Calcineurin inhibitors or cyclophosphamide in the treatment of membranous nephropathy superimposed with FSGS lesions: a retrospective study from China.

BACKGROUND: Cyclophosphamide (CTX) and calcineurin inhibitors (CNIs) based regimens are recommended as immunosuppressive therapies for patients with idiopathic membranous nephropathy (IMN). Focal and segmental glomerular sclerosis (FSGS) lesions, which are common in membranous nephropathy (MN), are poor predictors of outcome. This study compared the differences of prognosis between two regimens in patients with IMN combined with FSGS lesions. METHODS: This retrospective study enrolled 108 patients with biopsy-proven IMN, accompanied with FSGS lesions, nephrotic syndrome and an estimated glomerular filtration rate (eGFR)≥60 mL/min/1.73 m2 who were treated with CTX or CNIs. We used propensity score matching (PSM) for balancing the confounding variables. RESULTS: During follow-up, 10 patients (10/55 [18.2%]; nine males) in the CNIs group showed a 50% decline in eGFR; eight had a not otherwise specified variant. Patients initially treated with CNIs had a significantly higher risk of progression to the primary outcome and a lower probability of complete or total remission. The relapse rate was higher in patients who initially received CNIs- than in those who received CTX-based treatment. Before PSM, age and 24-h urine protein level differed significantly between the groups. The PSM model included data from 72 patients. Worse outcomes were also noted among patients who initially received CNIs than those who received CTX-based treatments after matching. CONCLUSIONS: Patients with MN combined with FSGS lesions have a higher risk of renal functional decline and a higher rate of relapse after CNIs than after CTX therapy.

Demonstrating a Comprehensive Wastewater-Based Surveillance Approach That Differentiates Globally Sourced Resistomes.

Wastewater-based surveillance (WBS) for disease monitoring is highly promising but requires consistent methodologies that incorporate predetermined objectives, targets, and metrics. Herein, we describe a comprehensive metagenomics-based approach for global surveillance of antibiotic resistance in sewage that enables assessment of 1) which antibiotic resistance genes (ARGs) are shared across regions/communities; 2) which ARGs are discriminatory; and 3) factors associated with overall trends in ARGs, such as antibiotic concentrations. Across an internationally sourced transect of sewage samples collected using a centralized, standardized protocol, ARG relative abundances (16S rRNA gene-normalized) were highest in Hong Kong and India and lowest in Sweden and Switzerland, reflecting national policy, measured antibiotic concentrations, and metal resistance genes. Asian versus European/US resistomes were distinct, with macrolide-lincosamide-streptogramin, phenicol, quinolone, and tetracycline versus multidrug resistance ARGs being discriminatory, respectively. Regional trends in measured antibiotic concentrations differed from trends expected from public sales data. This could reflect unaccounted uses, captured only by the WBS approach. If properly benchmarked, antibiotic WBS might complement public sales and consumption statistics in the future. The WBS approach defined herein demonstrates multisite comparability and sensitivity to local/regional factors.

The Prevalence of Integrons as the Carrier of Antibiotic Resistance Genes in Natural and Man-Made Environments.

Class 1 integrase intI1 has been considered as a good proxy for anthropogenic pollution because of being linked to genes conferring resistance to antibiotics. The gene cassettes of class 1 integrons could carry diverse antibiotic resistance genes (ARGs) and conduct horizontal gene transfer among microorganisms. The present study applied high-throughput sequencing technique combined with an intI1 database and genome assembly to quantify the abundance of intI1 in 64 environmental samples from 8 ecosystems, and to investigate the diverse arrangements of ARG-carrying gene cassettes (ACGCs) carried by class 1 integrons. The abundance of detected intI1 ranged from 3.83 × 10-4 to 4.26 × 10° intI1/cell. High correlation (Pearson's r = 0.852) between intI1 and ARG abundance indicated that intI1 could be considered as an important indicator of ARGs in environments. Aminoglycoside resistance genes were most frequently observed on gene cassettes, carried by 57% assembled ACGCs, followed by trimethoprim and beta-lactam resistance genes. This study established the pipeline for broad monitoring of intI1 in various environmental samples and scanning the ARGs carried by integrons. These findings supplemented our knowledge on the distribution of class 1 integrons and ARGs carried on mobile genetic elements, benefiting future studies on horizontal gene transfer of ARGs.

Cultivation-dependent and high-throughput sequencing approaches studying the co-occurrence of antibiotic resistance genes in municipal sewage system.

