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328600.9978Impact of Vegetative Treatment Systems on Multiple Measures of Antibiotic Resistance in Agricultural Wastewater. Wastewater is an important vector of antibiotic resistant bacteria and antibiotic resistance genes (ARB/G). While there is broad agreement that ARB/G from agricultural (ag) wastewaters can be transported through the environment and may contribute to untreatable infectious disease in humans and animals, there remain large knowledge gaps surrounding applied details on the types and amounts of ARB/G associated with different agricultural wastewater treatment options and different ag production systems. This study evaluates a vegetative treatment system (VTS) built to treat the wastewater from a beef cattle feedlot. Samples were collected for three years, and plated on multiple media types to enumerate tetracycline and cefotaxime-resistant bacteria. Enterobacteriaceae isolates (n = 822) were characterized for carriage of tetracycline resistance genes, and E. coli isolates (n = 673) were phenotyped to determine multi-drug resistance (MDR) profiles. Tetracycline resistance in feedlot runoff wastewater was 2-to-3 orders of magnitude higher compared to rainfall runoff from the VTS fields, indicating efficacy of the VTA for reducing ARB over time following wastewater application. Clear differences in MDR profiles were observed based on the specific media on which a sample was plated. This result highlights the importance of method, especially in the context of isolate-based surveillance and monitoring of ARB in agricultural wastewaters.201829933547
765410.9978Impact of fertilizing with raw or anaerobically digested sewage sludge on the abundance of antibiotic-resistant coliforms, antibiotic resistance genes, and pathogenic bacteria in soil and on vegetables at harvest. The consumption of crops fertilized with human waste represents a potential route of exposure to antibiotic-resistant fecal bacteria. The present study evaluated the abundance of bacteria and antibiotic resistance genes by using both culture-dependent and molecular methods. Various vegetables (lettuce, carrots, radish, and tomatoes) were sown into field plots fertilized inorganically or with class B biosolids or untreated municipal sewage sludge and harvested when of marketable quality. Analysis of viable pathogenic bacteria or antibiotic-resistant coliform bacteria by plate counts did not reveal significant treatment effects of fertilization with class B biosolids or untreated sewage sludge on the vegetables. Numerous targeted genes associated with antibiotic resistance and mobile genetic elements were detected by PCR in soil and on vegetables at harvest from plots that received no organic amendment. However, in the season of application, vegetables harvested from plots treated with either material carried gene targets not detected in the absence of amendment. Several gene targets evaluated by using quantitative PCR (qPCR) were considerably more abundant on vegetables harvested from sewage sludge-treated plots than on vegetables from control plots in the season of application, whereas vegetables harvested the following year revealed no treatment effect. Overall, the results of the present study suggest that producing vegetable crops in ground fertilized with human waste without appropriate delay or pretreatment will result in an additional burden of antibiotic resistance genes on harvested crops. Managing human exposure to antibiotic resistance genes carried in human waste must be undertaken through judicious agricultural practice.201425172864
765320.9977The impact of municipal sewage sludge stabilization processes on the abundance, field persistence, and transmission of antibiotic resistant bacteria and antibiotic resistance genes to vegetables at harvest. Biosolids were obtained from four Ontario municipalities that vary in how the sewage sludge is treated. These included a Class B biosolids that was anaerobically digested, a Class A biosolids that were heat treated and pelletized (Propell), and two Class A biosolids that were stabilized using either the N-Viro (N-Rich) or Lystek (LysteGro) processes. Viable enteric indicator or pathogenic bacteria in the biosolids were enumerated by plate count, gene targets associated with antibiotic resistance or horizontal gene transfer were detected by PCR, and a subset of these gene targets were quantified by qPCR. Following application at commercial rates to field plots, the persistence of enteric bacteria and gene targets in soil was followed during the growing season. Carrots, radishes and lettuce were sown into the amended and