Quantification Of Antibiotic Resistance Genes Research Articles

Comparative performance of electronegative membrane filtration and automated concentrating pipette for detection of antibiotic resistance genes and microbial markers in river water samples

The target viral and bacterial concentrations in river water are essential for environmental monitoring and public health studies. Filtration-based methods are commonly employed, yet challenges arise due to recoverability and filter pore size. This study aimed to compare the performance of electronegative membrane filtration (EMF) and automated Concentrating Pipette (CP) Select (InnovaPrep) methods for quantifying antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and bacterial and viral markers in river water samples. Fifty-four river water samples were collected from upstream and downstream locations in a river in Japan. The CP Select method was modified by adding MgCl2 and using different tips. The recovery efficiencies for total coliforms and Escherichia coli were assessed, and class 1 integron-integrase gene (intI1), 16S rRNA, gene encoding sulfonamide resistance (sul1), cross-assembly phage (crAssphage), pepper mild mottle virus (PMMoV), and Escherichia coli gene (sfmD) were detected. CP Select showed recovery efficiencies of 45 %–63 % for total coliforms and 17 %–35 % for E. coli. The intI1, 16S rRNA, sul1, crAssphage, PMMoV, and sfmD concentrations using the modified CP Select method were 10.1 ± 0.5, 8.7 ± 0.2, 7.7 ± 0.2, 6.7 ± 0.2, 5.4 ± 0.2, and 3.5 ± 0.5 log10 copies/L, respectively. Higher intI1 and sul1 concentrations were observed downstream, with the highest contribution percentage (22 % and 21 %) using CP Select or EMF. The modified CP Select method with 0.05 μm tips yielded more quantifiable results for all target genes and greater PMMoV concentrations (p < 0.05). Positive correlations were found among bacterial, ARG/MGE, and viral markers (Spearman's ρ = 0.71 for 16S rRNA and sfmD, 0.88 for intI1 and sul1, and 0.64 for PMMoV and crAssphage). The modified CP Select method demonstrated effective recovery of bacteria and quantification of ARGs, MGEs, and microbial markers in river water. Further studies are required to validate these methods and confirm their applicability in diverse environmental contexts.

Quantification of antibiotic resistance genes for environmental monitoring in the Peñas Blancas River Watershed: Insights from otter feces as bioindicators

Quantification of antibiotic resistance genes for environmental monitoring in the Peñas Blancas River Watershed: Insights from otter feces as bioindicators

Design and Validation of Primer Sets for the Detection and Quantification of Antibiotic Resistance Genes in Environmental Samples by Quantitative PCR

The high prevalence of antibiotic resistant bacteria (ARB) in several environments is a great concern threatening human health. Particularly, wastewater treatment plants (WWTP) become important contributors to the dissemination of ARB to receiving water bodies, due to the inefficient management or treatment of highly antibiotic-concentrated wastewaters. Hence, it is vital to develop molecular tools that allow proper monitoring of the genes encoding resistances to these important therapeutic compounds (antibiotic resistant genes, ARGs). For an accurate quantification of ARGs, there is a need for sensitive and robust qPCR assays supported by a good design of primers and validated protocols. In this study, eleven relevant ARGs were selected as targets, including aadA and aadB (conferring resistance to aminoglycosides); ampC, blaTEM, blaSHV, and mecA (resistance to beta-lactams); dfrA1 (resistance to trimethoprim); ermB (resistance to macrolides); fosA (resistance to fosfomycin); qnrS (resistance to quinolones); and tetA(A) (resistance to tetracyclines). The in silico design of the new primer sets was performed based on the alignment of all the sequences of the target ARGs (orthology grade > 70%) deposited in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, allowing higher coverages of the ARGs’ biodiversity than those of several primers described to date. The adequate design and performance of the new molecular tools were validated in six samples, retrieved from both natural and engineered environments related to wastewater treatment. The hallmarks of the optimized qPCR assays were high amplification efficiency (> 90%), good linearity of the standard curve (R2 > 0.980), repeatability and reproducibility across experiments, and a wide linear dynamic range. The new primer sets and methodology described here are valuable tools to upgrade the monitorization of the abundance and emergence of the targeted ARGs by qPCR in WWTPs and related environments.

