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Background: Neisseria meningitidis is a frequent colonizer of the human nasopharynx, with asymptomatic carriage providing the reservoir for invasive, disease-causing strains. Serogroup Y (MenY) strains are a major cause of meningococcal disease. High-resolution genetic analyses of carriage and disease isolates can establish epidemiological relationships and identify potential virulence factors., Methods: Whole-genome sequence data were obtained for 99 MenY carriage isolates recovered in the United Kingdom during 1997-2010. Sequences were compared to those of 73 MenY invasive isolates recovered during 2010-2011, using a gene-by-gene approach., Results: Comparisons across 1605 core genes resolved 91% of isolates into one of 8 clusters containing closely related disease and carriage isolates. Six clusters contained carried meningococci isolated during 1997-2001, suggesting temporal stability. One cluster of isolates, predominately sharing the designation Y: P1.5-1,10-1: F4-1: ST-1655 (cc23), was resolved into one subcluster with 86% carriage isolates and a second with 90% invasive isolates. These subclusters were defined by specific allelic differences in 5 core genes encoding glycerate kinase (glxK), valine-pyruvate transaminase (avtA), superoxide dismutase (sodB), and 2 hypothetical proteins., Conclusions: High-resolution genetic analyses detected long-term temporal stability and temporally overlapping carriage and disease populations for MenY clones but also evidence of a disease-associated clone., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Recombination enhances the adaptive potential of organisms by allowing genetic variants to be tested on multiple genomic backgrounds. Its distribution in the genome can provide insight into the evolutionary forces that underlie traits, such as the emergence of pathogenicity. Here, we examined landscapes of realized homologous recombination of 500 genomes from ten bacterial species and found all species have "hot" regions with elevated rates relative to the genome average. We examined the size, gene content, and chromosomal features associated with these regions and the correlations between closely related species. The recombination landscape is variable and evolves rapidly. For example in Salmonella, only short regions of around 1 kb in length are hot whereas in the closely related species Escherichia coli, some hot regions exceed 100 kb, spanning many genes. Only Streptococcus pyogenes shows evidence for the positive correlation between GC content and recombination that has been reported for several eukaryotes. Genes with function related to the cell surface/membrane are often found in recombination hot regions but E. coli is the only species where genes annotated as "virulence associated" are consistently hotter. There is also evidence that some genes with "housekeeping" functions tend to be overrepresented in cold regions. For example, ribosomal proteins showed low recombination in all of the species. Among specific genes, transferrin-binding proteins are recombination hot in all three of the species in which they were found, and are subject to interspecies recombination., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Cattle are the second most common source of human campylobacteriosis. However, routes to account for this scale of transmission have not been identified. In contrast to chicken, red meat is not heavily contaminated at point of sale. Although effective pasteurization prevents milk-borne infection, apparently sporadic infections may include undetected outbreaks from raw or perhaps incompletely pasteurized milk., Methods: A rise in Campylobacter gastroenteritis in an isolated population was investigated using whole-genome sequencing (WGS), an epidemiological study, and environmental investigations., Results: A single strain was identified in 20 cases, clearly distinguishable from other local strains and a reference population by WGS. A case-case analysis showed association of infection with the outbreak strain and milk from a single dairy (odds ratio, 8; Fisher exact test P value = .023). Despite temperature records indicating effective pasteurization, mechanical faults likely to lead to incomplete pasteurization of part of the milk were identified by further testing and examination of internal components of dairy equipment., Conclusions: Here, milk distribution concentrated on a small area, including school-aged children with low background incidence of campylobacteriosis, facilitated outbreak identification. Low-level contamination of widely distributed milk would not produce as detectable an outbreak signal. Such hidden outbreaks may contribute to the substantial burden of apparently sporadic Campylobacter from cattle where transmission routes are not certain. The effective discrimination of outbreak isolates from a reference population using WGS shows that integrating these data and approaches into surveillance could support the detection as well as investigation of such outbreaks., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

