Mechanisms of genome evolution of Streptococcus

Infection, Genetics and Evolution xxx (2014) xxx–xxx

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Mechanisms of genome evolution of Streptococcus Cheryl P. Andam, William P. Hanage ⇑ Department of Epidemiology, Harvard School of Public Health, Boston, MA 02115, USA

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Article history: Received 7 August 2014 Received in revised form 5 November 2014 Accepted 7 November 2014 Available online xxxx Keywords: Pneumococcus Genome evolution Recombination Selection Vaccination Antibiotics

a b s t r a c t The genus Streptococcus contains 104 recognized species, many of which are associated with human or animal hosts. A globally prevalent human pathogen in this group is Streptococcus pneumoniae (the pneumococcus). While being a common resident of the upper respiratory tract, it is also a major cause of otitis media, pneumonia, bacteremia and meningitis, accounting for a high burden of morbidity and mortality worldwide. Recent findings demonstrate the importance of recombination and selection in driving the population dynamics and evolution of different pneumococcal lineages, allowing them to successfully evade the impacts of selective pressures such as vaccination and antibiotic treatment. We highlight the ability of pneumococci to respond to these pressures through processes including serotype replacement, capsular switching and horizontal gene transfer (HGT) of antibiotic resistance genes. The challenge in controlling this pathogen also lies in the exceptional genetic and phenotypic variation among different pneumococcal lineages, particularly in terms of their pathogenicity and resistance to current therapeutic strategies. The widespread use of pneumococcal conjugate vaccines, which target only a small subset of the more than 90 pneumococcal serotypes, provides us with a unique opportunity to elucidate how the processes of selection and recombination interact to generate a remarkable level of plasticity and heterogeneity in the pneumococcal genome. These processes also play an important role in the emergence and spread of multi-resistant strains, which continues to pose a challenge in disease control and/or eradication. The application of population of genomic approaches at different spatial and temporal scales will help improve strategies to control this global pathogen, and potentially other pathogenic streptococci. Ó 2014 Published by Elsevier B.V.

1. Introduction The genus Streptococcus is a diverse group of 104 recognized species (http://www.bacterio.cict.fr/s/streptococcus.html as of July 21, 2014), whose interactions with host organisms vary from commensal to pathogenic. Many of the pathogenic species cause severe, invasive infections that account for a high burden of morbidity and mortality (Gray, 1998; Mitchell, 2003), associated with high economic and health care costs. One of the most clinically relevant Streptococcus species colonizing humans is Streptococcus pneumoniae (or pneumococcus), whose control remains a challenge despite the availability of highly effective vaccines. The pneumococcus is a quiescent colonizer of the upper respiratory tract and is carried asymptomatically by a fraction of the population, ranging from 21% to 94% in high burden settings such as Sub-Saharan Africa (Usuf et al., 2014). However, it can cause diseases such as otitis media, conjunctivitis, pneumonia, bacteremia and meningitis. Pneumococcal diseases are most prevalent in children, the elderly and immuno-compromised (French et al., 2007; ⇑ Corresponding author. Tel.: +1 617 432 1047. E-mail address: [email protected] (W.P. Hanage).

Gray, 1998; Rubins et al., 2008). The introduction of conjugate vaccination in children worldwide has had profound consequences for the burden of pneumococcal diseases as well as the population structure of carriage and invasive strains. However, the efficacy of these control measures has been limited by the pneumococcus’ exceptional ability to circumvent them. Years after the introduction of the first pneumococcal conjugate vaccine (PCV) in 2000, we are beginning to understand the long-term impact of these vaccines. Genomic methods have made an important contribution to our understanding of the biology and epidemiology of important bacterial pathogens, including the pneumococcus. Molecular methods historically important in pneumococcal epidemiology, and still widely used today, include multi-locus sequence typing (MLST) (Enright and Spratt, 1998), in which seven housekeeping genes are sequenced as a sample of genomic variation and are used to define the sequence type (ST). Another example of an historically important molecular method is pulsed field gel electrophoresis (PFGE) in which strains are distinguished on the basis of restriction digestion of their genomic DNA (Lefevre et al., 1993). Differences in the numbers and location of the restriction sites are then visualized by electrophoresis. By investigating genetic and phenotypic

http://dx.doi.org/10.1016/j.meegid.2014.11.007 1567-1348/Ó 2014 Published by Elsevier B.V.

