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1.  The extant World War 1 dysentery bacillus NCTC1: a genomic analysis 
Lancet  2014;384(9955):1691-1697.
Shigellosis (previously bacillary dysentery) was the primary diarrhoeal disease of World War 1, but outbreaks still occur in military operations, and shigellosis causes hundreds of thousands of deaths per year in developing nations. We aimed to generate a high-quality reference genome of the historical Shigella flexneri isolate NCTC1 and to examine the isolate for resistance to antimicrobials.
In this genomic analysis, we sequenced the oldest extant Shigella flexneri serotype 2a isolate using single-molecule real-time (SMRT) sequencing technology. Isolated from a soldier with dysentery from the British forces fighting on the Western Front in World War 1, this bacterium, NCTC1, was the first isolate accessioned into the National Collection of Type Cultures. We created a reference sequence for NCTC1, investigated the isolate for antimicrobial resistance, and undertook comparative genetics with S flexneri reference strains isolated during the 100 years since World War 1.
We discovered that NCTC1 belonged to a 2a lineage of S flexneri, with which it shares common characteristics and a large core genome. NCTC1 was resistant to penicillin and erythromycin, and contained a complement of chromosomal antimicrobial resistance genes similar to that of more recent isolates. Genomic islands gained in the S flexneri 2a lineage over time were predominately associated with additional antimicrobial resistances, virulence, and serotype conversion.
This S flexneri 2a lineage is a well adapted pathogen that has continued to respond to selective pressures. We have created a valuable historical benchmark for shigellae in the form of a high-quality reference sequence for a publicly available isolate.
The Wellcome Trust.
PMCID: PMC4226921  PMID: 25441199
2.  Genomic Investigations Unmask Mycoplasma amphoriforme, a New Respiratory Pathogen 
The results of high-resolution whole-genome sequencing data provide compelling evidence that Mycoplasma amphoriforme produces chronic relapsing infection and, importantly, is transmitted in a hospital environment.
Background. Mycoplasma amphoriforme has been associated with infection in patients with primary antibody deficiency (PAD). Little is known about the natural history of infection with this organism and its ability to be transmitted in the community.
Methods. The bacterial load was estimated in sequential sputum samples from 9 patients by quantitative polymerase chain reaction. The genomes of all available isolates, originating from patients in the United Kingdom, France, and Tunisia, were sequenced along with the type strain. Genomic data were assembled and annotated, and a high-resolution phylogenetic tree was constructed.
Results. By using high-resolution whole-genome sequencing (WGS) data, we show that patients can be chronically infected with M. amphoriforme manifesting as a relapsing-remitting bacterial load, interspersed by periods when the organism is undetectable. Importantly, we demonstrate transmission of strains within a clinical environment. Antibiotic resistance mutations accumulate in isolates taken from patients who received multiple courses of antibiotics.
Conclusions. Mycoplasma amphoriforme isolates form a closely related species responsible for a chronic relapsing and remitting infection in PAD patients in the United Kingdom and from immunocompetent patients in other countries. We provide strong evidence of transmission between patients attending the same clinic, suggesting that screening and isolation may be necessary for susceptible patients. This work demonstrates the critical role that WGS can play in rapidly unraveling the biology of a novel pathogen.
PMCID: PMC4293396  PMID: 25344534
Mycoplasma amphoriforme; whole genome sequencing; respiratory infection; infection control; primary antibody deficiency
3.  Genome Evolution and Plasticity of Serratia marcescens, an Important Multidrug-Resistant Nosocomial Pathogen 
Genome Biology and Evolution  2014;6(8):2096-2110.
