Resistance Genes In Wastewater Research Articles

Wastewater surveillance for antibiotic resistance genes during the late 2020 SARS-CoV-2 peak in two different populations

ABSTRACT The United States Centers for Disease Control and Prevention reported a rise in resistant infections after the coronavirus disease 2019 (COVID-19) pandemic started. How and if the pandemic contributed to antibiotic resistance in the larger population is not well understood. Wastewater treatment plants are good locations for environmental surveillance because they can sample entire populations. This study aimed to validate methods used for COVID-19 wastewater surveillance for bacterial targets and to understand how rising COVID-19 cases from October 2020 to February 2021 in Portugal (PT) and King County, Washington contributed to antibiotic resistance genes in wastewater. Primary influent wastewater was collected from two treatment plants in King County and five treatment plants in PT, and hospital effluent was collected from three hospitals in PT. Genomic extracts were tested with the quantitative polymerase chain reaction for antibiotic resistance genes conferring resistance against antibiotics under threat. Random-effect models were fit for log-transformed gene abundances to assess temporal trends. All samples collected tested positive for multiple resistance genes. During the sampling period, mecA statistically significantly increased in King County and PT. No statistical evidence exists of correlation between samples collected in the same Portuguese metro area.

Effects and Mechanisms of Polystyrene Microplastics on Extracellular Antibiotic Resistance Genes in Wastewater

Microplastics (MPs) and antibiotic resistance genes (ARGs) are typical co-existing emerging pollutants in wastewater treatment plants. MPs have been shown to alter the distribution pattern of ARGs in sludge, but their effects on free extracellular ARGs (feARGs) in wastewater remain unclear. In this study, we used fluorescence quantitative PCR to investigate the dynamics of feARGs (including tetC, tetO, sul1, and sul2) in wastewater and their transition mechanisms after 60 d of exposure to typical MPs (polystyrene, PS). The results showed that the absolute abundance of tetracycline feARGs decreased by 28.4 %-76.0 % and 35.2 %-96.2 %, respectively, under nm-level and mm-level PS exposure and changed by -55.4 %-122.4 % under μm-level PS exposure. The abundance of sul1 showed a trend of nm-level > μm-level > mm-level upon PS exposure, and the changes in sul1 abundance was greater with ρ(PS)=50 mg·L-1 exposure. The relative abundance of sul2 was reduced by 25.4 %-42.6 % and 46.1 %-90.3 % after μm-level and mm-level PS exposure, respectively, and increased by 1.9-3.9 times after nm-level PS exposure, and the sul2 showed a higher reduction at ρ (PS)=50 mg·L-1 exposure than that at ρ (PS)=0.5 mg·L-1. The Pearson correlation analysis showed that the relative abundance of feARGs during PS exposure was positively correlated with cell membrane permeability and typical mobile genetic elements (intI1) abundance and negatively correlated with reactive oxygen species level. Our findings elucidated the effects and corresponding mechanisms of PS on the growth and mobility of feARGs in wastewater, providing a scientific basis for the control of the combined MPs and ARGs pollution in wastewater.

Extended-spectrum β-lactamase-producing bacteria and their resistance determinants in different wastewaters and rivers in Nepal

Wastewaters serve as significant reservoirs of antibiotic resistant bacteria. Despite the evidence of antimicrobial resistance in wastewaters and river water in Kathmandu, direct linkage between them is not discussed yet. This study investigated the prevalence of extended-spectrum β-lactamase (ESBL)-producing bacteria and associated resistance genes in wastewaters and river water. Out of 246 bacteria from wastewaters, 57.72% were ESBL producers and 77.64% of them were multidrug resistant (MDR). ESBL producing E. coli was dominant in municipal and hospital wastewaters (HWW) as well as in river water while K. pneumoniae was common in pharmaceutical wastewater. The blaSHV and blaTEM genes were prevalent and commonly co-occurred with aac(6')-Ib-cr in K. pneumoniae isolated pharmaceutical wastewater. blaCTX-M carrying E. coli from hospital co-harbored aac(6')-Ib-cr while that from municipal influent and river water co-harbored qnrS. Whole genome sequencing data revealed the presence of diverse ARGs in bacterial isolates against multiple antibiotics. In average, an E. coli and a K. pneumoniae isolate contained 55.75 ± 0.96 and 40.2 ± 5.36 ARGs, respectively. Multi-locus sequence typing showed the presence of globally high-risk clones with wider host range such as E. coli ST10, and K. pneumoniae ST15 and ST307 in HWW and river indicating frequent dissemination of antimicrobial resistance in wastewater of Kathmandu. Whole genome sequence data aligned with phenotypic antibiograms and resistance genes detected by PCR in selected isolates. The presence of significant plasmid replicons (IncF, IncY) and mobile genetic elements (IS903, IS26) indicate high frequency of spreading antibiotic resistance. These findings indicate burden and dissemination of antimicrobial resistance in the environment and highlight the need for effective strategies to mitigate the antibiotic resistance.

