Summary

No single characterisation scheme currently encompasses all levels of bacterial diversity, from domain to strain. We propose Ribosomal Multi Locus Sequence Typing (rMLST), an approach which indexes variation of the 53 genes encoding the bacterial ribosome protein subunits (rps genes), as a means of integrated microbial taxonomy and typing. As with MLST, rMLST employs curated reference sequences to identify gene variants efficiently and rapidly. The rps loci are ideal targets for a universal characterization scheme as they are: (i) present in all bacteria; (ii) distributed around the chromosome; and (iii) encode proteins which are under stabilising selection for functional conservation. Collectively, the rps loci exhibit variation that resolves bacteria in to groups at all taxonomic and most typing levels providing significantly more resolution than 16S small subunit rRNA gene phylogenies. A web-accessible expandable database, comprising whole genome data from more than 1900 bacterial isolates, including 28 draft genomes assembled de novo from the EBI sequence read archive, has been assembled. The rps gene variation catalogued in this database permits rapid and computationally non-intensive identification of the phylogenetic position of any bacterial sequence at the domain, phylum, class, order, family, genus, species and strain levels. The groupings generated with rMLST data are consistent with current nomenclature schemes and independent of the clustering algorithm used. This approach is applicable to the other domains of life, potentially providing a rational and universal approach to the classification of life that is based on one of its fundamental features, the translation mechanism.

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

Background. 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.

Clostridium difficile infection (CDI) is an important cause of mortality and morbidity in healthcare settings. The major virulence determinants are large clostridial toxins, toxin A (tcdA) and toxin B (tcdB), encoded within the pathogenicity locus (PaLoc). Isolates vary in pathogenicity from hypervirulent PCR-ribotypes 027 and 078 with high mortality, to benign non-toxigenic strains carried asymptomatically. The relative pathogenicity of most toxigenic genotypes is still unclear, but may be influenced by PaLoc genetic variant. This is the largest study of C. difficile molecular epidemiology performed to date, in which a representative collection of recent isolates (n = 1290) from patients with CDI in Oxfordshire, UK, was genotyped by multilocus sequence typing. The population structure was described using NeighborNet and ClonalFrame. Sequence variation within toxin B (tcdB) and its negative regulator (tcdC), was mapped onto the population structure. The 69 Sequence Types (ST) showed evidence for homologous recombination with an effect on genetic diversification four times lower than mutation. Five previously recognised genetic groups or clades persisted, designated 1 to 5, each having a strikingly congruent association with tcdB and tcdC variants. Hypervirulent ST-11 (078) was the only member of clade 5, which was divergent from the other four clades within the MLST loci. However, it was closely related to the other clades within the tcdB and tcdC loci. ST-11 (078) may represent a divergent formerly non-toxigenic strain that acquired the PaLoc (at least) by genetic recombination. This study focused on human clinical isolates collected from a single geographic location, to achieve a uniquely high density of sampling. It sets a baseline of MLST data for future comparative studies investigating genotype virulence potential (using clinical severity data for these isolates), possible reservoirs of human CDI, and the evolutionary origins of hypervirulent strains.

The acquisition of superantigen-encoding genes by Streptococcus pyogenes has been associated with increased morbidity and mortality in humans, and the gain of four superantigens by Streptococcus equi is linked to the evolution of this host-restricted pathogen from an ancestral strain of the opportunistic pathogen Streptococcus equi subsp. zooepidemicus. A recent study determined that the culture supernatants of several S. equi subsp. zooepidemicus strains possessed mitogenic activity but lacked known superantigen-encoding genes. Here, we report the identification and activities of three novel superantigen-encoding genes. The products of szeF, szeN, and szeP share 59%, 49%, and 34% amino acid sequence identity with SPEH, SPEM, and SPEL, respectively. Recombinant SzeF, SzeN, and SzeP stimulated the proliferation of equine peripheral blood mononuclear cells, and tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) production, in vitro. Although none of these superantigen genes were encoded within functional prophage elements, szeN and szeP were located next to a prophage remnant, suggesting that they were acquired by horizontal transfer. Eighty-one of 165 diverse S. equi subsp. zooepidemicus strains screened, including 7 out of 15 isolates from cases of disease in humans, contained at least one of these new superantigen-encoding genes. The presence of szeN or szeP, but not szeF, was significantly associated with mitogenic activity in the S. equi subsp. zooepidemicus population (P < 0.000001, P < 0.000001, and P = 0.104, respectively). We conclude that horizontal transfer of these novel superantigens from and within the diverse S. equi subsp. zooepidemicus population is likely to have implications for veterinary and human disease.

