Genomic insights into the adaptation of Acinetobacter johnsonii RB2-047 to the heavy metal-contaminated subsurface mine environment.

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.

Genetic Basis of Acinetobacter sp. K1 Adaptation Mechanisms to Extreme Environmental Conditions.

Anthropogenic pollution often leads to the generation of technosols, technogenic soils with inhospitable conditions for all living organisms including microbiota. Aluminum production near Ziar nad Hronom (Slovakia) resulted in the creation of a highly alkaline and heavy-metal-rich brown mud landfill, from which a bacterial strain of a likely new species of the genus Acinetobacter, Acinetobacter sp. K1, was isolated. The whole-genome sequence analysis of this strain confirmed the presence of operon units enabling tolerance to the heavy metals copper, zinc, cobalt, cadmium, chromium, and metalloid arsenic, which are functionally active. Despite the predominance of plasmid-related sequences in the K1 genome, the results indicate that most of the resistance genes are chromosomally encoded. No significant alkali tolerance of Acinetobacter sp. K1 was observed in vitro, suggesting that community level mechanisms are responsible for the survival of this strain in the highly alkaline, brown mud bacterial community.

Antibiotic Resistance and Genetic Variability of Acinetobacter spp. from Wastewater Treatment Plant in Koksov-Baksa (Kosice, Slovakia).

This study investigated the genetic variability and antibiotic resistance of Acinetobacter community depending on the stage of wastewater treatment in Koksov-Baksa for the city of Kosice (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.

Bacterial Community Structure of Two Cold Sulfur Springs in Slovakia (Central Europe).

Sulfur-oxidizing bacteria, especially those from hot springs, have attracted the attention of microbiologists for more than 150 years. In contrast, the microbial diversity of cold sulfur springs remains largely unrecognized. Culture-dependent and culture-independent approaches were used to study the diversity of sulfur-oxidizing bacterial communities in two cold sulfur springs in Slovakia. Geological conditions and resulting spring water chemistry appear to be major factors influencing the composition of the sulfur-oxidizing bacterial community. Bacterial communities in both springs were found to be dominated by Proteobacteria with Epsilonproteobacteria being prevalent in the high-salinity Stankovany spring and Alpha- and Gammaproteobacteria in the low-salinity Jovsa spring. Limited overlap was found between culture-dependent and culture-independent approaches with multiple taxa of cultivated sulfur-oxidizing bacteria not being detected by the culture-independent metagenomics approach. Moreover, four cultivated bacterial isolates could represent novel taxa based on the low similarity of their 16S rRNA gene sequence (similarity lower than 98%) to sequences of known bacteria. Our study supports the current view that multiple approaches are required to assess the bacterial diversity in natural habitats and indicates that sulfur springs in Slovakia harbor unique, yet-undescribed microorganisms.

High frequency of antibiotic tolerance in deep subsurface heterotrophic cultivable bacteria from the Rozália Gold Mine, Slovakia.

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 Hodrusa-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.

The Faecal Microbiome of Building-Dwelling Insectivorous Bats (Myotis myotis and Rhinolophus hipposideros) also Contains Antibiotic-Resistant Bacterial Representatives.

The bacteriome of bat faeces and bat guano is still not well known, despite the increasing knowledge of it as a potential source of pathogenic and/or antibiotic-resistant species. In this study, the faecal microbiome composition of two building-dwelling insectivorous bat species (Myotis myotis and Rhinolophus hipposideros) was analysed using cultivation-based and non-cultivation-based approaches. The cultivation analyses indicate the dominance of Gram-positive bacteria, represented by the genera Enterococcus, Lactococcus and Lactobacillus. Non-cultivation analysis based on 16S rRNA DGGE assays provided a different pattern, with the genus Rahnella found to predominate in the faecal microbiome. The analyses showed bat species to be the most important factor influencing the structure of the guano-associated microbial population. The presence of several possible human pathogens (Hafnia alvei, Serratia fonticola, S. liquefaciens) and antibiotic-resistant bacteria (e.g. vancomycin resistant Staphylococcus nepalensis) was detected in faeces samples, indicating possible human health risks associated with bat guano. Although the bat-human transmission of infection caused by pathogenic bacteria has not been reliably confirmed to date, this possibility should not be underestimated. Given the very important ecosystem services of bats, particular those studied herein, it is time to think about appropriate management of bat populations inhabiting man-made buildings and potential conflicts with humans.