Characterisation of microbial communities and quantification of antibiotic resistance genes in Italian wastewater treatment plants using 16S rRNA sequencing and digital PCR

The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in humans, animals and environment is a growing threat to public health. Wastewater treatment plants (WWTPs) are crucial in mitigating the risk of environmental contamination by effectively removing contaminants before discharge. However, the persistence of ARB and ARGs even after treatment is a challenge for the management of water system.To comprehensively assess antimicrobial resistance dynamics, we conducted a one-year monitoring study in three WWTPs in central Italy, both influents and effluents. We used seasonal sampling to analyze microbial communities by 16S rRNA, as well as to determine the prevalence and behaviour of major ARGs (sul1, tetA, blaTEM, blaOXA-48, blaCTX-M-1 group, blaKPC) and the class 1 Integron (int1). Predominant genera included in order: Arcobacter, Acinetobacter, Flavobacterium, Pseudarcobacter, Bacteroides, Aeromonas, Trichococcus, Cloacibacterium, Pseudomonas and Streptococcus. A higher diversity of bacterial communities was observed in the effluents compared to the influents. Within these communities, we also identified bacteria that may be associated with antibiotic resistance and pose a significant threat to human health. The mean concentrations (in gene copies per liter, gc/L) of ARGs and int1 in untreated wastewater (absolute abundance) were as follows: sul1 (4.1 × 109), tetA (5.2 × 108), blaTEM (1.1 × 108), blaOXA-48 (2.1 × 107), blaCTX-M-1 group (1.1 × 107), blaKPC (9.4 × 105), and int1 (5.5 × 109). The mean values in treated effluents showed reductions ranging from one to three log. However, after normalizing to the 16S rRNA gene (relative abundance), it was observed that in 37.5 % (42/112) of measurements, the relative abundance of ARGs increased in effluents compared to influents. Furthermore, correlations were identified between ARGs and bacterial genera including priority pathogens. This study improves our understanding of the dynamics of ARGs and provides insights to develop more effective strategies to reduce their spread, protecting public health and preserving the future efficacy of antibiotics.

Metagenomic absolute quantification of antibiotic resistance genes and virulence factor genes-carrying bacterial genomes in anaerobic digesters

Sewage treatment works have been considered as hotspots for the dissemination of antibiotic resistance genes (ARGs). Anaerobic digestion (AD) has emerged as a promising approach for controlling the spread of ARGs while destroying biomass in sludge. Evaluating the impact of AD on ARG removal relies on the absolute quantification of ARGs. In this study, we quantified the ARG concentrations in both full-scale and lab-scale AD systems using a cellular spike-ins based absolute quantification approach. Results demonstrated that AD effectively removed 68 ± 18 %, 55 ± 12 %, and 57 ± 19 % of total ARGs in semi-continuous AD digesters, with solid retention times of 15, 20, and 25 days, respectively. The removal efficiency of total ARGs increased as the AD process progressed in the batch digesters over 40 days. A significant negative correlation was observed between digestion time and the concentrations of certain ARG types, such as beta-lactam, sulfonamide, and tetracycline. However, certain potential pathogenic antibiotic resistant bacteria (PARB) and multi-resistant high-risk ARGs-carrying populations robustly persisted throughout the AD process, regardless of the operating conditions. This study highlighted the influence of the AD process and its operating parameters on ARG removal, and revealed the broad spectrum and persistence of PARB in AD systems. These findings provided critical insights for the management of microbial hazards.

Comparison of Cefotaxime-Resistant Escherichia coli and sul1 and intI1 by qPCR for Monitoring of Antibiotic Resistance of Wastewater, Surface Water, and Recycled Water.