Background: Herd protection by meningococcal vaccines is conferred by population-level reduction of meningococcal nasopharyngeal colonization. Given the inverse epidemiological association between colonization by commensal Neisseria lactamica and meningococcal disease, we investigated whether controlled infection of human volunteers with N. lactamica prevents colonization by Neisseria meningitidis., Methods: In a block-randomized human challenge study, 310 university students were inoculated with 10(4) colony-forming units of N. lactamica or were sham-inoculated, and carriage was monitored for 26 weeks, after which all participants were reinoculated with N. lactamica and resampled 2 weeks later., Results: At baseline, natural N. meningitidis carriage in the control group was 22.4% (36/161), which increased to 33.6% (48/143) by week 26. Two weeks after inoculation of N. lactamica, 33.6% (48/143) of the challenge group became colonized with N. lactamica. In this group, meningococcal carriage reduced from 24.2% (36/149) at inoculation to 14.7% (21/143) 2 weeks after inoculation (-9.5%; P = .006). The inhibition of meningococcal carriage was only observed in carriers of N. lactamica, was due both to displacement of existing meningococci and to inhibition of new acquisition, and persisted over at least 16 weeks. Crossover inoculation of controls with N. lactamica replicated the result. Genome sequencing showed that inhibition affected multiple meningococcal sequence types., Conclusions: The inhibition of meningococcal carriage by N. lactamica is even more potent than after glycoconjugate meningococcal vaccination. Neisseria lactamica or its components could be a novel bacterial medicine to suppress meningococcal outbreaks. This observation explains the epidemiological observation of natural immunity conferred by carriage of N. lactamica., Clinical Trials Registration: NCT02249598., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N(6)-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N(6)-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5'-CGY M6A: G-3'), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5'-AC M6A: CC-3') and ModD1 (exemplified by M.Nme579I) (5'-CC M6A: GC-3'). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5'-CGY M6A: G-3'. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5'-GCGC M6A: GG-3' sites, to 100% at 5'-ACGT M6A: GG-3' sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The opportunistic pathogens Staphylococcus aureus and Staphylococcus epidermidis represent major causes of severe nosocomial infection, and are associated with high levels of mortality and morbidity worldwide. These species are both common commensals on the human skin and in the nasal pharynx, but are genetically distinct, differing at 24% average nucleotide divergence in 1,478 core genes. To better understand the genome dynamics of these ecologically similar staphylococcal species, we carried out a comparative analysis of 324 S. aureus and S. epidermidis genomes, including 83 novel S. epidermidis sequences. A reference pan-genome approach and whole genome multilocus-sequence typing revealed that around half of the genome was shared between the species. Based on a BratNextGen analysis, homologous recombination was found to have impacted on 40% of the core genes in S. epidermidis, but on only 24% of the core genes in S. aureus. Homologous recombination between the species is rare, with a maximum of nine gene alleles shared between any two S. epidermidis and S. aureus isolates. In contrast, there was considerable interspecies admixture of mobile elements, in particular genes associated with the SaPIn1 pathogenicity island, metal detoxification, and the methicillin-resistance island SCCmec. Our data and analysis provide a context for considering the nature of recombinational boundaries between S. aureus and S. epidermidis and, the selective forces that influence realized recombination between these species., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Herd immunity is important in the effectiveness of conjugate polysaccharide vaccines against encapsulated bacteria. A large multicenter study investigated the effect of meningococcal serogroup C conjugate vaccine introduction on the meningococcal population., Methods: Carried meningococci in individuals aged 15-19 years attending education establishments were investigated before and for 2 years after vaccine introduction. Isolates were characterized by multilocus sequence typing, serogroup, and capsular region genotype and changes in phenotypes and genotypes assessed., Results: A total of 8462 meningococci were isolated from 47 765 participants (17.7%). Serogroup prevalence was similar over the 3 years, except for decreases of 80% for serogroup C and 40% for serogroup 29E. Clonal complexes were associated with particular serogroups and their relative proportions fluctuated, with 12 statistically significant changes (6 up, 6 down). The reduction of ST-11 complex serogroup C meningococci was probably due to vaccine introduction. Reasons for a decrease in serogroup 29E ST-254 meningococci (from 1.8% to 0.7%) and an increase in serogroup B ST-213 complex meningococci (from 6.7% to 10.6%) were less clear., Conclusions: Natural fluctuations in carried meningococcal genotypes and phenotypes a can be affected by the use of conjugate vaccines, and not all of these changes are anticipatable in advance of vaccine introduction.

