The subsurface mine environments characterized by high levels of toxic metals and low nutrient availability represent an extreme threat to bacterial persistence. In recent study, the genomic analysis of the Acinetobacter johnsonii strain RB2-047 isolated from the Rozália Gold Mine in Slovakia was performed. As expected, the studied isolate showed a high level of heavy metal tolerance (minimum inhibitory concentrations were 500 mg/L for copper and nickel, 1,500 mg/L for lead, and 250 mg/L for zinc). The RB2-047 strain also showed noticeable resistance to several antibiotics (ampicillin, kanamycin, chloramphenicol, tetracycline and ciprofloxacin). The genomic composition analysis demonstrated a low number of antibiotic and metal resistance coding genes, but a high occurrence of efflux transporter genes located on the bacterial chromosome. The experimental inhibition of efflux pumps resulted in decreased tolerance to Zn and Ni (but not to Cu and Pb) and to all antibiotics tested. In addition, the H33342 dye-accumulation assay confirmed the high efflux activity in the RB2-047 isolate. These findings showed the important role of efflux pumps in the adaptation of Acinetobacter johsonii strain RB2-047 to metal polluted mine environment as well as in development of multi-antibiotic resistance.

The environment contaminated by antibiotics and heavy metals as a consequence of human activities is of great concern nowadays.Many pieces of research proved that the environment could act as a reservoir of antibiotic resistance determinants allowing themto spread among different bacterial species via the process called horizontal gene transfer. The result is antibiotic resistance even inpathogen microorganisms. Heavy metals act as important factors in this process because of their potential to select antibiotic resistantbacteria thanks to linkage among antibiotic resistance genes and heavy metals resistance genes.Thus, this experiment was conducted to screen the antibiotic tolerance profile of bacteria obtained from heavy metal contaminatedenvironment of mine, dump and the contaminated soil near the entry of mine.Several samples were collected from the only active gold mine in Slovakia in Hodruša – Hámre. The presence of cultivable bacteria wasproved via cultivation approaches with subsequent MALDI – TOF MS (Matrix – Assisted Laser Desorption/Ionisation Time of FlightMass Spectrometry) identification of selected isolates. Representative bacterial isolates were screened for their antibiotic toleranceagainst chosen antibiotics (ampicillin (AMP), chloramphenicol (CHLOR), tetracycline (TET) and kanamycine (KAN)) with the aimto define their minimal inhibitory concentration (MIC).The cultivable bacteria from studied environments were dominated by Gram-negative protebacteria of Pseudomonas and Rhizobiumgenera. Among more than 150 isolates the resistance to ampicillin (MIC>100µg/ml – 49% isolates), kanamycine (MIC>100µg/ml -18% isolates), and chloramphenicol (MIC>20µg/ml – 16% isolates) dominated. The resistance to tetracycline (MIC>20µg/ml) wasdetected in less than 1% of isolates. Overall counts of antibiotic resistance and multi-resistance were alarmingly high taking in accountthat industrial environments with no known antibiotic exposure were analysed.Our data indicate that heavy metals contaminated environment could influence the occurrence and the spread of antibiotic resistance.Possibly, metal contaminated environment act as a reservoir of antibiotic resistant bacteria.

Microorganisms actively participate in biogeochemical cycles of various elements in the environment, including gold. To explore the core microbiota associated with gold ore, this study examined the bacterial composition of mined rock from the Rozalia gold mine (two subsurface samples from the gold mine and one from the heap of mined ore) using a cultivation approach. Cultivation analyses showed the occurrence of bacteria with colony forming units (CFU) ranging from 2.18 x 10(5) to 3.16 x 10(5) per 1 gram of dry ore material, and the data analysis indicates that the type of cultivation medium used significantly influences the observed biodiversity of cultivable bacteria. Cultivated members of the microbial community were identified using a combination of MALDI-TOF MS and 16S rRNA gene sequencing. The cultivable microbiota of gold-bearing ore samples was predominantly composed of the phylum Proteobacteria. Identification of 473 isolates revealed the presence of 4 dominant genera: Rhizobium, Microbacterium, Pseudomonas, and Acinetobacter, which together accounted for 89% of the gold ore-associated cultivated bacteria. These results are consistent with previous studies on the microbiota of gold mines and suggest that these genera constitute the core microbiota of gold ore.

Heavy metal pollution caused by anthropogenic activity is a great concern of the present days. Widespread use of substances containingmetals inevitably lead to their deposition in soil affecting soil microbiota, which plays important role in maintaining soilfunctions. The aim of our study was to determine number of heavy metal resistant isolates acquired from the soil from heavy metalpolluted area of dump near Hnúšťa. Soil samples were obtained from two collection sites (48° 36´ 4,47502´´ N, 19° 57´32,654´´ Eand 48° 36´ 4,4634´´ N, 19° 57´ 32,67´´ E) and mixed together. The microorganisms used in this study were routinely cultivatedand screened for resistance to different concentrations of four heavy metals – Zn (2–500 mg/l), Cu, Ni, Pb (all three metals testedat concentrations from 0,5 to 125 mg/l). Very high resistance against Cu, Ni and Pb even at the highest tested concentrations wasfound at majority of tested bacterial strains. Almost all 89 from 89 isolates show resistance against these metals at tested concentrations.Only in case of Zn we determined the MIC (minimal inhibitory concentration) – 125 mg/l. The results point out very highresistance pattern in soil bacteria.

