Squalamine, an original chemosensitizer to combat antibiotic-resistant Gram-negative bacteria

Abstract

Sir, The worldwide dissemination of multidrug-resistant (MDR) pathogens has severely reduced the efficacy of our antibiotic arsenal and increased the frequency of therapeutic failure. To reduce the intracellular accumulation of antibiotics, a key bacterial response is to stringently control membrane transporters involved in the diffusion of drugs through the envelope into the cell and those involved in the expulsion of antibiotics, the so-called ‘in and out’ flux. In Gram-negative bacteria, the outer membrane is an additional barrier to antibiotic penetration. Numerous antibiotics that are active against Grampositive are much less active against Gram-negative bacteria. An attractive approach to circumvent bacterial resistance is the development of a chemosensitizer agent to promote an increase of the internal antibiotic concentration in resistant strains. This agent will be used in combination with classic antibiotics to bypass the mechanical and enzymatic barriers that reduce the intracellular concentration of the active antibacterial drug. Squalamine exhibits interesting antimicrobial activity, suggesting its potential application to combat resistant pathogens. It modifies membrane integrity by increasing permeability, as demonstrated by ATP release and dye entry in Gram-negative bacteria, and it has a moderate cytotoxicity at doses that kill MDR bacterial pathogens. The aim of this study was to evaluate the effect of subinhibitory concentrations of squalamine on the activity of various antibiotic classes against different resistant bacteria. The chemosensitizer activity of squalamine was studied by determining the MICs of various usual antibiotics in the presence of subinhibitory concentrations of squalamine. These concentrations corresponded to 1/5 and 1/10 of the respective squalamine MICs determined for each bacterial strain. A control was included by using the well-described efflux pump inhibitor phenylalanine arginine b-naphthylamide (PAbN) to evaluate the involvement of efflux. The strains were grown on Mueller–Hinton broth (Becton Dickinson) at 378C. The Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa strains used have been previously described. Results were read after 18 h at 378C and the MIC values are the means of three independent experiments. The results for documented E. coli strains exhibiting various porins and efflux pump profiles indicated that, even at the low subinhibitory concentration tested (MIC/10), squalamine had an effect on the activity of various different antibiotic classes (Table 1). A significant reduction in chloramphenicol, tetracycline and ciprofloxacin MICs was observed for AG100 and AG100Atet in the presence of squalamine. AG100Atet is an acrAB2 strain that overproduces several drug efflux pumps and down-regulates porin expression. An effect was also obtained on cefepime susceptibility, indicating that squalamine may favour the uptake of this b-lactam molecule via the permeabilizing effect previously reported. It is worthwhile mentioning that squalamine also increases the activity of tested antibiotics against susceptible cells, AG100 and AG100A, demonstrating that the effect is not focused on a resistance mechanism but is related to increased drug penetration. The MICs of erythromycin were not significantly modified by the addition of squalamine, indicating the prominent role of efflux mechanisms for this antibiotic group. In the K. pneumoniae KP55 clinical isolate, the MICs determined in the presence of squalamine were significantly decreased, e.g. 16–32-fold for chloramphenicol, ciprofloxacin, tetracycline and cefepime (Table 1). Squalamine was more efficient at increasing tetracycline and cefepime activity against E. aerogenes than PAbN, highlighting the role of uptake in susceptibility to the two antibiotics. The effect of squalamine on strains devoid of porin (KP63 and KP55) indicated an increase in penetration for cefepime and ciprofloxacin, which normally use porin channels to pass the membrane barrier. Regarding P. aeruginosa and E. aerogenes strains, the results similarly suggest that when the mechanical barrier, restricted uptake or efficient efflux pump participates in resistance, squalamine is able to significantly increase bacterial susceptibility. Squalamine has an effect on the susceptible strains (E. coli, E. aerogenes and P. aeruginosa) tested in this study, suggesting that squalamine may chemosensitize the membrane of nonresistant bacteria and promote the use of a decreased amount of antibiotics. We have previously demonstrated that the alteration of lipopolysaccharide involved in polymyxin-resistant clinical isolates only moderately changes squalamine susceptibility. Consequently, the use of squalamine against polymyxin-resistant isolates selected during colistin treatment, a therapy that is now proposed for the MDR phenotype, may be an attractive hypothesis for the development of future drug combinations. In the bacterial resistance context, and because of its action and its relative insensitivity to efflux resistance mechanisms, squalamine may be a fruitful partner for the development of combinations, such as b-lactams/b-lactamase inhibitors, to combat MDR pathogens. This use allows us to complete our new strategies by promoting the penetration (influx) of antibiotics, in parallel with inhibitors of efflux pumps andb-lactamase inhibitors. This aspect is especially important, since some recent molecules having an original antibacterial spectrum exhibit restricted diffusion through the Research letters