Magdalena Moryl

The Ukrainian brook lamprey (UBL) (Eudontomyzon mariae) spreads in Poland and often replaces the European brook lamprey (EBL) (Lampetra planeri) in rivers contaminated by wastewater effluents. We aimed to investigate whether this phenomenon is associated with differences in the intestinal microbiota of the two lamprey species. We analysed seasonal changes in the midgut microbial content of the UBL ammocoetes collected from a section of the River Gać affected by wastewater treatment plant effluent. The numbers of cultivable psychrophilic (autochthonous) and mesophilic (allochthonous) microorganisms, along with Escherichia coli and faecal streptococci, were compared to those found in the EBL from the same site. Higher levels of the faecal indicator microorganisms were observed in the UBL gut content compared to the EBL across most seasons, particularly in the winter (all mesophiles), in contrast to the levels of psychrophilic bacteria. This may suggest a relatively greater accumulation of mesophilic, sewage-associated bacteria in the UBL gut. Varying intestinal bacterial counts in the UBL gut did not reflect trends observed for the microorganisms in surrounding water during the studied seasons. These findings support the hypothesis that the UBL potentially benefits from the uptake of faecal bacteria. Such adaptation may contribute to its dominance in contaminated freshwater ecosystems, where EBL populations decline.

A phage–antibiotic synergy could be an alternative in urinary tract infection (UTI) therapy, as it leads to the elimination of bacteria and to the reduction in variants resistant to phages and antibiotics. The aims of the in vitro study were to determine whether phages vB_Efa29212_2e and vB_Efa29212_3e interact synergistically with selected antibiotics in the treatment of E. faecalis infections, to optimize antibiotic concentrations and phage titers for the most effective combinations, and to assess their impact on the number of spontaneous resistant variants and on the phages’ reproductive cycles. The modified double-layer disc diffusion method, checkboard, time–kill assays, one-step growth method and the double agar overlay plaque assay were implemented. Synergistic interactions were most often observed after the combined action of phages 2e or 3e and β-lactam antibiotics on E. faecalis strains. The beneficial effects depended on the bacterial strain, phage and antibiotic used. The lowest minimum inhibitory concentration (MIC₅₀) values of the antibiotics were recorded, after the application of low titers of phage 2e, and high titers of phage 3e. The combined use of the tested agents resulted in a significant reduction in the number of resistant variants and had an impact on the reproductive cycle of the tested phages, e.g., a 50% increase in burst size, and a 5 min reduction in the latency period of 2e were observed. The study confirmed beneficial interactions between phages and β-lactam antibiotics against E. faecalis growth.

This study investigates the impact of water faecal contamination on highly threatened European brook lamprey larvae (Lampetra planeri). Water samples and the midgut contents of lampreys collected from a small lowland river upstream (site 1) and downstream from a wastewater treatment plant (WWTP) discharge (site 2) were analysed to check how the faecal microbial load of the habitat is reflected in the intestines of larval lampreys. The counts of viable mesophiles, psychrophiles, Escherichia coli and faecal streptococci as bacterial indicators of microbial (including faecal) water contamination were estimated. Microbial composition and abundance in larval midgut contents depended on the numbers of various microorganisms in the water environment. At site 2, the water was heavily microbiologically contaminated throughout the year by sewage inflow from the WWTP, and the amounts of studied bacteria were also high in the midgut of lampreys inhabiting site 2 regardless of the season. At site 1, water quality was better, and the levels of tested microbial indicators were lower in the intestines of the lampreys living there. The numbers of bacteria dependent on water temperature were growing in warmer seasons both in water and in intestines. Sewage pollution negatively influenced the condition of lampreys in site 2, where they exhibited lower body condition than in site 1.

Biofilms are composed of multicellular communities of microbial cells and their self-secreted extracellular polymeric substances (EPS). The viruses named bacteriophages can infect and lyze bacterial cells, leading to efficient biofilm eradication. The aim of this study was to analyze how bacteriophages disrupt the biofilm structure by killing bacterial cells and/or by damaging extracellular polysaccharides, proteins, and DNA. The use of colorimetric and spectrofluorimetric methods and confocal laser scanning microscopy (CLSM) enabled a comprehensive assessment of phage activity against E. faecalis biofilms. The impact of the phages vB_Efa29212_2e and vB_Efa29212_3e was investigated. They were applied separately or in combination on 1-day and 7-day-old biofilms. Phages 2e effectively inhibited the growth of planktonic cells with a limited effect on the biofilm. They did not notably affect extracellular polysaccharides and proteins; however, they increased DNA levels. Phages 3e demonstrated a potent and dispersing impact on E. faecalis biofilms, despite being slightly less effective than bacteriophages 2e against planktonic cells. Phages 3e reduced the amount of extracellular polysaccharides and increased eDNA levels in both 1-day-old and 7-day-old biofilm cultures. Phage cocktails had a strong antimicrobial effect on both planktonic and biofilm-associated bacteria. A significant reduction in the levels of polysaccharides, proteins, and eDNA in 1-day-old biofilm samples was noted, which confirms that phages interfere with the structure of E. faecalis biofilm by killing bacterial cells and affecting extracellular polymer levels.

