Although the bacterial species that we included in this study are generally not virulent in healthy persons, these opportunists can cause severe and chronic respiratory tract infections in individuals with CF. Unremitting infection and the associated inflammation result in progressive lung disease that culminates in pulmonary failure, the leading cause of death for CF patients. Effective therapy of CF pulmonary infection is severely limited by the broad-spectrum antimicrobial resistance exhibited by these species, which are among the most drug-resistant bacteria encountered in human infection. The site of infection in CF presents another important obstacle to effective therapy. Infecting bacteria reside primarily within the airway lumen in sputum, the airway epithelial surface fluid, and the bronchial mucosa (3
). The penetration of systemically delivered antimicrobials to this infected site is generally poor. Treatment is further hampered by bacterial biofilm formation, which is believed to occur in the airways of infected patients (51
), and by the exceptionally viscous secretions that characterize the CF respiratory tract (40
). These challenges have driven an increase in recent efforts to develop antimicrobials for topical (i.e., inhalational) use in CF (8
The surfactant-stabilized oil-in-water nanoemulsion that we investigated in this study is similar to others that were previously found to be bactericidal against gram-positive bacteria and spores as well as some gram-negative bacilli and enveloped viruses (13
). The mechanism of bacterial and viral killing is believed to involve the fusion of the emulsion with microorganism lipid membranes, leading to rapid osmotic disruption and cell lysis (23
). The electrostatic attraction provided by the cationic surface charge of CPC-based nanoemulsions appears to overcome the lipopolysaccharide-mediated resistance of gram-negative bacteria to neutral and anionic detergents (23
). The nanoemulsion used in this study was not selected for specific activity against the bacterial species included in our test panel. However, in preliminary studies, we found that the bactericidal activity of NB-401 against gram-negative bacteria was enhanced by the addition of EDTA, which likely chelates divalent cations that stabilize outer membrane lipopolysaccharide, thereby facilitating interactions with the cationic emulsion. This interaction most likely leads to membrane permeabilization and lysis as well as the augmentation of the transmembrane diffusion of macromolecules (1
Inhaled nebulized hypertonic saline was recently shown to have clinical benefit in the management of CF (18
), possibly resulting from the osmotic restoration of airway surface fluid and improvement of airway mucus rheological properties (19
). The addition of 7% saline did not decrease the bactericidal activity of NB-401. In preliminary studies, we found NB-401 to be stable after nebulization (using a PARI LC Plus nebulizer) in 7% saline and well tolerated by mice as a single inhaled dose (data not shown).
We focused our investigation of NB-401 on species within the B. cepacia
complex, as infection with these species is particularly refractory to antimicrobial therapy and is associated with increased rates of morbidity and mortality in CF (7
). Importantly, infection with members of the B. cepacia
complex is also regarded by many CF care centers as an absolute contraindication to lung transplantation (34
). Because B. multivorans
and B. cenocepacia
account for the majority of B. cepacia
complex infections in CF (48
), we included relatively more strains from these two species. We also included Burkholderia gladioli
, which, although not a member of the B. cepacia
complex, is being recovered from CF patients with increasing frequency (4
). The numbers of strains included in our test panel from the remaining species were also chosen to reflect the relative proportion of these species recovered from CF patients. The exception is Acinetobacter
, which, although currently infrequently recovered in CF, appears to be an emerging pathogen in this patient population. The majority (94%) of isolates in the test panel were recovered from cultures of respiratory specimens from persons with CF. The panel also included one representative isolate from each of five previously described so-called epidemic lineages, each of which has been identified as infecting multiple CF patients. These included the B. cenocepacia
), PHDC (12
), and Midwest (16
) lineages as well as the B. multivorans
OHBM lineage (5
) and the B. dolosa
SLC6 lineage (5
). Finally, to ensure a stringent test of the activity of NB-401, one-third of the isolates in the test panel were multidrug resistant; 20 of these were panresistant. To avoid duplicate testing of the same strain, all isolates were confirmed as being distinct strains by genotyping analyses.
NB-401 showed very good activity in vitro against the strains studied. With the exception of two B. cenocepacia strains, all strains in the test panel were inhibited by an NB-401 concentration of ≤125 μg/ml, or a 1:16 dilution of the initial formulation; 59% of strains were inhibited by a concentration of ≤31.2 μg/ml, a 1:64 dilution of NB-401. NB-401 did not show decreased activity against multidrug-resistant (MIC90 of 125 μg/ml) or panresistant (MIC90 of 62.5 μg/ml) strains. In general, NB-401 showed slightly less activity against Burkholderia species than the other species examined, with 16 of the 18 highest MICs found among Burkholderia strains. Conversely, 33 of the 38 lowest MICs (15.6 μg/ml) were found among non-Burkholderia species. We observed no striking differences in NB-401 activity among the 10 Burkholderia species examined, although the two strains requiring the highest MICs (250 μg/ml and 500 μg/ml) were both B. cenocepacia. Among the non-Burkholderia species, NB-401 was most active against Ralstonia strains (MIC90 of ≤15.6 μg/ml) and relatively less active against Acinetobacter strains (MIC90 of 125 μg/ml). We found no evidence of tolerance to NB-401 among a subset of 34 strains for which both MICs and MBCs were determined. NB-401 killing of planktonically grown bacteria was time and concentration dependent; at a concentration eight times greater than the MIC, complete killing was achieved within 30 min. This suggests that a relatively brief exposure of bacteria to NB-401 could result in a significant decrease in levels of viable bacteria during therapy.