Horizontal genetic exchange strongly influences the evolution of many bacteria, substantially contributing to difficulties in defining their position in taxonomic groups. In particular, how clusters of related bacterial genotypes – currently classified as microbiological species – evolve and are maintained remains controversial. The nature and magnitude of gene exchange between two closely related (approx. 15 % nucleotide divergence) microbiologically defined species, Campylobacter jejuni and Campylobacter coli, was investigated by the examination of mosaic alleles, those with some ancestry from each population. A total of 1738 alleles from 2953 seven-locus housekeeping gene sequence types (STs) were probabilistically assigned to each species group with the model-based clustering algorithm structure. Alleles with less than 75 % assignment probability to one of the populations were confirmed as mosaics using the structure linkage model. For each of these, the putative source of the recombinant region was determined and the allele was mapped onto a clonalframe genealogy derived from concatenated ST sequences. This enabled the direction and frequency of introgression between the two populations to be established, with 8.3 % of C. coli clade 1 alleles having acquired C. jejuni sequence, compared to 0.5 % for the reciprocal process. Once generated, mosaic genes spread within C. coli clade 1 by a combination of clonal expansion and lateral gene transfer, with some evidence of erosion of the mosaics by reacquisition of C. coli sequence. These observations confirm previous analyses of the exchange of complete housekeeping alleles and extend this work by describing the processes of horizontal gene transfer and subsequent spread within recipient species.

Identification of clinical isolates of Neisseria meningitidis that are resistant to rifampin is important to avoid prophylaxis failure in contacts of patients, but it is hindered by the absence of a breakpoint for resistance, despite many efforts toward standardization. We examined a large number (n = 392) of clinical meningococcal isolates, spanning 25 years (1984 to 2009), that were collected in 11 European countries, Argentina, and the Central African Republic. The collection comprises all clinical isolates with MICs of ≥0.25 mg/liter (n = 161) received by the national reference laboratories for meningococci in the participating countries. Representative isolates displaying rifampin MICs of <0.25 mg/liter were also examined (n = 231). Typing of isolates was performed, and a 660-bp DNA fragment of the rpoB gene was sequenced. Sequences differing by at least one nucleotide were defined as unique rpoB alleles. The geometric mean of the MICs was calculated for isolates displaying the same allele. The clinical isolates displaying rifampin MICs of >1 mg/liter possessed rpoB alleles with nonsynonymous mutations at four critical amino acid residues, D542, H552, S548, and S557, that were absent in the alleles found in all isolates with MICs of ≤1 mg/liter. Rifampin-susceptible isolates could be defined as those with MICs of ≤1 mg/liter. The rpoB allele sequence and isolate data have been incorporated into the PubMLST Neisseria database (http://pubmlst.org/neisseria/). The rifampin-resistant isolates belonged to diverse genetic lineages and were associated with lower levels of bacteremia and inflammatory cytokines in mice. This biological cost may explain the lack of clonal expansion of these isolates.

A scientist in our laboratory was accidentally infected while working with Z5463, a Neisseria meningitidis serogroup A strain. She developed severe symptoms (fever, meningism, purpuric lesions) that fortunately evolved with antibiotic treatment to complete recovery. Pulse-field gel electrophoresis confirmed that the isolate obtained from the blood culture (Z5463BC) was identical to Z5463, more precisely to a fourth subculture of this strain used the week before the contamination (Z5463PI). In order to get some insights into genomic modifications that can occur in vivo, we sequenced these three isolates. All the strains contained a mutated mutS allele and therefore displayed an hypermutator phenotype, consistent with the high number of mutations (SNP, Single Nucleotide Polymorphism) detected in the three strains. By comparing the number of SNP in all three isolates and knowing the number of passages between Z5463 and Z5463PI, we concluded that around 25 bacterial divisions occurred in the human body. As expected, the in vivo passage is responsible for several modifications of phase variable genes. This genomic study has been completed by transcriptomic and phenotypic studies, showing that the blood strain used a different haemoglobin-linked iron receptor (HpuA/B) than the parental strains (HmbR). Different pilin variants were found after the in vivo passage, which expressed different properties of adhesion. Furthermore the deletion of one gene involved in LOS biosynthesis (lgtB) results in Z5463BC expressing a different LOS than the L9 immunotype of Z2491. The in vivo passage, despite the small numbers of divisions, permits the selection of numerous genomic modifications that may account for the high capacity of the strain to disseminate.