During the past years, antibiotic-resistant bacteria (ARB) leading for the spreading of antibiotic resistance genes (ARGs) became a global problem, especially multidrug-resistant (MDR) bacteria are considered the prime culprit of antibiotic resistance. However, the correlation between the antibiotic-resistant phenotype and the ARG profiles remains poorly understood. In the present study, metagenomic functional screening and metagenomic analysis of coliforms were combined to explore the phenotype and genotype of the ARBs from municipal sewage. Our results showed that the ARG co-occurrence was widespread in the municipal sewage. The present study also highlighted the high abundance of ARGs from antibiotic resistance coliforms especially the MDR coliforms with ARG level of 33.8 ± 4.2 copies per cell. The ARG profiles and the antibiotic resistance phenotypes of the isolated antibiotic resistant coliforms were also correlated and indicated that the resistance to the related antibiotic (ampicillin, kanamycin, erythromycin, chloramphenicol, and tetracycline) was mostly contributed by the ARGs belonging to the subtypes of ß-lactamase, aminoglycoside 3-phosphotransferase, phosphotransferase type 2, chloramphenicol acetyltransferase, tetA, etc.

Characteristics of the antibiotic resistance genes in the soil of medical waste disposal sites.

The inappropriate disposal of medical waste allows bacteria to acquire antibiotic resistance, which results in a threat to public health. Antibiotic resistance gene (ARG) profiles were determined for 45 different soil samples containing medical waste and 15 nearby soil samples as controls. Besides physical and chemical analyses (i.e., dry matter content, pH value, and metal content), the genomes of microorganisms from the soil samples were extracted for high-throughput sequencing. ARG abundances of these samples were obtained by searching the metagenomic sequences against the antibiotic resistance gene database and the copies of ARGs per copy of the 16S rRNA gene at different levels were assessed. The results showed medical waste accumulation significantly enriched the contents of Cu, Cr, Pb, and As in the tested soil samples. Compared to the controls, the samples collected from areas containing medical waste were significantly enriched (p < 0.05, t-test) with ARGs annotated as sulfonamide and multidrug resistance genes, and in particular, the subtypes sul1 and sul2 (sulfonamide resistance genes), and multidrug_transporter (multidrug resistance gene). Moreover, the ARGs of the samples from the polluted areas were more diverse than those of the control samples (p < 0.05, t-test). The comparatively higher abundance and diversity of ARGs in contaminated soil pose a potential risk to human health.

Mobile antibiotic resistome in wastewater treatment plants revealed by Nanopore metagenomic sequencing.

BACKGROUND: Wastewater treatment plants (WWTPs) are recognized as hotspots for horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). Despite our understanding of the composition and distribution of ARGs in WWTPs, the genetic location, host, and fate of ARGs remain largely unknown. RESULTS: In this study, we combined Oxford Nanopore and Illumina metagenomics sequencing to comprehensively uncover the resistome context of influent, activated sludge, and effluent of three WWTPs and simultaneously track the hosts of the ARGs. The results showed that most of the ARGs detected in all compartments of the WWTPs were carried by plasmids. Transposons and integrons also showed higher prevalence on plasmids than on the ARG-carrying chromosome. Notably, integrative and conjugative elements (ICEs) carrying five types of ARGs were detected, and they may play an important role in facilitating the transfer of ARGs, particularly for tetracycline and macrolide-lincosamide-streptogramin (MLS). A broad spectrum of ARGs carried by plasmids (29 subtypes) and ICEs (4 subtypes) was persistent across the WWTPs. Host tracking showed a variety of antibiotic-resistant bacteria in the effluent, suggesting the high potential for their dissemination into receiving environments. Importantly, phenotype-genotype analysis confirmed the significant role of conjugative plasmids in facilitating the survival and persistence of multidrug-resistant bacteria in the WWTPs. At last, the consistency in the quantitative results for major ARGs types revealed by Nanopore and Illumina sequencing platforms demonstrated the feasibility of Nanopore sequencing for resistome quantification. CONCLUSION: Overall, these findings substantially expand our current knowledge of resistome in WWTPs, and help establish a baseline analysis framework to study ARGs in the environment.

Puerarin prevents high-fat diet-induced obesity by enriching Akkermansia muciniphila in the gut microbiota of mice.

