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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]

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]