Metagenomic profiling of antibiotic resistance genes and mobile genetic elements in a tannery wastewater treatment plant.
ABSTRACT: Antibiotics are often used to prevent sickness and improve production in animal agriculture, and the residues in animal bodies may enter tannery wastewater during leather production. This study aimed to use Illumina high-throughput sequencing to investigate the occurrence, diversity and abundance of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in aerobic and anaerobic sludge of a full-scale tannery wastewater treatment plant (WWTP). Metagenomic analysis showed that Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria dominated in the WWTP, but the relative abundance of archaea in anaerobic sludge was higher than in aerobic sludge. Sequencing reads from aerobic and anaerobic sludge revealed differences in the abundance of functional genes between both microbial communities. Genes coding for antibiotic resistance were identified in both communities. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) further revealed that aerobic and anaerobic sludge contained various ARGs with high abundance, among which sulfonamide resistance gene sul1 had the highest abundance, occupying over 20% of the total ARGs reads. Tetracycline resistance genes (tet) were highly rich in the anaerobic sludge, among which tet33 had the highest abundance, but was absent in aerobic sludge. Over 70 types of insertion sequences were detected in each sludge sample, and class 1 integrase genes were prevalent in the WWTP. The results highlighted prevalence of ARGs and MGEs in tannery WWTPs, which may deserve more public health concerns.
Project description:To understand the diversity and abundance of antibiotic resistance genes (ARGs) in pharmaceutical wastewater treatment bioreactors, the ARGs in sludge from two full-scale pharmaceutical wastewater treatment plants (PWWTPs) were investigated and compared with sludge samples from three sewage treatment plants (STPs) using metagenomic approach. The results showed that the ARG abundances in PWWTP sludge ranged from 54.7 to 585.0 ppm, which were higher than those in STP sludge (27.2 to 86.4 ppm). Moreover, the diversity of ARGs in PWWTP aerobic sludge (153 subtypes) was higher than that in STP aerobic sludge (118 subtypes). In addition, it was found that the profiles of ARGs in PWWTP aerobic sludge were similar to those in STP aerobic sludge but different from those in PWWTP anaerobic sludge, suggesting that dissolve oxygen (DO) could be one of the important factors affecting the profiles of ARGs. In PWWTP aerobic sludge, aminoglycoside, sulfonamide and multidrug resistance genes were frequently detected. While, tetracycline, macrolide-lincosamide-streptogramin and polypeptide resistance genes were abundantly present in PWWTP anaerobic sludge. Furthermore, we investigated the microbial community and the correlation between microbial community and ARGs in PWWTP sludge. And, significant correlations between ARG types and seven bacterial genera were found. In addition, the mobile genetic elements (MGEs) were also examined and correlations between the ARGs and MGEs in PWWTP sludge were observed. Collectively, our results suggested that the microbial community and MGEs, which could be affected by DO, might be the main factors shaping the profiles of ARGs in PWWTP sludge.
Project description:Biological nitrification/denitrification is frequently used to remove nitrogen from tannery wastewater containing high concentrations of ammonia. However, information is limited about the bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants (WWTPs) due to the low-throughput of the previously used methods. In this study, 454 pyrosequencing and Illumina high-throughput sequencing, combined with molecular methods, were used to comprehensively characterize structures and functions of nitrification and denitrification bacterial communities in aerobic and anaerobic sludge of two full-scale tannery WWTPs. Pyrosequencing of 16S rRNA genes showed that Proteobacteria and Synergistetes dominated in the aerobic and anaerobic sludge, respectively. Ammonia-oxidizing bacteria (AOB) amoA gene cloning revealed that Nitrosomonas europaea dominated the ammonia-oxidizing community in the WWTPs. Metagenomic analysis showed that the denitrifiers mainly included the genera of Thauera, Paracoccus, Hyphomicrobium, Comamonas and Azoarcus, which may greatly contribute to the nitrogen removal in the two WWTPs. It is interesting that AOB and ammonia-oxidizing archaea had low abundance although both WWTPs demonstrated high ammonium removal efficiency. Good correlation between the qPCR and metagenomic analysis is observed for the quantification of functional genes amoA, nirK, nirS and nosZ, indicating that the metagenomic approach may be a promising method used to comprehensively investigate the abundance of functional genes of nitrifiers and denitrifiers in the environment.
