Survival of bacterial biofilms within neutrophil extracellular traps promotes nontypeable Haemophilus influenzae persistence in the chinchilla model for otitis media

Abstract

Nontypeable Haemophilus influenzae (NTHi) is a leading cause of acute and chronic otitis media, which are a major public health problem worldwide. The persistence of NTHi during chronic and recurrent otitis media infections involves multicellular biofilm communities formed within the middle-ear chamber. Bacterial biofilms resist immune clearance and antibiotic therapy due in part to encasement within a polymeric matrix. In this study, the contribution of biofilms to bacterial persistence in vivo and composition of the NTHi biofilm matrix during experimental otitis media were investigated. The presence of biofilms within the chinchilla middle-ear chamber was significantly correlated with increased bacterial load in middle-ear effusions and tissue. Examination of thin sections revealed polymorphonuclear cells within a DNA lattice containing elastase and histones, which is consistent with the definition of neutrophil extracellular traps. Viable multicellular biofilm communities with biofilm phenotypes were found within the DNA lattice throughout the biofilm. Further, NTHi was resistant to both phagocytic and extracellular neutrophil killing in vitro by means of lipooligosaccharide moieties that promote biofilm formation. These data support the conclusion that NTHi subverts neutrophil extracellular traps to persist in vivo. These data also indicate that a more inclusive definition for biofilms may be warranted.

Fig. 1

Biofilms formed by H. influenzae 86-020NP promote bacterial persistence in the chinchilla middle-ear chamber. Animals were infected via transbullar injection, and groups were euthanized at 7 and 14 days after infection. a Biofilm formation in the chinchilla middle-ear chamber. Photographic images in the top row show representative middle-ear chambers of mock-infected animal (left) as well as infected animals with macroscopically visible biofilms 7 days (center) and 14 days (right) after infection. Scanning electron micrographs (SEM) in the lower row show epithelial surface of the middle-ear chamber of a mock-infected animal (left) compared to infected animals 7 days (center) and 14 days (right) after infection. Note the visible biomass in the infected animals that contains embedded host cells in the 14-day image. b Bacterial counts obtained from middle-ear effusion fluids of animals with and without visible biofilm. Bars depict mean CFU counts from infected animals 7 days (left) or 14 days (right) after infection. Sample size: biofilm group, n = 31; no biofilm group, n = 9. Error bars show standard deviation. Statistical significance was assessed by Mann-Whitney nonparametric analysis and is denoted by an asterisk.

Fig. 2

Visualization of H. influenzae communities within cryosections of biofilms recovered from the chinchilla middle ear. Biofilms recovered from infected animals were sectioned and analyzed microscopically. a Histopathological analysis reveals neutrophils within the biofilm. Sections of biofilm from animals at 1 and 2 weeks after infection were stained with toluidine blue, and revealed many polymorphonuclear cells embedded within the biofilm structure. b Immunofluorescent staining to visualize H. influenzae bacteria within biofilm. Cryosections (5 μm) were stained with polyclonal rabbit antisera against H. influenzae 86-028NP and anti-rabbit Alexa488 conjugate and visualized by differential interference contrast (DIC)/Nomarski and fluorescence microscopy. H. influenzae communities are visible in green in fluorescent panel at both the 1- and 2-week postinfection time points. c Bacterial variants within communities within the biofilm. Cryosections were stained with polyclonal antiserum as above coupled with monoclonal antibody hyaluronan synthases specific for phosphorylcholine (PCho, left image) and secondary antibody Texas Red conjugate, or lectin LFA/rhodamine conjugate for sialic acid (NeuAc, right image). Fig. 2. d Quantitation of biofilm within cryosections. Graph depicts mean fluorescent pixel counts from 5 different fields of view of 20 different sections. Pixels with similar intensity (within 20%) were highlighted. Total pixel counts for images were comparable (within 15%). Error bars depict standard deviation.

Fig. 3

Viability staining reveals host cells and bacteria within a fibrous DNA lattice. Unfixed portions of biofilms were stained with Live/Dead BacLight^TM (Molecular Probes) and visualized by confocal laser-scanning microscopy. With this reagent, red staining (propidium iodide, PI) indicates nonviable cells or extracellular DNA, whereas green staining (Syto-9) indicates viable cells. Images depicted are merged from vertical Z sections (0.2 μm/slice, 5 sections per image). Biofilms were recovered 1 week (top row) or 2 weeks (bottom row) after infection. Viable bacteria and both viable and nonviable host cells were observed within fibrous DNA net in samples from both time points.

Fig. 4

Immunofluorescence microscopy reveals eukaryotic histones and elastase within the biofilm structure. Cryosections from biofilms obtained 1 or 2 weeks after infection (as indicated in labels on left margin) were stained with rabbit antisera against H. influenzae coupled with secondary antibody/Texas Red conjugate as above, coupled with monoclonal antibody specific for elastase and secondary antibody/Alexa488 conjugate (top) or deiminated histone/Alexa488 conjugate (bottom). The stained sections were visualized by confocal laser-scanning microscopy; the images depicted are merged from vertical Z sections (0.2 μm/slice, 10 sections per image).

Fig. 5

H. influenzae resists killing within preformed NETs. NET formation was elicited from primary human neutrophils by stimulation with PMA (see Materials and Methods), after which NTHi bacteria were added. After 30 min, the number of viable bacteria within the well was determined by plate count and normalized relative to mock-treated wells to which no neutrophils were added. Filled columns show means of total neutrophil killing (absence of cytochalasin), whereas open columns show means of extracellular killing (wells treated with cytochalasin). Error bars show standard deviation, while asterisks denote mean values that were significantly different from the parental NTHi 2019 strain as determined by analysis of variance with a post hoc test of significance. NET killing was defined as the percentage of decrease in total bacteria in the presence of neutrophils and cytochalasin D.