Antibiotic Resistance In Wastewater Research Articles

Integron-associated genes are reliable indicators of antibiotic resistance in wastewater despite treatment- and seasonality-driven fluctuations

The present study aims to characterize the bacterial community, resistome and integron abundance of a municipal wastewater treatment plant (WWTP) over the course of 12 months and evaluate the year-long performance of integron-related genes as potential indicators of antibiotic resistance mechanisms in influents and effluents. For that, total DNA was extracted and subjected to 16S rRNA-targeted metabarcoding, high-throughput (HT) qPCR (48 targets) and standard qPCR (5 targets). Targets included integrase genes, antibiotic resistance genes (ARGs) and putative pathogenic groups. A total of 16 physicochemical parameters determined in the wastewater samples were also considered. Results revealed that the WWTP treatment significantly impacted the bacterial community, as well as the content in ARGs and integrase genes. Indeed, there was a relative enrichment from influent to effluent of 13 pathogenic groups (e.g., Legionella and Mycobacterium) and genes conferring resistance to sulphonamides, aminoglycosides and disinfectants. Effluent samples (n=25) also presented seasonal differences, with an increase of the total ARGs’ concentration in summer, and differences between winter and summer on relative abundance of sulphonamide and disinfectant resistance mechanisms. From the eight putative integron-related genes selected, all were positively correlated with the total ARGs’ content in wastewater and the relative abundance of resistance to most of the specific antibiotic classes. The genes intI1, blaGES and qacE∆1 were the most strongly correlated with the total concentration of ARGs. Genes blaGES and blaVIM, were better correlated to resistance to beta-lactams, aminoglycosides and tetracyclines. This study supports the use of integron-related genes as powerful indicators of antibiotic resistance in wastewater, being robust despite the variability caused by wastewater treatment and seasonality.

Wastewater surveillance of antibiotic resistance and class 1 integron-integrase genes: potential impact of wastewater characteristics on genes profile

Antibiotic resistance (AR) is a major global health concern, but current surveillance efforts primarily focus on healthcare settings, leaving a lack of understanding about AR across all sectors of the One Health approach. To bridge this gap, wastewater surveillance provides a cost-effective and efficient method for monitoring AR within a population. In this study, we implemented a surveillance program by monitoring the wastewater effluent from two large-scale municipal treatment plants situated in Isfahan, a central region of Iran. These treatment plants covered distinct catchment regions and served a combined population about two million of residents. Furthermore, the effect of physicochemical and microbial characteristics of wastewater effluent including biological oxygen demand (BOD), chemical oxygen demand (COD), total suspended solids (TSS), temperature, total coliforms and Escherichia coli concentration on the abundance of ARGs (blaCTX-M, tetW, sul1, cmlA, and ermB) and class 1 integron-integrase gene (intI1) were investigated. Sul1 and blaCTX-M were the most and least abundant ARGs in the two WWTPs, respectively. Principal Component Analysis showed that in both of the WWTPs all ARGs and intI1 gene abundance were positively correlated with effluent temperature, but all other effluent characteristics (BOD, COD, TSS, total coliforms and E. coli) showed no significant correlation with ARGs abundance. Temperature could affect the performance of conventional activated sludge process, which in turn could affect the abundance of ARGs. The results of this study suggest that other factors than BOD, COD and TSS may affect the ARGs abundance. The predicted AR could lead to development of effective interventions and policies to combat AR in the clinical settings. However, further research is needed to determine the relationship between the AR in wastewater and clinical settings as well as the effect of other influential factors on ARGs abundance.

Mapping the spread and mobility of antibiotic resistance in wastewater due to COVID-19 surge.

