Imperial College London

Professor Angela Brueggemann

Faculty of MedicineDepartment of Medicine

Visiting Professor
 
 
 
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a.brueggemann

 
 
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Commonwealth BuildingHammersmith Campus

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Summary

 

Publications

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77 results found

Quirk SJ, Haraldsson G, Hjálmarsdóttir MÁ, van Tonder AJ, Hrafnkelsson B, Bentley SD, Haraldsson Á, Erlendsdóttir H, Brueggemann AB, Kristinsson KGet al., 2019, Vaccination of Icelandic children with the 10-valent pneumococcal vaccine leads to a significant herd effect among adults in Iceland, Journal of Clinical Microbiology, Vol: 57, ISSN: 0095-1137

The introduction of pneumococcal conjugate vaccines (PCV) into childhood vaccination programs has reduced carriage of vaccine serotypes and pneumococcal disease. The 10-valent PCV was introduced in Iceland in 2011. The aim of this study was to determine PCV impact on the prevalence of serotypes, genetic lineages and antimicrobial-resistant pneumococci isolated from the lower respiratory tract (LRT) of adults.Pneumococci isolated during 2009-2017 at the Landspitali University Hospital, were included (n=797). The hospital serves almost three-quarters of the Icelandic population. Isolates were serotyped and tested for antimicrobial susceptibility, and the genome of every other isolate from 2009-2014 was sequenced (n=275). Serotypes and multilocus sequence types (STs) were extracted from the genome data. Three study periods were defined: 2009-2011 (PreVac), 2012-2014 (PostVac-I) and 2015-2017 (PostVac-II).The total number of isolates and vaccine-type (VT) pneumococci decreased from PreVac to PostVac-II (n=314 vs n=230; p=0.002 and n=170 vs n=33; p<0.001, respectively), but nonvaccine-type (NVT) pneumococci increased among adults 18-64 years old (n=56 vs n=114; p=0.008). Serotype 19F decreased in the PostVac-II period: these isolates were all multidrug-resistant (MDR) and were members of the Taiwan19F-14 PMEN lineage. Serotype 6A decreased among adults ≥65 years old PostVac-II (p=0.037), while serotype 6C increased (p=0.021) and most serotype 6C isolates were MDR. Non-encapsulated S. pneumoniae (NESp) increased among adults 18-64 years old PostVac-II and the majority were MDR (p=0.028).An overall reduction in the number of LRT samples, pneumococcal-positive cultures, and significant changes in the serotype distribution became evident within four years thereby demonstrating a significant herd effect.

JOURNAL ARTICLE

van Tonder AJ, Bray JE, Jolley KA, van Rensburg MJ, Quirk SJ, Haraldsson G, Maidens MCJ, Bentley SD, Haraldsson A, Erlendsdottir H, Kristinsson KG, Brueggemann ABet al., 2019, Genomic analyses of >3,100 nasopharyngeal pneumococci revealed significant differences between pneumococci recovered in four different geographical regions, Frontiers in Microbiology, Vol: 10, ISSN: 1664-302X

Understanding the structure of a bacterial population is essential in order to understand bacterial evolution. Estimating the core genome (those genes common to all, or nearly all, strains of a species) is a key component of such analyses. The size and composition of the core genome varies by dataset, but we hypothesized that the variation between different collections of the same bacterial species would be minimal. To investigate this, we analyzed the genome sequences of 3,118 pneumococci recovered from healthy individuals in Reykjavik (Iceland), Southampton (United Kingdom), Boston (United States), and Maela (Thailand). The analyses revealed a “supercore” genome (genes shared by all 3,118 pneumococci) of 558 genes, although an additional 354 core genes were shared by pneumococci from Reykjavik, Southampton, and Boston. Overall, the size and composition of the core and pan-genomes among pneumococci recovered in Reykjavik, Southampton, and Boston were similar. Maela pneumococci were distinctly different in that they had a smaller core genome and larger pan-genome. The pan-genome of Maela pneumococci contained several >25 Kb sequence regions (flanked by pneumococcal genes) that were homologous to genomic regions found in other bacterial species. Overall, our work revealed that some subsets of the global pneumococcal population are highly heterogeneous, and our hypothesis was rejected. This is an important finding in terms of understanding genetic variation among pneumococci and is also an essential point of consideration before generalizing the findings from a single dataset to the wider pneumococcal population.

JOURNAL ARTICLE

Quirk SJ, Haraldsson G, Erlendsdóttir H, Hjálmarsdóttir MÁ, van Tonder AJ, Hrafnkelsson B, Sigurðsson S, Bentley SD, Haraldsson Á, Brueggemann AB, Kristinsson KGet al., 2018, Effect of vaccination on pneumococci isolated from the nasopharynx of healthy children and the middle ear of children with otitis media in Iceland, Journal of Clinical Microbiology, Vol: 56, ISSN: 0095-1137

