Source Of Antibiotic Resistance Genes Research Articles

Application of machine learning for antibiotic resistance in water and wastewater: A systematic review

Antibiotic resistance (AR) is considered one of the greatest global threats in the current century, which can only be overcome if all interconnected areas of humans, animals and the environment are taken into account as part of the One Health concept proposed by the World Health Organization (WHO). Water and wastewater are among the most important environmental media of AR sources, where the phenomena are generally non-linear. Therefore, the aim of this study was to investigate the application of machine learning-based methods (MLMs) to solve AR-induced problems in water and wastewater. For this purpose, most relevant databases were searched in the period between 1987 and 2023 to systematically analyze and categorize the applications. Accordingly, the results showed that out of 12 applications, 11 (91.6%) were for shallow learning and 1 (8.3%) for deep learning. In shallow learning category, n = 6, 50% of the applications were regression and n = 4, 33.3% were classification, mainly using artificial neural networks, decision trees and Bayesian methods for the following objectives: Predicting the survival of antibiotic-resistant bacteria (ARB), determining the order of influencing parameters on AR-based scores, and identifying the major sources of antibiotic resistance genes (ARGs). In addition, only one study (8.3%) was found for clustering and no study for association. Surprisingly, deep learning had been used in only one study (8.3%) to predict ARGs sequences. Therefore, working on the knowledge gaps of AR, especially using clustering, association and deep learning methods, would be a promising option to analyze more aspects of the related problems. However, there is still a long way to go to consider and apply MLMs as unique approaches to study different aspects of AR in water and wastewater.

Co-occurrence patterns of gut microbiome, antibiotic resistome and the perturbation of dietary uptake in captive giant pandas

The microbiome is a key source of antibiotic resistance genes (ARGs), significantly influenced by diet, which highlights the interconnectedness between diet, gut microbiome, and ARGs. Currently, our understanding is limited on the co-occurrence among gut microbiome, antibiotic resistome in the captive giant panda and the perturbation of dietary uptake, especially for the composition and forms in dietary nutrition. Here, a qPCR array with 384 primer sets and 16 S rRNA gene amplicon sequencing were used to characterize the antibiotic resistome and microbiomes in panda feces, dietary bamboo, and soil around the habitat. Diet nutrients containing organic and mineral substances in soluble and insoluble forms were also quantified. Organic and mineral components in water-unextractable fractions were 7.5 to 139 and 637 to 8695 times higher than those in water-extractable portions in bamboo and feces, respectively, while the latter contributed more to the variation (67.5 %) of gut microbiota. Streptococcus, Prevotellaceae, and Bacteroides were the dominant genera in giant pandas. The ARG patterns in panda guts showed higher diversity in old individuals but higher abundance in young ones, driven directly by the bacterial community change and mobile genetic element mediation and indirectly by dietary intervention. Our results suggest that dietary nutrition mainly accounts for the shift of gut microbiota, while bacterial community and mobile genetic elements influenced the variation of gut antibiotic resistome.

Distribution of antibiotic resistant bacteria and genes in sewage and surrounding environment of Tórshavn, Faroe Islands

Several studies have investigated the effects of swimming in sewage-polluted recreational beach water, highlighting the associated health hazards. To mitigate potential pathogen transmission, it is imperative that the polluted water is released away from recreational waters and foreshores, where children tend to play. At present, domestic sewage in the Faroe Islands solely undergoes primary wastewater treatment within primary settling tanks before being discharged into the ocean. Effluents are a major anthropogenic source of antibiotic resistance genes and antibiotic resistant bacteria, which are released into the environment. The aim of this study was to investigate antibiotic resistant Gram-negative bacteria and antibiotic resistance genes in influents and effluents of wastewater subjected solely to primary treatment, along with their release into the environment during both summer and winter. Water samples were collected from influents and effluents as well as with increasing distance away from the wastewater outlet and from nearby tidepools. Samples were cultured on MacConkey agar with four different antibiotics for detection of antibiotic-resistant bacteria and antibiotic resistance genes were quantified by droplet digital PCR. All multi-drug resistant bacteria were identified using the API 20E kit. We observed an overall decrease of the abundance of Gram-negative bacteria from the effluents compared to influents, however, we observed the opposite trend in the antibiotic resistance genes. Antibiotic resistant bacteria and antibiotic resistance genes in addition to multi-drug resistant bacteria were found in the surrounding oceanic and several terrestrial tidepool samples. Of the multi-drug resistant bacteria, we found, e.g., Escherichia coli, P. aeruginosa, and A. hydrophila species, which can be pathogenic, potentially causing an infection if encountering a host. These results indicate a relatively wide pollution range of the effluents from the septic tank and treated sewage released into the environment, posing a potential hazard for both humans and wildlife.

