# Investigation of antimicrobial synergism of actinomycin derivatives from Streptomyces parvus 35M1 against Escherichia coli ATCC 25922

## TL;DR

This study explores how actinomycin derivatives from a Streptomyces species work better with antibiotics against E. coli, showing potential for new combination therapies.

## Contribution

The first multimodel synergy analysis of actinomycin analogs against E. coli using multiple synergy evaluation models.

## Key findings

- Actinomycin D and X2 showed strong synergy with polymyxin B and kanamycin against E. coli.
- SEM analysis revealed significant morphological changes in E. coli cells under synergistic treatments.
- Actinomycin X2 also synergized with nalidixic acid, but not with rifampicin or ampicillin.

## Abstract

Limited drug development and increased antibiotic tolerance among Gram-negative bacteria have led researchers to consider combination therapy. Actinomycin derivatives, particularly actinomycin D, exhibit a wide range of bioactivities, including antibacterial effects. However, actinomycin D is less effective against Gram-negative pathogens. Therefore, it is essential to demonstrate the synergy of actinomycin D and its derivatives with clinically known antibiotics against Escherichia coli ATCC 25922 and investigate the effects on cellular morphology.

Streptomyces parvus 35M1 isolated from coastal sediment in İzmir/Türkiye was identified via genome sequencing. Large-scale (30 L) fermentation followed by chromatographic purification yielded compounds 1 (actinomycin D), 2 (actinomycin X2), and 3 (actinomycin X0ß), structurally confirmed by NMR spectroscopy. Later, checkerboard assays were used to assess combinatorial interactions with clinically relevant antibiotics in triplicate across two independent biological replicates. Synergistic interactions were evaluated in SynergyFinder using Zero Interaction Potency, Highest Single Agent, Loewe Additivity, and Bliss Independence models. Morphological alterations under synergistic treatments were examined via scanning electron microscopy.

Individual actinomycins exhibited weak antimicrobial activity (MICs > 100 μM). Nevertheless, strong synergistic effects were actinomycins D and X2 combinations with polymyxin B and kanamycin (Most Synergistic Area scores >10). Relatively high synergy scores were obtained for the actinomycin X2-polymyxin B combination, with values of 33.49, 32.69, 33.98, and 38.18 in the Loewe, Bliss, ZIP, and HSA models, respectively. Only actinomycin X2 synergized with nalidixic acid (MSAs ≥13.49 ± 2.63), while all actinomycins displayed additive/indifferent effects with rifampicin and ampicillin. Further, SEM analysis revealed cellular deformation, including elongation, membrane rupture, and dent formation.

This work represents the first multimodel synergy analysis of actinomycin analogs against E. coli, underscoring their potential. However, these findings are limited to in vitro assays on a single reference strain; thus, further validation with in vivo models will be necessary.