Vaccination with pneumococcal conjugate vaccines (PCVs) disrupts the pneumococcal population. Our aim was to determine the impact of the 10-valent PCV on the serotypes, genetic lineages and antimicrobial susceptibility of pneumococci isolated from children in Iceland. Pneumococci were collected between 2009-2017 from the nasopharynx of healthy children attending 15 day-care centres and from the middle ear (ME) of children with acute otitis media from the greater Reykjavik capital area. Isolates were serotyped and tested for antimicrobial susceptibility. Whole genome sequencing (WGS) was performed on alternate isolates from 2009-2014 and serotypes and multilocus sequence types (STs) were extracted from the WGS data. Two study periods were defined: 2009-2011 (PreVac) and 2012-2017 (PostVac). The overall nasopharyngeal carriage rate was similar between the two periods (67.3% PreVac and 61.5% PostVac, p=0.090). Vaccine-type (VT) pneumococci decreased and nonvaccine-type (NVT) pneumococci (serotypes 6C, 15A, 15B/C, 21, 22F, 23A, 23B, 35F, 35B) significantly increased post-PCV introduction in different age strata. The total number of pneumococci recovered from ME samples significantly decreased, as did the proportion that were VTs, although NVT pneumococci (6C, 15B/C, 23A and 23B) increased significantly. Most serotype 6C pneumococci were multidrug-resistant (MDR). Serotype 19F was the predominant serotype associated with ME and it significantly decreased post-PCV introduction: these isolates were predominantly MDR and of the Taiwan19F-14 PMEN lineage. Overall, the nasopharyngeal carriage rate remained constant and the number of ME-associated pneumococci decreased significantly post-PCV introduction; however, there was a concomitant and statistically significant shift from VTs to NVTs in both collections of pneumococci.

JOURNAL ARTICLE

Rezaei Javan R, van Tonder A, King J, Harrold C, Brueggemann ABet al., 2018, Genome sequencing reveals a large and diverse repertoire of antimicrobial peptides, Frontiers in Microbiology, Vol: 9, ISSN: 1664-302X

Competition among bacterial members of the same ecological niche is mediated by bacteriocins: antimicrobial peptides produced by bacterial species to kill other bacteria. Bacteriocins are also promising candidates for novel antimicrobials. Streptococcus pneumoniae (the “pneumococcus”) is a leading cause of morbidity and mortality worldwide and a frequent colonizer of the human nasopharynx. Here, 14 newly discovered bacteriocin gene clusters were identified among >6,200 pneumococcal genomes. The molecular epidemiology of the bacteriocin clusters was investigated using a large global and historical pneumococcal dataset dating from 1916. These analyses revealed extraordinary bacteriocin diversity among pneumococci and the majority of bacteriocin clusters were also found in other streptococcal species. Genomic hotspots for the integration of different bacteriocin gene clusters were discovered. Experimentally, bacteriocin genes were transcriptionally active when the pneumococcus was under stress and when two strains were co-cultured in broth. These findings reveal much more diversity among bacterial defense mechanisms than previously appreciated, which fundamentally broaden our understanding of bacteriocins relative to intraspecies and interspecies nasopharyngeal competition and bacterial population structure.

JOURNAL ARTICLE

Kurioka A, van Wilgenburg B, Javan RR, Hoyle R, van Tonder AJ, Harrold CL, Leng T, Howson LJ, Shepherd D, Cerundolo V, Brueggemann AB, Klenerman Pet al., 2018, Diverse Streptococcus pneumoniae strains drive a MAIT cell response through MR1-dependent and cytokine-driven pathways, Journal of Infectious Diseases, Vol: 217, Pages: 988-999, ISSN: 0022-1899

Mucosal Associated Invariant T (MAIT) cells represent an innate T-cell population which can recognize ligands generated by the microbial riboflavin synthesis pathway, presented via the major-histocompatibility-complex (MHC) class I-related molecule MR1. Streptococcus pneumoniae (the 'pneumococcus') is a major human pathogen that is also associated with commensal carriage, thus host control at the mucosal interface is critical. The recognition of pneumococci by MAIT cells has not been defined, nor have the genomics and transcriptomics of the riboflavin operon. We observed robust recognition of pneumococci by MAIT cells, using both MR1-dependent and independent pathways. The pathway used was dependent on the antigen-presenting cell. The riboflavin operon was highly conserved across a range of 571 pneumococci from 39 countries dating back to 1916, and different versions of the riboflavin operon were also identified in related Streptococcus species. These data indicate an important functional relationship between MAIT cells and pneumococci.

JOURNAL ARTICLE

Rezaei Javan R, van Tonder AJ, King JP, Harrold CL, Brueggemann ABet al., 2017, Genome sequencing reveals that Streptococcus pneumoniae possesses a large and diverse repertoire of antimicrobial toxins, Publisher: Cold Spring Harbor Laboratory

<jats:p>Streptococcus pneumoniae ('pneumococcus') is a leading cause of morbidity and mortality worldwide and a frequent coloniser of the nasopharynx. Competition among bacterial members of the nasopharynx is believed to be mediated by bacteriocins: antimicrobial toxins produced by bacteria to inhibit growth of other bacteria. Bacteriocins are also promising candidates for novel antimicrobials. Here, 14 newly-discovered bacteriocin gene clusters were identified among &gt;6,200 pneumococcal genomes. The molecular epidemiology of the bacteriocin clusters was investigated using a large global and historical pneumococcal dataset. The analyses revealed extraordinary bacteriocin diversity among pneumococci and the majority of bacteriocin clusters were also found in other streptococcal species. Genomic hotspots for the integration of bacteriocin genes were discovered. Experimentally, bacteriocin genes were transcriptionally active when the pneumococcus was under stress and when two strains were competing in broth co-culture. These findings fundamentally expand our understanding of bacteriocins relative to intraspecies and interspecies nasopharyngeal competition.</jats:p>

