Enhancing Colistin Activity against Colistin-Resistant Escherichia coli through Combination with Alginate Nanoparticles and Small Molecules.

Bacterial resistance to antibiotics has become a major public health problem worldwide, with the yearly number of deaths exceeding 700,000. To face this well-acknowledged threat, new molecules and therapeutic methods are considered. In this context, the application of nanotechnology to fight bacterial infection represents a viable approach and has experienced tremendous developments in the last decades. Escherichia coli (E. coli) is responsible for severe diarrhea, notably in the breeding sector, and especially in pig farming. The resulting infection (named colibacillosis) occurs in young piglets and could lead to important economic losses. Here, we report the design of several new formulations based on colistin loaded on alginate nanoparticles (Alg NPs) in the absence, but also in the presence, of small molecules, such as components of essential oils, polyamines, and lactic acid. These new formulations, which are made by concomitantly binding colistin and small molecules to Alg NPs, were successfully tested against E. coli 184, a strain resistant to colistin. When colistin was associated with Alg NPs, the minimal inhibition concentration (MIC) decreased from 8 to 1 µg/mL. It is notable that when menthol or lactic acid was co-loaded with colistin on Alg NPs, the MIC of colistin drastically decreased, reaching 0.31 or 0.62 µg/mL, respectively. These novel bactericidal formulations, whose innocuity towards eukaryotic HT-29 cells was established in vitro, are presumed to permeabilize the bacterial membrane and provoke the leakage of intracellular proteins. Our findings revealed the potentiating effect of the Alg NPs on colistin, but also of the small molecules mentioned above. Such ecological and economical formulations are easy to produce and could be proposed, after confirmation by in vivo and toxicology tests, as therapeutic strategies to replace fading antibiotics.

Enhanced Antibacterial Activity of Dermaseptin through Its Immobilization on Alginate Nanoparticles-Effects of Menthol and Lactic Acid on Its Potentialization.

Dermaseptin B2 (DRS-B2) is an antimicrobial peptide secreted by Phyllomedusa bicolor, which is an Amazonian tree frog. Here, we show that the adsorption of DRS-B2 on alginate nanoparticles (Alg NPs) results in a formulation (Alg NPs + DRS-B2) with a remarkable antibacterial activity against Escherichia coli ATCC 8739 and E. coli 184 strains, which are sensitive and resistant, respectively, to colistin. The antibacterial activity, obtained with this new formulation, is higher than that obtained with DRS-B2 alone. Of note, the addition of lactic acid or menthol to this new formulation augments its antibacterial activity against the aforementioned Gram-negative bacilli. The safety of DRS-B2, and also that of the new formulation supplemented or not with a small molecule such as lactic acid or menthol has been proven on the human erythrocytes and the eukaryotic cell line types HT29 (human) and IPEC-1 (animal). Similarly, their stability was determined under the conditions mimicking the gastrointestinal tract with different conditions: pH, temperature, and the presence of digestive enzymes. Based on all the obtained data, we assume that these new formulations are promising and could be suggested, after in vivo approval and completing regulation aspects, as alternatives to antibiotics to fight infections caused by Gram-negative bacilli such as E. coli.

Impact of colistin and colistin-loaded on alginate nanoparticles on pigs infected with a colistin-resistant enterotoxigenic Escherichia coli strain.

Colistin is frequently used for the control of post-weaning diarrhoea in pigs. Colistin resistance caused by plasmidic genes is a public health issue. We evaluated, in experimental animal facilities, whether free colistin or colistin-loaded on alginate nanoparticles (colistin/Alg NPs) could select a colistin-resistant Enterotoxigenic Escherichia coli. The Alg NPs were produced by a simple top-down approach through ball milling of sodium alginate polymer precursor, and colistin loading was achieved through physical adsorption. Colistin loading on Alg NPs was confirmed using various tools such Fourier transform infrared spectroscopy and dynamic light scattering measurements. Thirty-four piglets were orally inoculated or not with a mcr-1-positive, rifampicin-resistant enterotoxigenic E. coli strain, and the inoculated pigs were either treated or not during five days with commercial colistin (100,000 IU/kg) or colistin/Alg NPs (40,415 IU/kg). Clinical signs were recorded. Fecal and post-mortem samples were analyzed by culture. The result clearly indicated that colistin/Alg NPs had a slightly better therapeutic effect. Both treatments led to a transitory decrease of the total E. coli fecal population with a majority of colistin-resistant E. coli isolates during treatment, but the dominant E. coli population was found susceptible at the end of the trial. Further studies are needed to evaluate, in diverse experimental or field conditions, the therapeutic efficacy of colistin/Alg NPs for post-weaning diarrhoea.

New Bacteriocins from Lacticaseibacillus paracasei CNCM I-5369 Adsorbed on Alginate Nanoparticles Are Very Active against Escherichia coli.

Lacticaseibacillus paracasei CNCM I-5369, formerly Lactobacillus paracasei CNCM I-5369, produces bacteriocins that are remarkably active against Gram-negative bacteria, among which is the Escherichia coli-carrying mcr-1 gene that is involved in resistance to colistin. These bacteriocins present in the culture supernatant of the producing strain were extracted and semi-purified. The fraction containing these active bacteriocins was designated as E20. Further, E20 was loaded onto alginate nanoparticles (Alg NPs), leading to a highly active nano-antibiotics formulation named hereafter Alg NPs/E20. The amount of E20 adsorbed on the alginate nanoparticles was 12 wt.%, according to high-performance liquid chromatography (HPLC) analysis. The minimal inhibitory concentration (MIC) values obtained with E20 ranged from 250 to 2000 µg/mL, whilst those recorded for Alg NPs/E20 were comprised between 2 and 4 µg/mL, which allowed them to gain up to 500-fold in the anti-E. coli activity. The damages caused by E20 and/or Alg NPs/E20 on the cytology of the target bacteria were characterized by transmission electron microscopy (TEM) imaging and the quantification of intracellular proteins released following treatment of the target bacteria with these antimicrobials. Thus, loading these bacteriocins on Alg NPs appeared to improve their activity, and the resulting nano-antibiotics stand as a promising drug delivery system.