Understanding the emergence and spread of multidrug-resistant tuberculosis (MDR-TB) is crucial for its control. MDR-TB in previously treated patients is generally attributed to the selection of drug resistant mutants during inadequate therapy rather than transmission of a resistant strain. Traditional genotyping methods are not sufficient to distinguish strains in populations with a high burden of tuberculosis and it has previously been difficult to assess the degree of transmission in these settings. We have used whole genome analysis to investigate M. tuberculosis strains isolated from treatment experienced patients with MDR-TB in Uganda over a period of four years.
Methods and Findings
We used high throughput genome sequencing technology to investigate small polymorphisms and large deletions in 51 Mycobacterium tuberculosis samples from 41 treatment-experienced TB patients attending a TB referral and treatment clinic in Kampala. This was a convenience sample representing 69% of MDR-TB cases identified over the four year period. Low polymorphism was observed in longitudinal samples from individual patients (2-15 SNPs). Clusters of samples with less than 50 SNPs variation were examined. Three clusters comprising a total of 8 patients were found with almost identical genetic profiles, including mutations predictive for resistance to rifampicin and isoniazid, suggesting transmission of MDR-TB. Two patients with previous drug susceptible disease were found to have acquired MDR strains, one of which shared its genotype with an isolate from another patient in the cohort.
Whole genome sequence analysis identified MDR-TB strains that were shared by more than one patient. The transmission of multidrug-resistant disease in this cohort of retreatment patients emphasises the importance of early detection and need for infection control. Consideration should be given to rapid testing for drug resistance in patients undergoing treatment to monitor the emergence of resistance and permit early intervention to avoid onward transmission.
Global spread and genetic monomorphism are hallmarks of Mycobacterium tuberculosis, the agent of human tuberculosis. In contrast, Mycobacterium canettii, and related tubercle bacilli that also cause human tuberculosis and exhibit unusual smooth colony morphology, are restricted to East-Africa. Here, we sequenced and analyzed the genomes of five representative strains of smooth tubercle bacilli (STB) using Sanger (4-5x coverage), 454/Roche (13-18x coverage) and/or Illumina DNA sequencing (45-105x coverage). We show that STB are highly recombinogenic and evolutionary early-branching, with larger genome sizes, 25-fold more SNPs, fewer molecular scars and distinct CRISPR-Cas systems relative to M. tuberculosis. Despite the differences, all tuberculosis-causing mycobacteria share a highly conserved core genome. Mouse-infection experiments revealed that STB are less persistent and virulent than M. tuberculosis. We conclude that M. tuberculosis emerged from an ancestral, STB-like pool of mycobacteria by gain of persistence and virulence mechanisms and we provide genome-wide insights into the molecular events involved.
Whole genome sequencing of 616 asymptomatically carried pneumococci was used to study the impact of the 7-valent pneumococcal conjugate vaccine. Comparison of closely related isolates revealed the role of transformation in facilitating capsule switching to non-vaccine serotypes and the emergence of drug resistance. However, such recombination was found to occur at significantly different rates across the species, and the evolution of the population was primarily driven by changes in the frequency of distinct genotypes extant pre-vaccine. These alterations resulted in little overall effect on accessory genome composition at the population level, contrasting with the fall in pneumococcal disease rates after the vaccine’s introduction.
Whole genome sequencing of 616 asymptomatically carried pneumococci was
used to study the impact of the 7-valent pneumococcal conjugate vaccine.
Comparison of closely related isolates revealed the role of transformation in
facilitating capsule switching to non-vaccine serotypes and the emergence of
drug resistance. However, such recombination was found to occur at significantly
different rates across the species, and the evolution of the population was
primarily driven by changes in the frequency of distinct genotypes extant
pre-vaccine. These alterations resulted in little overall effect on accessory
genome composition at the population level, contrasting with the fall in
pneumococcal disease rates after the vaccine’s introduction.
Recurrence of tuberculosis after treatment makes management difficult and is a key factor for determining treatment efficacy. Two processes can cause recurrence: relapse of the primary infection or re-infection with an exogenous strain. Although re-infection can and does occur, its importance to tuberculosis epidemiology and its biological basis is still debated. We used whole-genome sequencing—which is more accurate than conventional typing used to date—to assess the frequency of recurrence and to gain insight into the biological basis of re-infection.