Large amounts of antibiotics have been discharged into wastewater during the COVID-19 pandemic due to overuse and misuse of antibiotics to treat patients. Wastewater-based surveillance can be used as an early warning for antibiotic resistance (AR) emergence. The present study analyzed municipal wastewater corresponding to the major pandemic waves (WW1, WW2, and WW3) in India along with hospital wastewater (Ho) taken as a benchmark for AR. Commonly prescribed antibiotics during a pandemic, azithromycin and cefixime residues, were found in the range of 2.1-2.6 μg/L in Ho and WW2. Total residual antibiotic concentration was less in WW2; however, the total antibiotic resistance gene (ARG) count was 1065.6 ppm compared to 85.2 ppm in Ho. Metagenome and RT-qPCR analysis indicated a positive correlation between antibiotics and non-corresponding ARGs (blaOXA, aadA, cat, aph3, and ere), where 7.2-7.5% was carried by plasmid in the bacterial community of WW1 and WW2. Moreover, as the abundance of the dfrA and int1 genes varied most among municipal wastewater, they can be suggested as AR markers for the pandemic. The common pathogens Streptococcus, Escherichia, Shigella, and Aeromonas were putative ARG hosts in metagenome-assembled genomes. The ARG profile and antibiotic levels varied between municipal wastewaters but were fairly similar for WW2 and Ho, suggesting the impact of the pandemic in shaping the resistome pattern. The study provides insights into the resistome dynamic, AR markers, and host-ARG association in wastewater during the COVID-19 surge. Continued surveillance and identification of intervention points for AR beyond the pandemic are essential to curbing the environmental spread of ARGs in the near future.

Antibiotic resistance in wastewater: Indian scenario

The surge of Antibiotic Resistant Bacteria (ARB) in the environment is poised to be the next health threat. World Health Organisation's (WHO's) Global Antimicrobial Surveillance System (GLASS) report indicates that developing countries may be at a greater risk. Among various factors, the major driver here could be untreated wastewater and poor sanitation. Bacteria are extremely adaptable to their surroundings and develop Antimicrobial Resistance (AMR) when exposed to antibiotics and other pollutants that cause microbial stress. Thus, untreated domestic wastewater drains could easily become hotspots for the occurrence of ARBs. This study reports surveillance of sewage-carrying drains across four urban cities in India and demonstrated the presence of ARBs in the bacterial community against 7 classes of antibiotics, namely, β-Lactams, Chloramphenicol, Glycopeptides, Macrolides, Tetracycline, Third Generation Cephalosporin, and Quinolones. Untreated domestic wastewater flowing in target drains was collected twice a month, for a period of six months and the microbial community was subjected to Antibiotic Susceptibility Testing (AST) by plate assays. The zone of inhibition was recorded and interpreted as per the interpretive chart of The Clinical & Laboratory Standards Institute (CLSI) & The European Committee on Antimicrobial Susceptibility Testing (EUCAST). The total number of samples showing resistance against antibiotics was used to define an Antibiotic Resistance Index (ARI), calculated for all 20 sampling sites (drains). Results demonstrated that the highest ARI was observed in Delhi and Mumbai, ranging from 0.81 to 0.92 in Delhi and 0.49–0.56 in Mumbai. This surveillance study reveals the antibiotic resistance pattern of the representative bacterial community in the drains and goes beyond few targeted bacterial species. The alarming presence of antibiotic resistant bacterial community highlights the concern of ARBs being the next looming health threat. This report aims to demonstrates the importance of considering sewage surveillance on routine basis by state authorities.

Bacterial cell lysis induced by pre-acidification greatly promoting the degradation of carried antibiotic resistance genes in zero-valent iron activated Fenton-like processes

Zero-valent iron (ZVI) activated Fenton-like processes have been proposed to inactivate ARB that carry diverse antibiotic resistance genes (ARGs) in wastewater, whereas they exhibit very low capacity of degrading ARB-carrying ARGs. Our previous study has revealed that pre-acidification can make cell inactivation and lysis. Thus, the present study investigated the effect of pre-acidification on the inactivation of a multi-drug resistant bacterium Pseudomonas putida MX-2 and the simultaneous degradation of carried seven ARGs by ZVI/H2O2 and ZVI/PDS processes. Results showed that the pre-acidification of bacterial solution to acidic pH greatly promoted the degradation of carried ARGs from 0.14 to 0.17 logs to 2.48–2.97 logs at the meanwhile of almost completely inactivating P. putida MX-2 in both ZVI/H2O2 and ZVI/peroxydisulfate (PDS) processes. Moreover, the pre-acidification seriously lysed the bacterial cells, released the carried ARGs into the extracellular environment, and facilitated the degradation of extracellular ARGs (e-ARGs) by the generated radicals. During the degradation of the resultant e-ARGs, they were fragmented to short fragments by ZVI/H2O2 and ZVI/PDS processes. The treatment of real wastewater containing P. putida MX-2 further validated the promoting effect of pre-acidification on the degradation of carried ARGs. Therefore, it is extremely crucial to lyse the cells of ARB efficiently via acidification or other methods to promote the degradation of carried ARGs greatly, which may also be applicable for other advanced oxidation processes to reduce antibiotic resistance in wastewater effectively.

