Acanthamoeba and other free-living amoebae in bat guano, an extreme habitat.

Several representatives of the so-called free-living amoebae (FLA) are of medical relevance, not only as facultative pathogens but also as vehicles for pathogenic bacteria. Some FLA can survive and even grow under extreme environmental conditions. Bat guano is an exceptional habitat, the conditions becoming gradually more extreme with aging. In the current study, samples of bat guano of different ages from five caves in Slovenia were screened for the presence of FLA. FLA were isolated from almost all guano samples, including guano with a pH of 3.5. Only the two samples that had been drawn from >20-year-old guano were negative for FLA. Generally, FLA diversity correlated to high concentrations of cultivable bacteria (∼10(8) CFU/g) and fungi (∼10(5) CFU/g). Interestingly, the absence of FLA in seasoned guanos was mirrored by the presence of dictyostelid slime moulds. The isolated amoebae were identified as belonging to the genera Acanthamoeba, Copromyxa, Naegleria, Sappinia, Tetramitus, Thecamoeba, Vahlkampfia, Vannella and Vermamoeba. To the best of our knowledge, this is the first study on the diversity of FLA in guano.

Microbiology of healing mud (fango) from Roman thermae aquae iasae archaeological site (Varazdinske Toplice, Croatia).

We found well-preserved, rocky artefacts that had been buried in the healing mud (fango) for more than 1,500 years at the Roman archaeological site at Varazdinske Toplice. This Roman pool with fango sediments and artefacts is fed from hot sulphidic springs. The fango exhibited nearly neutral pH, a high level of organic C, an elevated concentration of heavy metals and a high total microbial biomass, greater than 10(8) cells per gram of dry weight. The dominant microbes, assessed by molecular profiling (denaturing gradient gel electrophoresis), were affiliated with Thiobacillus, Sulfuricurvum, Polaromonas, and Bdellovibrio. Polymerase chain reaction screening for microbial functional guilds revealed the presence of sulphur oxidizers and methanogens but no sulphate reducers. The dominance of four Proteobacterial classes (α-, ß-, δ- and ε-Proteobacteria) was confirmed by fluorescence in situ hybridisation; Actinobacteria were less abundant. Cultivable bacteria represented up to 23.4 % of the total bacterial counts when cultivation media was enriched with fango. These bacteria represented the genera Acinetobacter, Aeromonas, Arthrobacter, Comamonas, Ewingella, Flavobacterium, Pseudomonas, Rahnella and Staphylococcus. This study showed that the heterogeneous nature of fango at neutral pH created various microniches, which largely supported microbial life based on sulphur-driven, autotrophic denitrification.

Low Bacterial Diversity and Nitrate Levels in Cores from Deep Boreholes in Pristine Karst.

This study investigates the nitrate gradients within the deep biosphere of karst carbonate rocks and their resident microbiota. Samples were taken from borehole cores at depths down to 350 m below the surface, collected during geological site investigations for proposed railway tunnels and analysed using 16S rRNA amplicon sequencing. 16S rRNA amplicon sequencing analysis revealed relatively low microbial diversity, which can serve as a reliable indicator of the pristine nature of deep karst. However, some local hotspots of diversity are independent of depth. Pseudomonadota dominated the samples, with Gammaproteobacteria dominating at the class level. The low nitrate content in deep karst, in contrast to higher values closer to the surface, serves as an additional marker of its undisturbed and unpolluted status. Based on the prediction of functional profiles from 16S rRNA sequencing data, nitrates remain low due to indigenous microbial denitrification and assimilatory nitrate reduction. Pathways related to nitrogen fixation, ammonia assimilation, and nitrification were not confirmed. When elevated nitrate levels are observed in karst, they are most probably related to anthropogenic activities. Environmental factors other than depth and nitrate content play an important role in shaping bacterial communities.

Molecular Screening for Cyanobacteria and Their Cyanotoxin Potential in Diverse Habitats.