unamended control plots, and the diversity and abundance of gene targets they carried at harvest determined. All three Class A biosolids carried fewer and less abundant antibiotic resistance genes than did the Class B biosolids, in particular the very alkaline N-Viro product (N-Rich). Following application, some gene targets (e.g. int1, sul1, strA/B, aadA) that are typically associated with mobile gene cassettes remained detectable throughout the growing season, whereas others (e.g. ermB, ermF, bla(OXA20)) that are not associated with cassettes became undetectable within three weeks or less. At harvest a larger number of gene targets were detected on the carrots and radishes than in the lettuce. Overall, land application of Class A biosolids will entrain fewer viable bacteria and genes associated with antibiotic resistance into crop ground than will amendment with Class B biosolids.201930316087
373030.9977Antibiotic-resistance and virulence-related genes in commercially bottled natural mineral waters. BACKGROUND: To date, the presence of antibiotics resistant genes (ARGs) and virulence-related genes (VRGs) has been evidenced in several surface waters, including natural surface water and wastewater, as well as drinking water. Bottled natural mineral waters, which are by law labelled as microbiologically pure at source, from underground aquifers, natural resurgence deposits or well suction pumps, do not undergo purification treatment, and do not experience any chemical decontamination or disinfection treatment, as in the case of drinking water from municipal conduits. The present study aimed to evaluate the presence of ARGs and VRGs, as well as the composition of microbial communities, in commercially bottled natural mineral drinking water by molecular methods. The study involved the analysis of bottled drinking water from four commercial brands. Moreover, an investigation was conducted into the potential association of known mobile elements or insertion sequences with the highlighted ARGs and VRGs. METHODS: Four commercial brands of drinking mineral bottled water were selected for analysis. A volume of 100 L from each brand was filtered to recover the microbes present in the water. The microbes successfully recovered on the filter, in conjunction with eventually other particles with a diameter of 0.22 μm or greater, or associated nucleic acids, underwent a process of DNA extraction using specific extraction kit. The extracted cell-DNA was subjected to shotgun sequencing. RESULTS: Sequence analysis revealed the presence of microbial communities associated with the water samples analyzed. Furthermore, several ARGs and VRGs were identified and, for some of them, a putative taxonomic assignment at genus level was defined. CONCLUSIONS: The results indicated that bottled drinking water may represent a potential reservoir of antibiotic resistance and virulence genes, which could persist and be transferred to other bacteria commonly found in the same water sample, as well as to microorganisms colonizing the human consumer. The use of the new molecular methods, such as next generation sequencing (NGS), could be useful for improving current methodologies for drinking water analysis, also considering their potential role of reservoir of antibiotic resistance and virulence genes, as well as the presence of potentially pathogenic microbes that cannot be detected by conventional cultural methods.202540993512
334640.9976Genes encoding antibiotic modifying enzymes conferring resistance against aminoglycosides in bacteria: Their identification and detection from wastewater. Global reporting of antibiotic resistant bacteria (ARB) bearing antibiotic resistance genes (ARGs) have increased in the past decade. Sewage systems act as breeding grounds for these pathogens. Dumping of untreated sewage effluent in river water systems have aided in their dissemination and spread. The molecular pathways circumventing antibiotics through ARGs is rising owing to overuse of these drugs. Use of aminoglycoside spectrum drugs has been increased exponentially. The genes providing resistance to these antibiotics are transferred through extra-chromosomal circular DNA elements. Polluted water bodies are ground zero for exchange of these genetic factors. Through literature survey, we shortlisted some clinically relevant genes which provide resistance against aminoglycosides and hold immense importance in present scenario. Initial screening for these genes was done on water samples collected from Swarna Rekha River channel in Gwalior District of Madhya Pradesh, India. A total of five identified genes were sequence verified using conventional PCR followed by targeted sequencing. Further, diagnostic platforms were designed for two reoccurring genes npmA & sat4(A) and their presence evaluated from wastewater samples collected from urban establishments of the district. Prevalence of these genes in sewage samples validated the broad impact of urban waste burden in polluting local water bodies. We were able to identify some indispensable and high risk aminoglycoside resistance providing genes, unreported in Indian context. This approach towards ARG screening could support risk assessment of future antibiotic resistance associated public health hazards.202539708933
