Sub-MICs of Azithromycin Decrease Biofilm Formation of Streptococcus suis and Increase Capsular Polysaccharide Content of S. suis

Abstract

Streptococcus suis (S. suis) caused serious disease symptoms in humans and pigs. S. suis is able to form thick biofilms and this increases the difficulty of treatment. After growth with 1/2 minimal inhibitory concentration (MIC) of azithromycin, 1/4 MIC of azithromycin, or 1/8 MIC of azithromycin, biofilm formation of S. suis dose-dependently decreased in the present study. Furthermore, scanning electron microscopy analysis revealed the obvious effect of azithromycin against biofilm formation of S. suis. Especially, at two different conditions (1/2 MIC of azithromycin non-treated cells and treated cells), we carried out comparative proteomic analyses of cells by using iTRAQ technology. Finally, the results revealed the existence of 19 proteins of varying amounts. Interestingly, several cell surface proteins (such as ATP-binding cassette superfamily ATP-binding cassette transporter (G7SD52), CpsR (K0FG35), Cps1/2H (G8DTL7), CPS16F (E9NQ13), putative uncharacterized protein (G7SER0), NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (G5L259), putative uncharacterized protein (G7S2D6), amino acid permease (B0M0G6), and NsuB (G5L351)) were found to be implicated in biofilm formation. More importantly, we also found that azithromycin affected expression of the genes cps1/2H, cpsR and cps16F. Especially, after growth with 1/2 MIC of azithromycin and 1/4 MIC of azithromycin, the capsular polysaccharide content of S. suis was significantly higher.

Comparative Proteomic Analysis Provides insight into the Key Proteins as Possible Targets Involved in Aspirin Inhibiting Biofilm Formation of Staphylococcus xylosus

“…Staphylococcus xylosus ATCC 700404 was grown in Tryptic Soy Broth (TSB; Summus Ltd., Harbin, Heilongjiang, China) in 100-mm polystyrene Petri dishes at 37°C for 24 h. Minimal inhibitory concentration (MIC) assays of aspirin were done three times (refer to Yang et al, 2016 ) with a few modifications. Briefly, S. xylosus ATCC700404 was grown aerobically at 37°C in TSB (Summus, Ltd., Harbin, Heilongjiang, China) overnight.…”

“…The procedure of Yang et al (2016) was followed. Briefly, cultures were diluted to an optical density of 0.1 at 600 nm (OD 600 ) and 2 mL was added to wells of a 6-well microplate containing an 10 mm × 10 mm sterilized rough organic membrane (Mosutech, Co., Ltd., Shanghai, China) respectively on the bottom.…”

“…After incubation without shaking for 24 h at 37°C, medium and planktonic bacteria on the organic membrane were removed with sterile PBS. The biofilms obtained from bacterial cells and prepared for analysis as described by Yang et al (2016) .…”

Analysis of multidrug resistance in Streptococcus suis ATCC 700794 under tylosin stress

“…To investigate the effect of tylosin on the expression of genes, six proteins were chosen ( Table 1). The 16S rRNA of S. suis strain was selected as a reference gene for all the experiments and the primers of target genes are listed in Table 2 [36]. The reaction conditions were 94℃ for 10 min followed by 40 cycles of amplification at 94℃ for 15 s and 60℃ for 60 s [36].…”

“…Our analysis detected 1501 proteins. A ratio of proteins with ＞ 1.2 or ＜ 0.8 Forward: 5' GATATATGGAGGAACACCG 3' [36] Reverse: 5' GACCCAACACCTAGCACT 3' [36] analysis (Figure 2), some proteins were found to have a significant change in their expression as visualized using the R studio software. The resistance-related proteins like elongation factor G (G7SMG3), elongation factor Ts (G7SBK2), elongation factor Tu (Q9EZW2) were up-regulated and had fold-change values of 3.67, 2.27 and 1.74, respectively.…”