WORKING PAPER

Brueggemann AB, Harrold CL, Javan RR, van Tonder AJ, McDonnell AJ, Edwards BAet al., 2017, Pneumococcal prophages are diverse, but not without structure or history, SCIENTIFIC REPORTS, Vol: 7, ISSN: 2045-2322

Bacteriophages (phages) infect many bacterial species, but little is known about the diversity of phages among the pneumococcus, a leading global pathogen. The objectives of this study were to determine the prevalence, diversity and molecular epidemiology of prophages (phage DNA integrated within the bacterial genome) among pneumococci isolated over the past 90 years. Nearly 500 pneumococcal genomes were investigated and RNA sequencing was used to explore prophage gene expression. We revealed that every pneumococcal genome contained prophage DNA. 286 full-length/putatively full-length pneumococcal prophages were identified, of which 163 have not previously been reported. Full-length prophages clustered into four major groups and every group dated from the 1930–40 s onward. There was limited evidence for genes shared between prophage clusters. Prophages typically integrated in one of five different sites within the pneumococcal genome. 72% of prophages possessed the virulence genes pblA and/or pblB. Individual prophages and the host pneumococcal genetic lineage were strongly associated and some prophages persisted for many decades. RNA sequencing provided clear evidence of prophage gene expression. Overall, pneumococcal prophages were highly prevalent, demonstrated a structured population, possessed genes associated with virulence, and were expressed under experimental conditions. Pneumococcal prophages are likely to play a more important role in pneumococcal biology and evolution than previously recognised.

JOURNAL ARTICLE

van Tonder AJ, Bray JE, Quirk SJ, Haraldsson G, Jolley KA, Maiden MC, Hoffmann S, Bentley SD, Haraldsson Á, Erlendsdóttir H, Kristinsson KG, Brueggemann ABet al., 2016, Putatively novel serotypes and the potential for reduced vaccine effectiveness: capsular locus diversity revealed among 5405 pneumococcal genomes., Microbial Genomics, Vol: 2, ISSN: 2057-5858

The pneumococcus is a leading global pathogen and a key virulence factor possessed by the majority of pneumococci is an antigenic polysaccharide capsule ('serotype'), which is encoded by the capsular (cps) locus. Approximately 100 different serotypes are known, but the extent of sequence diversity within the cps loci of individual serotypes is not well understood. Investigating serotype-specific sequence variation is crucial to the design of sequence-based serotyping methodology, understanding pneumococcal conjugate vaccine (PCV) effectiveness and the design of future PCVs. The availability of large genome datasets makes it possible to assess population-level variation among pneumococcal serotypes and in this study 5405 pneumococcal genomes were used to investigate cps locus diversity among 49 different serotypes. Pneumococci had been recovered between 1916 and 2014 from people of all ages living in 51 countries. Serotypes were deduced bioinformatically, cps locus sequences were extracted and variation was assessed within the cps locus, in the context of pneumococcal genetic lineages. Overall, cps locus sequence diversity varied markedly: low to moderate diversity was revealed among serogroups/types 1, 3, 7, 9, 11 and 22; whereas serogroups/types 6, 19, 23, 14, 15, 18, 33 and 35 displayed high diversity. Putative novel and/or hybrid cps loci were identified among all serogroups/types apart from 1, 3 and 9. This study demonstrated that cps locus sequence diversity varied widely between serogroups/types. Investigation of the biochemical structure of the polysaccharide capsule of major variants, particularly PCV-related serotypes and those that appear to be novel or hybrids, is warranted.

JOURNAL ARTICLE

van Tonder AJ, Bray JE, Roalfe L, White R, Zancolli M, Quirk SJ, Haraldsson G, Jolley KA, Maiden MCJ, Bentley SD, Haraldsson A, Erlendsdottir H, Kristinsson KG, Goldblatt D, Brueggemann ABet al., 2016, Correction for van Tonder et al., Genomics reveals the worldwide distribution of multidrug-resistant serotype 6E Pneumococci, Journal of Clinical Microbiology, Vol: 54, ISSN: 0095-1137

The pneumococcus is a leading pathogen infecting children and adults. Safe, effective vaccines exist, and they work by inducing antibodies to the polysaccharide capsule (unique for each serotype) that surrounds the cell; however, current vaccines are limited by the fact that only a few of the nearly 100 antigenically distinct serotypes are included in the formulations. Within the serotypes, serogroup 6 pneumococci are a frequent cause of serious disease and common colonizers of the nasopharynx in children. Serotype 6E was first reported in 2004 but was thought to be rare; however, we and others have detected serotype 6E among recent pneumococcal collections. Therefore, we analyzed a diverse data set of ∼1,000 serogroup 6 genomes, assessed the prevalence and distribution of serotype 6E, analyzed the genetic diversity among serogroup 6 pneumococci, and investigated whether pneumococcal conjugate vaccine-induced serotype 6A and 6B antibodies mediate the killing of serotype 6E pneumococci. We found that 43% of all genomes were of serotype 6E, and they were recovered worldwide from healthy children and patients of all ages with pneumococcal disease. Four genetic lineages, three of which were multidrug resistant, described ∼90% of the serotype 6E pneumococci. Serological assays demonstrated that vaccine-induced serotype 6B antibodies were able to elicit killing of serotype 6E pneumococci. We also revealed three major genetic clusters of serotype 6A capsular sequences, discovered a new hybrid 6C/6E serotype, and identified 44 examples of serotype switching. Therefore, while vaccines appear to offer protection against serotype 6E, genetic variants may reduce vaccine efficacy in the longer term because of the emergence of serotypes that can evade vaccine-induced immunity.

