Open Access

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

  • Bernardo Bello-Ortí1,
  • Vincent Deslandes2,
  • Yannick DN Tremblay2,
  • Josée Labrie2,
  • Kate J Howell3,
  • Alexander W Tucker3,
  • Duncan J Maskell3,
  • Virginia Aragon1, 4 and
  • Mario Jacques2Email author
Veterinary Research201445:104

DOI: 10.1186/s13567-014-0104-9

Received: 30 May 2014

Accepted: 26 September 2014

Published: 27 November 2014

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.

Introduction

Haemophilus parasuis is a Gram-negative bacterium and a commensal organism 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 [1]. Glasser’s disease is recognized as one of the main causes of economic loss in the pig industry.

The heterogeneity among H. parasuis strains has been demonstrated by multiple methods, including multilocus sequence typing (MLST) which has shown a genetic lineage associated with polyserositis (cluster A) and another lineage associated with nasal colonization (cluster C) [2]. Serum resistance [3], phagocytosis resistance [4], and invasion of endothelial cells [5],[6] have been associated with virulence of H. parasuis. Some putative virulence factors have been reported [7],[8]. Those include the capsule [4], lipooligosaccharide (LOS) [9]-[11] and other genes involved in polysaccharide production, such as galE and galU, which have been associated with serum resistance and biofilm production [12]. Genes involved in sialic acid utilization were identified as potential virulence factors [13]. The sialytransferase gene lsgB was predominantly present in systemic isolates and not in nasal isolates, and sialylation of the LOS was observed in the virulent strain H. parasuis Nagasaki. In addition, a family of trimeric autotransporters, designated virulence associated trimeric autotransporters (VtaA) has been described [14],[15] and these can be divided into three groups based on their translocation domains [14]. Group 3 vtaA gene is highly conserved among invasive and non-invasive strains, whilst groups 1 and 2 vtaA genes were detected only in virulent strains.

Bacterial biofilms are structured clusters of bacterial cells enclosed in a self-produced polymer matrix that are attached to a surface [16]-[18]. Bacteria can adhere to biotic surfaces (e.g. cells at the mucous layer) as well as 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. Biofilm formation by nasal strains of H. parasuis has been previously reported [19]. Although the role of biofilm in H. parasuis pathogenesis is not clear, expression of genes with putative function in biofilm formation was detected during pulmonary infection [20]. The aims of this study were to compare biofilm formation by well-characterized virulent and non-virulent strains of H. parasuis and to analyse the gene expression during biofilm growth. Such analyses would help explore the possible role of biofilms in the pathogenesis of H. parasuis.

Materials and methods

Bacterial strains

The H. parasuis strains used in this study and their main phenotypic and genotypic characteristics are listed in Table 1. Bacteria were grown on Brain Heart Infusion (BHI; Oxoid Ltd, Basingstoke, Hampshire, UK) agar containing 10 μg/mL NAD or on chocolate agar (Biomerieux, Madrid, Spain). Plates were incubated overnight at 37 °C with 5% CO2.
Table 1

Haemophilus parasuis strains used in the present study

Strains

Susceptibility1to

lsgB 2

group1vtaA2

MLST3

Serovar

Serum

Phagocytosis

Nasal strains

F9

S

S

-

-

C

6

ND14-1

S

S

-

-

C

7

SC14-1

S

S

-

-

C

15

MU21-2

S

S

-

-

C

7

SL3-2

S

S

-

+

B

10

Strains from lesions

Nagasaki

R

R

+

+

A

5

P015/96

R

R

+

+

A

5

ER-6P

R

R

+

+

A

15

264/99

R

R

+

+

A

10

IT29205

R

R

+

+

A

4

2725

S

R

+

+

A

10

373/03A

I

R

-

+

A

7

PV1-12

S

R

-

+

B

15

9904108

S

R

-

-

C

4

1(S) sensitive; (I) intermediate; (R) resistant.

2(+) gene present; (−) gene absent.

3Cluster associated with isolates from systemic lesions (A); with nasal isolation (C); or cluster with no statistical association with clinical origin (B) [2].

Biofilm assays

Static conditions

Biofilms were cultured in 96-well microtiter plates as described by Wu et al. [21], with some modifications. Briefly, colonies from overnight agar cultures were resuspended in BHI-NAD containing 5 μg/mL NAD, and the suspension was aliquoted (100 μL) in triplicate in a flat-bottom 96-well polystyrene plate (Costar® 3599, Corning, NY, USA) and incubated for 40 h at 37 °C. Wells containing sterile broth were used as negative control. Following incubation, biofilms were treated as described before [22] with some modifications. Briefly, the liquid medium was removed using a vacuum and unattached cells were removed by immersing the plate once in MilliQ water. The water was removed with a vacuum and excess water was removed by inverting plates onto a paper towel. Biofilms were then stained with 0.1% (w/v) crystal violet for 2 min. Biofilms were washed once with distilled water and then dried at 37 °C for 15 min. The stain was then released with 100 μL of 70% (v/v) ethanol and the amount of released stain was quantified by measuring the absorbance at 590 nm with a microplate reader (Powerwave; BioTek Instruments, Winooski, VT, USA).

