Demonstrating a Comprehensive Wastewater-Based Surveillance Approach That Differentiates Globally Sourced Resistomes.

Wastewater-based surveillance (WBS) for disease monitoring is highly promising but requires consistent methodologies that incorporate predetermined objectives, targets, and metrics. Herein, we describe a comprehensive metagenomics-based approach for global surveillance of antibiotic resistance in sewage that enables assessment of 1) which antibiotic resistance genes (ARGs) are shared across regions/communities; 2) which ARGs are discriminatory; and 3) factors associated with overall trends in ARGs, such as antibiotic concentrations. Across an internationally sourced transect of sewage samples collected using a centralized, standardized protocol, ARG relative abundances (16S rRNA gene-normalized) were highest in Hong Kong and India and lowest in Sweden and Switzerland, reflecting national policy, measured antibiotic concentrations, and metal resistance genes. Asian versus European/US resistomes were distinct, with macrolide-lincosamide-streptogramin, phenicol, quinolone, and tetracycline versus multidrug resistance ARGs being discriminatory, respectively. Regional trends in measured antibiotic concentrations differed from trends expected from public sales data. This could reflect unaccounted uses, captured only by the WBS approach. If properly benchmarked, antibiotic WBS might complement public sales and consumption statistics in the future. The WBS approach defined herein demonstrates multisite comparability and sensitivity to local/regional factors.

Carriage of ESBL-producing Enterobacterales in wastewater treatment plant workers and surrounding residents - the AWARE Study.

To investigate whether wastewater treatment plant (WWTP) workers and residents living in close proximity to a WWTP have elevated carriage rates of ESBL-producing Enterobacterales, as compared to the general population. From 2018 to 2020, we carried out a cross-sectional study in Germany, the Netherlands, and Romania among WWTP workers (N = 344), nearby residents (living ≤ 300 m away from WWTPs; N = 431) and distant residents (living ≥ 1000 m away = reference group; N = 1165). We collected information on potential confounders via questionnaire. Culture of participants' stool samples was performed with ChromID®-ESBL agar plates and species identification with MALDI-TOF-MS. We used logistic regression to estimate the odds ratio (OR) for carrying ESBL-producing E. coli (ESBL-EC). Sensitivity analyses included stratification by country and interaction models using country as secondary exposure. Prevalence of ESBL-EC was 11% (workers), 29% (nearby residents), and 7% (distant residents), and higher in Romania (28%) than in Germany (7%) and the Netherlands (6%). Models stratified by country showed that within the Romanian population, WWTP workers are about twice as likely (aOR = 2.34, 95% CI: 1.22-4.50) and nearby residents about three times as likely (aOR = 3.17, 95% CI: 1.80-5.59) to be ESBL-EC carriers, when compared with distant residents. In stratified analyses by country, we found an increased risk for carriage of ESBL-EC in Romanian workers and nearby residents. This effect was higher for nearby residents than for workers, which suggests that, for nearby residents, factors other than the local WWTP could contribute to the increased carriage.

Characterization of the First OXA-10 Natural Variant with Increased Carbapenemase Activity.

While carbapenem resistance in Gram-negative bacteria is mainly due to the production of efficient carbapenemases, ß-lactamases with a narrower spectrum may also contribute to resistance when combined with additional mechanisms. OXA-10-type class D ß-lactamases, previously shown to be weak carbapenemases, could represent such a case. In this study, two novel OXA-10 variants were identified as the sole carbapenem-hydrolyzing enzymes in meropenem-resistant enterobacteria isolated from hospital wastewater and found by next-generation sequencing to express additional ß-lactam resistance mechanisms. The new variants, OXA-655 and OXA-656, were carried by two related IncQ1 broad-host-range plasmids. Compared to the sequence of OXA-10, they both harbored a Thr26Met substitution, with OXA-655 also bearing a leucine instead of a valine in position 117 of the SAV catalytic motif. Susceptibility profiling of laboratory strains replicating the natural blaOXA plasmids and of recombinant clones expressing OXA-10 and the novel variants in an isogenic background indicated that OXA-655 is a more efficient carbapenemase. The carbapenemase activity of OXA-655 is due to the Val117Leu substitution, as shown by steady-state kinetic experiments, where the kcat of meropenem hydrolysis was increased 4-fold. In contrast, OXA-655 had no activity toward oxyimino-ß-lactams, while its catalytic efficiency against oxacillin was significantly reduced. Moreover, the Val117Leu variant was more efficient against temocillin and cefoxitin. Molecular dynamics indicated that Val117Leu affects the position 117-Leu155 interaction, leading to structural shifts in the active site that may alter carbapenem alignment. The evolutionary potential of OXA-10 enzymes toward carbapenem hydrolysis combined with their spread by promiscuous plasmids indicates that they may pose a future clinical threat.

