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Emerging, Non-PCV13 Serotypes 11A and 35B of Streptococcus pneumoniae Show High Potential for Biofilm Formation In Vitro

  • Mirian Domenech ,

    Contributed equally to this work with: Mirian Domenech, Diana Damián

    Affiliations Centro de Investigaciones Biológicas, CSIC, Madrid, Spain, CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain

  • Diana Damián ,

    Contributed equally to this work with: Mirian Domenech, Diana Damián

    Affiliation Centro de Investigaciones Biológicas, CSIC, Madrid, Spain

  • Carmen Ardanuy,

    Affiliations CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain, Hospital Universitari de Bellvitge–Universitat de Barcelona–Fundació Privada Institut d’Investigació Biomèdica de Bellvitge, Barcelona, Spain

  • Josefina Liñares,

    Affiliations CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain, Hospital Universitari de Bellvitge–Universitat de Barcelona–Fundació Privada Institut d’Investigació Biomèdica de Bellvitge, Barcelona, Spain

  • Asunción Fenoll,

    Affiliation Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain

  • Ernesto García

    Affiliations Centro de Investigaciones Biológicas, CSIC, Madrid, Spain, CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain

Emerging, Non-PCV13 Serotypes 11A and 35B of Streptococcus pneumoniae Show High Potential for Biofilm Formation In Vitro

  • Mirian Domenech, 
  • Diana Damián, 
  • Carmen Ardanuy, 
  • Josefina Liñares, 
  • Asunción Fenoll, 
  • Ernesto García



Since the use of pneumococcal conjugate vaccines PCV7 and PCV13 in children became widespread, invasive pneumococcal disease (IPD) has dramatically decreased. Nevertheless, there has been a rise in incidence of Streptococcus pneumoniae non-vaccine serotypes (NVT) colonising the human nasopharynx. Nasopharyngeal colonisation, an essential step in the development of S. pneumoniae-induced IPD, is associated with biofilm formation. Although the capsule is the main pneumococcal virulence factor, the formation of pneumococcal biofilms might, in fact, be limited by the presence of capsular polysaccharide (CPS).

Methodology/Principal Findings

We used clinical isolates of 16 emerging, non-PCV13 serotypes as well as isogenic transformants of the same serotypes. The biofilm formation capacity of isogenic transformants expressing CPSs from NVT was evaluated in vitro to ascertain whether this trait can be used to predict the emergence of NVT. Fourteen out of 16 NVT analysed were not good biofilm formers, presumably because of the presence of CPS. In contrast, serotypes 11A and 35B formed ≥45% of the biofilm produced by the non-encapsulated M11 strain.


This study suggest that emerging, NVT serotypes 11A and 35B deserve a close surveillance.


Streptococcus pneumoniae (pneumococcus) is a leading human pathogen that naturally inhabits the upper respiratory tract. It usually colonises the associated mucosal surfaces in early childhood, and persists as a symptomless commensal (carrier state) in the nasopharynx [1]. Carriage is higher in children less than 5 years and decreases with age and is generally higher in developing countries and among economically deprived populations. Once carriage is established, however, S. pneumoniae may invade several sterile sites, leading to what is known as invasive pneumococcal disease (IPD). Indeed, the pneumococcus is responsible for episodes of bacteraemic community-acquired pneumonia, bacteraemia and meningitis, mainly in children, the elderly, and immunocompromised patients [2]. In addition, pneumococci are the main ethiologic agent of non-bacteraemic community-acquired pneumonia and a major cause of other non-invasive diseases such as acute otitis media, sinusitis, and conjunctivitis.

The pneumococcal capsule is composed of polysaccharides (capsular polysaccharide or CPS) that, in most cases, are covalently bound to the cell wall. At least 95 different pneumococcal serotypes are currently known, each with a biochemically different CPS. The capsule is the main pneumococcal virulence factor since it allows the bacterium to evade the host immune system by blocking phagocytosis [2]. Pneumococcal vaccines [3] are based on combinations of CPSs, and include a) a 23-valent pneumococcal polysaccharide vaccine; b) a 7-valent conjugate vaccine (PCV7), directed at serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, but no longer available in the market; c) a 10-valent conjugate vaccine (PCV10), licensed in Europe (but not in the United States) and directed at serotypes 1, 5, and 7F in addition to the serotypes included in PCV7; and d) a 13-valent pneumococcal conjugate vaccine (PCV13) that was licensed five years ago; this subsequently replaced PCV7. PCV13 includes six conjugate CPSs of serotypes 1, 3, 5, 6A, 7F, and 19A in addition to the serotypes included in PCV7.