JOURNAL ARTICLE

Bogaardt C, van Tonder AJ, Brueggemann AB, 2015, Genomic analyses of pneumococci reveal a wide diversity of bacteriocins - including pneumocyclicin, a novel circular bacteriocin, BMC Genomics, Vol: 16, ISSN: 1471-2164

Background: One of the most important global pathogens infecting all age groups is Streptococcus pneumoniae(the ‘pneumococcus’). Pneumococci reside in the paediatric nasopharynx, where they compete for space andresources, and one competition strategy is to produce a bacteriocin (antimicrobial peptide or protein) to attackother bacteria and an immunity protein to protect against self-destruction. We analysed a collection of 336 diversepneumococcal genomes dating from 1916 onwards, identified bacteriocin cassettes, detailed their genetic compositionand sequence diversity, and evaluated the data in the context of the pneumococcal population structure.Results: We found that all genomes maintained a blp bacteriocin cassette and we identified several novel blp cassettesand genes. The composition of the ‘bacteriocin/immunity region’ of the blp cassette was highly variable: one cassettepossessed six bacteriocin genes and eight putative immunity genes, whereas another cassette had only one of each.Both widely-distributed and highly clonal blp cassettes were identified. Most surprisingly, one-third of pneumococcalgenomes also possessed a cassette encoding a novel circular bacteriocin that we called pneumocyclicin, whichshared a similar genetic organisation to well-characterised circular bacteriocin cassettes in other bacterial species.Pneumocyclicin cassettes were mainly of one genetic cluster and largely found among seven major pneumococcalclonal complexes.Conclusions: These detailed genomic analyses revealed a novel pneumocyclicin cassette and a wide variety of blpbacteriocin cassettes, suggesting that competition in the nasopharynx is a complex biological phenomenon.

JOURNAL ARTICLE

van Tonder AJ, Bray JE, Roalfe L, White R, Zancolli M, Quirk SJ, Haraldsson G, Jolley KA, Maiden MCJ, Bentley SD, Haraldsson A, Erlendsdottir H, Kristinsson KG, Goldblatt D, Brueggemann ABet al., 2015, Genomics Reveals the Worldwide Distribution of Multidrug-Resistant Serotype 6E Pneumococci, Journal of Clinical Microbiology, Vol: 53, Pages: 2271-2285, ISSN: 0095-1137

The pneumococcus is a leading pathogen infecting children and adults. Safe, effective vaccines exist, and they work by inducing antibodies to the polysaccharide capsule (unique for each serotype) that surrounds the cell; however, current vaccines are limited by the fact that only a few of the nearly 100 antigenically distinct serotypes are included in the formulations. Within the serotypes, serogroup 6 pneumococci are a frequent cause of serious disease and common colonizers of the nasopharynx in children. Serotype 6E was first reported in 2004 but was thought to be rare; however, we and others have detected serotype 6E among recent pneumococcal collections. Therefore, we analyzed a diverse data set of ∼1,000 serogroup 6 genomes, assessed the prevalence and distribution of serotype 6E, analyzed the genetic diversity among serogroup 6 pneumococci, and investigated whether pneumococcal conjugate vaccine-induced serotype 6A and 6B antibodies mediate the killing of serotype 6E pneumococci. We found that 43% of all genomes were of serotype 6E, and they were recovered worldwide from healthy children and patients of all ages with pneumococcal disease. Four genetic lineages, three of which were multidrug resistant, described ∼90% of the serotype 6E pneumococci. Serological assays demonstrated that vaccine-induced serotype 6B antibodies were able to elicit killing of serotype 6E pneumococci. We also revealed three major genetic clusters of serotype 6A capsular sequences, discovered a new hybrid 6C/6E serotype, and identified 44 examples of serotype switching. Therefore, while vaccines appear to offer protection against serotype 6E, genetic variants may reduce vaccine efficacy in the longer term because of the emergence of serotypes that can evade vaccine-induced immunity.

JOURNAL ARTICLE

Cox AJ, Brueggemann AB, Mitchell T, Brown JSet al., 2015, Progress towards understanding the pathology of the pneumococcus, PNEUMONIA, Vol: 7, Pages: 1-2, ISSN: 2200-6133

JOURNAL ARTICLE

van Tonder AJ, Mistry S, Bray JE, Hill DMC, Cody AJ, Farmer CL, Klugman KP, von Gottberg A, Bentley SD, Parkhill J, Jolley KA, Maiden MCJ, Brueggemann ABet al., 2014, Defining the Estimated Core Genome of Bacterial Populations Using a Bayesian Decision Model, PLOS COMPUTATIONAL BIOLOGY, Vol: 10, ISSN: 1553-734X