Awareness of the need for surveillance of antimicrobial resistance (AMR) in water environments is growing, but there is uncertainty regarding appropriate monitoring targets. Adapting culture-based fecal indicator monitoring to include antibiotics in the media provides a potentially low-tech and accessible option, while quantitative polymerase chain reaction (qPCR) targeting key genes of interest provides a broad, quantitative measure across the microbial community. The purpose of this study was to compare findings obtained from the culture of cefotaxime-resistant (cefR) Escherichia coli with two qPCR methods for quantification of antibiotic resistance genes across wastewater, recycled water, and surface waters. The culture method was a modification of US EPA Method 1603 for E. coli, in which cefotaxime is included in the medium to capture cefR strains, while qPCR methods quantified sul1 and intI1. A common standard operating procedure for each target was applied to samples collected by six water utilities across the United States and processed by two laboratories. The methods performed consistently, and all three measures reflected the same overarching trends across water types. The qPCR detection of sul1 yielded the widest dynamic range of measurement as an AMR indicator (7-log versus 3.5-log for cefR E. coli), while intI1 was the most frequently detected target (99% versus 96.5% and 50.8% for sul1 and cefR E. coli, respectively). All methods produced comparable measurements between labs (p < 0.05, Kruskal-Wallis). Further study is needed to consider how relevant each measure is to capturing hot spots for the evolution and dissemination of AMR in the environment and as indicators of AMR-associated human health risk.

Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

Distribution and quantification of antibiotic resistance genes in a large subalpine lake (Lugano Lake) and tributary rivers

Antibiotic resistance genes (ARGs) are causing increasing problems, especially in clinical settings. Nowadays, they are considered important environmental contaminants, but little is known about their fate in the environment or how they affect natural microbial populations. In the environment, especially in water affected by anthropic activities such as discharge of hospital, urban, and industrial wastewater treatment plant (WWTP) and agricultural runoff, antibiotic determinants may become part of the environmental gene pool, spread horizontally, and be ingested by humans and animals via contaminated food and drinking water. The aim of this work was to monitor long-term the presence of antibiotic resistance determinants in water samples collected from a subalpine lake and some tributary rivers located in the southern part of Switzerland, and to assess if anthropic activities could influence the distribution of antibiotic resistance genes present in water environments. We analysed water samples by qPCR to quantify five antibiotic resistance genes that confer resistance to the major classes of antibiotics used in clinical and veterinary settings (β-lactams, macrolides, tetracycline, quinolones, and sulphonamides). Water samples were collected from January 2016 to December 2021, from three rivers located in south Switzerland and from five different sites of Lugano Lake. The most abundant genes were sulII, followed by ermB, qnrS, and tetA; they were found especially in the river influenced by wastewater treatment plants and in the lake near the potable water uptake plant. We observed an overall decrease in the number of resistance genes during the three years. Our results indicate that the aquatic ecosystems monitored in this study are a reservoir of ARGs and could potentially be a setting for the transmission of resistance from the environment to humans.

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.

Quantification of antibiotic resistance genes (ARGs) in clouds at a mountain site (puy de Dôme, central France)

Antibiotic resistance in bacteria is becoming a major sanitary concern worldwide. The extensive use of large quantities of antibiotics to sustain human activity has led to the rapid acquisition and maintenance of antibiotic resistant genes (ARGs) in bacteria and to their spread into the environment. Eventually, these can be disseminated over long distances by atmospheric transport. Here, we assessed the presence of ARGs in clouds as an indicator of long-distance travel potential of antibiotic resistance in the atmosphere. We hypothesized that a variety of ARGs can reach the altitude of clouds mainly located within the free troposphere. Once incorporated in the atmosphere, they are efficiently transported and their respective concentrations should differ depending on the sources and the geographical origin of the air masses. We deployed high-flow rate impingers and collected twelve clouds between September 2019 and October 2021 at the meteorological station of the puy de Dôme summit (1465 m a.s.l., France). Total airborne bacteria concentration was assessed by flow cytometry, and ARGs subtypes of the main families of antibiotic resistance (quinolone, sulfonamide, tetracycline; glycopeptide, aminoglycoside, β-lactamase, macrolide) including one mobile genetic element (transposase) were quantified by qPCR. Our results indicate the presence of 29 different ARGs' subtypes at concentrations ranging from 1.01 × 103 to 1.61 × 104 copies m−3 of air. Clear distinctions could be observed between clouds in air masses transported over marine areas (Atlantic Ocean) and clouds influenced by continental surfaces. Specifically, quinolones (mostly qepA) resistance genes were prevalent in marine clouds (54 % of the total ARGs on average), whereas higher contributions of sulfonamide, tetracycline; glycopeptide, β-lactamase and macrolide were found in continental clouds. This study constitutes the first evidence for the presence of microbial ARGs in clouds at concentrations comparable to other natural environments. This highlights the atmosphere as routes for the dissemination of ARGs at large scale.

