Imperial College London

DrNicholasCroucher

Faculty of MedicineSchool of Public Health

Senior Lecturer in Bacterial Genomics
 
 
 
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Contact

 

+44 (0)20 7594 3820n.croucher

 
 
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Location

 

UG5Norfolk PlaceSt Mary's Campus

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Summary

 

Publications

Publication Type
Year
to

91 results found

D'Aeth JC, van der Linden MPG, McGee L, De Lencastre H, Turner P, Song J-H, Lo SW, Gladstone RA, Sa-Leao R, Ko KS, Hanage WP, Breiman RF, Beall B, Bentley SD, Croucher NJ, GPS Consortiumet al., 2021, The role of interspecies recombinations in the evolution of antibiotic-resistant pneumococci, eLife, Vol: 10, ISSN: 2050-084X

The evolutionary histories of the antibiotic-resistant Streptococcus pneumoniae lineages PMEN3 and PMEN9 were reconstructed using global collections of genomes. In PMEN3, one resistant clade spread worldwide, and underwent 25 serotype switches, enabling evasion of vaccine-induced immunity. In PMEN9, only 9 switches were detected, and multiple resistant lineages emerged independently and circulated locally. In Germany, PMEN9’s expansion correlated significantly with the macrolide:penicillin consumption ratio. These isolates were penicillin sensitive but macrolide resistant, through a homologous recombination that integrated Tn1207.1 into a competence gene, preventing further diversification via transformation. Analysis of a species-wide dataset found 183 acquisitions of macrolide resistance, and multiple gains of the tetracycline-resistant transposon Tn916, through homologous recombination, often originating in other streptococcal species. Consequently, antibiotic selection preserves atypical recom- bination events that cause sequence divergence and structural variation throughout the S. pneumoniae chromosome. These events reveal the genetic exchanges between species normally counter-selected until perturbed by clinical interventions.

Journal article

Gladstone RA, McNally A, Pontinen AK, Tonkin-Hill G, Lees JA, Skyten K, Cleon F, Christensen MOK, Haldorsen BC, Bye KK, Gammelsrud KW, Hjetland R, Kummel A, Larsen HE, Lindemann PC, Lohr IH, Marvik A, Nilsen E, Noer MT, Simonsen GS, Steinbakk M, Tofteland S, Vattoy M, Bentley SD, Croucher NJ, Parkhill J, Johnsen PJ, Samuelsen O, Corander Jet al., 2021, Emergence and dissemination of antimicrobial resistance in Escherichia coli causing bloodstream infections in Norway in 2002-17: a nationwide, longitudinal, microbial population genomic study, The Lancet Microbe, Vol: 2, Pages: E331-E341, ISSN: 2666-5247

BackgroundThe clonal diversity underpinning trends in multidrug resistant Escherichia coli causing bloodstream infections remains uncertain. We aimed to determine the contribution of individual clones to resistance over time, using large-scale genomics-based molecular epidemiology.MethodsThis was a longitudinal, E coli population, genomic, cohort study that sampled isolates from 22 512 E coli bloodstream infections included in the Norwegian surveillance programme on resistant microbes (NORM) from 2002 to 2017. 15 of 22 laboratories were able to share their isolates, and the first 22·5% of isolates from each year were requested. We used whole genome sequencing to infer the population structure (PopPUNK), and we investigated the clade composition of the dominant multidrug resistant clonal complex (CC)131 using genetic markers previously reported for sequence type (ST)131, effective population size (BEAST), and presence of determinants of antimicrobial resistance (ARIBA, PointFinder, and ResFinder databases) over time. We compared these features between the 2002–10 and 2011–17 time periods. We also compared our results with those of a longitudinal study from the UK done between 2001 and 2011.FindingsOf the 3500 isolates requested from the participating laboratories, 3397 (97·1%) were received, of which 3254 (95·8%) were successfully sequenced and included in the analysis. A significant increase in the number of multidrug resistant CC131 isolates from 71 (5·6%) of 1277 in 2002–10 to 207 (10·5%) of 1977 in 2011–17 (p<0·0001), was the largest clonal expansion. CC131 was the most common clone in extended-spectrum β-lactamase (ESBL)-positive isolates (75 [58·6%] of 128) and fluoroquinolone non-susceptible isolates (148 [39·2%] of 378). Within CC131, clade A increased in prevalence from 2002, whereas the global multidrug resistant clade C2 was not observed until 2007. Multiple de-n

Journal article

Croucher N, Harrow G, Lees J, Hanage W, Lipsitch M, Corander J, Colijn Cet al., 2021, Negative frequency-dependent selection and asymmetrical transformation stabilise multi-strain bacterial population structures, The ISME Journal: multidisciplinary journal of microbial ecology, Vol: 15, Pages: 1523-1538, ISSN: 1751-7362

