Protecting the Last Line of Defense: Analytical Approaches for Sample Preparation and Determination of the Reserve Group of Antibiotics in the Environment.

Drug resistance in microorganisms is a serious threat to life and health due to the limited number of antibiotics that show efficacy in treating infections and the difficulty in discovering new compounds with antibacterial activity. To address this issue, the World Health Organization created the AWaRe classification, a tool to support global and national antimicrobial stewardship programs. The AWaRe list categorizes antimicrobials into three groups - Access, Watch, and Reserve - according to their intended use. The Reserve group comprises "last resort" medicines used solely for treating infections caused by bacterial strains that are resistant to other treatments. It is therefore necessary to protect them, not only by using them as prudently as possible in humans and animals, but also by monitoring their subsequent fate. Unmetabolized antibiotics enter the environment through hospital and municipal wastewater or from manure, subsequently contaminating bodies of water and soils, thus contributing to the emergence and spread of antibiotic resistance. This article presents a review of determination methods for the Reserve group of antimicrobials in water, wastewater, and manure. Procedures for extracting and determining these substances in environmental samples are described, showing the limited research available, which is typically on a local level.

Assessment of legacy and emerging PFAS in the Oder River: Occurrence, distribution, and sources.

The purpose of the study was to evaluate the occurrence and distribution of emerging contaminants, poly- and perfluoroalkyl substances (PFAS), in the Polish Oder River, aiming to uncover new insights into their environmental impact. The research aimed to identify potential sources of PFAS, assess water quality levels, and verify compliance with European Union environmental quality standards. The concentrations of 25 PFAS (20 legacy and 5 emerging) in 20 samples from intakes upstream and downstream of urban areas were analyzed using novel, developed in these studies, environmental analytical procedures involving solid phase extraction and liquid chromatography-tandem mass spectrometry. The presence of 14 PFAS was confirmed, and the concentration of Σ14PFAS ranged from 7.6 to 68.0 ng/L. The main components were short-chain analogs. PFBA was the most abundant, accounting for about one-third of all PFAS detected. An exception was observed in the waters of the Gliwice Canal, where ADONA represented half of the detected Σ14PFAS. Alternative PFOS replacements were found in all samples. In 11 of 20 water samples, environmental quality standards for PFOS exceeded the limit of 0.65 ng/L. In 5 of 9 cases, the ability of urban areas to increase PFAS levels in the river was determined. 9.5%-54.4% share of alternative PFAS in relation to the sum of the targeted PFAS showing their increasing use as substitutes for phased-out PFOS. Hierarchical cluster analysis was used to identify potential sources of PFAS. Analysis revealed that PFAS in the Oder River most likely originated from domestic and agricultural wastewater, as well as chemical industry discharges. However, the occurrence of PFAS in the Oder River is low and comparable to other recent European studies. These findings provide valuable insights for environmental management to mitigate the risks associated with PFAS pollution in Polish rivers. Moreover, the developed analytical procedure provides a valuable tool that can be successfully applied by other researchers to monitor PFAS in rivers around the world.

Removal of selected sulfonamides and sulfonamide resistance genes from wastewater in full-scale constructed wetlands.

Sulfonamides are high-consumption antibiotics that reach the aquatic environment. The threat related to their presence in wastewater and the environment is not only associated with their antibacterial properties, but also with risk of the spread of drug resistance in bacteria. Therefore, the aim of this work was to evaluate the occurrence of eight commonly used sulfonamides, sulfonamide resistance genes (sul1-3) and integrase genes intI1-3 in five full-scale constructed wetlands (CWs) differing in design (including hybrid systems) and in the source of wastewater (agricultural drainage, domestic sewage/surface runoff, and animal runs runoff in a zoo). The CWs were located in low-urbanized areas in Poland and in Czechia. No sulfonamides were detected in the CW treating agricultural tile drainage water. In the other four systems, four sulfonamide compounds were detected. Sulfamethoxazole exhibited the highest concentration in those four CWs and its highest was 12,603.23 ± 1000.66 ng/L in a CW treating a mixture of domestic sewage and surface runoff. Despite the high removal efficiencies of sulfamethoxazole in the tested CWs (86 %-99 %), it was still detected in the treated wastewater. The sul1 genes occurred in all samples of raw and treated wastewater and their abundance did not change significantly after the treatment process and it was, predominantly, at the level 105 gene copies numbers/mL. Noteworthy, sul2 genes were only found in the influents, and sul3 were not detected. The sulfonamides can be removed in CWs, but their elimination is not complete. However, hybrid CWs treating sewage were superior in decreasing the relative abundance of genes and the concentration of SMX. CWs may play a role in the dissemination of sulfonamide resistance genes of the sul1 type and other determinants of drug resistance, such as the intI1 gene, in the environment, however, the magnitude of this phenomenon is a matter of further research.

