The influence of combined treatment of municipal wastewater and landfill leachate on the spread of antibiotic resistance in the environment - A preliminary case study.

Environmentally-friendly management of landfill leachate (LL) poses a challenge, and LL is usually co-treated with municipal wastewater in wastewater treatment plants (WWTPs). The extent to which the co-treatment of LL and municipal wastewater influences the spread of antibiotic resistance (AR) in the environment has not been examined to date. Two WWTPs with similar wastewater composition and technology were studied. Landfill leachate was co-treated with wastewater in one of the studied WWTPs. Landfill leachate, untreated and treated wastewater from both WWTPs, and river water sampled upstream and downstream from the wastewater discharge point were analyzed. Physicochemical parameters, microbial diversity, and antibiotic resistance genes (ARGs) abundance were investigated to determine the impact of LL co-treatment on chemical and microbiological contamination in the environment. Landfill leachate increased pollutant concentrations in untreated wastewater and river water. Cotreatment of LL and wastewater could affect the abundance and diversity of microbial communities and the interactions between microbial species. Co-treatment also decreased the stability of microbial co-occurrence networks in the examined samples. The mexF gene was identified as a potential marker of environmental pollution with LL. This is the first study to explore the impact of LL on the occurrence of AR determinants in wastewater and rivers receiving effluents.

Poultry manure-derived microorganisms as a reservoir and source of antibiotic resistance genes transferred to soil autochthonous microorganisms.

Animal husbandry is increasing yearly due to the growing demand for meat and livestock products, among other reasons. To meet these demands, prophylactic antibiotics are used in the livestock industry (i.e., poultry farming) to promote health and stimulate animal growth. However, antibiotics are not fully metabolized by animals, and they are evacuated to the environment with excreta. Animal manure is used as fertilizer to reduce the volume of waste generated in the livestock sector. However, manure often contains microorganisms harboring antibiotic resistance genes (ARGs). Then, the microbiome of manure applicate to the soil may contribute to the spread of antibiotic resistance in the environment, including autochthonous soil-dwelling microorganisms. The present study was conducted during the crops growing season in Poland (May to September 2019) to determine the influence of poultry manure as well as poultry manure supplemented with selected antibiotics on the diversity of the soil microbiome in treatments that had not been previously fertilized with manure and the ability of antibiotic-resistant bacteria to transfer ARGs to other soil bacteria. Antibiotic concentrations were elevated at the beginning of the study and decreased over time. Poultry manure induced significant changes in the structure of microbial communities in soil; the diversity of the soil microbiome decreased, and the abundance of bacterial genera Bradyrhizobium, Streptomyces, and Pseudomonas, which are characteristic of the analyzed manure, increased. Over time, soil microbial diversity was restored to the state observed before the application of manure. Genes conferring resistance to multiple drugs as well as genes encoding resistance to bacitracin and aminoglycosides were the most frequently identified ARGs in the analyzed bacteria, including on mobile genetic elements. Multidrug resistance was observed in 17 bacterial taxa, whereas ARGs were identified in 32 bacterial taxa identified in the soil microbiome. The results of the study conclude that the application of poultry manure supplemented with antibiotics initially affects soil microbiome and resistome diversity but finally, the soil shows resilience and returns to its original state after time, with most antibiotic resistance genes disappearing. This phenomenon is of great importance in sustainable soil health after manure application.

Uptake of Pharmaceutical Pollutants and Their Metabolites from Soil Fertilized with Manure to Parsley Tissues.

Manure is a major source of soil and plant contamination with veterinary drugs residues. The aim of this study was to evaluate the uptake of 14 veterinary pharmaceuticals by parsley from soil fertilized with manure. Pharmaceutical content was determined in roots and leaves. Liquid chromatography coupled with tandem mass spectrometry was used for targeted analysis. Screening analysis was performed to identify transformation products in the parsley tissues. A solid-liquid extraction procedure was developed combined with solid-phase extraction, providing recoveries of 61.9-97.1% for leaves and 51.7-95.6% for roots. Four analytes were detected in parsley: enrofloxacin, tylosin, sulfamethoxazole, and doxycycline. Enrofloxacin was detected at the highest concentrations (13.4-26.3 ng g-1). Doxycycline accumulated mainly in the roots, tylosin in the leaves, and sulfamethoxazole was found in both tissues. 14 transformation products were identified and their distribution were determined. This study provides important data on the uptake and transformation of pharmaceuticals in plant tissues.

