Journal: Infection and Immunity

Article Title: Haemophilus parainfluenzae Strain ATCC 33392 Forms Biofilms In Vitro and during Experimental Otitis Media Infections

doi: 10.1128/IAI.01070-16

Figure Lengend Snippet: Characterization of biofilm formation by H. parainfluenzae in vitro. (A) Microtiter plate biofilm assay indicates biofilm formation by H. parainfluenzae. Bacteria were cultured in 96-well plates. After increasing amounts of time, biofilms were washed and quantitated by crystal violet staining. Bars represent the mean absorbance. Error bars show the standard deviations. (B) Static biofilm assay with LIVE/DEAD staining to visualize H. parainfluenzae biofilms. Bacteria were cultured in 4-well chamber slides for 24 h. Biofilms formed on chamber slides were washed and stained with LIVE/DEAD BacLight reagents and visualized by CLSM. Syto 9 staining (green) labeled viable cells, whereas propidium iodide staining (red) indicated dead cells or cells with a damaged membrane. Z-series images were used to create representative volumetric views of H. parainfluenzae biofilms. Propidium iodide staining revealed that the majority of cells within 24-h biofilms were viable.

Article Snippet: Haemophilus parainfluenzae strain ATCC 33392 forms biofilms in vitro and during experimental otitis media infections.

Techniques: In Vitro, Biofilm Production Assay, Bacteria, Cell Culture, Staining, Labeling, Membrane

Journal: Infection and Immunity

Article Title: Haemophilus parainfluenzae Strain ATCC 33392 Forms Biofilms In Vitro and during Experimental Otitis Media Infections

doi: 10.1128/IAI.01070-16

Figure Lengend Snippet: H. parainfluenzae biofilms are susceptible to detachment by DNase I and proteinase K (PK). Bacterial biofilms formed in 96-well plates were washed and incubated with different concentrations of DNase I (A) or PK (B) in enzyme buffer for 1 h at 37°C. Biofilms were quantitated by crystal violet staining as described in Materials and Methods. Bars represent the mean absorbance. Error bars show the standard deviations. Asterisks indicate significant decreases in biofilm biomass following enzymatic treatment compared to the untreated biofilm biomass. P values were determined by Student's t test. *, P < 0.05; **, P < 0.01.

Article Snippet: Haemophilus parainfluenzae strain ATCC 33392 forms biofilms in vitro and during experimental otitis media infections.

Techniques: Incubation, Staining

Journal: Infection and Immunity

Article Title: Haemophilus parainfluenzae Strain ATCC 33392 Forms Biofilms In Vitro and during Experimental Otitis Media Infections

doi: 10.1128/IAI.01070-16

Figure Lengend Snippet: DNase I and proteinase K (PK) inhibit H. parainfluenzae biofilm formation. Bacteria were cultured with NBHI medium or NBHI medium supplemented with DNase I (1 mg ml−1) or proteinase K (1 mg ml−1) in 96-well plates, 24-well plates, or 4-well chamber slides for 24 h. (A) Biofilms formed in 96-well plates were washed and quantitated by crystal violet staining for measuring biofilm biomass. (B) Biofilms formed in 24-well plates were serially diluted and plated for viable bacterial counts. Biofilms formed in 4-well chamber slides were stained with Syto 9 alone for labeling all biofilm cells and visualized by CLSM. (C to E) Z-series images were used to create representative volumetric views of untreated (C), DNase I-treated (D), and proteinase K-treated (E) biofilms. (F to H) Z-series images were also exported to COMSTAT to obtain biofilm measurements, including average biofilm thickness (F), total biomass (G), and surface roughness coefficient (H). Error bars indicate standard deviations. Asterisks indicate a significant difference compared to the untreated biofilm as determined by Student's t test. **, P < 0.01.

Article Snippet: Haemophilus parainfluenzae strain ATCC 33392 forms biofilms in vitro and during experimental otitis media infections.