A proposed framework for the identification of indicator genes for monitoring antibiotic resistance in wastewater: Insights from metagenomic sequencing

Antibiotic resistance is one of the greatest threats to global human and animal health of our time. Municipal wastewater has been identified as a hotspot of antibiotic resistance contamination to water bodies. However, there are numerous potential antibiotic resistant pathogens and their associated antibiotic resistance genes (ARGs), making it difficult to implement routine monitoring that addresses the breadth of the problem. The objective of this study was to identify candidate indicator ARGs for monitoring antibiotic resistance in wastewater and receiving water bodies. We developed a framework to identify indicator ARGs that incorporated clinical relevance, abundance in wastewater, geographic ubiquity, environmental relevance, ARG mobility, associations with mobile genetic elements, and the availability of quantitative analytical methods. To identify indicator ARGs, published metagenomic sequencing data from 191 wastewater samples originating from 64 countries across the world were obtained from online public repositories. Through ARG annotation and network analysis, this framework revealed 56 candidate indicator ARGs distributed across four modules of strongly correlated ARGs, with one ARG from each module (oqxA, ermB, sul1, and mexE) proposed as a minimally redundant monitoring target. The results of this study provide the basis for antibiotic resistance surveillance and monitoring framework in wastewater and contaminated waterways.

Antibiotic resistance in wastewater from socio-spatially different communities

Abstract   Antibiotic resistance (ABR) and the spread of multidrug-resistant organisms (MDRO) via wastewater are of global health concern. Simultaneously, wastewater-based surveillance is a promising approach to gain an overview of the local ABR situation providing complementary public health data at lower costs. Research on the general community as one of the biggest dischargers is rather scarce. The objective of this study was to explore spatial and temporal differences in the occurrence of MDRO in community wastewater from socio-spatially different districts. Wastewater from three socio-spatially different districts in the Ruhr Metropolis, Germany, was sampled monthly over a year and analysed for extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli via culture-based methods. Isolates were validated with matrix assisted laser desorption ionization time of flight mass spectrometry. Antibiotic resistance profiles were analysed via microdilution. Phenotypic ESBL-producing E. coli could be isolated from every wastewater sample. The socio-spatially disadvantaged area displayed higher ESBL-producing E. coli loads adjusted for domestic discharge and inhabitants (median: 3.8e+8 CFU/d/inh.) compared to the other two areas (socio-spatially advantaged: 1.5e+8 CFU/d/inh. and socio-spatial average: 2.0e+8 CFU/d/inh.). Aggregated into seasons, values were highest for the winter season. Resistance profiles varied only for those antibiotics, which can be administered in outpatient care, showing lower resistance levels in the socio-spatially advantaged area. The general community is an important contributor of ESBL-producing E. coli in wastewater. Spatial and seasonal influences of ESBL-producing E. coli in wastewater between different communities, depicting the human outpatient sector, are identifiable and suggest a higher ABR burden in a socio-spatially disadvantaged area and during the winter season, as well as lower resistance levels in a socio-spatially advantaged area. Key messages The general community is a relevant source of multidrug-resistant bacteria in wastewater with observable spatial and seasonal influences between socio-spatially different communities. Findings suggest small-scale differences with a higher antibiotic resistance burden in a socio-spatially disadvantaged area and lower resistance levels in a socio-spatially advantaged area.