Mapping the profiles and underlying driving mechanisms of the antibiotic resistome and microbiome within a subtropical complex river watershed

Antibiotic resistance is an escalating global concern, leading to millions of annual deaths worldwide. Human activities can impact antibiotic resistance gene (ARG) prevalence in aquatic ecosystems, but the intricate interplay between anthropogenic disturbances and river system resilience, and their respective contributions to the dynamics of different river segments, remains poorly understood. In this study, we investigate the antibiotic resistome and microbiome in water and sediment samples from two distinct sub-watersheds within a specific watershed. Results show a decrease in the number of core ARGs downstream in water, while sediments near densely populated areas exhibit an increase. PCoA ordination reveals clear geographic clustering of resistome and microbiome among samples from strong anthropogenic disturbed areas, reservoir areas, and estuary area. Co-occurrence networks highlight a higher connectivity of mobile genetic elements (MGEs) in disturbed areas compared to reservoir areas, presenting a threat to densely populated areas. Water quality parameters and antibiotics concentration were the key factors shaping the ARG profiles in sediment samples from urban regions. Overall, our study reveals distinct patterns of ARGs in sediment and water samples, emphasizing the importance of considering both anthropogenic and natural factors in comprehending and managing ARG distribution in river systems.

A Review of Antibiotic Resistance: Global Reports, Sources, Incidents, Resistance Strategies, and Control Plans

Antimicrobial resistance is a major global issue that is only getting worse. Acquired resistance is defined by the emergence of coding genes for strategies of drug evasion from antimicrobial agents. The Enterobacteriaceae family has been linked to this behavior. Antibiotics like beta-lactams and carbapenems, which are the most used types, are used to treat bacterial infections. From a clinical perspective, research on antibiotic resistance is very important because of the effects it has on human health. Furthermore, one of the rare instances of evolution that can be researched in real time is antibiotic resistance. Therefore, doctors, evolutionary biologists, and ecologists are interested in understanding the general processes involved in the acquisition of antibiotic resistance. Environmental microbes are the source of antibiotic resistance genes that are currently found in human diseases. Therefore, research on both natural and medical environments is necessary to fully comprehend the emergence of antibiotic resistance. Recent findings about the evolutionary processes underlying resistance suggest that viability costs, the founder effect, and ecological connectivity are significant barriers that control the spread of resistance from environmental bacteria to diseases.

Microbial organic fertilizer prepared by co-composting of Trichoderma dregs mitigates dissemination of resistance, virulence genes, and bacterial pathogens in soil and rhizosphere

The use of manure, mycelium dregs and other waste as organic fertilizer is the main source of antibiotic resistance genes (ARGs) and pathogens in farmland. Composting of waste may effectively remove ARGs and pathogens. However, the profiles and drivers of changes in metal resistance genes (MRGs), biocide resistance genes (BRGs), and virulence genes (VGs) in soil-crop rhizosphere systems after compost application remain largely unknown. Here, we prepared two kinds of microbial organic fertilizers (MOF) by using Trichoderma dregs (TDs) and organic fertilizer mixing method (MOF1) and TDs co-composting method (MOF2). The effects of different types and doses of MOF on resistance genes, VGs and pathogens in soil-rhizosphere system and their potential mechanisms were studied. The results showed that co-composting of TDs promoted the decomposition of organic carbon and decreased the absolute abundance of ARGs and mobile genetic elements (MGEs) by 53.4–65.0%. MOF1 application significantly increased the abundance and diversity of soil ARGs, BRGs, and VGs, while low and medium doses of MOF2 significantly decreased their abundance and diversity in soil and rhizosphere. Patterns of positive co-occurrence between MGEs and VGs/MRGs/BRGs/ARGs were observed through statistical analysis and gene arrangements. ARGs/MRGs reductions in MOF2 soil were directly driven by weakened horizontal gene transfer triggered by MGEs. Furthermore, MOF2 reduced soil BRGs/VGs levels by shifting bacterial communities (e.g., reduced bacterial host) or improving soil property. Our study provided new insights into the rational use of waste to minimize the spread of resistomes and VGs in soil.