We assessed patients from the REMoxTB trial—a randomised controlled trial of tuberculosis treatment that enrolled previously untreated participants with Mycobacterium tuberculosis infection from Malaysia, South Africa, and Thailand. We did whole-genome sequencing and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing of pairs of isolates taken by sputum sampling: one from before treatment and another from either the end of failed treatment at 17 weeks or later or from a recurrent infection. We compared the number and location of SNPs between isolates collected at baseline and recurrence.
We assessed 47 pairs of isolates. Whole-genome sequencing identified 33 cases with little genetic distance (0–6 SNPs) between strains, deemed relapses, and three cases for which the genetic distance ranged from 1306 to 1419 SNPs, deemed re-infections. Six cases of relapse and six cases of mixed infection were classified differently by whole-genome sequencing and MIRU-VNTR. We detected five single positive isolates (positive culture followed by at least two negative cultures) without clinical evidence of disease.
Whole-genome sequencing enables the differentiation of relapse and re-infection cases with greater resolution than do genotyping methods used at present, such as MIRU-VNTR, and provides insights into the biology of recurrence. The additional clarity provided by whole-genome sequencing might have a role in defining endpoints for clinical trials.
Wellcome Trust, European Union, Medical Research Council, Global Alliance for TB Drug Development, European and Developing Country Clinical Trials Partnership.
Cytoplasmic incompatibility (CI) induced by the endosymbiont Wolbachia pipientis causes complex patterns of crossing sterility between populations of the Culex pipiens group of mosquitoes. The molecular basis of the phenotype is yet to be defined. In order to investigate what host changes may underlie CI at the molecular level, we examined the transcription of a homolog of the Drosophila melanogaster gene grauzone that encodes a zinc finger protein and acts as a regulator of female meiosis, in which mutations can cause sterility. Upregulation was observed in Wolbachia-infected C. pipiens group individuals relative to Wolbachia-cured lines and the level of upregulation differed between lines that were reproductively incompatible. Knockdown analysis of this gene using RNAi showed an effect on hatch rates in a Wolbachia infected Culex molestus line. Furthermore, in later stages of development an effect on developmental progression in CI embryos occurs in bidirectionally incompatible crosses. The genome of a wPip Wolbachia strain variant from Culex molestus was sequenced and compared with the genome of a wPip variant with which it was incompatible. Three genes in inserted or deleted regions were newly identified in the C. molestus wPip genome, one of which is a transcriptional regulator labelled wtrM. When this gene was transfected into adult Culex mosquitoes, upregulation of the grauzone homolog was observed. These data suggest that Wolbachia-mediated regulation of host gene expression is a component of the mechanism of cytoplasmic incompatibility.
Wolbachia are maternally inherited bacteria that manipulate invertebrate reproduction. Cytoplasmic incompatibility is embryo death that occurs when males carrying Wolbachia mate with females that do not, or that carry a different Wolbachia variant; its mechanism is poorly understood. In Culex mosquitoes, in the presence of Wolbachia a gene related to a Drosophila melanogaster gene, grauzone, which has been shown to act as a regulator of the meiotic cell cycle, showed an elevated level of expression. When lower levels of expression were achieved through RNA interference, embryo hatch rates were affected and the stage of development at which embryo death occurs was altered. To find Wolbachia genes that influence cytoplasmic incompatibility, we compared the genomes of two variants of Wolbachia from Culex that produce cytoplasmic incompatibility with one another. Although most segments of these genomes were very similar, one newly identified gene is predicted to be a regulator of gene transcription. We cloned this gene into a plasmid, expressed it in adult mosquitoes and found higher levels of expression of the Culex grauzone homolog. This suggests that the Wolbachia transcriptional regulator may play an important role in manipulating the host in order to induce cytoplasmic incompatibility.
Streptococcus pneumoniae of serotype 3 possess a mucoid capsule and cause disease associated with high mortality rates relative to other pneumococci. Phylogenetic analysis of a complete reference genome and 81 draft sequences from clonal complex 180, the predominant serotype 3 clone in much of the world, found most sampled isolates belonged to a clade affected by few diversifying recombinations. However, other isolates indicate significant genetic variation has accumulated over the clonal complex's entire history. Two closely related genomes, one from the blood and another from the cerebrospinal fluid, were obtained from a patient with meningitis. The pair differed in their behaviour in a mouse model of disease and in their susceptibility to antimicrobials, with at least some of these changes attributable to a mutation that up-regulated the patAB efflux pump. This indicates clinically important phenotypic variation can accumulate rapidly through small alterations to the genotype.