Bacteriophage therapy has emerged as a strategy supplementing traditional disinfection protocols to fight biofilms. The aim of the study was to isolate the phages against E. faecalis and to characterize its biological features, morphology, and lytic activity in a formed biofilm model. Methods: E. faecalis ATCC 29212 strain was used for the trial. Two novel vB_Efa29212_2e and vB_Efa29212_3e virulent phages were isolated from urban wastewater and characterized. The E. faecalis biofilm was established in 15 bovine teeth for 21 days. Transmission (TEM) and scanning electron (SEM) microscopes with the colony-forming unit (CFU) counting were used for assessment. Results: Isolated phages differed in morphology. Taxonomy for vB_Efa29212_2e (Siphoviridae, Efquatovirus) and for vB_Efa29212_3e (Herelleviridae, Kochikohdavirus) was confirmed. Both phages were stable at a temperature range of 4–50 °C and showed a different tolerance to chemicals: 15% EDTA, 1–3% sodium hypochlorite, and chlorhexidine. SEM analysis showed distortion of bacteria cells after phage inoculation, which proved the lytic activity against E. faecalis. A 54.6% reduction in the E. faecalis biofilm confirmed bacteriophage efficacy against isolates in the ex vivo model. Conclusions: Results strongly support the concept that phage therapy has a real therapeutic potential for the prevention and treatment of E. faecalis-associated infections.

Modification of outer membrane proteins (OMPs) is the first line of Gram-negative bacteria defence against antimicrobials. Here we point to Proteus mirabilis OMPs and their role in antibiotic and phage resistance. Protein profiles of amikacin (AMKrsv), phage (Brsv) and amikacin/phage (AMK/Brsv) resistant variants of P. mirabilis were compared to that obtained for a wild strain. In resistant variants there were identified 14, 1, 5 overexpressed and 13, 5, 1 downregulated proteins for AMKrsv, Brsv and AMK/Brsv, respectively. Application of phages with amikacin led to reducing the number of up- and downregulated proteins compared to single antibiotic treatment. Proteins isolated in AMKrsv are involved in protein biosynthesis, transcription and signal transduction, which correspond to well-known mechanisms of bacteria resistance to aminoglycosides. In isolated OMPs several cytoplasmic proteins, important in antibiotic resistance, were identified, probably as a result of environmental stress, e.g. elongation factor Tu, asparaginyl-tRNA and aspartyl-tRNA synthetases. In Brsv there were identified: NusA and dynamin superfamily protein which could play a role in bacteriophage resistance. In the resistant variants proteins associated with resistance mechanisms occurring in biofilm, e.g. polyphosphate kinase, flagella basal body rod protein were detected. These results indicate proteins important in the development of P. mirabilis antibiofilm therapies.

The impact of planktonic and biofilm lifestyles of the clinical isolate Proteus mirabilis 9B-m on its lipopolysaccharide (O-polysaccharide, core region, and lipid A) was evaluated. Proteus mirabilis bacteria are able to form biofilm and lipopolysaccharide is one of the factors involved in the biofilm formation. Lipopolysaccharide was isolated from planktonic and biofilm cells of the investigated strain and analyzed by SDS–PAGE with silver staining, Western blotting and ELISA, as well as NMR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry techniques. Chemical and NMR spectroscopic analyses revealed that the structure of the O-polysaccharide of P. mirabilis 9B-m strain did not depend on the form of cell growth, but the full-length chains of the O-antigen were reduced when bacteria grew in biofilm. The study also revealed structural modifications of the core region in the lipopolysaccharide of biofilm-associated cells—peaks assigned to compounds absent in cells from the planktonic culture and not previously detected in any of the known Proteus core oligosaccharides. No differences in the lipid A structure were observed. In summary, our study demonstrated for the first time that changes in the lifestyle of P. mirabilis bacteria leads to the modifications of their important virulence factor—lipopolysaccharide.

Urinary tract infections are a common disease in humans. Therefore, new methods are needed to destroy biofilms that are formed by uropathogens. Iturin A li

The aim of this study was to investigate the antimicrobial effect of lipopeptide biosurfactants from surfactin, iturin and fengycin families, synthesised by the Bacillus subtilis I'1a strain, on uropathogenic bacteria, including the effects on planktonic growth, processes of biofilm formation and dislodging. Antimicrobial activity was tested against 32 uropathogenic strains belonging to 12 different species of Gram-negative and Gram-positive bacteria. The sensitivity of 25 tested bacterial strains to the B. subtilis I'1a filtrate was confirmed by an agar diffusion assay. None of the strains seemed to be sensitive to pure surfactin at concentrations ranging from 0.1 mg × ml(-1) to 0.4 mg ml(-1). After the treatment of uropathogens with B. subtilis lipopeptides, the metabolic activity of planktonic cells was inhibited by 88.05±3.96% in the case of 21 studied uropathogens, the process of biofilm formation was reduced by 88.15±4.77% in the case of 24 uropathogens and mature biofilms of 18 strains were dislodged by about 81.20±4.72%. Ten strains of uropathogenic bacteria were selected to study the antimicrobial activity of surfactin (concentrations 0.1, 0.2 and 0.4 mg × ml(-1)). Surfactin had no influence on the metabolic activity of planktonic forms of uropathogens, however, biofilms of 5 tested strains were reduced by 64.77±9.05% in the presence of this biosurfactant at the concentration 0.1 mg × ml(-1). The negative effect of the compound on the biofilm formation process was observed at all concentrations used. The above-described results were fully confirmed by CLSM. It could suggest that synergistic application of biosurfactants could be efficient in uropathogen eradication.