The bacterial core genome is of intense interest and the volume of whole genome sequence data in the public domainavailable to investigate it has increased dramatically. The aim of our study was to develop a model to estimate the bacterialcore genome from next-generation whole genome sequencing data and use this model to identify novel genes associatedwith important biological functions. Five bacterial datasets were analysed, comprising 2096 genomes in total. We developeda Bayesian decision model to estimate the number of core genes, calculated pairwise evolutionary distances (p-distances)based on nucleotide sequence diversity, and plotted the median p-distance for each core gene relative to its genomelocation. We designed visually-informative genome diagrams to depict areas of interest in genomes. Case studiesdemonstrated how the model could identify areas for further study, e.g. 25% of the core genes with higher sequencediversity in theCampylobacter jejuniandNeisseria meningitidisgenomes encoded hypothetical proteins. The core gene withthe highest p-distance value inC. jejuniwas annotated in the reference genome as a putative hydrolase, but further workrevealed that it shared sequence homology with beta-lactamase/metallo-beta-lactamases (enzymes that provide resistanceto a range of broad-spectrum antibiotics) and thioredoxin reductase genes (which reduce oxidative stress and are essentialfor DNA replication) in otherC. jejunigenomes. Our Bayesian model of estimating the core genome is principled, easy to useand can be applied to large genome datasets. This study also highlighted the lack of knowledge currently available for manycore genes in bacterial genomes of significant global public health importance.

JOURNAL ARTICLE

Hanieh S, Hamaluba M, Kelly DF, Metz JA, Wyres KL, Fisher R, Pradhan R, Shakya D, Shrestha L, Shrestha A, Joshi A, Habens J, Maharjan BD, Thorson S, Bohler E, Yu L-M, Kelly S, Plested E, John T, Werno AM, Adhikari N, Murdoch DR, Brueggemann AB, Pollard AJet al., 2014, Streptococcus pneumoniae Carriage Prevalence in Nepal: Evaluation of a Method for Delayed Transport of Samples from Remote Regions and Implications for Vaccine Implementation, PLOS ONE, Vol: 9, ISSN: 1932-6203

Background:Pneumococcal disease is a significant cause of morbidity and mortality in young children in Nepal, andcurrently available pneumococcal conjugate vaccines offer moderate coverage of invasive disease isolates.Methods:A prevalence study of children aged 1.5 to 24 months in urban and rural Nepal was conducted. In the urbangroup, nasopharyngeal swabs (NPS) were transported using silica desiccant packages (SDP) with delayed processing (2weeks), or skim-milk-tryptone-glucose-glycerin (STGG) with immediate processing (within 8 hours). Pneumococcalnasopharyngeal carriage prevalence, serogroup/type distribution and isolate genotypes (as defined by multilocus sequencetyping) were determined.Results:1101 children were enrolled into the study: 574 in the urban group and 527 in the rural group. Overall carriageprevalence based on culture from specimens transported and stored in STGG was 58.7% (337/574), compared to 40.9%(235/574) in SDP. There was concordance of detection of pneumococcus in 67% of samples. Using the SDP method,pneumococcal carriage prevalence was higher in the rural population (69.2%; 364/526) compared to the urban population(40.9%; 235/574). Serogroup/type distribution varied with geographical location. Over half of the genotypes identified inboth the urban and rural pneumococcal populations were novel.Conclusion:The combination of delayed culture and transport using SDP underestimates the prevalence of pneumococcalcarriage; however, in remote areas, this method could still provide a useful estimate of carriage prevalence and serogroup/type distribution. Vaccine impact is unpredictable in a setting with novel genotypes and limited serotype coverage asdescribed here. Consequently, continued surveillance of pneumococcal isolates from carriage and disease in Nepali childrenfollowing the planned introduction of pneumococcal conjugate vaccines introduction will be essential.

JOURNAL ARTICLE

Domenech A, Ardanuy C, Grau I, Calatayud L, Pallares R, Fenoll A, Brueggemann AB, Linares Jet al., 2013, Evolution and genetic diversity of the Spain(23F)-ST81 clone causing adult invasive pneumococcal disease in Barcelona (1990-2012), Journal of Antimicrobial Chemotherapy, Vol: 69, Pages: 924-931, ISSN: 0305-7453

Objectives:We aimed to analyse the clinical epidemiology and genetic diversity of invasive pneumococcal disease (IPD) episodes attributed to the Spain23F-ST81 (PMEN1) clone.Methods:Fifty-eight (2.7%) of 2117 invasive pneumococci isolated from adult patients during the 1990–2012 period shared a PFGE pattern related to the PMEN1 clone. The genotype was confirmed by multilocus sequence typing. The pbp2x, pbp1a, pbp2b and pspA genes were PCR-amplified and sequenced. Polymorphisms in the pspC gene were identified by PCR restriction fragment length polymorphism. The presence of transposons with erythromycin and tetracycline resistance determinants was detected by PCR.Results:The prevalence of the PMEN1 clone increased from 0.8% in 1991 to 6.2% in 2001, and decreased to 0% in 2010–12, concomitant with the introduction of the seven-valent pneumococcal conjugate vaccine for children. A total of 93.1% of patients had pneumonia, meningitis or peritonitis; 87.9% of patients had associated underlying diseases, mainly cancer, chronic obstructive pulmonary disease and diabetes. Two closely related sequence types (STs) (ST81, n = 52; ST85, n = 6) were detected, with different serotypes: 23F (n = 42), 19A (n = 9) and 19F (n = 6). All the isolates were resistant to penicillin, co-trimoxazole and chloramphenicol. All the isolates also shared the same pbp1a allele, whereas multiple alleles of pbp2b, pbp2x, pspA and pspC were detected. Of the isolates, 89.7% were tetracycline resistant and 60.3% (n = 35) were macrolide resistant, and resistance was associated with different Tn916-like transposons.Conclusions:Adult IPD caused by this clone was mainly detected in patients with underlying conditions, and genetic variability was observed among PMEN1 isolates collected in our area over the past 20 years.