Aims: Crates used to transport live poultry can be contaminated with Campylobacter, despite periodic sanitization, and are potential vectors for transmission between flocks. We investigated the microbial contamination of standard and silver ion containing crates in normal use and the genetic structure of associated Campylobacter populations., Methods and Results: Bacteria from crates were enumerated by appropriate culture techniques, and multilocus sequence typing (MLST) was used to determine the genetic structure of Campylobacters isolated from standard and silver ion containing crates. Compared to standard crates, counts of bacteria, including Campylobacter, were consistently lower on silver ion containing crates throughout the decontamination process. In total, 16 different sequence types were identified from 89 Campylobacter jejuni isolates from crates. These were attributed to putative source population (chicken, cattle, sheep, the environment, wild bird) using the population genetic model, structure. Most (89%) were attributed to chicken, with 22% attribution to live chicken and 78% to retail poultry meat. MLST revealed a progressive shift in allele frequencies through the crate decontamination process. Campylobacter on crates survived for at least 3 h after sanitization, a period of time equivalent to the journey from the processing plant to the majority of farms in the catchment, showing the potential for involvement of crates in transmission., Conclusions: Inclusion of a silver ion biocide in poultry transportation crates to levels demonstrating acceptable antibacterial activity in vitro reduces the level of bacterial contamination during normal crate use compared to standard crates. Molecular analysis of Campylobacter isolates indicated a change in genetic structure of the population with respect to the poultry-processing plant sanitization practice., Significance and Impact of the Study: The application of a sustainable antimicrobial to components of poultry processing may contribute to reducing the levels of Campylobacter circulating in poultry., (© 2010 Biocote Ltd. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.)

Aims: To determine the diversity and population structure of Campylobacter jejuni (C. jejuni) isolates from Danish patients and to examine the association between multilocus sequence typing types and different clinical symptoms including gastroenteritis (GI), Guillain-Barré syndrome (GBS) and reactive arthritis (RA)., Methods and Results: Multilocus sequence typing (MLST) was used to characterize 122 isolates, including 18 from patients with RA and 8 from patients with GBS. The GI and RA isolates were collected in Denmark during 2002-2003 and the GBS isolates were obtained from other countries. In overall, 51 sequence types (STs) were identified within 18 clonal complexes (CCs). Of these three CCs, ST-21, ST-45 and ST-22 clonal complexes accounted for 64 percent of all isolates. The GBS isolates in this study significantly grouped into the ST-22 clonal complex, consistent with the PubMLST database isolates. There was no significant clustering of the RA isolates., Conclusions: Isolates from Denmark were found to be highly genetically diverse. GBS isolates grouped significantly with clonal complex ST-22, but the absence of clustering of RA isolates indicated that the phylogenetic background for this sequela could not be reconstructed using variation in MLST loci. Possibly, putative RA-associated genes may vary, by recombination or expression differences, independent of MLST loci., Significance and Impact of the Study: MLST typing of C. jejuni isolates from Danish patients with gastroenteritis confirmed that the diversity of clones in Denmark is comparable to that in other European countries. Furthermore, a verification of the grouping of GBS isolates compared to RA isolates provides information about evolution of the bacterial population resulting in this important sequela.

