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Caves are outstanding geomicrobiological, ecological, and evolutionary laboratories for the extreme conditions encountered in their deep passages: permanent absence of light, constant temperature, saturated air humidity, absence of plants, low nutrients, low biodiversity, and short food chains. The proposed research focused on understanding diversity, distribution patterns, and evolutionary interactions between environment, microorganisms, and invertebrates as part of the oligotrophic and chemoautotrophic food chains in caves, for future developments and applications in human health and other scientific fields. With the proposed research we intended to challenge the paradigm of the total dependance of cave ecosystems upon surface input and proved that at least part of the cave food chains is based on autochthonous microbial production. The advantage of studying simple cave food chains is that processes can be better understood than in complex surface food webs. The study of food webs in caves not only provides insights into the physical and behavioral adaptations of animals to low food, but the way they function can be used in understanding how surface ecosystems will be impacted by reduced food as a toll to climate change and human activities. 

1. In an unprecedented effort in caves 472 samples

of sediments, water, gut invertebrate,

mineralizations etc were collected from

Romanian and Brazilian caves.

Next generation sequencing and bioinformatic

analysis were provided data on the cave

different microbiomes (Bacteria and Archaea). 








2. The mineralogical and chemical composition of sediments in caves is

complex and variable. Ca was the central element in the sediments,

followed by K, Mg, P, Na, Al, and S. Movile Cave is a chemoautotrophic

system driven by in-house sulphur and methane oxidation and CO2 fixation

as primary production processes. These elements explain its separation from

the other oligotrophic caves. Muierilor Cave also separated and is known for 

the presence of hydroxyapatite, rich in phosphates. Ursilor Cave sediments

are characterized by high concentrations and variability of Ca followed by

Si, Al, Mg, and Fe. Differences between samples from Movile and the other

caves are even more evident in the water samples.

3. The sediments microbiome is dominated by Lysobacter, known to produce a variety of extracellular enzymes and antimicrobial compounds, an unknown genus, wb1-P19 (a sulfur- or nitrite-oxidizing autotrophic bacteria), and Pseudomonas, a generalist. More than 50% of the genera are unknown to science. Two unknown phyla were also obtained, one with 5% in relative abundance. Flavobacterium dominated the cave water microbiome, followed by Pseudomonas, and PolaromonasFlavobacterium and Polaromonas are chemoorganotrophs and aerobes. The unknown genera were ~17% in the water samples. Moonmilk, phosphate-rich crusts and other minerals were analyzed for their microbiome in relation to the substrate. Fossil bones, at different degrees of degradation were also considered.

4. A total of 106 medium- to high-quality Metagenome-Assembled Genomes were

recovered from the sediment metagenomes of Movile Cave, near the lake and in the

oligotrophic passages. In the lower gallery, near the lake, Chloroflexota was abundant

in two stations, while Proteobacteria (class Gammaproteobacteria) dominated the third

station. In the upper, dry gallery, Acidobacteriota was the most abundant phylum for

two stations.

5. Nineteen cave invertebrate taxa, each with several individuals, were studied for the gut microbiome. At a first glance, there is a significant separation between the taxa from Brazil and Romania. Within the Romanian clade, the separation between cave aquatics (stygobionts) and cave terrestrial (troglobionts) is also strongly supported. Many other individuals for extracted for the entire body microbiome. The results show that the gut microbiome in cave beetles was involved in adaptation to life in caves and to scarce food, and there are no similarities with the core bacteria in the sediments. On the contrary, the gut microbiome of amphipods show similarities with the water microbiome and less with the silt in the pools.

6. The gut microbiome was relatively conservative independent of the phyletic line and microclimatic conditions in cave beetles. However, the least troglomorphic species, showed the highest diversity of the microbiome.

This is an indication of the exploitation of different subterranean micro-niches, with different

degrees of influence from the soil microbiome above. For the cave amphipods some

bacteria are common in tropical and temperate caves and some of the core bacteria do not

reflect the habitat microbiome.


7. In Movile Cave, two antimicrobial resistance (AR) genes (ole(C), oqxB) encoding resistance to antibiotics (i.e. oleandomycin, chloramphenicol, ciprofloxacin, trimethoprim) and disinfectants were identified in MAGs affiliated with the class Actinomycetia and Gammaproteobacteria.

Other samples were collected from caves to performed the phenotypic characterization and establish the resistance phenotype of the isolated strains. The cave environment is unique and the contaminations with pollutants can be minimal in its deepest parts. We believe that a thorough analysis of the intrinsic resistance of bacterial strains inhabiting unpolluted environments where there is no antibiotic selection pressure will advance the understanding of nosocomial infections.  

8. The ratio between Firmicutes and Bacteroides, also dominant in most of the cave

samples, are of relevance in the diagnostic and treatment of gastrointestinal diseases.

The colon is the host for bacteria that can have as metabolic products short chain

fatty acids (SCFA) and menaquinones (MK) that released in the vascular system may

influence the physiological pathways at many levels.



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