Anti-Biofilm Effect of Tea Saponin on a Streptococcus agalactiae Strain Isolated from Bovine Mastitis

Abstract

Streptococcus agalactiae (GBS) is a highly contagious pathogen which not only can cause neonatal meningitis, pneumonia, and septicemia but is also considered to be a major cause of bovine mastitis (BM), leading to large economic losses to the dairy industry worldwide. Like many other pathogenic bacteria, GBS also has the capacity to form a biofilm structure in the host to cause persistent infection. Tea saponin (TS), is one of the main active agents extracted from tea ash powder, and it has good antioxidant and antibacterial activities. In this study, we confirmed that TS has a slight antibacterial activity against a Streptococcus agalactiae strain isolated from dairy cow with mastitis and inhibits its biofilm formation. By performing scanning electron microscopy (SEM) experiments, we observed that with addition of TS, the biofilm formed by this GBS strain exhibited looser structure and lower density. In addition, the results of real-time reverse transcription polymerase chain reaction (RT-PCR) experiments showed that TS inhibited biofilm formation by down-regulating the transcription of the biofilm-associated genes including srtA, fbsC, neuA, and cpsE.

Keywords: Streptococcus agalactiae; biofilm; bovine mastitis; tea saponin.

Figure 1

Effects of TS on the growth curve and survival ability of Streptococcus agalactiae strain GBS2 (GBS2). (A) Growth curves of GBS2 cultured in tryptic soy broth (TSB) medium without or with the corresponding concentration of tea saponin (TS). (B) Survival rate assays of GBS2. Colony counts of GBS2 were counted after 12 h of incubation at 38 °C without or with corresponding concentration of TS. The survival rates of the control groups without exposure to TS were designated as 100% (* represents p < 0.05 and ** represents p < 0.01).

Figure 2

Effects of TS on GBS2 biofilm. (A) The crystal-violet stained biofilms of GBS2 in the 96-well plates. (B) Results of the biofilm biomass measured by using a microplate reader at a wavelength of 560 nm. Every data point was obtained by averaging the absorbance data from at least four replicate wells (* represents p < 0.05 and ** represents p < 0.01).

Figure 3

Electron micrographs of GBS2 biofilm monitored by SEM. (A) The electron micrographs of GBS2 biofilm at low magnification (1500×) (a) without TS and (b) with 2 mg/mL TS. (B) The electron micrographs of GBS2 biofilm at high magnification (6000×) (a) without TS and (b) with 2 mg/mL TS.

Figure 4

Transcript levels of the biofilm-associated genes determined by RT-qPCR. The transcription of srtA, fbsC, csrR, neuA, cpsE, and luxS were measured in GBS2 without or with 2 mg/mL TS. The differences of gene expression were calculated by ΔΔCt (Ct = cycle threshold) method using the 16S rDNA gene as the housekeeping gene, normalized by subtracting the Ct value of 16S cDNA from target cDNA. All experiments were repeated at least three times with four parallels (* represents p < 0.05 and ** represents p < 0.01). Error bars indicate standard deviations.