The distribution of the hemoglobin receptor gene (hmbR) was investigated among disease and carriage Neisseria meningitidis isolates, revealing that the gene was detected at a significantly higher frequency among disease isolates than among carriage isolates. In isolates without hmbR, the locus was occupied by the cassettes exl2 or exl3 or by a "pseudo hmbR" gene, designated exl4. The hmbR locus exhibited characteristics of a pathogenicity island in published genomes of N. meningitidis, Neisseria gonorrhoeae, and Neisseria lactamica sequence type-640. These data are consistent with a role for the hmbR gene in meningococcal disease.

Background: Campylobacter species cause a high proportion of bacterial gastroenteritis cases and are a significant burden on health care systems and economies worldwide; however, the relative contributions of the various possible sources of infection in humans are unclear., Methods: National-scale genotyping of Campylobacter species was used to quantify the relative importance of various possible sources of human infection. Multilocus sequence types were determined for 5674 isolates obtained from cases of human campylobacteriosis in Scotland from July 2005 through September 2006 and from 999 Campylobacter species isolates from 3417 contemporaneous samples from potential human infection sources. These data were supplemented with 2420 sequence types from other studies, representing isolates from a variety of sources. The clinical isolates were attributed to possible sources on the basis of their sequence types with use of 2 population genetic models, STRUCTURE and an asymmetric island model., Results: The STRUCTURE and the asymmetric island models attributed most clinical isolates to chicken meat (58% and 78% of Campylobacter jejuni and 40% and 56% of Campylobacter coli isolates, respectively), identifying it as the principal source of Campylobacter infection in humans. Both models attributed the majority of the remaining isolates to ruminant sources, with relatively few isolates attributed to wild bird, environment, swine, and turkey sources., Conclusions: National-scale genotyping was a practical and efficient methodology for the quantification of the contributions of different sources to human Campylobacter infection. Combined with the knowledge that retail chicken is routinely contaminated with Campylobacter, these results are consistent with the view that the largest reductions in human campylobacteriosis in industrialized countries will come from interventions that focus on the poultry industry.

We show that a higher incidence of campylobacteriosis is found in young children (age, <5 years) living in rural, compared with urban, areas. Association of this difference with particular animal sources was evaluated using multilocus sequence typing. This evaluation was achieved by comparing Campylobacter isolates originating from these children, retail poultry, and a range of animal sources by use of source attribution and phylogenetic analysis methods. The results indicate that chicken is a major source of infection in young urban children, although not in their rural counterparts, for which ruminant and other avian sources are more important.

Background: In 1999, meningococcal serogroup C conjugate (MCC) vaccines were introduced in the United Kingdom for those under 19 years of age. The impact of this intervention on asymptomatic carriage of meningococci was investigated to establish whether serogroup replacement or protection by herd immunity occurred., Methods: Multicenter surveys of carriage were conducted during vaccine introduction and on 2 successive years, resulting in a total of 48,309 samples, from which 8599 meningococci were isolated and characterized by genotyping and phenotyping., Results: A reduction in serogroup C carriage (rate ratio, 0.19) was observed that lasted at least 2 years with no evidence of serogroup replacement. Vaccine efficacy against carriage was 75%, and vaccination had a disproportionate impact on the carriage of sequence type (ST)-11 complex serogroup C meningococci that (rate ratio, 0.06); these meningococci also exhibited high rates of capsule expression., Conclusions: The impact of vaccination with MCC vaccine on the prevalence of carriage of group C meningococci was consistent with herd immunity. The high impact on the carriage of ST-11 complex serogroup C could be attributed to high levels of capsule expression. High vaccine efficacy against disease in young children, who were not protected long-term by the schedule initially used, is attributed to the high vaccine efficacy against carriage in older age groups.