The widespread use of conjugate vaccines has been very effective in reducing cases of IPD, although increases in disease caused by non-vaccine serotypes (NVT) (“serotype replacement”) have subsequently offset some of these reductions. Although the PCV7 serotype 6B appears to offer cross-protection against the non-PCV7 serotype 6A, this is not the case for 19F and the non-PCV7 serotype 19A [4]. Indeed, the incidence of infections caused by 19A multiresistant pneumococcus has increased since PCV7 vaccination became common. NVT are also on the rise in the current post-PCV7/PCV13 era. Surveillance program results suggest that pneumococci of various serogroups/serotypes not included in PCV13 (e.g., 11, 12, 15, 22F, 23A, 23B, 33F, 24, 34, and 35B) are rapidly increasing in prevalence worldwide [58]. Serotype 6C pneumococci, which are good biofilm formers [9] and whose CPS is not included in PCV13, were not investigated further.

Concerns about serotype replacement have influenced recent policy discussions [10]. Predicting the amount of future replacement is difficult since the reasons underlying a particular NVT increases after vaccine introduction are not fully understood. In the years immediately preceding PCV13 introduction, no single non-PCV13 serotype seemed an obvious candidate for replacement, based on current rates of disease, antimicrobial resistance, and carriage data. We recently proposed that quantifying the in vitro biofilm formation capacity of isogenic S. pneumoniae transformants expressing different CPSs might help predict the emergence (and eventual expansion) of NVT that are prone to colonise the human nasopharynx [9]. In that work we showed that clinical isolates and isogenic pneumococcal transformants of serotypes 19F and 19A (but not those of serotypes 19B and 19C) are capable of forming substantial amounts of biofilm in vitro. Strains of serogroup 6 also showed significant biofilm-forming capacity.

In the present study, 16 isogenic pneumococcal transformants expressing CPSs of non-PCV13 serotypes were constructed to determine whether biofilm formation can reliably predict the emergence of NVT of S. pneumoniae that colonise the human nasopharynx.

Materials and Methods

Media, growth conditions, DNA purification, and genetic transformation

Table 1 shows the pneumococcal strains examined. Clinical isolates of S. pneumoniae were from the collection of the Spanish Pneumococcal Reference Laboratory (SPRL; Centro Nacional de Microbiología ISCIII, Majadahonda, Madrid, Spain). All strains were grown in liquid CpH8 medium supplemented (or not) with 0.08% yeast extract (C+Y medium), or in solid medium involving trypticase soy agar or D agar supplemented with 5% defibrinated sheep blood (Thermo Scientific; Hampshire, England) [11]. Growth was controlled by measuring the optical density at 550 nm (OD550).

S. pneumoniae chromosomal DNA was purified from clinical strains and encapsulated, isogenic transformants constructed by transformation of strain M11, as described elsewhere [9]. The selection of transformants was made by enriching those encapsulated by successive transfer to C+A medium (CpH8 containing 0.08% of bovine seroalbumin) supplemented with 0.5 μl/ml anti-R antiserum (which agglutinates only non-encapsulated pneumococci) [9]. Serotyping of S. pneumoniae was kindly performed by D. Vicioso at the SPRL.