Techniques: Bacteria, Cell Culture, Staining, Labeling

Journal: Infection and Immunity

Article Title: Haemophilus parainfluenzae Strain ATCC 33392 Forms Biofilms In Vitro and during Experimental Otitis Media Infections

doi: 10.1128/IAI.01070-16

Figure Lengend Snippet: H. parainfluenzae persists in the middle ear of the chinchilla model of OM. Chinchillas were infected via the middle ear superior bullae with either 104 (circles) or 108 (triangles) CFU of bacteria. Groups were harvested on days 3, 7, and 14 postinfection. (A) Photographs show a representative middle ear chamber of a mock-infected animal (left) and an infected animal with macroscopically visible biofilm and effusion (right) at day 3 after infection. (B and C) Bacterial counts were obtained from bullar homogenates (B) and effusions (C) of infected animals. Long horizontal lines show the limit of detection. Short horizontal lines represent the geometric mean of CFU in each group. Data represent the results from three independent experiments.

Article Snippet: Haemophilus parainfluenzae strain ATCC 33392 forms biofilms in vitro and during experimental otitis media infections.

Techniques: Infection, Bacteria

Journal: Infection and Immunity

Article Title: Haemophilus parainfluenzae Strain ATCC 33392 Forms Biofilms In Vitro and during Experimental Otitis Media Infections

doi: 10.1128/IAI.01070-16

Figure Lengend Snippet: Visualization of H. parainfluenzae and host cells within chinchilla middle ear biofilms. Chinchillas were infected via transbullar injection with 108 CFU of bacteria and euthanized at 3 days postinfection. (A) Immunofluorescence staining visualized bacterial communities within the biofilms. Cryosections of biofilms obtained from superior bullar surfaces were stained with rabbit anti-Haemophilus polyclonal antisera coupled with Alexa 488-conjugated anti-rabbit antibody and visualized by CLSM. Bacteria within the cryosections were visible in green (left; bar, 10 μm). Different interface contrast (DIC) image (right) indicates biofilm matrix. (B) Histopathological analysis revealed neutrophils within chinchilla middle ear biofilm. H&E staining of biofilm cryosections at low magnification revealed that many host cells were embedded within the biofilm structure (left; magnification, ×20) and at high magnification that the majority of them were polymorphonuclear cells (right; magnification, ×100). (C) LIVE/DEAD staining revealed viable host and bacterial cells within chinchilla middle ear biofilms. Unfixed portions of fresh chinchilla middle ear biofilms obtained from superior bullar surfaces at 3 days postinfection were immediately stained using the LIVE/DEAD BacLight kit and visualized by CLSM. White arrow indicates viable bacteria (green, left).

Article Snippet: Haemophilus parainfluenzae strain ATCC 33392 forms biofilms in vitro and during experimental otitis media infections.

Techniques: Infection, Injection, Bacteria, Immunofluorescence, Staining

Journal: Infection and Immunity

Article Title: Haemophilus parainfluenzae Strain ATCC 33392 Forms Biofilms In Vitro and during Experimental Otitis Media Infections

doi: 10.1128/IAI.01070-16

Figure Lengend Snippet: Polymicrobial infection promoted H. parainfluenzae clearance from biofilm communities in chinchilla model of OM. Chinchillas were infected with 103 CFU of NTHi 86-028 NP, 108 CFU of H. parainfluenzae, or a mixture of both species. Animals were harvested on days 3, 7, 14, and 21. After middle ear effusion fluids were removed, bullae were washed with PBS, removed, and homogenized for enumeration of viable NTHi (A) and H. parainfluenzae (B) cells by plating on SBHI or NBHI medium, respectively, with added vancomycin. Black circles represent CFU recovered from single-species bullar homogenates. Black triangles represent CFU recovered from polymicrobial-species bullar homogenates. Long horizontal lines show the limit of detection. Short horizontal lines represent the geometric mean of CFU in each group. Statistical significance was assessed by Mann-Whitney nonparametric analysis. *, P < 0.05 compared to the number of CFU from single-species bullar homogenates.

Article Snippet: Haemophilus parainfluenzae strain ATCC 33392 forms biofilms in vitro and during experimental otitis media infections.

Techniques: Infection, MANN-WHITNEY