The global surveillance of bacterial pathogens is particularly important for bacteria with diverse and dynamic populations that cause periodic epidemics or pandemics. The isolate characterization methods employed for surveillance should: (1) generate unambiguous data; (2) be readily implemented in a variety of scenarios and be reproducible among laboratories; (3) be scalable and preferably available in a high throughput format; and (4) be cost effective. Multilocus sequence typing (MLST) was designed to meet these criteria and has been implemented effectively for a wide range of microorganisms. The 'Impact of meningococcal epidemiology and population biology on public health in Europe (EU-MenNet)' project had amongst its objectives: (1) to disseminate meningococcal MLST and sequence-based typing throughout Europe by establishing a centre for training and data generation, and (2) to produce a comprehensive Europe-wide picture of meningococcal disease epidemiology for the first time. Data produced from the project have shown the distribution of a relatively small number of STs, clonal complexes and PorA types that account for a large proportion of the disease-associated isolates in Europe. The project demonstrates how molecular typing can be combined with epidemiological data via the Internet for global disease surveillance.

The diversity and dynamics of Neisseria meningitidis populations generate a requirement for high resolution, comprehensive, and portable typing schemes for meningococcal disease surveillance. Molecular approaches, specifically DNA amplification and sequencing, are the methods of choice for various reasons, including: their generic nature and portability, comprehensive coverage, and ready implementation to culture negative clinical specimens. The following target genes are recommended: (1) the variable regions of the antigen-encoding genes porA and fetA and, if additional resolution is required, the porB gene for rapid investigation of disease outbreaks and investigating the distribution of antigenic variants; (2) the seven multilocus sequence typing loci-these data are essential for the most effective national, and international management of meningococcal disease, as well as being invaluable in studies of meningococcal population biology and evolution. These targets have been employed extensively in reference laboratories throughout the world and validated protocols have been published. It is further recommended that a modified nomenclature be adopted of the form: serogroup: PorA type: FetA type: sequence type (clonal complex), thus: B: P1.19,15: F5-1: ST-33 (cc32).

Between 1999 and 2004, the European Union Invasive Bacterial Infections Surveillance Network (EU-IBIS) received c. 50,000 reports of meningococcal disease from 27 participating countries. Analysis has demonstrated a major decline in the incidence of invasive disease in those countries that have introduced routine vaccination against serogroup C infection. The establishment of rapid reporting of W135 and B2a/B2b strains has been able to provide early reassurance that these strains are not emerging as major public health problems in Europe. Between September 2001 and February 2005, the EU-MenNet project offered further opportunities for enhancing this data resource. Collaborative projects included: improving the EU-IBIS website; reviewing case ascertainment in Europe; reviewing cost-effectiveness studies for meningococcal serogroup C conjugate (MCC) vaccination; international comparisons of MCC vaccine efficacy; and mathematical modelling studies. In addition, linking of data from the European Meningococcal Multi-locus Sequence Type Centre to epidemiological data was performed. Particular clonal complexes were found to be preferentially associated with certain serogroups. Case fatality was also found to vary with clonal complex, suggesting that genotype can be a marker for hypervirulence. The importance of close collaboration between networks of epidemiologists, microbiologists, and the wider scientific and public health community is demonstrated.

Background: The relative invasiveness rates (attack rates) of Streptococcus pneumoniae of different capsular serotypes in children are not known. Estimates of capsular serotype invasiveness (designated "invasive odds ratios") that are based on cross-sectional prevalence carriage data have been published, but these estimates could be biased by variation in the duration of carriage., Methods: The relative attack rates of invasive pneumococci were measured using national UK surveillance data on invasive pneumococcal disease (IPD) incidence and data on incidence of pneumococcal acquisition from longitudinal studies of nasopharyngeal pneumococcal carriage., Results: We found significant differences in capsular serotype-specific attack rates. For example, capsular serotypes 4, 14, 7F, 9V, and 18C were associated with rates of >20 IPD cases/100,000 acquisitions, whereas capsular serotypes 23F, 6A, 19F, 16F, 6B, and 15B/C were associated with <10 IPD cases/100,000 acquisitions. There was an inverse relationship between duration of carriage and attack rate by capsular serotype (P<.0001). Attack rates were significantly correlated with invasive odds ratios (P<.0001)., Conclusions: The capsular serotype is a major determinant of both pneumococcal duration of carriage and attack rate. Published invasive odds ratios are a reliable and practical method of determining capsular serotype invasiveness and will be valuable for investigating and characterizing emerging capsular serotypes in the context of conjugate vaccination.