765250.9976Safely coupling livestock and crop production systems: how rapidly do antibiotic resistance genes dissipate in soil following a commercial application of swine or dairy manure? Animal manures recycled onto crop production land carry antibiotic-resistant bacteria. The present study evaluated the fate in soil of selected genes associated with antibiotic resistance or genetic mobility in field plots cropped to vegetables and managed according to normal farming practice. Referenced to unmanured soil, fertilization with swine or dairy manure increased the relative abundance of the gene targets sul1, erm(B), str(B), int1, and IncW repA. Following manure application in the spring of 2012, gene copy number decayed exponentially, reaching background levels by the fall of 2012. In contrast, gene copy number following manure application in the fall of 2012 or spring of 2013 increased significantly in the weeks following application and then declined. In both cases, the relative abundance of gene copy numbers had not returned to background levels by the fall of 2013. Overall, these results suggest that under conditions characteristic of agriculture in a humid continental climate, a 1-year period following a commercial application of raw manure is sufficient to ensure that an additional soil burden of antibiotic resistance genes approaches background. The relative abundance of several gene targets exceeded background during the growing season following a spring application or an application done the previous fall. Results from the present study reinforce the advisability of treating manure prior to use in crop production systems.201424632259
345660.9976Phage particles harboring antibiotic resistance genes in fresh-cut vegetables and agricultural soil. Bacteriophages are ubiquitously distributed prokaryotic viruses that are more abundant than bacteria. As a consequence of their life cycle, phages can kidnap part of their host's genetic material, including antibiotic resistance genes (ARGs), which released phage particles transfer in a process called transduction. The spread of ARGs among pathogenic bacteria currently constitutes a serious global health problem. In this study, fresh vegetables (lettuce, spinach and cucumber), and cropland soil were screened by qPCR for ten ARGs (bla(TEM), bla(CTX-M-1) group, bla(CTX-M-9) group, bla(OXA-48), bla(VIM), mecA, sul1, qnrA, qnrS and armA) in their viral DNA fraction. The presence of ARGs in the phage DNA was analyzed before and after propagation experiments in an Escherichia coli host strain to evaluate the ability of the phage particles to infect a host. ARGs were found in the phage DNA fraction of all matrices, although with heterogeneous values. ARG prevalence was significantly higher in lettuce and soil, and the most common overall were β-lactamases. After propagation experiments, an increase in ARG densities in phage particles was observed in samples of all four matrices, confirming that part of the isolated phage particles were infectious. This study reveals the abundance of free, replicative ARG-containing phage particles in vegetable matrices and cropland soil. The particles are proposed as vehicles for resistance transfer in these environments, where they can persist for a long time, with the possibility of generating new resistant bacterial strains. Ingestion of these mobile genetic elements may also favor the emergence of new resistances, a risk not previously considered.201829567433
