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Antimicrobial resistance (AMR) in drinking water has received less attention than its counterparts in the urban water cycle. While culture-based techniques or gene-centric PCR have been used to probe the impact of treatment approaches (e.g., disinfection) on AMR in drinking water, to our knowledge there is no systematic comparison of AMR trait distribution and prevalence between disinfected and disinfectant residual-free drinking water systems. We used metagenomics to assess the associations between disinfectant residuals and AMR prevalence and its host association in full-scale drinking water distribution systems (DWDSs) with and without disinfectant residuals. While the differences in AMR profiles between DWDSs were associated with the presence or absence of disinfectant, they were also associated with overall water chemistry and more importantly with microbial community structure. AMR genes and mechanisms differentially abundant in disinfected systems were primarily associated with nontuberculous mycobacteria (NTM). Finally, evaluation of metagenome assembled genomes (MAGs) also suggests that NTM possessing AMR genes conferring intrinsic resistance to key antibiotics were prevalent in disinfected systems, whereas such NTM genomes were not detected in disinfectant residual free DWDSs. Altogether, our findings provide insights into the drinking water resistome and its association with potential opportunistic pathogens, particularly in systems with disinfectant residual.

Limiting microbial growth during drinking water distribution is achieved either by maintaining a disinfectant residual or through nutrient limitation without the use of a disinfectant. The impact of these contrasting approaches on the drinking water microbiome is not systematically understood. We utilized genome-resolved metagenomics to compare the structure, metabolic traits, and population genomes of drinking water microbiomes across multiple full-scale drinking water systems utilizing these two-distinct microbial growth control strategies. Microbial communities cluster together at the structural- and functional potential-level based on the presence or absence of a disinfectant residual. Disinfectant residual concentrations alone explained 17 and 6.5% of the variance in structure and functional potential of the drinking water microbiome, respectively, despite including samples from multiple drinking water systems with variable source waters and source water communities, treatment strategies, and chemical compositions. The drinking water microbiome is structurally and functionally less diverse and less variable across disinfected systems as compared to non-disinfected systems. While bacteria were the most abundant domain, archaea and eukaryota were more abundant in non-disinfected and disinfected systems, respectively. Community-level differences in functional potential were driven by enrichment of genes associated with carbon and nitrogen fixation in non-disinfected systems and γ-aminobutyrate metabolism in disinfected systems which may be associated with the recycling of amino acids. Metagenome-assembled genome-level analyses for a subset of phylogenetically related microorganisms suggests that disinfection may select for microorganisms capable of using fatty acids, presumably from microbial decay products, via the glyoxylate cycle. Overall, we find that disinfection exhibits systematic and consistent selective pressures on the drinking water microbiome and may select for microorganisms able to utilize microbial decay products originating from disinfection inactivated microorganisms.

Antimicrobial resistance (AMR) in drinking water has received less attention than counterparts in the urban water cycle. While culture-based techniques or gene-centric PCR have been used to probe the impact of treatment approaches (e.g., disinfection) on AMR in drinking water, to our knowledge there is no systematic comparison of AMR traits between disinfected and disinfectant residual-free drinking water systems. We use metagenomics to assess the associations between disinfectant residuals and AMR prevalence and its host association in full-scale drinking water distribution systems (DWDSs). The differences in AMR profiles between DWDSs are associated with the presence or absence of disinfectant. Further, AMR genes and mechanisms enriched in disinfected systems are associated with drug classes primarily linked to nontuberculous mycobacteria (NTM). Finally, evaluation of metagenome assembled genomes (MAGs) of NTM indicates that they possess AMR genes conferring intrinsic resistance to key antibiotics, whereas such NTM genomes were not detected in disinfectant residual free DWDSs. Thus, disinfection may not only influence the AMR profiles of the drinking water microbiome but also select for NTM with intrinsic AMR.

BackgroundAmplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity, but is limited in genetic resolution due to short read lengths. While Oxford Nanopore or Pacific Biosciences platforms overcome this limitation, their application has been limited due to higher error rates or smaller data output.ResultsIn this study, we introduce an amplicon sequencing workflow, i.e., NanoAmpli-Seq, that builds on Intramolecular-ligated Nanopore Consensus Sequencing (INC-Seq) approach and demonstrate its application for full-length 16S rRNA gene sequencing. NanoAmpli-Seq includes vital improvements to the aforementioned protocol that reduces sample-processing time while significantly improving sequence accuracy. The developed protocol includes chopSeq software for fragmentation and read orientation correction of INC-Seq consensus reads while nanoClust algorithm was designed for read partitioning-based de novo clustering and within cluster consensus calling to obtain full-length 16S rRNA gene sequences.ConclusionsNanoAmpli-Seq accurately estimates the diversity of tested mock communities with average sequence accuracy of 99.5% for 2D and 1D2 sequencing on the nanopore sequencing platform. Nearly all residual errors in NanoAmpli-Seq sequences originate from deletions in homopolymer regions, indicating that homopolymer aware basecalling or error correction may allow for sequencing accuracy comparable to short-read sequencing platforms.