Biofilm formation assay, quantification, and statistical analysis

The optimal conditions for biofilm formation by pneumococcal cells on polystyrene microtitre plates have been previously described [11,14]. In short, pneumococcal cells were grown in C+Y medium at 37°C to an OD550 of 0.5–0.6 and diluted (1:100) in fresh C+Y medium. Aliquots (200 μl) were dispensed into each well in triplicate, and the plate incubated at 34°C for 6 h. Growth (OD595) was measured and the biofilm formed was stained with 1% crystal violet [11]. In the present study, a strain was considered as “good” or “intermediate” biofilm producer when it formed ≥45% or between 10% and 30% of the biofilm formed by the control, non-encapsulated M11 strain respectively. The data for biofilm formation include the mean ± standard error of at least three independent experiments, each performed in triplicate. Statistical significance was examined using the Student t test. For multiples comparisons, one-way analyses of variance (ANOVA) were performed, followed by Tukey’s post hoc test when the ANOVA rejected the null hypothesis. The SAS 9.3 statistical packge (SAS Institute, Cary, NC) was used for all analyses. Differences were considered statistically significant when P <0.05.


Fig 1 shows the biofilm formation capacity of clinical isolates of S. pneumoniae belonging to 16 non-PCV13 serotypes (Table 1). Two clinical isolates of each serotype were analysed in detail. Although pneumococci of every serotype tested produced less biofilm than the non-encapsulated strain M11, significant differences were noted in the biofilm formation capacity of several pairs of clinical strains of the same serotype: e.g., strains 2901 and 2963 (serotype 11A), 2948 and 2990 (serotype 15B/C), and 1544 and 3004 (serotype 23A). This confirms previous observations indicating that the genetic background, and not only the CPS, modulates pneumococcal attachment to the artificial substrate [9,11,15].

Fig 1. Biofilm formation.

Growth and biofilm formation by S. pneumoniae clinical isolates of the indicated serotypes, and their encapsulated M11 transformants. In all cases, open bars indicate bacterial growth. Hatched and blackened bars indicate, respectively, biofilm formation by either clinical isolates or their encapsulated M11 transformants. Grey bars correspond to biofilm formation by the M11 strain. Biofilm formation by the M11 strain was determined in all experiments with mean values between serotypes that were not significantly different (P >0.05 using one-way ANOVA).

Since in vitro biofilm formation by pneumococcus depends on many different genes and/or biochemical traits, the use of isogenic transformants (i.e., strains differing only by a single trait) is necessary when the influence of CPS in biofilm formation is to be measured. To this end, the non-encapsulated M11 strain was transformed with DNA prepared from the corresponding clinical isolates, and at least two independent transformants of each serotype were analysed. Since initial experiments revealed no significant differences between them, only one transformed strain of each serotype was studied further. The serotype 35B strains were the best biofilm producers; the P241 transformant strain produced ≈50% of the biofilm formed by M11. In addition, the P242 transformant (serotype 11A) was capable of synthesizing up to 45% of the biofilm produced by its non-encapsulated progenitor. Intermediate biofilm formation was noted for the isogenic S. pneumoniae transformants of serotype 23A. In contrast, serotype 33F pneumococci (whether clinical isolates or M11 transformant) were unable to produce any substantial quantity of biofilm in vitro (Fig 1).

Some variation was noted in the growth rate and/or biofilm formation capacity of the different strains (Fig 1). Consequently, the biofilm formation values for the encapsulated strains were normalised for the OD of the culture measured as stated in Materials and Methods, and the presented percentages were calculated in relation to the parental strain M11 [11]. Fig 2 shows the relative biofilm formation capacity of the isogenic transformants of the non-PCV13 serotypes compared with that of M11. Every serotype analysed formed significantly less biofilm than the non-encapsulated strain, although serotypes 35B and 11A formed significantly more biofilm than the other serotypes analysed.

Fig 2. Relative biofilm formation.

Relative biofilm formation capacity of M11 isogenic transformants with non-PCV13 serotypes compared to that of their parental non-encapsulated strain (M11). The percentages shown are the mean ± standard error of at least three independent experiments, each performed in triplicate. Relative biofilm formation was significantly different between serotypes (*, P <0.0001 in a one-way ANOVA). The Tukey’s post hoc test showed significant differences in biofilm formation between serotypes 35B and 11A, and the rest of serotypes. No significant differences were observed between the biofilm-forming capacity of serotypes 35B and 11A.


