Shigella are human-adapted Escherichia coli that have gained the ability to invade the human gut mucosa and cause dysentery1,2, spreading efficiently via low-dose fecal-oral transmission3,4. Historically, S. sonnei has been predominantly responsible for dysentery in developed countries, but is now emerging as a problem in the developing world, apparently replacing the more diverse S. flexneri in areas undergoing economic development and improvements in water quality4-6. Classical approaches have shown S. sonnei is genetically conserved and clonal7. We report here whole-genome sequencing of 132 globally-distributed isolates. Our phylogenetic analysis shows that the current S. sonnei population descends from a common ancestor that existed less than 500 years ago and has diversified into several distinct lineages with unique characteristics. Our analysis suggests the majority of this diversification occurred in Europe, followed by more recent establishment of local pathogen populations in other continents predominantly due to the pandemic spread of a single, rapidly-evolving, multidrug resistant lineage.

Background

Mycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate knowledge of the rate of change in the genome over relevant timescales is required.

Methods

We attempted to estimate a molecular clock by sequencing 199 isolates from epidemiologically linked tuberculosis cases, collected in the Netherlands spanning almost 16 years.

Results

Multiple analyses support an average mutation rate of ~0.3 SNPs per genome per year. However, all analyses revealed a very high degree of variation around this mean, making the confirmation of links proposed by epidemiology, and inference of novel links, difficult. Despite this, in some cases, the phylogenetic context of other strains provided evidence supporting the confident exclusion of previously inferred epidemiological links.

Conclusions

This in-depth analysis of the molecular clock revealed that it is slow and variable over short time scales, which limits its usefulness in transmission studies. However, the superior resolution of whole genome sequencing can provide the phylogenetic context to allow the confident exclusion of possible transmission events previously inferred via traditional DNA fingerprinting techniques and epidemiological cluster investigation. Despite the slow generation of variation even at the whole genome level we conclude that the investigation of tuberculosis transmission will benefit greatly from routine whole genome sequencing.

Summary

Background

Tuberculosis incidence in the UK has risen in the past decade. Disease control depends on epidemiological data, which can be difficult to obtain. Whole-genome sequencing can detect microevolution within Mycobacterium tuberculosis strains. We aimed to estimate the genetic diversity of related M tuberculosis strains in the UK Midlands and to investigate how this measurement might be used to investigate community outbreaks.

Methods

In a retrospective observational study, we used Illumina technology to sequence M tuberculosis genomes from an archive of frozen cultures. We characterised isolates into four groups: cross-sectional, longitudinal, household, and community. We measured pairwise nucleotide differences within hosts and between hosts in household outbreaks and estimated the rate of change in DNA sequences. We used the findings to interpret network diagrams constructed from 11 community clusters derived from mycobacterial interspersed repetitive-unit–variable-number tandem-repeat data.

Findings

We sequenced 390 separate isolates from 254 patients, including representatives from all five major lineages of M tuberculosis. The estimated rate of change in DNA sequences was 0·5 single nucleotide polymorphisms (SNPs) per genome per year (95% CI 0·3–0·7) in longitudinal isolates from 30 individuals and 25 families. Divergence is rarely higher than five SNPs in 3 years. 109 (96%) of 114 paired isolates from individuals and households differed by five or fewer SNPs. More than five SNPs separated isolates from none of 69 epidemiologically linked patients, two (15%) of 13 possibly linked patients, and 13 (17%) of 75 epidemiologically unlinked patients (three-way comparison exact p<0·0001). Genetic trees and clinical and epidemiological data suggest that super-spreaders were present in two community clusters.

Interpretation

Whole-genome sequencing can delineate outbreaks of tuberculosis and allows inference about direction of transmission between cases. The technique could identify super-spreaders and predict the existence of undiagnosed cases, potentially leading to early treatment of infectious patients and their contacts.

Funding

Medical Research Council, Wellcome Trust, National Institute for Health Research, and the Health Protection Agency.

Summary

Background

The emergence of meticillin-resistant Staphylococcus aureus (MRSA) that can persist in the community and replace existing hospital-adapted lineages of MRSA means that it is necessary to understand transmission dynamics in terms of hospitals and the community as one entity. We assessed the use of whole-genome sequencing to enhance detection of MRSA transmission between these settings.