Please cite this article in press as: Andam, C.P., Hanage, W.P. Mechanisms of genome evolution of Streptococcus. Infect. Genet. Evol. (2014), http:// dx.doi.org/10.1016/j.meegid.2014.11.007

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variation at different spatial and temporal scales, we can also elucidate the pace and patterns of pneumococcal diversification. The control of pneumococcal disease depends on our ability to anticipate the ways in which the organism responds to interventions, and genomic methods provide the best, most extensive data for the study of pneumococcal evolution. While genomics is a rapidly changing field, as of this writing, the pneumococcus can lay claim to be the best-studied organism from a population genomic standpoint, with multiple large samples already published (Chewapreecha et al., 2014; Croucher et al., 2014a, 2013). In this review, we discuss recent findings in pneumococcal genome evolution, with a focus on the roles of recombination and selection. We highlight the impacts of vaccine-induced selective pressures in driving the population dynamics and evolution of different pneumococcal lineages. We also discuss how the pneumococci respond to these pressures, mainly through recombination within the species as well as with other Streptococci. The contributions of mobile elements and phages to pneumococcal evolution will also be discussed. We conclude with future directions on how genomics can enhance our understanding of the role of vaccination and antibiotic use in pneumococcal evolution and disease control.

2. The promiscuous pneumococcus 2.1. Pneumococcal diversity, transformation and recombination Pneumococci are competent and naturally transformable, meaning that they readily take up exogenous DNA and can incorporate it into the genome by homologous recombination. This is not rare; a study of a single lineage [designated PMEN1 by the Pneumococcal Molecular Epidemiology Network (McGee et al., 2001)] estimated that 74% of its genome had been altered by recombination in at least one isolate in a sample estimated to have diversified from a most recent common ancestor in the early 1970s (Croucher et al., 2011). This was the result of a study of a global sample, but at the opposite scale, a study of six isolates obtained within a sevenmonth period from the same individual found a remarkable 23 recombination replacements (Hiller et al., 2010), resulting in the replacement of 7.8% of the genome. Horizontal transfer of genetic material in general, not only by homologous recombination but also by mobile genetic elements, can substantially amplify the heterogeneity of the common gene pool of the pneumococcus, as was observed in the study of PMEN1 isolates retrieved between 1994 and 2008 mentioned above. In this work, recombinant regions varied in size from 3 bp to 72,038 bp (Croucher et al., 2011). Such high levels of recombination ensure the success of a clone over the long term, particularly in the face of environmental changes, as was reported for the multi-drug resistant PMEN2 that predominated in Iceland in the 1990s. The decline in its prevalence following the reduction in antibiotic use was largely due to the lack of sequence import through recombination that prevented the modification of core sequences associated with resistance and antigenic diversification (Croucher et al., 2014b). Although the pneumococcus is known to be highly transformable and recombining, there is marked variation in these traits among isolates. An experimental study showed variation over four orders of magnitude (10 2 to 10 6) in transformation frequencies in isolates from asymptomatic carriage, and this variation does not correlate with genetic relatedness of isolates (Evans and Rozen, 2013). The causes of this variation, whether essential (resulting from differences in the mechanism of DNA uptake) or ecological (recombination is limited by the isolates rarely encountering one another in nature), remain poorly understood. One possible explanation is that these observed differences might be

