Fingolimod Promotes Antibacterial Effect of Doripenem against Carbapenem-Resistant Escherichia coli

Abstract

The aim of this study was to determine whether fingolimod could synergize the antibacterial activity of doripenem against carbapenem-resistant Escherichia coli (CREC) and its potential as an antibiotic adjuvant for doripenem. The E. coli used in this study had the bla_KPC gene and became resistant to many classes of antibiotics, particularly carbapenems. The minimum inhibitory concentrations (MICs) of fingolimod and doripenem were determined. To investigate the synergistic action between fingolimod and doripenem, synergy checkerboard, growth curve, and time-kill analyses were performed. A motility test was also performed using a semi-solid medium to determine whether fingolimod could inhibit the motility of E. coli, one of its virulence mechanisms. The expression levels of carbapenemase-, motility-, and efflux pump-related genes suppressed by fingolimod were analyzed by quantitative polymerase chain reaction (qPCR). Our study demonstrated that the combination of fingolimod and doripenem inhibited carbapenemase, biological activity and other CREC virulence factors. This study findings suggest the potential of fingolimod as an adjuvant to prevent antibiotic resistance in CREC.

Keywords: Escherichia coli; carbapenem; carbapenemase; doripenem; fingolimod; synergistic action.

Figure 1

The growth of E. coli KBN12P05816 was analyzed in the presence of fingolimod and doripenem. Each sample was plated on trypticase soy broth (TSB) plates and incubated at 37 °C for 24 h. (●) Untreated controls, (■) 4 μg/mL of fingolimod, (▲) 4 μg/mL of doripenem, and (▼) the combination of 4 μg/mL of fingolimod and 4 μg/mL of doripenem.

Figure 2

Synergistic action of fingolimod and doripenem against CREC. E. coli KBN12P05816 was treated with fingolimod and doripenem, plated with a spreader on Muller–Hinton agar (MHA), and incubated at 37 °C for 24 h. The CFU/mL values were recorded. (●) Untreated controls, (■) 4 μg/mL of fingolimod, (▲) 4 μg/mL of doripenem, and (▼) the combination of 4 μg/mL of fingolimod and 4 μg/mL of doripenem.

Figure 3

Effect of fingolimod and doripenem on the motility of CREC. (a) The motility of CREC KBN12P05816 was analyzed on semisolid agar plates containing fingolimod and doripenem. (b) Bar graph showing the mean migration distance (mm). The decrease in migration distance was significant (* p < 0.05 and ** p < 0.01).

Figure 4

Transcriptional changes involving the bla_KPC gene in CREC. After treatment with fingolimod alone (2 μg/mL F), doripenem alone (2 μg/mL D), or the combination of 2 μg/mL of doripenem and 2 μg/mL of fingolimod (F + D), qPCR analysis of the bla_KPC gene in E. coli KBN12P05816 was performed. 16S rRNA was used to normalize the transcriptional levels of the target gene. The error bars represent the means and standard deviation (SDs). Asterisks (*) indicate statistically significant differences (* p < 0.05 and ** p < 0.01) from the controls.

Figure 5

Transcriptional changes in virulence-related genes in E. coli. Expression levels of (a) efflux pump-related (acrB, acrD) genes and (b) motility-related (flhD, motA) genes are presented with a histogram. After treatment with fingolimod alone (2 μg/mL F), doripenem alone (2 μg/mL D), or the combination of 2 μg/mL of doripenem and 2 μg/mL of fingolimod (F + D), transcription levels of the target genes were normalized to that of 16S rRNA. Asterisks (*) indicate a significant decrease in expression levels in each gene (* p < 0.05 and ** p < 0.01, and *** p < 0.001).