Shear force conditions

Biofilms were cultured in a drip-flow apparatus (DFR 110 Biofilm Reactor, BioSurface Technologies Corp. Bozeman, MT, USA) as described by Goeres et al. [23] with some modifications [24]. Briefly, colonies of H. parasuis strains F9, MU21-2, ER-6P or Nagasaki on BHI-NAD agar were resuspended into 13 mL of fresh BHI-NAD to an OD600 of 0.1 and 12.5 mL of this inoculum was transferred into a channel containing a glass slide (Catalogue #48300-025, VWR, Ville Mont-Royal, QC, Canada). The apparatus was incubated for 24 h at 37 °C with 5% CO2 to allow the biofilm to form under static conditions. The apparatus legs were then attached to create a 10° downward slope. The apparatus was then connected to the nutrient system containing pre-warmed (37 °C) BHI-NAD. The flow (~25 mL per hour per channel) of the medium was then initiated and maintained for 24 h at 37 °C. After 24 h, the glass slide was removed and gently washed once with sterile MiliQ water. The biofilms were resuspended in 1.5 mL of MiliQ water, centrifuged and dried with a DNA 120 Speed Vac® (Thermo Scientific, Ottawa, ON, Canada). The weight of dry biofilms was then measured.

Biofilms were also cultured in a microfluidic system. Growth of biofilms in the BioFlux 200 device (Fluxion Biosciences, South San Francisco, CA, USA) was adapted from Benoit et al. [25] and the manufacturer’s recommendations. Briefly, colonies of H. parasuis strains F9, MU21-2, ER-6P or Nagasaki on BHI-NAD agar were resuspended into 2.5 mL of fresh BHI-NAD to an OD600 of 1.0 and this suspension was serially diluted to OD600 of 0.5 and 0.25. The microfluidic channels were wetted with BHI-NAD and were then inoculated with different bacterial suspensions (OD600 1.0, 0.5 or 0.25). The microfluidic plate was incubated for 2 h at 37 °C to allow bacteria to bind to the surface. The flow of fresh medium was then initiated and was set from 0.5 dyne/cm2 to 1.0 dyne/cm2. Growth of the biofilms was monitored for up to 24 h and, in some cases, fresh medium was added and the “waste” outlet was emptied to ensure that wells would not dry or spill. Images of BioFlux biofilms were obtained using an inverted fluorescence microscope (Olympus CKX41, Markham, ON, Canada), a digital camera (Retiga EX; Q Imaging, Surrey, BC, Canada), and the software provided with the BioFlux 200 device.

Confocal laser scanning microscopy (CLSM)

Biofilms were prepared under static conditions as described above and were stained with FilmTracer™ FM®1-43 Green biofilm cell stain (Molecular Probes, Eugene, OR, USA) according to manufacturer’s instructions. To determine the composition of the biofilm matrix, biofilms were stained with Wheat Germ Agglutinin (WGA-Oregon Green 488, Molecular Probes; binds to N-acetyl-D-glucosamine and N-acetylneuraminic acid residues), FilmTracer™ SYPRO® Ruby biofilm matrix stain (Molecular Probes; labels most classes of proteins) or BOBO™-3 iodide (Molecular Probes; stains extracellular DNA) according to manufacturer’s instructions. After a 30 min incubation at room temperature, the fluorescent marker solution was removed, biofilms were washed with water and the wells were then filled with 100 μL of water or PBS for WGA-stained biofilms. Stained biofilms were visualized by CLSM (Olympus FV1000 IX81, Markham, ON, Canada).

Dispersion of biofilm by enzymatic treatments

A biofilm dispersion assay was performed as described previously [26]. Briefly, biofilms were grown under static conditions as described above, and after the 40 h incubation, 50 μL of DNase I (500 μg/mL in 150 mM NaCl, 1 mM CaCl2), 50 μL of dispersin B (100 μg/mL in PBS; Kane Biotech Inc., Winnipeg, MB, Canada), or 50 μL of proteinase K (500 μg/mL in 50 mM Tris–HCl pH 7.5, 1 mM CaCl2) were added directly to the biofilms. Control wells were treated with 50 μL of the buffer without the enzyme. Wells treated with dispersin B were incubated for 5 min at 37 °C, and those treated with proteinase K or DNase I were incubated for 1 h at 37 °C. After the treatments, the biofilms were stained with crystal violet as described above.