The common zoonotic pathogen Campylobacter coli is an important cause of bacterial gastroenteritis worldwide but its evolution is incompletely understood. Using multilocus sequence type (MLST) data of 7 housekeeping genes from a national survey of Campylobacter in Scotland (2005/6), and a combined population genetic-phylogenetics approach, we investigated the evolutionary history of C. coli. Genealogical reconstruction of isolates from clinical infection, farm animals and the environment, revealed a three-clade genetic structure. The majority of farm animal, and all disease causing genotypes belonged to a single clade (clade 1) which had comparatively low synonymous sequence diversity, little deep branching genetic structure, and a higher number of shared alleles providing evidence of recent clonal decent. Calibration of the rate of molecular evolution, based on within-species genetic variation, estimated a more rapid rate of evolution than in traditional estimates. This placed the divergence of the clades at less than 2500 years ago, consistent with the introduction of an agricultural niche having had an effect upon the evolution of the C. coli clades. Attribution of clinical isolate genotypes to source, using an asymmetric island model, confirmed that strains from chicken and ruminants, and not pigs or turkeys, are the principal source of human C. coli infection. Taken together these analyses are consistent with an evolutionary scenario describing the emergence of agriculture-associated C. coli lineage that is an important human pathogen.

Background

The opportunities for bacterial population genomics that are being realised by the application of parallel nucleotide sequencing require novel bioinformatics platforms. These must be capable of the storage, retrieval, and analysis of linked phenotypic and genotypic information in an accessible, scalable and computationally efficient manner.

Results

The Bacterial Isolate Genome Sequence Database (BIGSDB) is a scalable, open source, web-accessible database system that meets these needs, enabling phenotype and sequence data, which can range from a single sequence read to whole genome data, to be efficiently linked for a limitless number of bacterial specimens. The system builds on the widely used mlstdbNet software, developed for the storage and distribution of multilocus sequence typing (MLST) data, and incorporates the capacity to define and identify any number of loci and genetic variants at those loci within the stored nucleotide sequences. These loci can be further organised into 'schemes' for isolate characterisation or for evolutionary or functional analyses. Isolates and loci can be indexed by multiple names and any number of alternative schemes can be accommodated, enabling cross-referencing of different studies and approaches. LIMS functionality of the software enables linkage to and organisation of laboratory samples. The data are easily linked to external databases and fine-grained authentication of access permits multiple users to participate in community annotation by setting up or contributing to different schemes within the database. Some of the applications of BIGSDB are illustrated with the genera Neisseria and Streptococcus.

The BIGSDB source code and documentation are available at http://pubmlst.org/software/database/bigsdb/.

Conclusions

Genomic data can be used to characterise bacterial isolates in many different ways but it can also be efficiently exploited for evolutionary or functional studies. BIGSDB represents a freely available resource that will assist the broader community in the elucidation of the structure and function of bacteria by means of a population genomics approach.

Chlamydia comprises a group of obligate intracellular bacterial parasites responsible for a variety of diseases in humans and animals, including several zoonoses. Chlamydia trachomatis causes diseases such as trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Chlamydia pneumoniae is a common cause of community-acquired respiratory tract infections. Chlamydia psittaci, causing zoonotic pneumonia in humans, is usually hosted by birds, while Chlamydia abortus, causing abortion and fetal death in mammals, including humans, is mainly hosted by goats and sheep. We used multi-locus sequence typing to asses the population structure of Chlamydia. In total, 132 Chlamydia isolates were analyzed, including 60 C. trachomatis, 18 C. pneumoniae, 16 C. abortus, 34 C. psittaci and one of each of C. pecorum, C. caviae, C. muridarum and C. felis. Cluster analyses utilizing the Neighbour-Joining algorithm with the maximum composite likelihood model of concatenated sequences of 7 housekeeping fragments showed that C. psittaci 84/2334 isolated from a parrot grouped together with the C. abortus isolates from goats and sheep. Cluster analyses of the individual alleles showed that in all instances C. psittaci 84/2334 formed one group with C. abortus. Moving 84/2334 from the C. psittaci group to the C. abortus group resulted in a significant increase in the number of fixed differences and elimination of the number of shared mutations between C. psittaci and C. abortus. C. psittaci M56 from a muskrat branched separately from the main group of C. psittaci isolates. C. psittaci genotypes appeared to be associated with host species. The phylogentic tree of C. psittaci did not follow that of its host bird species, suggesting host species jumps. In conclusion, we report for the first time an association between C. psittaci genotypes with host species.

