Dam inactivation in Neisseria meningitidis: prevalence among diverse hyperinvasive lineages

BACKGROUND: DNA adenine methyltransferase (Dam) activity is absent in many, but not all, disease isolates of Neisseria meningitidis, as a consequence of the insertion of a restriction endonuclease-encoding gene, the 'dam replacing gene' (drg) at the dam locus. Here, we report the results of a survey to assess the prevalence of drg in a globally representative panel of disease-associated meningococci. RESULTS: Of the known meningococcal hyper-invasive lineages investigated, drg was absent in all representatives of the ST-8 and ST-11 clonal complexes tested, but uniformly present in the representatives of the other hyper-invasive lineages present in the isolate collection (the ST-1, ST-4, ST-5, ST-32 and ST-41/44 clonal complexes). The patterns of sequence diversity observed in drg were consistent with acquisition of this gene from a source organism with a different G+C content, at some time prior to the emergence of present-day meningococcal clonal complexes, followed by spread through the meningococcal population by horizontal genetic exchange. During this spread a number of alleles have arisen by mutation and intragenic recombination. CONCLUSION: These findings are consistent with the idea that possession of the drg gene may contribute to the divergence observed among meningococcal clonal complexes, but does not have a direct mechanistic involvement in virulence

The domestication of the probiotic bacterium Lactobacillus acidophilus

Lactobacillus acidophilus is a Gram-positive lactic acid bacterium that has had widespread historical use in the dairy industry and more recently as a probiotic. Although L. acidophilus has been designated as safe for human consumption, increasing commercial regulation and clinical demands for probiotic validation has resulted in a need to understand its genetic diversity. By drawing on large, well-characterised collections of lactic acid bacteria, we examined L. acidophilus isolates spanning 92 years and including multiple strains in current commercial use. Analysis of the whole genome sequence data set (34 isolate genomes) demonstrated L. acidophilus was a low diversity, monophyletic species with commercial isolates essentially identical at the sequence level. Our results indicate that commercial use has domesticated L. acidophilus with genetically stable, invariant strains being consumed globally by the human population

A gene-by-gene population genomics platform: de novo assembly, annotation and genealogical analysis of 108 representative Neisseria meningitidis genomes

Background: Highly parallel,‘second generation’ sequencing technologies have rapidly expanded the number of bacterial whole genome sequences available for study, permitting the emergence of the discipline of population genomics. Most of these data are publically available as unassembled short-read sequence files that require extensive processing before they can be used for analysis. The provision of data in a uniform format, which can be easily assessed for quality, linked to provenance and phenotype and used for analysis, is therefore necessary. Results: The performance of de novo short-read assembly followed by automatic annotation using the pubMLST. orgNeisseriadatabase was assessed and evaluated for 108 diverse, representative, and well-characterisedNeisseria meningitidisisolates. High-quality sequences were obtained for >99% of known meningococcal genes among the de novoassembled genomes and four resequenced genomes and less than 1% of reassembled genes had sequence discrepancies or misassembled sequences. A core genome of 1600 loci, present in at least 95% of the population, was determined using the Genome Comparator tool. Genealogical relationships compatible with, but at a higher resolution than, those identified by multilocus sequence typing were obtained with core genome comparisons and ribosomal protein gene analysis which revealed a genomic structure for a number of previously described phenotypes. This unified system for cataloguing Neisseria genetic variation in the genome was implemented and used for multiple analyses and the data are publically available in the PubMLST Neisseria database. Conclusions: The de novo assembly, combined with automated gene-by-gene annotation, generates high quality draft genomes in which the majority of protein-encoding genes are present with high accuracy. The approach catalogues diversity efficiently, permits analyses of a single genome or multiple genome comparisons, and is a practical approach to interpreting WGS data for large bacterial population samples. The method generates novel insights into the biology of the meningococcus and improves our understanding of the whole population structure, not just disease causing lineages.</p

There are three major Neisseria gonorrhoeae β-lactamase plasmid variants which are associated with specific lineages and carry distinct TEM alleles

