Noise reduction strategies in metagenomic chromosome confirmation capture to link antibiotic resistance genes to microbial hosts

The gut microbiota is a reservoir for antimicrobial resistance genes (ARGs). With current sequencing methods, it is difficult to assign ARGs to their microbial hosts, particularly if these ARGs are located on plasmids. Metagenomic chromosome conformation capture approaches (meta3C and Hi-C) have recently been developed to link bacterial genes to phylogenetic markers, thus potentially allowing the assignment of ARGs to their hosts on a microbiome-wide scale. Here, we generated a meta3C dataset of a human stool sample and used previously published meta3C and Hi-C datasets to investigate bacterial hosts of ARGs in the human gut microbiome. Sequence reads mapping to repetitive elements were found to cause problematic noise in, and may importantly skew interpretation of, meta3C and Hi-C data. We provide a strategy to improve the signal-to-noise ratio by discarding reads that map to insertion sequence elements and to the end of contigs. We also show the importance of using spike-in controls to quantify whether the cross-linking step in meta3C and Hi-C protocols has been successful. After filtering to remove artefactual links, 87 ARGs were assigned to their bacterial hosts across all datasets, including 27 ARGs in the meta3C dataset we generated. We show that commensal gut bacteria are an important reservoir for ARGs, with genes coding for aminoglycoside and tetracycline resistance being widespread in anaerobic commensals of the human gut

Carbon induced extreme ultraviolet (EUV) reflectance loss characterized using visible-light ellipsometry

Carbon deposition on extreme ultraviolet (EUV) optics was observed due to photon-induced dissociation of hydrocarbons in a EUV lithography environment. The reflectance loss of the multilayer mirror is determined by the carbon layer thickness and density. To study the influence of various forms of carbon, EUV-induced carbon, hot filament and e-beam evaporated carbon were deposited on EUV multilayer mirrors. Spectroscopic ellipsometry was used to determine the carbon layer thickness and the optical constants ranging from ultraviolet to near infrared. The carbon density (and thus reflectance loss) was determined from the optical constants using both Bruggeman's effective medium approximation and the Clausius–Mosotti equation. Both approaches result in a similar EUV reflectance loss, with an accuracy of about 4%. The application of this process to ultrathin carbon films is further discussed

Integration of vanHAX downstream of a ribosomal RNA operon restores vancomycin resistance in a susceptible Enterococcus faecium strain

During the genomic characterisation of Enterococcus faecium strains (n = 39) collected in a haematology ward, we identified an isolate (OI25), which contained vanA-type vancomycin resistance genes but was phenotypically susceptible to vancomycin. OI25 could revert to resistance when cultured in the presence of vancomycin and was thus considered to be vancomycin-variable. Long-read sequencing was used to identify structural variations within the vancomycin resistance region of OI25 and to uncover its resistance reversion mechanism. We found that OI25 has a reduced ability to positively regulate expression of the vanHAX genes in the presence of vancomycin, which was associated with the insertion of an IS6-family element within the promoter region and the first 50 bp of the vanR gene. The vancomycin-resistant revertant isolates constitutively expressed vanHAX genes at levels up to 36,000-fold greater than OI25 via co-transcription with a ribosomal RNA operon. The vancomycin-resistant revertants did not exhibit a significant growth defect. During VRE outbreaks, attention should be paid to contemporaneous vancomycin-susceptible strains as these may carry silent vancomycin resistance genes that can be activated through genomic rearrangements

Recent Recombination Events in the Core Genome Are Associated with Adaptive Evolution in Enterococcus faecium

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