Occurrence and fate of antibiotics and ARGs in a wastewater treatment plant with JHB process in Guangzhou, South China: Removal performance, interaction and host analysis

Wastewater treatment plants (WWTPs) are recognized as sinks for antibiotics and sources of antibiotic resistance genes (ARGs). Thus, it is crucial to understand the occurrence and fate of antibiotics and ARGs in each treatment unit to propose an optimal regulation strategy for pollution control. In this study, the occurrence and fate of antibiotics and ARGs in a WWTP from Guangzhou, southern China with Johannesburg (JHB) process, a common WWTP treatment technique in China, were investigated. Occurrence and fate of a total of 17 common antibiotics and 14 ARGs types were determined in each treatment unit in the WWTP. The contribution of each treatment unit to the total removal efficiency of antibiotics and ARGs in the WWTP were investigated. Although a high removal rate (95.32 %) was observed for total antibiotics, risk quotient (RQ) analysis showed that chloramphenicol in effluent still presented notable ecological risk. We further found that disinfection dramatically increased ARGs abundance and risk of horizontal gene transfer (HGT). Contribution of various environmental factors to microbial community structure and ARGs variation were also quantified with variation partitioning analysis (VPA). Co-occurrence network analysis further revealed the potential hosts of ARGs. This study contributed to a specific understanding of the fate of antibiotics and ARGs within the WWTP with JHB process and provided insight into the removal of antibiotics and ARGs in the wastewater.

Mass-immigration shapes the antibiotic resistome of wastewater treatment plants

Wastewater treatment plants (WWTPs) are the hotspots for the spread of antibiotic resistance genes (ARGs) into the environment. Nevertheless, a comprehensive assessment of the city-level and short-term daily (inter-day) variations of ARG profiles in the whole process (influent-INF, activated sludge-AS and effluent-EF) of WWTPs is still lacking. Here, 285 ARGs and ten mobile gene elements were monitored in seven WWTPs in Xiamen for seven days via high-throughput qPCR. The average daily load of ARGs to WWTPs was about 1.32 × 1020 copies/d, and a total of 1.56 × 1018 copies/d was discharged to the environment across the entire city. Stochastic processes were the main force determining the assembly of ARG communities during sampling campaign, with their relative importance ranked in the order of INF > EFF > AS. There're little daily variations in ARG richness, abundance, β-diversity composition as well as assembly mechanisms. The results of SourceTracker, variation partitioning analysis, and hierarchical partitioning analysis indicated that bacteria and ARGs from upstream treatment processes played an increasingly dominant role in shaping ARG communities in AS and EFF, respectively, suggesting the importance of mass-immigration of bacteria and ARGs from the source on ARG transport in wastewater treatment processes. This emphasizes the need to revise the way we mitigate ARG contamination but focus on the source of ARGs in urban wastewater.

Portable biosensor combining CRISPR/Cas12a and loop-mediated isothermal amplification for antibiotic resistance gene ermB in wastewater

Wastewater is among the main sources of antibiotic resistance genes (ARGs) in the environment, but effective methods to quickly assess ARGs on-site in wastewater are lacking. Here, using the typical ARG ermB as the target, we report a portable biosensor combining CRISPR/Cas12a and loop-mediated isothermal amplification (LAMP) for the detection of ARGs. Six primers of LAMP and the crRNA of CRISPR/Cas12a were first designed to be preamplification with LAMP and lead Cas12a to recognize the ermB via base pairing. Due to the trans-cleavage activity of CRISPR/Cas12a after amplicon recognition, ssDNA probes modified with reporter molecules were used to implement a visual assay with lateral flow test strips and fluorescence. After a simple nucleic acid extraction with magnetic beads, the constructed biosensor possesses excellent sensitivity and selectivity as low as 2.75 × 103 copies/μL using fluorescence and later flow strips in wastewater. We further evaluated the community-wide prevalence of ermB in wastewater influent and found high mass loads of ermB during different months. This user-friendly and low-cost biosensor is applicable for rapid on-site ARG detection, providing a potential point-of-use method for rapid assessments of ARG abundance in wastewater from large city areas with many wastewater treatment plants and in resource-limited rural areas.

