In vitro characterization and inhibition of the interaction between ciprofloxacin and berberine against multidrug-resistant Klebsiella pneumoniae

Abstract

Ciprofloxacin is a quinolone antibiotic used to treat Klebsiella pneumoniae infections in the clinic. Previous studies have demonstrated that berberine exhibits antibacterial activity and less acquired resistance related to efflux pumps. The multidrug efflux pump acrAB-tolC can be stimulated to expel as much toxic material as possible from the cells, but a detrimental effect can be produced owing to an overcrowded periplasm with excess expression products, which inhibits bacterial growth. In this study, the in vitro antibacterial activities of ciprofloxacin in combination with berberine were evaluated and compared with those of ciprofloxacin and berberine alone by evaluating the MIC, MBC and summation fractional IC against 20 clinical multidrug-resistant K. pneumoniae isolates, 1 quality control bacterium and 1 induced-resistance bacterium. Susceptibility tests showed that the MIC for the combination of berberine and ciprofloxacin was 1/2 that of the individual agents or less. Antimicrobial activities of 18.18% synergy and 77.27% additivity were found. Furthermore, synergism was verified through a time-kill assay, which suggested that the synergistic antibacterial effect of the two-drug combination may, to some extent, be related to the high expression of the acrAB-tolC and acrR multidrug efflux pumps. Indeed, the expression of these genes was increased >14-fold in the isolates affected by ciprofloxacin-berberine combination synergism.

Figure 1

The time-kill curves (a−e) of K. pneumoniae isolates (R12, R5, R15, ATCC and RATCC); , growth control; , CIP (a, b, c and e: ciprofloxacin 32 μg ml⁻¹, d: ciprofloxacin 0.0625 μg ml⁻¹); , BER (berberine 1024 μg ml⁻¹); , CIP+BER (a, b, c and e: ciprofloxacin 32 μg ml⁻¹, d ciprofloxacin 0.0625 g ml⁻¹ combined with berberine 1024 μg ml⁻¹). Dose−response inhibition curve of cell growth (f) of 22 K. pneumoniae isolates. , berberine 1024 μg ml⁻¹ was combined with various concentrations of ciprofloxacin. , a sub-MIC level of ciprofloxacin was combined with various concentrations of berberine.

Figure 2

The relationship between the expression of acrA, acrB, tolC and acrR messenger RNA in five strains of multidrug-resistant K. pneumoniae (R5, R12, R15, ATCC and RATCC). (a) Analysis of the expression of acrA, (b) acrB, (c) tolC and (d) acrR. White bars indicate the control group in untreated culture; striped bars, CIP (ciprofloxacin 32 μg ml⁻¹ in all cases except 0.0625 μg ml⁻¹ against the ATCC strain); gray bars, BER (berberine 1024 μg ml⁻¹); black bars, CIP+BER (ciprofloxacin 32 μg ml⁻¹ in all cases except 0.0625 μg ml⁻¹ against the ATCC strain combined with berberine 1024 μg ml⁻¹). For the five strains not including RATCC, the expression of acrA, acrB, tolC and acrR was higher in the berberine and ciprofloxacin+berberine groups than in the controls. This effect was more pronounced in the ciprofloxacin+ berberine group (**P<0.05).