Serratia marcescens is an important nosocomial pathogen that can cause an array of infections, most notably of the urinary tract and bloodstream. Naturally, it is found in many environmental niches, and is capable of infecting plants and animals. The emergence and spread of multidrug-resistant strains producing extended-spectrum or metallo beta-lactamases now pose a threat to public health worldwide. Here we report the complete genome sequences of two carefully selected S. marcescens strains, a multidrug-resistant clinical isolate (strain SM39) and an insect isolate (strain Db11). Our comparative analyses reveal the core genome of S. marcescens and define the potential metabolic capacity, virulence, and multidrug resistance of this species. We show a remarkable intraspecies genetic diversity, both at the sequence level and with regards genome flexibility, which may reflect the diversity of niches inhabited by members of this species. A broader analysis with other Serratia species identifies a set of approximately 3,000 genes that characterize the genus. Within this apparent genetic diversity, we identified many genes implicated in the high virulence potential and antibiotic resistance of SM39, including the metallo beta-lactamase and multiple other drug resistance determinants carried on plasmid pSMC1. We further show that pSMC1 is most closely related to plasmids circulating in Pseudomonas species. Our data will provide a valuable basis for future studies on S. marcescens and new insights into the genetic mechanisms that underlie the emergence of pathogens highly resistant to multiple antimicrobial agents.
PMCID: PMC4231636  PMID: 25070509
Serratia marcescens; genome plasticity; virulence; multidrug resistance
4.  The Population Structure of Vibrio cholerae from the Chandigarh Region of Northern India 
Cholera infection continues to be a threat to global public health. The current cholera pandemic associated with Vibrio cholerae El Tor has now been ongoing for over half a century.
Methodology/Principal Findings
Thirty-eight V. cholerae El Tor isolates associated with a cholera outbreak in 2009 from the Chandigarh region of India were characterised by a combination of microbiology, molecular typing and whole-genome sequencing. The genomic analysis indicated that two clones of V. cholera circulated in the region and caused disease during this time. These clones fell into two distinct sub-clades that map independently onto wave 3 of the phylogenetic tree of seventh pandemic V. cholerae El Tor. Sequence analyses of the cholera toxin gene, the Vibrio seventh Pandemic Island II (VSPII) and SXT element correlated with this phylogenetic position of the two clades on the El Tor tree. The clade 2 isolates, characterized by a drug-resistant profile and the expression of a distinct cholera toxin, are closely related to the recent V. cholerae isolated elsewhere, including Haiti, but fell on a distinct branch of the tree, showing they were independent outbreaks. Multi-Locus Sequence Typing (MLST) distinguishes two sequence types among the 38 isolates, that did not correspond to the clades defined by whole-genome sequencing. Multi-Locus Variable-length tandem-nucleotide repeat Analysis (MLVA) identified 16 distinct clusters.
The use of whole-genome sequencing enabled the identification of two clones of V. cholerae that circulated during the 2009 Chandigarh outbreak. These clones harboured a similar structure of ICEVchHai1 but differed mainly in the structure of CTX phage and VSPII. The limited capacity of MLST and MLVA to discriminate between the clones that circulated in the 2009 Chandigarh outbreak highlights the value of whole-genome sequencing as a route to the identification of further genetic markers to subtype V. cholerae isolates.
Author Summary
Vibrio cholerae is a diarrheal pathogen that is responsible for substantial morbidity and mortality worldwide. Historically, seven pandemics of cholera have been recognized, with classical biotype strains associated with the sixth and the El Tor biotype with the seventh (current) pandemic. Recently multi-drug resistant El Tor variants expressing classical cholera toxin have replaced the original El Tor strains in many epidemics, and are sometimes associated with more severe diarrhea leading to a higher mortality rate. In regions that experience recurrent cholera outbreaks, such as Northern India, it is important to understand the nature of the circulating strains and establish how they are related to the strains circulating globally. Here, we have demonstrated that whole- genome sequencing is a valuable method to characterize V. cholerae isolates that circulated during the 2009 outbreak in the Northern Indian city of Chandigarh. Through comparative genomic analysis, we identified two clones that circulated during a single outbreak. Importantly, these clones contain significant differences in the structure of the cholera toxin gene and the Vibrio seventh pandemic island II. Our findings demonstrate the limitations of current molecular typing techniques and the importance of surveillance with whole-genome sequencing for identifying V. cholerae clades with distinct genomic signatures.
PMCID: PMC4109905  PMID: 25058483
5.  Structure, Diversity, and Mobility of the Salmonella Pathogenicity Island 7 Family of Integrative and Conjugative Elements within Enterobacteriaceae 
Journal of Bacteriology  2012;194(6):1494-1504.
