSSP in vitro [23]. Although PRSP isolates typically have reduced susceptibility to ertapenem, most isolates have MICs of 1–2 g/mL [23]. According to current NCCLS guidelines, pneumococci are considered susceptible to ertapenem when MICs are ≤1 g/mL and intermediately susceptible when the MIC is 2 g/mL. Although it was not our goal to validate these susceptibility breakpoints, our results are consistent with them. At a dosage of 200 mg/kg q6h, ertapenem had bacteriostatic activity against the PRSP isolate with an ertapenem MIC of 1 g/mL. These results are similar to those of other investigators who observed consistent bactericidal activity in a neutropenic thigh infection model against isolates for which MICs were <2 g/mL, but
only bacteriostatic activity against most isolates for which MICs were ≥2 g/mL [7]. These investigators administered an ertapenem dose of 50 mg/kg q6h to mice with renal impairment induced by uranyl nitrate and reduced clearance of the drug. This dosage best simulated ertapenem pharmacokinetics in humans for their model. Since the elimination half-life of ertapenem is expected to increase at least two- to three-fold in the setting of renal impairment induced by uranyl nitrate [7,24,25], our dosage of 200 mg/kg q6h is not excessive. Furthermore, both studies demonstrated that even doses without evident bactericidal activity reduced mortality in neutropenic mice. There are several legitimate criticisms of our model. First, pneumococcal pneumonia in humans follows aspiration of bacteria from the upper airways, not aerosol infection. Nevertheless, these aspiration events involve relatively small numbers of pneumococci that are expected to descend to the distal airways to incite infection. Our data show that the selective deposition of limited numbers of bacteria in the distal airways is more likely to result in a more indolent infection than intratracheal instillation, giving rise to an infection that is more representative of isolated, non-bacteraemic pneumonia. A second potential limitation of our model is the need to use mice that are neutropenic at the time of infection to obtain reliably lethal infections. Whilst a neutropenic mouse is not necessarily representative of most human hosts with pneumococcal pneumonia, it may afford the most accurate assessment of antimicrobial activity. Several groups of investigators have described enhancement of antimicrobial activity in non-neutropenic mice versus neutropenic mice [7,26,27]. The presence of neutrophils may, in fact, obscure significant differences in antimicrobial regimens. For example, in one of these studies, the use of non-neutropenic CBA/J mice did not allow differentiation of the effect of two different ertapenem doses, whilst the use of neutropenic mice did [7]. Furthermore, many drug-resistant strains come from pneumococcal serotypes such as 6, 14, 19 and 23 that are typically avirulent in mice [28]. Neutropenia provides a more level playing field for the evaluation of drug efficacy against such strains without necessarily facilitating dissemination of infection [29]. We originally hoped to avoid the use of immunosuppression and so chose to use inbred CBA/J mice, which are susceptible to infection with S. pneumoniae in the absence of neutropenia. However, many pneumococcal strains, especially drug-resistant isolates, will not cause a lethal infection in non-neutropenic mice via the aerosol route. In this report, we have demonstrated that effective antimicrobial drugs reduce lung CFU counts and improve survival in aerosol-infected mice. Differential efficacy was observed against infections with PSSP and PRSP strains. We therefore believe that the low-dose aerosol model of pneumococcal pneumonia in the mouse is a viable and time-saving alternative model for the evaluation of antimicrobial efficacy. It may be particularly useful in the evaluation of drugs that concentrate in the alveolar epithelial lining fluid or lung parenchyma.
E. Nuermberger et al. / International Journal of Antimicrobial Agents 26 (2005) 497–503
Acknowledgments Financial support was provided by Merck and Co., Inc. and NRSA# RR07002. Jim Dick, PhD, provided strain 1980 and supervised drug susceptibility testing. M. Chris Zink, DVM, PhD, gave valuable guidance on preparation of the manuscript.
References [1] Centers for Disease Control and Prevention. Premature deaths, monthly mortality and monthly physician contacts—United States. MMWR Morb Mortal Wkly Rep 1997;46:556–61. [2] Thornsberry C, Sahm DF, Kelly LJ, et al. Regional trends in antimicrobial resistance among clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in the United States: results from the TRUST Surveillance Program, 1999–2000. Clin Infect Dis 2002;34(Suppl. 1):S4–16. [3] Doern GV, Heilmann KP, Huynh HK, Rhomberg PR, Coffman SL, Brueggemann AB. Antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in the United States during 1999–2000, including a comparison of resistance rates since 1994–1995. Antimicrob Agents Chemother 2001;45:1721–9. [4] Karlowsky JA, Thornsberry C, Critchley IA, et al. Susceptibilities to levofloxacin in Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis clinical isolates from children: results from 2000–2001 and 2001–2002 TRUST studies in the United States. Antimicrob Agents Chemother 2003;47:1790–7. [5] Craig WA. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin Infect Dis 1998;26:1–10. [6] Andes D, Craig WA. In vivo activities of amoxicillin and amoxicillin–clavulanate against Streptococcus pneumoniae: application to breakpoint determinations. Antimicrob Agents Chemother 1998;42:2375–9. [7] Xuan D, Banevicius M, Capitano B, Kim MK, Nightingale C, Nicolau D. Pharmacodynamic assessment of ertapenem (MK-0826) against Streptococcus pneumoniae in a murine neutropenic thigh infection model. Antimicrob Agents Chemother 2002;46:2990–5. [8] Knudsen JD, Frimodt-Moller N, Espersen F. Experimental Streptococcus pneumoniae infection in mice for studying correlation of in vitro and in vivo activities of penicillin against pneumococci with various susceptibilities to penicillin. Antimicrob Agents Chemother 1995;39:1253–8. [9] Azoulay-Dupuis E, Vallee E, Veber B, Bedos JP, Bauchet J, Pocidalo JJ. In vivo efficacy of a new fluoroquinolone, sparfloxacin, against penicillin-susceptible and -resistant and multiresistant strains of Streptococcus pneumoniae in a mouse model of pneumonia. Antimicrob Agents Chemother 1992;36:2698–703. [10] Kim MK, Zhou W, Tessier PR, et al. Bactericidal effect and pharmacodynamics of cethromycin (ABT-773) in a murine pneumococcal pneumonia model. Antimicrob Agents Chemother 2002;46:3185–92. [11] Moine P, Vallee E, Azoulay-Dupuis E, et al. In vivo efficacy of a broad-spectrum cephalosporin, ceftriaxone, against penicillinsusceptible and -resistant strains of Streptococcus pneumoniae in a mouse pneumonia model. Antimicrob Agents Chemother 1994;38:1953–8. [12] Takashima K, Tateda K, Matsumoto T, et al. Establishment of a model of penicillin-resistant Streptococcus pneumoniae pneumonia in healthy CBA/J mice. J Med Microbiol 1996;45:319–22.