Population-level surveillance of antibiotic resistance in Escherichia coli through sewage analysis.

IntroductionThe occurrence of antibiotic resistance in faecal bacteria in sewage is likely to reflect the current local clinical resistance situation.AimThis observational study investigated the relationship between Escherichia coli resistance rates in sewage and clinical samples representing the same human populations.MethodsE. coli were isolated from eight hospital (n = 721 isolates) and six municipal (n = 531 isolates) sewage samples, over 1 year in Gothenburg, Sweden. An inexpensive broth screening method was validated against disk diffusion and applied to determine resistance against 11 antibiotics in sewage isolates. Resistance data on E. coli isolated from clinical samples from corresponding local hospital and primary care patients were collected during the same year and compared with those of the sewage isolates by linear regression.ResultsE. coli resistance rates derived from hospital sewage and hospital patients strongly correlated (r2 = 0.95 for urine and 0.89 for blood samples), as did resistance rates in E. coli from municipal sewage and primary care urine samples (r2 = 0.82). Resistance rates in hospital sewage isolates were close to those in hospital clinical isolates while resistance rates in municipal sewage isolates were about half of those measured in primary care isolates. Resistance rates in municipal sewage isolates were more stable between sampling occasions than those from hospital sewage.ConclusionOur findings provide support for development of a low-cost, sewage-based surveillance system for antibiotic resistance in E. coli, which could complement current monitoring systems and provide clinically relevant antibiotic resistance data for countries and regions where surveillance is lacking.

A Comprehensive Screening of Escherichia coli Isolates from Scandinavia's Largest Sewage Treatment Plant Indicates No Selection for Antibiotic Resistance.

There is concern that sewage treatment plants (STPs) serve as hotspots for emergence and selection of antibiotic resistant bacteria. However, field studies investigating resistance selection by comparing bacterial populations in influents and effluents have produced variable and sometimes contradictive results. Also, large taxonomic changes between influents and effluents make interpretation of studies measuring relative gene abundances ambiguous. The aim here was to investigate whether within-species selection occurs by conducting a comprehensive screening of Escherichia coli isolated from composite influent and effluent samples collected at Scandinavia's largest STP, accompanied by analyses of antibiotics residues. In total, 4028 isolates, collected on eight occasions during 18 months, were screened for resistance to seven antibiotics. Although differences in proportions of resistant E. coli between influent and effluent samples were detected for a few antibiotics on two occasions, aggregated data over time showed no such differences for any of the investigated antibiotics. Neither was there any enrichment of multiresistant or extended-spectrum beta-lactamase-producing isolates through the treatment process. Despite some antibiotics were detected at or close to concentrations predicted to provide some selective pressure, field observations of resistance profiles in E. coli do not provide support for systematic selection in the investigated STP.

Computational discovery and functional validation of novel fluoroquinolone resistance genes in public metagenomic data sets.

