In vitro Mixed Biofilm of Streptococcus suis and Actinobacillus pleuropneumoniae Impacts Antibiotic Susceptibility and Modulates Virulence Factor Gene Expression

Streptococcus suis (S. suis) and Actinobacillus pleuropneumoniae (A. pleuropneumoniae) are primary swine pathogens that have been frequently co-isolated from pigs suffering from severe respiratory disease. The purpose of this study was to investigate the biological impacts of the interactions between S. suis and A. pleuropneumoniae. A single- and dual-species culture model was established in vitro via S. suis HA9801 (serotype 2) and A. pleuropneumoniae CVCC265 (serotype 1). The single or mixed biofilms were imaged by confocal laser scanning microscopy. The biomass and viable cells in biofilms were quantified by crystal violet staining and determination of colony-forming units. The antibiotic susceptibility was determined by a microdilution broth method. The differences in gene transcription in pure- or mixed-species biofilms of S. suis and A. pleuropneumoniae was evaluated by quantitative PCR. S. suis and A. pleuropneumoniae formed two-species biofilms when co-cultured in vitro. When co-cultured with S. suis, biofilm formation by A. pleuropneumoniae was significantly increased with the absence of NAD that is necessary for the growth of A. pleuropneumoniae. Moreover, compared with monocultures, the antibiotic resistance of S. suis and A. pleuropneumoniae was both enhanced in the co-culture model. When grown in dual-species biofilms, for A. pleuropneumoniae, genes associated with virulence factors, including exotoxins and adhesins, were significantly upregulated. For S. suis, virulence factor-related genes cps2, gdh, mrp, and sly were highly induced. These results suggest that the interspecies interactions between S. suis and A. pleuropneumoniae may be cooperative under specific conditions and may play an important role in the disease progression and persistent infection.