Antibiotic Resistance in Wastewater and Its Impact on a Receiving River: A Case Study of WWTP Brno-Modřice, Czech Republic

Antibiotic resistance has become a global threat in which the anthropogenically influenced aquatic environment represents not only a reservoir for the spread of antibiotic resistant bacteria (ARB) among humans and animals but also an environment where resistance genes are introduced into natural microbial ecosystems. Wastewater is one of the sources of antibiotic resistance. The aim of this research was the evaluation of wastewater impact on the spread of antibiotic resistance in the water environment. In this study, qPCR was used to detect antibiotic resistance genes (ARGs)—blaCTX-M-15, blaCTX-M-32, ampC, blaTEM, sul1, tetM and mcr-1 and an integron detection primer (intl1). Detection of antibiotic resistant Escherichia coli was used as a complement to the observed qPCR results. Our results show that the process of wastewater treatment significantly reduces the abundances of ARGs and ARB. Nevertheless, treated wastewater affects the ARGs and ARB number in the receiving river.

Simultaneous inactivation of multidrug-resistant Escherichia coli and enterococci by peracetic acid in urban wastewater: Exposure-based kinetics and comparison with chlorine

The presence of antibiotic resistance in wastewater sparked a great interest in investigating the inactivation of antibiotic-resistant bacteria by disinfecting agents. In this study, the inactivation kinetics of multidrug-resistant E. coli and enterococci by an emerging environmentally-friendly disinfectant, peracetic acid (PAA), in wastewater and phosphate buffer at pH 6.5 and pH 7.5, were characterized. It was demonstrated that the inactivation of the studied multidrug-resistant bacteria was governed by their exposure to PAA, i.e., integral of the PAA concentration over time (integral CT or ICT). Both regimes of the PAA inactivation of bacteria, i.e., initial resistance followed by a faster inactivation, were described well by an ICT-based Chick-Watson inactivation kinetic model. In wastewater at pH 7.5, the model-predicted ICT requirements showed that the multidrug-resistant enterococci were less susceptible to PAA than E. coli, e.g., to achieve a 3-log reduction, an ICT of 32.7 mg min/L and 23.4 mg min/L was needed, respectively. No regrowth of the studied bacteria was observed after 72 h from PAA disinfection at 25 ± 1 °C. Soluble constituents of wastewater decreased the PAA inactivation of both multidrug-resistant bacteria, i.e., higher inactivation was observed in phosphate buffer than wastewater at the same pH of 7.5. In phosphate buffer, a lower pH of 6.5 resulted in higher inactivation of multidrug-resistant E. coli compared with pH 7.5, but it did not affect the PAA inactivation of multidrug-resistant enterococci. A comparison with the most commonly used chemical disinfectant, chlorine, showed higher inactivation of both multidrug-resistant bacteria by chlorine and higher chlorine decay than PAA. The results of the present study may have implications in designing a PAA disinfection process, aiming at controlling antibiotic resistance, in terms of selecting a suitable fecal indicator and optimizing disinfectant dosing.

Industrialization as a source of heavy metals and antibiotics which can enhance the antibiotic resistance in wastewater, sewage sludge and river water.

The spread of antibiotic resistance is closely related with selective pressure in the environment. Wastewater from industrialized regions is characterized by higher concentrations of these pollutants than sewage from less industrialized areas. The aim of this study was to compare the concentrations of contaminants such as antibiotics and heavy metals (HMs), and to evaluate their impact on the spread of genes encoding resistance to antimicrobial drugs in samples of wastewater, sewage sludge and river water in two regions with different levels of industrialization. The factors exerting selective pressure, which significantly contributed to the occurrence of the examined antibiotic resistance genes (ARGs), were identified. The concentrations of selected gene copy numbers conferring resistance to four groups of antibiotics as well as class 1 and 2 integron-integrase genes were determined in the analyzed samples. The concentrations of six HMs and antibiotics corresponding to genes mediated resistance from 3 classes were determined. Based on network analysis, only some of the analyzed antibiotics correlated with ARGs, while HM levels were correlated with ARG concentrations, which can confirm the important role of HMs in promoting drug resistance. The samples from a wastewater treatment plant (WWTP) located an industrialized region were characterized by higher HM contamination and a higher number of significant correlations between the analyzed variables than the samples collected from a WWTP located in a less industrialized region. These results indicated that treated wastewater released into the natural environment can pose a continuous threat to human health by transferring ARGs, antibiotics and HMs to the environment. These findings shed light on the impact of industrialization on antibiotic resistance dissemination.

