Dependence of cell's membrane potential on extracellular voltage observed in Chara globularis.

The membrane potential (Vm) of a cell results from the selective movement of ions across the cell membrane. Recent studies have revealed the presence of a gradient of voltage within a few nanometers adjacent to erythrocytes. Very notably this voltage is modified in response to changes in cell's membrane potential thus effectively extending the potential beyond the membrane and into the solution. In this study, using the microelectrode technique, we provide experimental evidence for the existence of a gradient of negative extracellular voltage (Vz) in a wide zone close to the cell wall of algal cells, extending over several micrometers. Modulating the ionic concentration of the extracellular solution with CO2 alters the extracellular voltage and causes an immediate change in Vm. Elevated extracellular CO2 levels depolarize the cell and hyperpolarize the zone of extracellular voltage (ZEV) by the same magnitude. This observation strongly suggests a coupling effect between Vz and Vm. An increase in the level of intracellular CO2 (dark respiration) leads to hyperpolarization of the cell without any immediate effect on the extracellular voltage. Therefore, the metabolic activity of a cell can proceed without inducing changes in Vz. Conversely, Vz can be modified by external stimulation without metabolic input from the cell. The evolution of the ZEV, particularly around spines and wounded cells, where ion exchange is enhanced, suggests that the formation of the ZEV may be attributed to the exchange of ions across the cell wall and cell membrane. By comparing the changes in Vm in response to external stimuli, as measured by electrodes and observed using a potential-sensitive dye, we provide experimental evidence demonstrating the significance of extracellular voltage in determining the cell's membrane potential. This may have implications for our understanding of cell membrane potential generation beyond the activities of ion channels.

Voltage and concentration gradients across membraneless interface generated next to hydrogels: relation to glycocalyx.

Next to many hydrophilic surfaces, including those of biological cells and tissues, a layer of water that effectively excludes solutes and particles can be generated. This interfacial water is the subject of research aiming for practical applications such as removal of salts, pathogens or manipulation of biomolecules. However, the exact mechanism of its creation is still elusive because its persistence and extension contradict hydrogen-bond dynamics and electric double layer predictions. The experimentally recorded negative voltage of this interfacial water remains to be properly explained. Even less is known about the nature of such water layers in biological systems. We present experimental evidence for ion and particle exclusion as a result of separation of ionic charges with distinct diffusion rates across a liquid junction at the gel/water interface and the subsequent repulsion of ions of a given sign by a like-charged gel surface. Gels represent features of biological interfaces (in terms of functional groups and porosity) and are subject to biologically relevant chemical triggers. Our results show that gels with -OSO3- and -COO- groups can effectively generate ion- and particle-depleted regions of water reaching over 100 µm and having negative voltage up to -30 mV. Exclusion distance and electric potential depend on the liquid junction potential at the gel/water interface and on the concentration gradient at the depleted region/bulk interface, respectively. The voltage and extension of these ion- and particle-depleted water layers can be effectively modified by CO2 (respiratory gas) or KH2PO4 (cell metabolite). Possible implications pertain to biologically unstirred water layers and a cell's bioenergetics.

An improved selective/differential medium for culturing the Bacteroides fragilis group from wastewater.

The aim of this study was to develop an effective selective/differential medium for culturing environmental strains of the Bacteroides fragilis group (BFG). This goal was achieved by modifying standard commercial Bacteroides Bile Esculin Agar (BBE Agar). Bacteroides Bile Esculin Agar was combined with substances that inhibit the growth of non-BFG bacteria, mostly Klebsiella pneumoniae and Fusobacterium mortiferum. The strains isolated from standard and modified BBE Agar were identified as BFG strains by PCR and 16S rRNA gene sequencing. The supplementation of standard BBE Agar with colistin (40 mg L-1), kanamycin (400 mg L-1) and vancomycin (7.5 mg L-1) increases the effectiveness of BFG bacteria isolation from <10% to 35%, and additional Gram staining improves the effectiveness of bacterial isolation five-fold relative to standard BBE Agar. The results of the present study also suggest that the presence of the bfr gene is not a reliable indicator for the identification of BFG strains.

Markers Specific to Bacteroides fragilis Group Bacteria as Indicators of Anthropogenic Pollution of Surface Waters.