Streptococcus pneumoniae can be divided into many strains, each a distinct set of isolates sharing similar core and accessorygenomes, which co-circulate within the same hosts. Previous analyses suggested the short-term vaccine-associated dynamicsof S. pneumoniae strains may be mediated through multi-locus negative frequency-dependent selection (NFDS), whichmaintains accessory loci at equilibrium frequencies. Long-term simulations demonstrated NFDS stabilised clonally-evolvingmulti-strain populations through preventing the loss of variation through drift, based on polymorphism frequencies,pairwise genetic distances and phylogenies. However, allowing symmetrical recombination between isolates evolving undermulti-locus NFDS generated unstructured populations of diverse genotypes. Replication of the observed data improvedwhen multi-locus NFDS was combined with recombination that was instead asymmetrical, favouring deletion of accessoryloci over insertion. This combination separated populations into strains through outbreeding depression, resulting fromrecombinants with reduced accessory genomes having lower fitness than their parental genotypes. Although simplisticmodelling of recombination likely limited these simulations’ ability to maintain some properties of genomic data asaccurately as those lacking recombination, the combination of asymmetrical recombination and multi-locus NFDS couldrestore multi-strain population structures from randomised initial populations. As many bacteria inhibit insertions into theirchromosomes, this combination may commonly underlie the co-existence of strains within a niche.

Journal article

Lochen A, Croucher N, Anderson R, 2020, Divergent serotype replacement trends and increasing diversity in pneumococcal disease in high income settings reduce the benefit of expanding vaccine valency, Scientific Reports, Vol: 10, ISSN: 2045-2322

Streptococcus pneumoniae is a significant cause of otitis media, pneumonia, and meningitis. Only seven of the approximately 100 serotypes were initially included in the pneumococcal conjugate vaccine (PCV) in 2000 before it was expanded in subsequent years. Although the invasive pneumococcal disease (IPD) incidence due to vaccine serotypes (VT) has declined, partial replacement by non-vaccine serotypes (NVT) was observed following widespread vaccine uptake. We conducted a trend analysis assembling all the available evidence for PCV impact on European, North American and Australian national IPD. Significant effectiveness against VT IPD in infants was observed, although the impact on national IPD incidence varied internationally due to serotype replacement. Currently, NVT serotypes 8, 9N, 15A and 23B are increasing in the countries assessed, although a variety of other NVTs are affecting each country and age group. Despite these common emerging serotypes, there has not been a dominant IPD serotype post-vaccination as there was pre-vaccination (serotype 14) or post-PCV7 (serotype 19A), suggesting that future vaccines with additional serotypes will be less effective at targeting and reducing IPD in global populations than previous PCVs. The rise of diverse NVTs in all settings’ top-ranked IPD-causing serotypes emphasizes the urgent need for surveillance data on serotype distribution and serotype-specific invasiveness post-vaccination to facilitate decision making concerning both expanding current vaccination programmes and increasing vaccine valency.

Journal article

Azarian T, Martinez PP, Arnold BJ, Qiu X, Grant LR, Corander J, Fraser C, Croucher NJ, Hammitt LL, Reid R, Santosham M, Weatherholtz RC, Bentley SD, O'Brien KL, Lipsitch M, Hanage WPet al., 2020, Frequency-dependent selection can forecast evolution in Streptococcus pneumoniae, PLOS BIOLOGY, Vol: 18, ISSN: 1544-9173

Journal article

Zamudio R, Haigh RD, Ralph JD, Croix MDS, Tasara T, Zurfluh K, Kwun MJ, Millard AD, Bentley SD, Croucher NJ, Stephan R, Oggioni MRet al., 2020, Lineage-specific evolution and gene flow inListeria monocytogenesare independent of bacteriophages, ENVIRONMENTAL MICROBIOLOGY, Vol: 22, Pages: 5058-5072, ISSN: 1462-2912

Journal article

Harrow GL, Lees JA, Hanage WP, Lipsitch M, Corander J, Colijn C, Croucher NJet al., 2020, Negative frequency-dependent selection and asymmetrical transformation stabilise multi-strain bacterial population structures, Publisher: Cold Spring Harbor Laboratory

<jats:title>Abstract</jats:title><jats:p><jats:italic>Streptococcus pneumoniae</jats:italic>can be split into multiple strains, each with a characteristic combination of core and accessory genome variation, able to co-circulate and compete within the same hosts. Previous analyses of epidemiological datasets suggested the short-term vaccine-associated dynamics of<jats:italic>S. pneumoniae</jats:italic>strains may be mediated through multi-locus negative frequency-dependent selection (NFDS), acting to maintain accessory loci at equilibrium frequencies. To test whether this model could explain how such multi-strain populations were generated, it was modified to incorporate recombination. The outputs of simulations featuring symmetrical recombination were compared with genomic data on locus frequencies and distributions between genotypes, pairwise genetic distances and tree shape. These demonstrated NFDS prevented the loss of variation through neutral drift, but generated unstructured populations of diverse isolates. Making recombination asymmetrical, favouring deletion of accessory loci over insertion, alongside multi-locus NFDS significantly improved the fit to genomic data. In a population at equilibrium, structuring into multiple strains was stable due to outbreeding depression, resulting from recombinants with reduced accessory genomes having lower fitness than their parental genotypes. As many bacteria inhibit the integration of insertions into their chromosomes, this combination of asymmetrical recombination and multi-locus NFDS may underlie the co-existence of strains within a single ecological niche.</jats:p>