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.
PMCID: PMC3294861  PMID: 22247511
6.  RNA-seq analysis of the influence of anaerobiosis and FNR on Shigella flexneri 
BMC Genomics  2014;15:438.
Shigella flexneri is an important human pathogen that has to adapt to the anaerobic environment in the gastrointestinal tract to cause dysentery. To define the influence of anaerobiosis on the virulence of Shigella, we performed deep RNA sequencing to identify transcriptomic differences that are induced by anaerobiosis and modulated by the anaerobic Fumarate and Nitrate Reduction regulator, FNR.
We found that 528 chromosomal genes were differentially expressed in response to anaerobic conditions; of these, 228 genes were also influenced by FNR. Genes that were up-regulated in anaerobic conditions are involved in carbon transport and metabolism (e.g. ptsG, manX, murQ, cysP, cra), DNA topology and regulation (e.g. ygiP, stpA, hns), host interactions (e.g. yciD, nmpC, slyB, gapA, shf, msbB) and survival within the gastrointestinal tract (e.g. shiA, ospI, adiY, cysP). Interestingly, there was a marked effect of available oxygen on genes involved in Type III secretion system (T3SS), which is required for host cell invasion and pathogenesis. These genes, located on the large Shigella virulence plasmid, were down regulated in anaerobiosis in an FNR-dependent manner. We also confirmed anaerobic induction of csrB and csrC small RNAs in an FNR-independent manner.
Anaerobiosis promotes survival and adaption strategies of Shigella, while modulating virulence plasmid genes involved in T3SS-mediated host cell invasion. The influence of FNR on this process is more extensive than previously appreciated, although aside from the virulence plasmid, this transcriptional regulator does not govern expression of genes on other horizontally acquired sequences on the chromosome such as pathogenicity islands.
PMCID: PMC4229854  PMID: 24907032
7.  Draft genome sequences of the type strains of Shigella flexneri held at Public Health England: comparison of classical phenotypic and novel molecular assays with whole genome sequence 
Gut Pathogens  2014;6:7.
Public Health England (PHE) holds a collection of Shigella flexneri Type strains isolated between 1949 and 1972 representing 15 established serotypes and one provisional type, E1037. In this study, the genomes of all 16 PHE Type strains were sequenced using the Illumina HiSeq platform. The relationship between core genome phylogeny and serotype was examined.
The most common target gene for the detection of Shigella species in clinical PCR assays, ipaH, was detected in all genomes. The type-specific target genes were correctly identified in each genome sequence. In contrast to the S. flexneri in serotype 5 strain described by Sun et al. (2012), the two PHE serotype 5 Type strains possessed an additional oac gene and were differentiated by the presence (serotype 5b) or absence (serotype 5a) of gtrX. The somatic antigen structure and phylogenetic relationship were broadly congruent for strains expressing serotype specific antigens III, IV and V, but not for those expressing I and II. The whole genome phylogenies of the 15 isolates sequenced showed that the serotype 6 Type Strain was phylogenetically distinct from the other S. flexneri serotypes sequenced. The provisional serotype E1037 fell within the serotype 4 clade, being most closely related to the Serotype 4a Type Strain.
The S. flexneri genome sequences were used to evaluate phylogenetic relationships between Type strains and validate genotypic and phenotypic assays. The analysis confirmed that the PHE S. flexneri Type strains are phenotypically and genotypically distinct. Novel variants will continue to be added to this archive.
PMCID: PMC3972513  PMID: 24684748
Shigella flexneri type strains; Next generation sequencing technology; Molecular serotyping
8.  Genomic Characterisation of Invasive Non-Typhoidal Salmonella enterica Subspecies enterica Serovar Bovismorbificans Isolates from Malawi 
Invasive Non-typhoidal Salmonella (iNTS) are an important cause of bacteraemia in children and HIV-infected adults in sub-Saharan Africa. Previous research has shown that iNTS strains exhibit a pattern of gene loss that resembles that of host adapted serovars such as Salmonella Typhi and Paratyphi A. Salmonella enterica serovar Bovismorbificans was a common serovar in Malawi between 1997 and 2004.