Recent years have seen continuous interest in the impact of pneumococcal conjugate vaccines on nasopharyngeal colonisation [16]. In fact, surveillance of colonisation has become an important component of the vaccination monitoring process in the post-licensure setting. Nasopharyngeal colonisation, which is associated with biofilm formation, is an essential step in the development of S. pneumoniae disease and prevention of colonisation may reduce host-to-host transmission [17,18]. Moreover, colonisation by multiple serotypes (co-colonisation), which appears to be much more prevalent than previously envisaged [19], is an important factor to consider as it facilitates horizontal gene transfer [20]. Thus, the ability of pneumococcal serotypes to form biofilms—which a previous publication [9] and the present study show can be rapidly tested in vitro—could be used to help predict the expansion of NVT. This method has recently shown isogenic transformants and clinical isolates of serotype 19A and serogroup 6, which were emerging during the PCV7 era, to be good biofilm-formers [9], and many studies have shown that the non-PCV13 serotypes analysed in the present study underwent a rapid increase in prevalence after PCV13 implementation. Nevertheless, no previous studies have assessed the ability of these serotypes to form biofilms in vitro.

The present results clearly show that most (14 out of 16) of the clinical isolates or isogenic transformants expressing the CPSs of NVT were unable to form substantial amounts of biofilm in vitro. Although an inverse relationship between CPS and biofilm formation exists [14], it has previously been shown that a minimum amount of CPS is necessary for efficient nasopharyngeal colonisation in mice [15]. The situation, however, is far from clear. For example, S. pneumoniae isolates of serotypes 8, 22F and 33F, which were found both in carriage and causing IPD, and which are potentially highly invasive [21], failed to produce substantial amounts of biofilms in vitro in the present study. This finding strongly suggests that in vitro biofilm formation is mostly unrelated to IPD-inducing capacity. It should be underlined that the CPSs of serotypes 22F and 33F are included in a 15-valent pneumococcal conjugate vaccine that is currently under pre-clinical evaluation [22].

The present results also show that the biofilm formation capacity of the corresponding isogenic transformants almost paralleled (and in some cases exceeded) that of the corresponding clinical isolates. One exception was seen, however: the clinical isolate 2948 (serotype 15B/C) (Fig 1). This strain showed significantly more biofilm formation capacity than strain P013, an M11 transformant of serotype 15B obtained using DNA from the reference strain SSISP15B/1 (Table 1). It may be that strain 2948 has a genetic background more prone to allow biofilm formation than strains M11 and 2990. It should be reminded that the serotypes of strains 2948 and 2990 were not determined; they might not, therefore, belong to the same serotype (Table 1, see footnote c).

In sharp contrast to the other serotypes tested here, isogenic transformants expressing the CPS of serotype 35B or 11A were capable of producing substantial amounts of biofilm in vitro, forming ≥45% of the amount of biofilm produced by the non-encapsulated M11 strain (Fig 2). This may explain the current prevalence of these serotypes in the human nasopharynx [23,24].

Many factors appear to influence the colonisation capacity of pneumococcal serotypes. For example, a previous study showed CPS (the major determinant of surface charge in S. pneumoniae) to be associated with colonisation capacity [25]. With the notable exceptions of serotypes 19A and 11A, higher net negative surface charge was associated with higher resistance to nonopsonic, neutrophil-mediated killing as well as higher carriage prevalence. It has also been suggested that a direct relationship exists between the success (in terms of relative numbers) of a serotype during carriage and the biochemistry of its CPS [26]. In fact, it has been recently shown that the CPSs of clinical isolates and isogenic transformants expressing the CPS of serotypes 19F and 19A, and all members of serogroup 6 (which are good biofilm producers), all include the disaccharides α-D-Glcp-(1→2)-α-L-Rhap-(1→ and α-D-Glcp-(1→3)-α-L-Rhap-(1→ [9]. However, neither of these disaccharides is present in the capsules of serotypes 11A or 35B (Fig 3). In addition, no chemical similarities between serotypes 11A and 35B CPSs are obvious—although both contain O-acetylated galactose residues. Recently, Calix et al. [27] showed that the newly discovered, invasive serotype 11E contains loss-of-function mutations in the capsule O-acetyltransferase gene wcjE, and does not express β-D-Galp6Ac at all. It is remarkably that, although frequently found in carriers, serotype 11A shows low invasiveness due to ficolin-2 recognition of O-acetylated capsule epitopes and the consequent activation of the lectin complement pathway activation [28]. It it well known that some serotypes are rarely found in carriage although they are known to cause disease, e.g., serotypes/serogroups 1, 3, 5, and 7 [29], but the reasons underlying how and when pneumococcal colonisation will result in dissemination and disease remain poorly understood [16,3032].

