Method to grow Actinobacillus pleuropneumoniaebiofilm on a biotic surface

Actinobacillus pleuropneumoniae, a Gram-negative bacterium belonging to the Pasteurellaceae family, is the causative agent of porcine pleuropneumonia. This severe and highly contagious infectious respiratory disease causes major economic losses in the swine industry [1, 2]. A. pleuropneumoniae is transmitted by means of aerosols or by direct contact with infected animals. The infection may result in rapid death or in severe pathology [1]. Animals exposed to A. pleuropneumoniae may develop chronic infections or become asymptomatic carriers and these animals may become the source of transmission of the disease to healthy animals or herds [1]. The virulence factors involved in colonization and lung lesions, which include type IV fimbriae, lipopolysaccharides, and the pore forming RTX toxins ApxI to IV, have been well characterized (for a recent review see [2]). The role of biofilm formation in the pathogenicity of A. pleuropneumoniae is gaining recognition.

Bacterial biofilms are structured clusters of bacterial cells enclosed in a self-produced polymer matrix that are attached to a surface [3–5]. Bacteria can adhere to a biotic surface (e.g. cells at the mucous layer) as well as to abiotic surfaces (e.g. floor or equipment found at a farm). The polymer matrix is often composed of exopolysaccharides, proteins and nucleic acids. The biofilm protects bacteria from hostile environmental conditions. Bacteria within a biofilm can resist attack from the host immune response, and are less sensitive than planktonic cells to desiccation and to the action of biocides.

It has been clearly shown that A. pleuropneumoniae has the ability to form biofilms under static growth conditions in polystyrene microtiter plates [6, 7] or with agitation in glass tubes [6, 8]. Using a 96-well microtiter plate assay, we screened mutants obtained by transposon mutagenesis for their biofilm phenotype and identified unique genetic determinants associated with biofilm formation in A. pleuropneumoniae[8]. We also demonstrated that the biofilms of A. pleuropneumoniae field isolates were more resistant than their planktonic counterpart to ampicillin, florfenicol, tiamulin and tilmicosin [9]. Recently, our group demonstrated that A. pleuropneumoniae can form a biofilm on a glass slide under low-shear force in a drip-flow apparatus [10]. The biofilm formation on abiotic surfaces (plastic and glass) depends on the production of a polymer of β-1,6-N-acetyl-D-glucosamine (PGA) [10–12]. Although in vitro models used in biofilm studies are extremely valuable to acquire knowledge regarding biofilm formation, the main limit of these in vitro models is that they do not mimic exactly the conditions found in the host such as the lung mucosa, which is a natural niche for A. pleuropneumoniae. Therefore, the aim of this study was to develop a method to form biofilms on a biotic surface. We selected the SJPL cell line because this cell line has been used extensively in our laboratory to study adherence of A. pleuropneumoniae and other porcine bacterial pathogens to cells [13]. In addition, this cell line was found to be permissive to porcine reproductive and respiratory syndrome virus (PRRSV) infection [14] and therefore represents a potential and powerful model to study viral-bacterial co-infections.

A non-hemolytic strain of A. pleuropneumoniae with deletions in the apxIC and apxIIC genes, named MBHPP147, was used in this study. We first determined the ability of this strain to form a biofilm using a standard microtiter plate assay combined with crystal violet staining (Figure 1). Strain MBHPP147 was able to form biofilms on the plastic surface and, as previously observed with the A. pleuropneumoniae S4074 parent strain [7], a robust biofilm was formed within 4 hours but rapidly dispersed afterwards. Such rapid dispersion has been observed previously with strain S4074 but the exact cause has yet to be identified [10]. The presence of the biofilm was confirmed by confocal laser scanning microscopy by staining the biofilm matrix with Wheat Germ Agglutinin (WGA)-Oregon green 488 (Figure 2). This lectin is known to bind to the PGA present in the biofilm matrix of A. pleuropneumoniae[11, 12].