JOURNAL ARTICLE

Brueggemann AB, Muroki BM, Kulohoma BW, Karani A, Wanjiru E, Morpeth S, Kamau T, Sharif S, Scott JAGet al., 2013, Population Genetic Structure of Streptococcus pneumoniae in Kilifi, Kenya, Prior to the Introduction of Pneumococcal Conjugate Vaccine, PLOS ONE, Vol: 8, ISSN: 1932-6203

Background: The 10-valent pneumococcal conjugate vaccine (PCV10) was introduced in Kenya in 2011.Introduction of any PCV will perturb the existing pneumococcal population structure, thus the aim was to genotypepneumococci collected in Kilifi before PCV10.Methods and Findings: Using multilocus sequence typing (MLST), we genotyped >1100 invasive and carriagepneumococci from children, the largest collection genotyped from a single resource-poor country and reported todate. Serotype 1 was the most common serotype causing invasive disease and was rarely detected in carriage; allserotype 1 isolates were members of clonal complex (CC) 217. There were temporal fluctuations in the majorcirculating sequence types (STs); and although 1-3 major serotype 1, 14 or 23F STs co-circulated annually, the twomajor serotype 5 STs mainly circulated independently. Major STs/CCs also included isolates of serotypes 3, 12F,18C and 19A and each shared ≤2 MLST alleles with STs that circulate widely elsewhere. Major CCs associated withnon-PCV10 serotypes were predominantly represented by carriage isolates, although serotype 19A and 12F CCswere largely invasive and a serotype 10A CC was equally represented by invasive and carriage isolates.Conclusions: Understanding the pre-PCV10 population genetic structure in Kilifi will allow for the detection ofchanges in prevalence of the circulating genotypes and evidence for capsular switching post-vaccine implementation.

JOURNAL ARTICLE

Wyres KL, van Tonder A, Lambertsen LM, Hakenbeck R, Parkhill J, Bentley SD, Brueggemann ABet al., 2013, Evidence of antimicrobial resistance-conferring genetic elements among pneumococci isolated prior to 1974, BMC GENOMICS, Vol: 14, ISSN: 1471-2164

Background:Antimicrobial resistance among pneumococci has greatly increased over the past two to threedecades. Resistance to tetracycline (tet(M)), chloramphenicol (cat) and macrolides (erm(B) and/ormef(A/E)) isgenerally conferred by acquisition of specific genes that are associated with mobile genetic elements, includingthose of the Tn916and Tn5252families. The first tetracycline-, chloramphenicol- and macrolide-resistantpneumococci were detected between 1962 and 1970; however, until now the oldest pneumococcus shown toharbour Tn916and/or Tn5252was isolated in 1974. In this study the genomes of 38 pneumococci isolated prior to1974 were probed for the presence oftet(M),cat, erm(B),mef(A/E) andint(integrase) to indicate the presence ofTn916/Tn5252-like elements.Results:Two Tn916-like,tet(M)-containing, elements were identified among pneumococci dated 1967 and 1968.The former element was highly similar to that of the PMEN1 multidrug-resistant, globally-distributed pneumococcalreference strain, which was isolated in 1984. The latter element was associated with a streptococcal phage. A third,novel genetic element, designated ICESpPN1, was identified in the genome of an isolate dated 1972. ICESpPN1contained a region of similarity to Tn5252, a region of similarity to a pneumococcal pathogenicity island and novellantibiotic synthesis/export-associated genes.Conclusions:These data confirm the existence of pneumococcal Tn916elements in the first decade within whichpneumococcal tetracycline resistance was described. Furthermore, the discovery of ICESpPN1 demonstrates thedynamic variability of pneumococcal genetic elements and is contrasted with the evidence for Tn916stability.

JOURNAL ARTICLE

Domenech A, Ardanuy C, Balsalobre L, Marti S, Calatayud L, De la Campa AG, Brueggemann AB, Linares Jet al., 2012, Pneumococci Can Persistently Colonize Adult Patients with Chronic Respiratory Disease, JOURNAL OF CLINICAL MICROBIOLOGY, Vol: 50, Pages: 4047-4053, ISSN: 0095-1137

JOURNAL ARTICLE

Wyres KL, Lambertsen LM, Croucher NJ, McGee L, von Gottberg A, Linares J, Jacobs MR, Kristinsson KG, Beall BW, Klugman KP, Parkhill J, Hakenbeck R, Bentley SD, Brueggemann ABet al., 2012, Pneumococcal Capsular Switching: A Historical Perspective, JOURNAL OF INFECTIOUS DISEASES, Vol: 207, Pages: 439-449, ISSN: 0022-1899

Background.Changes in serotype prevalence among pneumococcal populations result from both serotypereplacement and serotype (capsular) switching. Temporal changes in serotype distributions are well documented,but the contribution of capsular switching to such changes is unknown. Furthermore, it is unclear to what extentvaccine-induced selective pressures drive capsular switching.Methods.Serotype and multilocus sequence typing data for 426 pneumococci dated from 1937 through 2007 wereanalyzed. Whole-genome sequence data for a subset of isolates were used to investigate capsular switching events.Results.We identified 36 independent capsular switch events, 18 of which were explored in detail with whole-genome sequence data. Recombination fragment lengths were estimated for 11 events and ranged from approximately19.0 kb to≥58.2 kb. Two events took place no later than 1960, and the imported DNA included the capsular locusand the nearby penicillin-binding protein genespbp2xandpbp1a.Conclusions.Capsular switching has been a regular occurrence among pneumococcal populations throughout thepast 7 decades. Recombination of large DNA fragments (>30 kb), sometimes including the capsular locus and penicil-lin-binding protein genes, predated both vaccine introduction and widespread antibiotic use. This type of recombina-tion has likely been an intrinsic feature throughout the history of pneumococcal evolution.