Background: The incidence of serogroup C and Y meningococcal disease increased in the United States during the 1990s. The cyclical nature of endemic meningococcal disease remains unexplained. The purpose of this study was to investigate the mechanisms associated with the increase in the incidence of meningococcal disease., Methods: We characterized an increasing incidence of invasive serogroup C and Y meningococcal disease using population-based surveillance from 1992 through 2001. Isolates were characterized by multilocus sequence typing and antigen sequence typing of 3 outer membrane protein (OMP) genes: porA variable regions (VRs) 1 and 2, porB, and fetA VR., Results: For both serogroups, OMP antigenic shifts were associated with increased incidence of meningococcal disease. For serogroup Y, antigenic shift occurred through amino acid substitutions at all 3 OMPs--PorA VR 1 and 2, PorB, and FetA VR. For serogroup C, antigenic shift involved amino acid substitutions at FetA VR and, in some cases, deletion of the porA gene. On the basis of deduced amino acid sequences, the antigenic changes likely occurred by horizontal gene transfer., Conclusions: Antigenic shifts were associated with increased incidence of serogroup C and serogroup Y meningococcal disease. For serogroup Y, the changes involved all OMP genes that were studied. Increases in the incidence of meningococcal disease may be caused, in part, by antigenic shift.

Background: Neisseria meningitidis is a diverse commensal bacterium that occasionally causes severe invasive disease. The relationship between meningococcal genotype and capsular polysaccharide, the principal virulence factor and vaccine component, was investigated in carried meningococci isolated from 8000 children and young adults in Bavaria, Germany., Methods: Of the 830 meningococci isolated (carriage rate, 10.4%) by microbiological techniques, 822 were characterized by serogrouping, multilocus sequence typing, and genetic analysis of the capsule region. Statistical and population genetic analyses were applied to these data., Results: The rapid increase in carriage rates with age of carrier, the low prevalence of hyperinvasive meningococci, and the relative prevalence of the 4 disease-associated serogroups were consistent with earlier observations. There was no genetic structuring of the meningococcal population by age of carrier or sampling location; however, there was significant geographic structuring of the meningococci isolated in civil, but not military, institutions. The rate of capsule gene expression did not vary with age of carrier or meningococcal genotype, except for serogroup C, for which increased expression was associated with ST-11 (formerly ET-37) complex meningococci., Conclusions: Serogroup C capsule expression during carriage may contribute to the invasive character of ST-11 complex meningococci and to the high efficacy of meningococcal serogroup C conjugate polysaccharide vaccine.

Patterns of genetic diversity within populations of human pathogens, shaped by the ecology of host-microbe interactions, contain important information about the epidemiological history of infectious disease. Exploiting this information, however, requires a systematic approach that distinguishes the genetic signal generated by epidemiological processes from the effects of other forces, such as recombination, mutation, and population history. Here, a variety of quantitative techniques were employed to investigate multilocus sequence information from isolate collections of Neisseria meningitidis, a major cause of meningitis and septicemia world wide. This allowed quantitative evaluation of alternative explanations for the observed population structure. A coalescent-based approach was employed to estimate the rate of mutation, the rate of recombination, and the size distribution of recombination fragments from samples from disease-associated and carried meningococci obtained in the Czech Republic in 1993 and a global collection of disease-associated isolates collected globally from 1937 to 1996. The parameter estimates were used to reject a model in which genetic structure arose by chance in small populations, and analysis of molecular variation showed that geographically restricted gene flow was unlikely to be the cause of the genetic structure. The genetic differentiation between disease and carriage isolate collections indicated that, whereas certain genotypes were overrepresented among the disease-isolate collections (the "hyperinvasive" lineages), disease-associated and carried meningococci exhibited remarkably little differentiation at the level of individual nucleotide polymorphisms. In combination, these results indicated the repeated action of natural selection on meningococcal populations, possibly arising from the coevolutionary dynamic of host-pathogen interactions.