attributed to the environmental stresses faced by different pneumococcal lineages. Chemostat evolution experiments of the costs and benefits of transformation in pneumococcus grown for 1000 generations demonstrate that transformation can benefit cells living in stressful environments and can be costly in benign environments (Engelmoer et al., 2013). Rather than being selected by stress, recombination may be directly linked to it through the overlap of the stress response with the competence pathway responsible for taking up DNA (Dagkessamanskaia et al., 2004). It is easy to understand how interactions with the immune system, as well as interventions such as antimicrobials and vaccines, could create a stressful environment for the pathogen. Different pneumococcal lineages also demonstrate significant variation in recombination rates, measured as the rate at which nucleotide polymorphisms accumulate through recombination relative to mutation (r/m). For example, whole genome analyses of carried pneumococci from Massachusetts showed a wide range of recombination rates, with the highest value associated with a clade containing the highly resistant ST 320 and which has proliferated following vaccination despite originally having a vaccine serotype. The loci determining serotype have been replaced with those of a non-vaccine serotype (NVT) by recombination. This process, which can generate novel combinations of serotype and genomic backbone, is known as capsular switching (Croucher et al., 2013). Recombination, however, is not uniform throughout the pneumococcal population. It has been recently described to consist of two modes of action: micro-recombinations involve the frequent replacement of single, short DNA fragments, and macrorecombinations, though rarer, involve the acquisition of multiple, long fragments and is a associated with major phenotypic changes (Mostowy et al., 2014). In addition to variation in the uptake of DNA, some strains exhibit propensity to either donate or receive exogenous DNA more often than others. Chewapreecha et al. (2014) found that a lineage of pneumococci in their sample that lacked a capsule was more likely to both gain and donate DNA to other lineages. Again, a study of isolates from a single chronic childhood infection showed similar variation at the level of evolution within a single host (Hiller et al., 2010). During this time period, one particular strain, ST 2011v4, donated DNA extensively to the other infecting strains, all of which are closely related ST 13 strains. Unambiguously identifying donors is not easy, because in a recombinogenic organism like the pneumococcus, the sequence in question may be found in multiple lineages and the ultimate origin is hard to define. It is reasonable to think that some lineages may donate more often than others, if for no other reason than that some lineages are more commonly found in the population than others, and so will have more opportunity [(for instance, it is not clear the unencapsulated lineage discussed above (Chewapreecha et al., 2014) is a more common donor or recipient than one would expect given its frequency in the population]. Moreover, lineages that are over represented in a sample may be more readily identified as donors or recipients, and it is important to recognize such possible bias. The consequences of variation in recombination can be important; pneumococci with evidence of extensive recombination at housekeeping loci are significantly associated with antibiotic resistance (Hanage et al., 2009). Genomic sequencing in diverse environmental backgrounds can therefore help us define the causes and consequences of the variation in transformation, recombination and other phenotypic traits in pneumococcus. 2.2. Gene gain across species boundaries The ability of the pneumococcus to donate and receive genetic material is not restricted to its own species. Novel genes can be acquired from other taxa, many of which also inhabit the same

Please cite this article in press as: Andam, C.P., Hanage, W.P. Mechanisms of genome evolution of Streptococcus. Infect. Genet. Evol. (2014), http:// dx.doi.org/10.1016/j.meegid.2014.11.007

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mucosal surfaces of the oral cavity and nasopharynx as the pneumococcus, indicating that a shared habitat can greatly facilitate inter-species mobilization of genes (Muzzi and Donati, 2011). Horizontal gene transfer (HGT) that traverses boundaries of traditionally named species can enhance the pneumococcal gene pool and lead to exceptionally variable genomes of individual isolates. Other streptococcal species, such as Streptococcus mitis, Streptococcus infantis and Streptococcus oralis, are frequent exchange partners of pneumococcus (Donati et al., 2010). Genomic comparisons between these streptococcal species indicate that 30% of the sequence variability, which also includes pathogenicity genes, in pneumococcus is shared with S. mitis, making it an external genetic reservoir for the pneumococcus (Donati et al., 2010; Sanguinetti et al., 2012). HGT may also explain the large number of pneumococcal virulence genes, such as those encoding for pneumolysin, mitilysin and neuraminidase A, found in other members of the mitis group of Streptococci (Johnston et al., 2010). Frequent genetic transfer between and within streptococcal species can contribute to a rapid response to diverse and/or fluctuating environments, which are particularly useful for pathogens frequently bombarded with antibiotics. For example, large imports of genes from S. mitis and other mitis, anginosus, and salivarius group streptococci to the pneumococcus have been reported to contribute to the evolution and structural diversity of the pneumococcal capsule (Kilian et al., 2014).

3. Pneumococcal strategies against vaccine-induced selection pressures 3.1. Pneumococcal conjugate vaccines (PCVs) The primary virulence determinant of pneumococcus is its anti-phagocytic polysaccharide capsule. The capsular locus (cps) is transcribed as a single operon and all strains exhibit a similar organization with the genes coding for a specific capsule flanked by other genes (dexB and aliA) common to all strains (Bentley et al., 2006; García et al., 1999). Multiple and subtle polymorphisms in the capsular operon result in serologically distinct variants (or serotypes), of which there are currently 94 identified (Bentley et al., 2006; Bratcher et al., 2011). The serotypes vary considerably in carriage rates, disease potential, geographical distribution and the prevalence of antibiotic resistance (Hausdorff et al., 2008). PCVs are composed of capsular polysaccharides chemically linked to a carrier protein, making them more immunogenic in young children. Only a subset of these has been included in current vaccines. PCVs for routine immunization of infants and children started with PCV7, which was introduced in the US in 2000 (which targeted the serotypes 4, 6B, 9V, 14, 18C, 19F and 23F) and subsequently replaced with PCV13 in 2010 (for serotypes 1, 3, 5, 6A, 7F, 19A in addition to the PCV7 serotypes). These were designed to target the most dominant serotypes involved in invasive pneumococcal diseases in children; while PCV7 was targeted at the most common causes of disease in the US and Europe, PCV13 has expanded coverage to include serotypes like 1 and 5 that are important and frequent causes of disease in the rest of the world, especially sub-Saharan Africa (Hausdorff et al., 2000a,b). Other vaccines have also been manufactured, including PCV10 (for serotypes 1, 5 and 7F in addition to the PCV7 serotypes) and PPSV23 (for 23 serotypes; contains unattached polysaccharide antigens), with the latter approved for use in adults and patients with specific risk factors who are at least 2 years old. Conjugate vaccines are highly effective at preventing invasive disease (Dagan and Klugman, 2008; Feldman and Anderson, 2014), but the pneumococcus continues to be a formidable foe. Its ability to circumvent these selective pressures lies in its highly variable genome and intra-species