Streptococcus pneumoniae (‘the pneumococcus’) is a bacterium commonly found asymptomatically in the human nasopharynx that represents a common cause of diseases such as pneumonia, bacteraemia and meningitis. Some strains have been found to exchange DNA with other bacteria at a high rate. However, serotype 3 pneumococci are unusual both in not exhibiting much genetic variation and causing disease with a comparatively high relative rate of mortality. Here we used whole genome sequencing to characterise 82 serotype 3 pneumococci, finding that the majority of the population accumulate variation very slowly. However, comparing two isolates from a single case of disease revealed a small number of mutations had occurred over a short period of time. These resulted in differences in the activity of several genes, including two encoding a drug efflux pump. The pair of isolates was found to differ in their tolerance of different antimicrobial compounds and their behaviour in a mouse model of disease. However, moving the mutation that caused the change in resistance into a distantly-related pneumococcus failed to fully replicate the other changes in behaviour, which indicates that interpretation of the impact of mutations in different strains of diverse bacterial species will be difficult.
The soil-transmitted helminth (STH), Trichuris trichiura colonises the human large intestine where it may modify inflammatory responses, an effect possibly mediated through alterations in the intestinal microbiota. We hypothesised that patent T. trichiura infections would be associated with altered faecal microbiota and that anthelmintic treatment would induce a microbiota resembling more closely that observed in uninfected individuals.
Materials and Methods
School children in Ecuador were screened for STH infections and allocated to 3 groups: uninfected, T. trichiura only, and mixed infections with T. trichiura and Ascaris lumbricoides. A sample of uninfected children and those with T. trichiura infections only were given anthelmintic treatment. Bacterial community profiles in faecal samples were studied by 454 pyrosequencing of 16 S rRNA genes.
Microbiota analyses of faeces were done for 97 children: 30 were uninfected, 17 were infected with T. trichiura, and 50 with T. trichiura and A. lumbricoides. Post-treatment samples were analyzed for 14 children initially infected with T. trichiura alone and for 21 uninfected children. Treatment resulted in 100% cure of STH infections. Comparisons of the microbiota at different taxonomic levels showed no statistically significant differences in composition between uninfected children and those with T. trichiura infections. We observed a decreased proportional abundance of a few bacterial genera from the Clostridia class of Firmicutes and a reduced bacterial diversity among children with mixed infections compared to the other two groups, indicating a possible specific effect of A. lumbricoides infection. Anthelmintic treatment of children with T. trichiura did not alter faecal microbiota composition.
Our data indicate that patent human infections with T. trichiura may have no effect on faecal microbiota but that A. lumbricoides colonisation might be associated with a disturbed microbiota. Our results also catalogue the microbiota of rural Ecuadorians and indicate differences with individuals from more urban industrialised societies.
Two haemoglobin-binding proteins, HmbR and HpuAB, contribute to iron acquisition by Neisseria meningitidis. These receptors are subject to high frequency, reversible switches in gene expression - phase variation (PV) - due to mutations in homopolymeric (poly-G) repeats present in the open reading frame. The distribution and PV state of these receptors was assessed for a representative collection of isolates from invasive meningococcal disease patients of England, Wales and Northern Ireland. Most of the major clonal complexes had only the HmbR receptor whilst the recently expanding ST-275-centred cluster of the ST-269 clonal complex had both receptors. At least one of the receptors was in an ‘ON’ configuration in 76.3% of the isolates, a finding that was largely consistent with phenotypic analyses. As PV status may change during isolation and culture of meningococci, a PCR-based protocol was utilised to confirm the expression status of the receptors within contemporaneously acquired clinical specimens (blood/cerebrospinal fluid) from the respective patients. The expression state was confirmed for all isolate/specimen pairs with <15 tract repeats indicating that the PV status of these receptors is stable during isolation. This study therefore establishes a protocol for determining in vivo PV status to aid in determining the contributions of phase variable genes to invasive meningococcal disease. Furthermore, the results of the study support a putative but non-essential role of the meningococcal haemoglobin receptors as virulence factors whilst further highlighting their vaccine candidacy.