Background/Aims Studying the gut microbiota in unaffected relatives of people with Crohn’s disease (CD) may advance our understanding of the role of bacteria in disease aetiology. Methods Faecal microbiota composition (16S rRNA gene sequencing), genetic functional capacity (shotgun metagenomics) and faecal short chain fatty acids (SCFA) were compared in unaffected adult relatives of CD children (CDR, n = 17) and adult healthy controls, unrelated to CD patients (HUC, n = 14). The microbiota characteristics of 19 CD children were used as a benchmark of CD ‘dysbiosis’. Results The CDR microbiota was less diverse (p = 0.044) than that of the HUC group. Local contribution of β-diversity analysis showed no difference in community structure between the CDR and HUC groups. Twenty one of 1,243 (1.8%) operational taxonomic units discriminated CDR from HUC. The metagenomic functional capacity (p = 0.207) and SCFA concentration or pattern were similar between CDR and HUC (p>0.05 for all SCFA). None of the KEGG metabolic pathways were different between these two groups. Both of these groups (HUC and CDR) had a higher microbiota α-diversity (CDR, p = 0.026 and HUC, p

OBJECTIVES: Exploring associations between the gut microbiota and colonic inflammation and assessing sequential changes during exclusive enteral nutrition (EEN) may offer clues into the microbial origins of Crohn’s disease (CD).\ud METHODS: Fecal samples (n=117) were collected from 23 CD and 21 healthy children. From CD children fecal samples were collected before, during EEN, and when patients returned to their habitual diets. Microbiota composition and functional capacity were characterized using sequencing of the 16S rRNA gene and shotgun metagenomics.\ud RESULTS: Microbial diversity was lower in CD than controls before EEN (P=0.006); differences were observed in 36 genera, 141 operational taxonomic units (OTUs), and 44 oligotypes. During EEN, the microbial diversity of CD children further decreased, and the community structure became even more dissimilar than that of controls. Every 10 days on EEN, 0.6 genus diversity equivalents were lost; 34 genera decreased and one increased during EEN. Fecal calprotectin correlated with 35 OTUs, 14 of which accounted for 78% of its variation. OTUs that correlated positively or negatively with calprotectin decreased during EEN. The microbiota of CD patients had a broader functional capacity than healthy controls, but diversity decreased with EEN. Genes involved in membrane transport, sulfur reduction, and nutrient biosynthesis differed between patients and controls. The abundance of genes involved in biotin (P=0.005) and thiamine biosynthesis decreased (P=0.017), whereas those involved in spermidine/putrescine biosynthesis (P=0.031), or the shikimate pathway (P=0.058), increased during EEN.\ud CONCLUSIONS: Disease improvement following treatment with EEN is associated with extensive modulation of the gut microbiome.

Background The study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents. Results In this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination. Conclusions These results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised. Electronic supplementary material The online version of this article (doi:10.1186/s12915-014-0087-z) contains supplementary material, which is available to authorized users.

The study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents. In this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR based 16S rRNA gene surveys and shotgun metagenomics. These results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination. Concurrent sequencing of negative control samples is strongly advised.

Background Foodborne outbreaks of Salmonella remain a pressing public health concern. We recently detected a large outbreak of Salmonella enterica serovar Enteritidis phage type 14b affecting more than 30 patients in our hospital. This outbreak was linked to community, national and European-wide cases. Hospital patients with Salmonella are at high risk, and require a rapid response. We initially investigated this outbreak by whole-genome sequencing using a novel rapid protocol on the Illumina MiSeq; we then integrated these data with whole-genome data from surveillance sequencing, thereby placing the outbreak in a national context. Additionally, we investigated the potential of a newly released sequencing technology, the MinION from Oxford Nanopore Technologies, in the management of a hospital outbreak of Salmonella. Results We demonstrate that rapid MiSeq sequencing can reduce the time to answer compared to the standard sequencing protocol with no impact on the results. We show, for the first time, that the MinION can acquire clinically relevant information in real time and within minutes of a DNA library being loaded. MinION sequencing permits confident assignment to species level within 20 min. Using a novel streaming phylogenetic placement method samples can be assigned to a serotype in 40 min and determined to be part of the outbreak in less than 2 h. Conclusions Both approaches yielded reliable and actionable clinical information on the Salmonella outbreak in less than half a day. The rapid availability of such information may facilitate more informed epidemiological investigations and influence infection control practices. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0677-2) contains supplementary material, which is available to authorized users.

Background The effect that traditional and modern DNA extraction methods have on applications to study the role of gut microbiota in health and disease is a topic of current interest. Genomic DNA was extracted from three faecal samples and one probiotic capsule using three popular methods; chaotropic (CHAO) method, phenol/chloroform (PHEC) extraction, proprietary kit (QIAG). The performance of each of these methods on DNA yield and quality, microbiota composition using quantitative PCR, deep sequencing of the 16S rRNA gene, and sequencing analysis pipeline was evaluated. Results The CHAO yielded the highest and the QIAG kit the lowest amount of double-stranded DNA, but the purity of isolated nucleic acids was better for the latter method. The CHAO method yielded a higher concentration of bacterial taxa per mass (g) of faeces. Sequencing coverage was higher in CHAO method but a higher proportion of the initial sequencing reads were retained for assignments to operational taxonomic unit (OTU) in the QIAG kit compared to the other methods. The QIAG kit appeared to have longer trimmed reads and shorter regions of worse quality than the other two methods. A distinct separation of α-diversity indices between different DNA extraction methods was not observed. When compositional dissimilarities between samples were explored, a strong separation was observed according to sample type. The effect of the extraction method was either marginal (Bray–Curtis distance) or none (unweighted Unifrac distance). Taxon membership and abundance in each sample was independent of the DNA extraction method used. Conclusions We have benchmarked several DNA extraction methods commonly used in gut microbiota research and their differences depended on the downstream applications intended for use. Caution should be paid when the intention is to pool and analyse samples or data from studies which have used different DNA extraction methods. Electronic supplementary material The online version of this article (doi:10.1186/s13104-016-2171-7) contains supplementary material, which is available to authorized users.