BACKGROUND: Fluoroquinolones are broad-spectrum antibiotics used to prevent and treat a wide range of bacterial infections. Plasmid-mediated qnr genes provide resistance to fluoroquinolones in many bacterial species and are increasingly encountered in clinical settings. Over the last decade, several families of qnr genes have been discovered and characterized, but their true prevalence and diversity still remain unclear. In particular, environmental and host-associated bacterial communities have been hypothesized to maintain a large and unknown collection of qnr genes that could be mobilized into pathogens. RESULTS: In this study we used computational methods to screen genomes and metagenomes for novel qnr genes. In contrast to previous studies, we analyzed an almost 20-fold larger dataset comprising almost 13 terabases of sequence data. In total, 362,843 potential qnr gene fragments were identified, from which 611 putative qnr genes were reconstructed. These gene sequences included all previously described plasmid-mediated qnr gene families. Fifty-two of the 611 identified qnr genes were reconstructed from metagenomes, and 20 of these were previously undescribed. All of the novel qnr genes were assembled from metagenomes associated with aquatic environments. Nine of the novel genes were selected for validation, and six of the tested genes conferred consistently decreased susceptibility to ciprofloxacin when expressed in Escherichia coli. CONCLUSIONS: The results presented in this study provide additional evidence for the ubiquitous presence of qnr genes in environmental microbial communities, expand the number of known qnr gene variants and further elucidate the diversity of this class of resistance genes. This study also strengthens the hypothesis that environmental bacterial communities act as sources of previously uncharacterized qnr genes.

Isolation of novel IncA/C and IncN fluoroquinolone resistance plasmids from an antibiotic-polluted lake.

OBJECTIVES: Antibiotic-polluted environments may function as reservoirs for novel resistance plasmids not yet encountered in pathogens. The aims of this study were to assess the potential of resistance transfer between bacteria from such environments and Escherichia coli, and to characterize the conjugative elements involved. METHODS: Sediment samples from Kazipally lake and Asanikunta tank, two Indian lakes with a history of severe pollution with fluoroquinolones, were investigated. Proportions of resistant bacteria were determined by selective cultivation, while horizontal gene transfer was studied using a GFP-tagged E. coli as recipient. Retrieved transconjugants were tested for susceptibility by Etest(®) and captured conjugative resistance elements were characterized by WGS. RESULTS: The polluted lakes harboured considerably higher proportions of ciprofloxacin-resistant and sulfamethoxazole-resistant bacteria than did other Indian and Swedish lakes included for comparison (52% versus 2% and 60% versus 7%, respectively). Resistance plasmids were captured from Kazipally lake, but not from any of the other lakes; in the case of Asanikunta tank because of high sediment toxicity. Eight unique IncA/C and IncN resistance plasmids were identified among 11 sequenced transconjugants. Five plasmids were fully assembled, and four of these carried the quinolone resistance gene qnrVC1, which has previously only been found on chromosomes. Acquired resistance genes, in the majority of cases associated with class 1 integrons, could be linked to decreased susceptibility to several different classes of antibiotics. CONCLUSIONS: Our study shows that environments heavily polluted with antibiotics contain novel multiresistance plasmids transferrable to E. coli.

Fluoroquinolones and qnr genes in sediment, water, soil, and human fecal flora in an environment polluted by manufacturing discharges.

There is increasing concern that environmental antibiotic pollution promotes transfer of resistance genes to the human microbiota. Here, fluoroquinolone-polluted river sediment, well water, irrigated farmland, and human fecal flora of local villagers within a pharmaceutical industrial region in India were analyzed for quinolone resistance (qnr) genes by quantitative PCR. Similar samples from Indian villages farther away from industrial areas, as well as fecal samples from Swedish study participants and river sediment from Sweden, were included for comparison. Fluoroquinolones were detected by MS/MS in well water and soil from all villages located within three km from industrially polluted waterways. Quinolone resistance genes were detected in 42% of well water, 7% of soil samples and in 100% and 18% of Indian and Swedish river sediments, respectively. High antibiotic concentrations in Indian sediment coincided with high abundances of qnr, whereas lower fluoroquinolone levels in well water and soil did not. We could not find support for an enrichment of qnr in fecal samples from people living in the fluoroquinolone-contaminated villages. However, as qnr was detected in 91% of all Indian fecal samples (24% of the Swedish) it suggests that the spread of qnr between people is currently a dominating transmission route.

Evaluation of culture conditions for sewage-based surveillance of antibiotic resistance in Klebsiella pneumoniae.