Cholera remains a significant public health challenge in many sub-Saharan countries including Kenya. We have performed a combination of phylogenetic and phenotypic analysis based on whole genome DNA sequences derived from 40 environmental and 57 clinical V. cholerae from different regions of Kenya isolated between 2005 and 2010. Some environmental and all clinical isolates mapped back onto wave three of the monophyletic seventh pandemic V. cholerae El Tor phylogeny but other environmental isolates were phylogenetically very distinct. Thus, the genomes of the Kenyan V. cholerae O1 El Tor isolates are clonally related to other El Tor V. cholerae isolated elsewhere in the world and similarly harbour antibiotic resistance-associated STX elements. Further, the Kenyan O1 El Tor isolates fall into two distinct clades that may have entered Kenya independently.
Recently, a novel variant of mecA known as mecC (mecALGA251) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified a Staphylococcus xylosus isolate that harbors a new allotype of the mecC gene, mecC1. Whole-genome sequencing revealed that mecC1 forms part of a class E mec complex (mecI-mecR1-mecC1-blaZ) located at the orfX locus as part of a likely staphylococcal cassette chromosome mec element (SCCmec) remnant, which also contains a number of other genes present on the type XI SCCmec.
Salmonella enterica serovar Typhimurium definitive type 2 (DT2) is host restricted to Columba livia (rock or feral pigeon) but is also closely related to S. Typhimurium isolates that circulate in livestock and cause a zoonosis characterized by gastroenteritis in humans. DT2 isolates formed a distinct phylogenetic cluster within S. Typhimurium based on whole-genome-sequence polymorphisms. Comparative genome analysis of DT2 94-213 and S. Typhimurium SL1344, DT104, and D23580 identified few differences in gene content with the exception of variations within prophages. However, DT2 94-213 harbored 22 pseudogenes that were intact in other closely related S. Typhimurium strains. We report a novel in silico approach to identify single amino acid substitutions in proteins that have a high probability of a functional impact. One polymorphism identified using this method, a single-residue deletion in the Tar protein, abrogated chemotaxis to aspartate in vitro. DT2 94-213 also exhibited an altered transcriptional profile in response to culture at 42°C compared to that of SL1344. Such differentially regulated genes included a number involved in flagellum biosynthesis and motility.
Whereas Salmonella enterica serovar Typhimurium can infect a wide range of animal species, some variants within this serovar exhibit a more limited host range and altered disease potential. Phylogenetic analysis based on whole-genome sequences can identify lineages associated with specific virulence traits, including host adaptation. This study represents one of the first to link pathogen-specific genetic signatures, including coding capacity, genome degradation, and transcriptional responses to host adaptation within a Salmonella serovar. We performed comparative genome analysis of reference and pigeon-adapted definitive type 2 (DT2) S. Typhimurium isolates alongside phenotypic and transcriptome analyses, to identify genetic signatures linked to host adaptation within the DT2 lineage.
Vibrio cholerae is a globally important pathogen that is endemic in many areas of the world and causes 3-5 million reported cases of cholera every year (http://www.who.int/wer). Historically there have been seven acknowledged cholera pandemics; included in the 7th and ongoing pandemic are the recent outbreaks in Zimbabwe and Haiti1. Only serogroup O1 isolates (consisting of two biotypes known as ‘classical’ and ‘El Tor’) and the derivative O1392,3 can cause epidemic cholera2. It is believed that the first six cholera pandemics were caused by the classical biotype but El Tor has subsequently spread globally and replaced the classical biotype in the current pandemic1. Detailed molecular epidemiological mapping of cholera has been compromised by a reliance on sub-genomic regions such as mobile elements to infer relationships, making El Tor isolates associated with the 7th pandemic appear superficially diverse. To understand the underlying phylogeny of the lineage responsible for the current pandemic we identified high resolution markers (single nucleotide polymorphisms; SNPs) in 154 whole genome sequences of globally and temporally representative V. cholerae isolates. Using this phylogeny we show that the 7th pandemic has spread from the Bay of Bengal in at least three independent but overlapping waves with a common ancestor in the 1950’s and identify multiple transcontinental transmission events. Additionally, we show how the acquisition of the SXT family of antibiotic resistance elements has shaped the pandemic spread and show that it was first acquired at least 10 years prior to its discovery in V. cholerae.
