Duccio Medini

Microbiology in the post-genomic era

Genomics has revolutionized every aspect of microbiology. Now, 13 years after the first bacterial genome was sequenced, it is important to pause and consider what has changed in microbiology research as a consequence of genomics. In this article, we review the evolving field of bacterial typing and the genomic technologies that enable comparative analysis of multiple genomes and the metagenomes of complex microbial environments, and address the implications of the genomic era for the future of microbiology.

Adjuvanted H5N1 vaccine induces early CD4+ T cell response that predicts long-term persistence of protective antibody levels

Immune responses to vaccination are tested in clinical trials. This process usually requires years especially when immune memory and persistence are analyzed. Markers able to quickly predict the immune response would be very useful, particularly when dealing with emerging diseases that require a rapid response, such as avian influenza. To address this question we vaccinated healthy adults at days 1, 22, and 202 with plain or MF59-adjuvanted H5N1 subunit vaccines and tested both cell-mediated and antibody responses up to day 382. Only the MF59-H5N1 vaccine induced high titers of neutralizing antibodies, a large pool of memory H5N1-specific B lymphocytes, and H5-CD4+ T cells broadly reactive with drifted H5. The CD4+ response was dominated by IL-2+ IFN-γ− IL-13− T cells. Remarkably, a 3-fold increase in the frequency of virus-specific total CD4+ T cells, measurable after 1 dose, accurately predicted the rise of neutralizing antibodies after booster immunization and their maintenance 6 months later. We suggest that CD4+ T cell priming might be used as an early predictor of the immunogenicity of prepandemic vaccines.

Predicted strain coverage of a meningococcal multicomponent vaccine (4CMenB) in Europe: a qualitative and quantitative assessment.

BACKGROUND A novel multicomponent vaccine against meningococcal capsular group B (MenB) disease contains four major components: factor-H-binding protein, neisserial heparin binding antigen, neisserial adhesin A, and outer-membrane vesicles derived from the strain NZ98/254. Because the public health effect of the vaccine, 4CMenB (Novartis Vaccines and Diagnostics, Siena, Italy), is unclear, we assessed the predicted strain coverage in Europe. METHODS We assessed invasive MenB strains isolated mainly in the most recent full epidemiological year in England and Wales, France, Germany, Italy, and Norway. Meningococcal antigen typing system (MATS) results were linked to multilocus sequence typing and antigen sequence data. To investigate whether generalisation of coverage applied to the rest of Europe, we also assessed isolates from the Czech Republic and Spain. FINDINGS 1052 strains collected from July, 2007, to June, 2008, were assessed from England and Wales, France, Germany, Italy, and Norway. All MenB strains contained at least one gene encoding a major antigen in the vaccine. MATS predicted that 78% of all MenB strains would be killed by postvaccination sera (95% CI 63-90, range of point estimates 73-87% in individual country panels). Half of all strains and 64% of covered strains could be targeted by bactericidal antibodies against more than one vaccine antigen. Results for the 108 isolates from the Czech Republic and 300 from Spain were consistent with those for the other countries. INTERPRETATION MATS analysis showed that a multicomponent vaccine could protect against a substantial proportion of invasive MenB strains isolated in Europe. Monitoring of antigen expression, however, will be needed in the future. FUNDING Novartis Vaccines and Diagnostics.

Neisseria meningitidis is structured in clades associated with restriction modification systems that modulate homologous recombination

Molecular data on a limited number of chromosomal loci have shown that the population of Neisseria meningitidis (Nm), a deadly human pathogen, is structured in distinct lineages. Given that the Nm population undergoes substantial recombination, the mechanisms resulting in the evolution of these lineages, their persistence in time, and the implications for the pathogenicity of the bacterium are not yet completely understood. Based on whole-genome sequencing, we show that Nm is structured in phylogenetic clades. Through acquisition of specific genes and through insertions and rearrangements, each clade has acquired and remodeled specific genomic tracts, with the potential to impact on the commensal and virulence behavior of Nm. Despite this clear evidence of a structured population, we confirm high rates of detectable recombination throughout the whole Nm chromosome. However, gene conversion events were found to be longer within clades than between clades, suggesting a DNA cleavage mechanism associated with the phylogeny of the species. We identify 22 restriction modification systems, probably acquired by horizontal gene transfer from outside of the species/genus, whose distribution in the different strains coincides with the phylogenetic clade structure. We provide evidence that these clade-associated restriction modification systems generate a differential barrier to DNA exchange consistent with the observed population structure. These findings have general implications for the emergence of lineage structure and virulence in recombining bacterial populations, and they could provide an evolutionary framework for the population biology of a number of other bacterial species that show contradictory population structure and dynamics.