658270.9976Effective Treatment Strategies for the Removal of Antibiotic-Resistant Bacteria, Antibiotic-Resistance Genes, and Antibiotic Residues in the Effluent From Wastewater Treatment Plants Receiving Municipal, Hospital, and Domestic Wastewater: Protocol for a Systematic Review. BACKGROUND: The widespread and unrestricted use of antibiotics has led to the emergence and spread of antibiotic-resistant bacteria (ARB), antibiotic-resistance genes (ARGs), and antibiotic residues in the environment. Conventional wastewater treatment plants (WWTPs) are not designed for effective and adequate removal of ARB, ARGs, and antibiotic residues, and therefore, they play an important role in the dissemination of antimicrobial resistance (AMR) in the natural environment. OBJECTIVE: We will conduct a systematic review to determine the most effective treatment strategies for the removal of ARB, ARGs, and antibiotic residues from the treated effluent disposed into the environment from WWTPs that receive municipal, hospital, and domestic discharge. METHODS: We will search the MEDLINE, EMBASE, Web of Science, World Health Organization Global Index Medicus, and ProQuest Environmental Science Collection databases for full-text peer-reviewed journal articles published between January 2001 and December 2020. We will select only articles published in the English language. We will include studies that measured (1) the presence, concentration, and removal rate of ARB/ARGs going from WWTP influent to effluent, (2) the presence, concentration, and types of antibiotics in the effluent, and (3) the possible selection of ARB in the effluent after undergoing treatment processes in WWTPs. At least two independent reviewers will extract data and perform risk of bias assessment. An acceptable or narrative synthesis method will be followed to synthesize the data and present descriptive characteristics of the included studies in a tabular form. The study has been approved by the Ethics Review Board at the International Centre for Diarrhoeal Disease Research, Bangladesh (protocol number: PR-20113). RESULTS: This protocol outlines our proposed methodology for conducting a systematic review. Our results will provide an update to the existing literature by searching additional databases. CONCLUSIONS: Findings from our systematic review will inform the planning of proper treatment methods that can effectively reduce the levels of ARB, ARGs, and residual antibiotics in effluent, thus lowering the risk of the environmental spread of AMR and its further transmission to humans and animals. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/33365.202134842550
730480.9976Nanofiltration as an Efficient Tertiary Wastewater Treatment: Elimination of Total Bacteria and Antibiotic Resistance Genes from the Discharged Effluent of a Full-Scale Wastewater Treatment Plant. Wastewater reuse for agricultural irrigation still raises important public health issues regarding its safety, due to the increasing presence of emerging contaminants, such as antibiotic resistant bacteria and genes, in the treated effluents. In this paper, the potential for a commercial Desal 5 DK nanofiltration membrane to be used as a tertiary treatment in the wastewater treatment plants for a more effective elimination of these pollutants from the produced effluents was assessed on laboratory scale, using a stainless steel cross-flow cell. The obtained results showed high concentrations of total bacteria and target carbapenem and (fluoro)quinolone resistance genes (bla(KPC), bla(OXA-48), bla(NDM), bla(IMP), bla(VIM), qnrA, qnrB and qnrS) not only in the discharged, but also in the reused, effluent samples, which suggests that their use may not be entirely safe. Nevertheless, the applied nanofiltration treatment achieved removal rates superior to 98% for the total bacteria and 99.99% for all the target resistance genes present in both DNA and extracellular DNA fractions, with no significant differences for these microbiological parameters between the nanofiltered and the control tap water samples. Although additional studies are still needed to fully optimize the entire process, the use of nanofiltration membranes seems to be a promising solution to substantially increase the quality of the treated wastewater effluents.202235625274
495690.9976Rapid Identification of Plasmid Replicon Type and Coexisting Plasmid-Borne Antimicrobial Resistance Genes by S1-Pulsed-Field Gel Electrophoresis-Droplet Digital Polymerase Chain Reaction. Bacterial drug resistance is a significant food safety problem and public health threat. Plasmids carrying drug resistance genes may result in the rapid spread of resistance among different bacteria, hosts, and environments; therefore, antibiotic resistance monitoring and continuing research into the mechanisms of drug resistance are urgently needed. Southern blotting with probes for antibiotic resistance genes and even next-generation sequencing have been used previously to detect plasmid-borne resistance genes, but these approaches are complex and time-consuming. The next-generation sequencing requires strict laboratory conditions and bioinformatics analysis ability. In this study, we developed a simplified and sensitive