Fig 3. Primary structures of the capsular polysaccharides of S. pneumoniae serotypes 11A and 35B.

Data were taken from previous studies [13,27].

There is increasing interest in the interactions between the different species of the nasopharyngeal microbiota, because of the possibility that PCV vaccination might affect the ecology of the nasopharynx, which could have clinical consequences. This topic is subjected to debate; for example, an inverse relation between carriage of pneumococcus and Staphylococcus aureus has been found in some studies [33,34], but not in others [3537].

Evaluating vaccine efficacy for protection against colonisation with S. pneumoniae and other bacterial pathogens is an area of growing interest [38]. Conjugate vaccines lead to a reduction in the carriage prevalence of vaccine-serotype pneumococci in both vaccinated and unvaccinated individuals and a reduction in hospital admissions for invasive and non-invasive pneumococcal pneumonia in children younger than 5 years, as well as in some adult age groups, indicating herd protection [39]. Interestingly, it has been shown that estimating the adult burden of pneumococcal disease from bacteraemic pneumococcal pneumonia data alone significantly underestimates the true burden of disease in adults. For every case of bacteraemic pneumococcal pneumonia, it has been estimated that there are at least 3 additional cases of non-bacteraemic pneumococcal pneumonia [40]. Management of these infections is potentially being compromised by the increasing resistance of the pathogen to antibiotics commonly used to treat these infections [41]. Of note, increasing antibiotic resistance has been recently detected both in serotype 35B [42,43] and serotype 11A S. pneumoniae isolates [44]. However, since pneumococcal vaccines have been dessigned for the active prevention of IPD and pneumococci of serotypes 11A and 35B only seldom cause IPD [4547], it is questionable whether these serotypes should be included in future higher-valency PCVs. Yet, further studies of surveillance are needed in order to detect the emergence of non PCV-13 serotypes and allow rational vaccine design, implementation and continued effective control of pneumococcal disease.


The authors thank Pedro García for critically reading the manuscript, Guillermo Padilla for advice with statistical analysis, Adrian Burton for revising the English version, and Eloísa Cano and Susana Ruiz for skillful technical assistance.

Author Contributions

Conceived and designed the experiments: EG MD. Performed the experiments: MD DD. Analyzed the data: EG MD DD CA FL AF. Contributed reagents/materials/analysis tools: EG MD DD AF. Wrote the paper: EG MD DD CA FL AF JL.