Antibiotic Resistance in Wastewater Treatment Plants and Transmission Risks for Employees and Residents: The Concept of the AWARE Study.

Antibiotic resistance has become a serious global health threat. Wastewater treatment plants may become unintentional collection points for bacteria resistant to antimicrobials. Little is known about the transmission of antibiotic resistance from wastewater treatment plants to humans, most importantly to wastewater treatment plant workers and residents living in the vicinity. We aim to deliver precise information about the methods used in the AWARE (Antibiotic Resistance in Wastewater: Transmission Risks for Employees and Residents around Wastewater Treatment Plants) study. Within the AWARE study, we gathered data on the prevalence of two antibiotic resistance phenotypes, ESBL-producing E. coli and carbapenemase-producing Enterobacteriaceae, as well as on their corresponding antibiotic resistance genes isolated from air, water, and sewage samples taken from inside and outside of different wastewater treatment plants in Germany, the Netherlands, and Romania. Additionally, we analysed stool samples of wastewater treatment plant workers, nearby residents, and members of a comparison group living ≥1000 m away from the closest WWTP. To our knowledge, this is the first study investigating the potential spread of ESBL-producing E. coli, carbapenemase-producing Enterobacteriaceae, and antibiotic resistance genes from WWTPs to workers, the environment, and nearby residents. Quantifying the contribution of different wastewater treatment processes to the removal efficiency of ESBL-producing E. coli, carbapenemase-producing Enterobacteriaceae, and antibiotic resistance genes will provide us with evidence-based support for possible mitigation strategies.

Effects of tourist flows on antibiotic resistance in wastewater of a Greek island

Antibiotic resistance in wastewaters reflects the clinical resistome. This study aimed at investigating whether changes in the population size and composition in the popular tourist destination of Zakynthos island are associated with differentiation in the antibiotic resistance of environmental micro-organisms isolated from wastewater. The resistance profiles of four species (Escherichia coli, Staphylococcus sp., Pseudomonas sp. and Enterococcus sp.) were studied in 124 raw and treated municipal wastewaters from November 2015 to August 2016. Isolates were tested against their susceptibility to several antibiotics of different antimicrobial categories. Data concerning the alteration in the population size and composition, due to the tourist mobility, were collected and logistic regression models were used to examine the relationships between antimicrobial resistance and population changes. The decrease in resistance of E. coli to amoxicillin-clavulanate, of enterococci to norfloxacin and ampicillin, and of pseudomonads to almost all antibiotics tested was associated with population increase due to tourist flows. In contrast, the presence of tourists was linked to an increase in quinupristin-dalfopristin resistant enterococci. Resistance of staphylococci did not change during the tourist period. Changes in population composition in touristic areas are reflected in antibiotic resistance of environmental bacteria in wastewater. In touristic areas, where people carrying different resistomes are merged, medication should be adapted accordingly.

Nano-metal oxides naturally attenuate antibiotic resistance in wastewater: Killing antibiotic resistant bacteria by dissolution and decreasing antibiotic tolerance by attachment

Increasing discharge of waste nano metal oxides (NMOs) has become an emerging environmental issue. NMOs in wastewater co-occur with antibiotic resistant bacteria (ARB), changing their fate and associated risks in ways that are not yet well understood. In this study, the survival and antibiotic tolerance of ARB in wastewater were examined after exposure to three different NMOs at various retention times. Results indicated that NMOs killed 45.0% ~ 62.0% of ARB and decreased their antibiotic tolerance by 11.0% ~ 58.4%. The toxicity of NMOs was in the order of nZnO > nCuO > nTiO2 and was higher with longer retention times. Both direct attachment and dissolution of ions from NMOs led to their toxicities in wastewater. Notably, dissolution of ions was the main mechanism for ARB death by inducing reactive oxygen species (ROS) and destroying the cell membrane. Whereas, direct attachment was responsible for decreasing the antibiotic tolerance, causing antibiotic resistance gene mutations and modulating stress responses of transcriptome (mostly upregulation). These findings stress the role of NMOs in influencing the threat of antibiotic resistance in wastewater and provide insights for its natural attenuation by NMOs.