The aim of this study was to evaluate the applicability of markers specific to Bacteroides fragilis group (BFG) bacteria as indicators of anthropogenic pollution of surface waters. In addition, the impact of wastewater treatment plants (WWTPs) on the spread of genes specific to fecal indicator bacteria and genes encoding antimicrobial resistance in water bodies was also determined. Samples of hospital wastewater (HWW), untreated wastewater (UWW), and treated wastewater (TWW) evacuated from a WWTP were collected, and samples of river water were taken upstream (URW) and downstream (DRW) from the wastewater discharge point to determine, by qPCR, the presence of genes specific to BFG, Escherichia coli and Enterococcus faecalis, and the abundance of 11 antibiotic resistance genes (ARGs) and two integrase genes. The total number of bacterial cells (TCN) in the examined samples was determined by fluorescence in situ hybridization (FISH). Genes specific to BFG predominated among the analyzed indicator microorganisms in HWW, and their copy numbers were similar to those of genes specific to E. coli and E. faecalis in the remaining samples. The abundance of genes specific to BFG was highly correlated with the abundance of genes characteristic of E. coli and E. faecalis, all analyzed ARGs and intI genes. The results of this study indicate that genes specific to BFG can be used in analyses of human fecal pollution, and as indicators of environmental contamination with ARGs. A significant increase in the copy numbers of genes specific to BFG, E. coli, and seven out of the 11 analyzed ARGs was noted in samples of river water collected downstream from the wastewater discharge point, which suggests that WWTPs are an important source of these genes in riparian environments.

The impact of WWTP size and sampling season on the prevalence of antibiotic resistance genes in wastewater and the river system.

There is a growing concern about the fate of antibiotic resistance genes (ARGs) during wastewater treatment and their potential impacts on the receiving water bodies. We hypothesised that the quantity of ARGs in effluents may be related to the size of wastewater treatment plants (WWTPs) and sampling season. To date, only several attempts have been made to investigate the impact of the above factors at the catchment scale. Therefore, the goal of the present study was to explore possible differences in the quantity of ARGs in treated wastewater from small, medium-sized and large WWTPs in the catchment of the Pilica River (9258 km2). The impact of treated wastewater on the concentration of ARGs was also determined along the river continuum from upland to lowland segments to the point of confluence with the Vistula (342 km). Treated effluent was sampled in 17 WWTPs, and river water was sampled in 7 sampling sites in four seasons. The concentrations of blaTEM, tet(A), ermF, sul1 and aac(6')-Ib-cr genes, the integrase gene intI1 and the 16S rRNA gene were analysed by quantitative PCR. The physical and chemical parameters and nutrient concentrations (23 various parameters) in the analysed samples were determined. The highest absolute concentrations of the studied genes were noted in effluent samples from small WWTPs (p < 0.01). The concentration of ARGs (gene copies/mL) peaked in winter and spring samples (p < 0.04). The results of statistical analyses indicate that in small WWTPs, the absolute concentration of ARGs can be predicted based on the biochemical oxygen demand, in routine water analyses. However, none of the studied parameters supported predictions of ARG abundance in medium-sized and large WWTPs or in river water.

Environmental fate of Bacteroidetes, with particular emphasis on Bacteroides fragilis group bacteria and their specific antibiotic resistance genes, in activated sludge wastewater treatment plants.

The aim of this study was to determine the effect of the activated sludge process on the abundance of anaerobic bacteria of the phylum Bacteroidetes, with special emphasis on Bacteroides fragilis group (BFG) bacteria, in twelve full-scale wastewater treatment plants. The composition of bacterial phyla and classes in wastewater samples were analyzed by next-generation sequencing. The presence of specific to BFG bacteria genes and the abundance of ARGs and genes encoding class 1 integrase in wastewater samples were determined by qPCR. Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were dominant bacterial phyla in wastewater samples. Next-generation sequencing revealed similar proportions of Bacteroidia (<1.0-8.2 % of all bacteria) in wastewater influents and effluents, which suggest that these microorganisms are not completely eliminated in the activated sludge process. The average copy numbers of specific to BFG bacteria gene, were 106, and 104 copies in 1 mL of wastewater influents and effluents, respectively. The results revealed a correlation between the abundance of BFG bacteria and BFG-specific genes encoding resistance to antibiotics. The observed changes in the prevalence of BFG-specific genes and ARGs in untreated and treated wastewater indicate that the activated sludge process decreases the number of gene copies in the effluent evacuated to the environment.

Small-scale wastewater treatment plants as a source of the dissemination of antibiotic resistance genes in the aquatic environment.

Wastewater treatment plants (WWTPs) are significant source of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which can spread further in the environment by reaching rivers together with effluents discharged from WWTPs. In this study untreated and treated wastewater (UWW, TWW), upstream and downstream river water (URW, DRW) were collected from 4 WWTPs, in the winter and autumn seasons. The occurrence of ARB resistant to beta-lactams and tetracyclines as well as the presence of antibiotics from these classes were analysed in water and wastewater samples. Additionally, the amounts of 12 ARGs, 2 genes of mobile genetic elements (MGEs), gene uidA identifying E. coli and 16S rRNA were also determined. Resistance to beta-lactams prevailed among ARB in water and wastewater samples (constituting 82-88% of total counts of bacteria). The dominant genes in water and wastewater samples were blaTEM, tetA, sul1. The gene blaOXA demonstrated high variability of its concentration in samples collected in both seasons. Despite the high per cent reduction of ARB and ARGs concentration observed during the wastewater treatment processes, their large quantities are still transmitted into the environment. The research focuses on WWTPs' role in the dissemination of ARGs and MGEs in the aquatic environment.

