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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 β-lactamase, aminoglycoside 3-phosphotransferase, phosphotransferase type 2, chloramphenicol acetyltransferase, tetA, etc. [ABSTRACT FROM AUTHOR]

Emergence of new antibiotic resistance bacteria poses a serious threat to human health, which is largely attributed to the evolution and spread of antibiotic resistance genes (ARGs). In this work, a metagenomics-guided strategy consisting of metagenomic analysis and function validation was proposed for rapidly identifying novel ARGs from hot spots of ARG dissemination, such as wastewater treatment plants (WWTPs) and animal feces. We used an antibiotic resistance gene database to annotate 76 putative β-lactam resistance genes from the metagenomes of sludge and chicken feces. Among these 76 candidate genes, 25 target genes that shared 40~70% amino acid identity to known β-lactamases were cloned by PCR from the metagenomes. Their resistances to four β-lactam antibiotics were further demonstrated. Furthermore, the validated ARGs were used as the reference sequences to identify novel ARGs in eight environmental samples, suggesting the necessity of re-examining the profiles of ARGs in environmental samples using the validated novel ARG sequences. This metagenomics-guided pipeline does not rely on the activity of ARGs during the initial screening process and may specifically select novel ARG sequences for function validation, which make it suitable for the high-throughput screening of novel ARGs from environmental metagenomes. [ABSTRACT FROM AUTHOR]

The effectiveness and treatment conditions of FeCl- and AlCl-coagulated municipal sewage sludge from chemically enhanced primary treatment (CEPT) using anaerobic digestion (AD) and the structure of microbial community were investigated. The results based on 297 measurements under different operational conditions demonstrate good average AD performance of CEPT sludge, that is, percent volatile solid reduction of 58 %, specific biogas production (or biogas yield) of 0.92 m/kg volatile solids (VS) destroyed, and methane content of 65.4 %. FeCl dosing, organic loading rate, temperature, and hydraulic retention time all significantly affected AD performance. FeCl dosing greatly improved specific methane production (methane yield) by 38-54 % and significantly reduced hydrogen sulfide (HS) content in biogas (from up to 13,250 to <200 ppm), contributing to higher methane recovery and simplified biogas cleaning for power generation. Metagenomic analysis suggested that anaerobic digesters of both CEPT sludge and combined primary and secondary sludge were dominated by Bacteroidetes, Proteobacteria, Firmicutes, Actinobacteria, Thermotogae, and Chloroflexi. However, Methanomicrobia methanogens were better enriched in the anaerobic digesters of CEPT sludge than in the combined sludge. Further, different sources of CEPT sludge with various chemical properties nurtured shared and unique microbial community composition. Combined, this study supports AD as an efficient technology for CEPT sludge treatment and poses first insights into the microbial community structure. [ABSTRACT FROM AUTHOR]

Biofilms are widely used in wastewater treatment for their particular enhancement of nitrogen removal and other significant advantages. In this study, the diversity and potential functions of nitrogen removal bacteria in suspended activated sludge (AS) and biofilm of a full-scale hybrid reactor were uncovered by metagenomes (∼34 Gb), coupled with PCR-based 454 reads (>33 K reads). The results indicated that the diversity and abundance of nitrifiers and denitrifiers in biofilm did not surpass that in AS, while more nitrification and denitrification genes were indeed found in biofilm than AS, suggesting that the increased nitrogen removal ability by applying biofilm might be attributed to the enhancement of removal efficiency, rather than the biomass accumulation of nitrogen removal bacteria. The gene annotation and phylogenetic analysis results revealed that AS and biofilm samples consisted of 6.0 % and 9.4 % of novel functional genes for nitrogen removal and 18 % and 30 % of new Nitrospira species for nitrite-oxidizing bacteria, respectively. Moreover, the identification of Nitrospira-like amoA genes provided metagenomic evidence for the presence of complete ammonia oxidizer (comammox) with the functional potential to perform the complete oxidation of ammonia to nitrate. These findings have significant implications in expanding our knowledge of the biological nitrogen transformations in wastewater treatment. [ABSTRACT FROM AUTHOR]

