Infection, Genetics and Evolution 11 (2011) 1703–1708

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Infection, Genetics and Evolution

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Pherotypes of pneumococcal strains co-existing in healthy children

Didrik F. Vestrheim a,⇑, Peter Gaustad b,c, Ingeborg S. Aaberge a, Dominique A. Caugant a,d

a Department of Bacteriology and Immunology, Norwegian Institute of Public Health, Oslo, Norwayb Department of Microbiology, Oslo University Hospital, Oslo, Norwayc Department of Microbiology, University of Oslo, Oslo, Norwayd Department of Community Medicine, University of Oslo, Oslo, Norway

a r t i c l e i n f o

Article history:Received 15 April 2011Received in revised form 27 June 2011Accepted 3 July 2011Available online 7 July 2011

Keywords:Streptococcus pneumoniaePheromoneCompetence stimulating peptideNasopharyngeal carriageMultilocus sequence typing

1567-1348/$ - see front matter � 2011 Elsevier B.V. Adoi:10.1016/j.meegid.2011.07.003

⇑ Corresponding author. Address: Department of BNorwegian Institute of Public Health, PO BOX 440Norway. Tel.: +47 21 07 64 65; fax: +47 21 07 65 18.

E-mail address: didrik.frimann.vestrheim@fhi.no (

a b s t r a c t

Genetic diversity in the species Streptococcus pneumoniae is mainly driven by horizontal gene transfer.S. pneumoniae is naturally competent for transformation. Competence is induced by a pheromone termedcompetence stimulating peptide (CSP) by a quorum-sensing mechanism. Two CSP pherotypes predomi-nate amongst clinical isolates of S. pneumoniae, CSP-1 and CSP-2, with ability to trigger competence inbacteria of the homologue pherotype. Opposing theories on the effect of pherotypes on speciation havebeen proposed, either as a barrier for intra-pherotype gene transfer, or as a mechanism for fratricideresulting in lysis of non-competent bacterial cells.

The aim of the present study was to determine pherotype distribution in strains of S. pneumococci iso-lated from the nasopharynges of healthy children. We sequenced the locus encoding CSP, comC, in sets ofstrains obtained from children colonised by multiple pneumococcal strains simultaneously. The impact ofpherotype on co-colonisation was determined by comparing the observed distribution of pherotypes inco-colonising strains with the estimated pair-wise probability based on the overall pherotype distribu-tion in the sample set.

Five distinct comC alleles were identified, encoding CSP belonging to the two dominating pherotypes,CSP-1 (62.7%) and CSP-2 (37.3%). The observed distribution of pherotypes in sets of co-colonising pneu-mococcal strains did not differ from the probability estimate. Thus, co-colonisation of S. pneumoniae inhealthy children is not restricted by pherotype.

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1. Introduction

The species Streptococcus pneumoniae belongs to the pneumo-niae-mitis-pseudopneumoniae cluster of the genus Streptococcus.It may act both as a commensal bacterium colonising the nasopha-ryngeal epithelium, mainly of young children, and as the aetiologicagent of various infectious diseases including acute otitis media,pneumonia and meningitis (Bogaert et al., 2004; Kilian et al.,2008). S. pneumoniae is naturally competent for transformation,and horizontal gene transfer by homologous recombination is themajor source of genetic diversity within the species (Griffith,1928; Feil et al., 2001). From studies of housekeeping genes in car-ried pneumococci and whole genome sequencing of a highly suc-cessful antibiotic resistant clone the rate of recombinationrelative to mutation rate of S. pneumoniae has been estimated tobe approximately three and seven, respectively (Fraser et al.,

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acteriology and Immunology,4, Nydalen, NO-0403 Oslo,

D.F. Vestrheim).

2005; Croucher et al., 2011). This results in sexual speciation anda loosely genetically structured population (Fraser et al., 2007).