JOURNAL ARTICLE

Wyres KL, Lambertsen LM, Croucher NJ, McGee L, von Gottberg A, Linares J, Jacobs MR, Kristinsson KG, Beall BW, Klugman KP, Parkhill J, Hakenbeck R, Bentley SD, Brueggemann ABet al., 2012, The multidrug-resistant PMEN1 pneumococcus is a paradigm for genetic success, Genome Biology, Vol: 13, ISSN: 1474-7596

Background:Streptococcus pneumoniae, also called the pneumococcus, is a major bacterial pathogen. Since its introduction in the 1940s, penicillin has been the primary treatment for pneumococcal diseases. Penicillin resistance rapidly increased among pneumococci over the past 30 years, and one particular multidrug-resistant clone, PMEN1, became highly prevalent globally. We studied a collection of 426 pneumococci isolated between 1937 and 2007 to better understand the evolution of penicillin resistance within this species.Results:We discovered that one of the earliest known penicillin-nonsusceptible pneumococci, recovered in 1967 from Australia, was the likely ancestor of PMEN1, since approximately 95% of coding sequences identified within its genome were highly similar to those of PMEN1. The regions of the PMEN1 genome that differed from the ancestor contained genes associated with antibiotic resistance, transmission and virulence. We also revealed that PMEN1 was uniquely promiscuous with its DNA, donating penicillin-resistance genes and sometimes many other genes associated with antibiotic resistance, virulence and cell adherence to many genotypically diverse pneumococci. In particular, we describe two strains in which up to 10% of the PMEN1 genome was acquired in multiple fragments, some as long as 32 kb, distributed around the recipient genomes. This type of directional genetic promiscuity from a single clone to numerous unrelated clones has, to our knowledge, never before been described.Conclusions:These findings suggest that PMEN1 is a paradigm of genetic success both through its epidemiology and promiscuity. These findings also challenge the existing views about horizontal gene transfer among pneumococci.

JOURNAL ARTICLE

Golubchik T, Brueggemann AB, Street T, Gertz RE, Spencer CCA, Ho T, Giannoulatou E, Link-Gelles R, Harding RM, Beall B, Peto TEA, Moore MR, Donnelly P, Crook DW, Bowden Ret al., 2012, Pneumococcal genome sequencing tracks a vaccine escape variant formed through a multi-fragment recombination event, NATURE GENETICS, Vol: 44, Pages: 352-355, ISSN: 1061-4036

Streptococcus pneumoniae ('pneumococcus') causes an estimated 14.5 million cases of serious disease and 826,000 deaths annually in children under 5 years of age1. The highly effective introduction of the PCV7 pneumococcal vaccine in 2000 in the United States2,3 provided an unprecedented opportunity to investigate the response of an important pathogen to widespread, vaccine-induced selective pressure. Here, we use array-based sequencing of 62 isolates from a US national monitoring program to study five independent instances of vaccine escape recombination4, showing the simultaneous transfer of multiple and often large (up to at least 44 kb) DNA fragments. We show that one such new strain quickly became established, spreading from east to west across the United States. These observations clarify the roles of recombination and selection in the population genomics of pneumococcus and provide proof of principle of the considerable value of combining genomic and epidemiological information in the surveillance and enhanced understanding of infectious diseases.

JOURNAL ARTICLE

Harrison OB, Brueggemann AB, Caugant DA, van der Ende A, Frosch M, Gray S, Heuberger S, Krizova P, Olcen P, Slack M, Taha M-K, Maiden MCJet al., 2011, Molecular typing methods for outbreak detection and surveillance of invasive disease caused by Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, a review, MICROBIOLOGY-SGM, Vol: 157, Pages: 2181-2195, ISSN: 1350-0872

JOURNAL ARTICLE

Beall BW, Gertz RE, Hulkower RL, Whitney CG, Moore MR, Brueggemann ABet al., 2011, Shifting Genetic Structure of Invasive Serotype 19A Pneumococci in the United States, JOURNAL OF INFECTIOUS DISEASES, Vol: 203, Pages: 1360-1368, ISSN: 0022-1899

JOURNAL ARTICLE

Obando I, Munoz-Almagro C, Arroyo LA, Tarrago D, Sanchez-Tatay D, Moreno-Perez D, Dhillon SS, Esteva C, Hernandez-Bou S, Garcia-Garcia JJ, Hausdorff WP, Brueggemann ABet al., 2008, Pediatric parapneumonic empyema, Spain, EMERGING INFECTIOUS DISEASES, Vol: 14, Pages: 1390-1397, ISSN: 1080-6040