  1. 1. Crook DW, Brueggemann AB, Sleeman KL, Peto TEA. Pneumococcal carriage. In: Tuomanen E, Mitchell TJ, Morrison DA, Spratt BG, editors. The Pneumococcus. Washington, D.C.: ASM Press; 2004. pp. 136–147.
  2. 2. Henriques-Normark B, Tuomanen EI. The pneumococcus: epidemiology, microbiology, and pathogenesis. Cold Spring Harb Perspect Med. 2013; 3: a010215. pmid:23818515
  3. 3. Grabenstein JD, Klugman KP. A century of pneumococcal vaccination research in humans. Clin Microbiol Infect. 2012; 18: 15–24. pmid:22882735
  4. 4. Hausdorff W, Hoet B, Schuerman L. Do pneumococcal conjugate vaccines provide any cross-protection against serotype 19A? BMC Pediatr. 2010; 10: 4. pmid:20122261
  5. 5. Ardanuy C, Marimón JM, Calatayud L, Giménez M, Alonso M, Grau I, et al. Epidemiology of invasive pneumococcal disease in older people in Spain (2007–2009): implications for future vaccination strategies. PLoS One. 2012; 7: e43619. pmid:22928005
  6. 6. Steens A, Bergsaker MAR, Aaberge IS, Rønning K, Vestrheim DF. Prompt effect of replacing the 7-valent pneumococcal conjugate vaccine with the 13-valent vaccine on the epidemiology of invasive pneumococcal disease in Norway. Vaccine. 2013; 31: 6232–6238. pmid:24176490
  7. 7. Camilli R, Daprai L, Cavrini F, Lombardo D, D’Ambrosio F, Del Grosso M, et al. Pneumococcal carriage in young children one year after introduction of the 13-valent conjugate vaccine in Italy. PLoS One. 2013; 8: e76309. pmid:24124543
  8. 8. Kaplan SL, Barson WJ, Lin PL, Romero JR, Bradley JS, Tan TQ, et al. Early trends for invasive pneumococcal infections in children after the introduction of the 13-valent pneumococcal conjugate vaccine. Pediatr Infect Dis J. 2013; 32: 203–207. pmid:23558320
  9. 9. Domenech M, Araújo-Bazán L, García E, Moscoso M. In vitro biofilm formation by Streptococcus pneumoniae as a predictor of post-vaccination emerging serotypes colonizing the human nasopharynx. Environ Microbiol. 2014; 16: 1193–1201. pmid:24373136
  10. 10. World Health Organization. Review of serotype replacement in the setting of 7-valent pneumococcal conjugate vaccine (PCV-7) use and implications for the PCV10/PCV13 era. Wkly Epidemiol Rec. 2012; 87: 12–13.
  11. 11. Moscoso M, García E, López R. Biofilm formation by Streptococcus pneumoniae: role of choline, extracellular DNA, and capsular polysaccharide in microbial accretion. J Bacteriol. 2006; 188: 7785–7795. pmid:16936041
  12. 12. Muñoz R, Mollerach M, López R, García E. Characterization of the type 8 capsular gene cluster of Streptococcus pneumoniae. J Bacteriol. 1999; 181: 6214–6219. pmid:10498742
  13. 13. Kamerling JP. Pneumococcal polysaccharides: a chemical view. In: Tomasz A, editor. Streptococcus pneumoniae Molecular Biology & Mechanisms of Disease. Larchmont, NY: Mary Ann Liebert, Inc; 2000. pp. 81–114.
  14. 14. Domenech M, García E, Moscoso M. Versatility of the capsular genes during biofilm formation by Streptococcus pneumoniae. Environ Microbiol. 2009; 11: 2542–2555. pmid:19549167
  15. 15. Domenech M, García E, Moscoso M. Biofilm formation in Streptococcus pneumoniae. Microb Biotechnol. 2012; 5: 455–465. pmid:21906265
  16. 16. Goldblatt D, Ramakrishnan M, O’Brien K. Using the impact of pneumococcal vaccines on nasopharyngeal carriage to aid licensing and vaccine implementation; A Pneumocarr meeting report March 27–28, 2012, Geneva. Vaccine. 2013; 32: 146–152. pmid:23933374
  17. 17. Bogaert D, van Belkum A, Sluijter M, Luijendijk A, de Groot R, Rümke HC, et al. Colonisation by Streptococcus pneumoniae and Staphylococcus aureus in healthy children. Lancet. 2004; 363: 1871–1872. pmid:15183627