The sole ecological niche for S. pneumoniae is the human naso-pharynx, and inter- and intra-species horizontal gene transfer isbelieved to take place between streptococcal strains co-inhabitingthe same host. Horizontal gene transfer between S. pneumoniae andother commensal streptococcal species leads to mosaic genesequences, and has been demonstrated, for example, as a mecha-nism for the transfer of penicillin resistance determinants to pneu-mococci (Hakenbeck et al., 2001; Chi et al., 2007). Recombinationwithin S. pneumoniae resulting in switch of capsule type is wellknown (Griffith, 1928), and may significantly impair vaccine effec-tiveness (Nesin et al., 1998; Brueggemann et al., 2007).

Competence for genetic transformation in S. pneumoniae andrelated species in the Mitis group is regulated by an extracellularsecreted peptide (Tomasz and Hotchkiss, 1964), a pheromonetermed competence-stimulating peptide (CSP), encoded by thecomC gene (Håvarstein et al., 1995). Induction of the competentstate occurs when the extracellular concentration of CSP reachesa critical level and is sensed by the two-component regulatory sys-tem ComDE, a histidine kinase receptor and a response regulator,

1704 D.F. Vestrheim et al. / Infection, Genetics and Evolution 11 (2011) 1703–1708

encoded by comD and comE, respectively (Håvarstein et al., 1996;Pestova et al., 1996).

The comC gene is polymorphic within the pneumoniae-mitis-pseudopneumoniae cluster (Kilian et al., 2008). However,two allelic variants dominate amongst S. pneumoniae, comC1 andcomC2, resulting in two dominating pherotypes producing CSP-1and CSP-2, respectively (Pozzi et al., 1996; Whatmore et al.,1999). Induction of competence by CSP is believed to be restrictedby the ComD receptor (Iannelli et al., 2005), i.e. competence willonly occur within a pherotype. Therefore, it has been suggestedthat inter-pherotype recombination should be limited and sexualisolation and genetic subpopulations within the species would beassociated with the two dominating pherotypes (Håvarsteinet al., 1997; Tortosa and Dubnau, 1999).

Cells in the competent state are able to kill non-competent sib-ling cells, a mechanism termed fratricide (Guiral et al., 2005); dur-ing the competent state, a comM-encoded membrane proteinprotects competent cells from their own lysins (Håvarstein et al.,2006). It has thus been postulated that bacteria responding toextracellular CSP, i.e. bacteria belonging to the same pherotypeor with a promiscuous ComD receptor, will be protected duringthe competent state, while bacteria of a different pherotype willbe subjected to fratricide due to lack of protective ComM(Johnsborg et al., 2008). By this mechanism, extensive inter- andintra-species horizontal gene transfer is made possible with thepherotypes acting as a diversity reservoir, while gene transferwithin a pherotype is restricted.

Two recently published studies of pherotype distributionamongst clinical pneumococcal isolates have reached opposingconclusions regarding the role of pherotype in competition andspeciation, either as restricting recombination (Carrolo et al.,2009) or not (Cornejo et al., 2010). Indeed, Claverys et al. (2007)have suggested that both may be relevant, with the comC systembalancing genome stability and diversification.

Asymptomatic colonisation with pneumococci is commonamongst children, and co-colonisation with different serotypes oc-curs frequently (Lloyd-Evans et al., 1996; O’Brien et al., 2007;Vestrheim et al., 2010), offering an opportunity for horizontal genetransfer between pneumococcal strains. The aim of the presentstudy was to evaluate the impact of pherotypes on co-habitationof pneumococci in the nasopharynx. We characterised sets ofstrains co-colonising healthy children by serotype, multilocus se-quence type (MLST) and comC allele sequence. We used probabilitycalculations to determine whether co-habitation by pneumococcalpherotypes occurred by random.