BACKGROUND: Recent studies have shown promise in predicting clinical antibiotic resistance rates from sewage data. Few have focused on Klebsiella pneumoniae, despite its virulence and importance as carrier of antibiotic resistance. Several media have been suggested for the isolation of K. pneumoniae from complex samples. However, comprehensive evaluations of culture protocols for isolation of K. pneumoniae from sewage are lacking. METHODS: Here, influent samples from a major Swedish sewage treatment plant were used to evaluate ten culture conditions in parallel: cultivation on Brilliant green containing Inositol-Nitrate-Deoxycholate agar (BIND), Bruce agar, Klebsiella ChromoSelect Selective agar®, MacConkey-Inositol-Carbenicillin, or Simmons Citrate Agar with Inositol (SCAI) incubated at either 37°C or 42°C for 44 h. The culture conditions were compared based on colony counts of presumed K. pneumoniae and identification precision assessed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS: The sensitivity was lowest for BIND, whereas it was similar for the other media irrespective of incubation temperature. For four media, a better precision was observed after incubation at 42°C compared to 37°C, to a large extent explained by a lower frequency of captured Klebsiella oxytoca. SCAI incubated at 42°C showed the highest precision (84.4%). By combining this protocol with subsequent antibiotic resistance screening of collected isolates, low resistance rates in sewage K. pneumoniae were revealed, potentially reflecting the local resistance landscape. CONCLUSION: When combined with downstream analyses, SCAI incubated at 42°C could be a valuable culture protocol for sewage-based studies on various aspects of K. pneumoniae epidemiology including antibiotic resistance prevalence.

Comparison of CTX-M encoding plasmids present during the early phase of the ESBL pandemic in western Sweden.

Plasmids encoding blaCTX-M genes have greatly shaped the evolution of E. coli producing extended-spectrum beta-lactamases (ESBL-E. coli) and adds to the global threat of multiresistant bacteria by promoting horizontal gene transfer (HGT). Here we screened the similarity of 47 blaCTX-M -encoding plasmids, from 45 epidemiologically unrelated and disperse ESBL-E. coli strains, isolated during the early phase (2009-2014) of the ESBL pandemic in western Sweden. Using optical DNA mapping (ODM), both similar and rare plasmids were identified. As many as 57% of the plasmids formed five ODM-plasmid groups of at least three similar plasmids per group. The most prevalent type (28%, IncIl, pMLST37) encoded blaCTX-M-15 (n = 10), blaCTX-M-3 (n = 2) or blaCTX-M-55 (n = 1). It was found in isolates of various sequence types (STs), including ST131. This could indicate ongoing local HGT as whole-genome sequencing only revealed similarities with a rarely reported, IncIl plasmid. The second most prevalent type (IncFII/FIA/FIB, F1:A2:B20) harboring blaCTX-M-27, was detected in ST131-C1-M27 isolates, and was similar to plasmids previously reported for this subclade. The results also highlight the need for local surveillance of plasmids and the importance of temporospatial epidemiological links so that detection of a prevalent plasmid is not overestimated as a potential plasmid transmission event in outbreak investigations.

A double mutant heat-labile toxin from Escherichia coli, LT(R192G/L211A), is an effective mucosal adjuvant for vaccination against Helicobacter pylori infection.

Helicobacter pylori infection in the stomach is a common cause of peptic ulcer disease and is a strong risk factor for the development of gastric adenocarcinoma, yet no effective vaccine against H. pylori infection is available to date. In mice, mucosal vaccination with H. pylori antigens when given together with cholera toxin (CT) adjuvant, but not without adjuvant, can induce protective immune responses against H. pylori infection. However, the toxicity of CT precludes its use as a mucosal adjuvant in humans. We evaluated a recently developed, essentially nontoxic double mutant Escherichia coli heat-labile toxin, LT(R192G/L211A) (dmLT), as a mucosal adjuvant in an experimental H. pylori vaccine and compared it to CT in promoting immune responses and protection against H. pylori infection in mice. Immunization via the sublingual or intragastric route with H. pylori lysate antigens and dmLT resulted in a significant decrease in bacterial load after challenge compared to that in unimmunized infection controls and to the same extent as when using CT as an adjuvant. Cellular immune responses in the sublingually immunized mice known to correlate with protection were also fully comparable when using dmLT and CT as adjuvants, resulting in enhanced in vitro proliferative and cytokine responses from spleen and mesenteric lymph node cells to H. pylori antigens. Our results suggest that dmLT is an attractive adjuvant for inclusion in a mucosal vaccine against H. pylori infection.

