Antimicrobial Activity of a Synthetic Brevibacillin Analog Against Multidrug-Resistant Campylobacter spp

Abstract

Campylobacter spp. is one of the most prevalent causes of zoonotic foodborne infections associated with diarrhea in humans. The growing threat of antibiotic resistance calls for innovative approaches. The antimicrobial lipopeptide brevibacillin produced by Brevibacillus laterosporus and its synthetic analog brevibacillin Thr1 showed promising activity against Salmonella and E. coli. The latter is a 1602.13 Da positively charged (+3) synthetic peptide of 13 residues that showed reduced cytotoxicity (IC₅₀ of 32.2 µg/mL against Caco-2 cells) and hemolytic activity (1.2% hemolysis at 128 µg/mL) compared to the native peptide. It contains an N-terminal L-isoleucic fatty acid chain and four non-proteinogenic amino acids and ends with valinol at its C-terminus. One key structural modification is the substitution of α,β-dehydrobutyric acid with threonine. We investigated the antimicrobial potential of the synthetic brevibacillin Thr1 analog against a collection of 44 clinical Campylobacter spp. that were obtained from two reference laboratories. Susceptibility testing revealed marked resistance to ciprofloxacin, tetracycline, and ampicillin among the strains, with more than half expressing a multidrug-resistant phenotype. The genomes of the 44 strains were sequenced to study the genes responsible for their antimicrobial resistance. Tetracycline resistance was associated with tet(O), ciprofloxacin resistance with mutations in gyrA and regulatory sequences modulating the expression of an efflux system, and aminoglycoside resistance with genes of the aph family. The brevibacillin Thr1 analog was produced by chemical synthesis, and evaluation of its activity against a subset of clinical strains by microdilution revealed minimum inhibitory concentration and minimum bactericidal concentration ranging from 8 µg/mL to 64 µg/mL. The peptide was active against multidrug-resistant isolates with a bactericidal effect. Of note, despite numerous attempts, it proved impossible to select Campylobacter spp. for resistance to the brevibacillin Thr1 analog. These results underline the potential of lipopeptides, notably brevibacillin, as antimicrobial alternatives against antibiotic-resistant Campylobacter bacterial infections.

Keywords: Campylobacter spp.; WGS; antibiotics; brevibacillin; multidrug resistance.

Figure 1

Genomic organization and features of CmeR-CmeA intergenic region. The CmeABC operon is composed of three genes, a repressor, and a regulatory region that is negatively regulated by CmeR by binding to a 16-base-pair inverted repeat sequence in the promoter region (top). In our strains, we have identified point mutations that have been reported in the literature as causing overexpression of CmeABC. Alignment of the CmeR+CmeA region of Campylobacter jejuni strains (middle). Alignment of the CmeR+CmeA region of Campylobacter coli strains (bottom). Arrows indicate the start of the cmeA coding region, whose nucleotides are colored in red. Nucleotides in blue are from the R-box region upstream of cmeA.

Figure 2

Amino acid sequence, chemical structure, and activity of the brevibacillin Thr1 analog. (A) The chemical structures of native brevibacillin and the brevibacillin Thr1 analog. The Thr1 analog is made of 13 amino acids, it has a molecular weight of 1602.13 Da and a net charge of +3. (B) Agar diffusion assay against Campylobacter coli CIP.70.80, (a) inhibition of bacterial growth by brevibacillin Thr1 analog (512 µg/mL) (C-) negative control 10% DMSO in water.

Figure 3

Heatmap illustrating the MIC of the brevibacillin Thr1 analog according to species and antibiotic resistance. Species and MIC values (from 8–64 µg/mL) of the brevibacillin analog are shown on the left with color codes. The activity of the brevibacillin Thr1 analog was contrasted to antibiotic susceptibility to Amoxicillin + Clavulanic Acid (AMC), Gentamycin (GMN), Erythromycin (ERY), Ertapenem (ETP), Tetracycline (TET), Amoxicillin (AMP), and Ciprofloxacin (CIP) and shown in blue (sensitive) or red (resistant).