We sequenced the genomes of 14 Malawian bacteraemia and four veterinary isolates from the UK, to identify genomic variations and signs of host adaptation in the Malawian strains.
Principal Findings
Whole genome phylogeny of invasive and veterinary S. Bovismorbificans isolates showed that the isolates are highly related, belonging to the most common international S. Bovismorbificans Sequence Type, ST142, in contrast to the findings for S. Typhimurium, where a distinct Sequence Type, ST313, is associated with invasive disease in sub-Saharan Africa. Although genome degradation through pseudogene formation was observed in ST142 isolates, there were no clear overlaps with the patterns of gene loss seen in iNTS ST313 isolates previously described from Malawi, and no clear distinction between S. Bovismorbificans isolates from Malawi and the UK.
The only defining differences between S. Bovismorbificans bacteraemia and veterinary isolates were prophage-related regions and the carriage of a S. Bovismorbificans virulence plasmid (pVIRBov).
iNTS S. Bovismorbificans isolates, unlike iNTS S. Typhiumrium isolates, are only distinguished from those circulating elsewhere by differences in the mobile genome. It is likely that these strains have entered a susceptible population and are able to take advantage of this niche. There are tentative signs of convergent evolution to a more human adapted iNTS variant. Considering its importance in causing disease in this region, S. Bovismorbificans may be at the beginning of this process, providing a reference against which to compare changes that may become fixed in future lineages in sub-Saharan Africa.
Author Summary
Bacteraemia and meningitis caused by non-typhoidal Salmonella (including serovars Typhimurium, Enteritidis and Bovismorbificans) are a serious health issue in sub-Saharan Africa, particularly in young children and HIV-infected adults. Previous work has indicated that a distinct S. Typhimurium sequence type, ST313, has evolved and spread in these countries, and may be more human-adapted than isolates found in the developed world. We therefore investigated the genomes of Salmonella enterica serovar Bovismorbificans bacteraemia isolates from Malawi and compared them to genomes of veterinary S. Bovismorbificans isolates from the UK using Next Generation Sequencing Technology and subsequent genomic comparisons to establish if there is a genetic basis for this increase in invasive disease observed among African NTS. Contrary to the previous findings for S. Typhimurium, where a distinct ST is found only in sub-Saharan Africa, we discovered that the S. Bovismorbificans isolates from Malawi belong to the most common ST of the serovar and the genome is highly conserved across all sequenced isolates. The major differences between UK veterinary and African human isolates were due to prophage regions inserted into the genomes of African isolates, coupled with a higher prevalence of a virulence plasmid compared to the UK isolates.
PMCID: PMC3828162  PMID: 24244782
9.  Sequencing and Functional Annotation of Avian Pathogenic Escherichia coli Serogroup O78 Strains Reveal the Evolution of E. coli Lineages Pathogenic for Poultry via Distinct Mechanisms 
Infection and Immunity  2013;81(3):838-849.
Avian pathogenic Escherichia coli (APEC) causes respiratory and systemic disease in poultry. Sequencing of a multilocus sequence type 95 (ST95) serogroup O1 strain previously indicated that APEC resembles E. coli causing extraintestinal human diseases. We sequenced the genomes of two strains of another dominant APEC lineage (ST23 serogroup O78 strains χ7122 and IMT2125) and compared them to each other and to the reannotated APEC O1 sequence. For comparison, we also sequenced a human enterotoxigenic E. coli (ETEC) strain of the same ST23 serogroup O78 lineage. Phylogenetic analysis indicated that the APEC O78 strains were more closely related to human ST23 ETEC than to APEC O1, indicating that separation of pathotypes on the basis of their extraintestinal or diarrheagenic nature is not supported by their phylogeny. The accessory genome of APEC ST23 strains exhibited limited conservation of APEC O1 genomic islands and a distinct repertoire of virulence-associated loci. In light of this diversity, we surveyed the phenotype of 2,185 signature-tagged transposon mutants of χ7122 following intra-air sac inoculation of turkeys. This procedure identified novel APEC ST23 genes that play strain- and tissue-specific roles during infection. For example, genes mediating group 4 capsule synthesis were required for the virulence of χ7122 and were conserved in IMT2125 but absent from APEC O1. Our data reveal the genetic diversity of E. coli strains adapted to cause the same avian disease and indicate that the core genome of the ST23 lineage serves as a chassis for the evolution of E. coli strains adapted to cause avian or human disease via acquisition of distinct virulence genes.