Sewage sludge in agriculture - the effects of selected chemical pollutants and emerging genetic resistance determinants on the quality of soil and crops - a review.

In line with sustainable development principles and in order to combat climate change, which contributes to progressive soil depletion, various solutions are being sought to use treated sewage sludge as a soil amendment to improve soil quality and enrich arable soils with adequate amounts of biogenic compounds. This review article focuses on the effects of the agricultural use of biosolids on the environment. The article reviews the existing knowledge on selected emerging contaminants in treated sewage sludge and describes the impact of these pollutants on the environment and living organisms based on 183 publications selected from over 16,000 papers on related topics published over the last ten years. This study deals not only with chemical contaminants but also genetic determinants of resistance to these compounds. Current research has questioned the agricultural use of biosolids due to the presence of mutual interactions between antibiotics, heavy metals, the genetic determinants of resistance (antibiotic resistance genes - ARGs and heavy metal resistance genes - HMRGs) and non-steroidal anti-inflammatory drugs as well as the risks associated with their transfer to the environment. This study emphasizes the need for more extensive legal regulations that account for other pollutants of environmental concern (PEC), particularly in countries where sewage sludge is applied in agriculture most extensively. Future research should focus on more effective methods of eliminating PEC from sewage sludge, especially from the sludge that is used to fertilize agricultural land, because even small amounts of these micropollutants can have serious implications for the health and life of humans and animals.

Environmental Risk and Risk of Resistance Selection Due to Antimicrobials' Occurrence in Two Polish Wastewater Treatment Plants and Receiving Surface Water.

In this study, a screening of 26 selected antimicrobials using liquid chromatography coupled to a tandem mass spectrometry method in two Polish wastewater treatment plants and their receiving surface waters was provided. The highest average concentrations of metronidazole (7400 ng/L), ciprofloxacin (4300 ng/L), vancomycin (3200 ng/L), and sulfamethoxazole (3000 ng/L) were observed in influent of WWTP2. Ciprofloxacin and sulfamethoxazole were the most dominant antimicrobials in influent and effluent of both WWTPs. In the sludge samples the highest mean concentrations were found for ciprofloxacin (up to 28 µg/g) and norfloxacin (up to 5.3 µg/g). The removal efficiency of tested antimicrobials was found to be more than 50% for both WWTPs. However, the presence of antimicrobials influenced their concentrations in the receiving waters. The highest antimicrobial resistance risk was estimated in influent of WWTPs for azithromycin, ciprofloxacin, clarithromycin, metronidazole, and trimethoprim and in the sludge samples for the following antimicrobials: azithromycin, ciprofloxacin, clarithromycin, norfloxacin, trimethoprim, ofloxacin, and tetracycline. The high environmental risk for exposure to azithromycin, clarithromycin, and sulfamethoxazole to both cyanobacteria and eukaryotic species in effluents and/or receiving water was noted. Following the obtained results, we suggest extending the watch list of the Water Framework Directive for Union-wide monitoring with sulfamethoxazole.

Impact of Peat Mining and Restoration on Methane Turnover Potential and Methane-Cycling Microorganisms in a Northern Bog.

