Histologic and Genotypic Characterization of Lung Cancer in the Inuit Population of the Eastern Canadian Arctic.

Inuit are the Indigenous Arctic peoples and residents of the Canadian territory of Nunavut who have the highest global rate of lung cancer. Given lung cancer's mortality, histological and genomic characterization was undertaken to better understand the disease biology. We retrospectively studied all Inuit cases from Nunavut's Qikiqtani (Baffin) region, referred to the Ottawa Hospital Cancer Center between 2001 and 2011. Demographics were compiled from medical records and tumor samples underwent pathologic/histologic confirmation. Tumors were analyzed by next generation sequencing (NGS) with a cancer hotspot mutation panel. Of 98 patients, the median age was 66 years and 61% were male. Tobacco use was reported in 87%, and 69% had a history of lung disease (tuberculosis or other). Histological types were: non-small cell lung carcinoma (NSCLC), 81%; small cell lung carcinoma, 16%. Squamous cell carcinoma (SCC) represented 65% of NSCLC. NGS on 55 samples demonstrated mutation rates similar to public lung cancer datasets. In SCC, the STK11 F354L mutation was observed at higher frequency than previously reported. This is the first study to characterize the histologic/genomic profiles of lung cancer in this population. A high incidence of SCC, and an elevated rate of STK11 mutations distinguishes this group from the North American population.

Whole blood human transcriptome and virome analysis of ME/CFS patients experiencing post-exertional malaise following cardiopulmonary exercise testing.

Myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) is a syndrome of unknown etiology characterized by profound fatigue exacerbated by physical activity, also known as post-exertional malaise (PEM). Previously, we did not detect evidence of immune dysregulation or virus reactivation outside of PEM periods. Here we sought to determine whether cardiopulmonary exercise stress testing of ME/CFS patients could trigger such changes. ME/CFS patients (n = 14) and matched sedentary controls (n = 11) were subjected to cardiopulmonary exercise on 2 consecutive days and followed up to 7 days post-exercise, and longitudinal whole blood samples analyzed by RNA-seq. Although ME/CFS patients showed significant worsening of symptoms following exercise versus controls, with 8 of 14 ME/CFS patients showing reduced oxygen consumption ([Formula: see text]) on day 2, transcriptome analysis yielded only 6 differentially expressed gene (DEG) candidates when comparing ME/CFS patients to controls across all time points. None of the DEGs were related to immune signaling, and no DEGs were found in ME/CFS patients before and after exercise. Virome composition (P = 0.746 by chi-square test) and number of viral reads (P = 0.098 by paired t-test) were not significantly associated with PEM. These observations do not support transcriptionally-mediated immune cell dysregulation or viral reactivation in ME/CFS patients during symptomatic PEM episodes.

Immunosignature Analysis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

A random-sequence peptide microarray can interrogate serum antibodies in a broad, unbiased fashion to generate disease-specific immunosignatures. This approach has been applied to cancer detection, diagnosis of infections, and interrogation of vaccine response. We hypothesized that there is an immunosignature specific to ME/CFS and that this could aid in the diagnosis. We studied two subject groups meeting the Canadian Consensus Definition of ME/CFS. ME/CFS (n = 25) and matched control (n = 25) sera were obtained from a Canadian study. ME/CFS (n = 25) sera were obtained from phase 1/2 Norwegian trials (NCT01156909). Sera from six healthy controls from the USA were included in the analysis. Canadian cases and controls were tested for a disease immunosignature. By combining results from unsupervised and supervised analyses, a candidate immunosignature with 654 peptides was able to differentiate ME/CFS from controls. The immunosignature was tested and further refined using the Norwegian and USA samples. This resulted in a 256-peptide immunosignature with the ability to separate ME/CFS cases from controls in the international data sets. We were able to identify a 256-peptide signature that separates ME/CFS samples from healthy controls, suggesting that the hit-and-run hypothesis of immune dysfunction merits further investigation. By extending testing of both our signature and one previously reported in the literature to larger cohorts, and further interrogating the specific peptides we and others have identified, we may deepen our understanding of the origins of ME/CFS and work towards a clinically meaningful diagnostic biomarker.

Formalin fixation increases deamination mutation signature but should not lead to false positive mutations in clinical practice.

