Hyperbiofilm Formation by Bordetella pertussis Strains Correlates with Enhanced Virulence Traits

Abstract

Pertussis, or whooping cough, caused by the obligate human pathogen Bordetella pertussis is undergoing a worldwide resurgence. The majority of studies of this pathogen are conducted with laboratory-adapted strains which may not be representative of the species as a whole. Biofilm formation by B. pertussis plays an important role in pathogenesis. We conducted a side-by-side comparison of the biofilm-forming abilities of the prototype laboratory strains and the currently circulating isolates from two countries with different vaccination programs. Compared to the reference strain, all strains examined herein formed biofilms at high levels. Biofilm structural analyses revealed country-specific differences, with strains from the United States forming more structured biofilms. Bacterial hyperaggregation and reciprocal expression of biofilm-promoting and -inhibitory factors were observed in clinical isolates. An association of increased biofilm formation with augmented epithelial cell adhesion and higher levels of bacterial colonization in the mouse nose and trachea was detected. To our knowledge, this work links for the first time increased biofilm formation in bacteria with a colonization advantage in an animal model. We propose that the enhanced biofilm-forming capacity of currently circulating strains contributes to their persistence, transmission, and continued circulation.

Keywords: Bordetella pertussis; biofilms; hyperbiofilm; virulence.

FIG 1

Biofilm-forming capacity of B. pertussis strains. (A) Formation of a bacterial ring at the air-liquid interface of glass culture tubes. (B) Microtiter assay of biofilm formation at 96 h by B. pertussis strains. Each data point represents the average value of three independent experiments performed in quadruplicates; error bars indicate standard deviations. Significant differences were assessed by one-way ANOVA and Bonferroni posttest. ***, P <0.001.

FIG 2

Quantification of autoaggregation of B. pertussis strains. Each bar represents the mean value of at least three independent experiments performed in duplicate. Error bars represent standard deviations. Statistical differences were assessed by one-way ANOVA and Bonferroni posttest. ***, P <0.001.

FIG 3

Fluorescence microscopy and quantification of early bacterial attachment. (A) Attached GFP-labeled bacterial cells were observed by fluorescence microscopy. (B) Cells were counted by means of the ITCN plug-in for ImageJ. Data are average values of at least three independent experiments performed in duplicates. Four random regions were chosen for bacterial counting. Error bars indicate standard deviations.

FIG 4

CLSM micrographs of B. pertussis biofilms. GFP-labeled bacterial strains were grown on cover glasses in six-well plates for the designated time points. Biofilms were visualized in situ by CLSM microscopy. CLSM image stacks were acquired at 0.9-μm z-intervals; xy and xz representative focal planes are shown.

FIG 5

COMSTAT analyses of B. pertussis biofilms. CLSM image stacks were acquired at 0.9-μm z-intervals and analyzed by COMSTAT2. Average values of parameters from CLSM image stacks derived from at least three independent experiments are shown with standard errors. P values were determined using two-way ANOVA. Average thickness (A) and maximum thickness (B) were calculated only on the biomass (without counting the uncovered area). Biomass values (C) represent the biomass volume divided by the area of the substratum. Roughness coefficients (D) represent the variability in the heights of the biofilms. *, P <0.05; **, P <0.01; ***, P <0.001.

FIG 6

Biofilm dispersal by matrix-dissolving agents. Ninety-six-hour biofilms were treated with pronase E in Tris buffer (A), with 40 mM sodium metaperiodate (NaIO₄) in H₂O (B), and with DNase I in reaction buffer (C) for 2 h at 37°C (filled bars). Biofilms were treated with the respective reaction buffers as controls (open bars). Biofilm reduction is presented as a percentage of the value for the respective strain incubated with buffer only. Average values are shown from one representative assay of three independent replicates, with their respective standard deviations. Significance was assessed by two-way ANOVA. *, P <0.05; **, P <0.01; ***, P <0.001.

FIG 7

Determination of the levels of biofilm-associated factors/genes in B. pertussis strains. (A) Cell surface-associated FHA determination by ELISA. Average values of three replicates are presented with the respective standard deviations. (B) AC toxin activity quantification. AC toxin levels were assessed by enzymatic activity as described earlier (69). (C) bpsA expression and production. bpsA transcript levels were determined by qPCR and the Pfaffl method. *, P <0.05; **, P <0.01; ***, P <0.001. (D) Dot blot of Bps. Production of Bps was detected as described previously (27).

FIG 8

Adherence of B. pertussis strains to epithelial cells. Adhesion assays were performed with A549 epithelial cell lines. Each strain was incubated at a multiplicity of infection of 10. Results are expressed as the proportion of adherent bacteria to the amount of the original inoculum. Each data point is the average of three independent experiments performed in duplicate. Error bars indicate the standard deviations. Statistical differences were assessed by one-way ANOVA (P < 0.0001) and Student's t test with a Bonferroni post hoc correction. *, P <0.05; **, P <0.01; ***, P <0.001. Bp Bvg⁻, B. pertussis Bvg⁻ phase-locked strain.

FIG 9

Colonization of mouse respiratory tract by Bp536, Bp462, and STO1-SEAT0004. Groups of C57BL/6 mice were intranasally inoculated with approximately 5 × 10⁵ CFU in 50 μl of PBS. At 4 (A) and 7 (B) days postinoculation, animals were sacrificed, and bacterial loads were determined in nasal septum, trachea, and lung. Horizontal bars represent the average values for each group. Significance was analyzed by means of one-way ANOVA and Dunnett's posttest. *, P <0.05; **, P <0.01; ***, P <0.001.