Efficiency of phytoremediation and identification of biotransformation pathways of fluoroquinolones in the aquatic environment.

Phytoremediation is a low-cost and sustainable green technology that uses plants to remove organic and inorganic pollutants from aquatic environments. The aim of this study was to investigate the phytoextraction, phytoaccumulation, and phytotransformation of three fluoroquinolones (FQs) (ciprofloxacin [CIP], enrofloxacin [ENF], and levofloxacin [LVF]) by Japanese radish (Raphanus sativus var. longipinnatus) and duckweed (Lemma minor). Determination of FQs and identification of their transformation products (TPs) were performed using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Inter-tissue translocation of FQs in Japanese radish tissues depended on their initial concentration in the medium. CIP (IT = 14.4) and ENF (IT = 17.0) accumulated mainly in radish roots, while LVF in leaves (IT = 230.8) at an initial concentration of 10 µg g-1. CIP (2,104 ng g-1) was detected in the highest concentration, followed by ENF (426.3 ng g-1) and LVF (273.3 ng g-1) in the tissues of both plants. FQs' bioaccumulation factors were significantly higher for duckweed (1.490-18.240) than Japanese radish (0.027-0.103). The removal of FQs from water using duckweed was mainly due to their photolysis and hydrolysis than plant sorption. In the screening, analysis detected 29 FQ TPs. The biotransformation pathways of FQs are described in detail, and the factors that influence their formation are indicated.

This study has presented the efficiency of fluoroquinolone (FQ) residues phytoextraction from water by two plant species (water duckweed, Japanese radish). The use of two plant species allowed for a holistic study of the FQ phytoremediation process by determining the efficiency of extraction, tissue distribution, bioaccumulation tendency, and biotransformation. The research gap regarding FQ transformation products in the phytoremediation process and the factors determining their formation has been filled. This study indicated that duckweed can be used with great efficiency to purify water from FQ contamination.

Application of UHPLC-MS/MS method to monitor the occurrence of sulfonamides and their transformation products in soil in Silesia, Poland.

Sulfonamides circulating in the environment lead to disturbances in food chains and local ecosystems, but most importantly contribute to development of resistance genes, which generate problems with multidrug-resistant bacterial infections treatment. In urban areas, sources of sulfonamide distribution in soils have received comparatively less attention in contrast to rural regions, where animal-derived manure, used as a natural fertilizer, is considered the main source. The aim of this study was to determine eight sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxazole, sulfapyridine, sulfathiazole, and sulfisoxazole) in environmental soil samples collected from urbanized regions in Silesian Voivodeship with increased animal activity. These soils were grouped according to the organic carbon content. It was necessary to develop versatile and efficient extraction and determination method to analyze selected sulfonamides in various soil types. The developed LC-MS/MS method for sulfonamides analyzing was validated. The obtained recoveries exceeded 45% for soil with medium organic carbon content and 88% for sample with a very low organic carbon content (arenaceous quartz). The obtained results show the high impact of organic matter on analytes adsorption in soil, which influences recovery. All eight sulfa drugs were determined in environmental samples in the concentration range 1.5-10.5 ng g-1. The transformation products of the analytes were also identified, and 29 transformation products were detected in 24 out of 27 extracts from soil samples.

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.

Extraction, Purification, Quantification, and Stability of Bioactive Spilanthol from Acmella oleracea.