Cyanobacteria are adaptable and dominant organisms that exist in many harsh and extreme environments due to their great ecological tolerance. They produce various secondary metabolites, including cyanotoxins. While cyanobacteria are well studied in surface waters and some aerial habitats, numerous other habitats and niches remain underexplored. We collected 61 samples of: (i) biofilms from springs, (ii) aerial microbial mats from buildings and subaerial mats from caves, and (iii) water from borehole wells, caves, alkaline, saline, sulphidic, thermal, and iron springs, rivers, seas, and melted cave ice from five countries (Croatia, Georgia, Italy, Serbia, and Slovenia). We used (q)PCR to detect cyanobacteria (phycocyanin intergenic spacer-PC-IGS and cyanobacteria-specific 16S rRNA gene) and cyanotoxin genes (microcystins-mcyE, saxitoxins-sxtA, cylindrospermopsins-cyrJ), as well as amplicon sequencing and morphological observations for taxonomic identification. Cyanobacteria were detected in samples from caves, a saline spring, and an alkaline spring. While mcyE or sxtA genes were not observed in any sample, cyrJ results showed the presence of a potential cylindrospermopsin producer in a biofilm from a sulphidic spring in Slovenia. This study contributes to our understanding of cyanobacteria occurrence in diverse habitats, including rare and extreme ones, and provides relevant methodological considerations for future research in such environments.

Comparison of Culture-Dependent and Culture-Independent Methods for Routine Identification of Airborne Microorganisms in Speleotherapeutic Caves.

The effective identification of bacterial and fungal isolates is essential for microbiological monitoring in environments like speleotherapeutic caves. This study compares MALDI-TOF MS and the OmniLog ID System, two high-throughput culture-based identification methods. MALDI-TOF MS identified 80.0% of bacterial isolates to the species level, while the OmniLog ID System identified 92.9%. However, species-level matches between the methods were only 48.8%, revealing considerable discrepancies. For discrepant results, MALDI-TOF MS matched molecular identification at the genus level in 90.5% of cases, while the OmniLog ID System matched only in 28.6%, demonstrating MALDI-TOF MS's superiority. The OmniLog ID System had difficulties identifying genera from the order Micrococcales. Fungal identification success with MALDI-TOF MS was 30.6% at the species level, potentially improvable with a customised spectral library, compared to the OmniLog ID System's 16.7%. Metagenomic approaches detected around 100 times more microbial taxa than culture-based methods, highlighting human-associated microorganisms, especially Staphylococcus spp. In addition to Staphylococcus spp. and Micrococcus spp. as indicators of cave anthropisation, metagenomics revealed another indicator, Cutibacterium acnes. This study advocates a multi-method approach combining MALDI-TOF MS, the OmniLog ID System, culture-based, and metagenomic analyses for comprehensive microbial identification. Metagenomic sampling on nitrocellulose filters provided superior read quality and microbial representation over liquid sampling, making it preferable for cave air sample collection.

A comprehensive early warning system for karst water sources contamination risk, case study of the Unica springs, SW Slovenia.

Water suppliers should provide safe drinking water following preventive measures. This is especially important for karst water sources, as they are among the most vulnerable. Recently, there has been a strong focus on the early warning system, which mainly involves monitoring proxy parameters, but does not consider drainage area conditions and other monitoring recommendations. Here, we present an innovative strategy for assessing contamination risk of karst water sources that covers spatio-temporal dimensions and can be integrated into management practices. It is based on event-based monitoring and risk mapping and has been tested in a well-known study area. The holistic early warning system provides accurate spatial hazard and risk assessment and operational monitoring guidelines, including locations, indicator parameters, and temporal resolution and duration. In the study area, the high contamination risk, representing 0.5 % of the area, was spatially delineated. The highest probability of source contamination occurs during recharge events when proxy parameters such as bacteria, ATP, Cl, and Ca/Mg ratio should be monitored in addition to continuous monitoring of turbidity, EC, and T. Monitoring of sinking streams should serve as a preventive measure, since water transfer from ponors to springs has been shown to take about one day, and poor quality water is present for at least another day. Therefore, intensive monitoring should be conducted at intervals of a few hours for at least a week. Although hydrologic systems vary, the proposed strategy is particularly useful in systems where water flows rapidly and where remediation is not feasible.

Effects of Anthropogenic Disturbance and Seasonal Variation on Aerobiota in Highly Visited Show Caves in Slovenia.