Improvement on the bioconversion of cellulosic biomass depends much on the expanded knowledge on the underlying microbial structure and the relevant genetic information. In this study, metagenomic analysis was applied to characterize an enriched mesophilic cellulose-converting consortium, to explore its cellulose-hydrolyzing genes, and to discern genes involved in methanogenesis. Cellulose conversion efficiency of the mesophilic consortium enriched in this study was around 70 %. Apart from methane, acetate was the major fermentation product in the liquid phase, while propionate and butyrate were also detected at relatively high concentrations. With the intention to uncover the biological factors that might shape the varying cellulose conversion efficiency at different temperatures, results of this mesophilic consortium were then compared with that of a previously reported thermophilic cellulose-converting consortium. It was found that the mesophilic consortium harbored a larger pool of putative carbohydrate-active genes, with 813 of them in 54 GH modules and 607 genes in 13 CBM modules. Methanobacteriaceae and Methanosaetaceae were the two methanogen families identified, with a preponderance of the hydrogenotrophic Methanobacteriaceae. In contrast to its relatively high diversity and high abundance of carbohydrate-active genes, the abundance of genes involved in the methane metabolism was comparatively lower in the mesophilic consortium. A biological enhancement on the methanogenic process might serve as an effective option for the improvement of the cellulose bioconversion at mesophilic temperature. [ABSTRACT FROM AUTHOR]

As antibiotic resistance continues to spread globally, there is growing interest in the potential to limit the spread of antibiotic resistance genes (ARGs) from wastewater sources. In particular, operational conditions during sludge digestion may serve to discourage selection of resistant bacteria, reduce horizontal transfer of ARGs, and aid in hydrolysis of DNA. This study applied metagenomic analysis to examine the removal efficiency of ARGs through thermophilic and mesophilic anaerobic digestion using bench-scale reactors. Although the relative abundance of various ARGs shifted from influent to effluent sludge, there was no measureable change in the abundance of total ARGs or their diversity in either the thermophilic or mesophilic treatment. Among the 35 major ARG subtypes detected in feed sludge, substantial reductions (removal efficiency >90 %) of 8 and 13 ARGs were achieved by thermophilic and mesophilic digestion, respectively. However, resistance genes of aadA, macB, and sul1 were enriched during the thermophilic anaerobic digestion, while resistance genes of erythromycin esterase type I, sul1, and tetM were enriched during the mesophilic anaerobic digestion. Efflux pump remained to be the major antibiotic resistance mechanism in sludge samples, but the portion of ARGs encoding resistance via target modification increased in the anaerobically digested sludge relative to the feed. Metagenomic analysis provided insight into the potential for anaerobic digestion to mitigate a broad array of ARGs. [ABSTRACT FROM AUTHOR]

In this study, we utilized the Illumina high-throughput metagenomic approach to investigate diversity and abundance of both microbial community and copper resistance genes (CuRGs) in activated sludge (AS) which was enriched under copper selective stress up to 800 mg/L. The raw datasets (~3.5 Gb for each sample, i.e., the copper-enriched AS and the control AS) were merged and normalized for the BLAST analyses against the SILVA SSU rRNA gene database and self-constructed copper resistance protein database (CuRD). Also, the raw metagenomic sequences were assembled into contigs and analyzed based on Open Reading Frames (ORFs) to identify potentially novel copper resistance genes. Among the different resistance systems for copper detoxification under the high copper stress condition, the Cus system was the most enriched system. The results also indicated that genes encoding multi-copper oxidase played a more important role than those encoding efflux proteins. More significantly, several potentially novel copper resistance ORFs were identified by Pfam search and phylogenic analysis. This study demonstrated a new understanding of microbial-mediated copper resistance under high copper stress using high-throughput shotgun sequencing technique. [ABSTRACT FROM AUTHOR]

Here, shotgun metagenomic sequencing was conducted to reveal the hydrogen-oxidizing autotrophic-denitrifying metabolism in an enriched Thauera-dominated consortium. A draft genome named Thauera R4 of over 90 % completeness (3.8 Mb) was retrieved mainly by a coverage-defined binning method from 3.5 Gb paired-end Illumina reads. We identified 1,263 genes (accounting for 33 % of total genes in the finished genome of Thauera aminoaromatica MZ1T) with average nucleotide identity of 87.6 % shared between Thauera R4 and T. aminoaromatica MZ1T. Although Thauera R4 and T. aminoaromatica shared quite similar nitrogen metabolism and a high nucleotide similarity (98.8 %) in their 16S ribosomal RNA genes, they showed different functional potentials in several important environmentally relevant processes. Unlike T. aminoaromatica MZ1T, Thauera R4 carries an operon of [NiFe]-hydrogenase (EC 1.12.99.6) catalyzing molecular hydrogen oxidation in nitrate-rich solution. Moreover, Thauera R4 is a mixtrophic bacterium possessing key enzymes for autotrophic CO-fixation and heterotrophic acetate assimilation metabolism. This Thauera R4 bin provides another genetic reference to better understand the niches of Thauera and demonstrates a model pipeline to reveal functional profiles and reconstruct novel and dominant genomes from a simplified mixed culture in environmental studies. [ABSTRACT FROM AUTHOR]