Comparison of qPCR and metagenomic sequencing methods for quantifying antibiotic resistance genes in wastewater.

Surveillance methods of circulating antibiotic resistance genes (ARGs) are of utmost importance in order to tackle what has been described as one of the greatest threats to humanity in the 21st century. In order to be effective, these methods have to be accurate, quickly deployable, and scalable. In this study, we compare metagenomic shotgun sequencing (TruSeq DNA sequencing) of wastewater samples with a state-of-the-art PCR-based method (Resistomap HT-qPCR) on four wastewater samples that were taken from hospital, industrial, urban and rural areas. ARGs that confer resistance to 11 antibiotic classes have been identified in these wastewater samples using both methods, with the most abundant observed classes of ARGs conferring resistance to aminoglycoside, multidrug-resistance (MDR), macrolide-lincosamide-streptogramin B (MLSB), tetracycline and beta-lactams. In comparing the methods, we observed a strong correlation of relative abundance of ARGs obtained by the two tested methods for the majority of antibiotic classes. Finally, we investigated the source of discrepancies in the results obtained by the two methods. This analysis revealed that false negatives were more likely to occur in qPCR due to mutated primer target sites, whereas ARGs with incomplete or low coverage were not detected by the sequencing method due to the parameters set in the bioinformatics pipeline. Indeed, despite the good correlation between the methods, each has its advantages and disadvantages which are also discussed here. By using both methods together, a more robust ARG surveillance program can be established. Overall, the work described here can aid wastewater treatment plants that plan on implementing an ARG surveillance program.

Spread and persistence of antimicrobial resistance genes in wastewater from human and animal sources in São Paulo, Brazil.

The spread of antimicrobial resistance (AMR) through multiple reservoirs is a global concern. Wastewater is a critical AMR dissemination source, so this study aimed to assess the persistence of resistance genetic markers in wastewater using a culture-independent approach. Raw and treated wastewater samples (n = 121) from a wastewater treatment plant (WWTP), a human hospital, a veterinary hospital, and a pig farm were monthly collected and concentrated by filtration. DNA was extracted directly from filter membranes, and PCR was used in the qualitative search of 32 antimicrobial resistance genes (ARGs). Selected genes (blaCTX-M, blaKPC, qnrB, and mcr-1) were enumerated by quantitative real-time PCR (qPCR). Twenty-six ARGs were detected in the qualitative ARGs search, while quantitative data showed a low variation of the ARG's relative abundance (RA) throughout the months, especially at the human hospital and the WWTP. At the WWTP, despite significantly reducing the absolute number of gene copies/L after each treatment stage (p < 0.05), slight increases (p > 0.05) in the RAs of genes blaCTX-M, qnrB, and mcr-1 were observed in reused water (tertiary treatment) when compared with secondary effluent. Although the increase is not statistically significant, it is worth noting that there was some level of ARGs concentration after the disinfection process. No significant absolute or relative after-treatment quantification reductions were observed for any ARGs at the veterinary hospital or the pig farm. The spread of ARGs through sewage needs to be continuously addressed, because their release into natural environments may pose potential risks of exposure to resistant bacteria and impact local ecosystems.