Aerosols in caves are natural tracers and, together with climatic parameters, provide a detailed insight into atmospheric conditions, responses to climatic changes and anthropogenic influences in caves. Microbiological air monitoring in show caves is becoming increasingly useful to understand changes in cave ecosystems and to implement and review measures for sustainable cave use and tourism development. In 2017 and 2018, air along tourist trails in caves Postojnska jama and Skocjanske jame (Slovenia) was sampled before and after tourist visits. Samples were analysed using culture-dependent methods, flow cytometry, detection of ß-D-glucan and lipopolysaccharide and compared with CO2 and temperature data to measure anthropogenic influences and seasonality on aerobiota. While the presence of tourists significantly increased concentrations of airborne microorganisms (p < 0.05), ß-D-glucan and CO2 did not show such a trend and were more dependent on seasonal changes. Locally, concentrations of cultivable microorganisms above 1000 CFU/m3 were detected, which could have negative effects on the autochthonous microbiota and possibly on human health. A mixture of bacteria typically associated with humans was found in the air and identified with MALDI-TOF MS. Using MALDI-TOF MS, we achieved a 69.6% success rate in identification. Micrococcus luteus, Streptococcus mitis, Staphylococcus epidermidis and Moraxella spp. were recognized as good indicators of cave anthropisation.

Long-Term Monitoring of Bioaerosols in an Environment without UV and Desiccation Stress, an Example from the Cave Postojnska Jama, Slovenia.

A natural cave environment subject to regular human visitation was selected for aerobiological study to minimize the effects of severe temperature fluctuations, UV radiation, and desiccation stress on the aerobiome. The longer sampling period of bioaerosols, up to 22 months, was generally not associated with a proportionally incremental and cumulative increase of microbial biomass. The culture-independent biomass indicator ATP enabled quick and reliable determination of the total microbial biomass. Total airborne microbial biomass was influenced by human visitation to the cave, as confirmed by significantly higher concentrations being observed along tourist footpaths (p < 0.05). Airborne beta-glucans (BG) and lipopolysaccharide (LPS) are present in cave air, but their impact on the cave remains to be evaluated. Staphylococcus spp., as an indicator of human presence, was detected at all sites studied. Their long-term survival decrease is likely due to high relative humidity, low temperature, the material to which they adhere, and potentially natural elevated radon concentration. The most commonly recorded species were: S. saprophyticus, which was identified in 52% of the studied sites, S. equorum in 29%, and S. warneri in 24% of the studied sites. Only a few isolates were assigned to Risk group 2: S. aureus, S. epidermidis, S. haemolyticus, S. pasteuri, and S. saprophyticus.

Prokaryotic and eukaryotic airborne microorganisms as tracers of microclimatic changes in the underground (Postojna Cave, Slovenia).

Bioaerosols in cave air can serve as natural tracers and, together with physical parameters, give a detailed view of conditions in the cave atmosphere and responses to climatic changes. Airborne microbes in the Postojna Cave system indicated very dynamic atmospheric conditions, especially in the transitory seasonal periods between winter and summer. Physical parameters of cave atmosphere explained the highest variance in structure of microbial community in the winter and in the summer. The airborne microbial community is composed of different microbial groups with generally low abundances. At sites with elevated organic input, occasional high concentrations of bacteria and fungi can be expected of up to 1,000 colony-forming units/m(3) per individual group. The most abundant group of airborne amoebozoans were the mycetozoans. Along with movements of air masses, airborne algae also travel deep underground. In a cave passage with elevated radon concentration (up to 60 kBq/m(3)) airborne biota were less abundant; however, the concentration of DNA in the air was comparable to that in other parts of the cave. Due to seasonal natural air inflow, high concentrations of biological and inanimate particles are introduced underground. Sedimentation of airborne allochthonous material might represent an important and continuous source of organic material for cave fauna.

Characteristics and impact of aged coal ash with slag emplaced in a karst cave: the case of Divaska jama, Slovenia.

A mixture of coal bottom ash and slag, with a fraction of fly ash (CAFAS) from steam locomotives, was placed in the cave Divaska jama to delimit and level tourist trails. Emplacement began in 1914 and carried on for several decades. The CAFAS mixed with other cave material gradually changed its structure and appearance. Currently the concentration of some elements in the CAFAS (As, Cu, Hg, Ni, Pb, Zn), and also to a lesser extent in cave sediments (Cr, Cu, Ni), indicates a possibly harmful effect on sediment-associated biota based on ecotoxicological assays. Compared to the cave sediment, the CAFAS contains distinctly different mineral phases and presents a different source of radioactivity. Microbial metabolic activity of CAFAS is low, 0.22 µl O2/gDW h, but higher than that of cave sediment. The present environmental hazards from CAFAS are estimated to be low. Whereas the emplacement of CAFAS was seen initially a long-term solution for waste disposal and management of the cave, it turned out that CAFAS enriches the underground environment with inorganic and organic compounds and disperses pollution into the cave ecosystem. After its removal from the cave, the CAFAS should be investigated thoroughly due to its susceptibility to alteration.

