Whole-genome sequencing offers new insights into the evolution of bacterial pathogens and the etiology of bacterial disease. Staphylococcus aureus is a major cause of bacteria-associated mortality and invasive disease and is carried asymptomatically by 27% of adults. Eighty percent of bacteremias match the carried strain. However, the role of evolutionary change in the pathogen during the progression from carriage to disease is incompletely understood. Here we use high-throughput genome sequencing to discover the genetic changes that accompany the transition from nasal carriage to fatal bloodstream infection in an individual colonized with methicillin-sensitive S. aureus . We found a single, cohesive population exhibiting a repertoire of 30 single-nucleotide polymorphisms and four insertion/deletion variants. Mutations accumulated at a steady rate over a 13-mo period, except for a cluster of mutations preceding the transition to disease. Although bloodstream bacteria differed by just eight mutations from the original nasally carried bacteria, half of those mutations caused truncation of proteins, including a premature stop codon in an AraC -family transcriptional regulator that has been implicated in pathogenicity. Comparison with evolution in two asymptomatic carriers supported the conclusion that clusters of protein-truncating mutations are highly unusual. Our results demonstrate that bacterial diversity in vivo is limited but nonetheless detectable by whole-genome sequencing, enabling the study of evolutionary dynamics within the host. Regulatory or structural changes that occur during carriage may be functionally important for pathogenesis; therefore identifying those changes is a crucial step in understanding the biological causes of invasive bacterial disease.

Background Staphylococcus aureus is a major cause of healthcare associated mortality, but like many important bacterial pathogens, it is a common constituent of the normal human body flora. Around a third of healthy adults are carriers. Recent evidence suggests that evolution of S. aureus during nasal carriage may be associated with progression to invasive disease. However, a more detailed understanding of within-host evolution under natural conditions is required to appreciate the evolutionary and mechanistic reasons why commensal bacteria such as S. aureus cause disease. Therefore we examined in detail the evolutionary dynamics of normal, asymptomatic carriage. Sequencing a total of 131 genomes across 13 singly colonized hosts using the Illumina platform, we investigated diversity, selection, population dynamics and transmission during the short-term evolution of S. aureus.Principal FindingsWe characterized the processes by which the raw material for evolution is generated: micro-mutation (point mutation and small insertions/deletions), macro-mutation (large insertions/deletions) and the loss or acquisition of mobile elements (plasmids and bacteriophages). Through an analysis of synonymous, non-synonymous and intergenic mutations we discovered a fitness landscape dominated by purifying selection, with rare examples of adaptive change in genes encoding surface-anchored proteins and an enterotoxin. We found evidence for dramatic, hundred-fold fluctuations in the size of the within-host population over time, which we related to the cycle of colonization and clearance. Using a newly-developed population genetics approach to detect recent transmission among hosts, we revealed evidence for recent transmission between some of our subjects, including a husband and wife both carrying populations of methicillin-resistant S. aureus (MRSA).SignificanceThis investigation begins to paint a picture of the within-host evolution of an important bacterial pathogen during its prevailing natural state, asymptomatic carriage. These results also have wider significance as a benchmark for future systematic studies of evolution during invasive S. aureus disease.

Horizontal gene transfer is an important driver of bacterial evolution, but genetic exchange in the core genome of clonal species, including the major pathogen Staphylococcus aureus, is incompletely understood. Here we reveal widespread homologous recombination in S. aureus at the species level, in contrast to its near-complete absence between closely related strains. We discover a patchwork of hotspots and coldspots at fine scales falling against a backdrop of broad-scale trends in rate variation. Over megabases, homoplasy rates fluctuate 1.9-fold, peaking towards the origin-of-replication. Over kilobases, we find core recombination hotspots of up to 2.5-fold enrichment situated near fault lines in the genome associated with mobile elements. The strongest hotspots include regions flanking conjugative transposon ICE6013, the staphylococcal cassette chromosome (SCC) and genomic island νSaα. Mobile element-driven core genome transfer represents an opportunity for adaptation and challenges our understanding of the recombination landscape in predominantly clonal pathogens, with important implications for genotype–phenotype mapping.

IBD in children with disease onset before the age of 2 years is frequently unclassifiable into Crohn's disease and ulcerative colitis, particularly treatment resistant, and can be indistinguishable from monogenic diseases with IBD-like phenotype.