Acmella oleracea is an ethnobotanically significant plant with a relatiwely high content of spilanthol. Due to its broad spectrum of activity, including anti-inflammatory, antioxidant, analgesic, antifungal, and bacteriostatic properties, it is considered a valuable bioactive natural product. In addition, spilanthol as its main bioactive component inhibits facial muscle contractions, making it an attractive ingredient in anti-wrinkle and anti-aging cosmetics. Due to its muscle paralyzing effects, it is called herbal botox. The commercial interest in spilanthol encourages the development of effective methods of isolating it from plant material. The methodology used in this paper allows for the obtaining of extracts from Acmella oleracea with a relatively high content of spilanthol. An effective method of spilanthol extraction from all aerial parts of Acmella oleracea as well as methods of enriching spilanthol concentration in extracts achieved by removing polar and acidic substances from crude extracts was developed. To quantify the concentration of spilanthol, a simple, fast and economically feasible quantification protocol that uses nuclear magnetic resonance (HNMR) was developed. In addition, it has been proven, that oxidation of spilanthol by air gives (2E,7Z)-6,9-endoperoxy-N-(2-methylpropyl)-2,7-decadienamide. The studies on spilanthol solutions stability were carried out and the conditions for the long-time storage of spilanthol solutions have also been developed. Additionally, for confirmation of obtained results a sensitive (LOQ=1 ng/mL), precise (RSD lower than 7%) and accurate (RE lower than 7.5%), new HPLC-MS/MS method was applied.

Study of the Phytoextraction and Phytodegradation of Sulfamethoxazole and Trimethoprim from Water by Limnobium laevigatum.

Phytoremediation is an environmentally friendly and economical method for removing organic contaminants from water. The purpose of the present study was to use Limnobium laevigatum for the phytoremediation of water from sulfamethoxazole (SMX) and trimethoprim (TRI) residues. The experiment was conducted for 14 days, in which the loss of the pharmaceuticals in water and their concentration in plant tissues was monitored. Determination of SMX and TRI was conducted using liquid chromatography coupled with tandem mass spectrometry. The results revealed that various factors affected the removal of the contaminants from water, and their bioaccumulation coefficients were obtained. Additionally, the transformation products of SMX and TRI were identified. The observed decrease in SMX and TRI content after 14 days was 96.0% and 75.4% in water, respectively. SMX removal mainly involved photolysis and hydrolysis processes, whereas TRI was mostly absorbed by the plant. Bioaccumulation coefficients of the freeze-dried plant were in the range of 0.043-0.147 for SMX and 2.369-2.588 for TRI. Nine and six transformation products related to SMX and TRI, respectively, were identified in water and plant tissues. The detected transformation products stemmed from metabolic transformations and photolysis of the parent compounds.

Determination of antimicrobial agents and their transformation products in an agricultural water-soil system modified with manure.

Manure fertilization is the primary source of veterinary antimicrobials in the water-soil system. The research gap is the fate of antimicrobials after their release into the environment. This study aimed to provide a detailed and multi-faceted examination of fertilized cultivated fields using two types of manure (poultry and bovine) enriched with selected antimicrobials. The research focused on assessing the mobility and stability of antimicrobials in the water-soil system. Additionally, transformation products of antimicrobials in the environment were identified. The extraction (solid-phase extraction and/or solid-liquid extraction) and LC-MS/MS analysis procedures were developed to determine 14 antimicrobials in the soil and pore water samples. Ten out of fourteen antimicrobials were detected in manure-amended soil and pore water samples. The highest concentration in the soil was 109.1 ng g-1 (doxycycline), while in pore water, it was 186.6 ng L-1 (ciprofloxacin). Sixteen transformation products of antimicrobials were identified in the soil and soil-related pore water. The same transformation products were detected in both soil and soil pore water extracts, with significantly higher signal intensities observed in soil extracts than in water. Transformation products were formed in oxidation, carbonylation, and ring-opening reactions.

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.

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.

Suspect screening of antimicrobial agents transformation products in environmental samples development of LC-QTrap method running in pseudo MRM transitions.