Correction to: The Bacterial Population of Neutral Mine Drainage Water of Elizabeth's Shaft (Slovinky, Slovakia).

The article "The Bacterial Population of Neutral Mine Drainage Water of Elizabeth's Shaft (Slovinky, Slovakia)", written by Jana Kisková, Zuzana Perháèová, Ladislav Vlèko, Jana Sedláková, Simona Kvasnová and Peter Pristas, was originally published electronically on the publisher's internet portal on 12 March 2018 without open access. With the author(s)' decision to opt for Open Choice the copyright of the article changed on 9 April 2020 to © The Author(s) 2018 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ ), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The original article has been corrected.

Analysis of the bacterial community from high alkaline (pH > 13) drainage water at a brown mud disposal site near Ziar nad Hronom (Banská Bystrica region, Slovakia) using 454 pyrosequencing.

Brown mud, as a waste product of the industrial process of aluminum production, represents a great environmental burden due to its toxicity to living organisms. However, some microorganisms are able to survive in this habitat, and they can be used in bioremediation processes. Traditional cultivation methods have a limited capacity to characterize bacterial composition in environmental samples. Recently, next-generation sequencing methods have provided new perspectives on microbial community studies. The aim of this study was to analyze the bacterial community in the drainage water of brown mud disposal site near Ziar nad Hronom (Banská Bystrica region, Slovakia) using 454 pyrosequencing. We obtained 9964 sequences assigned to 163 operational taxonomic units belonging to 10 bacterial phyla. The phylum Proteobacteria showed the highest abundance (80.39%) within the bacterial community, followed by Firmicutes (13.05%) and Bacteroidetes (5.64%). Other bacterial phyla showed an abundance lower than 1%. The classification yielded 85 genera. Sulfurospirillum spp. (45.19%) dominated the bacterial population, followed by Pseudomonas spp. (13.76%) and Exiguobacterium spp. (13.02%). These results indicate that high heavy metals content, high pH, and lack of essential nutrients are the drivers of a dramatic reduction of diversity in the bacterial population in this environment.

The Bacterial Population of Neutral Mine Drainage Water of Elizabeth's Shaft (Slovinky, Slovakia).

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.

Occurrence of blood parasites and intensity of infection in Prunella modularis in the montane and subalpine zone in the Slovak Carpathians.

The objective of this study was to obtain primary information on the occurrence of blood parasites and intensity of infection in the Dunnock Prunella modularis in the montane region of Slovakia. Altogether 109 birds were examined during the years 2006-2010. The occurrence of Haemoproteus sp., Leucocytozoon fringillinarum and Trypanosoma sp. was documented. Blood parasite prevalences of 45% by microscopic examination and 55% by PCR diagnostics were found. The prevalence of Leucocytozoon sp. was found to be dependent on host sex with males showing a significantly higher intensity of infection with Leucocytozoon. Adult birds showed significantly higher infection prevalence than subadults. The prevalences of both Leucocytozoon sp. and Haemoproteus sp. were significantly dependent on bird age. The intensity of infection with Haemoproteus and Leucocytozoon was positively correlated and higher intensity of infection was confirmed in adult birds then in subadults birds. The prevalence of blood parasites in our samples was significantly higher in comparison to most other studies from different countries of Europe conducted at lower altitudes, indicating that the montane zone is especially favourable for the transmission of Leucocytozoon and Haemoproteus species.

Yersinia species in the dunnock (Prunella modularis) in sub-alpine habitats of the Western Carpathians.

The study presents the prevalence of Yersinia species in dunnok Prunella modularis from the sub-alpine zone of the Western Carpathians. Bacteria were detected from cloacal and pharyngeal swabs from 97 specimens using PCR assay. Yersinia enterocolitica showed the highest prevalence (47.4%) from among the determined Yersinia species. Yersinia species (except Y frederiksenii) were detected more frequently in pharyngeal than cloacal samples. The highest prevalence of yersiniosis was detected in April (Yersinia spp. - 80%, Y. enterocolitica - 70%). No statistically differences were observed in the prevalence of Yersinia spp. between males and females and between juveniles and adult birds. Bacterial contamination did not affect body weight or tarsus length.