method to detect plasmid-borne antimicrobial resistance genes and plasmid replicon types. Salmonella strains carrying plasmids of three different replicon types that contained mcr-1 and two ESBL-producing genes were used to verify the new method. The plasmids harbored by the Salmonella strains were separated by S1 nuclease treatment and pulsed-field gel electrophoresis (PFGE), then recovered and used as the templates for droplet digital polymerase chain reaction (ddPCR) to identify target genes. The target genes were present in significantly higher copy numbers on the plasmids than the background noise. These results were consistent with the plasmid sequencing results. This S1-PFGE-ddPCR method was less time-consuming to perform than Southern blot and complete plasmid sequencing. Therefore, this method represents a time-saving alternative for detecting plasmid-borne genes, and is likely to be a valuable tool for detecting coexisting plasmid-borne drug resistance genes.202133661029
7120100.9976Impact of liquid hog manure applications on antibiotic resistance genes concentration in soil and drainage water in field crops. Agricultural practices such as manure applications could contribute to the spread of antibiotic resistance genes (ARGs) within the environment. Our objective was to assess the impact of certain fertilization methods (mineral or manure) and tillage practices (reduced or conventional) on the presence of ARGs and bacteria in soil and drainage water under wheat and grain corn crops. Targeted ARGs tet(T), sul1, and bla(CTX-M-1) in liquid hog manure, soil, and water samples were quantified by qPCR. Conventional PCR was used to detect mcr-1 and mcr-2. ARGs in control plots were detected despite the absence of manure, representing an environmental reservoir of resistant microorganisms. The manure application rate higher than 39 m(3)/ha increased tet(T) and sul1 gene concentrations in soil for more than 180 days. Tillage practices had no impact on ARG concentrations in soil and water samples. The bla(CTX-M-1) gene was only detected in seven water samples in 2016, but no link was established with the treatments. The mcr-1 and mcr-2 genes were not detected in all tested samples. This study demonstrated that tet(T) and sul1 gene concentrations increased in soil after liquid hog manure application as well as in drainage water in the next weeks.202032330390
5072110.9976Integrated Sample to Detection of Carbapenem-Resistant Bacteria Extracted from Water Samples Using a Portable Gold Nanoparticle-Based Biosensor. Antimicrobial resistance (AMR) is a significant global threat and is driven by the overuse of antibiotics in both clinical and agricultural settings. This issue is further complicated by the lack of rapid surveillance tools to detect resistant bacteria in clinical, environmental, and food systems. Of particular concern is the rise in resistance to carbapenems, a critical class of beta-lactam antibiotics. Rapid detection methods are necessary for prevention and surveillance effort. This study utilized a gold nanoparticle-based plasmonic biosensor to detect three CR genes: bla(KPC-3), bla(NDM-1), and bla(OXA-1). Optical signals were analyzed using both a spectrophotometer and a smartphone app that quantified visual color changes using RGB values. This app, combined with a simple boiling method for DNA extraction and a portable thermal cycler, was used to evaluate the biosensor's potential for POC use. Advantages of the portable bacterial detection device include real time monitoring for immediate decision-making in critical situations, field and on-site testing in resource-limited settings without needing to transport samples to a centralized lab, minimal training required, automatic data analysis, storage and sharing, and reduced operational cost. Bacteria were inoculated into sterile water, river water, and turkey rinse water samples to determine the biosensor's success in detecting target genes from sample matrices. Magnetic nanoparticles were used to capture and concentrate bacteria to avoid time-consuming cultivation and separation steps. The biosensor successfully detected the target CR genes in all tested samples using three gene-specific DNA probes. Target genes were detected with a limit of detection of 2.5 ng/L or less, corresponding to ~10(3) CFU/mL of bacteria.202540942723