BackgroundApproximately 30–40% of children <1 year of age are Clostridium difficile colonized, and may represent a reservoir for adult C. difficile infections (CDI). Risk factors for colonization with toxigenic versus non-toxigenic C. difficile strains and longitudinal acquisition dynamics in infants remain incompletely characterized.MethodsPredominantly healthy infants (≤2 years) were recruited in Oxfordshire, UK, and provided ≥1 fecal samples. Independent risk factors for toxigenic/non-toxigenic C. difficile colonization and acquisition were identified using multivariable regression. Infant C. difficile isolates were whole-genome sequenced to assay genetic diversity and prevalence of toxin-associated genes, and compared with sequenced strains from Oxfordshire CDI cases.Results338/365 enrolled infants provided 1332 fecal samples, representing 158 C. difficile colonization or carriage episodes (107[68%] toxigenic). Initial colonization was associated with age, and reduced with breastfeeding but increased with pet dogs. Acquisition was associated with older age, Caesarean delivery, and diarrhea. Breastfeeding and pre-existing C. difficile colonization reduced acquisition risk. Overall 13% of CDI C. difficile strains were genetically related to infant strains. 29(18%) infant C. difficile sequences were consistent with recent direct/indirect transmission to/from Oxfordshire CDI cases (≤2 single nucleotide variants [SNVs]); 79(50%) shared a common origin with an Oxfordshire CDI case within the last ~5 years (0–10 SNVs). The hypervirulent, epidemic ST1/ribotype 027 remained notably absent in infants in this large study, as did other lineages such as STs 10/44 (ribotype 015); the most common strain in infants was ST2 (ribotype 020/014)(22%).ConclusionsIn predominantly healthy infants without significant healthcare exposure C. difficile colonization and acquisition reflect environmental exposures, with pet dogs identified as a novel risk factor. Genetic overlap between some infant strains and those isolated from CDI cases suggest common community reservoirs of these C. difficile lineages, contrasting with those lineages found only in CDI cases, and therefore more consistent with healthcare-associated spread.

A simple skin wash technique suitable for the quantitative and functional analysis of biomolecules in AD is described. Using this method we show that MMPs, and in particular MMP-8 and MMP-9, represent an important potential component of the pathology of AD. The method is expected to prove useful in advancing our understanding of AD and in identifying biomarkers for the evaluation of new therapies.

BackgroundStaphylococcal protein A (spa) is an important virulence factor which enables Staphylococcus aureus to evade host immune responses. Genotypes known as “spa-types”, based on highly variable Xr region sequences of the spa-gene, are frequently used to classify strains. A weakness of current spa-typing primers is that rearrangements in the IgG-binding region of the gene cause 1-2% of strains to be designated as “non-typeable”.ResultsWe developed an improved primer which enabled sequencing of all strains, containing any type of genetic rearrangement, in a large study among community carriers and hospital inpatients in Oxfordshire, UK (6110 isolates). We identified eight novel spa-gene variants, plus one previously described. Three of these rearrangements would be designated “non-typeable” using current spa-typing methods; they occurred in 1.8% (72/3905) asymptomatically carried and 0.6% (14/2205) inpatient S. aureus strains. Some individuals were simultaneously colonized by both formerly non-typeable and typeable strains; previously such patients would have been identified as carrying only currently typeable strains, underestimating mixed carriage prevalence and diversity. Formerly non-typeable strains were found in more spa-types associated with multilocus sequence type ST398 (35%), common among livestock, compared to other groups with any non-typeable strains (1-4%), suggesting particular spa-types may have been under-represented in previous human studies.ConclusionsThis improved method allows us to spa-type previously non-typeable strains with rearrangements in the spa-gene and to resolve cases of mixed colonization with deletions in one or more strains, thus accounting for hidden diversity of S. aureus in both community and hospital environments.

SummaryBackgroundStaphylococcus aureus nasal carriage increases infection risk. However, few studies have investigated S. aureus acquisition/loss over >1 year, and fewer still used molecular typing.Methods1123 adults attending five Oxfordshire general practices had nasal swabs taken. 571 were re-swabbed after one month then every two months for median two years. All S. aureus isolates were spa-typed. Risk factors were collected from interviews and medical records.Results32% carried S. aureus at recruitment (<1% MRSA). Rates of spa-type acquisition were similar in participants S. aureus positive (1.4%/month) and negative (1.8%/month, P = 0.13) at recruitment. Rates were faster in those carrying clonal complex (CC)15 (adjusted (a)P = 0.03) or CC8 (including USA300) (aP = 0.001) at recruitment versus other CCs. 157/274 (57%) participants S. aureus positive at recruitment returning ≥12 swabs carried S. aureus consistently, of whom 135 carried the same spa-type. CC22 (including EMRSA-15) was more prevalent in long-term than intermittent spa-type carriers (aP = 0.03). Antibiotics transiently reduced carriage, but no other modifiable risk factors were found.ConclusionsBoth transient and longer-term carriage exist; however, the approximately constant rates of S. aureus gain and loss suggest that ‘never’ or truly ‘persistent’ carriage are rare. Long-term carriage varies by strain, offering new explanations for the success of certain S. aureus clones.