Methods

We studied a putative MRSA outbreak on a special care baby unit (SCBU) at a National Health Service Foundation Trust in Cambridge, UK. We used whole-genome sequencing to validate and expand findings from an infection-control team who assessed the outbreak through conventional analysis of epidemiological data and antibiogram profiles. We sequenced isolates from all colonised patients in the SCBU, and sequenced MRSA isolates from patients in the hospital or community with the same antibiotic susceptibility profile as the outbreak strain.

Findings

The hospital infection-control team identified 12 infants colonised with MRSA in a 6 month period in 2011, who were suspected of being linked, but a persistent outbreak could not be confirmed with conventional methods. With whole-genome sequencing, we identified 26 related cases of MRSA carriage, and showed transmission occurred within the SCBU, between mothers on a postnatal ward, and in the community. The outbreak MRSA type was a new sequence type (ST) 2371, which is closely related to ST22, but contains genes encoding Panton-Valentine leucocidin. Whole-genome sequencing data were used to propose and confirm that MRSA carriage by a staff member had allowed the outbreak to persist during periods without known infection on the SCBU and after a deep clean.

Interpretation

Whole-genome sequencing holds great promise for rapid, accurate, and comprehensive identification of bacterial transmission pathways in hospital and community settings, with concomitant reductions in infections, morbidity, and costs.

Funding

UK Clinical Research Collaboration Translational Infection Research Initiative, Wellcome Trust, Health Protection Agency, and the National Institute for Health Research Cambridge Biomedical Research Centre.

Two Southeast Asian students attending the same school in the United Kingdom presented with pulmonary tuberculosis. An epidemiological investigation failed to link the two cases, and drug resistance profiles of the Mycobacterium tuberculosis isolates were discrepant. Whole-genome sequencing of the isolates found them to be genetically identical, suggesting a missed transmission event.

Objectives

Epidemiological investigations of Legionnaires’ disease outbreaks rely on the rapid identification and typing of clinical and environmental Legionella isolates in order to identify and control the source of infection. Rapid bacterial whole-genome sequencing (WGS) is an emerging technology that has the potential to rapidly discriminate outbreak from non-outbreak isolates in a clinically relevant time frame.

Methods

We performed a pilot study to determine the feasibility of using bacterial WGS to differentiate outbreak from non-outbreak isolates collected during an outbreak of Legionnaires’ disease. Seven Legionella isolates (three clinical and four environmental) were obtained from the reference laboratory and sequenced using the Illumina MiSeq platform at Addenbrooke's Hospital, Cambridge. Bioinformatic analysis was performed blinded to the epidemiological data at the Wellcome Trust Sanger Institute.

Results

We were able to distinguish outbreak from non-outbreak isolates using bacterial WGS, and to confirm the probable environmental source. Our analysis also highlighted constraints, which were the small number of Legionella pneumophila isolates available for sequencing, and the limited number of published genomes for comparison.

Conclusions

We have demonstrated the feasibility of using rapid WGS to investigate an outbreak of Legionnaires’ disease. Future work includes building larger genomic databases of L pneumophila from both clinical and environmental sources, developing automated data interpretation software, and conducting a cost–benefit analysis of WGS versus current typing methods.

Bacteriovorax marinus SJ is a predatory delta-proteobacterium isolated from a marine environment. The genome sequence of this strain provides an interesting contrast to that of the terrestrial predatory bacterium Bdellovibrio bacteriovorus HD100. Based on their predatory lifestyle, Bacteriovorax were originally designated as members of the genus Bdellovibrio but subsequently were re-assigned to a new genus and family based on genetic and phenotypic differences. B. marinus attaches to Gram-negative bacteria, penetrates through the cell wall to form a bdelloplast, in which it replicates, as shown using microscopy. Bacteriovorax is distinct, as it shares only 30% of its gene products with its closest sequenced relatives. Remarkably, 34% of predicted genes over 500 nt in length were completely unique with no significant matches in the databases. As expected, Bacteriovorax shares several characteristic loci with the other delta-proteobacteria. A geneset shared between Bacteriovorax and Bdellovibrio that is not conserved among other delta-proteobacteria such as Myxobacteria (which destroy prey bacteria externally via lysis), or the non-predatory Desulfo-bacteria and Geobacter species was identified. These 291 gene orthologues common to both Bacteriovorax and Bdellovibrio may be the key indicators of host-interaction predatory-specific processes required for prey entry. The locus from Bdellovibrio bacteriovorus is implicated in the switch from predatory to prey/host-independent growth. Although the locus is conserved in B. marinus, the sequence has only limited similarity. The results of this study advance understanding of both the similarities and differences between Bdellovibrio and Bacteriovorax and confirm the distant relationship between the two and their separation into different families.

