In vitro activity of antimicrobial peptide CDP-B11 alone and in combination with colistin against colistin-resistant and multidrug-resistant Escherichia coli.

Multidrug-resistant bacteria are a growing global concern, and with increasingly prevalent resistance to last line antibiotics such as colistin, it is imperative that alternative treatment options are identified. Herein we investigated the mechanism of action of a novel antimicrobial peptide (CDP-B11) and its effectiveness against multidrug-resistant bacteria including Escherichia coli #0346, which harbors multiple antibiotic-resistance genes, including mobilized colistin resistance gene (mcr-1). Bacterial membrane potential and membrane integrity assays, measured by flow cytometry, were used to test membrane disruption. Bacterial growth inhibition assays and time to kill assays measured the effectiveness of CDP-B11 alone and in combination with colistin against E. coli #0346 and other bacteria. Hemolysis assays were used to quantify the hemolytic effects of CDP-B11 alone and in combination with colistin. Findings show CDP-B11 disrupts the outer membrane of E. coli #0346. CDP-B11 with colistin inhibits the growth of E. coli #0346 at ≥ 10× lower colistin concentrations compared to colistin alone in Mueller-Hinton media and M9 media. Growth is significantly inhibited in other clinically relevant strains, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In rich media and minimal media, the drug combination kills bacteria at a lower colistin concentration (1.25 µg/mL) compared to colistin alone (2.5 µg/mL). In minimal media, the combination is bactericidal with killing accelerated by up to 2 h compared to colistin alone. Importantly, no significant red blood hemolysis is evident for CDP-B11 alone or in combination with colistin. The characteristics of CDP-B11 presented here indicate that it can be used as a potential monotherapy or as combination therapy with colistin for the treatment of multidrug-resistant infections, including colistin-resistant infections.

Circumventing colistin resistance by combining colistin and antimicrobial peptides to kill colistin-resistant and multidrug-resistant Gram-negative bacteria.

OBJECTIVES: Colistin is a 'last-line' antibiotic used to treat multidrug-resistant Gram-negative bacteria, but colistin resistance has emerged. Colistin normally binds to the lipid A moiety on the bacterial outer membrane, where it then destroys the bacterial membrane. Mobilize colistin resistance (MCR, encoded by mcr-1 and others) is a phosphoethanolamine transferase that modifies lipid A, preventing colistin binding. We hypothesized that combining pore-forming AMPs and colistin will circumvent this mechanism and reduce the minimum inhibitory concentration (MIC) of colistin for both colistin- and multidrug-resistant Gram-negative bacteria. METHODS: In vitro cultures were incubated for 18 h after combining bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa) with serially diluted colistin and a fixed concentration of peptide MSI-78 or OTD-244. RESULTS: When combined with either peptide, the colistin MIC decreased more than 4-fold for 88% of all tested isolates (n = 17; range, 4-64-fold reduction) and for 75% of colistin-resistant isolates (n = 8; range, 4-64-fold reduction). The concentrations used had no effect on red blood cells based on a conventional haemolysis assay. CONCLUSIONS: These findings are consistent with two membrane-damaging compounds having an additive effect on bacterial killing. Combining antimicrobial peptides with colistin is a promising strategy for bypassing MCR-mediated colistin resistance, but also for improving the susceptibility of other Gram-negative bacteria while potentially reducing the therapeutic concentration of colistin needed to treat infections.