The aim of the work was to develop a new HPLC-MS/MS method that allows for the simultaneous detection of antimicrobials agents (targeted analysis) and their transformation products (non-targeted analysis), which enabled the elucidation of their transformation pathways in the environment. Targeted analysis was performed for 16 selected antimicrobials agents (AMs) in wastewater collected at different stages of the treatment process and river water from sections before and after wastewater discharge. The samples were collected in the Lyna sewage treatment plant (Olsztyn, Poland) in three measuring periods at different seasons. Analytes were selected from tetracyclines, fluoroquinolones, ß-lactams, macrolides, glycopeptides, lincosamides and synthetic antibiotics. As a part of the targeted analysis, 13 AMs were detected in wastewater samples, and 7 of them in river water samples. However, their presence and concentrations were closely related to the type of the sample and the season in which the sample was taken. The highest concentrations of AMs were detected in samples collected in September (max. 1643.7 ng L-1 TRI), while the lowest AMs concentrations were found in samples collected in June (max. 136.1 ng L-1 CLR). The total content of AMs in untreated wastewater was in the range of 1.42-1644 ng L-1, while in the river water was for upstream 1.22-48.73 ng L-1 and for downstream 2.24-149 ng L-1. In the non-target analysis, 33 degradation products of the selected AMs were identified, and the transformation pathways of their degradation were speculated. In the course of the research, it was found that as a result of the processes taking place in wastewater treatment plant, the parent substances are transformed into a number of stable transformation products. Transformation products resulted from hydroxylation, ring opening, oxidation, methylation or demethylation, carboxylation, or cleavage of the CN bond of the parent AMs.

Heterogeneous Photocatalysis of Metronidazole in Aquatic Samples.

Metronidazole (MET) is a commonly detected contaminant in the environment. The compound is classified as poorly biodegradable and highly soluble in water. Heterogeneous photocatalysis is the most promoted water purification method due to the possibility of using sunlight and small amounts of a catalyst needed for the process. The aim of this study was to select conditions for photocatalytic removal of metronidazole from aquatic samples. The effect of catalyst type, mass, and irradiance intensity on the efficiency of metronidazole removal was determined. For this purpose, TiO2, ZnO, ZrO2, WO3, PbS, and their mixtures in a mass ratio of 1:1 were used. In this study, the transformation products formed were identified, and the mineralization degree of compound was determined. The efficiency of metronidazole removal depending on the type of catalyst was in the range of 50-95%. The highest MET conversion (95%) combined with a high degree of mineralization (70.3%) was obtained by using a mixture of 12.5 g TiO2-P25 + PbS (1:1; v/v) and running the process for 60 min at an irradiance of 1000 W m-2. Four MET degradation products were identified by untargeted analysis, formed by the rearrangement of the metronidazole and the C-C bond breaking.

Separation and Determination of Chemopreventive Phytochemicals of Flavonoids from Brassicaceae Plants.

The main aim of this study was to develop a method for the isolation and determination of polyphenols-in particular, flavonoids present in various morphological parts of plants belonging to the cabbage family (Brassicaceae). Therefore, a procedure consisting of maceration, acid hydrolysis and measurement of the total antioxidant capacity of plant extracts (using DPPH assay) was conducted. Qualitative analysis was performed employing thin-layer chromatography (TLC), which was presented to be a suitable methodology for the separation and determination of chemopreventive phytochemicals from plants belonging to the cabbage family. The study involved the analysis of 25 vegetal samples, including radish, broccoli, Brussels sprouts, kale, canola, kohlrabi, cabbage, Chinese cabbage, red cabbage, pak choi and cauliflower. In addition, selected flavonoids content in free form and bonded to glycosides was determined by using an RP-UHPLC-ESI-MS/MS method.

LC-MS/MS Analysis Elucidates a Daily Rhythm in Orexin A Concentration in the Rat Vitreous Body.

