16S and 18S rDNA Amplicon Sequencing Analysis of Aesthetically Problematic Microbial Mats on the Walls of the Petralona Cave: The Use of Essential Oils as a Cleaning Method.

The presence of microbial communities on cave walls and speleothems is an issue that requires attention. Traditional cleaning methods using water, brushes, and steam can spread the infection and cause damage to the cave structures, while chemical agents can lead to the formation of toxic compounds and damage the cave walls. Essential oils (EOs) have shown promising results in disrupting the cell membrane of bacteria and affecting their membrane permeability. In this study, we identified the microorganisms forming unwanted microbial communities on the walls and speleothems of Petralona Cave using 16S and 18S rDNA amplicon sequencing approaches and evaluated the efficacy of EOs in reducing the ATP levels of these ecosystems. The samples exhibited a variety of both prokaryotic and eukaryotic microorganisms, including Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, the SAR supergroup, Opisthokonta, Excavata, Archaeplastida, and Amoebozoa. These phyla are often found in various habitats, including caves, and contribute to the ecological intricacy of cave ecosystems. In terms of the order and genus taxonomy, the identified biota showed abundances that varied significantly among the samples. Functional predictions were also conducted to estimate the differences in expressed genes among the samples. Oregano EO was found to reduce ATP levels by 87% and 46% for black and green spots, respectively. Consecutive spraying with cinnamon EO further reduced ATP levels, with reductions of 89% for black and 88% for green spots. The application of a mixture solution caused a significant reduction up to 96% in ATP levels of both areas. Our results indicate that EOs could be a promising solution for the treatment of microbial communities on cave walls and speleothems.

Exploring the Bacterial Communities of the Kaiafas Thermal Spring Anigrides Nymphes in Greece Prior to Rehabilitation Actions.

Anigrides Nymphes of Lake Kaiafas is a thermal spring that is well known for its therapeutical properties, as the hot water (32-34 °C) is rich in sulfur compounds and minerals. Nowadays, efforts are made from the Hellenic Republic to modernize the existing facilities and infrastructure networks of the area. To study the complex ecosystem of the thermal spring, we collected water from four sampling points (Lake, and Caves 1, 2, and 3). Filtration method was used for microbial enumeration. In parallel, total bacterial DNA was extracted and subjected to next-generation sequencing (NGS). A total of 166 different bacterial families were detected. Differences in families, genera, and species abundances were detected between the different sampling points. Specifically, Comamonadaceae was the most common family detected in Lake and Cave 3. Similarly, in Caves 1 and 2, Rhodobacteraceae was detected at a higher percentage compared to the rest of the families. Moreover, the detection of sequences assigned to waterborne or opportunistic pathogens, i.e., Enterobacteriaceae, Legionellaceae, Coxiellaceae, and Clostridiaceae, as well as Enterococcus and Vibrio, is of great importance. Although the presence of pathogens was not examined by quantitative PCR, the detection of their sequences strengthens the need of the planned rehabilitation actions of this natural environment in order to allow human swimming.

Bioactive polar lipids from Chroococcidiopsis sp. (Cyanobacteria).

Many studies indicate that various bioactive metabolites subsist in cyanobacteria. Glycolipids of cyanobacteria are reported as molecules that exert specific bioactivities. In this study, total lipids of Chroococcidiopsissp., a coccoid cyanobacterium isolated from a Greek cave, were separated into neutral and polar-lipids and the latter were further fractionated by high-performance liquid chromatography (HPLC). Each polar lipid fraction was tested in vitro for its ability to inhibit platelet-activating factor (PAF)- and thrombin-induced washed rabbit platelet aggregation and/or to cause platelet aggregation. The structures of the most active fractions were elucidated by biological assays and identified by electrospray mass spectrometry. One fraction was a potent inhibitor of PAF-induced platelet aggregation. Structural studies of this fraction indicated the existence of phospho-glyco analog of ceramide. Another fraction that was a potent inhibitor of PAF- as well as of thrombin-induced platelet aggregation was structurally elucidated as a phospho-acetylated glyco-analog of diglyceride. The fraction that induced platelet aggregation was identified as a phospho-acetylated-glyco analog of ceramide. These novel bioactive polar lipids in cyanobacteria in regard to the structure and biological activity may contribute to the allergic character of cyanobacteria.

Characterization of bioactive glycolipids from Scytonema julianum (cyanobacteria).

Cyanobacteria serve as a rich source of novel bioactive metabolites. Few studies on glycolipids reported them as having specific biological activities. In this study, total lipids of Scytonema julianum, a filamentous cyanobacterium isolated from a Greek cave, were separated into neutral and phospho- and glycolipids, and the latter were further fractionated by high-performance liquid chromatography (HPLC). Each glycolipid fraction was tested in vitro for its ability to inhibit platelet-activating factor (PAF)- and thrombin-induced washed rabbit platelet aggregation and/or to cause platelet aggregation. The structures of the most active fractions were elucidated by biological assays, by chemical determinations and identified by electrospray mass spectrometry. One fraction was a potent inhibitor of PAF-induced platelet aggregation. Structural studies of this fraction indicated the existence of a phosphoglyco-analog of acyl-sphingosine. Two fractions causing platelet aggregation were detected and identified as phosphoglycolipids. The first one was identified as a phosphoglyco-analog of acyl-acetylated sphingosine and the second one as a glyco-analog of phosphatidylglycerol. The presence of the above bioactive compounds demonstrates new types of lipids in cyanobacteria in regard to the structure and biological activity. In addition, the identified bioactive lipids may contribute to the allergic character of cyanobacteria.

Isolation and structural elucidation of biologically active phospholipids from Scytonema julianum (cyanobacteria).

The role of platelet-activating factor (PAF) as a mediator appeared in rather primitive organisms like protozoans and was maintained in more evolved organisms. No reports exist for the presence of PAF or PAF analogues - or even compounds that exhibit PAF-like activity - in cyanobacteria, even though they belong to a a group of organisms at a low evolutionary level where the content of alkylacyl forms of ether lipids is expected to be high. In addition, cyanobacteria serve as a rich source of novel bioactive metabolites. In the present study the total lipids of a strain of Scytonema julianum, a filamentous cyanobacterium isolated from a Greek cave, were separated into neutral lipids and phospholipids, the latter being further fractionated by HPLC. Each phospholipid fraction was tested in vitro for its ability to inhibit PAF-, arachidonic acid- and ADP-induced washed-rabbit-platelet aggregation and/or to cause platelet aggregation. Two types of phospholipids causing platelet aggregation were detected and shown to be an acetylsphingomyelin and an acylacetylglycerol phosphoacetylated glycolipid. The existence of the sphingomyelin analogues is very important, since ceramides, cerebrosides and related lipids are intracellular second messengers. The identification of the phosphoglycoglycerolipid demonstrates a new type of lipid in cyanobacteria, namely one that exhibits a biological activity very similar to that of PAF. Its presence reinforces the concept that PAF is a member of a large family of lipid mediators, apparently having different physiological roles in prokaryotic and eukaryotic organisms. In addition, Scytonema julianum contains a phosphatidylcholine (C(16:0)/(18:2)), even though bacteria in general seldom contain choline-containing phosphoacylglycerols.