Wastewater irrigation may introduce antibiotic residues in the soil-plant systems. This study aimed to investigate the uptake of tetracyclines by spinach and collard greens and assess associated ecological and human health risks. Synthetic wastewater spiked with 1 ppm and 10 ppm of oxytetracycline, doxycycline, and tetracycline was used to grow vegetables in a greenhouse pot experiment. The uptake and accumulation of the tetracyclines were low and residual concentrations in the soil were negligible. All the tetracyclines were detected at concentrations ranging from 1.68 to 51.41 μg/g (spinach) and 1.94–30.95 μg/g (collard greens). The accumulation rate was in a dose-response scenario with a bioconcentration factor of 6.34 mL/kg (spinach) and 2.64 mL/kg (collard greens). Oxytetracycline had the highest accumulation in leaves, followed by doxycycline and tetracycline, and the residual concentrations followed the same order. The highest residual concentration was in soils receiving 10 ppm oxytetracycline. Residual concentrations in the soil were lower than accumulated levels and exerted negligible ecological risks. Tetracyclines accumulation in spinach significantly differed between the vegetables demonstrating a subspecies difference in uptake and accumulation. Ecological risk quotient (RQ) and human health risk quotient (HQ) were below thresholds that would exert toxicity and resistance selection impacts. Although RQs and HQs are low (<0.1), this study shows that the vegetables accumulate tetracyclines from irrigation water, posing plausible human health risks to allergic individuals. Similarly, the ecological risks cannot be ignored because the synergistic and antagonistic effects of sublethal concentrations can perturb ecosystem processes.

International audience | The reuse of treated wastewater (TWW) for irrigation is widely applied to alleviate pressure on freshwater resources. However, TWW contains antibiotics that once in soils, can exert selective pressure, promoting the emergence and spread of antimicrobial resistance (AMR) in the environment. Current environmental risk assessments for antibiotic residues rely on indicators such as Predicted No Effect Concentrations (PNECs), usually determined in liquid media. These PNECs aim to predict antibiotic concentrations that may promote resistance in the environment. Given the complexity of soil matrices, few studies have established PNEC values for soil, which likely differ significantly from aquatic environments.To address this gap, we developed a simplified experimental model using soil microcosms irrigated with TWW and the antibiotic sulfamethoxazole (SMX) to estimate threshold concentrations favouring resistance transfer or/and emergence within the soil microbiome. We identified SMX concentrations between 0.01 and 0.1 mg/kgdry soil that likely increased the abundance of sulfonamide resistance genes in soil. A time window of 1 to 7 days post-exposure showed a temporary rise in sul1 and intl1 gene abundance (over 1 log/soil 16S rDNA), the appearance of SMX transformation products, and an increase in some Rhodocyclaceae. After 1.5 months of incubation and complete SMX transformation, the relative abundance of sul1 and intl1 remained about 0.5 log higher than in SMX-free controls and soils with SMX levels below 0.1 mg/kg dry soil. A persistent transformation product, 4-N-glucuronide-SMX, was also observed.Here, the estimated PNEC for SMX in soil, between 0.01 and 0.1 mg/kg, exceeds typical SMX concentrations found in soils exposed to TWW. This may suggest low impact on resistance selection for this compound in the context of TWW exposure. However further studies on other soils, water, and antibiotics need to be conducted to expand our knowledge on soil PNECs.

International audience | Antimicrobial resistance (AMR) is a burgeoning environmental concern demanding a comprehensive One Health investigation to thwart its transmission to animals and humans, ensuring food safety. Seafood, housing bacterial AMR, poses a direct threat to consumer health, amplifying the risk of hospitalization, invasive infections, and death due to compromised antimicrobial treatments. The associated antimicrobial resistance genes (ARGs) in diverse marine species can amass and transmit through various pathways, including surface contact, respiration, and feeding within food webs. Our research, focused on the English Channel and North Sea, pivotal economic areas, specifically explores the occurrence of four proposed AMR indicator genes (tet(A), blaTEM, sul1, and intI1) in a benthic food web. Analyzing 350 flatfish samples' skin, gills, and gut, our quantitative PCR (qPCR) results disclosed an overall prevalence of 71.4% for AMR indicator genes. Notably, sul1 and intI1 genes exhibited higher detection in fish skin, reaching a prevalence of 47.5%, compared to gills and gut samples. Proximity to major European ports (Le Havre, Dunkirk, Rotterdam) correlated with increased AMR gene frequencies in fish, suggesting these ports' potential role in AMR spread in marine environments. We observed a broad dispersion of indicator genes in the English Channel and the North Sea, influenced by sea currents, maritime traffic, and flatfish movements. In conclusion, sul1 and intI1 genes emerge as robust indicators of AMR contamination in the marine environment, evident in seawater and species representing a benthic food web. Further studies are imperative to delineate marine species' role in accumulating and transmitting AMR to humans via seafood consumption. This research sheds light on the urgent need for a concerted effort in comprehending and mitigating AMR risks in marine ecosystems within the context of One Health.