Ombrotrophic peatlands are a recognized global carbon reservoir. Without restoration and peat regrowth, harvested peatlands are dramatically altered, impairing their carbon sink function, with consequences for methane turnover. Previous studies determined the impact of commercial mining on the physicochemical properties of peat and the effects on methane turnover. However, the response of the underlying microbial communities catalyzing methane production and oxidation have so far received little attention. We hypothesize that with the return of Sphagnum spp. postharvest, methane turnover potential and the corresponding microbial communities will converge in a natural and restored peatland. To address our hypothesis, we determined the potential methane production and oxidation rates in natural (as a reference), actively mined, abandoned, and restored peatlands over two consecutive years. In all sites, the methanogenic and methanotrophic population sizes were enumerated using quantitative PCR (qPCR) assays targeting the mcrA and pmoA genes, respectively. Shifts in the community composition were determined using Illumina MiSeq sequencing of the mcrA gene and a pmoA-based terminal restriction fragment length polymorphism (t-RFLP) analysis, complemented by cloning and sequence analysis of the mmoX gene. Peat mining adversely affected methane turnover potential, but the rates recovered in the restored site. The recovery in potential activity was reflected in the methanogenic and methanotrophic abundances. However, the microbial community composition was altered, being more pronounced for the methanotrophs. Overall, we observed a lag between the recovery of the methanogenic/methanotrophic activity and the return of the corresponding microbial communities, suggesting that a longer duration (>15 years) is needed to reverse mining-induced effects on the methane-cycling microbial communities.IMPORTANCE Ombrotrophic peatlands are a crucial carbon sink, but this environment is also a source of methane, an important greenhouse gas. Methane emission in peatlands is regulated by methane production and oxidation catalyzed by methanogens and methanotrophs, respectively. Methane-cycling microbial communities have been documented in natural peatlands. However, less is known of their response to peat mining and of the recovery of the community after restoration. Mining exerts an adverse impact on potential methane production and oxidation rates and on methanogenic and methanotrophic population abundances. Peat mining also induced a shift in the methane-cycling microbial community composition. Nevertheless, with the return of Sphagnum spp. in the restored site after 15 years, methanogenic and methanotrophic activity and population abundance recovered well. The recovery, however, was not fully reflected in the community composition, suggesting that >15 years are needed to reverse mining-induced effects.

The impact of urban areas on the water quality gradient along a lowland river.

The effects of five towns on river water pollution were examined along the Lyna River (southern watershed of the Baltic Sea, northern Poland). The relationships among the spatially derived indicators of urbanization, environmental variables, and physico-chemical and microbiological data (heterotrophic plate count at 22 and 37 °C, and fecal coli) obtained from longitudinal river profiling have been examined with the use of multivariate analyses such as principal component analysis with factor analysis (PCA/FA) and hierarchical cluster analysis (HCA). We recognized the river channel as an environmental path that links serial urban areas into an "urban river continuum." An overall increasing trend in nutrients and indicator bacteria from suburban headwaters to urbanized sections of the river was detected despite a significant decrease in those between the towns. We concluded that the role of a multicity is equally as important as a single urban area in predicting the impacts of man-made pollutants on river water quality.

Hospital and municipal wastewater as a source of carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa in the environment: a review.

The increase in the prevalence of carbapenem-resistant Gram-negative bacteria, in particular Acinetobacter baumannii (CRAB) and Pseudomonas aeruginosa (CRPA), poses a serious threat for public health worldwide. This article reviews the alarming data on the prevalence of infections caused by CRAB and CRPA pathogens and their presence in hospital and municipal wastewater, and it highlights the environmental impact of antibiotic resistance. The article describes the key role of antibiotic resistance genes (ARGs) in the acquisition of carbapenem resistance and sheds light on bacterial resistance mechanisms. The main emphasis was placed on the transfer of ARGs not only in the clinical setting, but also in the environment, including water, soil, and food. The aim of this review was to expand our understanding of the global health risks associated with CRAB and CRPA in hospital and municipal wastewater and to analyze the spread of these micropollutants in the environment. A review of the literature published in the last decade will direct research on carbapenem-resistant pathogens, support the implementation of effective preventive measures and interventions, and contribute to the development of improved strategies for managing this problem.

Genomic and metagenomic analysis reveals shared resistance genes and mobile genetic elements in E. coli and Klebsiella spp. isolated from hospital patients and hospital wastewater at intra- and inter-genus level.

