Macrophages in the healthy intestine are highly specialized and usually respond to the gut microbiota without provoking an inflammatory response. A breakdown in this tolerance leads to inflammatory bowel disease (IBD), but the mechanisms by which intestinal macrophages normally become conditioned to promote microbial tolerance are unclear. Strong epidemiological evidence linking disruption of the gut microbiota by antibiotic use early in life to IBD indicates an important role for the gut microbiota in modulating intestinal immunity. Here, we show that antibiotic use causes intestinal macrophages to become hyperresponsive to bacterial stimulation, producing excess inflammatory cytokines. Re-exposure of antibiotic-treated mice to conventional microbiota induced a long-term, macrophage-dependent increase in inflammatory T helper 1 (TH1) responses in the colon and sustained dysbiosis. The consequences of this dysregulated macrophage activity for T cell function were demonstrated by increased susceptibility to infections requiring TH17 and TH2 responses for clearance (bacterial Citrobacter rodentium and helminth Trichuris muris infections), corresponding with increased inflammation. Short-chain fatty acids (SCFAs) were depleted during antibiotic administration; supplementation of antibiotics with the SCFA butyrate restored the characteristic hyporesponsiveness of intestinal macrophages and prevented T cell dysfunction. Butyrate altered the metabolic behavior of macrophages to increase oxidative phosphorylation and also promoted alternative macrophage activation. In summary, the gut microbiota is essential to maintain macrophage-dependent intestinal immune homeostasis, mediated by SCFA-dependent pathways. Oral antibiotics disrupt this process to promote sustained T cell–mediated dysfunction and increased susceptibility to infections, highlighting important implications of repeated broad-spectrum antibiotic use.

Summary Supplementation with members of the early-life microbiota as “probiotics” is increasingly used in attempts to beneficially manipulate the preterm infant gut microbiota. We performed a large observational longitudinal study comprising two preterm groups: 101 infants orally supplemented with Bifidobacterium and Lactobacillus (Bif/Lacto) and 133 infants non-supplemented (control) matched by age, sex, and delivery method. 16S rRNA gene profiling on fecal samples (n = 592) showed a predominance of Bifidobacterium and a lower abundance of pathobionts in the Bif/Lacto group. Metabolomic analysis showed higher fecal acetate and lactate and a lower fecal pH in the Bif/Lacto group compared to the control group. Fecal acetate positively correlated with relative abundance of Bifidobacterium, consistent with the ability of the supplemented Bifidobacterium strain to metabolize human milk oligosaccharides into acetate. This study demonstrates that microbiota supplementation is associated with a Bifidobacterium -dominated preterm microbiota and gastrointestinal environment more closely resembling that of full-term infants.

BackgroundThe performance of RNA sequencing (RNA-seq) aligners and assemblers varies greatly across different organisms and experiments, and often the optimal approach is not known beforehand.ResultsHere, we show that the accuracy of transcript reconstruction can be boosted by combining multiple methods, and we present a novel algorithm to integrate multiple RNA-seq assemblies into a coherent transcript annotation. Our algorithm can remove redundancies and select the best transcript models according to user-specified metrics, while solving common artifacts such as erroneous transcript chimerisms.ConclusionsWe have implemented this method in an open-source Python3 and Cython program, Mikado, available on GitHub.

Accurate classification of a microbial mock community using MinION sequencing. We benchmarked MinION technology by profiling a bacterial mock community using R7.3 flow cells. Reads were analysed with NanoOK 18 and produced alignments to the 20 microbial reference sequences with 82-89% identity 19. Coverage ranged from almost 0 × (8 reads) of Actinomyces odontolyticus to 13 × (7,695 reads) of Streptococcus mutans, which is consistent with expected mock concentrations (Supplementary Table 1). Benchmarking to Illumina sequencing demonstrated high correlation with expected proportions (Fig. 1a, log-transformed Pearson's r = 0.94 for MinION and 0.97 for Illumina), and with each other (log-transformed Pearson's r = 0.98). Broadly similar abundance levels across both platforms were observed, with some differences in assignment to species versus genus/family (Fig. 1b). This is probable since the longer length Nanopore reads should provide

Clostridium perfringens is an important cause of animal and human infections, however information about the genetic makeup of this pathogenic bacterium is currently limited. In this study, we sought to understand and characterise the genomic variation, pangenomic diversity, and key virulence traits of 56 C. perfringens strains which included 51 public, and 5 newly sequenced and annotated genomes using Whole Genome Sequencing. Our investigation revealed that C. perfringens has an “open” pangenome comprising 11667 genes and 12.6% of core genes, identified as the most divergent single-species Gram-positive bacterial pangenome currently reported. Our computational analyses also defined C. perfringens phylogeny (16S rRNA gene) in relation to some 25 Clostridium species, with C. baratii and C. sardiniense determined to be the closest relatives. Profiling virulence-associated factors confirmed presence of well-characterised C. perfringens-associated exotoxins genes including α-toxin (plc), enterotoxin (cpe), and Perfringolysin O (pfo or pfoA), although interestingly there did not appear to be a close correlation with encoded toxin type and disease phenotype. Furthermore, genomic analysis indicated significant horizontal gene transfer events as defined by presence of prophage genomes, and notably absence of CRISPR defence systems in >70% (40/56) of the strains. In relation to antimicrobial resistance mechanisms, tetracycline resistance genes (tet) and anti-defensins genes (mprF) were consistently detected in silico (tet: 75%; mprF: 100%). However, pre-antibiotic era strain genomes did not encode for tet, thus implying antimicrobial selective pressures in C. perfringens evolutionary history over the past 80 years. This study provides new genomic understanding of this genetically divergent multi-host bacterium, and further expands our knowledge on this medically and veterinary important pathogen.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen particularly associated with the inherited disease cystic fibrosis (CF). Pseudomonas aeruginosa is well known to have a large and adaptable genome that enables it to colonise a wide range of ecological niches. Here, we have used a comparative genomics approach to identify changes that occur during infection of the CF lung. We used the mucoid phenotype as an obvious marker of host adaptation and compared these genomes to analyse SNPs, indels and islands within near-isogenic pairs. To commence the correction of the natural bias towards clinical isolates in genomics studies and to widen our understanding of the genomic diversity of P. aeruginosa, we included four environmental isolates in our analysis. Our data suggest that genome plasticity plays an important role in chronic infection and that the strains sequenced in this study are representative of the two major phylogenetic groups as determined by core genome SNP analysis.