Comparison of Culture- and Quantitative PCR-Based Indicators of Antibiotic Resistance in Wastewater, Recycled Water, and Tap Water.

Standardized methods are needed to support monitoring of antibiotic resistance in environmental samples. Culture-based methods target species of human-health relevance, while the direct quantification of antibiotic resistance genes (ARGs) measures the antibiotic resistance potential in the microbial community. This study compared measurements of tetracycline-, sulphonamide-, and cefotaxime-resistant presumptive total and fecal coliforms and presumptive enterococci versus a suite of ARGs quantified by quantitative polymerase chain reaction (qPCR) across waste-, recycled-, tap-, and freshwater. Cross-laboratory comparison of results involved measurements on samples collected and analysed in the US and Portugal. The same DNA extracts analysed in the US and Portugal produced comparable qPCR results (variation <28%), except for blaOXA-1 gene (0%–57%). Presumptive total and fecal coliforms and cefotaxime-resistant total coliforms strongly correlated with blaCTX-M and intI1 (0.725 ≤ R2 ≤ 0.762; p < 0.0001). Further, presumptive total and fecal coliforms correlated with the Escherichia coli-specific biomarkers, gadAB, and uidA, suggesting that both methods captured fecal-sourced bacteria. The genes encoding resistance to sulphonamides (sul1 and sul2) were the most abundant, followed by genes encoding resistance to tetracyclines (tet(A) and tet(O)) and β-lactams (blaOXA-1 and, blaCTX-M), which was in agreement with the culture-based enumerations. The findings can help inform future application of methods being considered for international antibiotic resistance surveillance in the environment.

Effects of UV disinfection on phenotypes and genotypes of antibiotic-resistant bacteria in secondary effluent from a municipal wastewater treatment plant

To elucidate the effects of UV disinfection on antibiotic resistance in biologically-treated wastewater, we investigated the antibiotic resistance profiles, species of cultivable heterotrophic bacteria, and antibiotic-resistance genes (ARGs) in antibiotic-resistant bacteria before and after treatment. UV disinfection greatly changed the bacterial community structure and the antibiotic resistance in wastewater. The antibiotic resistance in wastewater samples was strongly associated with the bacterial community. The proportions of Gram-positive bacteria gradually increased with increasing UV fluence. The proportions of bacteria resistant to cephalexin, penicillin, and vancomycin all greatly decreased after UV treatment in both sampling events (July 2018 and January 2019), and those for bacteria resistant to ofloxacin, ciprofloxacin, and sulfadiazine increased, resulting from the alternative antibiotic resistance profiles among different genera. UV disinfection induced the selection of multi-antibiotic resistant (MAR) bacteria. For example, the MAR indices of Aeromonas, the dominant genus during the treatments, were significantly increased after UV irradiation (P < 0.05). The MAR index was also markedly increased (P < 0.05) at a fluence of 5 mJ/cm2 in both events. In UV10 treatment, the bacterial community structure was greatly changed. The genera with relatively low MAR indices replaced that with high MAR indices, and became the dominant genera. As a result, the MAR indices of treated samples showed a decreased trend after 10 mJ/cm2 UV irradiation. The detection frequencies of ARGs located on the chromosome varied mainly due to the evolution of the microbial community. The occurrence of ARGs (tetA, tetC, tetM, tetW, tetX, and sul1) located on plasmid DNA decreased after UV disinfection, and the average detection frequencies of tet and sul genes decreased by 15% and 6%, respectively (P < 0.05). Generally speaking, the effect of UV disinfection on the enrichment of antibiotic resistance is limited in this study, and horizontal gene transfer via the plasmids in surviving bacteria might be impaired due to the decreased abundance of ARGs on the plasmids.

Characteristics of Carbapenemase-Producing Enterobacteriaceae in Wastewater Revealed by Genomic Analysis.