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diversity. Advances in the development of new generation vaccines focusing on recombinant protein and whole cell vaccines have the potential to provide serotype-independent protection. Such vaccines would provide an alternative, or at least complement, existing capsule-based vaccines in controlling drug resistance and eradicating pneumococcal diseases. 3.2. Serotype replacement With the elimination of previously dominant serotypes due to vaccine selection, expansion of NVTs can occur as they take advantage of the removal of competition from the vaccine types (VT). While there was initially some concern that changes in the prevalence of NVT could be ‘secular trends’ unrelated to the vaccine, it is now accepted that the increase in NVT has been too widespread and universally observed to be independent of PCV7. This population level change in the serotype distribution, most often involving pre-existing clones and serotypes that were already in circulation before vaccine implementation, is known as serotype replacement. In the United States, prevalence of PCV7 serotypes in all age groups decreased from 64% of invasive and 50% of noninvasive isolates in 1999–2000 to 3.8% and 4.2%, respectively, in 2010–2011 (Richter et al., 2013). After the initial PCV7 immunization, the diversity and population structure of carriage pneumococci was also altered (Hanage et al., 2010), even though the overall carriage prevalence has remained constant (Hanage et al., 2011; Wroe et al., 2012). Similar outcomes have been reported in Asia (Kim et al., 2012; Song et al., 2009), Australia (Lehmann et al., 2010), Africa (Nzenze et al., 2013) and Europe (Hanquet et al., 2010; Pichon et al., 2013). This observed decrease could be attributed to both direct vaccine effect and herd immunity in the community. The most noteworthy example of serotype replacement in the PCV7 era was the dramatic increase in the prevalence of serotype 19A. While it was initially anticipated that the serotype 19F component of PCV7 would provide cross-protection against 19A, this was not the case. In fact, 19A replacement occurred rapidly following high vaccine coverage. Initially, the majority of 19A in both carriage and disease were the result of the clonal expansion of one genotype, ST 199, already common prior to the introduction of PCV7 (Beall et al., 2011; Moore et al., 2008; Pai et al., 2005; Scott et al., 2012a). However, in time multiple unrelated genotypes were also found in increasing numbers, some of which exhibited highlevel resistance to multiple antibiotics, as in the case of the globally successful ST 320 (Clarke et al., 2004). ST 320 is an example of a lineage that is the product capsular switching, having originally been found with a PCV7 serotype: 19F. The 19A variant of this lineage was able to evade the vaccine and prosper. ST 320 has also been noted to have a very high rate of recombination across the genome (Croucher et al., 2013), which might plausibly contribute to its success in adapting to the post vaccine era. Another notable case is the replacement of 23F with relatives from 23A and 23B of the same serogroup (Croucher et al., 2013). Competitive interactions between strains, particularly in terms of colonization, are likely to drive serotype replacement in pneumococci (Mehtälä et al., 2013). While the long-term impacts of PCV13 on pneumococcal population structure and serotype composition remain to be seen, initial observations suggest patterns comparable to those observed for PCV7 (Kaplan et al., 2013). In carriage, although colonization with non-PCV13 serotypes appears to be increasing, the rate of overall pneumococcal colonization was not affected (Lee et al., 2014). In particular, the prevalence of the non-PCV13 serotype 35B appears to be increasing in both invasive and non-invasive diseases (Richter et al., 2013). Population genomic studies have allowed us to chart how the changing epidemiological and selective landscape imposed by vac-

Please cite this article in press as: Andam, C.P., Hanage, W.P. Mechanisms of genome evolution of Streptococcus. Infect. Genet. Evol. (2014), http:// dx.doi.org/10.1016/j.meegid.2014.11.007

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cination has influenced pneumococcal evolution. Hence, the prevalence of specific donors and recipients may also subsequently affect the genomic composition of individual serotypes. Whole genome analyses can provide insights into the long-term and broader impacts of serotype replacement, particularly in terms of the variation in the ecology and pathogenesis of different serotypes.

novel recombinant serotypes remains a concern. Through recombination, the pneumococcal population exhibits a large amount of standing variation such that, as we introduce new interventions, there is a good chance that a genotype capable of resisting them is already present, even if rare. Other resident streptococci, such as encapsulated and/or drug resistant S. mitis, can also contribute to this variation.