Background

Haemophilus influenzae is an important human commensal pathogen associated with significant levels of disease. High-throughput DNA sequencing was used to investigate differences in genome content within this species.

Results

Genomic DNA sequence was obtained from 85 strains of H. influenzae and from other related species, selected based on geographical site of isolation, disease association and documented genotypic and phenotypic differences. When compared by Mauve alignment these indicated groupings of H. influenzae that were consistent with previously published analyses; capsule expressing strains fell into two distinct groups and those of serotype b (Hib) were found in two closely positioned lineages. For 18 Hib strains representing both lineages we found many discrete regions (up to 40% of the total genome) displaying sequence variation when compared to a common reference strain. Evidence that this naturally occurring pattern of inter-strain variation in H. influenzae can be mediated by transformation was obtained through sequencing DNA obtained from a pool of 200 independent transformants of a recipient (strain Rd) using donor DNA from a heterologous Hib strain (Eagan).

Conclusion

Much of the inter-strain variation in genome sequence in H. influenzae is likely the result of inter-strain exchanges of DNA, most plausibly through transformation.

Background

Streptococcus pneumoniae, also called the pneumococcus, is a major bacterial pathogen. Since its introduction in the 1940s, penicillin has been the primary treatment for pneumococcal diseases. Penicillin resistance rapidly increased among pneumococci over the past 30 years, and one particular multidrug-resistant clone, PMEN1, became highly prevalent globally. We studied a collection of 426 pneumococci isolated between 1937 and 2007 to better understand the evolution of penicillin resistance within this species.

Results

We discovered that one of the earliest known penicillin-nonsusceptible pneumococci, recovered in 1967 from Australia, was the likely ancestor of PMEN1, since approximately 95% of coding sequences identified within its genome were highly similar to those of PMEN1. The regions of the PMEN1 genome that differed from the ancestor contained genes associated with antibiotic resistance, transmission and virulence. We also revealed that PMEN1 was uniquely promiscuous with its DNA, donating penicillin-resistance genes and sometimes many other genes associated with antibiotic resistance, virulence and cell adherence to many genotypically diverse pneumococci. In particular, we describe two strains in which up to 10% of the PMEN1 genome was acquired in multiple fragments, some as long as 32 kb, distributed around the recipient genomes. This type of directional genetic promiscuity from a single clone to numerous unrelated clones has, to our knowledge, never before been described.

Conclusions

These findings suggest that PMEN1 is a paradigm of genetic success both through its epidemiology and promiscuity. These findings also challenge the existing views about horizontal gene transfer among pneumococci.

Relapsing C. difficile disease in humans is linked to a pathological imbalance within the intestinal microbiota, termed dysbiosis, which remains poorly understood. We show that mice infected with epidemic C. difficile (genotype 027/BI) develop highly contagious, chronic intestinal disease and persistent dysbiosis characterized by a distinct, simplified microbiota containing opportunistic pathogens and altered metabolite production. Chronic C. difficile 027/BI infection was refractory to vancomycin treatment leading to relapsing disease. In contrast, treatment of C. difficile 027/BI infected mice with feces from healthy mice rapidly restored a diverse, healthy microbiota and resolved C. difficile disease and contagiousness. We used this model to identify a simple mixture of six phylogenetically diverse intestinal bacteria, including novel species, which can re-establish a health-associated microbiota and clear C. difficile 027/BI infection from mice. Thus, targeting a dysbiotic microbiota with a defined mixture of phylogenetically diverse bacteria can trigger major shifts in the microbial community structure that displaces C. difficile and, as a result, resolves disease and contagiousness. Further, we demonstrate a rational approach to harness the therapeutic potential of health-associated microbial communities to treat C. difficile disease and potentially other forms of intestinal dysbiosis.