Sequence-based typing is essential for understanding the epidemiology of Campylobacter infections, a major worldwide cause of bacterial gastroenteritis. We demonstrate the practical and rapid exploitation of whole-genome sequencing to provide routine definitive characterization of Campylobacter jejuni and Campylobacter coli for clinical and public health purposes. Short-read data from 384 Campylobacter clinical isolates collected over 4 months in Oxford, United Kingdom, were assembled de novo. Contigs were deposited at the pubMLST.org/campylobacter website and automatically annotated for 1,667 loci. Typing and phylogenetic information was extracted and comparative analyses were performed for various subsets of loci, up to the level of the whole genome, using the Genome Comparator and Neighbor-net algorithms. The assembled sequences (for 379 isolates) were diverse and resembled collections from previous studies of human campylobacteriosis. Small subsets of very closely related isolates originated mainly from repeated sampling from the same patients and, in one case, likely laboratory contamination. Much of the within-patient variation occurred in phase-variable genes. Clinically and epidemiologically informative data can be extracted from whole-genome sequence data in real time with straightforward, publicly available tools. These analyses are highly scalable, are transparent, do not require closely related genome reference sequences, and provide improved resolution (i) among Campylobacter clonal complexes and (ii) between very closely related isolates. Additionally, these analyses rapidly differentiated unrelated isolates, allowing the detection of single-strain clusters. The approach is widely applicable to analyses of human bacterial pathogens in real time in clinical laboratories, with little specialist training required.
The emergence of mecC methicillin-resistant Staphylococcus aureus (MRSA) poses a diagnostic challenge for clinical microbiology laboratories. Using the Vitek 2 system, we tested a panel of 896 Staphylococcus aureus isolates and found that an oxacillin-sensitive/cefoxitin-resistant profile had a sensitivity of 88.7% and a specificity of 99.5% for the identification of mecC MRSA isolates. The presence of the mecC gene, determined by bacterial whole-genome sequencing, was used as the gold standard. This profile could provide a zero-cost screening method for identification of mecC-positive MRSA strains.
Antimicrobial resistance among pneumococci has greatly increased over the past two to three decades. Resistance to tetracycline (tet(M)), chloramphenicol (cat) and macrolides (erm(B) and/or mef(A/E)) is generally conferred by acquisition of specific genes that are associated with mobile genetic elements, including those of the Tn916 and Tn5252 families. The first tetracycline-, chloramphenicol- and macrolide-resistant pneumococci were detected between 1962 and 1970; however, until now the oldest pneumococcus shown to harbour Tn916 and/or Tn5252 was isolated in 1974. In this study the genomes of 38 pneumococci isolated prior to 1974 were probed for the presence of tet(M), cat, erm(B), mef(A/E) and int (integrase) to indicate the presence of Tn916/Tn5252-like elements.
Two Tn916-like, tet(M)-containing, elements were identified among pneumococci dated 1967 and 1968. The former element was highly similar to that of the PMEN1 multidrug-resistant, globally-distributed pneumococcal reference strain, which was isolated in 1984. The latter element was associated with a streptococcal phage. A third, novel genetic element, designated ICESpPN1, was identified in the genome of an isolate dated 1972. ICESpPN1 contained a region of similarity to Tn5252, a region of similarity to a pneumococcal pathogenicity island and novel lantibiotic synthesis/export-associated genes.
These data confirm the existence of pneumococcal Tn916 elements in the first decade within which pneumococcal tetracycline resistance was described. Furthermore, the discovery of ICESpPN1 demonstrates the dynamic variability of pneumococcal genetic elements and is contrasted with the evidence for Tn916 stability.
Streptococcus pneumoniae; Antimicrobial resistance; Mobile genetic elements; Tetracycline resistance; ICE elements
Isolates of methicillin-resistant Staphylococcus aureus (MRSA) belonging to a single lineage are often indistinguishable by means of current typing techniques. Whole-genome sequencing may provide improved resolution to define transmission pathways and characterize outbreaks.
We investigated a putative MRSA outbreak in a neonatal intensive care unit. By using rapid high-throughput sequencing technology with a clinically relevant turnaround time, we retrospectively sequenced the DNA from seven isolates associated with the outbreak and another seven MRSA isolates associated with carriage of MRSA or bacteremia in the same hospital.