A robust high-throughput multilocus sequence typing (MLST) scheme for Clostridium difficile was developed and validated using a diverse collection of 50 reference isolates representing 45 different PCR ribotypes and 102 isolates from recent clinical samples. A total of 49 PCR ribotypes were represented overall. All isolates were typed by MLST and yielded 40 sequence types (STs). A web-accessible database was set up (http://pubmlst.org/cdifficile/) to facilitate the dissemination and comparison of C. difficile MLST genotyping data among laboratories. MLST and PCR ribotyping were similar in discriminatory abilities, having indices of discrimination of 0.90 and 0.92, respectively. Some STs corresponded to a single PCR ribotype (32/40), other STs corresponded to multiple PCR ribotypes (8/40), and, conversely, the PCR ribotype was not always predictive of the ST. The total number of variable nucleotide sites in the concatenated MLST sequences was 103/3,501 (2.9%). Concatenated MLST sequences were used to construct a neighbor-joining tree which identified four phylogenetic groups of STs and one outlier (ST-11; PCR ribotype 078). These groups apparently correlate with clades identified previously by comparative genomics. The MLST scheme was sufficiently robust to allow direct genotyping of C. difficile in total stool DNA extracts without isolate culture. The direct (nonculture) MLST approach may prove useful as a rapid genotyping method, potentially benefiting individual patients and informing hospital infection control.

In response to epidemic levels of serogroup B meningococcal disease in Cuba during the 1980s, the VA-MENGOC-BC vaccine was developed and introduced into the National Infant Immunization Program in 1991. Since then the incidence of meningococcal disease in Cuba has returned to the low levels recorded before the epidemic. A total of 420 Neisseria meningitidis strains collected between 1983 and 2005 in Cuba were analyzed by multilocus sequence typing (MLST). The set of strains comprised 167 isolated from disease cases and 253 obtained from healthy carriers. By MLST analysis, 63 sequence types (STs) were identified, and 32 of these were reported to be a new ST. The Cuban isolates were associated with 12 clonal complexes; and the most common were ST-32 (246 isolates), ST-53 (86 isolates), and ST-41/44 (36 isolates). This study also showed that the application of VA-MENGOC-BC, the Cuban serogroup B and C vaccine, reduced the frequency and diversity of hypervirulent clonal complexes ST-32 (vaccine serogroup B type-strain) and ST-41/44 and also affected other lineages. Lineages ST-8 and ST-11 were no longer found during the postvaccination period. The vaccine also affected the genetic composition of the carrier-associated meningococcal isolates. The number of carrier isolates belonging to hypervirulent lineages decreased significantly after vaccination, and ST-53, a sequence type common in carriers, became the predominant ST.

Most studies of bacterial pathogen populations have been based on isolates collected from individuals with disease, or their contacts, over short time periods. For commensal organisms that occasionally cause disease, such as Neisseria meningitidis, however, the analysis of isolates from long-term asymptomatic carriage is necessary to elucidate their evolution and population structure. Here, we use mathematical models to analyse the structuring and dynamics of three vaccine-candidate antigens among carried meningococcal isolates collected over nearly 30 years in the Czech Republic. The data indicate that stable combinations of antigenic alleles were maintained over this time period despite evidence for high rates of recombination, consistent with theoretical models in which strong immune selection can maintain non-overlapping combinations of antigenic determinants in the presence of recombination. We contrast this antigenic structure with the overlapping but relatively stable combinations of the housekeeping genes observed among the same isolates, and use a novel network approach to visualize these relationships.