Author Summary

Pathological imbalances within the intestinal microbiota, termed dysbiosis, are often associated with chronic Clostridium difficile infections in humans. We show that infection of mice with the healthcare pathogen C. difficile leads to persistent intestinal dysbiosis that is associated with chronic disease and a highly contagious state. Using this model we rationally designed a simple mixture of phylogenetically diverse intestinal bacteria that can disrupt intestinal dysbiosis and as a result resolve disease and contagiousness. Our results validate the microbiota as a viable therapeutic target and open the way to rationally design bacteriotherapy to treat chronic C. difficile infections and potentially other forms of persistent dysbiosis.

Next-generation sequencing has ushered in a new era of microbial genomics, enabling the detailed historical and geographical tracing of bacteria. This is helping to shape our understanding of bacterial evolution.

Summary

Mice lacking the transcription factor T-bet in the innate immune system develop microbiota-dependent colitis. Here, we show that interleukin-17A (IL-17A)-producing IL-7Rα+ innate lymphoid cells (ILCs) were potent promoters of disease in Tbx21−/−Rag2−/− ulcerative colitis (TRUC) mice. TNF-α produced by CD103−CD11b+ dendritic cells synergized with IL-23 to drive IL-17A production by ILCs, demonstrating a previously unrecognized layer of cellular crosstalk between dendritic cells and ILCs. We have identified Helicobacter typhlonius as a key disease trigger driving excess TNF-α production and promoting colitis in TRUC mice. Crucially, T-bet also suppressed the expression of IL-7R, a key molecule involved in controlling intestinal ILC homeostasis. The importance of IL-7R signaling in TRUC disease was highlighted by the dramatic reduction in intestinal ILCs and attenuated colitis following IL-7R blockade. Taken together, these data demonstrate the mechanism by which T-bet regulates the complex interplay between mucosal dendritic cells, ILCs, and the intestinal microbiota.

Highlights

► Chronic colitis in TRUC mice was mediated by IL-17-producing innate lymphoid cells ► TNF-α synergized with IL-23 to induce innate IL-17 production ► Helicobacter typhlonius triggered intestinal pathology in TRUC mice ► T-bet regulated IL-7R transcription, a key checkpoint in intestinal ILC homeostasis

Background

The classical Bordetella subspecies are phylogenetically closely related, yet differ in some of the most interesting and important characteristics of pathogens, such as host range, virulence and persistence. The compelling picture from previous comparisons of the three sequenced genomes was of genome degradation, with substantial loss of genome content (up to 24%) associated with adaptation to humans.

Results

For a more comprehensive picture of lineage evolution, we employed comparative genomic and phylogenomic analyses using seven additional diverse, newly sequenced Bordetella isolates. Genome-wide single nucleotide polymorphism (SNP) analysis supports a reevaluation of the phylogenetic relationships between the classical Bordetella subspecies, and suggests a closer link between ovine and human B. parapertussis lineages than has been previously proposed. Comparative analyses of genome content revealed that only 50% of the pan-genome is conserved in all strains, reflecting substantial diversity of genome content in these closely related pathogens that may relate to their different host ranges, virulence and persistence characteristics. Strikingly, these analyses suggest possible horizontal gene transfer (HGT) events in multiple loci encoding virulence factors, including O-antigen and pertussis toxin (Ptx). Segments of the pertussis toxin locus (ptx) and its secretion system locus (ptl) appear to have been acquired by the classical Bordetella subspecies and are divergent in different lineages, suggesting functional divergence in the classical Bordetellae.

Conclusions

Together, these observations, especially in key virulence factors, reveal that multiple mechanisms, such as point mutations, gain or loss of genes, as well as HGTs, contribute to the substantial phenotypic diversity of these versatile subspecies in various hosts.

