S. suis HA9801 (SS) was isolated from a diseased pig in Jiangsu Province, China, and identified to be S. suis serotype 2. All experiments in this study were approved by the Experimental Animal Monitoring Committee of Henan University of Science and Technology and carried out accordingly. A. pleuropneumoniae CVCC 265 (APP) was purchased from China Veterinary Culture Collection Center (CVCC). S. suis was grown in tryptic soy broth (TSB) or plated on TSA. A. pleuropneumoniae was cultivated in the same media supplemented with 0.1 μg/mL of β-nicotinamide-adenine-dinucleotide (NAD). Bacterial cultures were incubated at 37°C. All experiments described below were replicated in biological triplicate.

Bacterial colonies of S. suis and A. pleuropneumoniae were inoculated separately in solid media and grown overnight to mid-exponential phase in TSB + 0.1% NAD medium. The bacterial cultures were diluted in fresh medium to obtain an optical density at 660 (OD₆₆₀) of 0.03. Mono-culture of S. suis or A. pleuropneumoniae and co-culture of mixture (1:1 ratio) were inoculated in TSB + 0.1% NAD medium for 24 h. Samples were taken at 0, 2, 4, 6, 8, 10, 12, and 24 h, serially diluted in sterile PBS, and then plated on TSA or TSA + 0.1% NAD medium to discriminate S. suis or A. pleuropneumoniae, respectively, and the viable cell counts were enumerated.

For the mixed culture, the Competitive Index (CI) was calculated according to the formula: (A. pleuropneumoniae/S. suis)_output/(A. pleuropneumoniae/S. suis)_input. The output and input samples were assessed by plating onto selective medium at different time points, respectively. The Relative Increase Ratio (RIR) was similar to CI, and was calculated from the corresponding growth results obtained from single cultures of each strain (Macho et al., 2007). A positive CI value suggests a competitive advantage for A. pleuropneumoniae and vice versa. Only CIs that are statistically different from the RIRs at the same growth stages can be recognized as the result of prominent competition between species (Macho et al., 2007).

Biofilm production was quantified by crystal violet staining as described previously (Bragonzi et al., 2012). Briefly, overnight cultures of S. suis and A. pleuropneumoniae were diluted 1/100 in fresh TSB broth supplemented with NAD, and inoculated individually or at several different ratios in 96-well plates. After 24 h incubation, the medium and planktonic bacteria were removed, and each well was gently washed twice with sterile PBS. Methanol was used to fix the attached bacteria for 15 min. The plates were then air-dried and the biofilms were stained with crystal violet (0.1%). After 20 min, the excess dye was discarded and plates were washed twice with sterile PBS prior to adding 200 μl of 95% ethanol to the wells to dissolve the biofilms. The optical density at 620 nm (OD₆₂₀) was measured using a microplate reader.

Biofilms were prepared in the 96-well plates as described above. Biofilms were washed twice with sterile PBS, and then bacteria were detached and homogenized in 100 μl of sterile PBS by weak sonication for 4 min. TSA or TSA + 0.1% NAD medium was used to discriminate S. suis or A. pleuropneumoniae, respectively, and then viable cells were enumerated (Chan et al., 2017).

Chamber slides were inoculated with bacterial suspension (S. suis alone, A. pleuropneumoniae alone and combination in 1:1 ratio) for 24 h at 37°C. The slides were washed twice with PBS to remove the medium and unattached bacteria. Samples was stained with SYTO 9 solution following the manufacturer’s instructions from LIVE/DEAD^TM BacLight^TM Bacterial Viability Kit (Thermo Fischer Scientific, Inc., Waltham, MA, United States), and then washed with PBS (Tawakoli et al., 2013). Biofilms were observed using a Zeiss LSM800 CLSM (Carl Zeiss, Jena, Germany).

The in vitro antibiotic susceptibility of S. suis and A. pleuropneumoniae was determined by a twofold dilution method in microplates following the guidelines of Clinical and Laboratory Standards Institute. Antibiotic serial dilutions were made up in culture medium, and 100 μl was transferred to the wells. Overnight cultures of S. suis and A. pleuropneumoniae were diluted at 1:100 with TSB contain in 0.1% NAD, and 100 μl of S. suis, A. pleuropneumoniae, or combination in 1:1 were added to 96-well plates and incubated at 37°C for 24 h. The minimum inhibitory concentration (MIC) values were determined by reading the optical density and visual observation of the turbidity.