Working paper

Gladstone RA, Lo SW, Goater R, Yeats C, Taylor B, Hadfield J, Lees JA, Croucher NJ, van Tonder AJ, Bentley LJ, Quah FX, Blaschke AJ, Pershing NL, Byington CL, Balaji V, Hryniewicz W, Sigauque B, Ravikumar KL, Almeida SCG, Ochoa TJ, Ho PL, du Plessis M, Ndlangisa KM, Cornick JE, Kwambana-Adams B, Benisty R, Nzenze SA, Madhi SA, Hawkins PA, Pollard AJ, Everett DB, Antonio M, Dagan R, Klugman KP, von Gottberg A, Metcalf BJ, Li Y, Beall BW, McGee L, Breiman RF, Aanensen DM, Bentley SDet al., 2020, Visualizing variation within Global Pneumococcal Sequence Clusters (GPSCs) and country population snapshots to contextualize pneumococcal isolates, MICROBIAL GENOMICS, Vol: 6, ISSN: 2057-5858

Journal article

Lehtinen S, Chewapreecha C, Lees J, Hanage WP, Lipsitch M, Croucher NJ, Bentley SD, Turner P, Fraser C, Mostowy RJet al., 2020, Horizontal gene transfer rate is not the primary determinant of observed antibiotic resistance frequencies in Streptococcus pneumoniae, SCIENCE ADVANCES, Vol: 6, ISSN: 2375-2548

Journal article

Colijn C, Corander J, Croucher NJ, 2020, Designing ecologically optimized pneumococcal vaccines using population genomics, Nature Microbiology, Vol: 5, Pages: 473-485, ISSN: 2058-5276

Streptococcus pneumoniae (the pneumococcus) is a common nasopharyngeal commensal that can cause invasive pneumococcal disease (IPD). Each component of current protein–polysaccharide conjugate vaccines (PCVs) generally induces immunity specific to one of the approximately 100 pneumococcal serotypes, and typically eliminates it from carriage and IPD through herd immunity. Overall carriage rates remain stable owing to replacement by non-PCV serotypes. Consequently, the net change in IPD incidence is determined by the relative invasiveness of the pre- and post-PCV-carried pneumococcal populations. In the present study, we identified PCVs expected to minimize the post-vaccine IPD burden by applying Bayesian optimization to an ecological model of serotype replacement that integrated epidemiological and genomic data. We compared optimal formulations for reducing infant-only or population-wide IPD, and identified potential benefits to including non-conserved pneumococcal carrier proteins. Vaccines were also devised to minimize IPD resistant to antibiotic treatment, despite the ecological model assuming that resistance levels in the carried population would be preserved. We found that expanding infant-administered PCV valency is likely to result in diminishing returns, and that complementary pairs of infant- and adult-administered vaccines could be a superior strategy. PCV performance was highly dependent on the circulating pneumococcal population, further highlighting the advantages of a diversity of anti-pneumococcal vaccination strategies.

Journal article

Pensar J, Puranen S, Arnold B, MacAlasdair N, Kuronen J, Tonkin-Hill G, Pesonen M, Xu Y, Sipola A, Sanchez-Buso L, Lees JA, Chewapreechi C, Bentley SD, Harris SR, Parkhill J, Croucher NJ, Corander Jet al., 2019, Genome-wide epistasis and co-selection study using mutual information, NUCLEIC ACIDS RESEARCH, Vol: 47, ISSN: 0305-1048

Journal article

Dewe TCM, D'Aetht JC, Croucher NJ, 2019, Genomic epidemiology of penicillin-non-susceptible Streptococcus pneumoniae, Microbial Genomics, Vol: 5, Pages: 1-8, ISSN: 2057-5858

Penicillin-non-susceptible Streptococcus pneumoniae (PNSP) were first detected in the 1960s, and are now common worldwide, predominantly through the international spread of a limited number of strains. Extant PNSP are characterized by mosaic pbp2x, pbp2b and pbp1a genes generated by interspecies recombinations, with the extent of these alterations determining the range and concentrations of β-lactams to which the genotype is non-susceptible. The complexity of the genetics underlying these phenotypes has been the subject of both molecular microbiology and genome-wide association and epistasis analyses. Such studies can aid our understanding of PNSP evolution and help improve the already highly-performing bioinformatic methods capable of identifying PNSP from genomic surveillance data.