Orexins are two neuropeptides synthesised mainly in the brain lateral hypothalamic area. The orexinergic system provides arousal-dependent cues for a plethora of brain centres, playing a vital role in feeding behaviour, regulation of the sleep-wake cycle and circadian rhythms. Recently, orexins were found to be produced in the retina of an eye; however, their content in the vitreous body and possible daily pattern of expression have not yet been explored. In this manuscript, we describe the development and validation of a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method designed for quantitative bioanalysis of orexin in the rat vitreous body. Orexin was extracted from vitreous body samples with a water:acetonitrile:formic acid (80:20:0.1; v/v/v) mixture followed by vortexing and centrifuging. Separation was performed on a reverse-phase HPLC column under gradient conditions. Orexin was analysed via multiple-reaction monitoring (MRM) in the positive electrospray mode. The total analysis time for each sample was less than 5.0 min. Once the method was fully optimised, it was then validated, following the 2018 FDA guidance on bioanalytical method validations. The calibration curves for orexin (1-500 ng/mL) were constructed using a linear regression with a 1/x2 weighting. The lower limit of quantitation for orexin was 1.0 pg/mL for the vitreous body. Intra-day and inter-day estimates of accuracy and precision were within 10% of their nominal values, indicating that the method is reliable for quantitation of orexin in the rat vitreous body. From the physiological perspective, our results are the first to show daily rhythm of orexin synthesis by the retina with possible implications on the circadian regulation of vision.

Application of UHPLC-MS/MS method to study occurrence and fate of sulfonamide antibiotics and their transformation products in surface water in highly urbanized areas.

Sulfonamide antibiotics (SAs) are used on a large scale in human and veterinary medicine. The main goal of this study was to develop a method for the detection of selected SAs (sulfamethoxazole, sulfadiazine, sulfamethazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethiazole, and sulfisoxazole) in aqueous samples (targeted analysis), and then conduct a non-targeted analysis to determine the transformation products to elucidate their degradation pathways. These analyses were performed using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry. The procedure was used to detect selected antibiotics in water samples collected throughout a highly urbanized area. Among the studied compounds, sulfamethoxazole (max. 78.88 ng L-1) and sulfapyridine (max. 38.88 ng L -1) were the most common pollutants identified in surface waters. Trace amounts of sulfadiazine (below LOQ = 0.40 ng L-1) were also detected. Next, the samples were screened to detect the transformation products. Several sulfadiazine and sulfamethoxazole transformation products were detected and confirmed in the environmental samples.

Removal and transformation of sulfamethoxazole in acclimated biofilters with various operation modes - Implications for full-scale application.

The knowledge gaps regarding the degradation of sulfamethoxazole (SMX) in biofilters include the effect of aeration, constant feeding with readily biodegradable organic carbon and the presence of reactive media such as manganese oxides (MnOx). Thus, the goal of this study was to assess the removal of SMX in lab-scale biofilters with various operation variables: aeration, presence of MnOx as an amendment of filtering medium and the presence of readily biodegradable organic carbon (acetate). The sand used in the experiment as a filtering medium was previously exposed to the presence of SMX and acetate, which provided acclimation of the biomass. The removal of SMX was complete (>99%) with the exception of the unaerated columns fed with the influent containing acetate, due to apparent slower rate of SMX degradation. The obtained results suggest that bacteria were able to degrade SMX as a primary substrate and the degradation of this compound was subsequent to the depletion of acetate. The LC-MS/MS analysis of the effluents indicated several biotransformation reactions for SMX: (di)hydroxylation, acetylation, nitrosation, deamonification, S-N bond cleavage and isoxazole-ring cleavage. The relative abundance of transformation products was decreased in the presence of MnOx or acetate. Based on the Microtox assay, only the effluents from the unaerated columns filled with MnOx were classified as non-toxic. The results offer important implications for the design of biofilters for the elimination of SMX, namely that biofilters offer the greatest performance when fed with secondary wastewater and operated as non-aerated systems with a filtering medium containing MnOx.

Microbial and chemical pollutants on the manure-crops pathway in the perspective of "One Health" holistic approach.

This study determined the impact of poultry and bovine manure fertilization on the content of antibiotics, heavy metals (HMs), and the quantitative and qualitative composition of integrase and antibiotic resistance genes in soil, groundwater, and crops cultivated on manure-amended plots. Antibiotic concentration levels were analyzed using the HPLC-MS/MS, heavy metal concentration level were measured by HGAAS and ICP-OES, while the integrase genes and ARGs were quantified using Real-Time PCR (qPCR) method. Manure, soil, and crops samples contained the highest concentration of Zn (104-105 ng gdm-1) and Cu (103-105 ng gdm-1) of all HMs tested. Manure-supplemented soil was characterised by a high concentration of doxycycline and enrofloxacin. A high abundance of integrase genes was noted in samples of manure (109-1010 copies gdm-1) and soil (107-108 copies gdm-1). Among all the analyzed genes, sul1, sul2, blaTEM, and integrase genes were the most common. Results of the study demonstrate the selective character of ARGs transfer from poultry and bovine manure to plants. The only gene to occur in all studied environmental compartments was sul1 (from 102 - groundwater to 1011 - poultry manure). It was also found that animal manure may cause an increase in the HMs concentration in soil and their accumulation in crops, which may influence the health of humans and animals consuming crops grown on manure-amended soil. The high abundance of integrase genes and ARGs and their reciprocal correlations with HMs pose a serious risk of the rapid spread of antibiotic resistance in the environment. Moreover, unusual dependencies between integrase genes and selected ARGs indicate the possibility of changes in the mobility nature of genetic elements.