  18. 18. Simell B, Auranen K, Käyhty H, Goldblatt D, Dagan R, O’Brien KL, et al. The fundamental link between pneumococcal carriage and disease. Expert Rev Vaccines. 2012; 11: 841–855. pmid:22913260
  19. 19. Saha S, Modak JK, Naziat H, Al-Emran HM, Chowdury M, Islam M, et al. Detection of co-colonization with Streptococcus pneumoniae by algorithmic use of conventional and molecular methods. Vaccine. 2015; 33: 713–718. pmid:25523524
  20. 20. Marks LR, Reddinger RM, Hakansson AP. High levels of genetic recombination during nasopharyngeal carriage and biofilm formation in Streptococcus pneumoniae. mBio. 2012; 3: e00200–00212. pmid:23015736
  21. 21. van Hoek AJ, Sheppard CL, Andrews NJ, Waight PA, Slack MPE, Harrison TG, et al. Pneumococcal carriage in children and adults two years after introduction of the thirteen valent pneumococcal conjugate vaccine in England. Vaccine. 2014; 32: 4349–4355. pmid:24657717
  22. 22. Skinner JM, Indrawati L, Cannon J, Blue J, Winters M, MacNair J, et al. Pre-clinical evaluation of a 15-valent pneumococcal conjugate vaccine (PCV15-CRM197) in an infant-rhesus monkey immunogenicity model. Vaccine. 2011; 29: 8870–8876. pmid:21964055
  23. 23. Martin JM, Hoberman A, Paradise JL, Barbadora KA, Shaikh N, Bhatnagar S, et al. Emergence of Streptococcus pneumoniae serogroups 15 and 35 in nasopharyngeal cultures from young children with acute otitis media. Pediatr Infect Dis J. 2014; 33: e286–e290. pmid:24911895
  24. 24. McElligott M, Vickers I, Cafferkey M, Cunney R, Humphreys H. Non-invasive pneumococcal serotypes and antimicrobial susceptibilities in a paediatric hospital in the era of conjugate vaccines. Vaccine. 2014; 32: 3495–3500. pmid:24795223
  25. 25. Li Y, Weinberger DM, Thompson CM, Trzciński K, Lipsitch M. Surface charge of Streptococcus pneumoniae predicts serotype distribution. Infect Immun. 2013; 81: 4519–4524. pmid:24082068
  26. 26. Weinberger DM, Trzciński K, Lu Y- J, Bogaert D, Brandes A, Galagan J, et al. Pneumococcal capsular polysaccharide structure predicts serotype prevalence. PLoS Pathog. 2009; 5: e1000476. pmid:19521509
  27. 27. Calix JJ, Brady AM, Du VY, Saad JS, Nahm MH. Spectrum of pneumococcal serotype 11A variants results from incomplete loss of capsule O-acetylation. J Clin Microbiol. 2014; 52: 758–765. pmid:24352997
  28. 28. Brady AM, Calix JJ, Yu J, Geno KA, Cutter GR, Nahm MH. Low invasiveness of pneumococcal serotype 11A is linked to ficolin-2 recognition of O-acetylated capsule epitopes and lectin complement pathway activation. J Infect Dis. 2014; 210: 1155–1165. pmid:24683196
  29. 29. del Amo E, Selva L, de Sevilla MF, Ciruela P, Brotons P, Triviño M, et al. Estimation of the invasive disease potential of Streptococcus pneumoniae in children by the use of direct capsular typing in clinical specimens. Eur J Clin Microbiol Infect Dis. 2014: doi:0.1007/s10096-10014-12280-y.
  30. 30. Marks LR, Davidson BA, Knight PR, Hakansson AP. Interkingdom signaling induces Streptococcus pneumoniae biofilm dispersion and transition from asymptomatic colonization to disease. mBio. 2013; 4: e00438–00413. pmid:23882016
  31. 31. Charalambous BM, Leung MH. Pneumococcal sepsis and nasopharyngeal carriage. Curr Opin Pulm Med. 2012; 18: 222–227. pmid:22343427
  32. 32. Vernatter J, Pirofski L-a. Current concepts in host–microbe interaction leading to pneumococcal pneumonia. Curr Opin Infect Dis. 2013; 26: 277–283. pmid:23571695
  33. 33. van Gils EJM, Hak E, Veenhoven RH, Rodenburg GD, Bogaert D, Bruin JP, et al. Effect of seven-valent pneumococcal conjugate vaccine on Staphylococcus aureus colonisation in a randomised controlled trial. PLoS One. 2011; 6: e20229. pmid:21695210