Research Trends and Future Prospects of Constructed Wetland Treatment Technology in China

With the intensification of water pollution problems worldwide, constructed wetlands, as a green, efficient, and energy-saving wastewater treatment technology, have gradually attracted the wide attention of scholars at home and abroad. In order to better understand and master the research trends of constructed wetland treatment technology in China and promote its development, the literature from 2000 to 2023 in the CNKI database and the Web of Science (WoS) database (located in China) were selected as research objects. Then, CiteSpace software (6.2.R4) was used to visualize and analyze the literature, revealing the research trends and hot areas of constructed wetland treatment technology in China. Then, the optimized way of operation effect of constructed wetland was discussed to provide a theoretical and technical basis for the wide application of constructed wetland technology in our country. The results indicate that the annual publication volume of research on constructed wetlands in China is showing a rapid upward trend. Among them, the Chinese literature mainly focuses on how to improve the application effect of constructed wetlands on nitrogen and phosphorus removal of rural domestic wastewater by matching different wetland plants or developing combined processes. The English literature from the Web of Science (WoS) database mainly focuses on how to remove emerging pollutants, such as heavy metals and resistance genes in wastewater in China, by changing the filling matrix and microbial community structure or developing new processes, and the related mechanisms have been discussed. One of the hot spots for the future research of constructed wetlands in China is to vigorously develop microbial fuel cells, and try to overcome the problem of poor purification efficiency of constructed wetlands under complex conditions such as low temperature, low carbon-nitrogen ratio, and high pollution load. In order to strengthen its application, the specific optimization methods can be divided into two categories: self-optimization strategies such as increasing oxygen supply and transfer, providing electron donor matrix, preventing matrix blockage, and combination processes coupled with anaerobic treatment and other technologies.

The fate of antibiotic resistance genes in wastewater containing microalgae treated by chlorination, ultra-violet, and Fenton reaction

Antibiotic resistance genes (ARGs) and bacteria (ARBs) in the effluent of wastewater treatment plants (WWTPs) are of utmost importance for the dissemination of ARGs in natural aquatic environments. Therefore, there is an urgent need for effective technologies to eliminate WWTP ARGs/ARBs and mitigate the associated risks posed by the discharged ARG in aquatic environments. To test the effective technology for eliminating ARGs/ARBs, we compared the removal of ARGs and ARBs by three different tertiary treatments, namely ultra-violet (UV) disinfection, chlorination disinfection, and Fenton oxidation. Then, the treated wastewater was co-cultured with Chlorella vulgaris (representative of aquatic biota) to investigate the fate of discharged ARGs into the aquatic environment. The results demonstrated that chlorination (at a chlorine concentration of 15 mg/L) and Fenton (at pH 2.73, with 0.005 mol/L Fe2+ and 0.0025 mol/L H2O2) treatment showed higher efficacy in ARG removal (1.8 - 4.17 logs) than UV treatment (15 min) (1.29 - 3.87 logs). Moreover, chlorine at 15 mg/L and Fenton treatment effectively suppressed ARB regeneration while UV treatment for 15 min could not. Regardless of treatments tested in this study, the input of treated wastewater to the Chlorella system increased the number of ARGs and mobile genetic elements (MGEs), indicating the potential risk of ARG dissemination associated with WWTP discharge. Among the wastewater-Chlorella co-culture systems, chlorination resulted in less of an increase in the number of ARGs and MGEs compared to Fenton and UV treatment. When comparing the wastewater systems to the co-culture systems, it was observed that Chlorella vulgaris reduced the number of ARGs and MGEs in chlorination and UV-treated wastewater; however, Chlorella vulgaris promoted ARG survival in Fenton-treated water, suggesting that aquatic microalgae might act as a barrier to ARG dissemination. Overall, chlorination treatment not only effectively removes ARGs and inhibits ARB regeneration but also shows a lower risk of ARG dissemination. Therefore, chlorination is recommended for practical application in controlling the spread of discharged ARGs from WWTP effluent in natural aquatic environments.

Analysis of Antibiotic Resistance Genes in Water Reservoirs and Related Wastewater from Animal Farms in Central China.