Klebsiella spp. are frequently enriched in the gut microbiota of preterm neonates, and overgrowth is associated with necrotizing enterocolitis (NEC), nosocomial infections and late-onset sepsis. Little is known about the genomic and phenotypic characteristics of preterm-associated Klebsiella , as previous studies have focused on the recovery of antimicrobial-resistant isolates or culture-independent molecular analyses. The aim of this study was to better characterize preterm-associated Klebsiella populations using phenotypic and genotypic approaches. Faecal samples from a UK cohort of healthy and sick preterm neonates (n=109) were screened on MacConkey agar to isolate lactose-positive Enterobacteriaceae . Whole-genome sequences were generated for Klebsiella spp., and virulence and antimicrobial resistance genes identified. Antibiotic susceptibility profiling and in vitro macrophage and iron assays were undertaken for the Klebsiella strains. Metapangenome analyses with a manually curated genome dataset were undertaken to examine the diversity of Klebsiella oxytoca and related bacteria in a publicly available shotgun metagenome dataset. Approximately one-tenth of faecal samples harboured Klebsiella spp. ( Klebsiella pneumoniae , 7.3 %; Klebsiella quasipneumoniae , 0.9 %; Klebsiella grimontii , 2.8 %; Klebsiella michiganensis , 1.8 %). Isolates recovered from NEC- and sepsis-affected infants and those showing no signs of clinical infection (i.e. ‘healthy’) encoded multiple β-lactamases. No difference was observed between isolates recovered from healthy and sick infants with respect to in vitro siderophore production (all encoded enterobactin in their genomes). All K. pneumoniae , K. quasipneumoniae , K. grimontii and K. michiganensis faecal isolates tested were able to reside and persist in macrophages, indicating their immune evasion abilities. Metapangenome analyses of published metagenomic data confirmed our findings regarding the presence of K. michiganensis in the preterm gut. There is little difference in the phenotypic and genomic characteristics of Klebsiella isolates recovered from healthy and sick infants. Identification of β-lactamases in all isolates may prove problematic when defining treatment regimens for NEC or sepsis, and suggests that healthy preterm infants contribute to the resistome. Refined analyses with curated sequence databases are required when studying closely related species present in metagenomic data.

BackgroundInfants born prematurely, particularly extremely low birth weight infants (ELBW) have altered gut microbial communities. Factors such as maternal health, gut immaturity, delivery mode, and antibiotic treatments are associated with microbiota disturbances, and are linked to an increased risk of certain diseases such as necrotising enterocolitis. Therefore, there is a requirement to optimally characterise microbial profiles in this at-risk cohort, via standardisation of methods, particularly for studying the influence of microbiota therapies (e.g. probiotic supplementation) on community profiles and health outcomes. Profiling of faecal samples using the 16S rRNA gene is a cost-efficient method for large-scale clinical studies to gain insights into the gut microbiota and additionally allows characterisation of cohorts were sample quantities are compromised (e.g. ELBW infants). However, DNA extraction method, and the 16S rRNA region targeted can significantly change bacterial community profiles obtained, and so confound comparisons between studies. Thus, we sought to optimise a 16S rRNA profiling protocol to allow standardisation for studying ELBW infant faecal samples, with or without probiotic supplementation.MethodsUsing ELBW faecal samples, we compared three different DNA extraction methods, and subsequently PCR amplified and sequenced three hypervariable regions of the 16S rRNA gene (V1 + V2 + V3), (V4 + V5) and (V6 + V7 + V8), and compared two bioinformatics approaches to analyse results (OTU and paired end). Paired shotgun metagenomics was used as a ‘gold-standard’.ResultsResults indicated a longer bead-beating step was required for optimal bacterial DNA extraction and that sequencing regions (V1 + V2 + V3) and (V6 + V7 + V8) provided the most representative taxonomic profiles, which was confirmed via shotgun analysis. Samples sequenced using the (V4 + V5) region were found to be underrepresented in specific taxa including Bifidobacterium, and had altered diversity profiles. Both bioinformatics 16S rRNA pipelines used in this study (OTU and paired end) presented similar taxonomic profiles at genus level.ConclusionsWe determined that DNA extraction from ELBW faecal samples, particularly those infants receiving probiotic supplementation, should include a prolonged beat-beating step. Furthermore, use of the 16S rRNA (V1 + V2 + V3) and (V6 + V7 + V8) regions provides reliable representation of ELBW microbiota profiles, while inclusion of the (V4 + V5) region may not be appropriate for studies where Bifidobacterium constitutes a resident microbiota member.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4229-x) contains supplementary material, which is available to authorized users.