Effect of fibrinogen and fibronectin on biofilm formation

Biofilms were prepared under static conditions as described above with some modifications. Prior to inoculation, various concentrations of fibrinogen (bovine, porcine or human) (0.5, 1, 2 and 5 mg/mL) or fibronectin (human) (0.5, 1 and 2 mg/mL) were added to the biofilm medium. Plates were prepared in duplicate for each experiment: one plate was used to measure biofilm formation and the other plate was used to measure growth of the bacteria in the presence of proteins. Both plates were incubated and one plate was stained as described before. The unstained replicate plate was used to evaluate growth by measuring the absorbance at 600 nm.

Genome sequencing and assembly

Genomic DNA of the high biofilm producer strain F9 was prepared using the Blood and Tissue DNeasy kit (Qiagen) according to the manufacturer’s instructions. Illumina libraries were prepared using 500 ng of genomic DNA and modified Illumina protocols [27],[28]. Paired-end sequencing was performed on an Illumina HiSeq 2000 analyzer for 75 cycles at the Wellcome Trust Sanger Institute (Hinxton, Cambridge, UK). The fastq reads were mapped to the complete SH0165 genome using Stampy to check for quality and purity of the sequence before any analyses [29],[30].

A custom-made bioinformatics pipeline was used to assemble the draft genome sequence. Cutadapt was used to remove the adaptor sequences from the sequence reads that were previously introduced during the library preparation [31]. Any undetermined nucleotides (N’s) were removed from reads and the program sickle [32] was used to trim low-quality sequences found at the ends of paired-end sequence reads resulting in a minimum length of 31 bp, using the program’s default quality thresholds for the reads. Next, a custom Perl script was used to screen out low quality fastq reads and produce a single fastq file containing the good paired reads, and a separate file containing good quality single reads. Finally, we used Velvet [33] and VelvetOptimiser 2.2.0 [34] to assemble the fastq files into the de-novo genome assembly, made up of contiguous sequences (contigs). Assembly parameters were optimised to produce the highest quality assembly (i.e. highest n50 value) using VelvetOptimiser, which runs through all possible k-mer values from 19 to 71 in increments of 2. The draft genome sequence was annotated using the automatic annotation software Prokka [35], including the rfam option. This Whole Genome Shogun project has been deposited [DDBJ/EMBL/GenBank: JHQI00000000]. The version described in this paper is version JHQI01000000.

Transcriptomics analysis

The high biofilm producer strain F9 was chosen for transcriptomic analysis. Planktonic and biofilm samples were obtained under static conditions as described above with some modifications. Bacteria were cultured in 6-well microtiter plates and after 36 h, planktonic cultures were transferred to sterile tubes, whereas biofilm bacteria were collected by scraping the surface of the wells with lysis buffer (2% SDS in PBS). Bacteria from both samples, biofilm or planktonic, were recovered by centrifugation and pellets were used for RNA extraction. For comparison, F9 was grown with shaking (220 rpm) until the culture reached stationary phase and this culture was then processed for RNA extraction. This is considered to be the “stationary culture” sample.

To extract RNA, bacterial pellets were resuspended in 2% SDS, 16 mM EDTA, 10 mM Tris (pH 8.0) and incubated for 5 min at 100 °C. Afterwards, samples were processed by two hot acid phenol-chloroform extractions, followed by two chloroform/isoamyl alcohol extractions. RNA was then precipitated with 0.6 volumes of isopropanol, 0.1 volumes of 5 M ammonium acetate and 1 μL of glycogen. After centrifugation, the pellet was washed with 70% ethanol, dried and resuspended in warmed RNase-free water. To ensure that contaminating bacterial DNA was eliminated from the samples, treatment with RNase-free DNase (Qiagen) was performed. In addition, ribosomal RNA was eliminated with the Ribo-Zero rRNA removal kit (Epicentre Biotechnologies, Madison, WI, USA) following manufacturer instructions. PCR reactions using primers specific for H. parasuis 16S rRNA gene [36] were carried out to ensure that no bacterial DNA was left in the sample. Final RNA quality was verified with a Nanodrop spectrophotometer and the integrity was analyzed using Agilent Bioanalyzer 2100 (Agilent technologies). Libraries were generated using an Ion Torrent RNA-Seq v2 kit (Life Technologies) and sequenced using an Ion Torrent PGM (Life Technologies) with an Ion 318 chip (Life technologies) at the Centre for Research in Agricultural Genomics (CRAG, Campus de Bellaterra-UAB, Spain).

Bioinformatic analysis was performed following the count-based differential expression method. Briefly, reads quality control was performed using FastQC [37] and FASTX-Toolkit [38], and reads were then mapped to H. parasuis F9 genome using the recommended Torrent Mapping Alignment Program (TMAP) v3.4.1 with map2 setting [39]. Alignments were inspected using SAMtools [40] and Integrative Genomics Viewer (IGV) [41]. HTSeq v0.5.4p3 [42] was used for feature counting with intersection-nonempty setting and discarding non-protein coding CDS. Differentially expressed genes (DEGs) were identified with edgeR R package [43] with a 5% P-value cut-off, using an assigned dispersion value of 0.04.

