Effects of stigmata maydis on the methicillin resistant Staphylococus aureus biofilm formation

Abstract

Background: Mastitis is an inflammatory reaction of the mammary gland tissue, which causes huge losses to dairy farms throughout the world. Staphylococcus aureus is the most frequent agent associated with this disease. Staphylococcus aureus isolates, which have the ability to form biofilms, usually lead to chronic mastitis in dairy cows. Moreover, methicillin resistance of the bacteria further complicates the treatment of this disease. Stigmata maydis (corn silk), a traditional Chinese medicine, possess many biological activities.

Methods: In this study, we performed antibacterial activity assays, biofilm formation assays and real-time reverse transcription PCR experiments to investigate the effect of stigmata maydis (corn silk) on biofilm formation and vancomycin susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from dairy cows with mastitis.

Results: In this study, the aqueous extracts of stigmata maydis inhibited the biofilm formation ability of MRSA strains and increased the vancomycin susceptibility of the strains under biofilm-cultured conditions.

Conclusion: This study proves that the aqueous extracts of stigmata maydis inhibit the biofilm formation ability of MRSA strains and increase the vancomycin susceptibility of the MRSA strains under biofilm-cultured conditions.

Keywords: Biofilm; Bovine mastitis; MRSA; Stigmata maydis; Vancomycin.

Figure 1. Effect of stigmata maydis aqueous extract on growth of MRSA strains.

(A) The growth curves of MRSA strains SA2 and SA3 cultured in tryptic soy broth medium with or without specific concentrations of stigmata maydis extract. The results represent a mean of three independent experiments. (B) Colony-forming unit assays of MRSA strains SA2 and SA3. Colony counts of strains SA2 and SA3 were compared after 12 h of incubation at 37 °C with or without addition of stigmata maydis. The colony counts of the test group cultured with different concentrations of stigmata maydis were all compared with that of the control group (without stigmata maydis), the survival rate of which was designated as 100%.

Figure 2. Effect of stigmata maydis aqueous extract on biofilm formation of MRSA strains.

The cells of strains SA2 and SA3 were cultured in 96-well plates for 24 h at 37 °C, and the tigmata maydis extract was added in the tryptic soy broth at concentrations of zero, 0.5, one and two mg/mL, respectively. (A) Photographs of the 96-well plates were taken after staining with crystal violet. (B) The biomass that adhered to the plate after staining with crystal violet was measured by a MicroELISA auto-reader at a wavelength of 560 nm. The results represent a mean of three independent experiments. (** represents P < 0.01).

Figure 3. Comparison of the relative transcript levels of several biofilm-associated genes.

The transcript levels of icaA, icaD, icaB, icaC and icaR were measured by performing real-time reverse transcription-PCR in strains SA2 (A) and SA3 (B). The stigmata maydis extract was added to the culture medium at concentrations of zero, 0.5, one and two mg/mL, respectively. (** represents P < 0.01).

Figure 4. Colony-forming unit assays of the planktonic-cultured MRSA strains SA2 and SA3 in the presence of vancomycin.

The colony counts of the test group cultured with different concentrations of stigmata maydis were all compared with that of the control group (without stigmata maydis), the survival rate of which was designated as 100%. (A) MRSA strains were cultured with 1/4 MIC concentration of vancomycin (SA2: 0.125 μg/mL, SA3: 0.25 μg/mL). (B) MRSA strains were cultured with 1/2 MIC concentration of vancomycin (SA2: 0.25 μg/mL, SA3: 0.5 μg/mL).

Figure 5. Colony-forming unit assays of the biofilm-condition cultured MRSA strains SA2 and SA3 in the presence of vancomycin.

The colony counts of the test group cultured with different concentrations of stigmata maydis were all compared with that of the control group (without stigmata maydis), the survival rate of which was designated as 100%. (A) MRSA strains were cultured with 1/4 MIC concentration of vancomycin (SA2: 0.125 μg/mL, SA3: 0.25 μg/mL). (B) MRSA strains were cultured with 1/2 MIC concentration of vancomycin (SA2: 0.25 μg/mL, SA3: 0.5 μg/mL). (** represents P < 0.01).