Seas and oceans are a global reservoir of antibiotic resistance genes (ARGs). Only a few studies investigated the dynamics of ARGs along the water column of the Black Sea, a unique environment, with a peculiar geology, biology and history of anthropogenic pollution. In this study, we analyzed metagenomic data from two sampling campaigns (2013 and 2019) collected across three different sites in the Western Black Sea at depths ranging from 5 to 2000 m. The data were processed to annotate ARGs, metal resistance genes (MRGs) and integron integrase genes. The ARG abundance was significantly higher in the deep water layers and depth was the main driver of beta-diversity both for ARGs and MRGs. Moreover, ARG and MRG abundances strongly correlated (r = 0.95). The integron integrase gene abundances and composition were not influenced by the water depth and did not correlate with ARGs. The analysis of the obtained MAGs showed that some of them harbored intI gene together with several ARGs and MRGs, suggesting the presence of multidrug resistant bacteria and that MRGs and integrons could be involved in the selection of ARGs. These results demonstrate that the Black Sea is not only an important reservoir of ARGs, but also that they accumulate in the deep water layers where co-selection with MRGs could be assumed as a relevant mechanism of their persistence.

With the induction of various emerging environmental contaminants such as antibiotic resistance genes (ARGs), environment is considered as a key indicator for the spread of antimicrobial resistance (AMR). As such, the ARGs mediated environmental pollution raises a significant public health concern worldwide. Among various genetic mechanisms that are involved in the dissemination of ARGs, integrons play a vital role in the dissemination of ARGs. Integrons are mobile genetic elements that can capture and spread ARGs among environmental settings via transmissible plasmids and transposons. Most of the ARGs are found in Gram-negative bacteria and are primarily studied for their potential role in antibiotic resistance in clinical settings. As one of the most common microorganisms, Escherichia coli (E. coli) is widely studied as an indicator carrying drug-resistant genes, so this article aims to provide an in-depth study on the spread of ARGs via integrons associated with E. coli outside clinical settings and highlight their potential role as environmental contaminants. It also focuses on multiple but related aspects that do facilitate environmental pollution, i.e. ARGs from animal sources, water treatment plants situated at or near animal farms, agriculture fields, wild birds and animals. We believe that this updated study with summarized text, will facilitate the readers to understand the primary mechanisms as well as a variety of factors involved in the transmission and spread of ARGs among animals, humans, and the environment.

The spread of carbapenemase-producing bacteria is a worldwide concern as it challenges healthcare, especially considering the insufficient development of antimicrobials. These microorganisms have been described not only in hospitals, but also in several environmental settings including recreational waters. Community exposure to antimicrobial-resistant bacteria through recreation might be relevant for human health, but risk assessment studies are lacking. Absence of effective and feasible monitoring in recreational aquatic matrices contributes to such a knowledge gap. Here, we aimed at assessing predictors of occurrence of medically relevant carbapenemase-producing bacteria in coastal waters. We quantitatively assessed recovery of carbapenemase-producing Enterobacteriaceae, Pseudomonas spp., Acinetobacter spp. and Aeromonas spp. in superficial coastal waters showing distinct pollution history across one year, and registered data regarding tide regimen, 7-days pluviosity, salinity, pH, water temperature. We analyzed data using General Estimating Equation (GEE) to assess predictors of such occurrence. Our results suggest that the sampling site had the strongest effect over concentration of these antimicrobial-resistant microorganisms, followed by pollution indexes and tide regimen. Increased salinity, advanced sampling time, water temperature, rainfall and decrease of pH were related to decrease concentrations. We provide a list of factors that could be easily monitored and further included in models aiming at predicting occurrence of carbapenemase producers in coastal waters. Our study may encourage researchers to further improve this list and validate the model proposed, so that monitoring and future public policies can be developed to control the spread of antimicrobial resistance in the environment.

Antibiotic resistance genes (ARGs) in the environment are an exposure risk to humans and animals and is emerging as a global public health concern. In this study, mercury (Hg) driven co-selection of ARGs was investigated under controlled conditions in two Australian non-agricultural soils with differing pH. Soils were spiked with increasing concentrations of inorganic Hg and left to age for 5 years. Both soils contained ARGs conferring resistance to tetracycline (tetA, tetB), sulphonamides (sul1), trimethoprim (dfrA1) and the ARG indicator class 1 integron-integrase gene, intI1, as measured by qPCR. The last resort antibiotic vancomycin resistance gene, vanB and quinolone resistance gene, qnrS were not detected. Hg driven co-selection of several ARGs namely intI1, tetA and tetB were observed in the alkaline soil within the tested Hg concentrations. No co-selection of the experimental ARGs was observed in the neutral pH soil. 16S rRNA sequencing revealed proliferation of Proteobacteria and Bacteriodetes in Hg contaminated neutral and alkaline soils respectively. Multivariate analyses revealed a strong effect of Hg, soil pH and organic carbon content on the co-selection of ARGs in the experimental soils. Additionally, although aging caused a significant reduction in Hg content, agriculturally important bacterial phyla such as Nitrospirae did not regrow in the contaminated soils. The results suggest that mercury can drive co-selection of ARGs in contaminated non-agricultural soils over five years of aging which is linked to soil microbiota shift and metal chemistry in the soil.