This study aimed to explore the phenotype and relationship of drug resistance genes in livestock and poultry farm wastewater and drinking water reservoirs to provide evidence for the transmission mechanisms of drug resistance genes, in order to reveal the spread of drug resistance genes in wastewater from intensive farms in Central China to urban reservoirs that serve as drinking water sources and provide preliminary data for the treatment of wastewater from animal farms to reduce the threat to human beings. DNA extraction and metagenomic sequencing were performed on eight groups of samples collected from four water reservoirs and four related wastewaters from animal farms in Central China. Metagenomic sequencing showed that the top 20 AROs with the highest abundance were vanT_gene, vanY_gene, adeF, qacG, Mtub_rpsL_STR, vanY_gene_, vanW_gene, Mtub_murA_FOF, vanY_gene, vanH_gene, FosG, rsmA, qacJ, RbpA, vanW_gene, aadA6, vanY_gene, sul4, sul1, and InuF. The resistance genes mentioned above belong to the following categories of drug resistance mechanisms: antibiotic target replacement, antibiotic target protection, antibiotic inactivation, and antibiotic efflux. The resistomes that match the top 20 genes are Streptococcus agalactiae and Streptococcus anginosus; Enterococcus faecalis; Enterococcus faecium; Actinomyces viscosus and Bacillus cereus. Enterococcus faecium; Clostridium tetani; Streptococcus agalactiae and Streptococcus anginosus; Streptococcus agalactiae and Streptococcus anginosus; Acinetobacter baumannii, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Corynebacterium jeikeium, Corynebacterium urealyticum, Mycobacterium kansasii, Mycobacterium tuberculosis, Schaalia odontolytica, and Trueperella pyogenes; Mycobacterium avium and Mycobacterium tuberculosis; Aeromonas caviae, Enterobacter hormaechei, Vibrio cholerae, Vibrio metoecus, Vibrio parahaemolyticus, and Vibrio vulnificus; Pseudomonas aeruginosa and Pseudomonas fluorescens; Staphylococcus aureus and Staphylococcus equorum; M. avium, Achromobacter xylosoxidans, and Acinetobacter baumannii; Sphingobium yanoikuyae, Acinetobacter indicus, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, and Providencia stuartii. Unreported drug resistance genes and drug-resistant bacteria in Central China were identified in 2023. In the transmission path of drug resistance genes, the transmission path from aquaculture wastewater to human drinking water sources cannot be ignored. For the sake of human health and ecological balance, the treatment of aquaculture wastewater needs to be further strengthened, and the effective blocking of drug resistance gene transmission needs to be considered.

Prevalence of antibiotic-resistant Gram-negative bacteria having extended-spectrum β-lactamase phenotypes in polluted irrigation-purpose wastewaters from Indian agro-ecosystems.

Antibiotic resistance in bacteria has emerged as a serious public health threat worldwide. Aquatic environments including irrigation-purpose wastewaters facilitate the emergence and transmission of antibiotic-resistant bacteria and antibiotic resistance genes leading to detrimental effects on human health and environment sustainability. Considering the paramount threat of ever-increasing antibiotic resistance to human health, there is an urgent need for continuous environmental monitoring of antibiotic-resistant bacteria and antibiotic resistance genes in wastewater being used for irrigation in Indian agro-ecosystems. In this study, the prevalence of antibiotic resistance in Gram-negative bacteria isolated from irrigation-purpose wastewater samples from Sirmaur and Solan districts of Himachal Pradesh was determined. Bacterial isolates of genera Escherichia, Enterobacter, Hafnia, Shigella, Citrobacter, and Klebsiella obtained from 11 different geographical locations were found to exhibit resistance against ampicillin, amoxyclav, cefotaxime, co-trimoxazole, tobramycin, cefpodoxime and ceftazidime. However, all the isolates were sensitive to aminoglycoside antibiotic gentamicin. Enterobacter spp. and Escherichia coli showed predominance among all the isolates. Multidrug-resistance phenotype was observed with isolate AUK-06 (Enterobacter sp.) which exhibited resistant to five antibiotics. Isolate AUK-02 and AUK-09, both E. coli strains showed resistant phenotypes to four antibiotics each. Phenotypic detection revealed that six isolates were positive for extended-spectrum β-lactamases which includes two isolates from Enterobacter spp. and E. coli each and one each from Shigella sp. and Citrobacter sp. Overall, the findings revealed the occurrence of antibiotic resistant and ESBL-positive bacterial isolates in wastewaters utilized for irrigation purpose in the study area and necessitate continuous monitoring and precautionary interventions. The outcomes of the study would be of significant clinical, epidemiological, and agro-environmental importance in designing effective wastewater management and environmental pollution control strategies.