Aims: To identify and make available through the National Collection of Type Cultures (NCTC) a set of reference isolates for the clonal complexes of Campylobacter jejuni., Methods and Results: The development of a multilocus sequence typing scheme for C. jejuni enabled the genetic characterization of a large number of isolates (n = 814) from cases of human disease, animals, birds and their food products. The nucleotide sequence data were used to assign each isolate an allelic profile or sequence type (ST) and examine the C. jejuni population structure in terms of clonal complexes. The clonal complexes consisted of an abundant central or founder genotype (ST), after which the complex was named, together with very closely related, generally less abundant genotypes differing from the founder at one, two or three loci. The clonal complex is an informative unit for the study C. jejuni epidemiology. It provides data which enabled the choice of 13 C. jejuni founder isolates for submission to the NCTC as a representative cross-section of the C. jejuni population., Conclusions: These 13 isolates provide a defined resource for further research into aspects of C. jejuni biology such as genomic diversity, virulence and adaptation to particular hosts or environmental survival., Significance and Impact of Study: This isolate collection is available through the NCTC and provides a resource for further research.

Previous estimates of rates of synonymous (d(S)) and nonsynonymous (d(N)) substitution among Neisseria meningitidis gene sequences suggested that the surface loops of the variable outer membrane protein PorB were under only weak selection pressure from the host immune response. These findings were consistent with studies indicating that PorB variants were not always protective in immunological and microbiological assays and questioned the suitability of this protein as a vaccine component. PorB, which is expressed at high levels on the surface of the meningococcus, has been implicated in mechanisms of pathogenesis and has also been used as a typing target in epidemiological investigations. In this work, using more precise estimates of selection pressures and recombination rates, we have shown that some residues in the surface loops of PorB are under very strong positive selection, as great as that observed in human immunodeficiency virus-1 surface glycoproteins, whereas amino acids within the loops and the membrane-spanning regions of the protein are under purifying selection, presumably because of structural constraints. Congruence tests showed that recombination occurred at a rate that was not sufficient to erase all phylogenetic similarity and did not greatly bias selection analysis. Homology models of PorB structure indicated that many strongly selected sites encoded residues that were predicted to be exposed to host immune responses, implying that this protein is under strong immune selection and requires further examination as a potential vaccine candidate. These data show that phylogenetic inference can be used to complement immunological and biochemical data in the choice of vaccine candidates.

Unlabelled: The 32-bit Windows application START is implemented using Visual Basic and C(++) and performs analyses to aid in the investigation of bacterial population structure using multilocus sequence data. These analyses include data summary, lineage assignment, and tests for recombination and selection., Availability: START is available at http://outbreak.ceid.ox.ac.uk/software.htm., Contact: keith.jolley@ceid.ox.ac.uk

Motivation: Multi Locus Sequence Typing (MLST) is a newly developed typing method for bacteria based on the sequence determination of internal fragments of seven house-keeping genes. It has proved useful in characterizing and monitoring disease-causing and antibiotic resistant lineages of bacteria. The strength of this approach is that unlike data obtained using most other typing methods, sequence data are unambiguous, can be held on a central database and be queried through a web server., Results: A database-driven software system (mlstdb) has been developed, which is used by public health laboratories and researchers globally to query their nucleotide sequence data against centrally held databases over the internet. The mlstdb system consists of a set of perl scripts for defining the database tables and generating the database management interface and dynamic web pages for querying the databases., Availability: http://www.mlst.net.