  34. 34. van den Bergh MR, Biesbroek G, Rossen JWA, de Steenhuijsen Piters WA, Bosch AATM, van Gils EJM, et al. Associations between pathogens in the upper respiratory tract of young children: interplay between viruses and bacteria. PLoS One. 2012; 7: e47711. pmid:23082199
  35. 35. Hammitt LL, Akech DO, Morpeth SC, Karani A, Kihuha N, Nyongesa S, et al. Population effect of 10-valent pneumococcal conjugate vaccine on nasopharyngeal carriage of Streptococcus pneumoniae and non-typeable Haemophilus influenzae in Kilifi, Kenya: findings from cross-sectional carriage studies. Lancet Glob Health. 2014; 2: e397–e405. pmid:25103393
  36. 36. Nzenze SA, Shiri T, Nunes MC, Klugman KP, Kahn K, Twine R, et al. Temporal association of infant immunisation with pneumococcal conjugate vaccine on the ecology of Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus nasopharyngeal colonisation in a rural South African community. Vaccine. 2014; 32: 5520–5530. pmid:25101982
  37. 37. Nunes MC, Jones SA, Groome MJ, Kuwanda L, Van Niekerk N, von Gottberg A, et al. Acquisition of Streptococcus pneumoniae in South African children vaccinated with 7-valent pneumococcal conjugate vaccine at 6, 14 and 40 weeks of age. Vaccine. 2015; 33: 628–634. pmid:25541213
  38. 38. Auranen K, Rinta-Kokko H, Goldblatt D, Nohynek H, O'Brien KL, Satzke C, et al. Colonisation endpoints in Streptococcus pneumoniae vaccine trials. Vaccine. 2013; 32: 153–158. pmid:24016803
  39. 39. Simonsen L, Taylor RJ, Schuck-Paim C, Lustig R, Haber M, Klugman KP. Effect of 13-valent pneumococcal conjugate vaccine on admissions to hospital 2 years after its introduction in the USA: a time series analysis. Lancet Respir Med. 2014; 2: 387–394. pmid:24815804
  40. 40. Said MA, Johnson HL, Nonyane BA, Deloria-Knoll M, O'Brien KL, for the AGEDD Adult Pneumococcal Burden Study Team. Estimating the burden of pneumococcal pneumonia among adults: a systematic review and meta-analysis of diagnostic techniques. PLoS One. 2013; 8: e60273. pmid:23565216
  41. 41. Feldman C, Anderson R. Recent advances in our understanding of Streptococcus pneumoniae infection. F1000Prime Rep. 2014; 6: 82. pmid:25343039
  42. 42. Mendes RE, Biek D, Critchley IA, Farrell DJ, Sader HS, Jones RN. Decreased ceftriaxone susceptibility in emerging (35B and 6C) and persisting (19A) Streptococcus pneumoniae serotypes in the United States, 2011–2012: ceftaroline remains active in vitro among β-lactam agents. Antimicrob Agents Chemother. 2014; 58: 4923–4927. pmid:24867974
  43. 43. Richter SS, Diekema DJ, Heilmann KP, Dohrn CL, Riahi F, Doern GV. Changes in pneumococcal serotypes and antimicrobial resistance after introduction of the 13-valent conjugate vaccine in the United States. Antimicrob Agents Chemother. 2014; 58: 6484–6489. pmid:25136018
  44. 44. Domenech A, Tirado-Vélez JM, Fenoll A, Ardanuy C, Yuste J, Liñares J, et al. Fluoroquinolone-resistant pneumococci: dynamics of serotypes and clones in Spain in 2012 compared with those from 2002 and 2006. Antimicrob Agents Chemother. 2014; 58: 2393–2399. pmid:24514095
  45. 45. Hsu HE, Shutt KA, Moore MR, Beall BW, Bennett NM, Craig AS, et al. Effect of pneumococcal conjugate vaccine on pneumococcal meningitis. N Engl J Med. 2009; 360: 244–256. pmid:19144940
  46. 46. Chiba N, Morozumi M, Shouji M, Wajima T, Iwata S, Ubukata K, et al. Changes in capsule and drug resistance of pneumococci after introduction of PCV7, Japan, 2010–2013. Emerg Infect Dis. 2014; 20: 1132–1139. pmid:24960150
  47. 47. Grau I, Ardanuy C, Calatayud L, Schulze MH, Liñares J, Pallares R. Smoking and alcohol abuse are the most preventable risk factors for invasive pneumonia and other pneumococcal infections. Int J Infect Dis. 2014; 25: 59–64. pmid:24853638