Neisseria gonorrhoeae is a significant threat to global health with an estimated incidence of over 80 million cases each year and high levels of antimicrobial resistance. The gonococcal β-lactamase plasmid, pbla, carries the TEM β-lactamase, which requires only one or two amino acid changes to become an extended-spectrum β-lactamase (ESBL); this would render last resort treatments for gonorrhoea ineffective. Although pbla is not mobile, it can be transferred by the conjugative plasmid, pConj, found in N. gonorrhoeae. Seven variants of pbla have been described previously, but little is known about their frequency or distribution in the gonococcal population. We characterised sequences of pbla variants and devised a typing scheme, Ng_pblaST that allows their identification from whole genome short-read sequences. We implemented Ng_pblaST to assess the distribution of pbla variants in 15 532 gonococcal isolates. This demonstrated that only three pbla variants commonly circulate in gonococci, which together account for >99 % of sequences. The pbla variants carry different TEM alleles and are prevalent in distinct gonococcal lineages. Analysis of 2758 pbla-containing isolates revealed the co-occurrence of pbla with certain pConj types, indicating co-operativity between pbla and pConj variants in the spread of plasmid-mediated AMR in N. gonorrhoeae. Understanding the variation and distribution of pbla is essential for monitoring and predicting the spread of plasmid-mediated β-lactam resistance in N. gonorrhoeae

Long-term evolution of antigen repertoires among carried meningococci

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

Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications [version 1; referees: 2 approved]

The PubMLST.org website hosts a collection of open-access, curated databases that integrate population sequence data with provenance and phenotype information for over 100 different microbial species and genera.  Although the PubMLST website was conceived as part of the development of the first multi-locus sequence typing (MLST) scheme in 1998 the software it uses, the Bacterial Isolate Genome Sequence database (BIGSdb, published in 2010), enables PubMLST to include all levels of sequence data, from single gene sequences up to and including complete, finished genomes.  Here we describe developments in the BIGSdb software made from publication to June 2018 and show how the platform realises microbial population genomics for a wide range of applications.  The system is based on the gene-by-gene analysis of microbial genomes, with each deposited sequence annotated and curated to identify the genes present and systematically catalogue their variation.  Originally intended as a means of characterising isolates with typing schemes, the synthesis of sequences and records of genetic variation with provenance and phenotype data permits highly scalable (whole genome sequence data for tens of thousands of isolates) means of addressing a wide range of functional questions, including: the prediction of antimicrobial resistance; likely cross-reactivity with vaccine antigens; and the functional activities of different variants that lead to key phenotypes.  There are no limitations to the number of sequences, genetic loci, allelic variants or schemes (combinations of loci) that can be included, enabling each database to represent an expanding catalogue of the genetic variation of the population in question.  In addition to providing web-accessible analyses and links to third-party analysis and visualisation tools, the BIGSdb software includes a RESTful application programming interface (API) that enables access to all the underlying data for third-party applications and data analysis pipelines

Changes in serogroup and genotype prevalence among carried meningococci in the United Kingdom during vaccine implementation.

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

Development of the Pneumococcal Genome Library, a core genome multilocus sequence typing scheme, and a taxonomic life identification number barcoding system to investigate and define pneumococcal population structure

Investigating the genomic epidemiology of major bacterial pathogens is integral to understanding transmission, evolution, colonisation, disease, antimicrobial resistance, and vaccine impact. Furthermore, the recent accumulation of large numbers of whole genome sequences for many bacterial species enhances the development of robust genome-wide typing schemes to define the overall bacterial population structure and lineages within it. Using previously published data, we developed the Pneumococcal Genome Library (PGL), a curated dataset of 30,976 genomes and contextual data for carriage and disease pneumococci recovered between 1916-2018 in 82 countries. We leveraged the size and diversity of the PGL to develop a core genome multilocus sequence typing (cgMLST) scheme comprised of 1,222 loci. Finally, using multilevel single-linkage clustering, we stratified pneumococci into hierarchical clusters based on allelic similarity thresholds, and defined these with a taxonomic life identification number (LIN) barcoding system. The PGL, cgMLST scheme, and LIN barcodes represent a high-quality genomic resource and fine-scale clustering approaches for the analysis of pneumococcal populations, which support the genomic epidemiology and surveillance of this leading global pathogen. Impact statement Many thousands of pneumococcal genomes are available in the public domain, and this creates opportunities for the scientific community to re-use existing data; however, these data are most useful when the contextual data (provenance and phenotype) are also linked to the genomes. Therefore, we created a curated, open-access database in PubMLST that contained nearly 31,000 published pneumococcal genomes and the corresponding contextual data for each genome. This large and diverse pneumococcal database was used to create a novel cgMLST scheme and multilevel clustering method to define genetic lineages with high resolution and a standardised nomenclature. These are open-access resources for all to use and provide a unified framework for the characterisation of global pneumococcal populations

Multi Locus Sequence Typing of Chlamydia Reveals an Association between Chlamydia psittaci Genotypes and Host Species

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