Antimicrobial resistance (AMR) is a global problem that gives serious cause for concern. Hospital wastewater (HWW) is an important link between the clinical setting and the natural environment, and an escape route for pathogens that cause hospital infections, including urinary tract infections (UTI). Bacteria of the genera Escherichia and Klebsiella are common etiological factors of UTI, especially in children, and they can cause short-term infections, as well as chronic conditions. ESBL-producing Escherichia and Klebsiella have also emerged as potential indicators for estimating the burden of antimicrobial resistance under environmental conditions and the spread of AMR between clinical settings and the natural environment. In this study, whole-genome sequencing and the nanopore technology were used to analyze the complete genomes of ESBL-producing E.coli and Klebsiella spp. and the HWW metagenome, and to characterize the mechanisms of AMR. The similarities and differences in the encoded mechanisms of AMR in clinical isolates (causing UTI) and environmental strains (isolated from HWW and the HWW metagenome) were analyzed. Special attention was paid to the genetic context and the mobility of antibiotic resistance genes (ARGs) to determine the common sources and potential transmission of these genes. The results of this study suggest that the spread of drug resistance from healthcare facilities via HWW is not limited to the direct transmission of resistant clonal lines that are typically found in the clinical setting, but it also involves the indirect transfer of mobile elements carrying ARGs between bacteria colonizing various environments. Hospital wastewater could offer a supportive environment for plasmid evolution through the insertion of new ARGs, including typical chromosomal regions. These results indicate that interlined environments (hospital patients - HWW) should be closely monitored to evaluate the potential transmission routes of drug resistance in bacteria.

Antibiotic resistant Escherichia coli in hospital and municipal sewage and their emission to the environment.

The spreading of antibiotic resistant bacteria in the environment is a threat to human health but little is known about the transmission of extended-spectrum beta-lactamases (ESBL)-producing Escherichia coli from the hospital and municipal sewage to the water basin and to the air at the WWTPs (Waste Water Treatment Plants) area and their surroundings. Accordingly, it seems particularly interesting to trace the fate of these bacteria and their genes encoding antibiotic resistance in both untreated sewage from hospitals, and in sewage after different stages of purification, and finally to examine the degree of their emissions to environment. Although wastewater treatment processes reduce number of bacteria in sewage up to 99%, in the presented study it was reported that more than 2.7×10(3) CFU/mL E. coli reached the receiving water and contributed to dissemination of resistant bacteria into the environment. We received 395 E. coli strains from sewage and environmental samples and we investigated their antibiotic susceptibility and the presence of bla gene encoding TEM, CTX, OXA and SHV. From among 167 and 147 E. coli strains isolated from hospital effluents and municipal sewage in Olsztyn, Poland, up to 37.1% and 17.7%, respectively, were ESBL-positive. From among 38 and 43 strains isolated from river water and the air up to 18.4% and 27.9%, respectively, were ESBL-producers. The blaCTX-M (blaCTX-M-1, blaCTX-M-3, blaCTX-M-5, blaCTX-M-15) genes were the predominant group of the plasmid-coded ESBLs. More than 38% out of ESBL-producing isolates carried several bla genes. The multiple-antibiotic-resistant (MAR) indexes for ESBL-positive were higher than for ESBL-negative isolates and ranged from 0.45 to 0.63. The MAR indexes for E. coli from hospital effluents and air samples were greater than the indexes calculated for strains isolated from other samples. Presumably, the preliminary disinfection of hospital sewage before its inflow into the sewage system might minimize the spreading of antibiotic-resistant bacteria to the environment.

Extended-spectrum beta-lactamase (ESBL)-positive Enterobacteriaceae in municipal sewage and their emission to the environment.

The spread of Gram-negative bacteria with plasmid-borne extended-spectrum beta-lactamases (ESBLs) has become a worldwide problem. Their prevalence is increasing, both in hospitals and in the environment. The aim of this study was to investigate the presence of ESBL-positive Enterobacteriaceae in municipal sewage and their emission to the ambient air and the river receiving effluent from wastewater treatment plant (WWTP). In the group of 455 isolated strains, up to 19.8% (90 isolates) were phenotypic ESBL-producers. They were detected in the 63 (100%) of sewage samples analyzed, 7 (33.3%) of river water and in 10 (23.8%) of air samples collected at the WWTP area. The plasmid-mediated genes encoding beta-lactams resistance were detected in almost 10% out of bacteria of the WWTP's final effluents and in above 32% out of bacteria of air at the WWTP area. It confirms that those genes are released into the environment, which might facilitate further dissemination among environmental bacteria. Moreover, genes encoding antibiotic resistance were shown to be transferrable to an Escherichia coli recipient strain, which indicates a high possibility of horizontal gene transfer among strains of different genera within the sewage and environmental samples. This study demonstrated that despite the treatment, the municipal sewage may be a reservoir of antibiotic-resistant microorganisms and plasmid-mediated antibiotic resistance genes. This may pose a public health risk, which requires future evaluation and control.