Simultaneous removal of antibiotics and antibiotic resistance genes in wastewater by a novel nonthermal plasma/peracetic acid combination system: Synergistic performance and mechanism

In this study, a novel and green method combining plasma with peracetic acid (plasma/PAA) was developed to simultaneously remove antibiotics and antibiotic resistance genes (ARGs) in wastewater, which achieves significant synergistic effects in the removal efficiencies and energy yield. At a plasma current of 2.6 A and PAA dosage of 10 mg/L, the removal efficiencies of most detected antibiotics in real wastewater exceeded 90 % in 2 min, with the ARG removal efficiencies ranging from 6.3 % to 75.2 %. The synergistic effects of plasma and PAA could be associated with the motivated production of reactive species (including •OH, •CH3, 1O2, ONOO-, •O2- and NO•), which decomposed antibiotics, killed host bacteria, and inhibited ARG conjugative transfer. In addition, plasma/PAA also changed the contributions and abundances of ARG host bacteria and downregulated the corresponding genes of two-component regulatory systems, thus reducing ARG propagation. Moreover, the weak correlations between the removal of antibiotics and ARGs highlights the commendable performance of plasma/PAA in the simultaneous removal of antibiotics and ARGs. Therefore, this study affords an innovative and effective avenue to remove antibiotics and ARGs, which relies on the synergistic mechanisms of plasma and PAA and the simultaneous removal mechanisms of antibiotics and ARGs in wastewater.

Biodegradation of tylosin in swine wastewater by Providencia stuartii TYL-Y13: Performance, pathway, genetic background, and risk assessment

Microbial bioremediation offers a solution to the problem of residual antibiotics in wastewater associated with animal farms. Efficient degradation of antibiotic residues depends upon the genetic make-up of microbial degraders, which requires a comprehensive understanding of the degradation mechanisms. In this study, a novel, efficient tylosin (TYL)-degrading bacterium, Providencia stuartii TYL-Y13 (Y13) was isolated, which could completely degrade 100 mg/L TYL within 15 h under optimal operating conditions at 40 ℃, pH 7.0 %, and 1 % (v/v) bacterial inoculation rate. Whole genome sequencing revealed that strain Y13 consists of a circular chromosome and two plasmids. A new biodegradation pathway of TYL including desugarification, hydrolysis, and reduction reactions was proposed through the analysis of biodegradation products. It was demonstrated that strain Y13 gradually decreased the biotoxicity of TYL and its metabolites based on the results of the ecological structural activity relationships (ECOSAR) model analysis and toxicity assessment. Moreover, Y13 promoted the reduction of the target macrolide resistance genes in wastewater and disappeared within 84 h. These results shed new light on the mechanism of TYL biodegradation and better utilization of microbes to remediate TYL contamination.

Circulation of Antibiotic Resistance Genes in Wastewater in Mediterranean Basin Countries: A Review

Circulation of Antibiotic Resistance Genes in Wastewater in Mediterranean Basin Countries: A Review

Insight into using a novel ultraviolet/peracetic acid combination disinfection process to simultaneously remove antibiotics and antibiotic resistance genes in wastewater: Mechanism and comparison with conventional processes

In this study, the simultaneous removal mechanism of antibiotics and antibiotic resistance genes (ARGs) was investigated using the novel ultraviolet/peracetic acid (UV/PAA) combination disinfection process and conventional disinfection processes were also applied for comparison. The results showed that UV/PAA disinfection with a high UV dosage (UV/PAA-H) was most effective for the removal of tetracyclines, quinolones, macrolides and β-lactams; their average removal efficiencies ranged from 25.7% to 100%, while NaClO disinfection was effective for the removal of sulfonamides (∼81.6%). The majority of ARGs were well removed after the UV/PAA-H disinfection, while specific genes including tetB, tetC, ermA and blaTEM significantly increased after NaClO disinfection. In addition, β-lactam resistance genes (-35.9%) and macrolides resistance genes (-12.0%) remarkably augmented after UV/NaClO disinfection. The highly reactive oxidation species generated from UV/PAA process including hydroxyl radicals (•OH) and carbon-centered organic radicals (R-C•), were responsible for the elimination of antibiotics and ARGs. Correlation analysis showed that tetracycline, sulfonamide and macrolide antibiotics removal showed a positive correlation with the corresponding ARGs, and a low dose of antibiotic residues played an important role in the distribution of ARGs. Metagenomic sequencing analysis showed that UV/PAA disinfection could not only greatly decrease the abundance of resistant bacteria but also downregulate the expression of key functional genes involved in ARGs propagation and inhibit the signal transduction of the host bacteria, underlying that its removal mechanism was quite different from that of NaClO-based disinfection processes. Our study provides valuable information for understanding the simultaneous removal mechanism of antibiotics and ARGs in wastewater during the disinfection processes, especially for the novel UV/PAA combination process.