Improving the risk assessment of antimicrobial resistance (AMR) along the food/feed chain and from environmental reservoirs using qMRA and probabilistic modelling.

Efficient risk assessment of antimicrobial resistance (AMR) in environmental reservoirs, particularly agroecosystems, is critical for predicting threats to animal and human health due to infections unresponsive to antibiotic therapy. However, approaches currently employed for the risk assessment of AMR along the human food chain rarely rely on antibiotic resistance gene (ARG) environmental pathways connected to food production and related quantitative data. The present project aimed at improving the risk assessment related to the spread of AMR along the food/feed chain based on ARG quantification in agroecosystems and interconnected environments. The fellow received training and worked in close cooperation with the team on two ongoing research projects which involved: (i) the monitoring of ARGs in field soils and surface waters to identify and characterise food/feed chain‐associated environmental reservoirs of AMR relevant at the national level; (ii) the evaluation of ARG dynamics in relation to agricultural practice within an international project assessing biodiversity as an ecological barrier for the spread of clinically relevant ARGs in the environment. ARG quantification was performed using single/multiplex real‐time polymerase chain reaction (PCR) with tailor‐made primers/probe sets according to in‐house optimised and validated conditions. The assessment was completed by a comprehensive revision of available literature data for risk‐ranking of ARGs along with a literature review exploring AMR quantitative knowledge gaps and the role of certain AMR determinants encoded on free extracellular DNA (exDNA) in their environmental spread.

Quantification of antibiotic resistance genes in wildlife ungulates in Ngorongoro Conservation area and Mikumi National Park, Tanzania

Wild-animals can act as reservoirs for resistant bacteria and transfer of resistance genes in the environment. These genes can spread to livestock and human either directly by transmission of shared resistant bacteria, or by horizontal gene-transfer to environmental bacteria. To ascertain at what extent wild-animals carry resistance genes, eight faecal samples from buffalo, zebra and wildebeest from Ngorongoro Conservation Area (NCA) and Mikumi National Park (MNP), and four control samples from local zebu cattle grazing together with wildlife in NCA. The qPCR was carried on 14 antimicrobial resistance genes including tetracycline (tet(A), tet(B), 93 tet(C), tet(M), tet(O), tet(W), macrolide, lincosamide, streptogramin B (ermB, ermF), sulphonamide (sulI, sulII), beta-lactam (blaCTX-M-1 group, blaCMY-2, blaSHV) and glycopeptide (vanA). Samples from NCA, both wildlife and cattle were positive for 8 out of 14 resistance genes. The most prevalent genes were tet(W) and blaCMY-2 with the latter being of concern in encoding ESBL-type resistance. Three samples from Buffalo not interacting with cattle in MNP, were positive for tet(W) and blaCMY-2, and in addition for sulI. This suggests that wild ungulates on savannah, irrespective of contact with cattle, may constitute a reservoir for antimicrobial resistance determinants. Further studies are indicated to determine resistance gene-pool among wildlife animals

Antibiotic resistance and potential bacterial pathogens identified in red deer's faecal DNA.