Beta-lactamase-producing Enterobacteriaceae in hospital effluents.

Beta-lactams are widely used to treat bacterial infections in humans. In most countries, they are the largest group of antibiotics used by hospitals to treat infections caused by Gram-negative bacteria. Enterobacteriaceae, natural microbiota of the human gastrointestinal tract, represent a large part of bacterial communities colonizing hospital effluents, and they could be a source of genes encoding beta-lactamases and extended-spectrum-beta-lactamases (ESBLs). Those genes may be transmitted to other bacteria present in sewage and the environment. The prevalence of ESBL-producing Enterobacteriaceae was investigated in 63 sewage samples from three hospitals in Olsztyn, Poland. In the group of 310 randomly sampled strains isolated from hospital effluents, 295 (95.2%), 253 (81.6%) and 228 (73.5%) isolates were resistant to cefotaxime, ceftazidime and cefpodoxime, respectively. 150 of them were phenotypically ESBL-positive, but only 91 (29.4%) of those isolates harbored ESBL-genes. In the group of ESBL-producers, 54.9% (50/91), 39.6% (36/91), 24.2% (22/91) and 11.0% (10/91) carried blaCTx-M group 1, blaCTx-M group 9, blaSHV and blaTEM genes, respectively. More than 27.5% (25/91) of the analyzed isolates carried up to three bla genes. High minimum inhibitory concentration (MIC) values of cefotaxime and ceftazidime (≥512 µg/mL) were observed for the studied microorganisms. Escherichia coli and Citrobacter freundii were the most frequently identified ESBL-positive strains. A statistically significant correlation was observed between antibiotics consumption in each hospital and the incidence of ESBL-positive isolates in hospital effluents.

Chlorine disinfection modifies the microbiome, resistome and mobilome of hospital wastewater - A nanopore long-read metagenomic approach.

The aim of the present study was to analyze changes in the microbiome, resistome, and mobilome of hospital wastewater (HWW) induced by disinfection with chlorine compounds. Changes in bacterial communities and specific antibiotic resistance genes (ARGs) in HWW were determined with the use of a nanopore long-read metagenomic approach. The main hosts of ARGs in HWW were identified, and the mobility of resistance mechanisms was analyzed. Special attention was paid to the prevalence of critical-priority pathogens in the HWW microbiome, which pose the greatest threat to human health. The results of this study indicate that chlorine disinfection of HWW can induce significant changes in the structure of the total bacterial population and antibiotic resistant bacteria (ARB) communities, and that it can modify the resistome and mobilome of HWW. Disinfection favored the selection of ARGs, decreased their prevalence in HWW, while increasing their diversity. The mobility of the HWW resistome increased after disinfection. Disinfection led to the emergence of new drug resistance mechanisms in previously sensitive bacterial taxa. In conclusion, this study demonstrated that HWW disinfected with low (sublethal) concentrations of free chlorine significantly contributes to the mobility and transfer of drug resistance mechanisms (including critical mechanisms) between bacteria (including pathogens).

Digestate from Agricultural Biogas Plants as a Reservoir of Antimicrobials and Antibiotic Resistance Genes-Implications for the Environment.