The Rozália Mine, with its long mining history, could represent an environmental threat connected with metal contamination and associated antibiotic tolerance. Metal and antibiotic tolerance profiles of heterotrophic, cultivable bacteria isolated from the Rozália Gold Mine in Hodruša-Hámre, Slovakia, and the surrounding area were analysed. Subsurface samples were collected from different mine levels or an ore storage dump. As expected, heterotrophic cultivable bacteria showed high minimum inhibitory concentrations for metals (up to 1000 mg/l for zinc and nickel, 2000 mg/l for lead and 500 mg/l for copper). Surprisingly, very high minimum inhibitory concentrations of selected antibiotics were observed, e.g. > 10,000 μg/ml for ampicillin, up to 4800 μg/ml for kanamycin, 800 μg/ml for chloramphenicol and 50 μg/ml for tetracycline. Correlation analysis revealed a linkage between increased tolerance to the antibiotics ampicillin and chloramphenicol and metal tolerance to nickel and copper. A correlation was also observed between tetracycline-kanamycin tolerance and zinc-lead tolerance. Our data indicate that high levels of antibiotic tolerance occur in deep subsurface microbiota, which is probably connected with the increased level of metal concentrations in the mine environment.

Biogeochemical cycling of gold involves dispersion and reconcentration of gold (Au) due to physical, chemical and biological processesin Earth surface environments. These processes are evocated by a metabolic activity of different microbial taxa but many of them (andalso their biogeochemical potential) are still unexplored. Understanding the gold cycling is necessary for developing innovative, environmentally friendly gold processing techniques. Our experiments were aimed on isolation and identification of heterotrophic bacteriafrom ore and ore storage dump samples collected in Rozália gold mine in Hodruša-Hámre. Using culture-based approach followed bycombination of MALDI-TOF MS protein profiling and 16S rDNA sequencing, 18 different bacterial genera were identified in studiedmicrobiota. The participation of several representatives of these genera in individual gold cycling steps has already been reported. Thereal involvement of bacterial isolates in gold transformation reactions and their biogeochemical potential will be studied in subsequentexperiments.

Sulphur-oxidising autotrophic bacterial communities in deep biosphere from weathered ore samples from active gold mine Hodruša-Hámre, Slovakia were analysed using cultivation approach followed by DNA extraction, PCR amplification and 16S rRNA gene analyses. Indirect measurement of pH changes in cultivation media evidenced the presence of acidophilic bacteria with active production of acids. The decrease of pH was observed at the beginning of isolation and later pH in range of 1.5 – 2 was maintained in both, sulphuric acid and thiosulphate, media. The presence of homogenous population of gram-negative rods was proved by Gram staining. Molecular analyses have revealed that the population of sulphur-oxidising bacteria in gold mine is dominated by a single species of Aciditiobacillus genus, identified as A. albertensis, suggesting the low level of autotrophic bacteria diversity in deep deposits. For the first time this species was isolated from weathered rocks of a gold mine subsurface environment.

Heavy metal pollution is of great concern. Due to expansion of industrial activities, a large amount of metal is released into the environment, disturbing its fragile balance. Conventional methods of remediation of heavy metal-polluted soil and water are expensive and inefficient. Therefore, new techniques are needed to provide environmentally friendly and highly selective remediation. Streptomycetes, with their unique growth characteristics, ability to form spores and mycelia, and relatively rapid colonization of substrates, act as suitable agents for bioremediation of metals and organic compounds in polluted soil and water. A variety of mechanisms could be involved in reduction of metals in the environment, e.g., sorption to exopolymers, precipitation, biosorption and bioaccumulation. Studies performed on biosorption and bioaccumulation potential of streptomycetes could be used as a basis for further development in this field. Streptomycetes are of interest because of their ability to survive in environments contaminated by metals through the production of a wide range of metal ion chelators, such as siderophores, which provide protection from the negative effects of heavy metals or specific uptake for specialized metabolic processes. Many strains also have the equally important characteristic of resistance to high concentrations of heavy metals.

AbstractThe genus Acidithiobacillus comprises 7 species of Gram-negative obligatory acidophilic chemolithotrophic bacteria that derive energy mainly from the oxidation of reduced sulphur compounds. Four of the species also catalyse the dissimilatory oxidation of ferrous iron while three (A. thiooxidans, A. albertensis, and A. caldus) do not. Bacteria from the genus Acidithiobacillus are often associated with mineral biotechnologies (biomining) and acid mine drainage. While acceleration of mineral solubilisation is a positive aspect in environmental biotechnologies, it is undesirable in acid mine drainage with strong negative ecological impact and there is profound interest in genetics and genomics of these bacteria. Representatives of Acidithiobacillus genus occur world-wide, however there are limited data on Acidithiobacillus spp. variability from Slovakia. In our work the variability of Acidithiobacillus spp., from Slovakia was analysed and the presence of A ferrooxidans was detected. In addition, for the first time we report here on the occurrence of A. albertensis as well. Comparative analyses confirmed pronounced genetic and genomic diversity within the genus, especially within A. ferrooxidans and A. thioxidans complexes. Based on data presented, several Acidithiobacillus species could be considered as a complex species and the description of several new species is very probable in the near future.