To perform gene set enrichments, a custom Gene Ontology (GO) database was built. Protein coding genes were BLASTed using BLASTp (version 2.2.28) against the non-redundant NCBI database (April 2014), e value of 10−3 and keeping first 20 hits. GO terms were mapped to Blast hits using Blast2GO [44]. The most significantly up-regulated genes (P < 0.05) were identified as candidates for classic Fisher’s exact testing through Blast2GO. Tests were performed for biological process (BP), cellular component (CC) and molecular function (MF) with P < 0.05. Conservation of membrane-related genes among all 14 H. parasuis sequences available in GenBank [GenBank: APCA00000000.1, APBW00000000.1, ABKM00000000.2, APBX00000000.1, AZQU00000000.1, APBZ00000000.1, AOSU00000000.1, APBY00000000.1, APBV00000000.1, CP001321.1, APBT00000000.1, APBU00000000.1, APCB00000000.1, CP005384.1] was achieved as follows: whole F9 proteome was analyzed using Phobius [45] via Blast2GO, positively predicted membrane-related genes were blasted against the 14 H. parasuis genomic sequences using tBLASTn with the following settings: e value of 10−5, alignment length > 70% and match identity > 60%. Whole-genome BLASTp comparisons were performed between H. parasuis and Actinobacillus pleuroneumoniae serovar 5b strain L20 [GenBank: NC_009053.1], using the following settings: e value of 10−5, alignment length > 90% and match identity > 40%. All transcriptomic data were deposited in the Gene Expression Omnibus database [GEO: GSE56428].

Results

Biofilm formation under static conditions

The phenotypic and genotypic characteristics of the H. parasuis strains used in the present study are given in Table 1. Five non-virulent strains were recovered from the nasal cavities of healthy pigs while nine virulent strains were recovered from lesions of pigs with Glässer’s disease. The ability of H. parasuis to form biofilms at the solid–liquid interface in polystyrene microtiter plates was determined for each of the virulent and non-virulent strains (Figure 1A). Interestingly, the nasal, non-virulent strains formed significantly (p < 0.05) more biofilms than the virulent strains isolated from lesions of pigs with Glässer’s disease (Figure 1B). We also noticed that biofilm production was stronger in strains sensitive to serum compared to resistant strains (Additional file 1A, p = 0.059); in strains negative for vtaA group 1 genes compared to strains positive for these genes (Additional file 1B, p = 0.189); in strains belonging to MLST cluster C compared to strains belonging to MLST cluster A (Additional file 1C, p = 0.202), and in strains negative for the sialyltransferase gene lsgB compared to strains positive for lsgB (Additional file 1D, p = 0.228). These differences however were not statistically significant probably due to the small number of strains in some of the groups that were compared.
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).

Confocal laser scanning microscopy using different fluorescent probes was performed with the nasal, high-biofilm producer strains F9 and MU21-2 and the weak-biofilm producer virulent strains Nagasaki and ER-6P. The biofilm cells were stained with FilmTracer™FM®1-43, which becomes fluorescent once it is inserted in the cell membrane. The biofilms were also stained with fluorescent probes to label poly-N-acetylglucosamine (PGA), proteins, or extracellular DNA that could be present in the biofilm matrix. The data indicated that the biofilm matrix of these strains contains PGA, proteins, and eDNA; strain MU21-2 seemed however to produce less PGA than the other three strains (Figure 2). To further characterize the biofilms, 15 images of biofilm layers were recorded and stacked, and 3D-images of the biofilms were generated (Figure 3). Based on these reconstructions, the thickness as well as the biomass of the biofilm produced by each strain was evaluated. The thickness of the weak-biofilm producer strains Nagasaki and ER-6P was approximately 40 μm while the thickness of the high-biofilm producer strains MU21-2 and F9 was 50 μm and 70 μm, respectively. This was in agreement with the biomass (μm3/μm2) which was 17 for the weak-biofilm producer strain Nagasaki while the biomass of the high-biofilm producer strain F9 was 34.
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.

Biofilms of strains F9, MU21-2, Nagasaki and ER-6P were digested with enzymes to further characterize the composition of the matrix. All strains were resistant to dispersin B (more than 70% of the biofilm remained after treatment) (Figure 4A). All strains were however sensitive to the proteinase K treatment (Figure 4B), with strain Nagasaki showing the lowest sensitivity (~80% of the biofilm remaining after treatment) and strain ER-6P showing the highest sensitivity (less than 20% of the biofilm remaining after treatment). Most strains were not affected by DNase I treatment (more than 70% of the biofilm remained after treatment) except strain F9 which was highly sensitive (with less than 20% of the biofilm remaining after treatment) (Figure 4C). This suggested that proteins, and, in one strain, extracellular DNA play a larger role than PGA in H. parasuis biofilm formation.
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.