2. Materials and methods

2.1. Pneumococcal isolates

Co-colonising pneumococcal isolates were obtained from across-sectional study of nasopharyngeal carriage performed in2008 amongst children in day-care centres in Norway, followingintroduction of the seven-valent pneumococcal conjugate vaccine(PCV7). The study has been described in detail elsewhere(Vestrheim et al., 2010). Briefly, nasopharyngeal swabs were trans-ported in a serum broth, and, after plating onto blood agar, boththe broth and plate were incubated. Presence of pneumococciwas identified by serotyping directly from the broth, and con-firmed by growth of a-haemolytic and optochin sensitive colonieswith typical pneumococcal morphology on blood agar.

Of 602 samples obtained, 483 (80.2%) yielded growth of pneu-mococci. Multiple serotypes were identified in 72 (14.9%) of thepositive samples. In total, 152 pneumococcal isolates obtainedfrom 72 colonised children were subjected to further analyses in

the present study; in 8 samples three distinct strains were recov-ered (Vestrheim et al., 2010).

2.2. Phenotypic characterisation

The pneumococcal serotype was determined by latex agglutina-tion from the serum broth (Pneumotest-Latex kit, Statens SerumInstitut, Copenhagen, Denmark), and from isolates grown on bloodagar plates by the capsular reaction test (Quellung reaction) usingspecific antisera (Statens Serum Institut, Copenhagen, Denmark).

2.3. Genetic characterisation

Genomic DNA was prepared from isolate cultures by boiling a1-ll loopful of bacteria in 100 ll Tris–EDTA buffer for 10 min. Aftercentrifugation at 13,000 rpm for 5 min, the supernatants werestored at �20 �C.

All isolates were characterised by multilocus sequence typing asdescribed by Enright and Spratt (1998). Briefly, internal segments ofseven house-keeping genes (aroE, gdh, gki, recP, spi, ddl and xpt)were amplified and sequenced. Allele numbers and sequence types(STs) were assigned using the MLST database (http://www.mlst.net). Novel alleles and STs were submitted to the curatorof the database and assigned new designations. Clonal relationshipswere visualised using eBURST version 3 (http://eburst.mlst.net).

The comC gene was sequenced using two methods. In the firstmethod, primers complementary to Arg-tRNA and Glu-tRNA geneslocated upstream and downstream of the comCDE operon wereused for amplification as described by Håvarstein et al. (1996).The primers, 2tArg2: 50-CATAGCTCAGCTGGATAGAGCATTCGCCTTC-3 and 2tGlu: 50-GGCGGTGTCTTAACCCCTTGACCAACGGACC-3,were used in a standard PCR reaction with 30 cycles as follows:denaturation at 94� for 30 s, annealing at 60� for 30 s and elonga-tion at 72� for 2 min, yielding a 2.6 kb product. The comC sequencewas obtained using an internal sequencing primer, NPARG: 50-CGAACGGTCGCAGGTTCGAATCCTGCTGGGATC-3 (L. S. Håvarstein,personal communication).

If a comC sequence was not obtained using the above method,the protocol described by Cornejo et al. (2010), was employed.Briefly, the primers FOR: 50-CAATAACCGTCCCAAATCCA-30 andREV: 50-AAAAAGTACACTTTGGGAGAAAAA-30 were used in a stan-dard PCR reaction with 25 cycles as follows: denaturation at 94�for 30 s, annealing at 56� for 30 s and elongation at 72� for 1 min,yielding a product of 400 bp. The same primers were used forsequencing.

The comC sequences were translated into amino acid sequences,and the corresponding CSP and pherotype were assigned usingpublished sequences (Kilian et al., 2008).

2.4. Data analyses

We assumed that co-colonisation of pneumococcal pherotypesoccurs independently, and used the multiplicative rule for inde-pendent events,

ProbðA and BÞ ¼ ProbðAÞ � ProbðBÞ;

to postulate the distribution of concordant or discordant pairs of co-colonising pneumococci with respect to pherotype based on theoverall distribution in the sample set. E.g. the distribution of pairswith both strains belonging to pherotype CSP-1, Prob (CSP-1 andCSP-1), was calculated using the observed frequency distributionfor pherotype CSP-1.