Wastewater is considered a major source of antibiotic-resistant bacteria released into the environment. Here, we characterized carbapenemase-producing Enterobacteriaceae (CPE) in wastewater by whole-genome analysis. Wastewater samples (n = 40) were collected from municipal wastewater treatment plants and hospital wastewater in Japan and Taiwan. Samples were screened for CPE using selective media, and the obtained isolates were sequenced using an Illumina MiSeq. The isolates (n = 45) included the following microorganisms: Klebsiella quasipneumoniae (n = 12), Escherichia coli (n = 10), Enterobacter cloacae complex (n = 10), Klebsiella pneumoniae (n = 8), Klebsiella variicola (n = 2), Raoultella ornithinolytica (n = 1), Citrobacter freundii (n = 1), and Citrobacter amalonaticus (n = 1). Among the 45 isolates, 38 harbored at least one carbapenemase-encoding gene. Of these, the blaGES (blaGES-5, blaGES-6, and blaGES-24) genes were found in 29 isolates. The genes were situated in novel class 1 integrons, but the integron structures were different between the Japanese (In1439 with blaGES-24 and In1440 with blaGES-5) and Taiwanese (In1441 with blaGES-5 and In1442 with blaGES-6) isolates. Other carbapenemase-encoding genes (blaVIM-1, blaNDM-5, blaIMP-8, blaIMP-19, and blaKPC-2) were found in one to three isolates. Notably, class 1 integrons previously reported among clinical isolates obtained in the same regions as the present study, namely, In477 with blaIMP-19 and In73 with blaIMP-8, were found among the Japanese and Taiwanese isolates, respectively. The results indicate that CPE with various carbapenemase-encoding genes in different genetic contexts were present in biologically treated wastewater, highlighting the need to monitor for antibiotic resistance in wastewater.

Occurrence of Clinically Important Lineages, Including the Sequence Type 131 C1-M27 Subclone, among Extended-Spectrum-β-Lactamase-Producing Escherichia coli in Wastewater.

Contamination of environmental waters by extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli (ESBLEC) is of great concern. Wastewater treatment plants (WWTPs) and hospitals release large amounts of ESBLEC into the environment. In the present study, we isolated ESBLEC strains from wastewater collected from a WWTP and a hospital in Japan and performed whole-genome sequencing to characterize these strains. Genomic analysis of 54 strains (32 from the WWTP and 22 from hospital wastewater) revealed the occurrence of clinically important clonal groups with extraintestinal pathogenic E. coli status in the WWTP and hospital wastewater. Fine-scale phylogenetic analysis was performed to further characterize 15 sequence type 131 (ST131) complex strains (11 from the WWTP and 4 from hospital wastewater). These ST131 complex strains were comprised of the following different subgroups: clade A (n = 2), C1-M27 (n = 8), and C1 (non-C1-M27) (n = 1) for strains from the WWTP and clade A (n = 2), C1-M27 (n = 1), and C1 (non-C1-M27) (n = 1) for strains from hospital wastewater. The results indicate that ESBLEC strains belonging to clinically important lineages, including the C1-M27 clade, may disseminate into the environment through wastewater, highlighting the need to monitor for antibiotic resistance in wastewater.

Antibiotic resistance in wastewater: Occurrence and fate of Enterobacteriaceae producers of Class A and Class C β-lactamases