7104120.9976Antibiotic resistance genes load in an antibiotic free organic broiler farm. Antibiotic resistance is a serious concern for public health. Farm environments are relevant reservoirs of antibiotic resistant bacteria and antibiotic resistance genes (ARGs), thus strategies to limit the spread of ARGs from farms to the environment are needed. In this study a broiler farm, where antibiotics have never been used for any purpose, was selected to evaluate if this measure is effective in reducing the ARGs load in farm environment (FE) and in meat processing environment (MPE). Faecal samples from FE and MPE were processed for DNA extraction. Detection and quantification of the 16S rRNA gene and selected ARGs (bla(TEM), qnrS, sul2, and tetA) were carried out by PCR and digital droplet PCR (ddPCR), respectively. Generally, the relative abundance of the quantified ARGs in FE was similar or higher than that measured in intensive farms. Furthermore, apart for tetA, no differences in relative abundances of the other ARGs between FE and MPE were determined. These results suggest that the choice to not use antibiotics in broiler farming is not so effective to limit the ARGs spread in MPE and that further sources of ARGs should be considered including the preceding production phase with particular reference to the breeding stage.202235091251
7655130.9976Impact of manure fertilization on the abundance of antibiotic-resistant bacteria and frequency of detection of antibiotic resistance genes in soil and on vegetables at harvest. Consumption of vegetables represents a route of direct human exposure to bacteria found in soil. The present study evaluated the complement of bacteria resistant to various antibiotics on vegetables often eaten raw (tomato, cucumber, pepper, carrot, radish, lettuce) and how this might vary with growth in soil fertilized inorganically or with dairy or swine manure. Vegetables were sown into field plots immediately following fertilization and harvested when of marketable quality. Vegetable and soil samples were evaluated for viable antibiotic-resistant bacteria by plate count on Chromocult medium supplemented with antibiotics at clinical breakpoint concentrations. DNA was extracted from soil and vegetables and evaluated by PCR for the presence of 46 gene targets associated with plasmid incompatibility groups, integrons, or antibiotic resistance genes. Soil receiving manure was enriched in antibiotic-resistant bacteria and various antibiotic resistance determinants. There was no coherent corresponding increase in the abundance of antibiotic-resistant bacteria enumerated from any vegetable grown in manure-fertilized soil. Numerous antibiotic resistance determinants were detected in DNA extracted from vegetables grown in unmanured soil. A smaller number of determinants were additionally detected on vegetables grown only in manured and not in unmanured soil. Overall, consumption of raw vegetables represents a route of human exposure to antibiotic-resistant bacteria and resistance determinants naturally present in soil. However, the detection of some determinants on vegetables grown only in freshly manured soil reinforces the advisability of pretreating manure through composting or other stabilization processes or mandating offset times between manuring and harvesting vegetables for human consumption.201323851089
5073140.9976Parallel Detection of the Unamplified Carbapenem Resistance Genes bla(NDM-1) and bla(OXA-1) Using a Plasmonic Nano-Biosensor with a Field-Portable DNA Extraction Method. Antimicrobial resistance (AMR) is a rapidly growing global concern resulting from the overuse of antibiotics in agricultural and clinical settings. The challenge is exacerbated by the lack of rapid surveillance for resistant bacteria in clinical, environmental, and food supply settings. The increasing resistance to carbapenems, an important sub-class of beta-lactam antibiotics, is a major concern in the healthcare community. Carbapenem resistance (CR) has been found in the environment and food supply chain, where it has the potential to spread to pathogens, animals, and humans through direct or indirect contact. Rapid detection for preventative and control measures should be developed. This study utilized a gold nanoparticle-based plasmonic biosensor for the parallel detection of the CR genes bla(NDM-1) and bla(OXA-1). To explore the field portability, DNA was extracted using two methods: a commercial extraction kit and a boiling method. The results were compared between the two methods using a spectrophotometer and a cellphone application for RGB values to quantify the visual results. The results showed that the boiling method of extraction was more effective than extraction with a commercial kit for this analysis. The parallel detection of unamplified genes extracted via the boiling method is novel. When combined with other portable testing equipment, the approach has the potential to be an inexpensive, rapid, and simple on-site CR gene detection protocol.202539997014