Streptococcus oralis is a member of the normal human oral microbiota, capable of opportunistic pathogenicity; like related oral streptococci, it exhibits appreciable phenotypic and genetic variation. A multilocus sequence typing (MLST) scheme for S. oralis was developed and the resultant data analysed to examine the population structure of the species. Analysis of 113 isolates, confirmed as belonging to the S. oralis/mitis group by 16S rRNA gene sequencing, characterized the population as highly diverse and undergoing inter- and intra-species recombination with a probable clonal complex structure. ClonalFrame analysis of these S. oralis isolates along with examples of Streptococcus pneumoniae, Streptococcus mitis and Streptococcus pseudopneumoniae grouped the named species into distinct, coherent populations and did not support the clustering of S. pseudopneumoniae with S. mitis as reported previously using distance-based methods. Analysis of the individual loci suggested that this discrepancy was due to the possible hybrid nature of S. pseudopneumoniae. The data are available on the public MLST website (http://pubmlst.org/soralis/).

The continued evolution of bacterial pathogens has major implications for both human and animal disease, but the exchange of genetic material between host-restricted pathogens is rarely considered. Streptococcus equi subspecies equi (S. equi) is a host-restricted pathogen of horses that has evolved from the zoonotic pathogen Streptococcus equi subspecies zooepidemicus (S. zooepidemicus). These pathogens share approximately 80% genome sequence identity with the important human pathogen Streptococcus pyogenes. We sequenced and compared the genomes of S. equi 4047 and S. zooepidemicus H70 and screened S. equi and S. zooepidemicus strains from around the world to uncover evidence of the genetic events that have shaped the evolution of the S. equi genome and led to its emergence as a host-restricted pathogen. Our analysis provides evidence of functional loss due to mutation and deletion, coupled with pathogenic specialization through the acquisition of bacteriophage encoding a phospholipase A2 toxin, and four superantigens, and an integrative conjugative element carrying a novel iron acquisition system with similarity to the high pathogenicity island of Yersinia pestis. We also highlight that S. equi, S. zooepidemicus, and S. pyogenes share a common phage pool that enhances cross-species pathogen evolution. We conclude that the complex interplay of functional loss, pathogenic specialization, and genetic exchange between S. equi, S. zooepidemicus, and S. pyogenes continues to influence the evolution of these important streptococci.

Author Summary

Streptococci colonize a diverse range of animals and tissues, and this association is normally harmless. Occasionally some strains of streptococci have an increased ability to cause disease that is often associated with a reduction in the ability to colonize and the acquisition of new genes, which enable the strain to inhabit a new niche. S. equi is the causative agent of strangles, one of the most frequently diagnosed and feared infectious diseases of horses, which is believed to have evolved from the closely related and usually harmless S. zooepidemicus. We aim to understand the mechanisms by which S. equi causes disease by studying and comparing the genomes of these different strains. Here we identify specific genes that have been lost and gained by S. equi, which may have directed its transition from colonizer to invader. Several of the novel genes acquired by S. equi have also been identified in strains of the closely related bacterium S. pyogenes that are associated with increased morbidity and mortality in humans. Our research highlights the role of genetic exchange in cross-species bacterial evolution and argues that the evolution of human pathogens cannot be considered in isolation.

The meningococcal Opa proteins play an important role in pathogenesis by mediating invasion of human cells. The aim of this investigation was to determine whether carried and disease-associated meningococci possess different Opa repertoires and whether the diversity of these proteins is associated with clinical severity of disease. Opa repertoires in 227 disease-associated meningococci, isolated in the United Kingdom over a period of 6 years, were compared to the repertoires in 190 asymptomatically carried meningococci isolated in the United Kingdom from a contemporary, nonepidemic period. Multidimensional scaling (MDS) was employed to investigate the association between Opa repertoires and multilocus sequence typing (MLST) genotypes. Associations with clinical severity were also analyzed statistically. High levels of diversity were observed in opa alleles, variable regions, and repertoires, and MDS revealed that MLST genotypes were strongly associated with particular Opa repertoires. Individual Opa proteins or repertoires were not associated with clinical severity, though there was a trend toward an association with the opaD locus. Meningococcal Opa repertoire is strongly linked to MLST genotype irrespective of epidemiological sampling and therefore correlates with invasiveness. It is not, however, strongly associated with severity of meningococcal disease.