3.3. Capsular switching In pneumococci, genetic exchange at the capsular locus is greatly facilitated by the syntenic organization and homology of genes in the capsular locus (Hu et al., 2012). This process of substituting the genes encoding one type of capsule with genes encoding for another is referred to as serotype or capsular switching (Wyres et al., 2013a). Although capsular switching is not an unusual occurrence in pneumococcal biology, the primary concern lies in the VTto-NVT capsular switches because of the potential for strains that cause invasive diseases to evade limited-valency vaccines and become more virulent. Vaccine escape recombinants emerged after the introduction of PCV7, exemplified by the case of ST 320 described above. The first record of ST 320 exhibiting a 19A capsule in the MLST database (www.mlst.net) is from South Korea in 1998, in other words well prior to the implementation of PCV7 in the US in 2000. Several years after PCV7 was first used, surveillance programs showed that this novel 19A variant and its descendants have successfully spread in many parts of the US as well as in other continents (Ansaldi et al., 2011; Golubchik et al., 2012). ST 320 is not the only example of VTs switching to NVTs and surviving into the post-vaccine era. PMEN1 (ST 81), initially a 23F clone, has also produced 19A variants, the genomes of some of which have been determined along with multiple other capsular switching events (Croucher et al., 2011). Additionally ST 695, yet another 19A clone, was produced from a serotype 4 ancestor targeted by PCV7 and became increasingly often isolated from carriage and disease. Similar outcomes were also reported for other serotypes (Croucher et al., 2011; Everett et al., 2012; Hu et al., 2012). The properties of the novel recombinant strains produced by capsular switching may be quite different from their ancestors, and not necessarily easy to predict. For example, a recent experimental study of switch variants with a serotype 6A background carrying a 6C capsule showed that the new recombinants can acquire enhanced virulence in an animal model of otitis media (Sabharwal et al., 2013). Although the implementation of PCV13 is expected to reduce the prevalence of 19A, the remaining 80 non-vaccine serotypes provide a ready pool of antigenic diversity that the vaccine will be unable to affect and which may allow VTs to survive. In other words, recombination can result to lineages surviving PCV introduction that will be NVT by definition, but their genetic background will be that of earlier VTs. The capsular switch from serotype 4 that produced the 19A variant of ST 695 was also associated with the simultaneous transfer of multiple DNA fragments from other parts of the genome, some of which are as long as 44 kb (Golubchik et al., 2012; Wyres et al., 2013a). This means that multiple genes can be acquired in a single step. For example, the capsular locus is in close proximity to the penicillin binding protein genes pbp2x and pbp1a, and so resistance can be acquired along with a new capsule (Golubchik et al., 2012; Trzcin´ski et al., 2004; Wyres et al., 2013a). For the novel 19A variant, a single recombination event led to the switching of the capsule locus as well as with non-susceptibility to penicillin (Brueggemann et al., 2007). Although capsular switching appears to be a common enough feature of pneumococci to have been widespread prior to vaccination (and probably antibiotic use) (Wyres et al., 2013a), uncertainty over the evolution of these