PMCID: PMC3584874  PMID: 23275093
10.  Genome and Transcriptome Adaptation Accompanying Emergence of the Definitive Type 2 Host-Restricted Salmonella enterica Serovar Typhimurium Pathovar 
mBio  2013;4(5):e00565-13.
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.
PMCID: PMC3760250  PMID: 23982073
11.  Antagonistic coevolution accelerates molecular evolution 
Nature  2010;464(7286):275-278.
The Red Queen hypothesis proposes that coevolution of interacting species (such as hosts and parasites) should drive molecular evolution through continual natural selection for adaptation and counter-adaptation1–3. Although the divergence observed at some host-resistance4–6 and parasite-infectivity7–9 genes is consistent with this, the long time periods typically required to study coevolution have so far prevented any direct empirical test. Here we show, using experimental populations of the bacterium Pseudomonas fluorescens SBW25 and its viral parasite, phage Φ2 (refs 10, 11), that the rate of molecular evolution in the phage was far higher when both bacterium and phage coevolved with each other than when phage evolved against a constant host genotype. Coevolution also resulted in far greater genetic divergence between replicate populations, which was correlated with the range of hosts that coevolved phage were able to infect. Consistent with this, the most rapidly evolving phage genes under coevolution were those involved in host infection. These results demonstrate, at both the genomic and phenotypic level, that antagonistic coevolution is a cause of rapid and divergent evolution, and is likely to be a major driver of evolutionary change within species.
PMCID: PMC3717453  PMID: 20182425
12.  Global Phylogeny of Shigella sonnei Strains from Limited Single Nucleotide Polymorphisms (SNPs) and Development of a Rapid and Cost-Effective SNP-Typing Scheme for Strain Identification by High-Resolution Melting Analysis 
Journal of Clinical Microbiology  2013;51(1):303-305.
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.
PMCID: PMC3536230  PMID: 23115259
13.  Public Health Value of Next-Generation DNA Sequencing of Enterohemorrhagic Escherichia coli Isolates from an Outbreak 
Journal of Clinical Microbiology  2013;51(1):232-237.
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.
PMCID: PMC3536255  PMID: 23135946
14.  Horizontally Acquired Glycosyltransferase Operons Drive Salmonellae Lipopolysaccharide Diversity 
PLoS Genetics  2013;9(6):e1003568.
The immunodominant lipopolysaccharide is a key antigenic factor for Gram-negative pathogens such as salmonellae where it plays key roles in host adaptation, virulence, immune evasion, and persistence. Variation in the lipopolysaccharide is also the major differentiating factor that is used to classify Salmonella into over 2600 serovars as part of the Kaufmann-White scheme. While lipopolysaccharide diversity is generally associated with sequence variation in the lipopolysaccharide biosynthesis operon, extraneous genetic factors such as those encoded by the glucosyltransferase (gtr) operons provide further structural heterogeneity by adding additional sugars onto the O-antigen component of the lipopolysaccharide. Here we identify and examine the O-antigen modifying glucosyltransferase genes from the genomes of Salmonella enterica and Salmonella bongori serovars. We show that Salmonella generally carries between 1 and 4 gtr operons that we have classified into 10 families on the basis of gtrC sequence with apparent O-antigen modification detected for five of these families. The gtr operons localize to bacteriophage-associated genomic regions and exhibit a dynamic evolutionary history driven by recombination and gene shuffling events leading to new gene combinations. Furthermore, evidence of Dam- and OxyR-dependent phase variation of gtr gene expression was identified within eight gtr families. Thus, as O-antigen modification generates significant intra- and inter-strain phenotypic diversity, gtr-mediated modification is fundamental in assessing Salmonella strain variability. This will inform appropriate vaccine and diagnostic approaches, in addition to contributing to our understanding of host-pathogen interactions.
