intractable. Staphylococcus aureus and Pseudomonas aeruginosa are two of the most common biofilm-forming pathogens. Problems are complicated by widely applied antibiotics in animal food. In this study, S. aureus biofilm formation under sub-MIC antibiotics, as well as the polymicrobial interaction between S. aureus and P. aeruginosa were investigated.

Viability and biomass of 12 S. aureus strains were determined during biofilm formation under sub-MIC antibiotics. The sub-MIC antibiotics induced biofilms were further analyzed by RNA-seq and Q-PCR. Two S. aureus and 6 P. aeruginosa strains with different biofilm formation abilities were co-cultivated in planktonic and biofilm states. During co-cultivation, the culturability, viability, biofilm biomass, and morphology of both cells were identified and RNA-seq on typical models were performed.

Dramatical increase in biofilm formation was obtained under 1/4 MIC of certain antibiotics. Based on RNA-seq and bioinformatics analyses, differentially expressed genes were enriched to TCS which plays important role in the response of microbes to adverse environment. Combined with Q-PCR results, tagA, lytR, arlR, hssR, clfB, and altA genes were hypothesized to be critical during the biofilm formation under sub-MIC antibiotics environment. P. aeruginosa strains were classified into kill group (AT) and inhibit group (HQ) based on the interaction with S. aureus. AT killed S. aureus in 2 to 4 days, while HQ only suppressed S. aureus growth. RNA-seq results revealed that AT upregulated T6SS gene clusters during co-cultivation with S. aureus, whereas HQ expressed differently in secretion systems. Different expression of lrg operon with AT and HQ may lead to difference in S. aureus autolysis rate and thus inhibition intensity. Also, various S. aureus virulence factors were upregulated, including staphylocoagulase, clumping factor and protease