Multilocus sequence typing (MLST) is a recently developed nucleotide sequence-based method for the definitive assignment of isolates within bacterial populations to specific clones. MLST uses the same principles as multilocus enzyme electrophoresis and provides data that can be used to investigate aspects of the population genetics and evolution of bacterial species. We used an MLST data set consisting of the sequences of approximately 450-bp fragments from seven housekeeping loci from a large strain collection of Neisseria meningitidis to estimate the relative impact of recombination compared with point mutation in the diversification of N. meningitidis clonal complexes. 126 meningococcal isolates were assigned to 10 clonal complexes, 9 of which contained minor clonal variants. The allelic variation within each complex was classified as a recombinational exchange or a putative point mutation through a comparison of the sequences of each variant allele with that of the allele typically found in the clonal complex. The nine clonal complexes contained a total of 23 allelic variants, and analysis of the sequences of these variant alleles revealed that a single nucleotide site in a meningococcal housekeeping gene is at least 80-fold more likely to change as a result of recombination than as a result of mutation. This value is estimated to be 10-50-fold for Escherichia coli and approximately 50-fold for Streptococcus pneumoniae.

The extent to which recombination disrupts the bifurcating treelike phylogeny and clonal structure imposed by binary fission on bacterial populations remains contentious. Here, we address this question with a study of nucleotide sequence data from 107 isolates of the human pathogen Neisseria meningitidis. Gene fragments from 12 house-keeping loci distributed around the meningococcal chromosome were analyzed, showing that (1) identical alleles are disseminated among genetically diverse isolates, with no evidence for linkage disequilibrium; (2) different loci give distinct and incongruent phylogenetic trees; and (3) allele sequences are incompatible with a bifurcating treelike phylogeny at all loci. These observations are consistent with the hypothesis that meningococcal populations comprise organisms assembled from a common gene pool, with alleles and allele fragments spreading independently, together with the occasional importation of genetic material from other species. Further, they support the view that recombination is an important genetic mechanism in the generation new meningococcal clones and alleles. Consequently, for anything other than the short-term evolution of this species, a bifurcating treelike phylogeny is not an appropriate model.

Some transformable bacteria have acquired target-mediated antibiotic resistance by horizontal genetic exchange of fragments of chromosomal genes. The resistant strains express variants of the antibiotic target that are metabolically active but exhibit a lowered affinity for the antibiotic. The alleles encoding these resistant proteins are mosaics comprising DNA derived from the host and other bacteria, often members of a different species. Examples include penicillin-resistant penicillin-binding proteins (PBPs) in Streptococcus pneumoniae and the pathogenic Neisseria species and sulfonamide-resistant dihydropterate synthase in Neisseria meningitidis. Distinct mosaic alleles encoding antibiotic resistance have arisen on multiple occasions, indicating the mobility of chromosomal genes in these species. Mosaic genes can arise at any chromosomal locus, and S. pneumoniae organisms with high-level penicillin resistance have acquired mosaic PBP genes at three bacterial bpb loci. Furthermore, horizontal genetic exchange permits movement of alleles among bacterial lineages, increasing the opportunities for the spread of antibiotic resistance.

Information of the biochemistry and genetics of bacterial species, usually obtained by the study of single isolates, is enhanced by studies of populations of bacteria. Recent advances in molecular technology, particularly polymerase chain reaction-based nucleotide sequence analysis, provide powerful tools for the study of population genetics. Data obtained by such techniques indicate that, while some bacterial species have a clonal population structure, others are non-clonal or panmictic. Clonal populations are a consequence of asexual reproduction by binary fission; panmictic population structures result from 'horizontal' exchange of genetic material between clones. A consequence of horizontal genetic exchange is mosaic gene structures, recognisable by comparisons of nucleotide sequences. In transformable bacteria, for example the human pathogen Neisseria meningitidis, several different genes, including the gene encoding the class 1 outer membrane protein, a major surface antigen, are mosaics. This genetic process has implications both for vaccine design and in the interpretation of epidemiological data.