Biofilm formation by virulent and non-virulent strains of Haemophilus parasuis

Abstract

Haemophilus parasuis is a commensal bacterium of the upper respiratory tract of healthy pigs. It is also the etiological agent of Glässer's disease, a systemic disease characterized by polyarthritis, fibrinous polyserositis and meningitis, which causes high morbidity and mortality in piglets. The aim of this study was to evaluate biofilm formation by well-characterized virulent and non-virulent strains of H. parasuis. We observed that non-virulent strains isolated from the nasal cavities of healthy pigs formed significantly (p < 0.05) more biofilms than virulent strains isolated from lesions of pigs with Glässer's disease. These differences were observed when biofilms were formed in microtiter plates under static conditions or formed in the presence of shear force in a drip-flow apparatus or a microfluidic system. Confocal laser scanning microscopy using different fluorescent probes on a representative subset of strains indicated that the biofilm matrix contains poly-N-acetylglucosamine, proteins and eDNA. The biofilm matrix was highly sensitive to degradation by proteinase K. Comparison of transcriptional profiles of biofilm and planktonic cells of the non-virulent H. parasuis F9 strain revealed a significant number of up-regulated membrane-related genes in biofilms, and genes previously identified in Actinobacillus pleuropneumoniae biofilms. Our data indicate that non-virulent strains of H. parasuis have the ability to form robust biofilms in contrast to virulent, systemic strains. Biofilm formation might therefore allow the non-virulent strains to colonize and persist in the upper respiratory tract of pigs. Conversely, the planktonic state of the virulent strains might allow them to disseminate within the host.

Figure 1

Biofilm formation by H. parasuis isolates. (A) Biofilm formation under static conditions in microtiter plates for Haemophilus parasuis nasal strains (n = 5) and strains isolated from lesions of pigs with Glässer’s disease (n = 9). (B) Medians of biofilm formation for H. parasuis strains isolated from the nasal cavities of healthy pigs (n = 5) or for strains isolated from the lesions of pigs with Glasser’s disease (n = 9). Difference between the median of the two groups of strains was statistically significant (p < 0.05).

Figure 2

Images of H. parasuis biofilms obtained by CLSM. Confocal laser scanning microscopy of Haemophilus parasuis strains F9, MU21-2, Nagasaki and ER-6P biofilms formed under static conditions in wells of microtiter plates. Biofilms were stained with FilmTracer^™ FM 1–43, wheat-germ agglutinin (WGA)-Oregon green 488 (for poly-N-acetyl glucosamine), SYPRO Ruby (for proteins) and BOBO-3 (for eDNA).

Figure 3

3D images of H. parasuis strain F9 obtained by CLSM. Confocal laser scanning microscopy three-dimensional images of Haemophilus parasuis strain F9 biofilm formed under static conditions in wells of a microtiter plate. Biofilm was stained with wheat-germ agglutinin (WGA)-Oregon green 488. Stack of sections of the X-Z plane of the biofilm.

Figure 4

Effect of enzymatic treatments on H. parasuis biofilms. Dispersion of Haemophilus parasuis biofilms formed under static conditions in microtiter plates by (A) dispersin B, (B) proteinase K, and (C) DNase I.

Figure 5

Biofilm formation by H. parasuis in a drip-flow apparatus. Biofilm formation under low shear force in a drip-flow apparatus. Images of typical biofilms for Haemophilus parasuis strains MU21-2, F9, ER-6P, and Nagasaki visible after 24 h of incubation with continuous flow (25 mL/h).

Figure 6

Biofilm formation by H. parasuis in a microfluidic system. Biofilm formation under controlled shear force in a BioFlux 200 microfluidic system. Phase-contrast images of typical biofilms of Haemophilus parasuis non-virulent strains MU21-2 and F9, and virulent strains ER-6P and Nagasaki obtained after 24 h of incubation with an inoculum of OD₆₀₀ of 0.25 and a shear force of 0.5 dyne/cm².

Figure 7

Effects of fibrinogen on biofilm formation by H. parasuis . Effects of various concentrations of fibrinogen added to the culture medium on biofilm formation by Haemophilus parasuis strains F9, MU21-2, Nagasaki and ER-6P under static conditions in microtiter plates. Assays were performed in triplicate, and the means ± standard deviations are indicated.

Figure 8

Identification of H. parasuis genes differentially expressed. MA plots generated by EdgeR showing transcript expression profiles in the three comparisons performed: biofilm vs planktonic (A), biofilm vs stationary phase (B) and planktonic vs stationary phase (C). For each gene, log₂(fold change) between the two conditions is plotted (M, y axis) against the gene’s log₂(average expression) in the two samples (A, x axis). The blue lines indicate 2-fold changes. Red dots highlight the genes at 5% P-value.

Figure 9

Comparison of H. parasuis genes that were up- or down-regulated. Venn diagrams of Haemophilus parasuis genes identified as up- (A) and down-regulated (B) under different growth states.