Biofilm formation under shear force conditions

Previous experiments with A. pleuropneumoniae and the drip-flow apparatus indicated that this biofilm reactor could be used to grow biofilm of fastidious Pasteurellaceae isolated from the upper respiratory tract [24]. The conditions (50 mL/channel/hour; 50% BHI-NAD) used to grow A. pleuropneumoniae biofilms in the drip-flow apparatus were used for the first test. However, these did not support the growth of H. parasuis biofilms. Given that H. parasuis is more fastidious than A. pleuropneumoniae, a full strength BHI-NAD was used and the speed of the flow was reduced by half. These conditions allowed the two strains with a strong-biofilm phenotype, MU21-2 and F9, to produce visible biofilms (Figure 5). Although a thin film was observable for Nagasaki and ER-6P, which are strains with a weak-biofilm phenotype under static conditions, the conditions of the drip-flow apparatus did not enhance the biofilm formation by these strains (Figure 5). As indicated by the biofilm dry weight, strain MU21-2 produced a larger biomass than strain F9 under these flow conditions (Table 2).
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).

Table 2

Average dry weight (in mg) for drip-flow biofilms formed after 24 h of continuous flow by 4 different strains of Haemophilus parasuis

Strain

Biofilm dry weight (±Standard error of the mean)

MU21-2

8.33 (±3.23)

F9

3.37 (±0.38)

Nagasaki

ND1

ER-6P

ND

1The amount of biofilm was below the detection limit.

The BioFlux 200 flowthrough device is a high throughput microfluidic system that has been recently tested for the growth of bacterial biofilms [25]. This system has yet to be tested with Pasteurellaceae and could provide certain advantages over the drip-flow apparatus. For example, the system requires smaller volumes, which range in μL to mL, and can be used for high throughput screens. Initial density of the inoculum (OD600 of 1.0, 0.5 and 0.25) and the incubation time (2 h and 4 h) for the initial attachment were the first parameters tested with a relatively low shear force (0.5 dyne/cm2). An OD600 of 0.25 and an incubation time of 2 h for the initial attachment were sufficient for strains MU21-2 and F9 to form biofilms that block the microfluidic channel after 6 h (Figure 6); however, strains Nagasaki and ER-6P did not form biofilms (Figure 6). To prevent blocking of the channel, a range of shear force was tested with an inoculum at OD600 of 0.25 and an incubation time of 2 h for the initial attachment. Both strains MU21-2 and F9 were not able to form a biofilm when the shear force was equal or above 0.7 dyne/cm2. Between 0.5 and 0.7 dyne/cm2, both strains always blocked the channel within 12 h but it took longer as the shear force was increased. Therefore, in our hands, this microfluidic system can only be used to study biofilm formation of H. parasuis during short incubation periods.
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.

Effect of fibrinogen and fibronectin on biofilm formation

It has been shown that supplementing the culture medium with fibrinogen induces biofilm formation of Streptococcus suis, another important swine pathogen [46]. Thus, we evaluated the effect of supplemental fibrinogen on H. parasuis biofilm formation. As shown in Figure 7, fibrinogen (at a concentration of 1 mg/mL) inhibited biofilm formation by all four strains. Fibronectin had no effect on bioflm formation (data not shown). Inhibition of biofilm formation by fibrinogen was not related to an inhibition of growth since fibrinogen did not affect growth of H. parasuis (data not shown).
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.

Transcriptional profile of H. parasuis F9 grown in biofilm

Given that the genome of strain F9 was not previously sequenced, the genome of this high-biofilm producer was sequenced to facilitate transcriptional analysis. The assembly size of the F9 genome was 2.49 Mb, with an n50 of 44 023 and 644 contigs, with a G + C content of 39%, which is comparable to that of the draft and complete genomes of H. parasuis[30],[47]. Transcriptomic analysis was performed with an average of 2 M sequence reads per mRNA sample. More than 75% of the reads for each sample were mapped. The majority of reads mapped with a mapping quality (MAPQ) ≥ 20, and only reads mapping with a MAPQ ≥ 10 were further processed for differential gene expression analysis (Additional file 2A). Some reads mapped in non-protein coding sequences, mainly in tRNA gene sequences, and were particularly high in the stationary culture sample (Additional file 2B). Differential expression analysis found 425 DEGs in biofilm (B) when compared to planktonic growth (P) (Table 3). When B or P condition was compared to the stationary-phase culture (S), a notable increase in the number of up-regulated genes was observed (Figure 8 and Table 3). Filtered lists of DEGs for B vs P, P vs S and B vs S are shown in Additional files 3, 4 and 5, respectively. A large number of up- and down-regulated genes were shared between B vs S and P vs S comparisons (Figure 9A and B), although a considerable amount was unique to each condition. Fifty-five genes were up-regulated in all three comparisons, which included 8 ribosomal proteins. On the other hand, 56 up-regulated genes were unique to the biofilm and included, among others, six transcriptional regulators, possibly involved in biofilm formation (Additional file 6).
Table 3

Summary of the differential expression analysis performed with edgeR tool ( P -value < 0.05)

Comparison

Up

Down

Total

4Genome (%)

1B vs 2P

212

213

425

19

B vs 3S

538

571

1109

49

P vs S

376

417

793

35

1Biofilm.