The emergence of antimicrobial resistance in environmental strains of the Bacteroides fragilis group.

Anaerobic bacteria of the genus Bacteroides are a large group of commensal microorganisms that colonize the human and animal digestive tract. The genus Bacteroides and the closely related genus Parabacteroides include the Bacteroides fragilis group (BFG) of potentially pathogenic bacteria which are frequently isolated from patients with anaerobic infections. The aim of this study was to assess the antimicrobial resistance of environmental strains of the Bacteroides fragilis group. Strains were isolated from human feces, hospital wastewater, influent (UWW) and effluent (TWW) wastewater from a wastewater treatment plant (WWTP), and from the feces of lab rats as a negative control to monitor the entire route of transmission of BFG strains from humans to the environment. The resistance of 123 environmental BFG strains to six antibiotic groups was analyzed with the use of culture-dependent methods. Additionally, the presence of 25 genes encoding antibiotic resistance was determined by PCR. The analyzed environmental BFG strains were highly resistant to the tested antibiotics. The percentage of resistant strains differed between the analyzed antibiotics and was determined at 97.56% for ciprofloxacin, 49.59% for erythromycin, 44.71% for ampicillin, 35.77% for tetracycline, 32.52% for amoxicillin/clavulanic acid, 26.83% for chloramphenicol, 26.01% for clindamycin, 11.38% for moxifloxacin, and 8.94% for metronidazole. The highest drug-resistance levels were observed in the strains isolated from UWW and TWW samples. The mechanisms of antibiotic-resistance were determined in phenotypically resistant strains of BFG. Research has demonstrated the widespread presence of genes encoding resistance to chloramphenicol (100% of all chloramphenicol-resistant strains), tetracyclines (97.78% of all tetracycline-resistant strains), macrolides, lincosamides and streptogramins (81.97% of all erythromycin-resistant strains). Genes encoding resistance to ß-lactams and fluoroquinolones were less prevalent. None of the metronidazole-resistant strains harbored the gene encoding resistance to nitroimidazoles. BFG strains isolated from UWW and TWW samples were characterized by the highest diversity of antibiotic-resistance genes and were most often drug-resistant and multidrug-resistant. The present study examines the potential negative consequences of drug-resistant and multidrug-resistant BFG strains that are evacuated with treated wastewater into the environment. The transmission of these bacteria to surface water bodies can pose potential health threats for humans and animals; therefore, the quality of treated wastewater should be strictly monitored.

The prevalence and characterization of antibiotic-resistant and virulent Escherichia coli strains in the municipal wastewater system and their environmental fate.

Antibiotics are widely used in human and veterinary medicine and in animal production, which increases their concentrations in aquatic ecosystems and contributes to selective pressure on environmental microorganisms. The objective of this study was to identify antibiotic resistance determinants in Escherichia coli strains isolated from untreated and treated wastewater (UWW and TWW) and from river water sampled downstream and upstream (URW and DRW) from the effluent discharge point. The analyzed antibiotic groups were beta-lactams, tetracyclines and fluoroquinolones which are widely used in human and veterinary medicine. The virulence of the isolated E. coli strains was also analyzed, and their clonal relatedness was determined by ERIC (enterobacterial repetitive intergenic consensus sequence) PCR. The highest counts of bacteria resistant to beta-lactams, tetracyclines and fluoroquinolones were noted in UWW at 6.4×104, 4.2×104 and 3.1×103CFU/mL, respectively. A total of 317 E. coli isolates resistant to at least one group of antibiotics were selected among bacterial isolates from river water and wastewater samples. Nearly 38% of those isolates were resistant to all of the tested antibiotics. The highest percent (43%) of multidrug-resistant isolates was noted in UWW samples. Isolates resistant to beta-lactams most frequently harbored blaTEM and blaOXA genes. The group of genes encoding resistance to tetracyclines was most frequently represented by tetA, tetB and tetK, whereas the qnrS gene was noted in isolates resistant to fluoroquinolones. Virulence genes bfpA (65%), ST (56%) and eae (39%) were most widely distributed in all isolates, regardless of their origin. The results of this experiment reveal the dangers associated with environmental contamination by drug-resistant and virulent E. coli strains distributed with treated wastewater. Multidrug resistance was determined more frequently in strains isolated from DRW than in isolates from URW samples. Our findings provide valuable inputs for evaluating public health hazards associated with bacterial contamination.