Anthropogenic waste streams can be major sources of antibiotic resistant microbes within the environment, creating a potential risk to public health. We examined patterns in the occurrence of a suite of antibiotic resistance genes (ARGs) and their links to enteric bacteria at a popular swimming beach in Australia that experiences intermittent contamination by sewage, with potential points of input including stormwater drains and a coastal lagoon. Samples were collected throughout a significant rainfall event (40.8 mm over 3 days) and analysed using both qPCR and 16S rRNA amplicon sequencing. Before the rainfall event, low levels of faecal indicator bacteria and a microbial source tracking human faeces (sewage) marker (Lachno3) were observed. These levels increased over 10x following rainfall. Within lagoon, drain and seawater samples, levels of the ARGs sulI, dfrA1 and qnrS increased by between 1 and 2 orders of magnitude after 20.4 mm of rain, while levels of tetA increased by an order of magnitude after a total of 40.8 mm. After 40.8 mm of rain sulI, tetA and qnrS could be detected 300 m offshore with levels remaining high five days after the rain event. Highest levels of sewage markers and ARGs were observed adjacent to the lagoon (when opened) and in-front of the stormwater drains, pinpointing these as the points of ARG input. Significant positive correlations were observed between all ARGs, and a suite of Amplicon Sequence Variants that were identified as stormwater drain indicator taxa using 16S rRNA amplicon sequencing data. Of note, some stormwater drain indicator taxa, which exhibited correlations to ARG abundance, included the human pathogens Arcobacter butzleri and Bacteroides fragilis. Given that previous research has linked high levels of ARGs in recreationally used environments to antimicrobial resistant pathogen infections, the observed patterns indicate a potentially elevated human health risk at a popular swimming beach following significant rainfall events.

Antimicrobial resistance (AMR) is a recognised threat to global health. Obtaining data on the prevalence of AMR in environmental bacteria is key to understanding drivers and routes of transmission. Here, 325 Shiga toxin negative deer faecal samples—gathered from across the Scottish mainland—were screened for the presence of AMR Escherichia coli and investigated for potential risk factors associated with AMR occurrence. E. coli with resistance to antimicrobials of clinical health concern, including carbapenems and 3rd generation cephalosporins, were targeted. Ninety-nine percent of samples yielded E. coli, and the prevalence of resistant E. coli at the level of faecal samples was 21.8% (n = 71) for tetracycline, 6.5% (n = 21) for cefpodoxime, 0.3% for ciprofloxacin (n = 1), with no recorded resistance to meropenem. Potential risk factors for tetracycline and cefpodoxime resistance were investigated. The presence of broadleaved woodlands was significantly associated with both AMR phenotypes, which may relate to land use within or around such woodlands. Associated risk factors varied across resistance phenotype and deer species, with proximity or density of horses an indicator of significantly decreased and increased risk, respectively, or tetracycline and cefpodoxime resistance in E. coli from roe deer, but not from red deer. Distance from wastewater treatment plants was a significant risk factor for tetracycline resistance in E. coli from red deer but not from roe deer. Data indicated that AMR E. coli can occur in wild deer populations that are not directly exposed to the selective pressure exerted by antimicrobial treatment. Overall, resistance to critically important antimicrobials was found to be low in the studied population, suggesting no immediate cause for concern regarding human health. Utilising existing culling frameworks, wild deer in Scotland could function well as a sentinel species for the surveillance of AMR in the Scottish environment.

Wastewater treatment plants (WWTPs) are among the main hotspots of antibiotic resistance genes (ARGs) in the environment. Previously, we demonstrated that, by increasing anthropogenic pollution, the antibiotic resistome persisted in the microbial community of rivers and lakes, independently by changes in community composition. In this study, we reanalysed the data to test for the relation of metal resistance genes (MRGs), plasmids, and integrons to the persistence of the antibiotic resistome. The experiment consisted in replicated co-cultures of riverine or lacustrine microbial communities and WWTP effluents in different proportions. Samples before (T0) and after a short period of incubation (TF) were collected and community metagenomic data were obtained by shotgun sequencing. The data were processed to annotate MRGs, plasmids, and integrases. The integrases stabilized in the aquatic environment following the degree of contamination with effluent water (in particular in one site), whereas MRGs and plasmids showed stochastic trajectories. These results confirm the potential correlation between integrons and anthropogenic pollution, and the reliability of intI1 as a pollution marker. Only in one site MRGs, plasmids, and ARGs were correlated, highlighting their partial contribution to the persistence of ARGs in surface waters.