Journal article

Furi L, Crawford LA, Rangel-Pineros G, Manso AS, Croix MDS, Haigh RD, Kwun MJ, Fjelland KE, Gilfillan GD, Bentley SD, Croucher NJ, Clokie MR, Oggioni MRet al., 2019, Methylation Warfare: Interaction of Pneumococcal Bacteriophages with Their Host, JOURNAL OF BACTERIOLOGY, Vol: 201, ISSN: 0021-9193

Journal article

Kwun M, Oggioni MR, Bentley SD, Fraser C, Croucher Net al., 2019, Synergistic activity of mobile genetic element defences in Streptococcus pneumoniae, Genes, Vol: 10, ISSN: 2073-4425

A diverse set of mobile genetic elements (MGEs) transmit between Streptococcus pneumoniae cells, but many isolates remain uninfected. The best-characterised defences against horizontal transmission of MGEs are restriction-modification systems (RMSs), of which there are two phase-variable examples in S. pneumoniae. Additionally, the transformation machinery has been proposed to limit vertical transmission of chromosomally integrated MGEs. This work describes how these mechanisms can act in concert. Experimental data demonstrate RMS phase variation occurs at a sub-maximal rate. Simulations suggest this may be optimal if MGEs are sometimes vertically inherited, as it reduces the probability that an infected cell will switch between RMS variants while the MGE is invading the population, and thereby undermine the restriction barrier. Such vertically inherited MGEs can be deleted by transformation. The lack of between-strain transformation hotspots at known prophage att sites suggests transformation cannot remove an MGE from a strain in which it is fixed. However, simulations confirmed that transformation was nevertheless effective at preventing the spread of MGEs into a previously uninfected cell population, if a recombination barrier existed between co-colonising strains. Further simulations combining these effects of phase variable RMSs and transformation found they synergistically inhibited MGEs spreading, through limiting both vertical and horizontal transmission.

Journal article

Nasher F, Kwun MJ, Croucher NJ, Heller M, Hathaway LJet al., 2019, Peptide Occurring in Enterobacteriaceae Triggers Streptococcus pneumoniae Cell Death, FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY, Vol: 9, ISSN: 2235-2988

Journal article

De Ste Croix M, Chen Y, Vacca I, Manso AS, Johnston C, Polard P, Kwun MJ, Bentley SD, Croucher NJ, Bayliss CD, Haigh RD, Oggioni MRet al., 2019, Recombination of the phase variable spnIII locus is independent of all known pneumococcal site-specific recombinases, Journal of Bacteriology, Vol: 201, ISSN: 0021-9193

Streptococcus pneumoniae is one of the world's leading bacterial pathogens, causing pneumonia, septicaemia and meningitis. In recent years it has been shown that genetic rearrangements in a type I restriction-modification system (SpnIII) can impact colony morphology and gene expression. By generating a large panel of mutant strains, we have confirmed a previously reported result that the CreX (also known as IvrR and PsrA) recombinase found within the locus is not essential for hsdS inversions. In addition, mutants of homologous recombination pathways also undergo hsdS inversions. In this work we have shown that these genetic rearrangements, which result in different patterns of genome methylation, occur across a wide variety of serotypes and sequence types including two strains (a 19F and a 6B strain) naturally lacking CreX. Our gene expression analysis, by RNAseq, confirm that the level of creX expression is impacted by these genomic rearrangements. In addition, we have shown that the frequency of hsdS recombination is temperature dependent. Most importantly we have demonstrated that the other known pneumococcal site-specific recombinases XerD, XerS and SPD_0921 are not involved in spnIII recombination, suggesting a currently unknown mechanism is responsible for the recombination of these phase variable type I systems.ImportanceStreptococcus pneumoniae is a leading cause of pneumonia, septicaemia and meningitis. The discovery that genetic rearrangements in a type I restriction modification locus can impact gene regulation and colony morphology have led to a new understanding of how this pathogen switches from harmless coloniser to invasive pathogen. These rearrangements, which alter the DNA specificity of the type I restriction modification enzyme, occur across many different pneumococcal serotypes and sequence types, and in the absence of all known pneumococcal site-specific recombinases. This finding suggests that this is a truly global mechanism of pneumococcal

Journal article

Lehtinen S, Chewapreecha C, Lees J, Hanage WP, Lipsitch M, Croucher NJ, Bentley SD, Turner P, Fraser C, Mostowy RJet al., 2019, Horizontal gene transfer rate is not the primary determinant of observed antibiotic resistance frequencies in Streptococcus pneumoniae