Poultry manure-derived microorganisms as a reservoir and source of antibiotic resistance genes transferred to soil autochthonous microorganisms

Animal husbandry is increasing yearly due to the growing demand for meat and livestock products, among other reasons. To meet these demands, prophylactic antibiotics are used in the livestock industry (i.e., poultry farming) to promote health and stimulate animal growth. However, antibiotics are not fully metabolized by animals, and they are evacuated to the environment with excreta. Animal manure is used as fertilizer to reduce the volume of waste generated in the livestock sector. However, manure often contains microorganisms harboring antibiotic resistance genes (ARGs). Then, the microbiome of manure applicate to the soil may contribute to the spread of antibiotic resistance in the environment, including autochthonous soil-dwelling microorganisms. The present study was conducted during the crops growing season in Poland (May to September 2019) to determine the influence of poultry manure as well as poultry manure supplemented with selected antibiotics on the diversity of the soil microbiome in treatments that had not been previously fertilized with manure and the ability of antibiotic-resistant bacteria to transfer ARGs to other soil bacteria. Antibiotic concentrations were elevated at the beginning of the study and decreased over time. Poultry manure induced significant changes in the structure of microbial communities in soil; the diversity of the soil microbiome decreased, and the abundance of bacterial genera Bradyrhizobium, Streptomyces, and Pseudomonas, which are characteristic of the analyzed manure, increased. Over time, soil microbial diversity was restored to the state observed before the application of manure. Genes conferring resistance to multiple drugs as well as genes encoding resistance to bacitracin and aminoglycosides were the most frequently identified ARGs in the analyzed bacteria, including on mobile genetic elements. Multidrug resistance was observed in 17 bacterial taxa, whereas ARGs were identified in 32 bacterial taxa identified in the soil microbiome. The results of the study conclude that the application of poultry manure supplemented with antibiotics initially affects soil microbiome and resistome diversity but finally, the soil shows resilience and returns to its original state after time, with most antibiotic resistance genes disappearing. This phenomenon is of great importance in sustainable soil health after manure application.

Identifying sources of antibiotic resistance genes in the environment using the microbial Find, Inform, and Test framework.

Antimicrobial resistance (AMR) is an increasing public health concern for humans, animals, and the environment. However, the contributions of spatially distributed sources of AMR in the environment are not well defined. To identify the sources of environmental AMR, the novel microbial Find, Inform, and Test (FIT) model was applied to a panel of five antibiotic resistance-associated genes (ARGs), namely, erm(B), tet(W), qnrA, sul1, and intI1, quantified from riverbed sediment and surface water from a mixed-use region. A one standard deviation increase in the modeled contributions of elevated AMR from bovine sources or land-applied waste sources [land application of biosolids, sludge, and industrial wastewater (i.e., food processing) and domestic (i.e., municipal and septage)] was associated with 34-80% and 33-77% increases in the relative abundances of the ARGs in riverbed sediment and surface water, respectively. Sources influenced environmental AMR at overland distances of up to 13 km. Our study corroborates previous evidence of offsite migration of microbial pollution from bovine sources and newly suggests offsite migration from land-applied waste. With FIT, we estimated the distance-based influence range overland and downstream around sources to model the impact these sources may have on AMR at unsampled sites. This modeling supports targeted monitoring of AMR from sources for future exposure and risk mitigation efforts.

Spatiotemporal dynamics, traceability analysis, and exposure risks of antibiotic resistance genes in PM2.5 in Handan, China.