3.4. Emergence and spread of multi-drug resistant pneumococci Antibiotic resistance in pneumococci is widespread, with numerous clones identified that have acquired resistance on separate occasions (McGee et al., 2001), and selective pressures due to vaccination can change resistance patterns temporally and geographically (McCormick et al., 2003). An excellent example of this is the increased prevalence of penicillin-nonsusceptible pneumococci (PNSP) among NVT (Gertz et al., 2010; Gherardi et al., 2012). The pool of pbp genes capable of conferring diminished susceptibility to penicillin in pneumococci is large and diverse, largely as a result of a history of extensive recombination that includes loci originating from other species (Hakenbeck et al., 2012; Izdebski et al., 2008). Other factors are also known to influence the rapid emergence and evolution of resistance in pneumococcal populations. Horizontal transfer involving mobile elements has led to increasing rates of macrolide resistance in pneumococci, as exemplified by the macrolide-resistant ST 320, carrying the mefE and ermB genes, both of which are carried by the transposon Tn2010 (Bowers et al., 2012; Croucher et al., 2013). Changes in the frequencies of specific serotypes following PCV immunization can also affect patterns of antibiotic resistance. In the United States, changes in the sequences of resistance determinants against macrolides and b-lactams occurred after PCV7 introduction (Bowers et al., 2012; Moore et al., 2008; Pelton et al., 2007). For some pneumococcal lineages, multidrug resistance and exceptional levels of genetic diversity arise through ‘soft’ selective sweeps in response to a change in selection pressure, as was observed in the highly resistant PMEN14 after PCV7 introduction. Soft selective sweeps result from selection on standing diversity (Hermisson and Pennings, 2005), or migration importing multiple beneficial alleles (Pennings and Hermisson, 2006). In soft selective sweeps multiple adaptive alleles sweep across the population at the same time, in contrast to hard selective sweeps wherein only a single adaptive allele spreads (Messer and Petrov, 2013). Multiple adaptive alleles in a soft selective sweep are derived from different independent origins, resulting in an extensive pool of standing genetic variation and greater preservation of the ancestral diversity (Croucher et al., 2014a). The long term use of conjugate vaccines is expected to lead to successive rounds of serotype replacement, unless a formulation can be arrived at that contains all the more than 90 distinct capsular types. The alternative is to develop vaccines based on protein antigens that will target any pneumococcal strain regardless of its capsule type, although protein antigens are also genetically variable and subject to the same evolutionary processes including recombination as the capsule. However, the use of different antigens in vaccine development will greatly improve control strategies of pneumococcal diseases. This relies on genomic approaches that will provide valuable insights into the diversity and evolution of different molecular targets for protective immunity. Genomics can also provide a more profound understanding of the range in variation in adapting to antibiotics by different pneumococcal lineages and how we can potentially manipulate this variation to reduce the potential for multidrug resistance.

Please cite this article in press as: Andam, C.P., Hanage, W.P. Mechanisms of genome evolution of Streptococcus. Infect. Genet. Evol. (2014), http:// dx.doi.org/10.1016/j.meegid.2014.11.007

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4. Evolution and genomic contributions of non-encapsulated pneumococci Nontypeable pneumococci (NTPn) fall into several categories (Salter et al., 2012): those that do not possess capsule genes (Hanage et al., 2006; Marsh et al., 2010), pneumococci that are phenotypically non-encapsulated but possess down-regulated or defective capsule genes (Hathaway et al., 2004), and a population of atypical non-encapsulated isolates that are genetically divergent from most pneumococci (Ing et al., 2012; Rolo et al., 2013). Nonencapsulated pneumococci are plainly a successful part of the carriage population (Chewapreecha et al., 2014; Marsh et al., 2010) but have also been reported in mucosal infections and conjunctivitis (Haas et al., 2011) as well as rare cases of invasive disease (Scott et al., 2012b). These NTPn variants are also highly diverse, with some strains having a widespread inter-continental distribution. Some NTPn isolates in Brazil were found to be genetically similar to the global, PNSP clone Spain 9V-3 (Andrade et al., 2010). A lineage of true NTPn isolated from carriage was reported to have been found in different continents (Hanage et al., 2006) and subsequent studies have designated this as the clone NorwayNTST344 (Simões et al., 2011). In the genome analyses with the highest number of NTPn sequenced, NTPn were the most prevalent capsule phenotype group, out of the more than 3000 carriage isolates sampled in their study (Chewapreecha et al., 2014). One lineage of NTPn forms one of the biggest clusters in their sample group, while many others are distributed within encapsulated pneumococcal clusters. This study highlights the remarkable diversity of NTPn in pneumococcal populations that remains poorly recognized and characterized. The lack of capsule has been shown to facilitate colonization of the nasopharynx through enhanced adherence to epithelial cells and biofilm formation (Domenech et al., 2009; Marks et al., 2012). Recently, some isolates have been found to possess novel and/or alternative capsule-independent survival mechanisms. Of noteworthy example is the finding that some NTPn isolates have been reported to possess enhanced binding to epithelial cells via a pspK-dependent mechanism (Keller et al., 2013; Park et al., 2012). These do not appear to be a transient subpopulation in carriage isolates; rates of colonization and adherence are comparable between them and encapsulated isolates (Keller et al., 2013; Park et al., 2012). Interestingly, pspK is located at and has replaced the capsule locus, and recombination of flanking homologous genes has the potential to spread this gene between encapsulated and non-encapsulated isolates. Sporadic or random switching between encapsulated and nonencapsulated states may play an important role in pneumococcal population dynamics and antibiotic resistance. Frequent loss and subsequent gain of capsule loci may partly explain the observed variation in recombination rates and antibiotic resistance among pneumococcal lineages. A possible model is illustrated schematically in Fig. 1. Consider a group of closely related encapsulated strains at time 1 (t1). By chance, one strain (labeled C) loses its ability to produce a capsule through gene loss, deletion or insertion of a stop codon within the capsule locus at t2. Capsule gene loss may be caused by a single nucleotide polymorphism or indel that can lead to a nonsynonymous substitution, or a transposon insertion. As noted above, the capsule is not an absolute requirement for carriage or transmission, and so the strain persists in a non-encapsulated state in which it exhibits a higher transformation efficiency (Chewapreecha et al., 2014; Pearce et al., 2002; Weiser and Kapoor, 1999) and subsequently acquires exogenous DNA, including potential virulence and resistance genes at a higher rate than its encapsulated relatives. The strain may eventually regain its original capsule type or another capsule of a different serotype (t3).