<jats:p>The extent to which evolution is constrained by the rate at which horizontal gene transfer (HGT) allows DNA to move between genetic lineages is an open question, which we address in the context of antibiotic resistance in <jats:italic>Streptococcus pneumoniae</jats:italic>. We analyze microbiological, genomic and epidemiological data from the largest-to-date sequenced pneumococcal carriage study in 955 infants from a refugee camp on the Thailand-Myanmar border. Using a unified framework, we simultaneously test prior hypotheses on rates of HGT and a key evolutionary covariate (duration of carriage) as determinants of resistance frequencies. We conclude that in this setting, there is only weak evidence for the rate of HGT playing a role in the evolutionary dynamics of resistance. Instead, observed resistance frequencies are best explained as the outcome of selection acting on a pool of variants, irrespective of the rate at which resistance determinants move between genetic lineages.</jats:p>

Journal article

Lees JA, Ferwerda B, Kremer PHC, Wheeler NE, Seron MV, Croucher NJ, Gladstone RA, Bootsma HJ, Rots NY, Wijmega-Monsuur AJ, Sanders EAM, Trzcinski K, Wyllie AL, Zwinderman AH, van den Berg LH, van Rheenen W, Veldink JH, Harboe ZB, Lundbo LF, de Groot LCPGM, van Schoor NM, van der Velde N, Angquist LH, Sorensen TIA, Nohr EA, Mentzer AJ, Mills TC, Knight JC, du Plessis M, Nzenze S, Weiser JN, Parkhill J, Madhi S, Benfield T, von Gottberg A, van der Ende A, Brouwer MC, Barrett JC, Bentley SD, van de Beek Det al., 2019, Joint sequencing of human and pathogen genomes reveals the genetics of pneumococcal meningitis, NATURE COMMUNICATIONS, Vol: 10, ISSN: 2041-1723

Journal article

Lehtinen S, Blanquart F, Lipsitch M, Fraser C, Bentley SD, Croucher NJ, Lees JA, Turner Pet al., 2019, On the evolutionary ecology of multidrug resistance in bacteria, PLoS Pathogens, Vol: 15, ISSN: 1553-7366

Resistance against different antibiotics appears on the same bacterial strains more oftenthan expected by chance, leading to high frequencies of multidrug resistance. There are multiple explanations for this observation, but these tend to be specific to subsets of antibioticsand/or bacterial species, whereas the trend is pervasive. Here, we consider the questionin terms of strain ecology: explaining why resistance to different antibiotics is often seen onthe same strain requires an understanding of the competition between strains with differentresistance profiles. This work builds on models originally proposed to explain another aspectof strain competition: the stable coexistence of antibiotic sensitivity and resistance observedin a number of bacterial species. We first identify a partial structural similarity in these models: either strain or host population structure stratifies the pathogen population into evolutionarily independent sub-populations and introduces variation in the fitness effect of resistancebetween these sub-populations, thus creating niches for sensitivity and resistance. We thengeneralise this unified underlying model to multidrug resistance and show that models withthis structure predict high levels of association between resistance to different drugs andhigh multidrug resistance frequencies. We test predictions from this model in six bacterialdatasets and find them to be qualitatively consistent with observed trends. The higher thanexpected frequencies of multidrug resistance are often interpreted as evidence that thesestrains are out-competing strains with lower resistance multiplicity. Our work provides analternative explanation that is compatible with long-term stability in resistance frequencies.

Journal article

Gladstone RA, Lo SW, Lees JA, Croucher NJ, van Tonder AJ, Corander J, Page AJ, Marttinen P, Bentley LJ, Ochoa TJ, Ho PL, du Plessis M, Cornick JE, Kwambana-Adams B, Benisty R, Nzenze SA, Madhi SA, Hawkins PA, Everett DB, Antonio M, Dagan R, Klugman KP, von Gottberg A, McGee L, Breiman RF, Bentley SDet al., 2019, International genomic definition of pneumococcal lineages, to contextualise disease, antibiotic resistance and vaccine impact, EBioMedicine, Vol: 43, Pages: 338-346, ISSN: 2352-3964