503
[13] Hoffman HL, Klepser ME, Ernst EJ, Petzold CR, Sa’adah LM, Doern GV. Influence of macrolide susceptibility on efficacies of clarithromycin and azithromycin against Streptococcus pneumoniae in a murine lung infection model. Antimicrob Agents Chemother 2003;47:739–46. [14] Bergeron Y, Ouellet N, Deslauriers AM, Simard M, Olivier M, Bergeron MG. Cytokine kinetics and other host factors in response to pneumococcal pulmonary infection in mice. Infect Immun 1998;66:912–22. [15] Dallaire F, Ouellet N, Bergeron Y, et al. Microbiological and inflammatory factors associated with the development of pneumococcal pneumonia. J Infect Dis 2001;184:292–300. [16] Tateda K, Takashima K, Miyazaki H, Matsumoto T, Hatori T, Yamaguchi K. Noncompromised penicillin-resistant pneumococcal pneumonia CBA/J mouse model and comparative efficacies of antibiotics in this model. Antimicrob Agents Chemother 1996;40:1520–5. [17] Coil JA, Dickerman JD, Boulton E. Increased susceptibility of splenectomized mice to infection after exposure to an aerosolized suspension of type III Streptococcus pneumoniae. Infect Immun 1978;21:412–6. [18] Ortiz-Ruiz G, Caballero-Lopez J, Friedland IR, Woods GL, Carides A. A study evaluating the efficacy, safety, and tolerability of ertapenem versus ceftriaxone for the treatment of communityacquired pneumonia in adults. Clin Infect Dis 2002;34:1076–83. [19] Vetter N, Cambronero-Hernandez E, Rohlf J, et al. A prospective, randomized, double-blind multicenter comparison of parenteral ertapenem and ceftriaxone for the treatment of hospitalized adults with community-acquired pneumonia. Clin Ther 2002;24: 1770–85. [20] Musher DM. Streptococcus pneumoniae. In: Mandell GL, Bennett JE, Dolin R, editors. Principles and practice of infectious diseases. Philadelphia, PA: Churchill Livingstone; 2000. p. 2128–47. [21] Musher DM, Alexandraki I, Graviss EA, et al. Bacteremic and nonbacteremic pneumococcal pneumonia. A prospective study. Medicine (Baltimore) 2000;79:210–21. [22] Maglio D, Capitano B, Banevicius MA, Geng Q, Nightingale CH, Nicolau DP. Differential efficacy of clarithromycin in lung versus thigh infection models. Chemotherapy 2004;50:63–6. [23] Livermore DM, Sefton AM, Scott GM. Properties and potential of ertapenem. J Antimicrob Chemother 2003;52:331–44. [24] Van Ogtrop ML, Andes D, Craig WA. In vivo antimicrobial activity of MK-0826, a new carbapenem, against various penicillin-resistant pneumococci. In: Program and Abstracts of the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. F-48. [25] Gill CJ, Jackson JJ, Gerckens LS, et al. In vivo activity and pharmacokinetic evaluation of a novel long-acting carbapenem antibiotic, MK-826 (L-749,345). Antimicrob Agents Chemother 1998;42:1996–2001. [26] Mattoes HM, Banevicius M, Li D, et al. Pharmacodynamic assessment of gatifloxacin against Streptococcus pneumoniae. Antimicrob Agents Chemother 2001;45:2092–7. [27] Craig WA. Post-antibiotic effects in experimental infection models: relationship to in-vitro phenomena and to treatment of infections in man. J Antimicrob Chemother 1993;31(Suppl. D):149–58. [28] Briles DE, Crain MJ, Gray BM, Forman C, Yother J. Strong association between capsular type and virulence for mice among human isolates of Streptococcus pneumoniae. Infect Immun 1992;60: 111–6. [29] Wang E, Simard M, Ouellet N, Bergeron Y, Beauchamp D, Bergeron MG. Pathogenesis of pneumococcal pneumonia in cyclophosphamide-induced leukopenia in mice. Infect Immun 2002;70:4226–38.