Antimicrobials and antibiotic resistance genes (ARGs) in substrates processed during anaerobic digestion in agricultural biogas plants (BPs) can reach the digestate (D), which is used as fertilizer. Antimicrobials and ARGs can be transferred to agricultural land, which increases their concentrations in the environment. The concentrations of 13 antibiotics in digestate samples from biogas plants (BPs) were investigated in this study. The abundance of ARGs encoding resistance to beta-lactams, tetracyclines, sulfonamides, fluoroquinolones, macrolide-lincosamide-streptogramin antibiotics, and the integrase genes were determined in the analyzed samples. The presence of cadmium, lead, nickel, chromium, zinc, and mercury was also examined. Antimicrobials were not eliminated during anaerobic digestion. Their concentrations differed in digestates obtained from different substrates and in liquid and solid fractions (ranging from 62.8 ng/g clarithromycin in the solid fraction of sewage sludge digestate to 1555.9 ng/L doxycycline in the liquid fraction of cattle manure digestate). Digestates obtained from plant-based substrates were characterized by high concentrations of ARGs (ranging from 5.73 × 102 copies/gDcfxA to 2.98 × 109 copies/gDsul1). The samples also contained mercury (0.5 mg/kg dry mass (dm)) and zinc (830 mg/kg dm). The results confirmed that digestate is a reservoir of ARGs (5.73 × 102 to 8.89 × 1010 copies/gD) and heavy metals (HMs). In addition, high concentrations of integrase genes (105 to 107 copies/gD) in the samples indicate that mobile genetic elements may be involved in the spread of antibiotic resistance. The study suggested that the risk of soil contamination with antibiotics, HMs, and ARGs is high in farms where digestate is used as fertilizer.

The challenges in the identification of Escherichia coli from environmental samples and their genetic characterization.

Escherichia coli bacteria are an essential indicator in evaluations of environmental pollution, which is why they must be correctly identified. This study aimed to determine the applicability of various methods for identifying E. coli strains in environmental samples. Bacterial strains preliminary selected on mFc and Chromocult media as E. coli were identified using MALDI Biotyper techniques, based on the presence of genes characteristic of E. coli (uidA, uspA, yaiO), as well as by 16S rRNA gene sequencing. The virulence and antibiotic resistance genes pattern of bacterial strains were also analyzed to investigate the prevalence of factors that may indicate adaptation to unsupportive environmental conditions and could have any significance in further identification of E. coli. Of the strains that had been initially identified as E. coli with culture-based methods, 36-81% were classified as E. coli with the use of selected techniques. The value of Cohen's kappa revealed the highest degree of agreement between the results of 16S rRNA gene sequencing, the results obtained in the MALDI Biotyper system, and the results of the analysis based on the presence of the yaiO gene. The results of this study could help in the selection of more accurate and reliable methods which can be used in a preliminary screening and more precise identification of E. coli isolated from environmental samples.

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.

The Impact of Chlorine Disinfection of Hospital Wastewater on Clonal Similarity and ESBL-Production in Selected Bacteria of the Family Enterobacteriaceae.

Hospitals are regarded as ecological niches of antibiotic-resistant bacteria (ARB). ARB can spread outside the hospital environment via hospital wastewater (HWW). Therefore, HWW is often disinfected in local stations to minimize that risk. Chlorine-based treatment is the most popular method of HWW disinfection around the world, however, recent research has suggested that it can contribute to the spread of antimicrobial resistance (AMR). The aim of this study is to determine the impact of HWW disinfection on the clonal similarity of Enterobacteriaceae species and their ability to produce extended-spectrum beta-lactamases (ESBLs). The study was conducted in a hospital with a local chlorine-based disinfection station. Samples of wastewater before disinfection and samples of disinfected wastewater, collected in four research seasons, were analyzed. Bacteria potentially belonging to the Enterobacteriaceae family were isolated from HWW. The Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) method was used to generate DNA fingerprints of all bacterial isolates. The isolates were phenotypically tested for the production of ESBLs. Antibiotic resistance genes (blaSHV, blaTEM, and blaOXA, blaCTX-M-1-group, blaCTX-M-2-group, blaCTX-9-group and blaCTX-M-8/25-group) were detected by PCR in strains with confirmed phenotypic ability to produce ESBLs. The ESBL+ isolates were identified by the sequencing of 16S rDNA. In the present study, the same bacterial clones were isolated from HWW before and after disinfection and HWW was sampled in different seasons. Genetic and phenotypic variations were observed in bacterial clones. ESBL+ strains were isolated significantly more often from disinfected than from non-disinfected HWW. The blaOXA gene was significantly more prevalent in isolates from disinfected than non-disinfected HWW. Enterobacter hormaechei and Klebsiella pneumoniae were the dominant species in ESBL+ strains isolated from both sampling sites. The results of this study indicate that chlorine-based disinfection promotes the survival of ESBL-producing bacteria and/or the transmission of genetic determinants of antimicrobial resistance. As a result, chlorination increases the proportion of ESBL-producing Enterobacteriaceae in disinfected wastewater. Consequently, chlorine-based disinfection practices may pose a risk to the environment and public health by accelerating the spread of antimicrobial resistance.