Fine particulate matter (PM2.5) seriously affects environmental air quality and human health, and antibiotic resistance genes (ARGs) in PM2.5 posed a great challenge to clinical medicine. The year of 2013-2017 was an important 5-year period for the implementation of Air Pollution Prevention and Control Action Plan (APPCAP) in China. Here, we took Handan, a PM2.5 polluted city in northern China, as the research object and analyzed ARGs in PM2.5 in winter (January) from 2013 to 2017. The results showed that the abundance of ARGs was the highest in 2013 (3.7 × 10-2 copies/16S rRNA), and ARGs were positively correlated with air quality index (AQI) (r = 0.328, P < 0.05) and PM2.5 concentration (r = 0.377, P = 0.020 < 0.05) in the 5-year period. The ARGs carried by PM2.5 in four functional regions of sewage treatment plant, steel works, university, and park showed that sul1 and qepA had higher abundance in each functional region, and the total ARG abundance in sewage treatment plant (1.3 × 10-1 copies/16S rRNA) was the highest, while lowest in park (2.0 × 10-3 copies/16S rRNA). Potential source contribution function (PSCF) and concentration-weighted trajectory (CWT) model were used to trace the pollutants at the sampling points, which indicated that the surrounding cities contributed more than quarter to the sampling points. Therefore, regional transportation reduces the spatial distribution difference of ARGs in PM2.5. The exposure dose of ARGs in different functional regions illustrated that the total inhaled dose of ARGs in sewage treatment plant (1.7 × 105 copies/d) was the highest, while lowest in park (3.2 × 104 copies/d). This study is of great significance for assessing the distribution and sources of ARGs under the clean air initiative in China.

Hydrogen peroxide treatment mitigates antibiotic resistance gene and mobile genetic element propagation in mariculture sediment

Mariculture sediments have been exchange and propagation sources of antibiotic resistance genes (ARGs). However, no efficient methods have been generated to remove ARGs from sediments. Here, we explored the impact of hydrogen peroxide (H2O2) and aeration on the efficient removal of ARGs and mobile genetic elements (MGEs) in mariculture sediments. When compared with the aeration group, the ARG abundance was 3.8–32.3% lower in the H2O2 group during the first 14 days. ARG and MGE abundances were also significantly associated with reduced total bacterial population and diversity (P < 0.05). Based on partial squares path modeling, reduction of MGEs had important roles in ARG removal from H2O2 treatments, while in the aeration group, ARG reductions were mainly determined by changes in bacterial community composition. These results suggested that H2O2 treatment represent a promising method for controlling ARG abundance after dosing feed stuff and limit the spread of ARGs in aquaculture environments.

Degradation of Antibiotic Resistance Genes by VADER with CRISPR-Cas Immunity.

The evolution and dissemination of antibiotic resistance genes (ARGs) are prompting severe health and environmental issues. While environmental processes, e.g., biological wastewater treatment, are key barriers to prevent the spread of ARGs, they are often sources of ARGs at the same time, requiring upgraded biotechnology. Here, we present VADER, a synthetic biology system for the degradation of ARGs based on CRISPR-Cas immunity, an archaeal and bacterial immune system for eliminating invading foreign DNAs, to be implemented for wastewater treatment processes. Navigated by programmable guide RNAs, VADER targets and degrades ARGs depending on their DNA sequences, and by employing an artificial conjugation machinery, IncP, it can be delivered via conjugation. The system was evaluated by degrading plasmid-borne ARGs in Escherichia coli and further demonstrated via the elimination of ARGs on the environmentally relevant RP4 plasmid in Pseudomonas aeruginosa. Next, a prototype conjugation reactor at a 10-mL scale was devised, and 100% of the target ARG was eliminated in the transconjugants receiving VADER, giving a proof of principle for the implementation of VADER in bioprocesses. By generating a nexus of synthetic biology and environmental biotechnology, we believe that our work is not only an enterprise for tackling ARG problems but also a potential solution for managing undesired genetic materials in general in the future. IMPORTANCE Antibiotic resistance has been causing severe health problems and has led to millions of deaths in recent years. Environmental processes, especially those of the wastewater treatment sector, are an important barrier to the spread of antibiotic resistance from the pharmaceutical industry, hospitals, or civil sewage. However, they have been identified as a nonnegligible source of antibiotic resistance at the same time, as antibiotic resistance with its main cause, antibiotic resistance genes (ARGs), may accumulate in biological treatment units. Here, we transplanted the CRISPR-Cas system, an immune system via programmable DNA cleavage, to tackle the antibiotic resistance problem raised in wastewater treatment processes, and we propose a new sector specialized in ARG removal with a conjugation reactor to implement the CRISPR-Cas system. Our study provides a new angle for resolving public health issues via the implementation of synthetic biology in environmental contexts at the process level.