Project description:The presence of emerging biological pollutants in treated wastewater effluents has gained attention due to increased interest in water reuse. To evaluate the effectiveness of the removal of such contaminants by the conventional wastewater treatment process, the fate and decay kinetics of NDM-1-positive <i>Escherichia coli</i> strain PI7 and its plasmid-encoded antibiotic resistance genes (ARGs) were assessed in microcosms of anaerobic and aerobic sludge. Results showed that <i>E. coli</i> PI7 decayed at a significantly lower rate under anaerobic conditions. Approximate half-lives were 32.4 ± 1.4 h and 5.9 ± 0.9 h in the anaerobic and aerobic microcosms, respectively. In the aerobic microcosms, after 72 h of operation, <i>E. coli</i> PI7 remained detectable, but no further decay was observed. Instead, 1 in every 10,000 <i>E. coli</i> cells was identified to be recalcitrant to decay and persist indefinitely in the sludge. ARGs associated with the <i>E. coli</i> PI7 strain were detected to have transferred to other native microorganisms in the sludge or were released to the liquid fraction upon host decay. Extracellular DNA quickly degraded in the liquid fraction of the aerobic sludge. In contrast, no DNA decay was detected in the anaerobic sludge water matrix throughout the 24-h sampling period. This study suggests an increased likelihood of environmental dispersion of ARGs associated with anaerobically treated wastewater effluents and highlights the potential importance of persister cells in the dissemination of <i>E. coli</i> in the environment during reuse events of treated wastewater.<b>IMPORTANCE</b> This study examines the decay kinetics of a pathogenic and antibiotic resistant strain of <i>Escherichia coli</i> in microcosms simulating biological treatment units of aerobic and anaerobic sludge. The results of this study point at a significantly prolonged persistence of the <i>E. coli</i> and the associated antibiotic resistance gene in the anaerobic sludge. However, horizontal transfer of the plasmid encoding the antibiotic resistance gene was detected in the aerobic sludge by a cultivation method. A subpopulation of persister <i>E. coli</i> cells was also detected in the aerobic sludge. The findings of this study suggest potential areas of concern arising from pathogenic and antibiotic-resistant <i>E. coli</i> during both anaerobic and aerobic sludge treatment processes.
Project description:The overuse or misuse of antibiotics has accelerated antibiotic resistance, creating a major challenge for the public health in the world. Sewage treatment plants (STPs) are considered as important reservoirs for antibiotic resistance genes (ARGs) and activated sludge characterized with high microbial density and diversity facilitates ARG horizontal gene transfer (HGT) via mobile genetic elements (MGEs). However, little is known regarding the pool of ARGs and MGEs in sludge microbiome. In this study, the transposon aided capture (TRACA) system was employed to isolate novel plasmids from activated sludge of one STP in Hong Kong, China. We also used Illumina Hiseq 2000 high-throughput sequencing and metagenomics analysis to investigate the plasmid metagenome. Two novel plasmids were acquired from the sludge microbiome by using TRACA system and one novel plasmid was identified through metagenomics analysis. Our results revealed high levels of various ARGs as well as MGEs for HGT, including integrons, transposons and plasmids. The application of the TRACA system to isolate novel plasmids from the environmental metagenome, coupled with subsequent high-throughput sequencing and metagenomic analysis, highlighted the prevalence of ARGs and MGEs in microbial community of STPs.