Author Summary
Bacterial pathogens frequently evolve mechanisms to vary the composition of their surface structures. The consequence is enhanced long-term survival by facilitating persistence and evasion of the host immune system. Salmonella sp., cause severe infections in a range of mammalian hosts and guard themselves with a protective coat, termed the O-antigen. Through genome sequence analyses we found that Salmonella have acquired an unprecedented repertoire of genetic sequences for modifying their O-antigen coat. There is strong evidence that these genetic factors have a dynamic evolutionary history and are spread through the bacterial population by bacteriophage. In addition to this genetic repertoire, we determined that Salmonella can and often do employ stochastic mechanisms for expression of these genetic factors. This means that O-antigen coat diversity can be generated within a Salmonella population that otherwise has a common genome. Our data significantly enhance our appreciation of the genetic and regulatory characteristics underpinning Salmonella O-antigen diversity. The role attributed to bacteriophage in generating this diversity highlights that Salmonella are acquiring an extensive repertoire of O-antigen modifying traits that may enhance the pathogen's ability to persist and cause disease in mammalian hosts. Such genetic traits may make useful markers for defining new epidemiological and diagnostic tools.
PMCID: PMC3688519  PMID: 23818865
15.  Shigella sonnei genome sequencing and phylogenetic analysis indicate recent global dissemination from Europe 
Nature genetics  2012;44(9):1056-1059.
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.
PMCID: PMC3442231  PMID: 22863732
16.  Genome Sequence of Chlamydia psittaci Strain 01DC12 Originating from Swine 
Genome Announcements  2013;1(1):e00078-12.
Chlamydia psittaci is the etiological agent of psittacosis and is a zoonotic pathogen infecting birds and a variety of mammalian hosts. Here we report the genome sequence of the porcine strain 01DC12 which is representative of a novel clade of C. psittaci belonging to ompA genotype E.
PMCID: PMC3569293  PMID: 23405306
17.  Whole genome analysis of diverse Chlamydia trachomatis strains identifies phylogenetic relationships masked by current clinical typing 
Nature genetics  2012;44(4):413-S1.
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.
PMCID: PMC3378690  PMID: 22406642
18.  Fitness of Escherichia coli strains carrying expressed and partially silent IncN and IncP1 plasmids 
BMC Microbiology  2012;12:53.
Understanding the survival of resistance plasmids in the absence of selective pressure for the antibiotic resistance genes they carry is important for assessing the value of interventions to combat resistant bacteria. Here, several poorly explored questions regarding the fitness impact of IncP1 and IncN broad host range plasmids on their bacterial hosts are examined; namely, whether related plasmids have similar fitness impacts, whether this varies according to host genetic background, and what effect antimicrobial resistance gene silencing has on fitness.
For the IncP1 group pairwise in vitro growth competition demonstrated that the fitness cost of plasmid RP1 depends on the host strain. For the IncN group, plasmids R46 and N3 whose sequence is presented for the first time conferred remarkably different fitness costs despite sharing closely related backbone structures, implicating the accessory genes in fitness. Silencing of antimicrobial resistance genes was found to be beneficial for host fitness with RP1 but not for IncN plasmid pVE46.
These findings suggest that the fitness impact of a given plasmid on its host cannot be inferred from results obtained with other host-plasmid combinations, even if these are closely related.
PMCID: PMC3347995  PMID: 22475035
19.  Genome Sequences of Salmonella enterica Serovar Typhimurium, Choleraesuis, Dublin, and Gallinarum Strains of Well- Defined Virulence in Food-Producing Animals ▿ 
Journal of Bacteriology  2011;193(12):3162-3163.
Salmonella enterica is an animal and zoonotic pathogen of worldwide importance and may be classified into serovars differing in virulence and host range. We sequenced and annotated the genomes of serovar Typhimurium, Choleraesuis, Dublin, and Gallinarum strains of defined virulence in each of three food-producing animal hosts. This provides valuable measures of intraserovar diversity and opportunities to formally link genotypes to phenotypes in target animals.