Antibiotic resistance genes in farm animal slaughterhouse wastes in Al-Dewanyiah province, Iraq

Environment represents as a reservoir for emerging and disseminating of antibiotic resistance genes. Slaughterhouse waste is one of the important sources of antibiotic resistance genes (ARGs) even after treatment processes. This study was conducted to evaluate role of farm animal slaughterhouse in dissemination of antibiotics resistance in Al-Dewanyiah, Iraq. A total of eighty samples were collected from the central farm animal slaughterhouse. The detection was based on three mobile genetic elements and nine antibiotic resistance genes. The results showed that tetO &amp; tetK are common resistance genes in the tested samples with great relative abundance 60%. While, MGE transposon (Tn3) was detected in 80% of the tested samples. Gene encoding resistance to quinolone, methicillin, aminoglycoside and β-lactamases were also detected in the tested samples. Presence of three class of integrons as a mobile genetic were tested and the results of type 1 recorded high abundance (P&gt;0.05) as a compare with type 2 and type 3 integrons. Furthermore, concentrations of ARGs and MGEs per gram of sample were tested using qPCR. The genes encoding for tetracycline resistance and transposon (Tn3) were found in higher concentration (P&gt;0.05) (copy number) per gram of slaughterhouse sediments comparing with the selected genes. Quantification of ARGs and MGEs in the slaughterhouse wastes indicates that those wastes represent as a hotspot for dissemination of antibiotic resistance to the environment. Low darning and no treatment for slaughterhouse wastes might increase abundance of ARGs and resistant bacteria in the natural environment.

Soil antibiotic resistance genes accumulate at different rates over four decades of manure application.

Manure can be a source of antibiotic resistance genes (ARGs) that enter the soil. However, previous studies assessing ARG persistence in soil have generally lacked continuity over sampling times, consistency of location, and assessing the impact of discontinuing manure application. We evaluated both short- and long-term ARG accumulation dynamics in soil with a 40-year known history of manure use. Manure application caused a greater abundance of tetracycline, macrolide, and sulfonamide ARGs in the soil. There was an initial spike in ARG abundance resulting from manure bacteria harboring ARGs being introduced to soil, followed by resident soil bacteria out-competing them, which led to ARG dissipation within a year. However, over four decades, annual manure application caused linear or exponential ARG accumulation, and bacteria associated with ARGs differed compared to those in the short term. Eleven years after discontinuing manure application, most soil ARG levels declined but remained elevated. We systematically explored the historical accumulation of ARGs in manured soil, and provide insight into factors that affect their persistence.

Antibiotic Resistance Genes in Interconnected Surface Waters as Affected by Agricultural Activities.

Pastures have become one of the most important sources of antibiotic resistance genes (ARGs) pollution, bringing risks to human health through the environment and the food that is grown there. Another significant source of food production is greenhouse horticulture, which is typically located near pastures. Through waterways, pasture-originated ARGs may transfer to the food in greenhouses. However, how these pasture-originated ARGs spread to nearby waterways and greenhouses has been much less investigated, while this may pose risks to humans through agricultural products. We analyzed 29 ARGs related to the most used antibiotics in livestock in the Netherlands at 16 locations in an agricultural area, representing pastures, greenhouses and lakes. We found that ARGs were prevalent in all surface waters surrounding pastures and greenhouses and showed a similar composition, with sulfonamide ARGs being dominant. This indicates that both pastures and greenhouses cause antibiotic resistance pressures on neighboring waters. However, lower pressures were found in relatively larger and isolated lakes, suggesting that a larger water body or a non-agricultural green buffer zone could help reducing ARG impacts from agricultural areas. We also observed a positive relationship between the concentrations of the class 1 integron (intl1 gene)-used as a proxy for horizontal gene transfer-and ARG concentration and composition. This supports that horizontal gene transfer might play a role in dispersing ARGs through landscapes. In contrast, none of the measured four abiotic factors (phosphate, nitrate, pH and dissolved oxygen) showed any impact on ARG concentrations. ARGs from different classes co-occurred, suggesting simultaneous use of different antibiotics. Our findings help to understand the spatial patterns of ARGs, specifically the impacts of ARGs from pastures and greenhouses on each other and on nearby waterways. In this way, this study guides management aiming at reducing ARGs' risk to human health from agricultural products.