Functional verification of computationally predicted qnr genes.

BACKGROUND: The quinolone resistance (qnr) genes are widely distributed among bacteria. We recently developed and applied probabilistic models to identify tentative novel qnr genes in large public collections of DNA sequence data including fragmented metagenomes. FINDINGS: By using inducible recombinant expressions systems the functionality of four identified qnr candidates were evaluated in Escherichia coli. Expression of several known qnr genes as well as two novel candidates provided fluoroquinolone resistance that increased with elevated inducer concentrations. The two novel, functionally verified qnr genes are termed Vfuqnr and assembled qnr 1. Co-expression of two qnr genes suggested non-synergistic action. CONCLUSION: The combination of a computational model and recombinant expression systems provides opportunities to explore and identify novel antibiotic resistance genes in both genomic and metagenomic datasets.

Sampling Considerations for Wastewater Surveillance of Antibiotic Resistance in Fecal Bacteria.

Wastewaters can be analyzed to generate population-level data for public health surveillance, such as antibiotic resistance monitoring. To provide representative data for the contributing population, bacterial isolates collected from wastewater should originate from different individuals and not be distorted by a selection pressure in the wastewater. Here we use Escherichia coli diversity as a proxy for representativeness when comparing grab and composite sampling at a major municipal wastewater treatment plant influent and an untreated hospital effluent in Gothenburg, Sweden. All municipal samples showed high E. coli diversity irrespective of the sampling method. In contrast, a marked increase in diversity was seen for composite compared to grab samples from the hospital effluent. Virtual resampling also showed the value of collecting fewer isolates on multiple occasions rather than many isolates from a single sample. Time-kill tests where individual E. coli strains were exposed to sterile-filtered hospital wastewater showed rapid killing of antibiotic-susceptible strains and significant selection of multi-resistant strains when incubated at 20 °C, an effect which could be avoided at 4 °C. In conclusion, depending on the wastewater collection site, both sampling method and collection/storage temperature could significantly impact the representativeness of the wastewater sample.

Extensive screening reveals previously undiscovered aminoglycoside resistance genes in human pathogens.

Antibiotic resistance is a growing threat to human health, caused in part by pathogens accumulating antibiotic resistance genes (ARGs) through horizontal gene transfer. New ARGs are typically not recognized until they have become widely disseminated, which limits our ability to reduce their spread. In this study, we use large-scale computational screening of bacterial genomes to identify previously undiscovered mobile ARGs in pathogens. From ~1 million genomes, we predict 1,071,815 genes encoding 34,053 unique aminoglycoside-modifying enzymes (AMEs). These cluster into 7,612 families (<70% amino acid identity) of which 88 are previously described. Fifty new AME families are associated with mobile genetic elements and pathogenic hosts. From these, 24 of 28 experimentally tested AMEs confer resistance to aminoglycoside(s) in Escherichia coli, with 17 providing resistance above clinical breakpoints. This study greatly expands the range of clinically relevant aminoglycoside resistance determinants and demonstrates that computational methods enable early discovery of potentially emerging ARGs.

The resistome and microbiome of wastewater treatment plant workers - The AWARE study.

Urban wastewater treatment plants harbor a large collection of antibiotic resistant enteric bacteria. It is therefore reasonable to hypothesize that workers at such plants would possess a more diverse set of resistant enteric bacteria, compared to the general population. To address this hypothesis, we have compared the fecal microbiome and resistome of 87 workers at wastewater treatment plants (WWTPs) from Romania and the Netherlands to those of 87 control individuals, using shotgun metagenomics. Controlling for potential confounders, neither the total antibiotic resistance gene (ARG) abundance, nor the overall bacterial composition were significantly different between the two groups. If anything, the ARG richness was slightly lower in WWTP workers, and in a stratified analysis the total ARG abundance was significantly lower in Dutch workers compared to Dutch control participants. We identified country of residence, together with recent antibiotic intake in the Dutch population, as the largest contributing factors to the total abundance of ARGs. A striking side-finding was that sex was associated with carriage of disinfectant resistance genes, with women in both Romania and the Netherlands having significantly higher abundance compared to men. A follow up investigation including an additional 313 publicly available samples from healthy individuals from three additional countries showed that the difference was significant for three genes conferring resistance to chemicals commonly used in cosmetics and cleaning products. We therefore hypothesize that the use of cosmetics and, possibly, cleaning products leads to higher abundance of disinfectant resistance genes in the microbiome of the users. Altogether, this study shows that working at a WWTP does not lead to a higher abundance or diversity of ARGs and no large shifts in the overall gut microbial composition in comparison to participants not working at a WWTP. Instead, other factors such as country of residence, recent antibiotic intake and sex seem to play a larger role.