PMCID: PMC3133203  PMID: 21478351
20.  Sequence-Based Analysis Uncovers an Abundance of Non-Coding RNA in the Total Transcriptome of Mycobacterium tuberculosis 
PLoS Pathogens  2011;7(11):e1002342.
RNA sequencing provides a new perspective on the genome of Mycobacterium tuberculosis by revealing an extensive presence of non-coding RNA, including long 5’ and 3’ untranslated regions, antisense transcripts, and intergenic small RNA (sRNA) molecules. More than a quarter of all sequence reads mapping outside of ribosomal RNA genes represent non-coding RNA, and the density of reads mapping to intergenic regions was more than two-fold higher than that mapping to annotated coding sequences. Selected sRNAs were found at increased abundance in stationary phase cultures and accumulated to remarkably high levels in the lungs of chronically infected mice, indicating a potential contribution to pathogenesis. The ability of tubercle bacilli to adapt to changing environments within the host is critical to their ability to cause disease and to persist during drug treatment; it is likely that novel post-transcriptional regulatory networks will play an important role in these adaptive responses.
Author Summary
Tuberculosis bacteria are able to hide quietly inside the body for years or decades before reawakening to cause disease. If we knew more about how the bacteria change from a harmless persistent form to an aggressive disease-causing form, we could develop drugs that would be more effective in treating active tuberculosis and may also allow us to eliminate the infection before it erupts into disease. The key to this is in knowing how the bacteria determine which of their genes to express at different times. By applying modern sequencing technologies we have discovered a new putative network of gene regulation in Mycobacterium tuberculosis that is based on RNA molecules rather than protein molecules. We anticipate that this finding will open the way for new research that will allow us to understand the fundamental mechanisms underlying this deadly human disease, and that will help us to design better tools for prevention and treatment of TB.
PMCID: PMC3207917  PMID: 22072964
21.  Retrospective Application of Transposon-Directed Insertion Site Sequencing to a Library of Signature-Tagged Mini-Tn5Km2 Mutants of Escherichia coli O157:H7 Screened in Cattle▿ †  
Journal of Bacteriology  2011;193(7):1771-1776.
Massively parallel sequencing of transposon-flanking regions assigned the genotype and fitness score to 91% of Escherichia coli O157:H7 mutants previously screened in cattle by signature-tagged mutagenesis (STM). The method obviates the limitations of STM and markedly extended the functional annotation of the prototype E. coli O157:H7 genome without further animal use.
PMCID: PMC3067669  PMID: 21278291
22.  Genome Sequence of the Zoonotic Pathogen Chlamydophila psittaci▿  
Journal of Bacteriology  2010;193(5):1282-1283.
We present the first genome sequence of Chlamydophila psittaci, an intracellular pathogen of birds and a human zoonotic pathogen. A comparison with previously sequenced Chlamydophila genomes shows that, as in other chlamydiae, most of the genome diversity is restricted to the plasticity zone. The C. psittaci plasmid was also sequenced.
PMCID: PMC3067587  PMID: 21183672
23.  Salmonella bongori Provides Insights into the Evolution of the Salmonellae 
PLoS Pathogens  2011;7(8):e1002191.