Chlamydia trachomatis is responsible for both trachoma and sexually transmitted infections causing substantial morbidity and economic cost globally. Despite this, our knowledge of its population and evolutionary genetics is limited. Here we present a detailed whole genome phylogeny from representative strains of both trachoma and lymphogranuloma venereum (LGV) biovars from temporally and geographically diverse sources. Our analysis demonstrates that predicting phylogenetic structure using the ompA gene, traditionally used to classify Chlamydia, is misleading because extensive recombination in this region masks true relationships. We show that in many instances ompA is a chimera that can be exchanged in part or whole, both within and between biovars. We also provide evidence for exchange of, and recombination within, the cryptic plasmid, another important diagnostic target. We have used our phylogenetic framework to show how genetic exchange has manifested itself in ocular, urogenital and LGV C. trachomatis strains, including the epidemic LGV serotype L2b.

Summary: Spoligotyping is a well-established genotyping technique based on the presence of unique DNA sequences in Mycobacterium tuberculosis (Mtb), the causal agent of tuberculosis disease (TB). Although advances in sequencing technologies are leading to whole-genome bacterial characterization, tens of thousands of isolates have been spoligotyped, giving a global view of Mtb strain diversity. To bridge the gap, we have developed SpolPred, a software to predict the spoligotype from raw sequence reads. Our approach is compared with experimentally and de novo assembly determined strain types in a set of 44 Mtb isolates. In silico and experimental results are identical for almost all isolates (39/44). However, SpolPred detected five experimentally false spoligotypes and was more accurate and faster than the assembling strategy. Application of SpolPred to an additional seven isolates with no laboratory data led to types that clustered with identical experimental types in a phylogenetic analysis using single-nucleotide polymorphisms. Our results demonstrate the usefulness of the tool and its role in revealing experimental limitations.

Availability and implementation: SpolPred is written in C and is available from www.pathogenseq.org/spolpred.

Contact: francesc.coll@lshtm.ac.uk

Supplementary information: Supplementary data are available at Bioinformatics Online.

Background

Malawi commenced the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into the routine infant immunisation schedule in November 2011. Here we have tested the utility of high throughput whole genome sequencing to provide a high-resolution view of pre-vaccine pneumococcal epidemiology and population evolutionary trends to predict potential future change in population structure post introduction.

Methods

One hundred and twenty seven (127) archived pneumococcal isolates from randomly selected adults and children presenting to the Queen Elizabeth Central Hospital, Blantyre, Malawi underwent whole genome sequencing.

Results

The pneumococcal population was dominated by serotype 1 (20.5% of invasive isolates) prior to vaccine introduction. PCV13 is likely to protect against 62.9% of all circulating invasive pneumococci (78.3% in under-5-year-olds). Several Pneumococcal Molecular Epidemiology Network (PMEN) clones are now in circulation in Malawi which were previously undetected but the pandemic multidrug resistant PMEN1 lineage was not identified. Genome analysis identified a number of novel sequence types and serotype switching.

Conclusions

High throughput genome sequencing is now feasible and has the capacity to simultaneously elucidate serotype, sequence type and as well as detailed genetic information. It enables population level characterization, providing a detailed picture of population structure and genome evolution relevant to disease control. Post-vaccine introduction surveillance supported by genome sequencing is essential to providing a comprehensive picture of the impact of PCV13 on pneumococcal population structure and informing future public health interventions.

Background

The anaerobic spirochaete Brachyspira pilosicoli causes enteric disease in avian, porcine and human hosts, amongst others. To date, the only available genome sequence of B. pilosicoli is that of strain 95/1000, a porcine isolate. In the first intra-species genome comparison within the Brachyspira genus, we report the whole genome sequence of B. pilosicoli B2904, an avian isolate, the incomplete genome sequence of B. pilosicoli WesB, a human isolate, and the comparisons with B. pilosicoli 95/1000. We also draw on incomplete genome sequences from three other Brachyspira species. Finally we report the first application of the high-throughput Biolog phenotype screening tool on the B. pilosicoli strains for detailed comparisons between genotype and phenotype.