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Once the capsule has been regained, the result will be variation in recombination rates even at unlinked loci among them, as has been observed (Hanage et al., 2009), and which may also correlate with increased resistance for strain C that previously lacked a capsule. Since recombination is an important mechanism to generate genomic variation in response to selective pressures from the environment, it is likely that the environment can lead to significant changes in capsule production in pneumococcus. Oxygen concentration in the environment has been reported to influence capsule production (Weiser et al., 2001). More recently, Carvalho et al. reported that inactivation of the gene coding for pyruvate oxidase resulted to enhanced capsule production (Carvalho et al., 2013). The authors proposed that this enzyme acts as a sensor of oxygen levels in the environment to signal the pneumococcus to determine the amount of capsule it needs to produce (Carvalho et al., 2013). 5. The role of mobile elements and phages in pneumococcal evolution Mobile elements and phages play an important role in pneumococcal evolution, both as a selective pressure and as an additional means of mobilizing genetic material. Integrative conjugative elements (ICEs) are autonomously mobile entities that can be excised and subsequently integrated into the genome within the same cell at a different location or in a new host cell. These elements are transferred mainly through conjugation between cells. The Tn5253 and Tn916 ICE families are commonly found in pneumococcus and are important vehicles for the dissemination of antibiotic resistance in pneumococci and between Streptococcus species (Roberts and Mullany, 2011). The ICE family of Tn916 is transmissible in many Gram-positive bacteria and can therefore easily mobilize genetic elements from diverse sources. These transposons are particularly known for carrying and transferring the tetracycline resistance determinant tet(M). Some Tn916-like elements also carry other resistance genes, such as erm(B)-mediated erythromycin resistance, and genes associated with other accessory functions (Cochetti et al., 2007). Genomic studies of the highly successful PMEN1 clone showed a diverse array of Tn916-like elements contributing to its extensive antibiotic resistant phenotype (Croucher et al., 2011; Domenech et al., 2013). The persistence of antibiotic resistance for many years may also be attributed to ICEs, as in the case of Tn916 elements from pneumococci dated between 1967 and 1972 that exhibit high similarity to the Tn916 circulating in pneumococcal populations today (Wyres et al., 2013b). The reason why these elements should be so conserved is not clear, but the lack of variation is in marked contrast with other ICE (Wyres et al., 2013b). Tn5253 and its relatives are large composite transposons consisting of Tn5252 and a Tn916-like Tn5251 (Ayoubi et al., 1991; Iannelli et al., 2014). Although the majority of the ORFs in Tn5253 are of unknown function (Iannelli et al., 2014), Tn5252 is known to possess a chloramphenicol resistance gene (cat). Hence, because of the composite nature of Tn5253, resistance to both tetracycline and chloramphenicol can be easily disseminated in bacterial populations. Tn5253-like elements in pneumococci are also extremely diverse in gene content (Henderson-Begg et al., 2009; Mingoia et al., 2011), further expanding their genetic repertoire. Bacteriophages also play an important role in pneumococcal evolution and pathogenicity by rapidly disseminating genetic material, as in the case of the PMEN1 lineage discussed previously, which carries a 39.1 kb intact prophage and a 6.4 kb prophage remnant (Croucher et al., 2009). Comparative analyses of ten temperate pneumococcal phages also indicate the presence of different