BackgroundPneumococcal conjugate vaccines have reduced the incidence of invasive pneumococcal disease, caused by vaccine serotypes, but non-vaccine-serotypes remain a concern. We used whole genome sequencing to study pneumococcal serotype, antibiotic resistance and invasiveness, in the context of genetic background.MethodsOur dataset of 13,454 genomes, combined with four published genomic datasets, represented Africa (40%), Asia (25%), Europe (19%), North America (12%), and South America (5%). These 20,027 pneumococcal genomes were clustered into lineages using PopPUNK, and named Global Pneumococcal Sequence Clusters (GPSCs). From our dataset, we additionally derived serotype and sequence type, and predicted antibiotic sensitivity. We then measured invasiveness using odds ratios that relating prevalence in invasive pneumococcal disease to carriage.FindingsThe combined collections (n = 20,027) were clustered into 621 GPSCs. Thirty-five GPSCs observed in our dataset were represented by >100 isolates, and subsequently classed as dominant-GPSCs. In 22/35 (63%) of dominant-GPSCs both non-vaccine serotypes and vaccine serotypes were observed in the years up until, and including, the first year of pneumococcal conjugate vaccine introduction.Penicillin and multidrug resistance were higher (p < .05) in a subset dominant-GPSCs (14/35, 9/35 respectively), and resistance to an increasing number of antibiotic classes was associated with increased recombination (R2 = 0.27 p < .0001). In 28/35 dominant-GPSCs, the country of isolation was a significant predictor (p < .05) of its antibiogram (mean misclassification error 0.28, SD ± 0.13).We detected increased invasiveness of six genetic backgrounds, when compared to other genetic backgrounds expressing the same serotype. Up to 1.6-fold changes in invasiveness odds ratio were observed.InterpretationWe define GPSCs that can be assigned to any pneumococcal genomic dataset, to aid international comparisons. Existing n

Journal article

McNally A, Kallonen T, Connor C, Abudahab K, Aanensen DM, Horner C, Peacock SJ, Parkhill J, Croucher NJ, Corander Jet al., 2019, Diversification of colonization factors in a multidrug-resistant escherichia coli lineage evolving under negative frequency-dependent selection, mBio, Vol: 10, ISSN: 2150-7511

Escherichia coli is a major cause of bloodstream and urinary tract infections globally. The wide dissemination of multidrug-resistant (MDR) strains of extraintestinal pathogenic E. coli (ExPEC) poses a rapidly increasing public health burden due to narrowed treatment options and increased risk of failure to clear an infection. Here, we present a detailed population genomic analysis of the ExPEC ST131 clone, in which we seek explanations for its success as an emerging pathogenic strain beyond the acquisition of antimicrobial resistance (AMR) genes. We show evidence for evolution toward separate ecological niches for the main clades of ST131 and differential evolution of anaerobic metabolism, key colonization, and virulence factors. We further demonstrate that negative frequency-dependent selection acting across accessory loci is a major mechanism that has shaped the population evolution of this pathogen.IMPORTANCE Infections with multidrug-resistant (MDR) strains of Escherichia coli are a significant global public health concern. To combat these pathogens, we need a deeper understanding of how they evolved from their background populations. By understanding the processes that underpin their emergence, we can design new strategies to limit evolution of new clones and combat existing clones. By combining population genomics with modelling approaches, we show that dominant MDR clones of E. coli are under the influence of negative frequency-dependent selection, preventing them from rising to fixation in a population. Furthermore, we show that this selection acts on genes involved in anaerobic metabolism, suggesting that this key trait, and the ability to colonize human intestinal tracts, is a key step in the evolution of MDR clones of E. coli.

Journal article

Mitchell PK, Azarian T, Croucher NJ, Callendrello A, Thompson CM, Pelton S, Lipsitch M, Hanage WPet al., 2019, Population genomics of pneumococcal carriage in Massachusetts children following introduction of PCV-13, Microbial Genomics, Vol: 5, ISSN: 2057-5858

The 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced in the United States in 2010. Using a large paediatric carriage sample collected from shortly after the introduction of PCV-7 to several years after the introduction of PCV-13, we investigate alterations in the composition of the pneumococcal population following the introduction of PCV-13, evaluating the extent to which the post-vaccination non-vaccine type (NVT) population mirrors that from prior to vaccine introduction and the effect of PCV-13 on vaccine type lineages. Draft genome assemblies from 736 newly sequenced and 616 previously published pneumococcal carriage isolates from children in Massachusetts between 2001 and 2014 were analysed. Isolates were classified into one of 22 sequence clusters (SCs) on the basis of their core genome sequence. We calculated the SC diversity for each sampling period as the probability that any two randomly drawn isolates from that period belong to different SCs. The sampling period immediately after the introduction of PCV-13 (2011) was found to have higher diversity than preceding (2007) or subsequent (2014) sampling periods {Simpson’s D 2007: 0.915 [95 % confidence interval (CI) 0.901, 0.929]; 2011:  0.935 [0.927, 0.942]; 2014 :  0.912 [0.901, 0.923]}. Amongst NVT isolates, we found the distribution of SCs in 2011 to be significantly different from that in 2007 or 2014 (Fisher’s exact test P=0.018, 0.0078), but did not find a difference comparing 2007 to 2014 (Fisher’s exact test P=0.24), indicating greater similarity between samples separated by a longer time period than between samples from closer time periods. We also found changes in the accessory gene content of the NVT population between 2007 and 2011 to have been reduced by 2014. Amongst the new serotypes targeted by PCV-13, four were present in our sample. The proportion of our sample composed of PCV-13-only vaccine serotypes 19A, 6C and 7F decreased between 2007 and 2014, but no

