Strong and widespread cycloheximide resistance in Stichococcus-like eukaryotic algal taxa

Nur Hidayu Syuhada,Syazana Zaki,Peter Convey,Faradina Merican,Paul A Broady,Narongrit Muangmai

doi:10.1038/s41598-022-05116-y

Abstract

This study was initiated following the serendipitous discovery of a unialgal culture of a Stichococcus-like green alga (Chlorophyta) newly isolated from soil collected on Signy Island (maritime Antarctica) in growth medium supplemented with 100 µg/mL cycloheximide (CHX, a widely used antibiotic active against most eukaryotes). In order to test the generality of CHX resistance in taxa originally identified as members of Stichococcus (the detailed taxonomic relationships within this group of algae have been updated since our study took place), six strains were studied: two strains isolated from recent substrate collections from Signy Island (maritime Antarctica) (“Antarctica” 1 and “Antarctica” 2), one isolated from this island about 50 years ago (“Antarctica” 3) and single Arctic (“Arctic”), temperate (“Temperate”) and tropical (“Tropical”) strains. The sensitivity of each strain towards CHX was compared by determining the minimum inhibitory concentration (MIC), and growth rate and lag time when exposed to different CHX concentrations. All strains except “Temperate” were highly resistant to CHX (MIC > 1000 µg/mL), while “Temperate” was resistant to 62.5 µg/mL (a concentration still considerably greater than any previously reported for algae). All highly resistant strains showed no significant differences in growth rate between control and treatment (1000 µg/mL CHX) conditions. Morphological examination suggested that four strains were consistent with the description of the species Stichococcus bacillaris while the remaining two conformed to S. mirabilis. However, based on sequence analyses and the recently available phylogeny, only one strain, “Temperate”, was confirmed to be S. bacillaris, while “Tropical” represents the newly erected genus Tetratostichococcus, “Antarctica 1” Tritostichococcus, and “Antarctica 2”, “Antarctica 3” and “Arctic” Deuterostichococcus. Both phylogenetic and CHX sensitivity analyses suggest that CHX resistance is potentially widespread within this group of algae.

Highlights

This study was initiated following the serendipitous discovery of a unialgal culture of a Stichococcuslike green alga (Chlorophyta) newly isolated from soil collected on Signy Island in growth medium supplemented with 100 μg/mL cycloheximide (CHX, a widely used antibiotic active against most eukaryotes)
Cycloheximide (CHX) is an antibiotic originally discovered in studies of the bacterium Streptomyces griseus[4], where it was found to be effective in killing fungal pathogens at a concentration as low as 0.2 μg/mL but possessed little or no antibiotic activity against bacteria[4]
The data presented here conclusively demonstrate that multiple Stichococcus-like algal strains obtained across a global range of locations are able to grow in the presence of the widely used eukaryotic growth inhibitor cycloheximide

Summary

Introduction

This study was initiated following the serendipitous discovery of a unialgal culture of a Stichococcuslike green alga (Chlorophyta) newly isolated from soil collected on Signy Island (maritime Antarctica) in growth medium supplemented with 100 μg/mL cycloheximide (CHX, a widely used antibiotic active against most eukaryotes). Based on sequence analyses and the recently available phylogeny, only one strain, “Temperate”, was confirmed to be S. bacillaris, while “Tropical” represents the newly erected genus Tetratostichococcus, “Antarctica 1” Tritostichococcus, and “Antarctica 2”, “Antarctica 3” and “Arctic” Deuterostichococcus Both phylogenetic and CHX sensitivity analyses suggest that CHX resistance is potentially widespread within this group of algae. Complete lysis of cells of Euglena gracilis occurred within seven days in broth containing 100 μg/mL CHX9 These investigations have led to the standard and widespread use of CHX at 20–200 μg/mL in bacterial and cyanobacterial cultures to eliminate eukaryotic algae and fungi[10,11,12,13,14] against which it is regarded as one of the most effective antibiotics[9]. This was resistant to only very low CHX concentration (0.5 μg/mL) after an extended lag time in culture of up to 10 days

Methods

Results

Conclusion