Growing evidence indicates that the gut microbiota plays a significant role in the pathophysiological processes of obesity and its related metabolic symptoms in the host. Puerarin, an active ingredient in the root of Pueraria lobate has been suggested to have a potent anti-obesity effect. Herein, we tested whether this effect of puerarin is associated with changes in the gut microbiota. In addition to reducing body weight, inflammation, and insulin resistance, puerarin administration significantly altered the composition of the gut microbiota. Notably, puerarin treatment greatly increased the abundance of Akkermansia muciniphila, a mucin-degrading bacterium known to be beneficial for host metabolism and significantly downregulated in high-fat diet-fed mice. Further experiments revealed that puerarin increased intestinal expression levels of Muc2 and Reg3g and protected intestinal barrier function (normal permeability) by increasing the expression of ZO-1 and occludin in vivo and in vitro. These data suggest that puerarin's enriching effect on A. muciniphila is mediated, at least in part, by a host cellular response to protect the host from diet-induced metabolic disorders and other diseases.

Effects of sample preservation and DNA extraction on enumeration of antibiotic resistance genes in wastewater.

With the growing application of high-throughput sequencing-based metagenomics for profiling antibiotic resistance genes (ARGs) in wastewater treatment plants (WWTPs), comparison of sample pretreatment and DNA extraction methods are needed to move toward standardized comparisons among laboratories. Three widely employed DNA extraction methods (FastDNA® Spin Kit for Soil, PowerSoil® DNA Isolation Kit and ZR Fecal DNA MiniPrep), with and without preservation in 50% ethanol and freezing, were applied to the influent, activated sludge and effluent of two WWTPs, in Hong Kong and in the USA. Annotated sequences obtained from the DNA extracted using the three kits shared similar taxonomy and ARG profiles. Overall, it was found that the DNA yield and purity, and diversity of ARGs captured were all highest when applying the FastDNA SPIN Kit for Soil for all three WWTP sample types investigated here (influent, activated sludge, effluent). Quantitative polymerase chain reaction of 16S rRNA genes confirmed the same trend as DNA extraction yields and similar recovery of a representative Gram-negative bacterium (Escherichia coli). Moreover, sample fixation in ethanol, deep-freezing and overseas shipment had no discernable effect on ARG profiles, as compared to fresh samples. This approach serves to inform future efforts toward global comparisons of ARG distributions in WWTPs.

Catalogue of antibiotic resistome and host-tracking in drinking water deciphered by a large scale survey.

BACKGROUND: Excesses of antibiotic resistance genes (ARGs), which are regarded as emerging environmental pollutants, have been observed in various environments. The incidence of ARGs in drinking water causes potential risks to human health and receives more attention from the public. However, ARGs harbored in drinking water remain largely unexplored. In this study, we aimed at establishing an antibiotic resistome catalogue in drinking water samples from a wide range of regions and to explore the potential hosts of ARGs. RESULTS: A catalogue of antibiotic resistome in drinking water was established, and the host-tracking of ARGs was conducted through a large-scale survey using metagenomic approach. The drinking water samples were collected at the point of use in 25 cities in mainland China, Hong Kong, Macau, Taiwan, South Africa, Singapore and the USA. In total, 181 ARG subtypes belonging to 16 ARG types were detected with an abundance range of 2.8 × 10-2 to 4.2 × 10-1 copies of ARG per cell. The highest abundance was found in northern China (Henan Province). Bacitracin, multidrug, aminoglycoside, sulfonamide, and beta-lactam resistance genes were dominant in drinking water. Of the drinking water samples tested, 84% had a higher ARG abundance than typical environmental ecosystems of sediment and soil. Metagenomic assembly-based host-tracking analysis identified Acidovorax, Acinetobacter, Aeromonas, Methylobacterium, Methyloversatilis, Mycobacterium, Polaromonas, and Pseudomonas as the hosts of ARGs. Moreover, potential horizontal transfer of ARGs in drinking water systems was proposed by network and Procrustes analyses. CONCLUSIONS: The antibiotic resistome catalogue compiled using a large-scale survey provides a useful reference for future studies on the global surveillance and risk management of ARGs in drinking water. .

MinION Nanopore Sequencing Enables Correlation between Resistome Phenotype and Genotype of Coliform Bacteria in Municipal Sewage.