Mitigating Antibiotic Resistance Genes in Wastewater by Sequential Treatment with Novel Nanomaterials.

Wastewater (WW) has been widely recognized as the major sink of a variety of emerging pathogens (EPs), antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which may disseminate and impact wider environments. Improving and maximizing WW treatment efficiency to remove these microbial hazards is fundamentally imperative. Despite a variety of physical, biological and chemical treatment technologies, the efficiency of ARG removal is still far from satisfactory. Within our recently accomplished M-ERA.NET project, novel functionalized nanomaterials, i.e., molecularly imprinted polymer (MIP) films and quaternary ammonium salt (QAS) modified kaolin microparticles, were developed and demonstrated to have significant EP removal effectiveness on both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB) from WW. As a continuation of this project, we took the further step of exploring their ARG mitigation potential. Strikingly, by applying MIP and QAS functionalized kaolin microparticles in tandem, the ARGs prevalent in wastewater treatment plants (WWTPs), e.g., blaCTXM, ermB and qnrS, can be drastically reduced by 2.7, 3.9 and 4.9 log (copies/100 mL), respectively, whereas sul1, tetO and mecA can be eliminated below their detection limits. In terms of class I integron-integrase I (intI1), a mobile genetic element (MGE) for horizontal gene transfer (HGT), 4.3 log (copies/100 mL) reduction was achieved. Overall, the novel nanomaterials exhibit outstanding performance on attenuating ARGs in WW, being superior to their control references. This finding provides additional merit to the application of developed nanomaterials for WW purification towards ARG elimination, in addition to the proven bactericidal effect.

Prevalence of antibiotic resistance genes in wastewater collected from ornamental fish market in northern China.

Large amounts of antibiotics/disinfectants are used in the farming of ornamental fish so as to prevent and treat bacterial infection. This may exert considerable selection pressures on the prevalence and propagation of antibiotic resistance genes (ARGs). However, the levels of ARGs and their potential prevalence mechanism in the wastewater of the ornamental fish industry remains unclear. In this work, wastewater is collected from a representative ornamental fish market (OFM) that is located at the northern China to study the occurrence and abundance of 21 ARGs and 2 integrase genes. Results indicated that 15 different ARGs and 2 integrase genes are existent and prevalent in the wastewater of OFM, whereby concentrations range from 2.01 to 10.34 copies/L. Proteobacteria, Bacteroidetes, Verrucomicrobia, and Firmicutes are the predominant phyla in the wastewater samples. 17 species of human opportunistic pathogens are present with relative abundance of up to 0.01%, which suggests a considerable risk of pathogens acquiring and disseminating ARGs. Moreover, oxytetracycline, ciprofloxacin, norfloxacin, sulfadiazine, and chloramphenicol are most frequently detected in wastewater, with concentrations of up to 1150, 877, 514, 1970, and 1700ng/L, respectively. Notably, good correlations have been determined among ARGs and antibiotics, non-antibiotic environmental factors in wastewater of OFM. This current study reveals, for the first time, that OFM is a previously unperceived reservoir for ARG prevalence in aquatic environment and water environmental factors (particularly antibiotics), and their induced shifts in the microbial communities are the key factors for distribution of ARGs in OFM.

Integrated metagenomic and metatranscriptomic analyses of ultraviolet disinfection effects on antibiotic resistance genes and bacterial communities during wastewater treatment.

Ultraviolet (UV) disinfection is now widely implemented in wastewater treatment plants (WWTPs) worldwide, but its effect on antibiotic resistome of the surviving bacteria remains unclear. In this study, we employed high-throughput sequencing-based metagenomic and metatranscriptomic approaches to comprehensively elucidate the effects of UV disinfection on the shifts of bacterial community and antibiotic resistance genes (ARGs) on both DNA and mRNA levels in one WWTP. Metagenomic analyses revealed an insignificant change in the bacterial community after UV disinfection, while metatranscriptomic analyses showed that UV disinfection significantly changed the abundance of 13.79% of phyla and 10.32% of genera. In total, 38 ARG-like open reading frames (ORFs) and 327 ARG-like transcripts were identified in the DNA and RNA samples, respectively. The relative abundances of the total ARGs, each ARG type, and each ARG subtype also varied after UV disinfection. Additionally, UV disinfection significantly reduced the expression of total ARGs from 49.40 transcripts per kilobase of exon model per million mapped reads (TPM) to 47.62 TPM, and significantly changed the expression of 10.75% of ARG subtypes in wastewater (p < 0.05). Notably, the significant increase in the expression and obvious increase in the relative abundance of macrolide-lincosamide-streptogramin B (MLSB) resistance genes revealed that UV disinfection increases the potential health risk of MLSB resistance genes in wastewater. Moreover, potential host analyses of ARGs revealed the different preferences of antibiotic resistant bacteria (ARB) to ARGs. This study may shed new light on the underlying mechanism of the UV disinfection effect on antibiotic resistance.