3148150.9976Analysis of antibiotic resistance genes in pig feces during the weaning transition using whole metagenome shotgun sequencing. Antibiotics have been used in livestock production for not only treatment but also for increasing the effectiveness of animal feed, aiding animal growth, and preventing infectious diseases at the time when immunity is lowered due to stress. South Korea and the EU are among the countries that have prohibited the use of antibiotics for growth promotion in order to prevent indiscriminate use of antibiotics, as previous studies have shown that it may lead to increase in cases of antibiotic-resistant bacteria. Therefore, this study evaluated the number of antibiotic resistance genes in piglets staging from pre-weaning to weaning. Fecal samples were collected from 8 piglets just prior to weaning (21 d of age) and again one week after weaning (28 d of age). Total DNA was extracted from the 200 mg of feces collected from the 8 piglets. Whole metagenome shotgun sequencing was carried out using the Illumina Hi-Seq 2000 platform and raw sequence data were imported to Metagenomics Rapid Annotation using Subsystem Technology (MG-RAST) pipeline for microbial functional analysis. The results of this study did not show an increase in antibiotic-resistant bacteria although confirmed an increase in antibiotic-resistant genes as the consequence of changes in diet and environment during the experiment.202337093913
4177160.9976Statement on how to interpret the QPS qualification on 'acquired antimicrobial resistance genes'. The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of microorganisms intended for use in the food or feed chains. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications' which should be assessed at strain and/or product level by EFSA's Scientific Panels. The generic qualification 'the strains should not harbour any acquired antimicrobial resistance (AMR) genes to clinically relevant antimicrobials' applies to all QPS bacterial TUs. The different EFSA risk assessment areas use the same approach to assess the qualification related to AMR genes. In this statement, the terms 'intrinsic' and 'acquired' AMR genes were defined for the purpose of EFSA's risk assessments, and they apply to bacteria used in the food and feed chains. A bioinformatic approach is proposed for demonstrating the 'intrinsic'/'acquired' nature of an AMR gene. All AMR genes that confer resistance towards 'critically important', 'highly important' and 'important' antimicrobials, as defined by the World Health Organisation (WHO), found as hits, need to be considered as hazards (for humans, animals and environment) and need further assessment. Genes identified as responsible for 'intrinsic' resistance could be considered as being of no concern in the frame of the EFSA risk assessment. 'Acquired' AMR genes resulting in a resistant phenotype should be considered as a concern. If the presence of the 'acquired' AMR gene is not leading to phenotypic resistance, further case-by-case assessment is necessary.202337915981
7651170.9975Antibiotic resistance gene profile changes in cropland soil after manure application and rainfall. Land application of manure introduces gastrointestinal microbes into the environment, including bacteria carrying antibiotic resistance genes (ARGs). Measuring soil ARGs is important for active stewardship efforts to minimize gene flow from agricultural production systems; however, the variety of sampling protocols and target genes makes it difficult to compare ARG results between studies. We used polymerase chain reaction (PCR) methods to characterize and/or quantify 27 ARG targets in soils from 20 replicate, long-term no-till plots, before and after swine manure application and simulated rainfall and runoff. All samples were negative for the 10 b-lactamase genes assayed. For tetracycline resistance, only source manure and post-application soil samples were positive. The mean number of macrolide, sulfonamide, and integrase genes increased in post-application soils when compared with source manure, but at plot level only, 1/20, 5/20, and 11/20 plots post-application showed an increase in erm(B), sulI, and intI1, respectively. Results confirmed the potential for temporary blooms of ARGs after manure application, likely linked to soil moisture levels. Results highlight uneven distribution of ARG targets, even within the same soil type and at the farm plot level. This heterogeneity presents a challenge for separating effects of manure application from background ARG noise under field conditions and needs to be considered when designing studies to evaluate the impact of best management practices to reduce ARG or for surveillance. We propose expressing normalized quantitative PCR (qPCR) ARG values as the number of ARG targets per 100,000 16S ribosomal RNA genes for ease of interpretation and to align with incidence rate data.202033016404