2Planktonic.

3Stationary phase.

42259 of the 2317 annotated protein-coding genes were taken as total (at least one count per million (cpm) in at least two samples).

Percentages of differentially expressed genes are also shown for each comparison.

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.

Blast2GO allowed 76% of GO term assignment to H. parasuis proteome, with a good GO level distribution (mean level = 6.8; SD = 2.7) and more than 8 K annotations. Enrichment analysis of the DEGs (P < 0.05) identified a large number of up- or down-regulated pathways (Additional file 7). Analysis was visualized performing Venn diagrams (Additional files 8 and 9). All three comparisons shared the enriched up-regulated GO term “structural constituent of ribosome”. Enrichment of B vs S and P vs S shared the following GO terms: activation of response to oxidative stress, iron ion binding, ribosome biogenesis, unfolded protein binding and peroxidase activity pathways, indicating the importance of these functions for both biofilm and planktonic growth. Interestingly, protein folding, cell outer membrane, protein secretion, and sequence-specific DNA binding (transcriptional regulators) GO terms were up-regulated specifically in B vs S and B vs P comparisons, which could indicate specific biofilm pathways. Other GO terms were specific to each comparison. A total of 20 up-regulated GO terms were specific to B vs P comparison, such as lipopolysaccharide transport, response to stress or DNA-mediated regulation of transcription (transcriptional regulators) (Additional file 9). The same analysis was performed for down-regulated genes and showed that membrane-related genes were over-represented among these genes in all three comparisons. These membrane-related genes were related to transport, more especially to sodium ion transport or phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS). B vs S and P vs S detected down-regulated genes related to nutrient transport, such as ions, amino acids or monosaccharides, which indicate common pathways in biofilm and planktonic conditions. On the other hand, some enriched GO terms were specific to biofilm, as evidenced by the B vs P comparisons. For instance, GOs related to translation (ribosomal proteins) were among the most significantly down-regulated, which suggest a possible low metabolic state, but these enriched GO terms were caused by different ribosomal proteins than the ones responsible for the shared enriched up-regulated GO term (Additional file 9).

The F9 surface-associated proteins were predicted using Phobius and the conservation of the up-regulated ones was examined by tBLASTn in the 14 H. parasuis genomic sequences available in GenBank. The number of predicted membrane proteins up-regulated in F9 grown under biofilm conditions (B vs S) was 148 from a total of 538. The majority of the predicted membrane proteins were well conserved among the majority of strains, including a 28 kDa outer membrane protein (HS372_00711), Omp P5 precursor (HS372_01222), PilQ (HS372_02002) and Omp85 (HS372_00165) (Additional file 10). Others were found exclusively in non-virulent isolates, such as OmpW (HS372_00498), prophage CP4-57 integrase (HS372_00596), filamentous hemagglutinin FhaB (HS372_01074) or five hypothetical proteins (HS372_00147, HS372_01332, HS372_02390, HS372_00611 and HS372_02391) (Additional file 10). Additionally, FhaC (annotated as TpsB) was also found only in the non-virulent strains (Additional file 10).

H. parasuis gene expression during biofilm and planktonic growth was compared to transcriptomic data of A. pleuropneumoniae, another swine pathogen member of the Pasteurellaceae family, from a previous study [24]. Although preliminary analysis showed that A. pleuropneumoniae and H. parasuis F9 shared only 60% of the genome, some common up-regulated genes were found under biofilm condition (Table 4), but no predominant pathways were detected, suggesting different regulatory networks for these two species. Notably, proteins related to anaerobic metabolism such as cytochrome c-type protein NapC (HS372_02085) or putative electron transport protein yccM (HS372_02091) and some lipoproteins (HS372_01222 and HS372_00366) were found in both bacteria.
Table 4

Common up-regulated genes between Haemophilus parasuis F9 and Actinobacillus pleuropneumoniae when grown in biofilm condition