Fisher’s exact test (GraphPad Prism) was used to evaluatewhether the observed distribution differed from the estimateddistribution.

D.F. Vestrheim et al. / Infection, Genetics and Evolution 11 (2011) 1703–1708 1705

3. Results

3.1. Serotypes and genotypes

Two isolates were excluded from the study as no comC PCRproduct was obtained (see Section 3.2). The remaining 150 isolatesbelonged to 27 different serotypes in addition to non-typeable (NT)isolates. PCV7 serotypes, i.e. serotypes 4, 6B, 9V, 14, 18C, 19F, and23F, accounted for 33 (22%) isolates.

By MLST the 150 isolates were assigned to 57 unique STs, ofwhich 7 STs were novel; ST4054, ST4055, ST4058, ST4110,ST4112, ST4114, ST4115. When using identity at 5 of 7 loci asgroup definition, the isolates were grouped into 13 clonal com-plexes (CC) and 25 singletons (Fig. 1).

3.2. comC alleles and pherotypes

The comC allele was sequenced for 150 of 152 pneumococcalisolates. For two isolates, a serotype 19F (ST476) and a serotype34 (ST3922), we were unable to obtain a product following re-peated PCR amplification using the two methods described. Thesewere the only isolates representing these two very divergent STs inour material. These isolates were excluded from further analyses inthe study, and consequently two of the 72 sample sets were ex-cluded from the concordance analysis.

Five comC alleles were identified, of which one had not previ-ously been described. Two allelic variants of comC1 were translatedto the same peptide, CSP-1; the codon for the 4th amino acid wasCTG in CSP-1 and TTG in CSP-1⁄, both encoding leucine. Two vari-ants of comC2 were identified, comC2.1 and comC2.2, differing onlyin the leading sequence and both being translated to CSP-2. In

Fig. 1. Population snapshot based on eBURST analysis of 150 pneumococcal strains.corresponds to the number of isolates. STs belonging to the CSP-1 pherogroup are numbelocus variants are linked by solid lines, constituting clonal complexes.

addition, a novel and unassigned comC allele, derivated fromcomC2.1, was identified and termed comC2.1b; a base deletion re-sulted in a Lys! Ser substitution in the 17th amino acid of the ma-ture CSP heptapenta-peptide. As a result of this deletion, the stopcodon terminating the translation was lost. This mutation was seenin all three ST393 isolates, serotype 38, recovered from three chil-dren attending two day-care centres in two distinct municipalities.In total, 94 (62.7%) of the isolates belonged to pherotype CSP-1,while 56 (37.3%) belonged to pherotype CSP-2 (Table 1).

In all cases, STs within a CC belonged to the same pherotype(Table 2, Fig. 1).

3.3. Pherotypes in sets of co-colonising pneumococci

The probability distribution of pherotypes in sets of co-colonis-ing pneumococci was estimated using Bayes rule of probability onthe overall distribution for all 150 available isolates. The CSP-coding sequence was available for all isolates in 70 sets of co-col-onising pneumococci. Sets with more than two isolates wereregarded concordant if all isolates belonged to the same pherotype,or discordant if two pherotypes were identified. Isolates with thecomC2.1b-allele variant were assigned to pherotype 2. Theestimated and observed frequency distribution of concordant anddiscordant sample sets were compared (Table 3). The observedand estimated distributions were not significantly different(p = 0.3, Fisher’s exact test).

3.4. Novel STs in sets of co-colonising pneumococci

The MLST allele sequences of 7 novel STs were evaluated andcompared with that of the most closely related ST in the MLST

Each sequence type (ST) is represented by a numbered circle, the size of whichred in black, and STs belonging to CSP-2 pherogroup are numbered in green. Single

Table 1Distribution of CSP pherotypes amongst 150 pneumococcal isolates from nasopharyngeal carriage. Amino acid differences within mainpherotypes are shown in bold.

