Vaccination Drives Changes in Metabolic and Virulence Profiles of Streptococcus pneumoniae.

The bacterial pathogen, Streptococcus pneumoniae (the pneumococcus), is a leading cause of life-threatening illness and death worldwide. Available conjugate vaccines target only a small subset (up to 13) of >90 known capsular serotypes of S. pneumoniae and, since their introduction, increases in non-vaccine serotypes have been recorded in several countries: a phenomenon termed Vaccine Induced Serotype Replacement (VISR). Here, using a combination of mathematical modelling and whole genome analysis, we show that targeting particular serotypes through vaccination can also cause their metabolic and virulence-associated components to transfer through recombination to non-vaccine serotypes: a phenomenon we term Vaccine-Induced Metabolic Shift (VIMS). Our results provide a novel explanation for changes observed in the population structure of the pneumococcus following vaccination, and have important implications for strain-targeted vaccination in a range of infectious disease systems.

Identifying Neisseria species by use of the 50S ribosomal protein L6 (rplF) gene.

The comparison of 16S rRNA gene sequences is widely used to differentiate bacteria; however, this gene can lack resolution among closely related but distinct members of the same genus. This is a problem in clinical situations in those genera, such as Neisseria, where some species are associated with disease while others are not. Here, we identified and validated an alternative genetic target common to all Neisseria species which can be readily sequenced to provide an assay that rapidly and accurately discriminates among members of the genus. Ribosomal multilocus sequence typing (rMLST) using ribosomal protein genes has been shown to unambiguously identify these bacteria. The PubMLST Neisseria database (http://pubmlst.org/neisseria/) was queried to extract the 53 ribosomal protein gene sequences from 44 genomes from diverse species. Phylogenies reconstructed from these genes were examined, and a single 413-bp fragment of the 50S ribosomal protein L6 (rplF) gene was identified which produced a phylogeny that was congruent with the phylogeny reconstructed from concatenated ribosomal protein genes. Primers that enabled the amplification and direct sequencing of the rplF gene fragment were designed to validate the assay in vitro and in silico. Allele sequences were defined for the gene fragment, associated with particular species names, and stored on the PubMLST Neisseria database, providing a curated electronic resource. This approach provides an alternative to 16S rRNA gene sequencing, which can be readily replicated for other organisms for which more resolution is required, and it has potential applications in high-resolution metagenomic studies.

Role of stochastic processes in maintaining discrete strain structure in antigenically diverse pathogen populations.

Many highly diverse pathogen populations appear to exist stably as discrete antigenic types despite evidence of genetic exchange. It has been shown that this may arise as a consequence of immune selection on pathogen populations, causing them to segregate permanently into discrete nonoverlapping subsets of antigenic variants to minimize competition for available hosts. However, discrete antigenic strain structure tends to break down under conditions where there are unequal numbers of allelic variants at each locus. Here, we show that the inclusion of stochastic processes can lead to the stable recovery of discrete strain structure through loss of certain alleles. This explains how pathogen populations may continue to behave as independently transmitted strains despite inevitable asymmetries in allelic diversity of major antigens. We present evidence for this type of structuring across global meningococcal isolates in three diverse antigens that are currently being developed as vaccine components.

Metabolic shift in the emergence of hyperinvasive pandemic meningococcal lineages.

Hyperinvasive lineages of Neisseria meningitidis, which persist despite extensive horizontal genetic exchange, are a major cause of meningitis and septicaemia worldwide. Over the past 50 years one such lineage of meningococci, known as serogroup A, clonal complex 5 (A:cc5), has caused three successive pandemics, including epidemics in sub-Saharan Africa. Although the principal antigens that invoke effective immunity have remained unchanged, distinct A:cc5 epidemic clones have nevertheless emerged. An analysis of whole genome sequence diversity among 153 A:cc5 isolates identified eleven genetic introgression events in the emergence of the epidemic clones, which primarily involved variants of core genes encoding metabolic processes. The acquired DNA was identical to that found over many years in other, unrelated, hyperinvasive meningococci, suggesting that the epidemic clones emerged by acquisition of pre-existing metabolic gene variants, rather than 'virulence' associated or antigen-encoding genes. This is consistent with mathematical models which predict the association of transmission fitness with the emergence and maintenance of virulence in recombining commensal organisms.