Pediatric parapneumonic empyema (PPE) has been increasing in several countries including Spain. Strepto-coccus pneumoniae is a major PPE pathogen; however, antimicrobial pretreatment before pleural fl uid (PF) sam-pling frequently results in negative diagnostic cultures, thus greatly underestimating the contribution of pneumo-cocci, especially pneumococci susceptible to antimicrobial agents, to PPE. The study aim was to identify the sero-types and genotypes that cause PPE by using molecular diagnostics and relate these data to disease incidence and severity. A total of 208 children with PPE were pro-spectively enrolled; blood and PF samples were collected. Pneumococci were detected in 79% of culture-positive and 84% of culture-negative samples. All pneumococci were genotyped by multilocus sequence typing. Serotypes were determined for 111 PPE cases; 48% were serotype 1, of 3 major genotypes previously circulating in Spain. Variance in patient complication rates was statistically signifi cant by serotype. The recent PPE increase is principally due to nonvaccine serotypes, especially the highly invasive serotype 1.

JOURNAL ARTICLE

Sanchez-Tatay D, Arroyo LA, Tarrago D, Lirola MJ, Porras A, Fenoll A, Hausdorff WP, Brueggemann AB, Obando Iet al., 2008, Antibiotic susceptibility and molecular epidemiology of nasopharyngeal pneumococci from Spanish children, CLINICAL MICROBIOLOGY AND INFECTION, Vol: 14, Pages: 797-801, ISSN: 1198-743X

JOURNAL ARTICLE

Moore MR, Gertz RE, Woodbury RL, Barkocy-Gallagher GA, Schaffner W, Lexau C, Gershman K, Reingold A, Farley M, Harrison LH, Hadler JL, Bennett NM, Thomas AR, McGee L, Pilishvili T, Brueggemann AB, Whitney CG, Jorgensen JH, Beall Bet al., 2008, Population snapshot of emergent streptococcus pneumoniae serotype 19A in the United States, 2005, 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Publisher: UNIV CHICAGO PRESS, Pages: 1016-1027, ISSN: 0022-1899

CONFERENCE PAPER

Foster D, Knox K, Walker AS, Griffiths DT, Moore H, Haworth E, Peto T, Brueggemann AB, Crook DWet al., 2008, Invasive pneumococcal disease: epidemiology in children and adults prior to implementation of the conjugate vaccine in the Oxfordshire region, England, JOURNAL OF MEDICAL MICROBIOLOGY, Vol: 57, Pages: 480-487, ISSN: 0022-2615

JOURNAL ARTICLE

Brueggemann AB, Pai R, Crook DW, Beall Bet al., 2007, Vaccine escape recombinants emerge after pneumococcal vaccination in the united states, PLOS PATHOGENS, Vol: 3, Pages: 1628-1636, ISSN: 1553-7366

The heptavalent pneumococcal conjugate vaccine (PCV7) was introduced in the United States (US) in 2000 and hassignificantly reduced invasive pneumococcal disease; however, the incidence of nonvaccine serotype invasive disease,particularly due to serotype 19A, has increased. The serotype 19A increase can be explained in part by expansion of agenotype that has been circulating in the US prior to vaccine implementation (and other countries since at least 1990),but also by the emergence of a novel‘‘vaccine escape recombinant’’pneumococcal strain. This strain has a genotypethat previously was only associated with vaccine serotype 4, but now expresses a nonvaccine serotype 19A capsule.Based on prior evidence for capsular switching by recombination at the capsular locus, the genetic event that resultedin this novel serotype/genotype combination might be identifiable from the DNA sequence of individual pneumococcalstrains. Therefore, the aim of this study was to characterise the putative recombinational event(s) at the capsular locusthat resulted in the change from a vaccine to a nonvaccine capsular type. Sequencing the capsular locus flankingregions of 51 vaccine escape (progeny), recipient, and putative donor pneumococci revealed a 39 kb recombinationalfragment, which included the capsular locus, flanking regions, and two adjacent penicillin-binding proteins, and thusresulted in a capsular switch and penicillin nonsusceptibility in a single genetic event. Since 2003, 37 such vaccineescape strains have been detected, some of which had evolved further. Furthermore, two new types of serotype 19Avaccine escape strains emerged in 2005. To our knowledge, this is the first time a single recombinational event hasbeen documented in vivo that resulted in both a change of serotype and penicillin nonsusceptibility. Vaccine escape bygenetic recombination at the capsular locus has the potential to reduce PCV7 effectiveness in the longer term.

JOURNAL ARTICLE

Jefferies JMC, Johnston CHG, Kirkham L-AS, Cowan GJM, Ross KS, Smith A, Clarke SC, Brueggemann AB, George RC, Pichon B, Pluschke G, Pflueger V, Mitchell TJet al., 2007, Presence of nonhemolytic pneumolysin in serotypes of Streptococcus pneumoniae associated with disease outbreaks, JOURNAL OF INFECTIOUS DISEASES, Vol: 196, Pages: 936-944, ISSN: 0022-1899

JOURNAL ARTICLE

Chung A, Perera R, Brueggemann AB, Elamin AE, Harnden A, Mayon-White R, Smith S, Crook DW, Mant Det al., 2007, Effect of antibiotic prescribing on antibiotic resistance in individual children in primary care: prospective cohort study, BRITISH MEDICAL JOURNAL, Vol: 335, Pages: 429-431, ISSN: 0959-8146

JOURNAL ARTICLE

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