Results

Feature and sequence genome comparisons revealed a high degree of similarity between the three B. pilosicoli strains, although the genomes of B2904 and WesB were larger than that of 95/1000 (~2,765, 2.890 and 2.596 Mb, respectively). Genome rearrangements were observed which correlated largely with the positions of mobile genetic elements. Through comparison of the B2904 and WesB genomes with the 95/1000 genome, features that we propose are non-essential due to their absence from 95/1000 include a peptidase, glycine reductase complex components and transposases. Novel bacteriophages were detected in the newly-sequenced genomes, which appeared to have involvement in intra- and inter-species horizontal gene transfer. Phenotypic differences predicted from genome analysis, such as the lack of genes for glucuronate catabolism in 95/1000, were confirmed by phenotyping.

Conclusions

The availability of multiple B. pilosicoli genome sequences has allowed us to demonstrate the substantial genomic variation that exists between these strains, and provides an insight into genetic events that are shaping the species. In addition, phenotype screening allowed determination of how genotypic differences translated to phenotype. Further application of such comparisons will improve understanding of the metabolic capabilities of Brachyspira species.

Integrative and conjugative elements (ICEs) are self-mobile genetic elements found in the genomes of some bacteria. These elements may confer a fitness advantage upon their host bacteria through the cargo genes that they carry. Salmonella pathogenicity island 7 (SPI-7), found within some pathogenic strains of Salmonella enterica, possesses features indicative of an ICE and carries genes implicated in virulence. We aimed to identify and fully analyze ICEs related to SPI-7 within the genus Salmonella and other Enterobacteriaceae. We report the sequence of two novel SPI-7-like elements, found within strains of Salmonella bongori, which share 97% nucleotide identity over conserved regions with SPI-7 and with each other. Although SPI-7 within Salmonella enterica serovar Typhi appears to be fixed within the chromosome, we present evidence that these novel elements are capable of excision and self-mobility. Phylogenetic analyses show that these Salmonella mobile elements share an ancestor which existed approximately 3.6 to 15.8 million years ago. Additionally, we identified more distantly related ICEs, with distinct cargo regions, within other strains of Salmonella as well as within Citrobacter, Erwinia, Escherichia, Photorhabdus, and Yersinia species. In total, we report on a collection of 17 SPI-7 related ICEs within enterobacterial species, of which six are novel. Using comparative and mutational studies, we have defined a core of 27 genes essential for conjugation. We present a growing family of SPI-7-related ICEs whose mobility, abundance, and cargo variability indicate that these elements may have had a large impact on the evolution of the Enterobacteriaceae.

The increase in the capacity and reduction in cost of whole-genome sequencing methods present the imminent prospect of such data being used routinely in real time for investigations of bacterial disease outbreaks. For this to be realized, however, it is necessary that generic, portable, and robust analysis frameworks be available, which can be readily interpreted and used in real time by microbiologists, clinicians, and public health epidemiologists. We have achieved this with a set of analysis tools integrated into the PubMLST.org website, which can in principle be used for the analysis of any pathogen. The approach is demonstrated with genomic data from isolates obtained during a well-characterized meningococcal disease outbreak at the University of Southampton, United Kingdom, that occurred in 1997. Whole-genome sequence data were collected, de novo assembled, and deposited into the PubMLST Neisseria BIGSdb database, which automatically annotated the sequences. This enabled the immediate and backwards-compatible classification of the isolates with a number of schemes, including the following: conventional, extended, and ribosomal multilocus sequence typing (MLST, eMLST, and rMLST); antigen gene sequence typing (AGST); analysis based on genes conferring antibiotic susceptibility. The isolates were also compared to a reference isolate belonging to the same clonal complex (ST-11) at 1,975 loci. Visualization of the data with the NeighborNet algorithm, implemented in SplitsTree 4 within the PubMLST website, permitted complete resolution of the outbreak and related isolates, demonstrating that multiple closely related but distinct strains were simultaneously present in asymptomatic carriage and disease, with two causing disease and one responsible for the outbreak itself.