The next step was to evaluate the ability of strain A. pleuropneumoniae MBHPP147 to form biofilm on a biotic surface (i.e. on a SJPL cell monolayer). Therefore, A. pleuropneumoniae MBHPP147 was incubated with SJPL cells for up to 48 hours. Confocal laser scanning microscopy and staining with WGA-Oregon green 488 was used to visualize the biofilms (Figure 3). Uninfected SJPL cells were used as a negative control, and, as expected, were devoid of any adherent bacteria (Figure 3A). The cells were stained with WGA because this lectin binds to N-acetyl-glucosamine and N-acetyl-neuraminic residues. There was no obvious biofilm formation after 3 h (Figure 3B) and 6 h (Figure 3C) of incubation despite the attachment of A. pleuropneumoniae MBHPP147 on SJPL cells (Figure 4). A. pleuropneumoniae MBHPP147 clearly formed robust biofilms at the surface of SJPL cells after 24 hours (Figure 3D) and 48 hours (Figure 3E) of incubation. The formation of biofilms was associated with an increased in the number of bacteria attached to SJPL cells (Figure 4). The 24 h and 48 h biofilms were stained with WGA suggesting the presence of PGA in the matrix as observed previously with abiotic surfaces [7, 10–12]. The presence of PGA was confirmed by the dispersion of the biofilm (Figure 3F), and the decrease in the number of bacteria attached to SJPL cells (Figure 4) when the biofilms were treated with Dispersin B, a glycoside hydrolase that catalyzes the hydrolysis of poly-N-acetylglucosamine. To further ensure that the WGA staining observed was associated with the biofilm of A. pleuropneumoniae MBHPP147, the assay was repeated with a bacterial strain expressing a plasmid encoded GFP from a constitutive promoter [15]. A GFP signal was detected for SJPL cells infected with A. pleuropneumoniae and it was not detected when cells were not infected (Figure 5). This indicated that bacteria were attached to the cells. Furthermore, the GFP pattern exhibited by the bacteria on SJPL cells was very similar to the one observed with WGA staining (Figure 5). Additionally, the WGA staining observed in the uninfected cells appeared to follow the pattern of the phalloidin staining of the cytoskeleton (Figure 5). Taken together, this indicated that the observed change in WGA-staining pattern (Figures 3 and 5) is clearly due to the formation of biofilm by A. pleuropneumoniae and not due to a structural change on the surface of SJPL cells.

Unlike biofilms formed on an abiotic surface [10], the biofilms formed on SJPL cells was present after 24 and 48 hours. Furthermore, A. pleuropneumoniae S4074 and MBHPP147 were not able to form biofilms in the culture medium used to grow SJPL cells in the absence of SJPL cells (data not shown). Taken together, these observations underline the importance of bacterium-animal cell interactions in biofilm formation and the use of a biotic surface. This conclusion is also supported by the fact that exposing Streptococcus pneumoniae to the airway epithelial cells 1HAEo-before a biofilm assay resulted in a significant increase in biofilm formation in microtiter plates [16].

Only a few co-culture models have been described in the literature to study human respiratory tract pathogens. For example, S. pneumoniae can form biofilms on NCI-H292 (mucoepidermoid bronchial carcinoma cells) and primary human bronchial epithelial cells [17]. While the majority of primary bronchial epithelial cells remained intact after 24 hours of incubation with S. pneumoniae, the NCI-H292 cells were rapidly killed by the bacteria. Recently, Vidal et al. [18] developed a biofilm reactor with living cultures of human-derived lung cells A549 and a continuous flow of nutrients to avoid the cytotoxic effect of S. pneumoniae. Another example is Pseudomonas aeruginosa that can form biofilms on cystic fibrosis-derived human airway epithelial cells [19]. However, the biofilm disrupted the monolayer after an overnight incubation in the absence of antibiotics. To the best of our knowledge, this is the first report of biofilm formation by A. pleuropneumoniae on a biotic surface and for long incubation periods. In previous works [13], we have determined the SJPL cells’protein profile after a short incubation of 3 hours with A. pleuropneumoniae as well as the transcriptomic profile of A. pleuropneumoniae during that interaction. It will be interesting in future studies to use this new protocol to investigate the SJPL cell’s response to bacterial biofilms present after 24 and 48 hours of co-culture. It will also be interesting to compare A. pleuropneumoniae gene expression in a mature biofilm formed on a biotic surface with the genes expression determined with mature biofilms on plastic (polystyrene microtiter plate) or glass (drip-flow apparatus) surfaces [10]. Additionally, the protocol could be adapted and design to screen for anti-adherence and/or anti-biofilm molecules.

The use of a non-hemolytic, non-cytotoxic strain of A. pleuropneumoniae allows for longer incubation periods with an in vitro cell line model. Others have used paraformaldehyde-fixed epithelial cells to avoid the cytotoxic effect of a bacterial pathogen [17]. Although cell monolayers do not perfectly reproduce the in vivo conditions, the use of a biotic surface is certainly more physiologically relevant to study biofilm formation than a plastic or a glass surface. This model could be easily adapted to evaluate inhibitors of bacterial adherence or inhibitors of biofilm formation, or the efficacy of antibiotics in the presence of a biofilm. Finally, this model can also be adapted to study bacterial-viral co-infections knowing that the SJPL cells are permissive to PRRSV [14].