The genus Salmonella contains two species, S. bongori and S. enterica. Compared to the well-studied S. enterica there is a marked lack of information regarding the genetic makeup and diversity of S. bongori. S. bongori has been found predominantly associated with cold-blooded animals, but it can infect humans. To define the phylogeny of this species, and compare it to S. enterica, we have sequenced 28 isolates representing most of the known diversity of S. bongori. This cross-species analysis allowed us to confidently differentiate ancestral functions from those acquired following speciation, which include both metabolic and virulence-associated capacities. We show that, although S. bongori inherited a basic set of Salmonella common virulence functions, it has subsequently elaborated on this in a different direction to S. enterica. It is an established feature of S. enterica evolution that the acquisition of the type III secretion systems (T3SS-1 and T3SS-2) has been followed by the sequential acquisition of genes encoding secreted targets, termed effectors proteins. We show that this is also true of S. bongori, which has acquired an array of novel effector proteins (sboA-L). All but two of these effectors have no significant S. enterica homologues and instead are highly similar to those found in enteropathogenic Escherichia coli (EPEC). Remarkably, SboH is found to be a chimeric effector protein, encoded by a fusion of the T3SS-1 effector gene sopA and a gene highly similar to the EPEC effector nleH from enteropathogenic E. coli. We demonstrate that representatives of these new effectors are translocated and that SboH, similarly to NleH, blocks intrinsic apoptotic pathways while being targeted to the mitochondria by the SopA part of the fusion. This work suggests that S. bongori has inherited the ancestral Salmonella virulence gene set, but has adapted by incorporating virulence determinants that resemble those employed by EPEC.
Author Summary
The bacterial genus Salmonella consists of two species: Salmonella enterica and Salmonella bongori. Salmonella are common causes of food poisoning in humans and can also cause more severe disease such as typhoid fever. Most of the Salmonella that cause disease in humans and animals are members of S. enterica. On the other hand S. bongori, is largely associated with reptiles but can cause disease in humans, albeit rarely. We have determined genomes for S. bongori isolates representing its known diversity. Using this, and existing genome information for a large number of different members of S. enterica, we were able to identify functions found in both species, and therefore likely to be ancestral, and differentiate them from those that have been more recently acquired. This information gives us more perspective on how pathogens evolve over the longer-term and allows us to identify functions that are associated exclusively with isolates that commonly cause disease in humans. Our analysis suggests that when S. bongori and S. enterica diverged they evolved to occupy very different niches.
PMCID: PMC3158058  PMID: 21876672
24.  Phylogenetic diversity and historical patterns of pandemic spread of Yersinia pestis 
Nature genetics  2010;42(12):1140-1143.
Pandemic infectious diseases have accompanied humans since their origins1, and have shaped the form of civilizations2. Of these, plague is possibly historically the most dramatic. We reconstructed historical patterns of plague transmission through sequence variation in 17 complete genome sequences and 933 single nucleotide polymorphisms (SNPs) within a global collection of 286 Yersinia pestis isolates. Y. pestis evolved in or near China, and has been transmitted via multiple epidemics that followed various routes, probably including transmissions to West Asia via the Silk Road and to Africa by Chinese marine voyages. In 1894, Y. pestis spread to India and radiated to diverse parts of the globe, leading to country-specific lineages that can be traced by lineage-specific SNPs. All 626 current isolates from the U.S.A. reflect one radiation and 82 isolates from Madagascar represent a second. Subsequent local microevolution of Y. pestis is marked by sequential, geographically-specific SNPs.
PMCID: PMC2999892  PMID: 21037571
Genomic comparisons; SNP typing; phylogeography; neutral evolution; epidemic spread
25.  A Commensal Gone Bad: Complete Genome Sequence of the Prototypical Enterotoxigenic Escherichia coli Strain H10407▿ †  
Journal of Bacteriology  2010;192(21):5822-5831.
In most cases, Escherichia coli exists as a harmless commensal organism, but it may on occasion cause intestinal and/or extraintestinal disease. Enterotoxigenic E. coli (ETEC) is the predominant cause of E. coli-mediated diarrhea in the developing world and is responsible for a significant portion of pediatric deaths. In this study, we determined the complete genomic sequence of E. coli H10407, a prototypical strain of enterotoxigenic E. coli, which reproducibly elicits diarrhea in human volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains, revealing that the chromosome is closely related to that of the nonpathogenic commensal strain E. coli HS and to those of the laboratory strains E. coli K-12 and C. Furthermore, these analyses demonstrated that there were no chromosomally encoded factors unique to any sequenced ETEC strains. Comparison of the E. coli H10407 plasmids with those from several ETEC strains revealed that the plasmids had a mosaic structure but that several loci were conserved among ETEC strains. This study provides a genetic context for the vast amount of experimental and epidemiological data that have been published.
PMCID: PMC2953697  PMID: 20802035

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