Please cite this article in press as: Andam, C.P., Hanage, W.P. Mechanisms of genome evolution of Streptococcus. Infect. Genet. Evol. (2014), http:// dx.doi.org/10.1016/j.meegid.2014.11.007

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Fig. 1. Switching between encapsulated and non-encapsulated states in a closely related group of pneumococci. Labels A–E represent different strains. Different capsule colors indicate different serotypes. tn indicates point in time. Colored arrows represent different genes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

virulence-associated genes and extensive genetic exchange between phages (Romero et al., 2009). Hence, phage-mediated recombination can significantly enhance the genetic diversity and ability of the pneumococcus to evade selection pressures. The contributions of ICEs and phages to pneumococcal biology and pathogenicity further highlight the remarkable ability of these bacteria to exchange genes through different transfer mechanisms (transformation, transduction and conjugation). However, the diversity of these pneumococcal mobile elements and phages has not been fully explored and remain under-appreciated. We anticipate that genome sequencing of pneumococcal samples from diverse geographical origins will provide additional insight into the extent of the genetic diversity of ICEs and phages, and how these combine with other loci to make up the accessory genome of pneumococci.

6. Conclusions and future directions Genomic data can offer unprecedented resolution in the understanding of pathogen evolution, adaptation and pathogenicity. For pneumococcus, this approach has been especially informative in defining how the pathogen responds to interventions such as antibiotic use or conjugate vaccination, and can better inform us of the likely consequences of variation among different pneumococcal lineages in the face of any new selective pressure. Moreover, genomic studies have given us a deeper insight into the contributions of genetic exchange as a driving factor in pneumococcal evolution and as a mechanism for evading selection pressures. The use of a population genomic dataset is also crucial in identifying the association of clusters of orthologous genes (COGs) with specific environments, such as specific host characteristics (age, immunosuppression, antibiotic consumption, temporal association with other respiratory bacterial or viral diseases) and geography. The precise COGs found in any population in question may greatly vary under different ecological conditions. The implications of these changes, particularly in light of the re-structuring of pneumococcal populations in response to clinical interventions, remain unclear. For example, the composition of the core genome may likely reflect the resulting niche differentiation that would result in strains adapted to particular host characteristics, as has been suggested for antigen COGs that were stably associated with

specific genotypes and host ages (Croucher et al., 2013). Although this line of investigation is still in its infancy, we can expect that such information will be vital in understanding the ecological aspects of pneumococcal pathogenicity. Recent work has shown that the relationship between SNP divergence in the ‘core’ genome and divergence in gene content, at least in the population studied in (Croucher et al., 2013), is approximately linear (Croucher et al., Nature Communications, in press). This work also shows that the major lineages previously identified are approximately equidistant in terms of how much they differ in gene content. How homologous recombination and other processes, such as phage or conjugative elements, combine to produce this pattern is presently unclear. Although it can be argued that the core genome should not include any niche-specific genes, the definition of the core genome of any microbial population will vary depending on the number and/or identity of strains included, sampling scheme and the ecological setting on which the populations rests, and may therefore include adaptive and niche-specific genes. Thus, the core genome is not an inflexible property of a population, although it encounters the same evolutionary processes as the accessory genome. Continued surveillance is critical as novel genotypes can emerge from obscurity and exhibit characteristics that are poorly understood. Likewise, a more profound understanding of the evolution, geographic distribution and transmission of multi-drug resistant strains can also be attained with genomics (Croucher et al., 2014a,b, 2013, 2011). Population genomic approaches at different spatial and temporal scales are a valuable tool to enhance our ability to detect cryptic outbreaks and to trace the origins and historical patterns of spread, thereby improving our current strategies in controlling this global pathogen as well as other pathogenic species of this genus. The present challenge is combining genomics with rigorous experimental methods and to ask how we can robustly link observed genomic variation to phenotype. This is the work of the next few years, and will be the fruit of the systematic application of evolutionary biology and genomic methods.

Acknowledgements This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under

Please cite this article in press as: Andam, C.P., Hanage, W.P. Mechanisms of genome evolution of Streptococcus. Infect. Genet. Evol. (2014), http:// dx.doi.org/10.1016/j.meegid.2014.11.007

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Award Number R01 AI106786-01 (to WPH). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Please cite this article in press as: Andam, C.P., Hanage, W.P. Mechanisms of genome evolution of Streptococcus. Infect. Genet. Evol. (2014), http:// dx.doi.org/10.1016/j.meegid.2014.11.007