In the last decades, the wildlife-human interface has been increasing due to several anthropogenic factors. Therefore, it is crucial to be aware of the impact of these new dynamics on the health of wild animals and their associated zoonotic disease risks. This study aimed to characterize the faecal microbiota of two populations of red deer (Cervus elaphus) by metabarcoding, with a particular focus on potential human and veterinary pathogens, and to perform an assessment of antibiotic resistance genes (ARGs) occurrence. The faecal microbiota of red deer was assessed by metabarcoding using the 16S rRNA marker, and OTUs of the genera Treponema, Yersinia, Clostridium, Mycobacterium, and Rickettsia were identified. Two of them affiliated with species more commonly regarded as pathogens (Clostridiumpiliforme and Yersinia enterocolitica). The quantification of ARGs was performed by quantitative real-time PCR, using a metagenomic approach, and the most abundant genes were found to be blaTEM , sul1, tetracycline resistance genes (tetW, tetO, and tetQ) and ermF. From these, tetO and tetW are rank II ARGs, which were recently considered future threats for human health. Our results suggest the need for screening programs for the occurrence of pathogens and ARGs in wildlife and particularly in-game species.

Quantification of Antibiotic Resistance Genes (Args) in Clouds at a Mountain Site (Puy De Dôme, Central France)

Quantification of Antibiotic Resistance Genes (Args) in Clouds at a Mountain Site (Puy De Dôme, Central France)

Quantification of antibiotic resistance genes and mobile genetic in dairy manure.

BackgroundAntibiotic resistance genes (ARGs) are considered to be emerging environmental contaminants of concern potentially posing risks to human and animal health, and this research studied the prevalence of antimicrobial resistance in dairy manure.MethodsThis study is focused on investigating prevalence of ARGs in California dairy farm manure under current common different manure management. A total of 33 manure samples were collected from multiple manure treatment conditions: (1) flushed manure (FM), (2) fresh pile (FP), (3) compost pile (CP), (4) primary lagoon (PL), and (5) secondary lagoon (SL). After DNA extraction, all fecal samples were screened by PCR for the presence of eight ARGs: four sulfonamide ARGs (sulI, sulII, sulIII, sulA), two tetracycline ARGs (tetW, tetO), two macrolide-lincosamide-streptogramin B (MLSB) ARGs (ermB, ermF). Samples were also screened for two mobile genetic elements (MGEs) (intI1, tnpA), which are responsible for dissemination of ARGs. Quantitative PCR was then used to screen all samples for five ARGs (sulII, tetW, ermF, tnpA and intI1).ResultsPrevalence of genes varied among sample types, but all genes were detectable in different manure types. Results showed that liquid-solid separation, piling, and lagoon conditions had limited effects on reducing ARGs and MGEs, and the effect was only found significant on tetW (p = 0.01). Besides, network analysis indicated that sulII was associated with tnpA (p < 0.05), and Psychrobacter and Pseudomonas as opportunistic human pathogens, were potential ARG/MGE hosts (p < 0.05). This research indicated current different manure management practices in California dairy farms has limited effects on reducing ARGs and MGEs. Improvement of different manure management in dairy farms is thus important to mitigate dissemination of ARGs into the environment.

Simultaneous sulfamethoxazole degradation with electricity generation by microbial fuel cells using Ni-MOF-74 as cathode catalysts and quantification of antibiotic resistance genes

Antibiotic wastewater presents serious challenges in water treatment. Metal-organic frameworks (MOFs) have received significant attention as promising precursors and sacrificial templates in the preparation of porous carbon-supported catalysts. Herein, we investigated the sulfamethoxazole (SMX) degradation and electrochemical performance of microbial fuel cells (MFCs) that applied as-prepared Ni-MOF-74 and Ni–N–C (Ni-MOF-74 underwent pyrolysis treatment at different temperatures) as air-cathode catalyst. Firstly, the electrocatalytic activity towards oxygen reduction reaction (ORR) of the catalyst was investigated by rotating disk electrode. The results showed that electron transfer number for Ni-MOF-74 was 2.12, while that of 800Ni–N–C was 3.44, which was close to four-electron reduction. Applying Ni-MOF-74 in MFCs, a maximum power density of 446 mW/m2 was obtained, which was close to that of 800Ni–N–C. Besides, using Ni-MOF-74 as cathode catalyst, a chemical oxygen demand removal rate of about 84% was obtained, and the degradation rate of 10 mg/L SMX was 61%. The degradation rate decreased with increasing antibiotic concentration, but the average degradation efficiency increased stepwise. Additionally, the relative abundance of resistant gene sul1 in the reactors of the new catalytic material was about 62% lower than that of sul1 in the control (Pt/C) reactors, and the relative abundance of sul2 was about 73% lower. Moreover, cost assessments related to the catalyst performance are presented. The findings of this study demonstrated that Ni-MOF-74 could be considered as a two-electron transfer ORR catalyst, and offers a promising technique for preparation of Ni–N–C for use as four-electron transfer ORR catalysts. In comparison, Ni-MOF-74 could be a promising ORR catalyst of MFCs for antibiotic degradation.