Background

B. bronchiseptica infections are usually associated with wild or domesticated animals, but infrequently with humans. A recent phylogenetic analysis distinguished two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Complex IV isolates appear to have a bias for infecting humans; however, little is known regarding their epidemiology, virulence properties, or comparative genomics.

Results

Here we report a characterization of the virulence of human-associated complex IV B. bronchiseptica strains. In in vitro cytotoxicity assays, complex IV strains showed increased cytotoxicity in comparison to a panel of complex I strains. Some complex IV isolates were remarkably cytotoxic, resulting in LDH release levels in A549 cells that were 10- to 20-fold greater than complex I strains. In vivo, a subset of complex IV strains was found to be hypervirulent, with an increased ability to cause lethal pulmonary infections in mice. Hypercytotoxicity in vitro and hypervirulence in vivo were both dependent on the activity of the bsc T3SS and the BteA effector. To clarify differences between lineages, representative complex IV isolates were sequenced and their genomes were compared to complex I isolates. Although our analysis showed there were no genomic sequences that can be considered unique to complex IV strains, there were several loci that were predominantly found in complex IV isolates.

Conclusion

Our observations reveal a T3SS-dependent hypervirulence phenotype in human-associated complex IV isolates, highlighting the need for further studies on the epidemiology and evolutionary dynamics of this B. bronchiseptica lineage.

Transformation is an important mechanism of microbial evolution through which bacteria have been observed to rapidly adapt in response to clinical interventions; examples include facilitating vaccine evasion and the development of penicillin resistance in the major respiratory pathogen Streptococcus pneumoniae. To characterise the process in detail, the genomes of 124 S. pneumoniae isolates produced through in vitro transformation were sequenced and recombination events detected. Those recombinations importing the selected marker were independent of unselected events elsewhere in the genome, the positions of which were not significantly affected by local sequence similarity between donor and recipient or mismatch repair processes. However, both types of recombinations were sometimes mosaic, with multiple non-contiguous segments originating from the same molecule of donor DNA. The lengths of the unselected events were exponentially distributed with a mean of 2.3 kb, implying that recombinations are stochastically resolved with a fixed per base probability of 4.4×10−4 bp−1. This distribution of recombination sizes, coupled with an observed under representation of large insertions within transferred sequence, suggests transformation has the potential to reduce the size of bacterial genomes, and is unlikely to act as an efficient mechanism for the uptake of accessory genomic loci.

Author Summary

Transformation is the process by which cells take up DNA from the environment and integrate it into their genome. It was first observed in the bacterium Streptococcus pneumoniae, a common cause of pneumonia and meningitis. This ability has allowed S. pneumoniae to evolve resistance to penicillin and to change its surface antigens to evade vaccines. To characterise this process in detail, we transformed an S. pneumoniae strain in the laboratory and sequenced the genomes of the resultant mutants. This showed that multiple recombinations, arising from different molecules of imported DNA, could occur around the genome at the same time. Some individual imported molecules donated multiple segments of sequence into the recipient. The positions of the recombinations were not significantly affected by the level of sequence similarity between donor and recipient, as had previously been observed for transfers between species, or by the mismatch repair process, which had previously been found to inhibit the transfer of small numbers of mutations. The recombinations' lengths were exponentially distributed with a mean length of 2.3 kb. This implies that the smallest version of a genetic locus will become the most common in the bacterial population in the absence of selection for a longer alternative.

Objective

To identify, compare and contrast the microbiota in patients with and without pouchitis after RPC for UC and FAP.

Summary Background Data

Pouchitis is the most common complication following restorative proctocolectomy (RPC). An abnormal host-microbial interaction has been implicated. We investigated the pouch microbiota in patients with and without pouchitis undergoing restorative proctocolectomy for UC and familial adenomatous polyposis (FAP).

Methods

Mucosal pouch biopsies, taken from 16 UC (pouchitis 8) and 8 FAP (pouchitis 3) patients were analysed to the species (or phylotype) level by cloning and sequencing of 3,184 full-length bacterial 16S rRNA genes.