Ten years of pan-genome analyses.

Next generation sequencing technologies have engendered a genome sequence data deluge in public databases. Genome analyses have transitioned from single or few genomes to hundreds to thousands of genomes. Pan-genome analyses provide a framework for estimating the genomic diversity of the dataset at hand and predicting the number of additional whole genomes sequences that would be necessary to fully characterize that diversity. We review recent implementations of the pan-genome approach, its impact and limits, and we propose possible extensions, including analyses at the whole genome multiple sequence alignment level.

Bactericidal antibody against a representative epidemiological meningococcal serogroup B panel confirms that MATS underestimates 4CMenB vaccine strain coverage

BACKGROUND 4CMenB (Bexsero), a vaccine developed against invasive meningococcal disease caused by capsular group B strains (MenB), was recently licensed for use by the European Medicines Agency. Assessment of 4CMenB strain coverage in specific epidemiologic settings is of primary importance to predict vaccination impact on the burden of disease. The Meningococcal Antigen Typing System (MATS) was developed to predict 4CMenB strain coverage, using serum bactericidal antibody assay with human complement (hSBA) data from a diverse panel of strains not representative of any specific epidemiology. OBJECTIVE To experimentally validate the accuracy of MATS-based predictions against strains representative of a specific epidemiologic setting. METHODS AND RESULTS We used a stratified sampling method to identify a representative sample from all MenB disease isolates collected from England and Wales in 2007-2008, tested the strains in the hSBA assay with pooled sera from infant and adolescent vaccinees, and compared these results with MATS. MATS predictions and hSBA results were significantly associated (P=0.022). MATS predicted coverage of 70% (95% CI, 55-85%) was largely confirmed by 88% killing in the hSBA (95% CI, 72-95%). MATS had 78% accuracy and 96% positive predictive value against hSBA. CONCLUSION MATS is a conservative predictor of strain coverage by the 4CMenB vaccine in infants and adolescents.

Comparative genomics of the genus Bifidobacterium

Whole-genome sequencing efforts have revolutionized the study of bifidobacterial genetics and physiology. Unfortunately, the sequence of a single genome does not provide information on bifidobacterial genetic diversity and on how genetic variability supports improved adaptation of these bacteria to the environment of the human gastrointestinal tract (GIT). Analysis of nine genomes from bifidobacterial species showed that such genomes display an open pan-genome structure. Mathematical extrapolation of the data indicates that the genome reservoir available to the bifidobacterial pan-genome consists of more than 5000 genes, many of which are uncharacterized, but which are probably important to provide adaptive abilities pertinent to the human GIT. We also define a core bifidobacterial gene set which will undoubtedly provide a new baseline from which one can examine the evolution of bifidobacteria. Phylogenetic investigation performed on a total of 506 orthologues that are common to nine complete bifidobacterial genomes allowed the construction of a Bifidobacterium supertree which is largely concordant with the phylogenetic tree obtained using 16S rRNA genes. Moreover, this supertree provided a more robust phylogenetic resolution than the 16S rRNA gene-based analysis. This comparative study of the genus Bifidobacterium thus presents a foundation for future functional analyses of this important group of GIT bacteria.

Diversity of Canadian meningococcal serogroup B isolates and estimated coverage by an investigational meningococcal serogroup B vaccine (4CMenB).