Journal article

Lees JA, Harris SR, Tonkin-Hill G, Gladstone RA, Lo SW, Weiser JN, Corander J, Bentley SD, Croucher NJet al., 2019, Fast and flexible bacterial genomic epidemiology with PopPUNK, Genome Research, Vol: 29, Pages: 304-316, ISSN: 1088-9051

The routine use of genomics for disease surveillance provides the opportunity for high-resolution bacterial epidemiology. Current whole-genome clustering and multilocus typing approaches do not fully exploit core and accessory genomic variation, and they cannot both automatically identify, and subsequently expand, clusters of significantly similar isolates in large data sets spanning entire species. Here, we describe PopPUNK (Population Partitioning Using Nucleotide K -mers), a software implementing scalable and expandable annotation- and alignment-free methods for population analysis and clustering. Variable-length k-mer comparisons are used to distinguish isolates' divergence in shared sequence and gene content, which we demonstrate to be accurate over multiple orders of magnitude using data from both simulations and genomic collections representing 10 taxonomically widespread species. Connections between closely related isolates of the same strain are robustly identified, despite interspecies variation in the pairwise distance distributions that reflects species' diverse evolutionary patterns. PopPUNK can process 103-104 genomes in a single batch, with minimal memory use and runtimes up to 200-fold faster than existing model-based methods. Clusters of strains remain consistent as new batches of genomes are added, which is achieved without needing to reanalyze all genomes de novo. This facilitates real-time surveillance with consistent cluster naming between studies and allows for outbreak detection using hundreds of genomes in minutes. Interactive visualization and online publication is streamlined through the automatic output of results to multiple platforms. PopPUNK has been designed as a flexible platform that addresses important issues with currently used whole-genome clustering and typing methods, and has potential uses across bacterial genetics and public health research.

Journal article

Cremers AJH, Mobegi FM, van der Gaast-de Jongh C, van Weert M, van Opzeeland FJ, Vehkala M, Knol MJ, Bootsma HJ, Valimaki N, Croucher NJ, Meis JF, Bentley S, van Hijum SAFT, Corander J, Zomer AL, Ferwerda G, de Jonge MIet al., 2019, The Contribution of Genetic Variation of Streptococcus pneumoniae to the Clinical Manifestation of Invasive Pneumococcal Disease, CLINICAL INFECTIOUS DISEASES, Vol: 68, Pages: 61-69, ISSN: 1058-4838

Journal article

Campo JJ, Le TQ, Pablo JV, Hung C, Teng AA, Tettelin H, Tate A, Hanage WP, Alderson MR, Liang X, Malley R, Lipsitch M, Croucher NJet al., 2018, Panproteome-wide analysis of antibody responses to whole cell pneumococcal vaccination, Elife, Vol: 7, ISSN: 2050-084X

Pneumococcal whole cell vaccines (WCVs) could cost-effectively protect against a greater strain diversity than current capsule-based vaccines. Immunoglobulin G (IgG) responses to a WCV were characterised by applying longitudinally-sampled sera, available from 35 adult placebo-controlled phase I trial participants, to a panproteome microarray. Despite individuals maintaining distinctive antibody 'fingerprints', responses were consistent across vaccinated cohorts. Seventy-two functionally distinct proteins were associated with WCV-induced increases in IgG binding. These shared characteristics with naturally immunogenic proteins, being enriched for transporters and cell wall metabolism enzymes, likely unusually exposed on the unencapsulated WCV's surface. Vaccine-induced responses were specific to variants of the diverse PclA, PspC and ZmpB proteins, whereas PspA- and ZmpA-induced antibodies recognised a broader set of alleles. Temporal variation in IgG levels suggested a mixture of anamnestic and novel responses. These reproducible increases in IgG binding a limited, but functionally diverse, set of conserved proteins indicate WCV could provide species-wide immunity.

Journal article

Didelot X, Croucher NJ, Bentley SD, Harris SR, Wilson DJet al., 2018, Bayesian inference of ancestral dates on bacterial phylogenetic trees, Nucleic Acids Research, Vol: 46, Pages: 1-11, ISSN: 0305-1048

The sequencing and comparative analysis of a collection of bacterial genomes from a single species or lineage of interest can lead to key insights into its evolution, ecology or epidemiology. The tool of choice for such a study is often to build a phylogenetic tree, and more specifically when possible a dated phylogeny, in which the dates of all common ancestors are estimated. Here, we propose a new Bayesian methodology to construct dated phylogenies which is specifically designed for bacterial genomics. Unlike previous Bayesian methods aimed at building dated phylogenies, we consider that the phylogenetic relationships between the genomes have been previously evaluated using a standard phylogenetic method, which makes our methodology much faster and scalable. This two-step approach also allows us to directly exploit existing phylogenetic methods that detect bacterial recombination, and therefore to account for the effect of recombination in the construction of a dated phylogeny. We analysed many simulated datasets in order to benchmark the performance of our approach in a wide range of situations. Furthermore, we present applications to three different real datasets from recent bacterial genomic studies. Our methodology is implemented in a R package called BactDating which is freely available for download at https://github.com/xavierdidelot/BactDating.