Antibiotics have been intensively used over the last decades in human and animal therapy and livestock, resulting in serious environmental and public health problems, namely due to the antibiotic residues concentration in wastewaters and to the development of antibiotic-resistant bacteria. This study aimed to access the contribution of some anthropological activities, namely urban household, hospital and a wastewater treatment plant, to the spread of antibiotic resistances in the treated wastewater released into the Mondego River, Coimbra, Portugal. Six sampling sites were selected in the wastewater network and in the river. The ampicillin-resistant Enterobacteriaceae of the water samples were enumerated, isolated and phenotypically characterized in relation to their resistance profile to 13 antibiotics. Some isolates were identified into species level and investigated for the presence of class A and class C -lactamases. Results revealed high frequency of resistance to the -lactam group, cefoxitin (53.5%), amoxicillin/clavulanic acid combination (43.5%), cefotaxime (22.7%), aztreonam (21.3) cefpirome (19.2%), ceftazidime (16.2%) and to the non--lactam group, trimethoprim/sulfamethoxazol (21.1%), tetracycline (18.2%), followed by ciprofloxacin (14.1%). The hospital effluent showed the higher rates of resistance to all antibiotic, except two (chloramphenicol and gentamicin). Similarly, higher resistance rates were detected in the wastewater treatment plant (WWTP) effluent compared with the untreated affluent. Regarding the multidrug resistance, the highest incidence was recorded in the hospital sewage and the lowest in the urban waste. The majority of the isolates altogether are potentially extended-spectrum -lactamases positive (ESBL+) (51.9%), followed by AmpC+ (44.4%) and ESBL+/AmpC+ (35.2%). The most prevalent genes among the potential ESBL producers were blaOXA (33.3%), blaTEM (24.1%) and blaCTX-M (5.6%) and among the AmpC producers were blaEBC (38.9%), blaFOX (1.9%) and blaCIT (1.9%). In conclusion, the hospital and the WWTP activities revealed to have the highest contribution to the spread of multidrug resistant bacteria in the study area. Such data is important for future management of the environmental and public health risk of these contaminants. This is the first embracing study in the water network of Coimbra region on the dissemination of antibiotic resistance determinants. Moreover, it is also the first report with the simultaneous detection of multiresistant bacteria producers of AmpC and ESBLs -lactamases in aquatic systems in Portugal.

Surveillance of antimicrobial resistance among Escherichia coli in wastewater in Stockholm during 1 year: does it reflect the resistance trends in the society?

The resistance patterns of Escherichia coli in untreated (raw) urban wastewater (UW) was monitored by repeated sampling during 1 year. Comparison with data from wastewater samples collected from hospital wastewater (HW) in the same urban area was made. A total of 1326 E. coli isolates from 17 UW samples and 451 isolates from six HW samples were analysed by typing using the PhenePlate™ system, and their susceptibility towards 10 antibiotics was determined. Resistance to at least one antibiotic was observed in 34% of the UW isolates and 55% of the HW isolates. For UW isolates, phenotypic diversity was lower among antibiotic-susceptible than among antibiotic-resistant isolates, indicating a higher presence of clonal groups among susceptible isolates. Total antibiotic resistance measured as the Multiple Antibiotic Resistance (MAR) index was 0.08 for UW compared with 0.19 for HW, and increased over time for UW isolates, indicating increasing resistance among E. coli in the urban population during the studied time period. Resistance to all included β-lactam antibiotics was detected in 2.4% of UW isolates and 14.0% of HW isolates, and 73/75 (97%) analysed isolates were confirmed to be extended-spectrum β-lactamase (including plasmid-mediated AmpC)-producing E. coli. Thus, by cultivating samples from wastewater and analysing many independent isolates per sample, increasing frequencies of antibiotic resistance in UW were detected during 1 year that may reflect increasing faecal carriage of resistant bacteria in the society. Surveillance of antibiotic resistance in wastewater could be a valuable tool for screening of resistance trends on a population level.

Antibiotic resistance in wastewater: origins, fate, and risks

Antimicrobial resistance is increasing worldwide and threatens our capacity to treat infectious diseases. Recently, special attention has been given to the environment in the cycling of antibiotic resistance from and to humans. In urban areas, wastewater treatment plants represent a critical point of resistance propagation, since the discharge of domestic, hospital, and industrial effluents into municipal collectors mixes together human commensal, pathogenic, and environmental bacteria and high loads of antibiotic residues. Today we observe that wastewater bacteria have a high prevalence of resistance to antibiotics used in the last few decades. In parallel, resistance determinants recently detected in clinical settings are regularly observed in wastewater, suggesting a rapid propagation to the environment. The residues of antimicrobial compounds and some heavy metals play an important role in the propagation of resistance, not only during wastewater treatment but also after the discharge into the environment. Currently, it is not possible to estimate the probability of resistant bacteria or genes reaching humans, but direct transmission seems highly unlikely. However, the contamination of surface water and soil may represent a source of transmission to humans, via the food chain, drinking water, or leisure activities.