H. parasuis(this study) vsA. pleuropneumoniae[[22]]

H. parasuisF9

App

Product

HS372_02083

APL_1425

Cytochrome c-type protein NapC

HS372_02085

APL_1427

Putative electron transport protein yccM

HS372_02091

APL_1821

50S ribosomal protein L31 type B

HS372_00945

APL_1440

High-affinity zinc uptake system protein znuA precursor

HS372_00147

APL_1894

hypothetical protein

HS372_02009

APL_1423

Putative esterase

HS372_02012

APL_0433

Peptide methionine sulfoxide reductase MsrB

HS372_01222

APL_0460

Outer membrane protein P5 precursor

HS372_00060

APL_1206

putative ribonuclease FitB

HS372_01900

APL_1173

Nicotinamide riboside transporter pnuC

HS372_00666

APL_0442

vancomycin high temperature exclusion protein

HS372_01587

APL_0484

Alpha-aminoadipate--lysW ligase lysX

HS372_02062

APL_0038

hypothetical protein

HS372_00364

APL_0222

Putative lipoprotein/NMB1162 precursor

HS372_01892

APL_0133

Cys regulon transcriptional activator

HS372_00988

APL_1295

Arginine repressor

HS372_02252

APL_1873

Succinyl-diaminopimelate desuccinylase

HS372_02064

APL_0036

hypothetical protein

HS372_01893

APL_0134

hypothetical protein

HS372_01521

APL_1320

Thiamine import ATP-binding protein ThiQ

HS372_02387

APL_1059

Integrase core domain protein

HS372_00061

APL_1207

prevent-host-death family protein

HS372_00916

APL_0423

Ribonuclease HI

HS372_01200

APL_0593

Inosine-5’-monophosphate dehydrogenase

HS372_01950

APL_1574

Putative hydrolase ydeN

HS372_02244

APL_0254

Cytosol non-specific dipeptidase

HS372_01281

APL_1230

Phosphoserine phosphatase

HS372_01385

APL_0967

Glutamate permease

HS372_01220

APL_0463

Putative phosphinothricin acetyltransferase YwnH

HS372_01490

APL_0928

hypothetical protein

HS372_01179

APL_1499

Threonine synthase

HS372_02366

APL_0395

Sigma-E factor negative regulatory protein

HS372_01208

APL_0895

Formate dehydrogenase-N subunit gamma

HS372_01221

APL_0461

putative phosphatase YwpJ

HS372_02265

APL_0687

D-lactate dehydrogenase

HS372_01342

APL_1448

Spermidine/putrescine import ATP-binding protein PotA

HS372_00366

APL_0220

Putative lipoprotein/NMB1164 precursor

HS372_00099

APL_1853

Ketol-acid reductoisomerase

Discussion

In the present study, we used well-characterized virulent and non-virulent strains of H. parasuis from our collection [13]. We found that most nasal strains (4/5) were strong-biofilm producers whereas most systemic strains (8/9) formed no biofilm or were very weak-biofilm producers. Similarly, a strong association was found between MLST clusters [2] and biofilm formation. Strains of MLST cluster C (which are associated with nasal isolation) formed more biofilms than strains of MLST cluster A (which are isolated from systemic lesions). Similarly, the presence of autotransporter vtaA group 1 genes and of sialyltransferase lsgB gene associated with virulent strains [13],[48], and was not associated with strong biofilm formation. Overall, our results are in agreement with Jin et al. [19] who observed that, generally, serovars considered non-virulent showed a higher degree of biofilm formation than virulent serovars. Although it is important to note that a strict relationship between serovars and virulence in H. parasuis has not been demonstrated [1].

The use of CLSM and fluorescent probes showed the presence of PGA, proteins, and eDNA in the biofilm matrix of H. parasuis nasal, high-biofilm producer strains F9 and MU21-2 and of the weak-biofilm producer strains Nagasaki and ER-6P recovered from lesions of pigs with Glässer’s disease. This is, to the best of our knowledge, the first report of the presence of PGA and proteins in the biofilm matrix of H. parasuis. PGA is the major component of the biofilm matrix of several bacteria including other Pasteurellaceae members such as A. pleuropneumoniae and Aggregatibacter actinomycetemcomitans[18],[49]. Nevertheless all 4 strains tested were resistant to dispersin B. All strains were however sensitive to a proteinase K treatment suggesting that proteins play a larger role than PGA in H. parasuis biofilm formation. Tang et al. [50] have shown that treatment with the staphylococcal nuclease NUC1 decreased slightly the biofilm formation of H. parasuis strain 0322. This is in agreement with results of this study showing the presence of eDNA in the biofilm matrix of H. parasuis and the high sensitivity of strain F9 biofilm to DNase I digestion.

