Evaluating the Potential and Synergetic Effects of Microcins against Multidrug-Resistant Enterobacteriaceae

Abstract

The advent of multidrug-resistant bacteria has hampered the development of new antibiotics, exacerbating their morbidity and mortality. In this context, the gastrointestinal tract reveals a valuable source of novel antimicrobials. Microcins are bacteriocins produced by members of the family Enterobacteriaceae, which are endowed with a wide diversity of structures and mechanisms of action, and exert potent antibacterial activity against closely related bacteria. In this study, we investigated the antibacterial activities of four microcins against 54 Enterobacteriaceae isolates from three species (Escherichia coli, Klebsiella pneumoniae, and Salmonella enterica). The selected microcins, microcin C (McC, nucleotide peptide), microcin J25 (MccJ25, lasso peptide), microcin B17 (MccB17, linear azol(in)e-containing peptide), and microcin E492 (MccE492, siderophore peptide) carry different post-translational modifications and have distinct mechanisms of action. MICs and minimal bactericidal concentrations (MBC) of the microcins were measured and the efficacy of combinations of the microcins together or with antibiotics was assessed to identify potential synergies. Every isolate showed sensitivity to at least one microcin with MIC values ranging between 0.02 μM and 42.5 μM. Among the microcins tested, McC exhibited the broadest spectrum of inhibition with 46 strains inhibited, closely followed by MccE492 with 38 strains inhibited, while MccJ25 showed the highest activity. In general, microcin activity was observed to be independent of antibiotic resistance profile and strain genus. Of the 42 tested combinations, 20 provided enhanced activity (18 out of 20 being microcin-antibiotic combinations), with two being synergetic. IMPORTANCE With their wide range of structures and mechanisms of action, microcins are shown to exert antibacterial activities against Enterobacteriaceae resistant to antibiotics together with synergies with antibiotics and in particular colistin.

Keywords: RiPPs; antimicrobial activity; bacteriocins; microcins; multidrug resistance; synergy.

FIG 1

Mechanisms of action of McC, MccJ25, MccB17, and MccE492. MccE492 (A) and MccJ25 (D) gain entry to the periplasm through siderophore receptors FepA/Fiu/Cir (A) or FhuA (D) coupled to the TonB-ExbB-ExbD complex for translocation. McC (B) and MccB17 (C) enter the periplasm through the porin OmpF. McC gains access to the cytoplasm through YejABEF, whereas MccB17 and MccJ25 use SbmA. When in the cytoplasm, MccJ25, MccB17, and McC target RNA polymerase, DNA gyrase, and aspartyl tRNA synthetase, respectively, to kill the cells. For MccE492, it does not enter the cytoplasm but inserts itself into the inner-membrane by stably associating with ManYZ and inducing depolarization of the inner membrane and perturbation of the mannose transport. *, Inside bacteria McC is processed by a deformylase and the peptidases A, B, and N, resulting in the formation of a non-hydrolysable Asp-tRNA mime, thus blocking translation. Figure made with Biorender.

FIG 2

Agar diffusion assays for McC, MccJ25, MccB17, and MccE492 against E. coli, K. pneumoniae, and S. enterica strains. Microcins were deposited at the concentration of 100 μg/mL.

FIG 3

Efficacy of microcins against Enterobacteriaceae isolates. McC, MccJ25, MccB17, and MccE492 were tested against a collection of 54 Enterobacteriaceae isolates. (A) Heatmap representing the MIC values (in μg/mL). High, medium, and low sensitivity correspond to MIC ≤ 10, 10 < MIC ≤ 50, and MIC > 50 μg/mL, respectively. The corresponding values in μM are provided in Tables S6 to S8. Strains are noted EC for E. coli, K for K. pneumoniae, and S for S. enterica. (B) Susceptibility to microcins per bacterial species. (C) Inhibition type observed per microcin.

FIG 4

MFA correlations identified between microcin and antibiotic susceptibilities. (A) Representation of the strains in the first two dimensions, colored per species. (B) Representation of the susceptibility categories in the first two dimensions. In red: susceptibility to microcins (H, high, MIC ≤ 10 μg/mL, L, low, 10 < MIC ≤ 50 μg/mL, N, no activity up to 50 μg/mL); in black: susceptibility to antibiotics (R, resistant, S, susceptible).

FIG 5

FIC values measured for all consortia tested in the study against E. coli and Salmonella indicator strains. (A) shows the FIC values for the antibiotic microcin-consortia. (B) shows the FIC values for the microcin-microcin consortia. FIC ≤ 0.5: synergy, 0.5 < FIC ≤ 0.75: partial synergy, 0.75 < FIC < 1: additivity and 1 ≤ FIC ≤ 2 = indifference.