Results

There was a significant increase in Proteobacteria (p= 0.019) and a significant decrease in Bacteroidetes (p= 0.001) and Faecalibacterium prausnitzii (p=0.029) in the total UC compared to the total FAP cohort, but only limited differences were found between the UC non-pouchitis and pouchitis groups and the FAP pouchitis and non-pouchitis groups. Bacterial diversity in the FAP non-pouchitis group was significantly greater than in UC non-pouchitis (p= 0.019) and significantly greater in UC non-pouchitis compared to UC pouchitis (p= 0.009). No individual species or phylotype specifically associated with either UC or FAP pouchitis was found.

Conclusions

UC pouch patients have a different, less diverse, gut microbiota than FAP patients. A further reduction in bacterial diversity but no significant dysbiosis occurs in those with pouchitis. The study suggests that a dysbiosis occurs in the ileal pouch of UC RPC patients which predisposes to, but may not directly cause, pouchitis.

Novel adhesions, including trimeric autotransporters, might contribute to virulence.

An emergent clone of Haemophilus influenzae biogroup aegyptius (Hae) is responsible for outbreaks of Brazilian purpuric fever (BPF). First recorded in Brazil in 1984, the so-called BPF clone of Hae caused a fulminant disease that started with conjunctivitis but developed into septicemic shock; mortality rates were as high as 70%. To identify virulence determinants, we conducted a pan-genomic analysis. Sequencing of the genomes of the BPF clone strain F3031 and a noninvasive conjunctivitis strain, F3047, and comparison of these sequences with 5 other complete H. influenzae genomes showed that >77% of the F3031 genome is shared among all H. influenzae strains. Delineation of the Hae accessory genome enabled characterization of 163 predicted protein-coding genes; identified differences in established autotransporter adhesins; and revealed a suite of novel adhesins unique to Hae, including novel trimeric autotransporter adhesins and 4 new fimbrial operons. These novel adhesins might play a critical role in host–pathogen interactions.

Background

M. africanum West African 2 constitutes an ancient lineage of the M. tuberculosis complex that commonly causes human tuberculosis in West Africa and has an attenuated phenotype relative to M. tuberculosis.

Methodology/Principal Findings

In search of candidate genes underlying these differences, the genome of M. africanum West African 2 was sequenced using classical capillary sequencing techniques. Our findings reveal a unique sequence, RD900, that was independently lost during the evolution of two important lineages within the complex: the “modern” M. tuberculosis group and the lineage leading to M. bovis. Closely related to M. bovis and other animal strains within the M. tuberculosis complex, M. africanum West African 2 shares an abundance of pseudogenes with M. bovis but also with M. africanum West African clade 1. Comparison with other strains of the M. tuberculosis complex revealed pseudogenes events in all the known lineages pointing toward ongoing genome erosion likely due to increased genetic drift and relaxed selection linked to serial transmission-bottlenecks and an intracellular lifestyle.

Conclusions/Significance

The genomic differences identified between M. africanum West African 2 and the other strains of the Mycobacterium tuberculosis complex may explain its attenuated phenotype, and pave the way for targeted experiments to elucidate the phenotypic characteristic of M. africanum. Moreover, availability of the whole genome data allows for verification of conservation of targets used for the next generation of diagnostics and vaccines, in order to ensure similar efficacy in West Africa.

Author Summary

Mycobacterium africanum, a close relative of M. tuberculosis, is studied for the following reasons: M. africanum is commonly isolated from West African patients with tuberculosis yet has not spread beyond this region, it is more common in HIV infected patients, and it is less likely to lead to tuberculosis after one is exposed to an infectious case. Understanding this organism's unique biology gets a boost from the decoding of its genome, reported in this issue. For example, genome analysis reveals that M. africanum contains a region shared with “ancient” lineages in the M. tuberculosis complex and other mycobacterial species, which was lost independently from both M. tuberculosis and M. bovis. This region encodes a protein involved in transmembrane transport. Furthermore, M. africanum has lost genes, including a known virulence gene and genes for vitamin synthesis, in addition to an intact copy of a gene that may increase its susceptibility to antibiotics that are insufficiently active against M. tuberculosis. Finally, the genome sequence and analysis reported here will aid in the development of new diagnostics and vaccines against tuberculosis, which need to take into account the differences between M. africanum and other species in order to be effective worldwide.