While we found NB-401 to be highly active against a rigorously assembled panel of CF-associated bacteria, the relevance of standard susceptibility testing of planktonically grown bacteria to the treatment of infections involving bacteria within biofilms has been questioned recently. Several species involved in CF infection, including P. aeruginosa
species, have the capacity to produce biofilms in vitro, and emerging evidence strongly suggests that biofilm formation within the airways of infected patients contributes to disease progression and persistence of infection (20
). Furthermore, sessile bacteria within biofilms demonstrate increased antimicrobial resistance relative to their planktonic counterparts (9
). We therefore sought to assess the activity of NB-401 against bacteria grown in vitro as biofilms. Using a relatively strict definition of in vitro biofilm formation to ensure a stringent test of NB-401 activity, 12 biofilm-forming strains representing several species and a range of planktonic susceptibilities to NB-401 were identified. As there is currently no standardized protocol for susceptibility testing of bacteria in biofilm, we considered the merits of various previously reported methods (9
) and tested a number of variables to develop a combination of steps that we believed were best suited for our study, particularly considering the opacity of the nanoemulsion. These steps included comparing inoculum volumes and concentrations, peg versus well surfaces for biofilm growth, durations of biofilm growth, spectrophotometric versus colorimetric measurements, and various methods to suspend biofilm bacteria after treatment including sonication, centrifugation, and scraping of surfaces to release cells (data not shown). Although the MBIC and MBEC of NB-401 were increased compared to the respective MIC and MBC for each strain tested (median eightfold increase in MBIC compared to MIC), all 12 strains were inhibited or killed by NB-401 when grown as biofilms. Only a single strain required undiluted NB-401 (2,000 μg/ml) for the eradication of viable biofilm bacteria. We assessed relative biomasses among the biofilm-forming strains spectrophotometrically with crystal violet staining and observed no correlation between biomass and MBIC/MBEC results (data not shown). In fact, although B. gladioli
strain AU10529 produced the greatest biomass among the 12 strains tested, the MBIC and MBEC of NB-401 for this strain were relatively low (both 62.5 μg/ml). Conversely, B. cenocepacia
strain J2315 produced relatively little biomass yet required an NB-401 MBEC of 1,000 μg/ml. Similarly, we observed no correlation between colony counts from untreated biofilm wells and MBIC/MBEC results (data not shown). Intra- and interexperimental reproducibilities of colony counts from the untreated biofilm controls validated the uniformity of the scraping method and the overall biofilm assay.
CF sputum, like biofilm, is reported to antagonize the activity of antibacterial drugs (28
). Glycoproteins and high-molecular-weight DNA are present at elevated levels, resulting in exceptionally viscous sputum that provides a physical barrier protecting bacteria. In addition, these macromolecules bind and sequester antibiotics while small cationic molecules, and the decreased pH of CF sputum blocks drug penetration into bacteria and reduces drug bioactivity. The strategy of increasing drug dosing to overcome these obstacles is limited by drug toxicity. To assess the impact of sputum on the antibacterial activity of NB-401, we repeated the standard planktonic testing for the 12 biofilm-forming strains in the presence of CF sputum. We used a mixture of sputum from 15 CF patients to avoid interpatient variation in macromolecule and high-molecular-weight DNA compositions and in ionic conditions and applied mechanical shearing only to minimize changes to the native microenvironment (22
). The activity of NB-401 against bacteria suspended in medium containing 43% sputum (the maximum sputum concentration achieved in our test system) was decreased with bactericidal concentrations 2- to 32-fold greater than the respective planktonic MBCs without sputum. The sputum MBCs were identical to or within 1 dilution of the MBECs obtained with biofilm-grown bacteria. Although the activity of NB-401 was similarly antagonized by both CF sputum and biofilm growth, it remained bactericidal for all the strains tested under both test conditions.
In summary, the results of this study support a potential role for NB-401 as an antimicrobial treatment for infection due to CF-related opportunistic pathogens. Most strains tested, including many multi- and panresistant strains, were inhibited in vitro by ≤125 μg/ml of NB-401 (a 16-fold dilution of the nanoemulsion). This emulsion was rapidly bactericidal and was active against bacteria whether grown planktonically, as a biofilm, or in the presence of CF sputum. In ongoing work, we have found nanoemulsions to be exceptionally stable, unchanged after nebulization, and broadly microbicidal. Importantly, we have not observed the development of resistance to NB-401 by any bacterial species examined to date. Comprehensive phase II clinical safety, tolerability, and pharmacokinetic studies of similar nanoemulsion formulations for use as topical therapeutics for herpes labilis and onychomycosis have shown these formulations (NB-001 and NB-002, respectively) to be safe and well tolerated, as described previously (30
). Nasal application of similar nanoemulsions as vaccine delivery agents was well tolerated and did not induce inflammation in mice (6
), and a recent preclinical safety evaluation of a nanoemulsion vaccine adjuvant similarly showed no inflammation, cytotoxicity, or systemic toxicity with mucosal application (36
). The potential role of NB-401 as an inhaled antimicrobial is supported by preliminary studies showing that multiple daily inhaled doses of undiluted NB-401 (2,000 μg/ml) are well tolerated in mice, with no apparent pulmonary pathology observed upon postmortem examination (our unpublished data). These observations raise the possibility that inhaled NB-401 and hypertonic saline might be useful as a combination inhalational therapy that would have broad-spectrum antimicrobial effects without increasing the already high treatment burden for CF patients. Clearly, however, more comprehensive preclinical efficacy and toxicity testing is required, and these studies are under way. Future studies will assess the pharmacokinetics of NB-401 in CF patients and the concentration of NB-401 achievable within the airway lumen and lung parenchyma following inhalation. Such studies will further characterize the potential utility of NB-401 as an inhaled antimicrobial for the management of CF.