This is also the first description of H. parasuis biofilm formation under shear force in a drip-flow reactor and a microfluidic system. Interestingly, similar biofilm phenotypes (i.e. high-producer vs weak-producer) were observed when static or controlled flow conditions were used. The drip-flow apparatus is a system that it is thought to create an environment with an air-liquid interface which closely resembles the lung environment [23]. Under the conditions we selected, the two strains with a strong-biofilm phenotype, MU21-2 and F9, produced visible biofilms while a thin film was observable for Nagasaki and ER-6P, which are strains with a weak-biofilm phenotype under static conditions. We also used the BioFlux flowthrough device, a high throughput microfluidic system that has been recently tested for the growth of dental plaque bacteria biofilms [51] and Pseudomonas aeruginosas biofilms [25]. Again, strains Nagasaki and ER-6P did not form biofilms under the conditions tested but strains MU21-2 and F9 formed biofilms that rapidly blocked the microfluidic channel. This microfluidic system can therefore be used to study biofilm formation of H. parasuis but for short incubation periods. This potentially can be used to investigate genes involved in the early steps of biofilm formation and, if the appropriate tools are combined, to study real-time gene expression during the early steps as demonstrated in Staphylococcus aureus[52].

The genes involved in H. parasuis biofilm formation are currently not known. Recently, it was reported that both galU and galE genes seemed to play a role in biofilm formation of H. parasuis[12]. A galU mutant was unable to form biofilm in a glass tube while a galE mutant produced more biofilm than the parent wild-type strain. Surprisingly however, the galU mutant also showed an increased tendency to autoagglutinate which is usually associated with a greater ability to form biofilm. Here, the transcriptome of H. parasuis F9 strain showed that static biofilm and planktonic cultures are in similar biological states but not identical, whereas greater differences in gene expression were evident when compared to stationary-phase culture. Subtle differences in gene expression between biofilm and planktonic cells have been also reported for other bacterial species [53],[54]. In addition to identifying DEGs related to metabolism, gene enrichment allowed the identification of a large proportion of membrane-related genes among the up-regulated genes in biofilm, including some that have been reported for other bacterial species [53],[54]. The conservation of some highly expressed membrane-protein genes in biofilms among the 14 H. parasuis genomes available indicates that those may not specifically associate with biofilm formation. A sub-set of these up-regulated membrane-protein genes, such as the type IV pilus biogenesis protein pilQ, may be involved in adhesion to different abiotic and biotic surfaces. Interestingly, some up-regulated genes, such as fhaB and fhaC (tpsB) or ompW, were only found in the genome of non-virulent strains. This finding, together with the fact that non-virulent strains formed stronger biofilms, suggests a possible role of these genes in biofilm formation. In fact, Fha and OmpW, together with type IV pilus, have been shown to play a role in biofilm formation in other bacteria [55],[56]. Additionally, some lipoproteins, such as lipoprotein Plp4, as well as signs of anaerobic metabolism were evidenced in the biofilms of H. parasuis and A. pleuropneumoniae, which may indicate a potential role in biofilm formation of both bacterial species. The function of specific genes in biofilm formation will need further confirmation.

Interestingly, experimental infections in snatch-farrowed, colostrum-deprived piglets showed that the H. parasuis strains that proceeded to invade the host were not maintained well in the nasal cavities of the piglets [57]. This suggests changes in the bacteria from a “colonizing state” to an “invasive state”, which could be modulated by the ability of each animal to control the infection. In the light of the results of the present study, these changes from a “colonizing state” to an “invasive state” could also be due to the ability of a given bacterial strain to form or not a robust biofilm. We propose that biofilm formation might allow the non-virulent strains to colonize and persist in the upper respiratory tract of pigs. Conversely, the predominant planktonic state of the virulent strains might allow them to disseminate within the host. This latter statement is supported by the inhibition of biofilm formation by fibrinogen. It is worth noting that a recent study on the human pathogen Streptococcus pneumoniae showed that biofilm formation in vivo is associated with reduced invasiveness and a dampened cytokine response [58]. High-biofilm production phenotype might therefore not always be linked to virulence.

Additional files

Declarations

Acknowledgements

This work was supported in part by a discovery grant from the Natural Sciences and Engineering Research Council of Canada to MJ and by a grant AGL2010-15232 from the Ministerio de Economía y Competitividad of Spain to VA. VD was a recipient of a doctoral scholarship and of an international training award from Fonds de recherche du Québec – Nature et technologies. BBO is a recipient of a FPI fellowship from the Spanish Ministerio de Economía y Competitividad. The authors would also like to acknowledge the support of the Gouvernement du Québec, Ministère des Relations internationales, de la Francophonie et du Commerce extérieur (VIIe rencontre du Comité mixte Québec-Catalogne, projet 07.304).

Authors’ Affiliations

(1)
Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Autònoma de Barcelona
(2)
Groupe de recherche sur les maladies infectieuses du porc, Faculté de médecine vétérinaire, Université de Montréal
(3)
Department of Veterinary Medicine, University of Cambridge
(4)
Institut de Recerca i Tecnologia Agroalimentàries (IRTA)

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© Bello-Orti et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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