Impact of Anthropogenic Activities on the Dissemination of ARGs in the Environment-A Review.

Over the past few decades, due to the excessive consumption of drugs in human and veterinary medicine, the antimicrobial resistance (AR) of microorganisms has risen considerably across the world, and this trend is predicted to intensify. Many worrying research results indicate the occurrence of pools of AR, both directly related to human activity and environmental factors. The increase of AR in the natural environment is mainly associated with the anthropogenic activity. The dissemination of AR is significantly stimulated by the operation of municipal facilities, such as wastewater treatment plants (WWTPs) or landfills, as well as biogas plants, agriculture and farming practices, including animal production and land application of manure. These activities entail a risk to public health by spreading bacteria resistant to antimicrobial products (ARB) and antibiotic resistance genes (ARGs). Furthermore, subinhibitory concentrations of antimicrobial substances additionally predispose microbial consortia and resistomes to changes in particular environments that are permeated by these micropollutants. The current state of knowledge on the fate of ARGs, their dissemination and the complexity of the AR phenomenon in relation to anthropogenic activity is inadequate. This review summarizes the state-of-the-art knowledge on AR in the environment, in particular focusing on AR spread in an anthropogenically altered environment and related environmental consequences.

The Resistome of ESKAPEE Pathogens in Untreated and Treated Wastewater: A Polish Case Study.

The aim of this study was to quantify ESKAPEE bacteria, genes encoding resistance to antibiotics targeting this group of pathogens, as well as integrase genes in municipal wastewater and river water. Environmental DNA was extracted from the collected samples and used in deep sequencing with the Illumina TruSeq kit. The abundance of bacterial genera and species belonging to the ESKAPEE group, 400 ARGs associated with this microbial group, and three classes of integrase genes were determined. A taxonomic analysis revealed that Acinetobacter was the dominant bacterial genus, whereas Acinetobacter baumannii and Escherichia coli were the dominant bacterial species. The analyzed samples were characterized by the highest concentrations of the following ARGs: blaGES, blaOXA-58, blaTEM, qnrB, and qnrS. Acinetobacter baumannii, E. coli, and genes encoding resistance to ß-lactams (blaVEB-1, blaIMP-1, blaGES, blaOXA-58, blaCTX-M, and blaTEM) and fluoroquinolones (qnrS) were detected in samples of river water collected downstream from the wastewater discharge point. The correlation analysis revealed a strong relationship between A. baumannii (bacterial species regarded as an emerging human pathogen) and genes encoding resistance to all tested groups of antimicrobials. The transmission of the studied bacteria (in particular A. baumannii) and ARGs to the aquatic environment poses a public health risk.

Solar-light driven photodegradation of antimicrobials, their transformation by-products and antibiotic resistance determinants in treated wastewater.

This study aimed to assess the possibility of using solar light-driven photolysis and TiO2-based photocatalysis to remove (1) antibiotic residues, (2) their transformation products (TPs), (3) antibiotic resistance determinants, and (4) genes identifying the indicator bacteria in a treated wastewater (secondary effluent). 16 antimicrobials belonging to the different classes and 45 their transformation by-products were selected for the study. The most susceptible to photochemical decomposition was tetracycline, which was completely removed in the photocatalysis process and in more than 80% in the solar light-driven photolysis. 83.8% removal (on average) was observed using photolysis and 89.9% using photocatalysis in the case of the tested genes, among which the genes sul1, uidA, and intI1 showed the highest degree of removal by both methods. The study revealed that applied methods promisingly remove the tested antibiotics, their TPs and genes even in such a complex matrix including treated wastewater and photocatalysis process had a higher removal efficiency of antibiotics, TPs and genes tested. Moreover, the high percentage removal of the intI1 gene (>93%) indicates the possibilities of use of the solar light-driven photolysis and TiO2-based photocatalysis in minimizing the antibiotic resistance genes transfer by mobile genetic elements.