Project description:Wastewater treatment plants (WWTPs) release treated effluent containing mobile genetic elements (MGEs), antibiotic resistance genes (ARGs), and microorganisms into the environment, yet little is known about their influence on nearby microbial communities and the retention of these factors in receiving water bodies. Our research aimed to characterize the genes and organisms from two different WWTPs that discharge into Lake Michigan, as well as from surrounding lake sediments to determine the dispersal and fate of these factors with respect to distance from the effluent outfall. Shotgun metagenomics coupled to distance-decay analyses showed a higher abundance of genes identical to those in WWTP effluent genes in sediments closer to outfall sites than in sediments farther away, indicating their possible WWTP origin. We also found genes attributed to organisms, such as those belonging to Helicobacteraceae, Legionellaceae, Moraxellaceae, and Neisseriaceae, in effluent from both WWTPs and decreasing in abundance in lake sediments with increased distance from WWTPs. Moreover, our results showed that the WWTPs likely influence the ARG composition in lake sediments close to the effluent discharge. Many of these ARGs were located on MGEs in both the effluent and sediment samples, indicating a relatively broad propensity for horizontal gene transfer (HGT). Our approach allowed us to specifically link genes to organisms and their genetic context, providing insight into WWTP impacts on natural microbial communities. Overall, our results suggest a substantial influence of wastewater effluent on gene content and microbial community structure in the sediments of receiving water bodies.IMPORTANCE Wastewater treatment plants (WWTPs) release their effluent into aquatic environments. Although treated, effluent retains many genes and microorganisms that have the potential to influence the receiving water in ways that are poorly understood. Here, we tracked the genetic footprint, including genes specific to antibiotic resistance and mobile genetic elements and their associated organisms, from WWTPs to lake sediments. Our work is novel in that we used metagenomic data sets to comprehensively evaluate total gene content and the genetic and taxonomic context of specific genes in environmental samples putatively impacted by WWTP inputs. Based on two different WWTPs with different treatment processes, our findings point to an influence of WWTPs on the presence, abundance, and composition of these factors in the environment.
Project description:Wastewater treatment plants (WWTPs) are an effective barrier in the protection of human and environment health around the world, although WWTPs also are suggested to be selectors and-or reservoirs of antibiotic resistance genes (ARGs) before entering the environment. The dogma about WWTPs as "ARG selectors" presumes that biotreatment compartments (e.g., activated sludge; AS) are single densely populated ecosystems with elevated horizontal gene transfer. However, recent work has suggested WWTP biotreatment compartments may be different than previously believed relative to antibiotic resistance (AR) fate, and other process factors, such as bacterial separation and specific waste sources, may be key to ARGs released to the environment. Here we combined 16S rRNA metagenomic sequencing and high-throughput qPCR to characterise microbial communities and ARGs across a wastewater network in Spain that includes both community (i.e., non-clinical urban) and hospital sources. Contrary to expectations, ARGs found in downstream receiving waters were not dominated by AS biosolids (RAS), but more resembled raw wastewater sources. In fact, ARGs and microbial communities in liquid-phase WWTP effluents and RAS were significantly different (Bray-Curtis dissimilarity index?=?0.66?±?0.11), with a consequential fraction of influent ARGs and organisms passing directly through the WWTP with limited association with RAS. Instead, ARGs and organisms in the RAS may be more defined by biosolids separation and biophysical traits, such as flocculation, rather than ARG carriage. This explains why RAS has significantly lower ARG richness (47?±?4 ARGs) than liquid-phase effluents (104?±?5 ARGs), and downstream water column (135?±?4 ARGs) and river sediments (120?±?5 ARGs) (Tukey's test, p?<?0.001). These data suggest RAS and liquid-phase WWTP effluents may reflect two parallel ecosystems with potentially limited ARG exchange. As such, ARG mitigation in WWTPs should more focus on removing bacterial hosts from the liquid phase, AR source reduction, and possibly disinfection to reduce ARG releases to the environment.
Project description:Organic fertilizer is a major carrier that stores and transmits antibiotic resistance genes (ARGs). In the environment, due to the application of organic fertilizers in agriculture, the increasing diversity and abundance of ARGs poses a potential threat to human health and environmental safety. In this paper, the microbial community structure and ARGs in different types of organic fertilizer treated with composting were examined. We found that the abundance and diversity of ARGs in earthworm cast organic fertilizer were the lowest and the highest in chicken manure organic fertilizer. Interestingly, the abundance and diversity of ARGs, especially beta-lactam resistance genes, sulfonamide resistance genes, and macrolide-lincosamide-streptogramin B (MLSB) resistance genes, in organic fertilizers were reduced significantly, while composting caused no significant change in mobile genetic elements (MGEs), where antibiotic deactivation and the use of efflux pumps were the two most dominant mechanisms. It was clear that removal of ARGs became more efficient with increasing reduction in the bacterial abundances and diversity of potential ARG hosts, and integron-mediated horizontal gene transfers (HGTs) played an important role in the proliferation of most ARG types. Therefore, the reduction in ARGs was mainly driven by changes in bacterial community composition caused by composting. Furthermore, rather than HGTs, the diversity and abundance of bacterial communities affected by compost physical and chemical properties were the main drivers shaping and altering the abundance and diversity of ARGs, which was indicated by a correlation analysis of these properties, antibiotic residues, microbial community structure, and ARGs. In general, high-temperature composting effectively removed antibiotic residues and ARGs from these organic fertilizers; however, it cannot prevent the proliferation of MGEs. The insights gained from these results may be of assistance in the safe and rational use of organic fertilizers by indicating the changes in microbial community structure and ARGs in different types of organic fertilizer treated with composting.