Genomic analysis of cancer tissues is an essential aspect of personalized oncology treatment. Though it has been suggested that formalin fixation of patient tissues may be suboptimal for molecular studies, this tissue processing approach remains the industry standard. Therefore clinical molecular laboratories must be able to work with formalin fixed, paraffin embedded (FFPE) material. This study examines the effects of pre-analytic variables introduced by routine pathology processing on specimens used for clinical reports produced by next-generation sequencing technology. Tissue resected from three colorectal cancer patients was subjected to 2, 15, 24, and 48 hour fixation times in neutral buffered formalin. DNA was extracted from all tissues twice, once with uracil-N-glycosylase (UNG) treatment to counter deamination effects, and once without. Of note, deamination events at methylated cytosine, as found at CpG sites, remains unaffected by UNG. After extraction a two-step PCR targeted sequencing method was performed using the Illumina MiSeq and the data was analyzed via a custom-built bioinformatics pipeline, including filtration of reads with mapping quality <30. A larger baseline group of samples (n = 20) was examined to establish if there was a sample performance difference between the two DNA extraction methods, with/without UNG treatment. There was no statistical difference between sequencing performance of the two extraction methods when comparing read counts (raw, mapped, and filtered) and read quality (% mapped, % filtered). Analyzing mutation type, there was no significant difference between mutation calls until the 48 hour fixation treatment. At 48 hours there is a significant increase in C/G->T/A mutations that is not represented in DNA treated with UNG. This suggests these errors may be due to deamination events triggered by a longer fixation time. However the allelic frequency of these events remained below the limit of detection for reportable mutations in this assay (<2%). We do however recommend that suspected intratumoral heterogeneity events be verified by re-sequencing the same FFPE block.

RNA-Seq Analysis of Gene Expression, Viral Pathogen, and B-Cell/T-Cell Receptor Signatures in Complex Chronic Disease.

Background: Chronic fatigue syndrome (CFS) remains poorly understood. Although infections are speculated to trigger the syndrome, a specific infectious agent and underlying pathophysiological mechanism remain elusive. In a previous study, we described similar clinical phenotypes in CFS patients and alternatively diagnosed chronic Lyme syndrome (ADCLS) patients­individuals diagnosed with Lyme disease by testing from private Lyme specialty laboratories but who test negative by reference 2-tiered serologic analysis. Methods: Here, we performed blinded RNA-seq analysis of whole blood collected from 25 adults diagnosed with CFS and 13 ADCLS patients, comparing these cases to 25 matched controls and 11 patients with well-controlled systemic lupus erythematosus (SLE). Samples were collected at patient enrollment and not during acute symptom flares. RNA-seq data were used to study host gene expression, B-cell/T-cell receptor profiles (BCR/TCR), and potential viral infections. Results: No differentially expressed genes (DEGs) were found to be significant when CFS or ADCLS cases were compared to controls. Forty-two DEGs were found when SLE cases were compared to controls, consistent with activation of interferon signaling pathways associated with SLE disease. BCR/TCR repertoire analysis did not show significant differences between CFS and controls or ADCLS and controls. Finally, viral sequences corresponding to anelloviruses, human pegivirus 1, herpesviruses, and papillomaviruses were detected in RNA-seq data, but proportions were similar (P = .73) across all genus-level taxonomic categories. Conclusions: Our observations do not support a theory of transcriptionally mediated immune cell dysregulation in CFS and ADCLS, at least outside of periods of acute symptom flares.

Metagenomic Investigation of Plasma in Individuals with ME/CFS Highlights the Importance of Technical Controls to Elucidate Contamination and Batch Effects.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease causing indefinite fatigue. ME/CFS has long been hypothesised to have an infectious cause; however, no specific infectious agent has been identified. We used metagenomics to analyse the RNA from plasma samples from 25 individuals with ME/CFS and compare their microbial content to technical controls as well as three control groups: individuals with alternatively diagnosed chronic Lyme syndrome (N = 13), systemic lupus erythematosus (N = 11), and healthy controls (N = 25). We found that the majority of sequencing reads were removed during host subtraction, thus there was very low microbial RNA content in the plasma. The effects of sample batching and contamination during sample processing proved to outweigh the effects of study group on microbial RNA content, as the few differences in bacterial or viral RNA abundance we did observe between study groups were most likely caused by contamination and batch effects. Our results highlight the importance of including negative controls in all metagenomic analyses, since there was considerable overlap between bacterial content identified in study samples and control samples. For example, Proteobacteria, Firmicutes, Actinobacteria, and Bacteriodes were found in both study samples and plasma-free negative controls. Many of the taxonomic groups we saw in our plasma-free negative control samples have previously been associated with diseases, including ME/CFS, demonstrating how incorrect conclusions may arise if controls are not used and batch effects not accounted for.

Genomic Analysis of a Serotype 5 Streptococcus pneumoniae Outbreak in British Columbia, Canada, 2005-2009.