BACKGROUND In collaboration with the Canadian Immunization Monitoring Program Active (IMPACT), the National Microbiology Laboratory, the UK Health Protection Agency and Novartis Vaccines, we tested the potential of an investigational 4-component meningococcal B vaccine (4CMenB) to cover Canadian strains circulating from 2006 to 2009. METHODS IMPACT meningococcal surveillance is population based and includes over 50% of Canadian adults and children. All isolates were characterized by Meningococcal Antigen Typing System (MATS) and sequencing for factor H-binding protein (fHbp), Neisseria Heparin Binding Antigen (NHBA) and Neisserial adhesin A (NadA). RESULTS In total, 157 isolates were tested. Overall, 4CMenB MATS predicted strain coverage was 66% (95% CI: 46-78%), with 26%, 29% and 11% of strains covered by one, two and three vaccine antigens, respectively. The coverage of each antigen was as follows: 13% PorA, 1% NadA, 52% fHbp and 51% NHBA. The majority of strains for clonal complex (cc) 41/44 and cc60 were covered by NHBA; the majority of strains for cc269 and cc32 were covered by fHbp and NHBA. Coverage for two prevalent strains (sequence type (ST)-269 and ST-154) was 95% and 100%, respectively. CONCLUSIONS 4CMenB has the potential to protect against a significant proportion of Canadian invasive MenB strains.

Interlaboratory Standardization of the Sandwich Enzyme-Linked Immunosorbent Assay Designed for MATS, a Rapid, Reproducible Method for Estimating the Strain Coverage of Investigational Vaccines

ABSTRACT The meningococcal antigen typing system (MATS) sandwich enzyme-linked immunosorbent assay (ELISA) was designed to measure the immunologic cross-reactivity and quantity of antigens in target strains of a pathogen. It was first used to measure the factor H-binding protein (fHbp), neisserial adhesin A (NadA), and neisserial heparin-binding antigen (NHBA) content of serogroup B meningococcal (MenB) isolates relative to a reference strain, or “relative potency” (RP). With the PorA genotype, the RPs were then used to assess strain coverage by 4CMenB, a multicomponent MenB vaccine. In preliminary studies, MATS accurately predicted killing in the serum bactericidal assay using human complement, an accepted correlate of protection for meningococcal vaccines. A study across seven laboratories assessed the reproducibility of RPs for fHbp, NadA, and NHBA and established qualification parameters for new laboratories. RPs were determined in replicate for 17 MenB reference strains at laboratories A to G. The reproducibility of RPs among laboratories and against consensus values across laboratories was evaluated using a mixed-model analysis of variance. Interlaboratory agreement was very good; the Pearson correlation coefficients, coefficients of accuracy, and concordance correlation coefficients exceeded 99%. The summary measures of reproducibility, expressed as between-laboratory coefficients of variation, were 7.85% (fHbp), 16.51% (NadA), and 12.60% (NHBA). The overall within-laboratory measures of variation adjusted for strain and laboratory were 19.8% (fHbp), 28.8% (NHBA), and 38.3% (NadA). The MATS ELISA was successfully transferred to six laboratories, and a further laboratory was successfully qualified.

Protein Homology Network Families Reveal Step-Wise Diversification of Type III and Type IV Secretion Systems

From the analysis of 251 prokaryotic genomes stored in public databases, the 761,260 deduced proteins were used to reconstruct a complete set of bacterial proteic families. Using the new Overlap algorithm, we have partitioned the Protein Homology Network (PHN), where the proteins are the nodes and the links represent homology relationships. The algorithm identifies the densely connected regions of the PHN that define the families of homologous proteins, here called PHN-Families, recognizing the phylogenetic relationships embedded in the network. By direct comparison with a manually curated dataset, we assessed that this classification algorithm generates data of quality similar to a human expert. Then, we explored the network to identify families involved in the assembly of Type III and Type IV secretion systems (T3SS and T4SS). We noticed that, beside a core of conserved functions (eight proteins for T3SS, seven for T4SS), a variable set of accessory components is always present (one to nine for T3SS, one to five for T4SS). Each member of the core corresponds to a single PHN-Family, while accessory proteins are distributed among different pure families. The PHN-Family classification suggests that T3SS and T4SS have been assembled through a step-wise, discontinuous process, by complementing the conserved core with subgroups of nonconserved proteins. Such genetic modules, independently recruited and probably tuned on specific effectors, contribute to the functional specialization of these organelles to different microenvironments.