Journal article

Kwun MJ, Oggioni MR, De Ste Croix M, Bentley SD, Croucher NJet al., 2018, Excision-reintegration at a pneumococcal phase-variable restriction-modification locus drives within- and between-strain epigenetic differentiation and inhibits gene acquisition., Nucleic Acids Research, Vol: 46, Pages: 11438-11453, ISSN: 0305-1048

Phase-variation of Type I restriction-modification systems can rapidly alter the sequence motifs they target, diversifying both the epigenetic patterns and endonuclease activity within clonally descended populations. Here, we characterize the Streptococcus pneumoniae SpnIV phase-variable Type I RMS, encoded by the translocating variable restriction (tvr) locus, to identify its target motifs, mechanism and regulation of phase variation, and effects on exchange of sequence through transformation. The specificity-determining hsdS genes were shuffled through a recombinase-mediated excision-reintegration mechanism involving circular intermediate molecules, guided by two types of direct repeat. The rate of rearrangements was limited by an attenuator and toxin-antitoxin system homologs that inhibited recombinase gene transcription. Target motifs for both the SpnIV, and multiple Type II, MTases were identified through methylation-sensitive sequencing of a panel of recombinase-null mutants. This demonstrated the species-wide diversity observed at the tvr locus can likely specify nine different methylation patterns. This will reduce sequence exchange in this diverse species, as the native form of the SpnIV RMS was demonstrated to inhibit the acquisition of genomic islands by transformation. Hence the tvr locus can drive variation in genome methylation both within and between strains, and limits the genomic plasticity of S. pneumoniae.

Journal article

Abudahab K, Prada JM, Yang Z, Bentley SD, Croucher NJ, Corander J, Aanensen DMet al., 2018, PANINI: Pangenome Neighbour Identification for Bacterial Populations., Microbial Genomics, Vol: 4, ISSN: 2057-5858

The standard workhorse for genomic analysis of the evolution of bacterial populations is phylogenetic modelling of mutations in the core genome. However, a notable amount of information about evolutionary and transmission processes in diverse populations can be lost unless the accessory genome is also taken into consideration. Here, we introduce panini (Pangenome Neighbour Identification for Bacterial Populations), a computationally scalable method for identifying the neighbours for each isolate in a data set using unsupervised machine learning with stochastic neighbour embedding based on the t-SNE (t-distributed stochastic neighbour embedding) algorithm. panini is browser-based and integrates with the Microreact platform for rapid online visualization and exploration of both core and accessory genome evolutionary signals, together with relevant epidemiological, geographical, temporal and other metadata. Several case studies with single- and multi-clone pneumococcal populations are presented to demonstrate the ability to identify biologically important signals from gene content data. panini is available at http://panini.pathogen.watch and code at http://gitlab.com/cgps/panini.

Journal article

Azarian T, Martinez PP, Arnold BJ, Grant LR, Corander J, Fraser C, Croucher NJ, Hammitt LL, Reid R, Santosham M, Weatherholtz RC, Bentley SD, OBrien KL, Lipsitch M, Hanage WPet al., 2018, Predicting evolution using frequency-dependent selection in bacterial populations

<jats:title>Abstract</jats:title><jats:p>Predicting how pathogen populations will change over time is challenging. Such has been the case with <jats:italic>Streptococcus pneumoniae</jats:italic>, an important human pathogen, and the pneumococcal conjugate vaccines (PCVs), which target only a fraction of the strains in the population. Here, we use the frequencies of accessory genes to predict changes in the pneumococcal population after vaccination, hypothesizing that these frequencies reflect negative frequency-dependent selection (NFDS) on the gene products. We find that the standardized predicted fitness of a strain estimated by an NFDS-based model at the time the vaccine is introduced enables to predict whether the strain increases or decreases in prevalence following vaccination. Further, we are able to forecast the equilibrium post-vaccine population composition and assess the invasion capacity of emerging lineages. Overall, we provide a method for predicting the impact of an intervention on pneumococcal populations with potential application to other bacterial pathogens in which NFDS is a driving force.</jats:p>

Thesis dissertation

Croucher NJ, Lochen A, Bentley SD, 2018, Pneumococcal Vaccines: Host Interactions, Population Dynamics, and Design Principles, ANNUAL REVIEW OF MICROBIOLOGY, VOL 72, Vol: 72, Pages: 521-549, ISSN: 0066-4227

Journal article

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