High-throughput profiling of antibiotic resistance genes in wastewater: comparison between a pond system in Namibia and an activated sludge treatment in Germany.

There are increasing concerns about wastewater treatment plants (WWTPs) acting as hotspots for antibiotic resistance genes (ARG). However, their role largely depends upon the treatment methods and antibiotics in the wastewater. To better understand these influences, we compared the occurrence and fate of ARG between a pond system in a developing country (Namibia) and an advanced WWTP (activated sludge system) in a developed country (Germany). A targeted metagenomic approach was used to investigate the wide-spectrum profiles of ARGs and their co-occurrence patterns at both locations. In total, 93 ARG subtypes were found in the German influent wastewater, 277 in the Namibian influent wastewater. The abundant ARG types found in Namibia and Germany differed, especially for multidrug resistance genes. The differences in occurrence and reduction can help to understand the performance of simple WWTP such as pond systems common in Namibia, where direct contact with wastewater is a potential risk for contamination.

Occurrence of Fluoroquinolones and Sulfonamides Resistance Genes in Wastewater and Sludge at Different Stages of Wastewater Treatment: A Preliminary Case Study

This study identified differences in the prevalence of antibiotic resistance genes (ARGs) between wastewater treatment plants (WWTPs) processing different proportions of hospital and municipal wastewater as well as various types of industrial wastewater. The influence of treated effluents discharged from WWTPs on the receiving water bodies (rivers) was examined. Genomic DNA was isolated from environmental samples (river water, wastewater and sewage sludge). The presence of genes encoding resistance to sulfonamides (sul1, sul2) and fluoroquinolones (qepA, aac(6′)-Ib-cr) was determined by standard polymerase chain reaction (PCR). The effect of the sampling season (summer – June, fall – November) was analyzed. Treated wastewater and sewage sludge were significant reservoirs of antibiotic resistance and contained all of the examined ARGs. All wastewater samples contained sul1 and aac(6′)-lb-cr genes, while the qepA and sul2 genes occurred less frequently. These observations suggest that the prevalence of ARGs is determined by the type of processed wastewater. The Warmia and Mazury WWTP was characterized by higher levels of the sul2 gene, which could be attributed to the fact that this WWTP processes agricultural sewage containing animal waste. However, hospital wastewater appears to be the main source of the sul1 gene. The results of this study indicate that WWTPs are significant sources of ARGs, contributing to the spread of antibiotic resistance in rivers receiving processed wastewater.

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.

Identification of Selected Antibiotic Resistance Genes in Two Different Wastewater Treatment Plant Systems in Poland: A Preliminary Study.

Antibiotic resistance is a growing problem worldwide. The emergence and rapid spread of antibiotic resistance determinants have led to an increasing concern about the potential environmental and public health endangering. Wastewater treatment plants (WWTPs) play an important role in this phenomenon since antibacterial drugs introduced into wastewater can exert a selection pressure on antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Therefore, WWTPs are perceived as the main sources of antibiotics, ARB and ARG spread in various environmental components. Furthermore, technological processes used in WWTPs and its exploitation conditions may influence the effectiveness of antibiotic resistance determinants’ elimination. The main aim of the present study was to compare the occurrence of selected tetracycline and sulfonamide resistance genes in raw influent and final effluent samples from two WWTPs different in terms of size and applied biological wastewater treatment processes (conventional activated sludge (AS)-based and combining a conventional AS-based method with constructed wetlands (CWs)). All 13 selected ARGs were detected in raw influent and final effluent samples from both WWTPs. Significant ARG enrichment, especially for tet(B, K, L, O) and sulIII genes, was observed in conventional WWTP. The obtained data did not show a clear trend in seasonal fluctuations in the abundance of selected resistance genes in wastewaters.