This study investigated the genetic variability and antibiotic resistance of Acinetobacter community depending on the stage of wastewater treatment in Kokšov-Bakša for the city of Košice (Slovakia). After cultivation, bacterial isolates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and their sensitivity to ampicillin, kanamycin, tetracycline, chloramphenicol and ciprofloxacin was examined. Acinetobacter spp. and Aeromonas spp. dominated bacterial populations in all wastewater samples. We identified 12 different groups based on protein profiling, 14 genotypes by amplified ribosomal DNA restriction analysis and 11 Acinetobacter species using 16S rDNA sequence analysis within Acinetobacter community, which showed significant variability in their spatial distribution. While Acinetobacter population structure changed during the wastewater treatment, the prevalence of antibiotic-resistant strains did not significantly vary depending on the stage of wastewater treatment. The study highlights the role of a highly genetically diverse Acinetobacter community surviving in wastewater treatment plants as an important environmental reservoir assisting in the further dissemination of antibiotic resistance in aquatic systems.

Although neutral mine drainage is the less frequent subject of the interest than acid mine drainage, it can have adverse environmental effects caused mainly by precipitation of dissolved Fe. The aim of the study was to characterize the composition of bacterial population in environment with high concentration of iron and sulfur compounds represented by neutral mine drainage water of Elizabeth’s shaft, Slovinky (Slovakia). Direct microscopic observations, cultivation methods, and 454 pyrosequencing of the 16S rRNA gene amplicons were used to examine the bacterial population. Microscopic observations identified iron–oxidizing Proteobacteria of the genera Gallionella and Leptothrix which occurrence was not changed during the years 2008–2014. Using 454 pyrosequencing, there were identified members of 204 bacterial genera that belonged to 25 phyla. Proteobacteria (69.55%), followed by Chloroflexi (10.31%) and Actinobacteria (4.24%) dominated the bacterial community. Genera Azotobacter (24.52%) and Pseudomonas (14.15%), followed by iron–oxidizing Proteobacteria Dechloromonas (11%) and Methyloversatilis (8.53%) were most abundant within bacterial community. Typical sulfur bacteria were detected with lower frequency, e.g., Desulfobacteraceae (0.25%), Desulfovibrionaceae (0.16%), or Desulfobulbaceae (0.11%). Our data indicate that the composition of bacterial community of the Elizabeth’s shaft drainage water reflects observed neutral pH, high level of iron and sulfur ions in this aquatic habitat.

The bacterial evolution and the emergence of new species are likely influenced by multiple forces, including long-term environmental pressure such as living in extreme conditions. In this study, the genomes of two potentially new Streptomyces species isolated from a former mine heap in Tarnowskie Góry in Poland, were analyzed. A bioinformatic approach revealed notable phylogenetic and metabolic differences between the studied Streptomyces strains, despite originating from the same environment. While both strains are characterized by genetic features common to actinomycetes, additional unique biosynthetic gene clusters were also predicted in their genomes. The comparative genomic analysis with other Streptomyces spp. revealed a high conservation in heavy metal adaptive mechanisms, indicating a preadaptation to extreme conditions. The difference observed in the cad and mer operons could be attributed to the specific adaptations to heavy metal contamination. The high metal tolerance of examined strains was also confirmed by an agar dilution assay in the presence of several heavy metals. The confirmed siderophore production represents an additional mechanism allowing streptomycetes to survive in extreme conditions. On the other hand, both of studied genomes show significant differences in energy acquisition processes and the production of putative novel secondary metabolites. The isolates showed these differences not only among themselves but also compared to other Streptomyces species, indicating their uniqueness. Our results demonstrate that extreme environmental conditions can lead to the development of various adaptation mechanisms in the Streptomyces spp. Furthermore, the results indicate that diverse Streptomyces species have developed conserved adaptation mechanisms against the heavy metals under extreme conditions, indicating the emergence of preadaptations that allow bacteria to respond rapidly to polluted environments and evolve their genomes accordingly up to the evolution of new species.

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.