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.

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.

CYP3A gene variability and cancer cells response to the treatment.

The treatment of cancer depends on the activity of the cytochrome P450 enzyme family, which is essentially carried out by the CYP3A4 and CYP3A5 enzymes. The aim of our study was to investigate whether the CYP3A4 polymorphism could contribute to protein activity and their influence to the response of cancer cells to treatment. The variability of CYP3A4 cDNA profiles between the cancer cell lines parental HT-29 and resistant HT-29-OxR adenocarcinoma was detected using denaturing gradient gel electrophoresis (DGGE). Subsequently, sequence analysis of CYP3A family members (CYP3A4, CYP3A5) confirmed polymorphism of the CYP3A4 gene in studied cancer cell lines. Variations at the gene expression level, the protein level and the activity of CYP3A4 protein in 12 cancer cell lines were observed, also different response to drug treatments between cell line HT-29 and oxaliplatin-resistant cell line HT-29-OxR. The variability of CYP3A might affect the efficiency of anti-cancer drugs in general and have an impact on metabolism.

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.

New Endophytic Fusarium spp. from Fraxinus excelsior Leaves in Slovakia.

Fraxinus excelsior is currently suffering from ash dieback disease caused by the fungus Hymenoscyphus fraxineus. Co-occurrence of large numbers of other fungi with endophytic as well as pathogenic lifestyles has been observed on F. excelsior. In this study, new endophytic Fusarium spp. isolate Fraxinus excelsior Fusarium from Slovak territory, associated with F. excelsior, was characterised. Using morphology-based approaches, the fungus was assigned to the genus Fusarium. The isolate formed thick-walled hyaline, fusiform, slightly curved macroconidia, produced from monophialides in sporodochia, pointed at the tip, mostly 3 - 5 septate, occasionally 6 septate, and 40 - 68 × 3.8 - 5.0 µm in size with basal pedicellate cells. For more accurate taxonomic affiliation, molecular sequence analyses of the internal transcribed spacer, translation elongation factor 1-alpha sequences, and partial ß-tubulin gene were performed. While the internal transcribed spacer sequence of the isolate showed the highest similarity to the Fraxinus oxysporum species, translation elongation factor 1-alpha and partial ß-tubulin sequences were distantly related to the Fraxinus avenaceum species. Based on these data, the Fraxinus excelsior Fusarium isolate could probably be considered a new species of the Fusarium genus.

Candidatus Cryptoplasma Associated with Green Lizards and Ixodes ricinus Ticks, Slovakia, 2004-2011.

During 2004-2011, we collected green lizards and Ixodes ricinus ticks in Slovak Karst National Park in Slovakia; 90% (36/40) of lizards and 37% of ticks removed from lizards were infected with family Anaplasmataceae bacteria. Only Candidatus Cryptoplasma sp. REP (reptile) was identified in these samples. Green lizards transmit this bacterium.

Peptidoglycan hydrolase enterolysin a recognizes lipoteichoic acid chains in the cell walls of sensitive bacteria.

C-terminal domain of peptidoglycan hydrolase enterolysin A (EnlA) is involved in specific recognition and binding to the target cell envelopes and represents true cell wall binding (CWB) domain. Sensitivity/resistance to EnlA is dependent on binding ability/disability of its CWB domain. We assume that main mechanism of resistance against EnlA is absence of the specific receptor on the cell surface, which is necessary for binding of the enzyme molecule. Using competitive and enzymatic assays we have uncovered the chemical nature of the EnlA receptor, which is a lipoteichoic acid.