The opa genes of the Gram negative bacterium Neisseria meningitidis encode Opacity-associated outer membrane proteins whose role is to promote adhesion to the human host tissue during colonisation and invasion. Each meningococcus contains 3–4 opa loci, each of which may be occupied by one of a large number of alleles. We analysed the Opa repertoire structure in a large, well-characterised collection of asymptomatically carried meningococci. Our data show an association between Opa repertoire and meningococcal lineages similar to that observed previously for meningococci isolated from cases of invasive disease. Furthermore, these Opa repertoires exhibit discrete, non-overlapping structure at a population level, and yet low within-repertoire diversity. These data are consistent with the predictions of a mathematical model of strong immune selection upon a system where identical alleles may occupy different loci.

Author Summary

Neisseria meningitidis is a globally important pathogen that causes 2,000–3,000 cases of invasive meningococcal disease annually in the United Kingdom. The meningococcal Opa proteins are important in mediating adhesion to and invasion of human tissues, and are important for evasion of the host immune response. They are encoded by a repertoire of 3–4 genomic loci in each meningococcus and exhibit high levels of sequence diversity. Here we analyzed the Opa repertoires of a large, well-characterised, asymptomatically carried meningococcal isolate collection. We found that the Opa repertoires were specific to individual meningococcal genotypes, similar to that observed in isolates from cases of invasive disease. These repertoires exhibited discrete, non-overlapping structure at a population level, and yet low within-repertoire diversity. These data were consistent with the predictions of a mathematical model of strong immune selection, suggesting that the collective immune response of the host population shapes the antigenic diversity of the meningococcal Opa repertoire. This study provides new insights into Opa-mediated meningococcal pathogenesis and the effect of host population immunity on the biodiversity and population structure of bacterial pathogens. These data may also have implications for the design of new meningococcal vaccines based on surface proteins.

Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, PenI) harbor alterations in the penA gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3′ half of penA was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different penA alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101 penA alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the PenI phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work (http://neisseria.org/nm/typing/penA). These data argue for the use of penA sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among Neisseria species.

Background

Various typing methods have been developed for Neisseria gonorrhoeae, but none provide the combination of discrimination, reproducibility, portability, and genetic inference that allows the analysis of all aspects of the epidemiology of this pathogen from a single data set. Multilocus sequence typing (MLST) has been used successfully to characterize the related organisms Neisseria meningitidis and Neisseria lactamica. Here, the same seven locus Neisseria scheme was used to characterize a diverse collection of N. gonorrhoeae isolates to investigate whether this method would allow differentiation among isolates, and to distinguish these three species.

Results

A total of 149 gonococcal isolates were typed and submitted to the Neisseria MLST database. Although relatively few (27) polymorphisms were detected among the seven MLST loci, a total of 66 unique allele combinations (sequence types, STs), were observed, a number comparable to that seen among isolate collections of the more diverse meningococcus. Patterns of genetic variation were consistent with high levels of recombination generating this diversity. There was no evidence for geographical structuring among the isolates examined, with isolates collected in Liverpool, UK, showing levels of diversity similar to a global collection of isolates. There was, however, evidence that populations of N. meningitidis, N. gonorrhoeae and N. lactamica were distinct, with little support for frequent genetic recombination among these species, with the sequences from the gdh locus alone grouping the species into distinct clusters.

Conclusion

The seven loci Neisseria MLST scheme was readily adapted to N. gonorrhoeae isolates, providing a highly discriminatory typing method. In addition, these data permitted phylogenetic and population genetic inferences to be made, including direct comparisons with N. meningitidis and N. lactamica. Examination of these data demonstrated that alleles were rarely shared among the three species. Analysis of variation at a single locus, gdh, provided a rapid means of identifying misclassified isolates and determining whether mixed cultures were present.

