Antimicrobial activity of innate immune molecules against Streptococcus pneumoniae, Moraxella catarrhalis and nontypeable Haemophilus influenzae

Abstract

Background: Despite its direct connection to the nasopharynx which harbors otitis media pathogens as part of its normal flora, the middle ear cavity is kept free of these bacteria by as yet unknown mechanisms. Respiratory mucosal epithelia, including those of the middle ear and eustachian tube, secrete antimicrobial effectors including lysozyme, lactoferrin and beta defensins-1 and -2. To elucidate the role of these innate immune molecules in the normal defense and maintenance of sterility of respiratory mucosa such as that of the middle ear, we assessed their effect on the respiratory pathogens nontypeable Haemophilus influenzae (NTHi) 12, Moraxella catarrhalis 035E, and Streptococcus pneumoniae 3, and 6B.

Methods: Two assay methods, the radial assay and the liquid broth assay, were employed for testing the antimicrobial activity of the molecules. This was done in order to minimize the possibility that the observed effects were artifacts of any single assay system employed. Also, transmission electron microscopy (TEM) was employed to evaluate the effect of antimicrobial innate immune molecules on OM pathogens. For the statistical analysis of the data, Student's t-test was performed.

Results: Results of the radial diffusion assay showed that beta defensin-2 was active against all four OM pathogens tested, while treatment with beta defensin-1 appeared to only affect M. catarrhalis. The radial assay results also showed that lysozyme was quite effective against S. pneumoniae 3 and 6B and was partially bacteriostatic/bactericidal against M. catarrhalis. Lysozyme however, appeared not to affect the growth of NTHi. Thus, lysozyme seems to have a more pronounced impact on the growth of the Gram-positive S. pneumoniae as compared to that of Gram-negative pathogens. Lactoferrin on the other hand, enhanced the growth of the bacteria tested. The results of the radial assays were confirmed using liquid broth assays for antimicrobial activity, and showed that lysozyme and beta defensin-2 could act synergistically against S. pneumoniae 6B. Moreover, in the liquid broth assay, beta defensin-1 showed a modest inhibitory effect on the growth of S. pneumoniae 6B. As assessed by ultrastructural analysis, lysozyme and beta defensin-2, and to a much lesser extent, beta defensin-1, appeared to be able to cause damage to the bacterial membranes.

Conclusions: Here we report that lysozyme and the beta defensins can inhibit the growth of clinical isolates of otitis media pathogens - namely NTHi strain 12, S. pneumoniae strains 3 and 6B and M. catarrhalis strain 035E - and cause ultrastructural damage to these pathogens. Moreover, we demonstrate that lysozyme and beta defensin-2 can act synergistically against S. pneumoniae. These findings are consistent with the concept that secreted antimicrobial peptides and other components of innate immunity constitute the first line of defense protecting host mucosal surfaces, including the tubotympanal (eustachian tube and middle ear cavity) mucosa, against pathogens.

Figure 1

Radial inhibition assay results demonstrating the effect of innate immune molecules on otitis media pathogens. Representative radial inhibition assay for the measurement of the activity of innate immune molecules against NTHi 12, M. catarrhalis 035E (M cat), S. pneumoniae 3 (Sp 3) and S. pneumoniae 6B (Sp 6B). Panel A shows the results of testing three doses (50 μg, 100 μg and 200 μg) of human lysozyme (Lz) against the bacteria. Panel B shows the results of testing the lactoferrin (Lf) and the defensins against the bacteria. Three doses (4 μg, 10 μg and 40 μg) of human lactoferrin (Lf) were tested, as were two doses (4 μg and 10 μg) each of human β defensin-1 (hBD-1) and β defensin-2 (hBD-2). Each dose was delivered in a total volume of 4 μl. The control well received only solvent (0.01% acetic acid). The diameter of the inhibition zone was measured and averaged in three separate experiments.

Figure 2

Liquid broth assay results demonstrating the susceptibility of S. pneumoniae to lysozyme and the β defensins. Antibacterial liquid broth assays for the measurement of the activity of lysozyme (Lz) and β defensins-1 and -2 (HBD1 and HBD2), alone (A) and in combination (B) (Lz+HBD2), against S. pneumoniae 6B (Sp 6B). The combination of Lz and HBD1 was not tested. The combination of lysozyme (1000 μg/ml) and β defensin-2 (1 μg/ml or 2.5 μg/ml) was significantly more effective than lysozyme (1000 μg/ml), or β defensin-2 (1 μg/ml or 2.5 μg/ml) alone. (P = 0.008 and 0.005, respectively, for the difference in the effect of the Lz-HBD2 combinations versus Lz (1 mg/ml) alone. P= 0.0007 and 0.03 for the difference in the effect of the Lz-HBD2 combinations versus 2.5 μg/ml of β defensin-2 alone). Four concentrations (1 μg/ml, 2.5 μg/ml, 5 μg/ml and 10 μg) of human β defensin-2 (HBD-2) and one concentration (1 mg/ml) of lysozyme (Lz) were tested against the bacteria – either alone, or in combination.

Figure 3

Ultrastructural changes in OM pathogens treated with innate immune molecules. Panels A, B and C are electron micrographs showing the experimental results with NTHi. Untreated NTHi are shown in panel A. Bacteria were treated for 30 minutes with β defensin-2 (10 μg/ml) (panel B), or with 1 mg/ml human milk lysozyme (panel C). Bar = 0.5 micron. Panels D, E and F show results of experiments with S. pneumoniae 3. Untreated bacteria are shown in panel D. Bacteria were treated for 30 minutes with human β defensin-2 (10 μg/ml) (panel E), or with 1 mg/ml human milk lysozyme (panel F). Bar = 0.5 micron. Panels G, H and I show the results of experiments S. pneumoniae 6B. Untreated bacteria are shown in panel G. Bacteria treated for 30 minutes with human β defensin-2 (10 μg/ml) are shown in panel H and those treated for 3 hours with human β defensin-1 (10 μg/ml) are shown in panel I. Bar = 0.5 micron. Panels J, K and L show experiments results with M. catarrhalis. Untreated bacteria are shown in panel J. Results of a 30-minute incubation with β defensin-2 are shown in panel K and those of a 30 minute treatment of the bacteria with lysozyme are shown in panel L. Bar = 0.5 micron.