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.

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 β-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.

Wastewater treatment plants (WWTPs) are an important reservoir in the development of drug resistance phenomenon and they provide a potential route of antibiotic resistance gene (ARGs) dissemination in the environment. The aim of this study was to assess the role of WWTPs in the spread of ARGs. Untreated and treated wastewater samples that were collected from thirteen Polish WWTPs (applying four different modifications of activated sludge–based treatment technology) were analyzed. The quantitative occurrence of genes responsible for the resistance to beta-lactams and tetracyclines was determined using the real-time PCR method. Such genes in the DNA of both the total bacterial population and of the E. coli population were analyzed. Among the tested genes that are responsible for the resistance to beta-lactams and tetracyclines, bla_OXA and bla_TEM and tetA were dominant, respectively. This study found an insufficient reduction in the quantity of the genes that are responsible for antibiotic resistance in wastewater treatment processes. The results emphasize the need to monitor the presence of genes determining antibiotic resistance in the wastewater that is discharged from treatment plants, as they can help to identify the hazard that treated wastewater poses to public health.

E3S Web of Conferences, open access proceedings in environment, energy and earth sciences

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.