Wastewater treatment plants, an "escape gate" for ESCAPE pathogens.

Antibiotics are an essential tool of modern medicine, contributing to significantly decreasing mortality and morbidity rates from infectious diseases. However, persistent misuse of these drugs has accelerated the evolution of antibiotic resistance, negatively impacting clinical practice. The environment contributes to both the evolution and transmission of resistance. From all anthropically polluted aquatic environments, wastewater treatment plants (WWTPs) are probably the main reservoirs of resistant pathogens. They should be regarded as critical control points for preventing or reducing the release of antibiotics, antibiotic-resistant bacteria (ARB), and antibiotic-resistance genes (ARGs) into the natural environment. This review focuses on the fate of the pathogens Enterococcus faecium, Staphylococcus aureus, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae spp. (ESCAPE) in WWTPs. All ESCAPE pathogen species, including high-risk clones and resistance determinants to last-resort antibiotics such as carbapenems, colistin, and multi-drug resistance platforms, were detected in wastewater. The whole genome sequencing studies demonstrate the clonal relationships and dissemination of Gram-negative ESCAPE species into the wastewater via hospital effluents and the enrichment of virulence and resistance determinants of S. aureus and enterococci in WWTPs. Therefore, the efficiency of different wastewater treatment processes regarding the removal of clinically relevant ARB species and ARGs, as well as the influence of water quality factors on their performance, should be explored and monitored, along with the development of more effective treatments and appropriate indicators (ESCAPE bacteria and/or ARGs). This knowledge will allow the development of quality standards for point sources and effluents to consolidate the WWTP barrier role against the environmental and public health AR threats.

The subcellular location of antigen expressed by adenoviral vectors modifies adaptive immunity but not dependency on cross-presenting dendritic cells.

Adenoviral (Ad) vaccine vectors can generate protective immunity to various pathogens in animal studies. However, recent failures in clinical vaccine trials have underscored the need for a better understanding of how mucosal immune responses to Ad-encoded vaccine Ags are generated in vivo. In this study, we addressed whether directing Ad-encoded ovalbumin (OVA) to different subcellular compartments influences the generation of OVA-specific acquired immunity and the APCs required following i.n. immunization of mice. We show that both secreted and membrane-anchored OVA activate CD4(+) T cells, induce cytotoxic CD8(+) T lymphocytes (CTLs) and generate serum IgG. Additionally, vaginal IgG is induced when OVA is expressed at these subcellular locations, but only the secreted form generates a significant IgA response in the lungs. On the contrary, intracellular expression of OVA efficiently expands CD8(+) T cells but fails to activate CD4(+) T cells, results in poor CTL activity, and does not generate Abs. Finally, we show that regardless of the subcellular localization of OVA, conventional DCs (cDCs) are required for the activation of T cells. However, the direct transduction of conventional DCs is not essential. These findings have important implications for the improvement of Ad vector design and vaccine-induced mucosal immunity.

Antibiotic resistance in the environment.