An inventory of 44 qPCR assays using hydrolysis probes operating with a unique amplification condition for the detection and quantification of antibiotic resistance genes

Early antibiotic resistance determinants (ARDs) detection in humans or animals is crucial to counteract their propagation. The ARDs quantification is fundamental to understand the perturbation caused by disruptors, such as antibiotics, during therapies. Forty-three qPCRs on the most diffused ARDs and integrons among human and animal Enterobacterales, and one on the 16S rDNA for bacteria quantification, were developed. The qPCRs, using hydrolysis probes, operated with a unique amplification condition and were tested analytically and diagnostically performing 435 reactions on five positive and negative controls for each qPCR. Diagnostic sensitivity and specificity were confirmed by PCR and genome sequencing of control isolates, demonstrating 100% performance for all qPCRs. An easy and rapid discrimination method for the epidemiologically relevant blaCTX-Ms is provided. This large, noncommercial qPCRs inventory could serve for precise quantification of ARDs, but also as a rapid screening tool for surveillance purposes, providing the basis for further high-throughput developments.

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

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

Bacterial community changes and antibiotic resistance gene quantification in microbial electrolysis cells during long-term sulfamethoxazole treatment

In this study, sulfamethoxazole served as the electron donor for microbial electrolysis cells. After 6 months of operation, the removal efficiencies of sulfamethoxazole in three microbial electrolysis cells were 77.60%, 87.55%, and 92.53% for a 3-day period and were directly proportional to the initial added concentrations. However, the removal efficiencies in the microbial electrolysis cells with open circuits and without microorganisms were only 51% and 8%, respectively. Higher sulfamethoxazole concentrations and sustained electrical stimulation caused faster bioelectrochemical reactions, thereby enhancing sulfamethoxazole degradation. Bacterial community analysis revealed that Proteobacteria and Synergistetes, which are the main functional phyla, proliferated with increased antibiotic concentrations. The qPCR results indicated that the copy numbers of antibiotic resistance genes and integrons in microbial electrolysis cell biofilms and effluents were distinctly lower than those in traditional biological treatment systems. Thus, the generation and dissemination of antibiotic resistance genes might be a diminished challenge in microbial electrolysis cells.

Use of synthesized double-stranded gene fragments as qPCR standards for the quantification of antibiotic resistance genes

Pollution of various environmental matrices by antibiotic resistance genes (ARGs) has become a growing threat to human health. For the quantitative analysis of the presence of ARGs, there is a need for sensitive and robust qPCR assays which can detect various genes from different types of DNA extracts. Fourteen ARGs were selected as target genes in this study including: blaTEM, blaOXA-1 and blaCTX-M coded for resistance to β-lactams; ermB for macrolides; tetA, tetG, tetM, tetQ, tetW and tetX for tetracyclines; sul I and sul II for sulfonamides; drfA1 and drfA12 d for trimethoprim; and integron gene intI 1 and intI 2. Chemically synthesized double-stranded gene fragments were modified using molecular biology methods and used as real-time PCR standards as well as to establish in-house qPCR assays. The ermB gene from a naturally occurring plasmid was used to compare the performance of qPCR assay with the chemically synthesized ermB. Additionally, environmental water, soil and faeces samples were used to validate the established qPCR assays. Importantly, the study proves the usefulness of rapidly synthesized oligonucleotides serving as qPCR standards for ARG analysis and provides comparable sensitivity and reliability to a traditional amplicon standard.