We constructed a phylogenetic tree by comparing single-nucleotide polymorphisms (SNPs) in the core genome to a reference genome (an epidemic MRSA clone, EMRSA-15 [sequence type 22]). This revealed a distinct cluster of outbreak isolates and clear separation between these and the nonoutbreak isolates. A previously missed transmission event was detected between two patients with bacteremia who were not part of the outbreak. We created an artificial “resistome” of antibiotic-resistance genes and demonstrated concordance between it and the results of phenotypic susceptibility testing; we also created a “toxome” consisting of toxin genes. One outbreak isolate had a hypermutator phenotype with a higher number of SNPs than the other outbreak isolates, highlighting the difficulty of imposing a simple threshold for the number of SNPs between isolates to decide whether they are part of a recent transmission chain.
Whole-genome sequencing can provide clinically relevant data within a time frame that can influence patient care. The need for automated data interpretation and the provision of clinically meaningful reports represent hurdles to clinical implementation. (Funded by the U.K. Clinical Research Collaboration Translational Infection Research Initiative and others.)
We sought to determine the prevalence of nasal colonisation with methicillin-resistant Staphylococcus aureus among cattle veterinarians in the UK. There was particular interest in examining the frequency of colonisation with MRSA harbouring mecC, as strains with this mecA homologue were originally identified in bovine milk and may represent a zoonotic risk to those in contact with dairy livestock. Three hundred and seven delegates at the British Cattle Veterinarian Association (BCVA) Congress 2011 in Southport, UK were screening for nasal colonisation with MRSA. Isolates were characterised by whole genome sequencing and antimicrobial susceptibility testing. Eight out of three hundred and seven delegates (2.6%) were positive for nasal colonisation with MRSA. All strains were positive for mecA and none possessed mecC. The time since a delegate’s last visit to a farm was significantly shorter in the MRSA-positive group than in MRSA-negative counterparts. BCVA delegates have an increased risk of MRSA colonisation compared to the general population but their frequency of colonisation is lower than that reported from other types of veterinarian conference, and from that seen in human healthcare workers. The results indicate that recent visitation to a farm is a risk factor for MRSA colonisation and that mecC-MRSA are rare among BCVA delegates (<1% based on sample size). Contact with livestock, including dairy cattle, may still be a risk factor for human colonisation with mecC-MRSA but occurs at a rate below the lower limit of detection available in this study.
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 B. bacteriovorus HD100. Based on their predatory lifestyle, Bacteriovorax were originally designated 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, attaches, penetrates through the cell wall to form a bdelloplast, in which it replicates, as shown using microscopy.
Bacteriovorax is distinct, since 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 shared geneset Bacteriovorax and Bdellovibrio that is not conserved amongst 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 key indicators of predatory-specific processes required for prey entry. The hit locus from Bd. bacteriovorus is implicated in the switch from predatory to prey/host-independent (HI) 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 genera.
The current Shigella sonnei pandemic involves geographically associated, multidrug-resistant clones. This study has demonstrated that S. sonnei phylogeny can be accurately defined with limited single nucleotide polymorphisms (SNPs). By typing 6 informative SNPs using a high-resolution melting (HRM) assay, major S. sonnei lineages/sublineages can be identified as defined by whole-genome variation.
In 2009, an outbreak of enterohemorrhagic Escherichia coli (EHEC) on an open farm infected 93 persons, and approximately 22% of these individuals developed hemolytic-uremic syndrome (HUS). Genome sequencing was used to investigate outbreak-derived animal and human EHEC isolates. Phylogeny based on the whole-genome sequence was used to place outbreak isolates in the context of the overall E. coli species and the O157:H7 sequence type 11 (ST11) subgroup. Four informative single nucleotide polymorphisms (SNPs) were identified and used to design an assay to type 122 other outbreak isolates. The SNP phylogeny demonstrated that the outbreak strain was from a lineage distinct from previously reported O157:H7 ST11 EHEC and was not a member of the hypervirulent clade 8. The strain harbored determinants for two Stx2 verotoxins and other putative virulence factors. When linked to the epidemiological information, the sequence data indicate that gross contamination of a single outbreak strain occurred across the farm prior to the first clinical report of HUS. The most likely explanation for these results is that a single successful strain of EHEC spread from a single introduction through the farm by clonal expansion and that contamination of the environment (including the possible colonization of several animals) led ultimately to human cases.
