Characterization of antimicrobial activity of culturable bacteria isolated from Krubera-Voronja Cave

Abstract

In the present study we aimed to perform the first analysis of antimicrobial activity of bacteria isolated from Krubera-Voronja Cave, with the main focus on their activity against Grampositive bacteria, including Gram-positive pathogens. Using five different media, in total 874 heterotrophic cultures were isolated from water and sediment samples collected in Krubera-Voronja Cave at a depth from 220 m to 1640 m. 14.0% of all isolates demonstrated antibacterial activity against Gram-positive and Gram-negative test microorganisms. Our results show that this percentage was not uniform; it increased with the sampling depth and was the highest in the lower part of the cave. 24 isolates were active exclusively against Gram-positive test strains Micrococcus luteus and Bacillus thuringiensis. Two isolates, namely strains 1350R2-TSA30-6 and 1410WF1-TSA30-2, were chosen for the further work because of the high and comparable activity against both Gram-positive test microorganisms. It was determined that both strains belong to the family Bacillaceae in phylum Firmicutes. The detailed bioactivity analysis of these two Gram-positive strains revealed the different mixtures of volatile compounds with antibacterial activity. The main antibacterial compounds of the strain 1350R2-TSA30-6 are pyrrolopyrazines pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-and pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)-. The main antibacterial compound of the strain 1410WF1-TSA30-2 is 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester. Mixtures of the volatile antimicrobial compounds of both strains were antagonistic against Gram-positive strains isolated from Krubera-Voronja Cave, and their activity against Gram-positive pathogenic bacteria substantially differed. antimicrobial, Bacillus, cave microorganisms, Krubera-Voronja Cave, volatile compound

Screening and Transcriptional Analysis of Polyketide Synthases and Non-ribosomal Peptide Synthetases in Bacterial Strains From Krubera–Voronja Cave

“…Phenotypic antibacterial activity of these strains against the test strains of Micrococcus luteus Sar1, Bacillus thuringiensis TL8, Escherichia coli BL21(DE3), and Pseudomonas sp. VR1 was determined previously (Klusaite et al, 2016). For the current work, three groups of Krubera–Voronja Cave bacterial strains were chosen: 54 strains without phenotypic antibacterial activity (a test group), 27 strains with phenotypic antibacterial activity (a control group), and 10 randomly selected strains with unknown antibacterial activity.…”

“…Genomic DNA was extracted from fresh grown cultures using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, United States). BOX-PCR was performed as described in Klusaite et al (2016). Products of amplification were analyzed by electrophoresis using 1% agarose gel.…”

“…Amplification of 16S rRNA genes was done according to Kuisiene et al (2002). Cloning of 16S rRNA genes as well as sequence analysis were performed as described by Klusaite et al (2016). 16S rRNA gene sequences were deposited in the GenBank under the acc.…”

“…Krubera–Voronja Cave (43.4184 N 40.3083 E, Western Caucasus) is one of the deepest caves in the world. Antibacterial activity of the cave bacterial isolates was phenotypically characterized previously and was found to be lower than expected for such a deep cave (Klusaite et al, 2016). It could be that the screening system and/or conditions were inappropriate or these isolates do not indeed encode enzymes that are necessary for the synthesis of some bioactive compounds, particularly polyketides and non-ribosomal peptides.…”

High Levels of Antibiotic Resistance but No Antibiotic Production Detected Along a Gypsum Gradient in Great Onyx Cave, KY, USA

“…The percent of cultures that showed antibiosis to indicator bacteria using the cross-streak method ranged from 8.62% to 21.1%. A study from a cave in the Western Caucasus [26] showed 14% of 87 isolates produced antimicrobials. The proportion increased with sampling depth into the cave, and was highest in the most remote lower levels of the cave.…”