CSP (pherotype) Amino acid sequence of CSPA No. of isolates (%)

CSP1 94 (62.7)CSP-1.0a mkntvkleqfvalkekdlqkikgg EMRLSKFFRDFILQRKK 64 (42.7)CSP-1.0⁄B mkntvkleqfvalkekdlqkikgg EMRLSKFFRDFILQRKK 30 (20.0)

CSP 2 53 (37.3)CSP-2.1 mkntvkleqfvalkekdlqkikgg EMRISRIILDFLFLRKK 47 (31.3)CSP-2.1bC mkntvkleqfvalkekdlqkikgg EMRISRIILDFLFLRKS 3 (2.0)

CSP-2.2 mkntvkleqfvalkekdlqnikgg EMRISRIILDFLFLRKK 6 (4.0)

A Lower case; leading sequence. Upper case; mature CSP.B CSP-1.0⁄; silent mutation.C CSP-2.1b; proposed terminology for novel comC allele.

Table 2Distribution of pherotypes within serotypes and genotypes amongst 150 pneumococcal isolates from nasopharyngeal carriage.

CSP(pherotype)

Serotype (STA [no. of isolates])

CSP1CSP-1.0a 6B (ST176 [3], ST2547 [1], ST2779 [2]), 18C (ST113 [2]), 19F (ST423 [1], ST1173 [1], ST3016 [1]), 23F (ST36 [3],

ST440 [7]), 1 (ST306 [2]), 6A (ST138 [1]), 7F (ST3795 [2]), 8 (ST53 [3], ST3778 [1]), 9N (ST405 [1]), 11A (ST62 [1],ST2549 [1]), 15B/C (ST1262 [9]), 16F (ST30 [2], ST3273 [1]), 21 (1877 [6], ST4055 [1]), 23A (ST439 [1]), 33F (ST100[3], ST673 [2], ST4115 [2]), 35B (ST198 [3]), NT (ST124 [1])

CSP-1.0⁄B 3 (ST180 [11], ST4054 [1]), 19A (ST199 [2], ST667 [1]), 11A (ST62 [1]), 15B/C (ST3976 [1], ST200 [2], ST199 [8]),22F (ST433 [1], ST4110 [2])

CSP2CSP-2.1 4 (ST205 [1]), 19F (ST177 [7], ST179 [3], ST462 [1]), 3 (ST1377 [1]), 6A (ST460 [3], ST1692 [1]),10A (ST97 [1],

ST2068 [1]), 17F (ST4112 [1]), 31 (ST444 [1]), 35B (ST452 [4], ST4114 [1]), 35F (ST446 [3], ST1635 [4]), 38 (ST4058[1]), NT (ST448 [1], ST449 [2], ST1054 [10])

CSP-2.1bC 38 (ST393 [3])CSP-2.2 24F (ST72 [6])

A ST; sequence type.B CSP-1.0⁄; silent mutation.C CSP-2.1b; proposed terminology for novel comC allele.

Table 3Estimated and observed frequency distribution of pneumococcal pherotypes in 70 sample sets.

Pherotypes in sample sets Estimated Observed

Probability proportion (%) No. of sample sets Proportion (%)

CSP-1 and CSP-1 39.7 23 32.9CSP-2 and CSP-2 13.7 7 10.0CSP-1 and CSP-2 46.6 40 57.1Concordant 53.4 30 42.9Discordant 46.6 40 57.1

Table 4Allele combinations in two novel sequence types (STs), their single locus variant (SLV)and co-colonising strain. Alleles for which within host recombination are suggestedare marked in bold. The suggested donor strains are marked in italic.

