In Vitro and In Vivo Biological Activity of Berberine Chloride against Uropathogenic E. coli Strains Using Galleria mellonella as a Host Model

Abstract

Berberine is an alkaloid of the protoberberine type used in traditional oriental medicine. Its biological activities include documented antibacterial properties against a wide variety of microorganisms; nonetheless, its use against Escherichia coli strains isolated from urinary infections has not yet been widely investigated in vivo. The emergence of antimicrobial resistance requires new therapeutic approaches to ensure the continued effectiveness of antibiotics for the treatment and prevention of urinary infections. Moreover, uropathogenic Escherichia coli (UPEC) has developed several virulence factors and resistance to routine antibiotic therapy. To this end, several in vitro and in vivo tests were conducted to assess the activity of berberine on uropathogenic E. coli strains. Galleria mellonella as an infection model was employed to confirm the in vivo translatability of in vitro data on berberine activity and its influence on adhesion and invasion proprieties of E. coli on human bladder cells. In vitro pre-treatment with berberine was able to decrease the adhesive and invasive UPEC ability. In vivo treatment increased the larvae survival infected with UPEC strains and reduced the number of circulating pathogens in larvae hemolymph. These preliminary findings demonstrated the efficacy and reliability of G. mellonella as in vivo model for pre-clinical studies of natural substances.

Keywords: Galleria mellonella; UPEC; antimicrobial activity; berberine chloride; host–pathogen interactions.

Figure 1

Influence of Berberine chloride on in vitro growth curves of E. coli strains by Cell Counting. Black continuous lines: untreated controls; black dashed lines: nonlinear fit of untreated controls; continuous orange lines: strains treated with berberine 1/4 MIC; orange dashed lines: nonlinear fit of strains treated with berberine 1/4 MIC; continuous red lines: strains treated with berberine 1/2 MIC; red dashed lines: nonlinear fit of strains treated with berberine ½ MIC. (a) E. coli ATCC 11775. (b) E. coli DSM 22312. (c) E. coli CL1 (CA-UTI). (d) E. coli CL2 (CA-UTI). The bars represent means ± SD of independent experiments performed in triplicate.

Figure 2

Effect of berberine on E. coli strains adhesion 5637 ATCC (HTB-9) cell line. Black histograms: strains not treated with berberine. Light grey histograms with large squares: strains pre-treated with 1/2 MIC berberine. Light grey histograms with small squares: strains pre-treated with 1/4 MIC berberine. Dark grey histograms with wide lines: strains co-incubated with 1/2 MIC berberine. Dark grey histograms with narrow lines: strains co-incubated with 1/4 MIC berberine. (a) E. coli ATCC 11775. (b) E. coli DSM 22312. (c) E. coli CL1 (CA-UTI). (d) E. coli CL2 (CA-UTI). Results are expressed as CFU/well. The bars represent means ± SD of three independent experiments performed in triplicate. Statistically significant differences, determined by one-way analysis of variance ANOVA (* indicates a significant difference at p < 0.05 versus control/untreated, ** indicates a significant difference at p < 0.01 versus control/untreated).

Figure 3

Effect of berberine on E. coli strains invasion ability against 5637 ATCC (HTB-9) cell line. Black histograms: strains not treated with berberine. Light grey histograms with large squares: strains pre-treated with 1/2 MIC berberine. Light grey histograms with small squares: strains pre-treated with 1/4 MIC berberine. Dark grey histograms with wide lines: strains co-incubated with 1/2 MIC berberine. Dark grey histograms with narrow lines: strains co-incubated with 1/4 MIC berberine. (a) E. coli ATCC 11775. (b) E. coli DSM 22312. (c) E. coli (CA-UTI) CL1. (d) E. coli CL2 (CA-UTI). Results are expressed as CFU/well. The bars represent means ± SD of three independent experiments performed in triplicate. Statistically significant differences, determined by one-way analysis of variance ANOVA (* indicates a significant difference at p < 0.05 versus control/untreated, ** indicates a significant difference at p < 0.01 versus control/untreated).

Figure 4

Kaplan–Meier survival plots of G. mellonella larvae by E. coli strains infection. Light grey dashed line: larvae inoculated with saline; Yellow line: Berberine toxicity control; Black dashed line: larvae inoculated with 10⁵ CFU/larvae; orange line: larvae infected with berberine pre-treated E. coli at 1/2MIC; red line: larvae infected with berberine pre-treated E. coli at 1/4MIC; blue line: larvae infected with E. coli and co-incubated with 2 MIC berberine. (a) E. coli ATCC 11775. (b) E. coli DSM 22312. (c) E. coli CL1 (CA-UTI). (d) E. coli CL2 (CA-UTI). All groups of each treatment were compared with the control group using the log-rank test; the p values are reported for each group.

Figure 5

CFU/mL of E. coli strains recovered from G. mellonella larvae haemolymph infected with 10⁵ CFU/larvae and treated with berberine at different concentration. Light grey circles: saline inoculated larvae; Yellow squares: Berberine control; Black triangles: E. coli 10⁵ CFU/larvae inoculated; Orange triangles: E. coli pre-treated berberine 1/2MIC infected larvae; Red squares: E. coli berberine 1/4MIC pre-treated infected larvae; blue circles: berberine co-incubated infected larvae. (a) E. coli ATCC 11775. (b) E. coli DSM 22312. (c) E. coli CL1 (CA-UTI). (d) E. coli CL2 (CA-UTI). Data expressed as the mean ± standard deviation (log10 CFU/mL of hemolymph) of three independent experiments. Statistical analysis was performed using the two-way ANOVA with Bonferroni multiple comparison post-test. *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 6

Hemocyte count of G. mellonella larvae infected with E. coli 10⁵ CFU/larvae and treated with berberine. Larvae treated with: saline (White histograms) or berberine. (Black histograms). Dark grey histograms with small squares: larvae infected with E. coli strains. Light grey histograms: larvae infected with E. coli pre-treated with 1/2 MIC and with 1/4 MIC berberine. Dark grey histograms: larvae infected and co-incubated with berberine. (a) E. coli ATCC 11775. (b) E. coli DSM 22312. (c) E. coli (CA-UTI) CL1. (d) E. coli CL2 (CA-UTI). Results are expressed as cells/mL. The bars represent means ± SD of three independent experiments performed in triplicate. Statistically significant differences, determined by one-way analysis of variance ANOVA (* indicates a significant difference at p < 0.05 versus control/untreated, ** indicates a significant difference at p < 0.01 versus control/untreated).