Wastewater treatment plants (WWTPs) functioned as the intersection between the human society and nature environment, are receiving increasingly more attention on risk assessment of the acquisition of environmental antibiotic resistance genes (ARGs) by pathogenetic populations during treatment. However, because of the general lack of robust resistome profiling methods, genotype, and resistance phenotype is still poorly correlated in human pathogens of sewage samples. Here we applied MinION sequencing to quantify the resistance genes of multiple antibiotic resistant (MAR) coliform bacteria, a common indicator for human enteric pathogens in sewage samples. Our pipeline could deliver the results within 30 h from sample collection and the resistome quantification was consistent to that based on the Illumina platform. Additionally, the long nanopore reads not only enabled a simultaneous identification of the carrier populations of ARGs detected, but also facilitated the genome reconstruction of a representative MAR strain, from which we identified an instance of chromosomal integration of environmental resistance gene obtained by plasmid exchange with a porcine pathogen. This study demonstrated the utilization of MinION sequencing in quick monitoring and simultaneous phylogenetic tracking of environmental ARGs to address potential health risk associated with them.

Discovery of new cellulases from the metagenome by a metagenomics-guided strategy.

BACKGROUND: Energy shortage has become a global problem. Production of biofuels from renewable biomass resources is an inevitable trend of sustainable development. Cellulose is the most abundant and renewable resource in nature. Lack of new cellulases with unique properties has become the bottleneck of the efficient utilization of cellulose. Environmental metagenomes are regarded as huge reservoirs for a variety of cellulases. However, new cellulases cannot be obtained easily by functional screening of metagenomic libraries. RESULTS: In this work, a metagenomics-guided strategy for obtaining new cellulases from the metagenome was proposed. Metagenomic sequences of DNA extracted from the anaerobic beer lees converting consortium enriched at thermophilic conditions were assembled, and 23 glycoside hydrolase (GH) sequences affiliated with the GH family 5 were identified. Among the 23 GH sequences, three target sequences (designated as cel7482, cel3623 and cel36) showing low identity with those known GHs were chosen as the putative cellulase genes to be functionally expressed in Escherichia coli after PCR cloning. The three cellulases were classified into endo-ß-1,4-glucanases by product pattern analysis. The recombinant cellulases were more active at pH 5.5 and within a temperature range of 60-70 °C. Computer-assisted 3D structure modeling indicated that the active residues in the active site of the recombinant cellulases were more similar to each other compared with non-active site residues. The recombinant cel7482 was extremely tolerant to 2 M NaCl, suggesting that cel7482 may be a halotolerant cellulase. Moreover, the recombinant cel7482 was shown to have an ability to resist three ionic liquids (ILs), which are widely used for cellulose pretreatment. Furthermore, active cel7482 was secreted by the twin-arginine translocation (Tat) pathway of Bacillus subtilis 168 into the culture medium, which facilitates the subsequent purification and reduces the formation of inclusion body in the context of overexpression. CONCLUSIONS: This study demonstrated a simple and efficient method for direct cloning of new cellulase genes from environmental metagenomes. In the future, the metagenomics-guided strategy may be applied to the high-throughput screening of new cellulases from environmental metagenomes.

Exploring antibiotic resistance genes and metal resistance genes in plasmid metagenomes from wastewater treatment plants.

Plasmids operate as independent genetic elements in microorganism communities. Through horizontal gene transfer (HGT), they can provide their host microorganisms with important functions such as antibiotic resistance and heavy metal resistance. In this study, six metagenomic libraries were constructed with plasmid DNA extracted from influent, activated sludge (AS) and digested sludge (DS) of two wastewater treatment plants (WWTPs). Compared with the metagenomes of the total DNA extracted from the same sectors of the wastewater treatment plant, the plasmid metagenomes had significantly higher annotation rates, indicating that the functional genes on plasmids are commonly shared by those studied microorganisms. Meanwhile, the plasmid metagenomes also encoded many more genes related to defense mechanisms, including ARGs. Searching against an antibiotic resistance genes (ARGs) database and a metal resistance genes (MRGs) database revealed a broad-spectrum of antibiotic (323 out of a total 618 subtypes) and MRGs (23 out of a total 23 types) on these plasmid metagenomes. The influent plasmid metagenomes contained many more resistance genes (both ARGs and MRGs) than the AS and the DS metagenomes. Sixteen novel plasmids with a complete circular structure that carried these resistance genes were assembled from the plasmid metagenomes. The results of this study demonstrated that the plasmids in WWTPs could be important reservoirs for resistance genes, and may play a significant role in the horizontal transfer of these genes.