Background. Streptococcus pneumoniae can cause a wide spectrum of disease, including invasive pneumococcal disease (IPD). From 2005 to 2009 an outbreak of IPD occurred in Western Canada, caused by a S. pneumoniae strain with multilocus sequence type (MLST) 289 and serotype 5. We sought to investigate the incidence of IPD due to this S. pneumoniae strain and to characterize the outbreak in British Columbia using whole-genome sequencing. Methods. IPD was defined according to Public Health Agency of Canada guidelines. Two isolates representing the beginning and end of the outbreak were whole-genome sequenced. The sequences were analyzed for single nucleotide variants (SNVs) and putative genomic islands. Results. The peak of the outbreak in British Columbia was in 2006, when 57% of invasive S. pneumoniae isolates were serotype 5. Comparison of two whole-genome sequenced strains showed only 10 SNVs between them. A 15.5 kb genomic island was identified in outbreak strains, allowing the design of a PCR assay to track the spread of the outbreak strain. Discussion. We show that the serotype 5 MLST 289 strain contains a distinguishing genomic island, which remained genetically consistent over time. Whole-genome sequencing holds great promise for real-time characterization of outbreaks in the future and may allow responses tailored to characteristics identified in the genome.

Whole-Genome Sequencing of Three Clonal Clinical Isolates of B. cenocepacia from a Patient with Cystic Fibrosis.

Burkholderia cepacia complex bacteria are amongst the most feared of pathogens in cystic fibrosis (CF). The BCC comprises at least 20 distinct species that can cause chronic and unpredictable lung infections in CF. Historically the species B. cenocepacia has been the most prevalent in CF infections and has been associated in some centers with high rates of mortality. Modeling chronic infection by B. cenocepacia in the laboratory is challenging and no models exist which effectively recapitulate CF disease caused by BCC bacteria. Therefore our understanding of factors that contribute towards the morbidity and mortality caused by this organism is limited. In this study we used whole-genome sequencing to examine the evolution of 3 clonal clinical isolates of B. cenocepacia from a patient with cystic fibrosis. The first isolate was from the beginning of infection, and the second two almost 10 years later during the final year of the patients' life. These isolates also demonstrated phenotypic heterogeneity, with the first isolate displaying the mucoid phenotype (conferred by the overproduction of exopolysaccharide), while one of the later two was nonmucoid. In addition we also sequenced a nonmucoid derivative of the initial mucoid isolate, acquired in the laboratory by antibiotic pressure. Examination of sequence data revealed that the two late stage isolates shared 20 variant nucleotides in common compared to the early isolate. However, despite their isolation within 10 months of one another, there was also considerable variation between the late stage isolates, including 42 single nucleotide variants and three deletions. Additionally, no sequence differences were identified between the initial mucoid isolate and its laboratory acquired nonmucoid derivative, however transcript analysis indicated at least partial down regulation of genes involved in exopolysaccharide production. Our study examines the progression of B. cenocepacia throughout chronic infection, including establishment of sub-populations likely evolved from the original isolate, suggestive of parallel evolution. Additionally, the lack of sequence differences between two of the isolates with differing mucoid phenotypes suggests that other factors, such as gene regulation, come into play in establishing the mucoid phenotype.

Lyme Disease Diagnosed by Alternative Methods: A Phenotype Similar to That of Chronic Fatigue Syndrome.

BACKGROUND: A subset of patients reporting a diagnosis of Lyme disease can be described as having alternatively diagnosed chronic Lyme syndrome (ADCLS), in which diagnosis is based on laboratory results from a nonreference Lyme specialty laboratory using in-house criteria. Patients with ADCLS report symptoms similar to those reported by patients with chronic fatigue syndrome (CFS). METHODS: We performed a case-control study comparing patients with ADCLS and CFS to each other and to both healthy controls and controls with systemic lupus erythematosus (SLE). Subjects completed a history, physical exam, screening laboratory tests, 7 functional scales, reference serology for Lyme disease using Centers for Disease Control and Prevention criteria, reference serology for other tick-associated pathogens, and cytokine expression studies. RESULTS: The study enrolled 13 patients with ADCLS (12 of whom were diagnosed by 1 alternative US laboratory), 25 patients with CFS, 25 matched healthy controls, and 11 SLE controls. Baseline clinical data and functional scales indicate significant disability among ADCLS and CFS patients and many important differences between these groups and controls, but no significant differences between each other. No ADCLS patient was confirmed as having positive Lyme serology by reference laboratory testing, and there was no difference in distribution of positive serology for other tick-transmitted pathogens or cytokine expression across the groups. CONCLUSIONS: In British Columbia, a setting with low Lyme disease incidence, ADCLS patients have a similar phenotype to that of CFS patients. Disagreement between alternative and reference laboratory Lyme testing results in this setting is most likely explained by false-positive results from the alternative laboratory.

Submaximal exercise testing with near-infrared spectroscopy in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome patients compared to healthy controls: a case-control study.