Lineage structure of Streptococcus pneumoniae may be driven by immune selection on the groEL heat-shock protein.

Populations of Streptococcus pneumoniae (SP) are typically structured into groups of closely related organisms or lineages, but it is not clear whether they are maintained by selection or neutral processes. Here, we attempt to address this question by applying a machine learning technique to SP whole genomes. Our results indicate that lineages evolved through immune selection on the groEL chaperone protein. The groEL protein is part of the groESL operon and enables a large range of proteins to fold correctly within the physical environment of the nasopharynx, thereby explaining why lineage structure is so stable within SP despite high levels of genetic transfer. SP is also antigenically diverse, exhibiting a variety of distinct capsular serotypes. Associations exist between lineage and capsular serotype but these can be easily perturbed, such as by vaccination. Overall, our analyses indicate that the evolution of SP can be conceptualized as the rearrangement of modular functional units occurring on several different timescales under different pressures: some patterns have locked in early (such as the epistatic interactions between groESL and a constellation of other genes) and preserve the differentiation of lineages, while others (such as the associations between capsular serotype and lineage) remain in continuous flux.

Towards understanding the epidemiology of Neisseria meningitidis in the African meningitis belt: a multi-disciplinary overview.

OBJECTIVES: Neisseria meningitidis is the major cause of seasonal meningitis epidemics in the African meningitis belt. In the changing context of a reduction in incidence of serogroup A and an increase in incidence of serogroups W and C and of Streptococcus pneumoniae, a better understanding of the determinants driving the disease transmission dynamics remains crucial to improving bacterial meningitis control. METHODS: The literature was searched to provide a multi-disciplinary overview of the determinants of meningitis transmission dynamics in the African meningitis belt. RESULTS: Seasonal hyperendemicity is likely predominantly caused by increased invasion rates, sporadic localized epidemics by increased transmission rates, and larger pluri-annual epidemic waves by changing population immunity. Carriage likely involves competition for colonization and cross-immunity. The duration of immunity likely depends on the acquisition type. Major risk factors include dust and low humidity, and presumably human contact rates and co-infections; social studies highlighted environmental and dietary factors, with supernatural explanations. CONCLUSIONS: Efforts should focus on implementing multi-country, longitudinal seroprevalence and epidemiological studies, validating immune markers of protection, and improving surveillance, including more systematic molecular characterizations of the bacteria. Integrating climate and social factors into disease control strategies represents a high priority for optimizing the public health response and anticipating the geographic evolution of the African meningitis belt.

Metabolic competition as a driver of bacterial population structure.

Understanding the processes whereby diversity arises and is maintained in pathogen populations is pivotal for designing disease control interventions. A particular problem is the maintenance of strain structure in bacterial pathogen populations despite frequent genetic exchange. Although several theoretical frameworks have been put forward to explain this widespread phenomenon, few have focused on the role of genes encoding metabolic functions, despite an increasing recognition of their importance in pathogenesis and transmission. In this article, we review the literature for evidence of metabolic niches within the host and discuss theoretical frameworks which examine ecological interactions between metabolic genes. We contend that metabolic competition is an important phenomenon which contributes to the maintenance of population structure and diversity of many bacterial pathogens.

Pharyngeal carriage of Neisseria species in the African meningitis belt.

OBJECTIVES: Neisseria meningitidis, together with the non-pathogenic Neisseria species (NPNs), are members of the complex microbiota of the human pharynx. This paper investigates the influence of NPNs on the epidemiology of meningococcal infection. METHODS: Neisseria isolates were collected during 18 surveys conducted in six countries in the African meningitis belt between 2010 and 2012 and characterized at the rplF locus to determine species and at the variable region of the fetA antigen gene. Prevalence and risk factors for carriage were analyzed. RESULTS: A total of 4694 isolates of Neisseria were obtained from 46,034 pharyngeal swabs, a carriage prevalence of 10.2% (95% CI, 9.8-10.5). Five Neisseria species were identified, the most prevalent NPN being Neisseria lactamica. Six hundred and thirty-six combinations of rplF/fetA_VR alleles were identified, each defined as a Neisseria strain type. There was an inverse relationship between carriage of N. meningitidis and of NPNs by age group, gender and season, whereas carriage of both N. meningitidis and NPNs was negatively associated with a recent history of meningococcal vaccination. CONCLUSION: Variations in the prevalence of NPNs by time, place and genetic type may contribute to the particular epidemiology of meningococcal disease in the African meningitis belt.