Extremophilic Microorganisms in Central Europe.

Extremophiles inhabit a wide variety of environments. Here we focus on extremophiles in moderate climates in central Europe, and particularly in Slovenia. Although multiple types of stress often occur in the same habitat, extremophiles are generally combined into groups according to the main stressor to which they are adapted. Several types of extremophiles, e.g., oligotrophs, are well represented and diverse in subsurface environments and karst regions. Psychrophiles thrive in ice caves and depressions with eternal snow and ice, with several globally distributed snow algae and psychrophilic bacteria that have been discovered in alpine glaciers. However, this area requires further research. Halophiles thrive in salterns while thermophiles inhabit thermal springs, although there is little data on such microorganisms in central Europe, despite many taxa being found globally. This review also includes the potential use of extremophiles in biotechnology and bioremediation applications.

Microbiota entrapped in recently-formed ice: Paradana Ice Cave, Slovenia.

Paradana is one of the biggest ice caves in Slovenia, with an estimated ice volume of 8,000 m3. Reflecting climatological conditions, the cave ice undergoes repeated freeze-thaw cycles and regular yearly deposition of fresh ice. Three distinct ice block samples, collected from the frozen lake in May 2016, were analysed to obtain data on ice physicochemical properties and the composition of associated microbiota. Isotopic composition of the ice samples (18O, 2H) and a local meteoric water line (LMWL) constructed for monthly precipitation at Postojna were used to estimate the isotopic composition of the water that formed the ice, which had high values of deuterium excess and low concentrations of chloride, sulphate and nitrate. The values of total organic carbon (1.93-3.95 mg/l) within the ice blocks fall within the range of those measured in karst streams. Total cell count in the ice was high and the proportion of cell viability increased along the depth gradient and ranged from 4.67 × 104 to 1.52 × 105 cells/ml and from 51.0 to 85.4%, respectively. Proteobacteria represented the core of the cave-ice microbiome (55.9-79.1%), and probably play an essential role in this ecosystem. Actinobacteria was the second most abundant phylum (12.0-31.4%), followed in abundance by Bacteroidetes (2.8-4.3%). Ice phylotypes recorded amounted to 442 genera, but only 43 genera had abundances greater than 0.5%. Most abundant were Pseudomonas, a well-known ice dweller, and Lysobacter, which previously was not reported in this context. Finally, two xanthophytes, Chloridella glacialis and Ellipsoidion perminimum, known from polar environments, were cultured from the ice. This indicates that the abundance and ecological role of phototrophs in such environments might be greater than previously deduced.

F-like plasmid sequences in enteric bacteria of diverse origin, with implication of horizontal transfer and plasmid host range.

Seventy-eight bacterial isolates from human, animal, and plant hosts, representing eight species of the family Enterobacteriaceae, were screened for F-like plasmid sequences. Of the examined human Escherichia coli strains, 28% harbored one or two of the three F-like, RepFI replication regions, while 35% of the examined animal and all phytopathogenic strains harbored RepFIA-specific sequences. Comparative analysis of Salmonella, Shigella, Erwinia, and E. coli plasmid RepFI sequences showed 100% or very high homology, indicating frequent and recent interspecies gene transfer. The high incidence of RepFIA sequences in enteric bacterial species, including Klebsiella and Erwinia, showed that F-like plasmids are successful in avoiding natural barriers to establishment of horizontally transferred DNA and that in the natural environment conjugal transfer is efficient in diverse ecological niches.

A cka-gfp transcriptional fusion reveals that the colicin K activity gene is induced in only 3 percent of the population.

In prokaryotes, only a few examples of differential gene expression in cell populations have been described. Colicin production in natural populations of Escherichia coli, while providing a competitive advantage in the natural habitat, also leads to lysis of the toxin-producing cell. Colicin K synthesis has been found to be induced due to an increase in ppGpp (I. Kuhar, J. P. van Putten, D. Zgur-Bertok, W. Gaastra, and B. J. Jordi, Mol. Microbiol. 41:207-216). Using two transcriptional fusions, cka-gfp and cki-gfp, we show that at the single-cell level, the colicin K activity gene cka is expressed in only 3% of the bacterial population upon induction by nutrient starvation. In contrast, the immunity gene cki is expressed in the large majority of the cells. Expression of the cka-gfp fusion in a lexA-defective strain and in a relA spoT mutant strain indicates that differential expression of cka is established primarily at the level of transcription.