Wastewater treatment plants as a reservoir of integrase and antibiotic resistance genes - An epidemiological threat to workers and environment.

Conventional mechanical and biological wastewater treatment is unable to completely eliminate all pollutants, which can therefore enter surface water bodies together with treated wastewater. In addition, bioaerosols produced during wastewater treatment can pose a threat to the health of the wastewater treatment plant staff. In order to control the impact of a wastewater treatment plant (WWTP) on the surrounding environment, including its employees, samples of wastewater and water from a river which received treated wastewater were analysed in terms of their content of antibiotics and heavy metals, levels of selected physiochemical parameters, concentrations of antibiotic-resistance genes (ARGs) and genes of integrases. Furthermore, a quantitative analysis of ARGs in the metagenomic DNA from nasal and throat swabs collected from the WWPT employees was made. Both untreated and treated wastewater samples were dominated by genes of resistance to sulphonamides (sul1, sul2), MLS group of drugs (ermF, ermB) and beta-lactams (blaOXA). A significant increase in the quantities of ARGs and concentrations of antibiotics was observed in the river following the discharge of treated wastewater in comparison to their amounts in the river water upstream from the point of discharge. Moreover, a higher concentration of ARGs was detected in the DNA from swabs obtained from the wastewater treatment plant employees than from ones collected from the control group. Many statistically significant (p < 0.05) correlations between the concentration of the gene of resistance to heavy metals cnrA versus ARGs, and between the ARGs content and the concentrations of heavy metals in both wastewater and river water samples were observed. The study has demonstrated that the mechanical and biological methods of wastewater treatment are not efficient and may affect the transmission of hazardous pollutants to the aquatic environment and to the atmospheric air. It has been shown that an activated sludge bioreactor can be a potential source of the presence of multi-drug resistant microorganisms in the air, which is a health risk to persons working in WWTPs. It has also been found that an environment polluted with heavy metals is where co-selection of antibiotic resistance may occur, in the development of which integrase genes play an essential role.

The impact of antimicrobials on the efficiency of methane fermentation of sewage sludge, changes in microbial biodiversity and the spread of antibiotic resistance.

The study was designed to simultaneously evaluate the influence of high doses (512-1024 µg/g) the most commonly prescribed antimicrobials on the efficiency of anaerobic digestion of sewage sludge, qualitative and quantitative changes in microbial consortia responsible for the fermentation process, the presence of methanogenic microorganisms, and the fate of antibiotic resistance genes (ARGs). The efficiency of antibiotic degradation during anaerobic treatment was also determined. Metronidazole, amoxicillin and ciprofloxacin exerted the greatest effect on methane fermentation by decreasing its efficiency. Metronidazole, amoxicillin, cefuroxime and sulfamethoxazole were degraded in 100%, whereas ciprofloxacin and nalidixic acid were least susceptible to degradation. The most extensive changes in the structure of digestate microbiota were observed in sewage sludge exposed to metronidazole, where a decrease in the percentage of bacteria of the phylum Bacteroidetes led to an increase in the proportions of bacteria of the phyla Firmicutes and Proteobacteria. The results of the analysis examining changes in the concentration of the functional methanogen gene (mcrA) did not reflect the actual efficiency of methane fermentation. In sewage sludge exposed to antimicrobials, a significant increase was noted in the concentrations of ß-lactam, tetracycline and fluoroquinolone ARGs and integrase genes, but selective pressure was not specific to the corresponding ARGs.