Epidemic Clostridium difficile (027/BI/NAP1) rapidly emerged in the past decade as the leading cause of antibiotic-associated diarrhea worldwide. However, the key moments in the evolutionary history leading to its emergence and subsequent patterns of global spread remain unknown. Here we define the global population structure of C. difficile 027/BI/NAP1 based on whole-genome sequencing and phylogenetic analysis. We demonstrate that two distinct epidemic lineages, FQR1 and FQR2, not one as previously thought, emerged in North America within a relatively short period after acquiring the same fluoroquinolone resistance mutation and a highly-related conjugative transposon. The two epidemic lineages displayed distinct patterns of global spread, and the FQR2 lineage spread more widely leading to healthcare outbreaks in the UK, continental Europe and Australia. Our analysis identifies key genetic changes linked to the rapid trans-continental dissemination of epidemic C. difficile 027/BI/NAP1 and highlights the routes by which it spreads through the global healthcare system.
Ruminococcus flavefaciens is an important fibre-degrading bacterium found in the mammalian gut. Cellulolytic strains from the bovine rumen have been shown to produce complex cellulosome structures that are associated with the cell surface. R. flavefaciens 007 is a highly cellulolytic strain whose ability to degrade dewaxed cotton, but not Avicel cellulose, was lost following initial isolation in the variant 007S. The ability was recovered after serial subculture to give the cotton-degrading strain 007C. This has allowed us to investigate the factors required for degradation of this particularly recalcitrant form of cellulose.
The major proteins associated with the bacterial cell surface and with the culture supernatant were analyzed for R. flavefaciens 007S and 007C grown with cellobiose, xylan or Avicel cellulose as energy sources. Identification of the proteins was enabled by a draft genome sequence obtained for 007C. Among supernatant proteins a cellulosomal GH48 hydrolase, a rubrerthyrin-like protein and a protein with type IV pili N-terminal domain were the most strongly up-regulated in 007C cultures grown on Avicel compared with cellobiose. Strain 007S also showed substrate-related changes, but supernatant expression of the Pil protein and rubrerythrin in particular were markedly lower in 007S than in 007C during growth on Avicel.
This study provides new information on the extracellular proteome of R. flavefaciens and its regulation in response to different growth substrates. Furthermore it suggests that the cotton cellulose non-degrading strain (007S) has altered regulation of multiple proteins that may be required for breakdown of cotton cellulose. One of these, the type IV pilus was previously shown to play a role in adhesion to cellulose in R. albus, and a related pilin protein was identified here for the first time as a major extracellular protein in R. flavefaciens.
Tuberculosis (TB) is caused by gram-positive bacteria known as the Mycobacterium tuberculosis complex (MTBC). MTBC include several human-associated lineages and several variants adapted to domestic and, more rarely, wild animal species. We report an M. tuberculosis strain isolated from a wild chimpanzee in Côte d’Ivoire that was shown by comparative genomic and phylogenomic analyses to belong to a new lineage of MTBC, closer to the human-associated lineage 6 (also known as M. africanum West Africa 2) than to the other classical animal-associated MTBC strains. These results show that the general view of the genetic diversity of MTBC is limited and support the possibility that other MTBC variants exist, particularly in wild mammals in Africa. Exploring this diversity is crucial to the understanding of the biology and evolutionary history of this widespread infectious disease.
tuberculosis and other mycobacteria; bacteria; Mycobacterium tuberculosis; MTBC; wild chimpanzee; zoonoses; nonhuman primate; infection; lineage; M. tuberculosis; tuberculosis; TB; Africa; mammals
Epidemiological studies of the naturally transformable bacterial pathogen Streptococcus pneumoniae have previously been confounded by high rates of recombination. Sequencing 240 isolates of the PMEN1 (Spain23F-1) multidrug-resistant lineage enabled base substitutions to be distinguished from polymorphisms arising through horizontal sequence transfer. Over 700 recombinations were detected, with genes encoding major antigens frequently affected. Among these were ten capsule switching events, one of which accompanied a population shift as vaccine-escape serotype 19A isolates emerged in the USA following the introduction of the conjugate polysaccharide vaccine. The evolution of resistance to fluoroquinolones, rifampicin and macrolides was observed to occur on multiple occasions. This study details how genomic plasticity within lineages of recombinogenic bacteria can permit adaptation to clinical interventions over remarkably short timescales.