Unraveling the influence of human fecal pollution on antibiotic resistance gene levels in different receiving water bodies using crAssphage indicator gene.

Discharged wastewater treatment plant (WWTP) effluents can contaminate receiving water bodies with human feces and alter the abundance of antibiotic resistance genes (ARGs). In this study, we examined the co-occurrence of ARGs, human fecal pollution indicator crAssphage, and antibiotics in human feces and a series of connected receiving water bodies affected by human feces, including water from different treatment units of a WWTP, river, lake, and tap waters. Results showed that crAssphage was detected in 68.2 % of the studied water bodies, confirming widespread human fecal contamination. Both ARG and crAssphage abundances exhibited a distance-decay effect from the emission source to the receiving environment. Interestingly, the detected ARG abundance in the water bodies was significantly correlated with crAssphage abundance but not with the residual antibiotic concentration, demonstrating that the presence of ARG could largely be explained by the extent of fecal pollution, with no clear signs of antibiotic selection. In addition, 14 ARGs co-shared by human feces and water bodies were significantly correlated with crAssphage. Furthermore, a close evolutionary relationship was observed between the blaTEM-1 gene from human feces and aquatic environments. These results imply a potential ARG exchange between human feces and receiving water bodies. Overall, this study provides important insights into the distribution and sources of ARGs in water bodies affected by human fecal contamination.

Industrial effluents boosted antibiotic resistome risk in coastal environments

Wastewater treatment plants (WWTPs) have been regarded as an important source of antibiotic resistance genes (ARGs) in environment, but out of municipal domestic WWTPs, few evidences show how environment is affected by industrial WWTPs. Here we chose Hangzhou Bay (HZB), China as our study area, where land-based municipal and industrial WWTPs discharged their effluent into the bay for decades. We adopted high-throughput metagenomic sequencing to examine the antibiotic resistome of the WWTP effluent and coastal sediment samples. And we proposed a conceptual framework for the assessment of antibiotic resistome risk, and a new bioinformatic pipeline for the evaluation of the potential horizontal gene transfer (HGT) frequency. Our results revealed that the diversity and abundance of ARGs in the WWTP’s effluent were significantly higher than those in the sediment. Furthermore, the antibiotic resistome in the effluent-receiving area (ERA) showed significant difference from that in HZB. For the first time, we identified that industrial WWTP effluent boosted antibiotic resistome risk in coastal sediment. The crucial evidences included: 1) the proportion of ARGs derived from WWTP activated sludge (WA) was higher (14.3 %) and two high-risky polymyxin resistance genes (mcr-4 and mcr-5) were enriched in the industrial effluent receiving area; 2) the HGT potential was higher between resistant microbiome of the industrial effluent and its ERA sediment; and 3) the highest resistome risk was determined in the industrial effluent, and some biocide resistance genes located on high-risky contigs were related to long-term stress of industrial chemicals. These findings highlight the important effects of industrial activities on the development of environmental antimicrobial resistance.

Differential impacts of salinity on antibiotic resistance genes during cattle manure stockpiling are linked to mobility potentials revealed by metagenomic sequencing

Livestock manure is an important source of antibiotic resistance genes (ARGs), and its salinity level can change during stockpiling. To understand how the salinity changes affect the fate of ARGs, cattle manure was adjusted of salinity and stockpiled in laboratory microcosms at low (0.3% salt), moderate (3.0%) and high salinity levels (10.0%) for 44 days. Amongst the five ARGs (tetO, blaTEM, sul1, tetM, and ermB) and the first-class integrase (intI1) monitored by qPCR, the relative abundance of tetO and blaTEM exhibited no clear trend in response to salinity levels, while that of sul1, tetM, ermB and intI1 showed clear downward trends over time at the lower salinity levels (0.3% and 3%) but not at the high salinity level (10%). Metagenomic contig construction of cattle manure samples revealed that sul1, tetM and ermB genes were more likely to associate with mobile genetic elements (MGEs) than tetO and blaTEM, suggesting that their slower decay at higher salinity levels was either caused by horizontal gene transfer or co-selection of ARGs and osmotic stress resistant determinants. Further analysis of metagenomic contigs showed that osmotic stress resistance can also be located on MGEs or in conjunction with ARGs.