Contrasting within- and between-host immune selection shapes Neisseria Opa repertoires.

Pathogen evolution is influenced strongly by the host immune response. Previous studies of the effects of herd immunity on the population structure of directly transmitted, short-lived pathogens have primarily focused on the impact of competition for hosts. In contrast, for long-lived infections like HIV, theoretical work has focused on the mechanisms promoting antigenic variation within the host. In reality, successful transmission requires that pathogens balance both within- and between-host immune selection. The Opa adhesins in the bacterial Neisseria genus provide a unique system to study the evolution of the same antigens across two major pathogens: while N. meningitidis is an airborne, respiratory pathogen colonising the nasopharynx relatively transiently, N. gonorrhoeae can cause sexually transmitted, long-lived infections. We use a simple mathematical model and genomic data to show that trade-offs between immune selection pressures within- and between-hosts can explain the contrasting Opa repertoires observed in meningococci and gonococci.

Methods for identifying Neisseria meningitidis carriers: a multi-center study in the African meningitis belt.

OBJECTIVE: Detection of meningococcal carriers is key to understanding the epidemiology of Neisseria meningitidis, yet no gold standard has been established. Here, we directly compare two methods for collecting pharyngeal swabs to identify meningococcal carriers. METHODS: We conducted cross-sectional surveys of schoolchildren at multiple sites in Africa to compare swabbing the posterior pharynx behind the uvula (U) to swabbing the posterior pharynx behind the uvula plus one tonsil (T). Swabs were cultured immediately and analyzed using molecular methods. RESULTS: One thousand and six paired swab samples collected from schoolchildren in four countries were analyzed. Prevalence of meningococcal carriage was 6.9% (95% CI: 5.4-8.6%) based on the results from both swabs, but the observed prevalence was lower based on one swab type alone. Prevalence based on the T swab or the U swab alone was similar (5.2% (95% CI: 3.8-6.7%) versus 4.9% (95% CI: 3.6-6.4%) respectively (p=0.6)). The concordance between the two methods was 96.3% and the kappa was 0.61 (95% CI: 0.50-0.73), indicating good agreement. CONCLUSIONS: These two commonly used methods for collecting pharyngeal swabs provide consistent estimates of the prevalence of carriage, but both methods misclassified carriers to some degree, leading to underestimates of the prevalence.

Persistence of hyperinvasive meningococcal strain types during global spread as recorded in the PubMLST database.

Neisseria meningitidis is a major cause of septicaemia and meningitis worldwide. Most disease in Europe, the Americas and Australasia is caused by meningococci expressing serogroup B capsules, but no vaccine against this polysaccharide exists. Potential candidates for 'serogroup B substitute' vaccines are outer membrane protein antigens including the typing antigens PorA and FetA. The web-accessible PubMLST database (www.pubmlst.org) was used to investigate the temporal and geographical patterns of associations among PorA and FetA protein variants and lineages defined by combinations of housekeeping genes, known as clonal complexes. The sample contained 3460 isolates with genotypic information from 57 countries over a 74 year period. Although shifting associations among antigen variants and clonal complexes were evident, a subset of strain types associated with several serogroups persisted for decades and proliferated globally. Genetic stability among outer membrane proteins of serogroup A meningococci has been described previously, but here long-lived genetic associations were also observed among meningococci belonging to serogroups B and C. The patterns of variation were consistent with behaviour predicted by models that invoke inter-strain competition mediated by immune selection. There was also substantial geographic and temporal heterogeneity in antigenic repertoires, providing both opportunities and challenges for the design of broad coverage protein-based meningococcal vaccines.