Synergy between the clavanins as a weapon against multidrug-resistant Enterobacter cloacae

Abstract

Finding new antibiotics that can act synergistically with each other offers many benefits such as lower dosages used for each drug, improved pathogen clearance, and ability to act against multi-drug resistant strains. In this study, six peptides isolated from the tunicate Styela clava were evaluated for their synergistic interaction using the checkerboard assay and the time kill kinetics assay. Using two different tests, we report synergy between clavanin D and clavaspirin in both tests and synergy between clavanin A and B only in the checkerboard test when used against the multidrug resistant E. cloacae 0136. This work demonstrates the possible cooperativity between homologous AMPs from a single organism and the advantage of using two susceptibility tests instead of one when testing synergistic combinations.

Fig. 1. CD spectroscopy data for all the clavanins taken at 50 μM concentration in 50% TFE solution showing the typical α-helix signals.

Fig. 2. Isobolograms for all possible pairs of the clavanins. The dashed line illustrates the model additive interaction expected for Loewe additivity. The lowest FIC is listed at the top of each isobologram. Synergy is defined as having an FIC of ≤0.5 which is only seen in the combination of Clav A + B and Clav D and S.

Fig. 3. Time-kill kinetics curve of synergistic clavanin pairs against E. cloacae 0136. Each peptide was tested at the concentration that gave the lowest FIC in the checkerboard assay. ClavA and B were tested at 16 μM each, while ClavD and ClavS were tested at 2 and 1 μM, respectively.

Fig. 4. Confocal microscopy images of E. cloacae treated with (A) ClavD-FAM, (B) ClavS-TAMRA, and (C) combined treatment of ClavD-FAM and ClavS-TAMRA. Bacteria were incubated with the peptides for 30 min before mounting on glass slides. Images were taken using Nikon A1R confocal microscope with 60× oil immersion objective (scale bars = 5 μm).

Fig. 5. TEM images of E. cloacae. Top images represent untreated cells (OM = outer membrane, PS = periplasmic space, IM = internal membrane, N = nucleoid). Bottom images represent cells treated with 1 μM ClavD and 0.5 μM ClavS showing electron-lucent region (red circle) and electron-dense regions (red arrows). Scale bars = 200 nm.