BACKGROUND: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating illness. Symptoms include profound fatigue and distinctive post-exertional malaise (PEM). We asked whether a submaximal exercise test would prove useful for identifying different patterns of tissue oxygen utilization in individuals with ME/CFS versus healthy subjects. Such a test has potential to aid with ME/CFS diagnosis, or to characterize patients' illness. METHODS: A case-control study of 16 patients with ME/CFS compared to 16 healthy controls completing a 3-min handgrip protocol was performed. Response was measured using near-infrared spectroscopy, resulting in measurements of oxygenated (O2Hb) and deoxygenated hemoglobin (HHb) over wrist extensors and flexors. Changes in O2Hb (delta (d)O2Hb) and HHb (dHHb) absorbance between the first and last contraction were calculated, as were the force-time product of all contractions, measured as tension-time index (TTI), and ratings of perceived exertion (RPE). RESULTS: Individuals with ME/CFS demonstrated smaller dO2Hb and dHHb than controls. However, after adjusting for TTI and change in total hemoglobin (delta (d)tHb), differences in dO2Hb and dHHb were reduced, with large overlapping variances. RPE was significantly higher for cases than controls, particularly at rest. CONCLUSIONS: Relative to controls, participants with ME/CFS demonstrated higher RPE, lower TTI, and reduced dO2Hb and dHHb during repetitive handgrip exercise, although considerable variance was observed. With further study, submaximal exercise testing may prove useful for stratifying patients with a lower propensity for inducing PEM, and have the ability to establish baseline intensities for exercise prescription.

Mobile elements drive recombination hotspots in the core genome of Staphylococcus aureus.

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.

Prevalence of Staphylococcus aureus protein A (spa) mutants in the community and hospitals in Oxfordshire.

BACKGROUND: Staphylococcal 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". RESULTS: We 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. CONCLUSIONS: This 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.

Carriage of methicillin-resistant Staphylococcus aureus by wild urban Norway rats (Rattus norvegicus).

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of multi-drug-resistant infections in people, particularly indigent populations. MRSA can be transmitted between people and domestic animals, but the potential for transmission between people and commensal pests, particularly rodents, had not been investigated. The objective of this study was to identify the presence and characterize the ecology of MRSA in rats (Rattus spp.) from in an impoverished, inner-city neighborhood. Oropharyngeal swabs were collected from rats trapped in 33 city blocks and one location within the adjacent port. Bacterial culture was performed and MRSA isolates were characterized using a variety of methods, including whole-genome sequencing (WGS). The ecology of MRSA in rats was described using phylogenetic analysis, geospatial analysis, and generalized linear mixed models. MRSA was identified 22 of 637 (3.5%) rats tested, although prevalence varied from 0 - 50% among blocks. Isolates belonged to 4 clusters according to WGS, with the largest cluster (n = 10) containing isolates that were genetically indistinguishable from community-acquired USA300 MRSA strains isolated from people within the study area. MRSA strains demonstrated both geographic clustering and dispersion. The odds of an individual rat carrying MRSA increased with increased body fat (OR = 2.53, 95% CI = 1.33-4.82), and in the winter (OR = 5.29, 95% CI = 1.04-26.85) and spring (OR = 5.50, 95% CI = 1.10-27.58) compared to the fall. The results show that urban rats carried the same MRSA lineages occurring in local human and/or animal populations, supporting recent transmission from external sources. MRSA carriage was influenced by season, most likely as a result of temporal variation in rat behavior and rat-human interactions.

Dynamics of acquisition and loss of carriage of Staphylococcus aureus strains in the community: the effect of clonal complex.

BACKGROUND: Staphylococcus aureus nasal carriage increases infection risk. However, few studies have investigated S. aureus acquisition/loss over >1 year, and fewer still used molecular typing. METHODS: 1123 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. RESULTS: 32% 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. CONCLUSIONS: Both 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.

Metagenomics for pathogen detection in public health.

Traditional pathogen detection methods in public health infectious disease surveillance rely upon the identification of agents that are already known to be associated with a particular clinical syndrome. The emerging field of metagenomics has the potential to revolutionize pathogen detection in public health laboratories by allowing the simultaneous detection of all microorganisms in a clinical sample, without a priori knowledge of their identities, through the use of next-generation DNA sequencing. A single metagenomics analysis has the potential to detect rare and novel pathogens, and to uncover the role of dysbiotic microbiomes in infectious and chronic human disease. Making use of advances in sequencing platforms and bioinformatics tools, recent studies have shown that metagenomics can even determine the whole-genome sequences of pathogens, allowing inferences about antibiotic resistance, virulence, evolution and transmission to be made. We are entering an era in which more novel infectious diseases will be identified through metagenomics-based methods than through traditional laboratory methods. The impetus is now on public health laboratories to integrate metagenomics techniques into their diagnostic arsenals.

Within-host evolution of Staphylococcus aureus during asymptomatic carriage.

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 FINDINGS: We 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). SIGNIFICANCE: This 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.

Evolutionary dynamics of Staphylococcus aureus during progression from carriage to disease.

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.