Antibiotic resistance is a global health challenge, involving the transfer of bacteria and genes between humans, animals and the environment. Although multiple barriers restrict the flow of both bacteria and genes, pathogens recurrently acquire new resistance factors from other species, thereby reducing our ability to prevent and treat bacterial infections. Evolutionary events that lead to the emergence of new resistance factors in pathogens are rare and challenging to predict, but may be associated with vast ramifications. Transmission events of already widespread resistant strains are, on the other hand, common, quantifiable and more predictable, but the consequences of each event are limited. Quantifying the pathways and identifying the drivers of and bottlenecks for environmental evolution and transmission of antibiotic resistance are key components to understand and manage the resistance crisis as a whole. In this Review, we present our current understanding of the roles of the environment, including antibiotic pollution, in resistance evolution, in transmission and as a mere reflection of the regional antibiotic resistance situation in the clinic. We provide a perspective on current evidence, describe risk scenarios, discuss methods for surveillance and the assessment of potential drivers, and finally identify some actions to mitigate risks.

Antibiotic resistance genes of emerging concern in municipal and hospital wastewater from a major Swedish city.

The spread of antibiotic resistance among bacterial pathogens is to a large extent mediated by mobile antibiotic resistance genes (ARGs). The prevalence and geographic distribution of several newly discovered ARGs, as well as some clinically important ARGs conferring resistance to last resort antibiotics, are largely unknown. Targeted analysis of wastewater samples could allow estimations of carriage in the population connected to the sewers as well as release to the environment. Here we quantified ARGs conferring resistance to linezolid (optrA and cfr(A)) and colistin (mcr-1, -2, -3, -4 and -5) and the recently discovered gar (aminoglycoside ARG) and sul4 (sulphonamide ARG) in raw hospital and municipal wastewater as well as treated municipal wastewater during five years in a low antibiotic resistance prevalence setting (Gothenburg, Sweden). Additionally, variations in bacterial composition of the wastewaters characterized by 16S rRNA sequencing were related to the variations of the ARGs in an attempt to reveal if the presence of known or suspected bacterial host taxa could explain the presence of the ARGs in wastewater. The mcr-1, mcr-3, mcr-4, mcr-5, sul4 and gar genes were detected regularly in all types of wastewater samples while optrA and cfr(A) were detected only in hospital wastewater. The most abundant genes were mcr-3 and mcr-5, especially in municipal wastewater. The detection of optrA was restricted to a peak during one year. Most of the ARGs correlated with taxa previously described as bacterial hosts and associated with humans. Although some of the tentative hosts may include bacteria also thriving in wastewater environments, detection of the ARGs in the wastewaters could reflect their presence in the gut flora of the contributing populations. If so, they could already today or in the near future hinder treatment of bacterial infections in a setting where they currently are rarely targeted/detected during clinical surveillance.

Long-read metagenomic sequencing reveals shifts in associations of antibiotic resistance genes with mobile genetic elements from sewage to activated sludge.

BACKGROUND: There is concern that the microbially rich activated sludge environment of wastewater treatment plants (WWTPs) may contribute to the dissemination of antibiotic resistance genes (ARGs). We applied long-read (nanopore) sequencing to profile ARGs and their neighboring genes to illuminate their fate in the activated sludge treatment by comparing their abundance, genetic locations, mobility potential, and bacterial hosts within activated sludge relative to those in influent sewage across five WWTPs from three continents. RESULTS: The abundances (gene copies per Gb of reads, aka gc/Gb) of all ARGs and those carried by putative pathogens decreased 75-90% from influent sewage (192-605 gc/Gb) to activated sludge (31-62 gc/Gb) at all five WWTPs. Long reads enabled quantification of the percent abundance of ARGs with mobility potential (i.e., located on plasmids or co-located with other mobile genetic elements (MGEs)). The abundance of plasmid-associated ARGs decreased at four of five WWTPs (from 40-73 to 31-68%), and ARGs co-located with transposable, integrative, and conjugative element hallmark genes showed similar trends. Most ARG-associated elements decreased 0.35-13.52% while integrative and transposable elements displayed slight increases at two WWTPs (1.4-2.4%). While resistome and taxonomic compositions both shifted significantly, host phyla for chromosomal ARG classes remained relatively consistent, indicating vertical gene transfer via active biomass growth in activated sludge as the key pathway of chromosomal ARG dissemination. CONCLUSIONS: Overall, our results suggest that the activated sludge process acted as a barrier against the proliferation of most ARGs, while those that persisted or increased warrant further attention. Video abstract.