Project description:The activated sludge in wastewater treatment plants (WWTP) designed for enhanced biological phosphorus removal (EBPR) experiences periodically changing nutrient and oxygen availability. Tetrasphaera is the most abundant genus in Danish WWTP and represents up to 20-30% of the activated sludge community based on 16S rRNA amplicon sequencing and quantitative fluorescence in situ hybridization analyses, although the genus is in low abundance in the influent wastewater. Here we investigated how Tetrasphaera can successfully out-compete most other microorganisms in such highly dynamic ecosystems. To achieve this, we analyzed the physiological adaptations of the WWTP isolate T. elongata str. LP2 during an aerobic to anoxic shift by label-free quantitative proteomics and NMR-metabolomics. Escherichia coli was used as reference organism as it shares several metabolic capabilities and is regularly introduced to wastewater treatment plants without succeeding there. When compared to E. coli, only minor changes in the proteome of T. elongata were observed after the switch to anoxic conditions. This indicates that metabolic pathways for anaerobic energy harvest were already expressed during the aerobic growth. This allows continuous growth of Tetrasphaera immediately after the switch to anoxic conditions. Metabolomics furthermore revealed that the substrates provided were exploited far more efficiently by Tetrasphaera than by E. coli. These results suggest that T. elongata prospers in the dynamic WWTP environment due to adaptation to the changing environmental conditions.
Project description:Ready-to-eat salad harbors microorganisms that may carry various antibiotic resistance genes (ARGs). However, few studies have focused on the prevalence of ARGs on salad, thus underestimating the risk of ARGs transferring from salad to consumers. In this small-scale study, high-throughput quantitative PCR was used to explore the presence, prevalence and abundance of ARGs associated with serving salad sourced from two restaurant types, fast-food chain and independent casual dining. A total of 156 unique ARGs and nine mobile genetic elements (MGEs) were detected on the salad items assessed. The abundance of ARGs and MGEs were significantly higher in independent casual dining than fast-food chain restaurants. Absolute copies of ARGs in salad were 1.34 × 107 to 2.71 × 108 and 1.90 × 108 to 4.87 × 108 copies per g salad in fast-food and casual dining restaurants, respectively. Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes were the dominant bacterial phyla detected from salad samples. Pseudomonas, Acinetobacter, Exiguobacterium, Weissella, Enterobacter, Leuconostoc, Pantoea, Serratia, Erwinia, and Ewingella were the 10 most dominant bacterial genera found in salad samples. A significant positive correlation between ARGs and MGEs was detected. These results integrate knowledge about the ARGs in ready-to-eat salad and highlight the potential impact of ARGs transfer to consumers.
Project description:In this study, compound microbial inoculants, including three <i>Bacillus</i> strains and one Yeast strain, were inoculated into swine manure composting to explore the effects on antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), microbial community structure, and pathogenic bacteria. The results indicated that the abundances of the detected ARGs ranged from 3.6 × 10<sup>3</sup> to 1.13 × 10<sup>8</sup> copies/g. The ARGs with the highest abundance was <i>sul2</i>, and the lowest was <i>blaCTX</i>. Composting removes most of the ARGs and MGEs by 22.8-99.7%. These ARGs were significantly reduced during the thermophilic phase of compost. The removal rate of ARGs at the different layers of compost pile was different as follows: middle layer > upper layer > lower layer. But some ARGs proliferated significantly in the maturation phase of compost, especially the sulfonamide resistance genes. Compound microbial inoculants increased the temperature of compost, accelerated water loss, nitrogen fixation, and increased the removal rate of ?-lactamase resistance genes, the transposon gene <i>tn916</i> and part of tetracycline resistance genes by 3.7-23.8% in compost. Compound microbial inoculants changed the community structure and increased the <i>Bacillus</i> abundance in the thermophilic phase of compost. And it was helpful for removing pathogens during composting. The addition of compound microbial inoculants causes the decrease of <i>Firmicutes</i> and the increase of <i>Bacteroidetes</i>, which may be related to the removal and proliferation of ARGs.