The eubacterial genus Wolbachia comprises one of the most abundant groups of obligate intracellular bacteria, and it has a host range that spans the phyla Arthropoda and Nematoda. Here we developed a multilocus sequence typing (MLST) scheme as a universal genotyping tool for Wolbachia. Internal fragments of five ubiquitous genes (gatB, coxA, hcpA, fbpA, and ftsZ) were chosen, and primers that amplified across the major Wolbachia supergroups found in arthropods, as well as other divergent lineages, were designed. A supplemental typing system using the hypervariable regions of the Wolbachia surface protein (WSP) was also developed. Thirty-seven strains belonging to supergroups A, B, D, and F obtained from singly infected hosts were characterized by using MLST and WSP. The number of alleles per MLST locus ranged from 25 to 31, and the average levels of genetic diversity among alleles were 6.5% to 9.2%. A total of 35 unique allelic profiles were found. The results confirmed that there is a high level of recombination in chromosomal genes. MLST was shown to be effective for detecting diversity among strains within a single host species, as well as for identifying closely related strains found in different arthropod hosts. Identical or similar allelic profiles were obtained for strains harbored by different insect species and causing distinct reproductive phenotypes. Strains with similar WSP sequences can have very different MLST allelic profiles and vice versa, indicating the importance of the MLST approach for strain identification. The MLST system provides a universal and unambiguous tool for strain typing, population genetics, and molecular evolutionary studies. The central database for storing and organizing Wolbachia bacterial and host information can be accessed at http://pubmlst.org/wolbachia/.

The opacity (Opa) proteins mediate a variety of interactions between the bacterium Neisseria meningitidis and its human host. These interactions are thought to be of central importance in both the asymptomatic colonization of the nasopharynx and the sporadic occurrence of meningococcal disease. The receptor specificities of a limited number of Opa protein variants have been explored, but the high level of amino acid sequence diversity among variants has complicated the assignment of specific roles to individual Opa variants or combinations of variants. In addition, the distribution of Opa protein variants among diverse meningococci, information that is potentially informative for studies of Opa function, is poorly understood. A systematic survey of the genetic diversity in the four opa gene loci in each of 77 meningococcal isolates was undertaken. These isolates were representative of the seven hyperinvasive meningococcal clonal complexes that caused the majority of meningococcal disease over the last 50 years. Consistent with previous studies, a high level of sequence diversity was observed among the opa genes and the proteins that they encoded; however, particular sets of Opa protein variants were consistently associated with each of the clonal complexes over time periods often spanning decades and during global spread. These observations were consistent with the postulate that particular combinations of Opa proteins confer fitness advantages to individual clonal complexes and have implications for studies of Opa function and the inclusion of Opa proteins in novel meningococcal vaccines.

Background

Bacterial typing schemes based on the sequences of genes encoding surface antigens require databases that provide a uniform, curated, and widely accepted nomenclature of the variants identified. Due to the differences in typing schemes, imposed by the diversity of genes targeted, creating these databases has typically required the writing of one-off code to link the database to a web interface. Here we describe agdbNet, widely applicable web database software that facilitates simultaneous BLAST querying of multiple loci using either nucleotide or peptide sequences.

Results

Databases are described by XML files that are parsed by a Perl CGI script. Each database can have any number of loci, which may be defined by nucleotide and/or peptide sequences. The software is currently in use on at least five public databases for the typing of Neisseria meningitidis, Campylobacter jejuni and Streptococcus equi and can be set up to query internal isolate tables or suitably-configured external isolate databases, such as those used for multilocus sequence typing. The style of the resulting website can be fully configured by modifying stylesheets and through the use of customised header and footer files that surround the output of the script.

Conclusion

The software provides a rapid means of setting up customised Internet antigen sequence databases. The flexible configuration options enable typing schemes with differing requirements to be accommodated.

Intramammary infection with Streptococcus uberis is a common cause of bovine mastitis throughout the world. Several procedures to differentiate S. uberis isolates have been proposed. However, all are prone to interlaboratory variation, and none is suitable for the description of the population structure. We describe here the development of a multilocus sequence typing (MLST) scheme for S. uberis to help address these issues. The sequences of seven housekeeping gene fragments from each of 160 United Kingdom milk isolates of S. uberis were determined. Between 5 and 17 alleles were obtained per locus, giving the potential to discriminate between 1.3 × 107 sequence types. In this study, 57 sequence types (STs) were identified. Statistical comparisons between the maximum-likelihood trees constructed by using the seven housekeeping gene fragments showed that the congruence was no better than that between each tree and trees of random topology, indicating there had been significant recombination within these loci. The population contained one major lineage (designated the ST-5 complex). This dominated the population, containing 24 STs and representing 112 isolates. The other 33 STs were not assigned to any clonal complex. All of the isolates in the ST-5 lineage carried hasA, a gene that is essential for capsule production. There was no clear association between ST or clonal complex and disease. The S. uberis MLST system offers researchers a valuable tool that allows further investigation of the population biology of this organism and insights into the epidemiology of this disease on a global scale.