Combination of berberine and ciprofloxacin reduces multi-resistant Salmonella strain biofilm formation by depressing mRNA expressions of luxS, rpoE, and ompR

Abstract

Bacterial biofilms have been demonstrated to be closely related to clinical infections and contribute to drug resistance. Berberine, which is the main component of Coptis chinensis, has been reported to have efficient antibacterial activity. This study aimed to investigate the potential effect of a combination of berberine with ciprofloxacin (CIP) to inhibit Salmonella biofilm formation and its effect on expressions of related genes (rpoE, luxS, and ompR). The fractional inhibitory concentration (FIC) index of the combination of berberine with CIP is 0.75 showing a synergistic antibacterial effect. The biofilm's adhesion rate and growth curve showed that the multi-resistant Salmonella strain had the potential to form a biofilm relative to that of strain CVCC528, and the antibiofilm effects were in a dose-dependent manner. Biofilm microstructures were rarely observed at 1/2 × MIC/FIC concentrations (MIC, minimal inhibition concentration), and the combination had a stronger antibiofilm effect than each of the antimicrobial agents used alone at 1/4 × FIC concentration. LuxS, rpoE, and ompR mRNA expressions were significantly repressed (p ＜ 0.01) at 1/2 × MIC/FIC concentrations, and the berberine and CIP combination repressed mRNA expressions more strongly at the 1/4 × FIC concentration. The results indicate that the combination of berberine and CIP has a synergistic effect and is effective in inhibiting Salmonella biofilm formation via repression of luxS, rpoE, and ompR mRNA expressions.

Keywords: Salmonella; berberine; biofilm; drug combinations; multidrug resistance.

Fig. 1. Effects of ciprofloxacin (CIP) (A), berberine (B), and their combination (C) on the biofilm formation by No. 5 multi-resistant Salmonella strain and the CVCC528 strain. (D) Comparison of antibiofilm effects of CIP, berberine, and the combination on the multi-resistant Salmonella strain. Data are presented as mean ± SD. Marked difference (^*p < 0.05), significant difference (^**p < 0.01) compared to the non-treated group. MIC, minimal inhibition concentration; FIC, fractional inhibitory concentration.

Fig. 2. The biofilm growth curves of the multi-resistant Salmonella strain and the CVCC528 strain without antimicrobial agents (A) and with ciprofloxacin (CIP) (B), berberine (C), and the combination (D). OD, optical density; MIC, minimal inhibition concentration; FIC, fractional inhibitory concentration.

Fig. 3. Biofilm microstructure observations of Salmonella strains under scanning electron microscopy. (A) Non-treated multi-resistant Salmonella strain. (B) Non-treated CVCC528 strain. (C) Multi-resistant Salmonella strain with a 1/2 × MIC concentration of CIP. (D) Multi-resistant Salmonella strain with a 1/4 × MIC concentration of CIP. (E) Multi-resistant Salmonella strain with a 1/2 × MIC concentration of berberine. (F) Multi-resistant Salmonella strain with a 1/4 × MIC concentration of berberine. (G) Multi-resistant Salmonella strain with a 1/2 × FIC concentration of the berberine and CIP combination. (H) Multi-resistant Salmonella strain with a 1/4 × FIC concentration of the combination. MIC, minimal inhibition concentration; CIP, ciprofloxacin; FIC, fractional inhibitory concentration. Scale bars = 100 µm (A–H).

Fig. 4. The effects of the antimicrobial agents on luxS, rpoE, and ompR mRNA expression levels in the multi-resistant Salmonella strain (A) and in strain CVCC528 (B). Marked difference (^*p < 0.05), significant difference (^**p < 0.01) compared to the non-treated group. CIP, ciprofloxacin; FIC, fractional inhibitory concentration.