ST aroE gdh gki recP spi xpt ddl CSP

Set 1Co-colonising 199 8 13 14 4 17 4 14 CSP-1Novel STA 4054 7 15 2 10 6 4 22 CSP-1SLVB 180 7 15 2 10 6 1 22 CSP-1

Set 21Co-colonising 1262 7 41 2 6 10 26 1 CSP-1Novel STA 4058 10 43 41 18 10 49 6 CSP-2SLVB 393 10 43 41 18 13 49 6 CSP-2

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database, and with that of the co-colonising strain. No homologybetween novel ST and the co-colonising strain at allele level couldbe identified for ST4055, ST4110, ST4112, ST4114 and ST4115. Inthree of these novel STs, ST4110, ST4114 and ST4115, novel ddl al-leles were identified. For ST4112, no single locus variant (SLV) ordouble locus variant (DLV) had been entered in the database.ST4055 differed from its SLVs by diversity of the xpt allele; thesexpt alleles differed from those in the co-colonising strain.

On the other hand, in two novel STs, i.e. ST4054 and ST4058, theallele separating the novel ST from its SLV, xpt and spi, respectively,was shared with its co-colonising strain, suggesting within hostrecombination (Table 4). In one case, the proposed donor and reci-pient belonged to the same pherotype, while they differed in thesecond case.

A ST; sequence type.B SLV; single locus variant.

4. Discussion

Polymorphisms in the comC locus within S. pneumoniae appearto be limited, with only two dominating pherotypes (Pozzi et al.,

1996; Whatmore et al., 1999). The CSP polymorphism have beenproposed to have impact on the genetic differentiation of S. pneu-moniae by two opposing models, either resulting in sexual isolation

D.F. Vestrheim et al. / Infection, Genetics and Evolution 11 (2011) 1703–1708 1707

within pherotypes, or acting as a reservoir for genetic diversityacross pherotypes (Claverys and Håvarstein, 2007; Carrolo et al.,2009; Cornejo et al., 2010). Furthermore, if both ability to inducecompetence and evasion of fratricide differ between pneumococcalstrains, this might substantially impact the dynamics and evolu-tion of pneumococcal populations.

The human nasopharynx is the scene of action for horizontalgene transfer between pneumococcal strains or related species inthe Mitis group. However, it is unknown to what extent compe-tence is induced during asymptomatic colonisation, and to whatextent the resulting fratricide impacts the colonising strains. Com-petition between serotypes has been demonstrated during stablecolonisation in a mouse model, although the underlying mecha-nism has not been identified (Lipsitch et al., 2000). In a recent epi-demiologic study by Auranen et al. (2010), competition betweenserotypes is suggested to occur mainly at the stage of acquisition.

In pneumococci, development of competence occurs duringearly logarithmic growth in vitro, and in vivo competence resultingin horizontal gene transfer might be a stress response caused byexternal pH or antibiotic-induced stress (Claverys et al., 2006;Prudhomme et al., 2006; Claverys and Håvarstein, 2007). As de-scribed by Claverys and Håvarstein (2007), under laboratory condi-tions competence in pneumococci is induced in times of feastrather than famine. The pneumococcal strains analysed in the pres-ent study were obtained from asymptomatic and healthy childrenwith a limited history of consumption of antibiotics and with noongoing antibiotic treatment.

To our knowledge, the present study is the first study of CSP-distribution amongst colonising pneumococci, and co-colonisingstrains in particular. The distribution of pherotypes amongst theisolates analysed in the present study, 62% CSP-1 and 38% CSP2,was comparable to what has been observed in other studies basedon invasive disease isolates (Carrolo et al., 2009; Cornejo et al.,2010). However, a trend towards a slightly higher proportion ofstrains belonging to the CSP2 pherotype was noted. This mightbe due to a different serotype distribution in carried strains com-pared to invasive disease isolates.