6581180.9975Do wastewater treatment plants increase antibiotic resistant bacteria or genes in the environment? Protocol for a systematic review. BACKGROUND: Antibiotic resistance is a global public health threat. Water from human activities is collected at wastewater treatment plants where processes often do not sufficiently neutralize antibiotic resistant bacteria and genes, which are further shed into the local environment. This protocol outlines the steps to conduct a systematic review based on the Population, Exposure, Comparator and Outcome (PECO) framework, aiming at answering the question "Are antimicrobial-resistant enterobacteriaceae and antimicrobial resistance genes present (O) in air and water samples (P) taken either near or downstream or downwind or down-gradient from wastewater treatment plants (E), as compared to air and water samples taken either further away or upstream or upwind or up-gradient from such wastewater treatment plant (C)?" Presence of antimicrobial-resistant bacteria and genes will be quantitatively measured by extracting their prevalence or concentration, depending on the reviewed study. METHODS: We will search PubMed, EMBASE, the Cochrane database and Web of Science for original articles published from 1 Jan 2000 to 3 Sep 2018 with language restriction. Articles will undergo a relevance and a design screening process. Data from eligible articles will be extracted by two independent reviewers. Further, we will perform a risk of bias assessment using a decision matrix. We will synthesize and present results in narrative and tabular form and will perform a meta-analysis if heterogeneity of results allows it. DISCUSSION: Antibiotic resistance in environmental samples around wastewater treatment plants may pose a risk of exposure to workers and nearby residents. Results from the systematic review outlined in this protocol will allow to estimate the extend of exposure, to inform policy making and help to design future studies.201931806019
7126190.9975Longitudinal characterization of antimicrobial resistance genes in feces shed from cattle fed different subtherapeutic antibiotics. BACKGROUND: Environmental transmission of antimicrobial-resistant bacteria and resistance gene determinants originating from livestock is affected by their persistence in agricultural-related matrices. This study investigated the effects of administering subtherapeutic concentrations of antimicrobials to beef cattle on the abundance and persistence of resistance genes within the microbial community of fecal deposits. Cattle (three pens per treatment, 10 steers per pen) were administered chlortetracycline, chlortetracycline plus sulfamethazine, tylosin, or no antimicrobials (control). Model fecal deposits (n = 3) were prepared by mixing fresh feces from each pen into a single composite sample. Real-time PCR was used to measure concentrations of tet, sul and erm resistance genes in DNA extracted from composites over 175 days of environmental exposure in the field. The microbial communities were analyzed by quantification and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S-rRNA. RESULTS: The concentrations of 16S-rRNA in feces were similar across treatments and increased by day 56, declining thereafter. DGGE profiles of 16S-rRNA differed amongst treatments and with time, illustrating temporal shifts in microbial communities. All measured resistance gene determinants were quantifiable in feces after 175 days. Antimicrobial treatment differentially affected the abundance of certain resistance genes but generally not their persistence. In the first 56 days, concentrations of tet(B), tet(C), sul1, sul2, erm(A) tended to increase, and decline thereafter, whereas tet(M) and tet(W) gradually declined over 175 days. At day 7, the concentration of erm(X) was greatest in feces from cattle fed tylosin, compared to all other treatments. CONCLUSION: The abundance of genes coding for antimicrobial resistance in bovine feces can be affected by inclusion of antibiotics in the feed. Resistance genes can persist in feces from cattle beyond 175 days with concentrations of some genes increasing with time. Management practices that accelerate DNA degradation such as frequent land application or composting of manure may reduce the extent to which bovine feces serves as a reservoir of antimicrobial resistance.201121261985