The minimum biofilm eradication concentration (MBEC) values were also determined. Biofilms were prepared in the 96-well plates as described above. Wells were washed twice with PBS and antibiotic serial dilutions made up in TSB containing 0.1% NAD were transferred (200 μl) to the wells. The plates were incubated for another 24 h. The MBEC values were determined by plating samples on culture medium plates.

Real-time PCR was used to assess the relative expression of virulence factor genes in A. pleuropneumoniae or S. suis when grown alone or in mixed biofilms. Biofilms were prepared in 24-well plates as described above, washed twice with PBS, detached by sonicating, and then collected. RNA was extracted by the Trizol method (Pompilio et al., 2015). gDNA removal and cDNA synthesis were performed using PrimeScript^TM RT reagent Kit with gDNA Eraser (TaKaRa) according to the manufacturer’s protocol. Real-time PCR assay was performed with TB Green^® Premix Ex Taq^TM (TaKaRa) following the manufacturer’s instructions. The primers used are listed in Table 1. The 16S rRNA was used as the house-keeping gene for normalization. Relative expression levels were determined by the (ΔΔCt) method.

GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, United States) was used for data analysis. Results were obtained from three independent experiments and all values were expressed as means ± standard deviation. Differences between mean values were evaluated by Student’s t-test. P-value of 0.05 or less was considered statistically significant.

To study the interactions between S. suis and A. pleuropneumoniae in planktonic co-cultures, the growth curves of single and mixed cultures were compared, and the results are shown in Figure 1A. The kinetics analysis showed that the growth of S. suis in mixed culture was negatively affected from 8 to 24 h, while A. pleuropneumoniae growth was not clearly affected when co-cultured with S. suis. To further estimate the differences in growth kinetics between S. suis and A. pleuropneumoniae in single or mixed cultures, CI and RIR indexes were calculated. As shown in Figure 1B, a positive CI index of A. pleuropneumoniae versus S. suis was always observed, indicating a competitive advantage for A. pleuropneumoniae over S. suis in co-cultures. The CI was significantly higher than the RIR between 8 and 24 h (p < 0.05), suggesting a noticeable negative effect of A. pleuropneumoniae on S. suis growth.

Biofilm formation by S. suis and A. pleuropneumoniae in single and dual culture in 96-well plate was assessed by crystal violet staining and viable count, and the results are shown in Figure 2. Both S. suis and A. pleuropneumoniae formed important biofilms when grown in TSB supplemented with NAD (Figure 2A). However, in the absence of NAD, A. pleuropneumoniae couldn’t form biofilms (Figure 2B). Under favorable growth conditions(supplement with NAD)for A. pleuropneumoniae, mixed biofilms with S. suis were formed (Figure 2C). Moreover, based on results of crystal violet staining and determination of CFU, A. pleuropneumoniae was able to grow and form a dual-species biofilm without the addition of NAD when grown in the presence of S. suis (Figure 2D).

By using confocal laser scanning microscopy (Figure 3), it could be confirmed that both S. suis and A. pleuropneumoniae were able to form robust single- and dual-species biofilms in vitro.

The in vitro antibiotic susceptibility of S. suis and A. pleuropneumoniae, individually and in combination to several antimicrobial drugs, was determined (Table 2). For tylosin tartrate, when in co-culture with the other, the MBEC of S. suis increased from 1.25 to 80 μg/ml, and the MBEC of A. pleuropneumoniae increased from 40 to 80 μg/ml. For tilmicosin, when in co-culture, the MIC and MBEC of S. suis or A. pleuropneumoniae were twice as high as that in pure culture. In general, S. suis and A. pleuropneumoniae in mixed biofilms showed increased resistance to tylosin tartrate and tilmicosin with higher values of MBEC in comparison to single-species biofilms.

As shown in Figure 4, in comparison with the single species biofilms, the virulence factor genes in the two-species biofilms were overall upregulated. For A. pleuropneumoniae, compared with mono-species biofilms, ApxI and ApxII, codifying for exotoxin, were upregulated by 10.65- and 22.58-fold, respectively, afuB involved in biofilm formation was upregulated by 8.69-fold, apaI associated with adhesin was upregulated by 5.73-fold, and hgbA involved in iron uptake was upregulated by 18.3-fold (Figure 4A). For S. suis, virulence factor related genes cps2, gdh, mrp, and sly were significantly upregulated by 2. 61-, 2. 23-, 3. 81-, and 2.26-fold, respectively, with no statistical differences of fbps gene (Figure 4B). The results may indicate that co-culture may modulate the bacterial virulence in mixed biofilm.