The distribution of pherotypes in sets of co-colonising pneumo-coccal strains was not different from the expected proportionsbased on the overall pherotype distribution in the strain collection.This demonstrates that pneumococci belonging to distinct phero-types can co-exist in a stable and independent way in the naso-pharynx. Hence, fratricide committed by competent cells doesnot appear to impact the within-host competition between strainsof different pherotypes.

It may be argued that a cross-sectional study with a limitednumber of co-colonising strains as the one performed here willonly catch a glimpse of a dynamic situation during colonisation.However, in order to yield growth of a pneumococcal strain froma nasopharyngeal swab, the colonising strain must have persistedlong enough to multiply to a density sufficient for detection byour sampling. The co-colonisation observed thus requires at leasta level of stability in order to be detected. However, studies usinga longitudinal design would be of value to confirm the results ofthe present study. Although a sensitive culture method was used,pneumococci present in small proportions might be undetected.Studies of larger sample collections and with sensitive methodswould be preferable in order to confirm the level of co-colonisa-tion, and the proportions of comC-alleles.

The finding that all members of a CC belonged to the samepherotype indicates that pherotype is a stable clonal property.However, the eBURST snapshot does not provide detailed informa-tion on deep genetic relationships between clones.

Amongst seven novel STs described, two cases were identifiedin which acquisition of DNA from a co-habiting strain might haveoccurred resulting in novel STs (ST 4054 and ST 4058); the MLST

allele differing from the SLV corresponded to that of the co-colon-ising strain. Hence, the observed novel STs might be the result of arecombinational event between two clones present during co-col-onisation in the nasopharynx of these hosts.

Of the MLST genes, the ddl allele is highly divergent. The gene islocated close to the penicillin-binding protein 2b gene, and allelicdivergence is explained by hitch-hiking of ddl with pbp2b, a geneunder antibiotic selection (Enright and Spratt, 1999). Three novelSTs carried by five children were identified due to novel ddl alleles.Of these children, one had received amoxicillin during the threemonths preceding sampling. However, the strain was fully suscep-tible to penicillin, and it is doubtful whether this selective pressuremay have impacted the evolution of the new clone.

A novel comC allele sequence was identified, termed comC2.1bas it is closely related to the comC2.1 allele. In the novel allelethe 17th amino acid in the mature CSP is altered, and the stop co-don following the CSP-coding sequence is lost. This might lead to adefective CSP, and further study of the corresponding comD se-quence would be of interest in order to determine whether compe-tence would be expected to be induced by CSP-2.1 or CSP-2.1b.

Both environmental factors and regulatory proteins may impactdevelopment of competence (Johnsborg and Håvarstein, 2009).Furthermore, other mechanisms that might influence competitionduring colonisation and limit horizontal gene flow have been pro-posed. The ability to induce competence resulting in transforma-tion differs between serotypes, e.g. the level of competence islower for serotype 3 compared to serotype 14 and 19F (Hsiehet al., 2006). Blp bacteriocins, small antimicrobial peptides with atwo-component regulatory system similar to CSP, have been dem-onstrated to confer inter-species competition (Dawid et al., 2007).A recent experimental colonisation study by Lysenko et al. (2010)identified relative resistance to opsonophagocytosis conferred bypneumococcal capsules as a selective determinant for survival dur-ing co-colonisation. Thus, competition and genetic exchange dur-ing in vivo co-colonisation is complex and might be influencedby numerous factors in addition to CSP.

5. Conclusions

CSP polymorphism is limited amongst carried pneumococcalisolates, with only two dominating pherotypes. The pherotype dis-tribution in pairs of co-colonising strains indicates that pneumo-coccal pherotypes co-exist in the nasopharynx, and that theimpact of competence induced fratricide on competition is limited.

Acknowledgements

We are indebted to Leiv Sigve Håvarstein for valuable discus-sion during initial stages of the study, and for guidance on comCsequencing.

Martha Langedok Bjørnstad and Jan Oksnes are thanked forexcellent technical assistance.

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