Streptococcus pneumoniae, or pneumococcus, is a leading cause of infections, such as pneumonia and meningitis, among children > 5 years old. In addition, this microorganism is also commonly found colonizing the human upper respiratory tract, a niche considered as its major reservoir and the main entry for the establishment of invasive pneumococcal disease (IPD) (Lynch and Zhanel, 2009; Weiser, 2010; Tan, 2012; Donkor, 2013).

This pathogen presents a polysaccharide capsule as the most important virulence factor (Bogaert et al., 2004; Kadioglu et al., 2008; Hyams et al., 2010). The pneumococcal capsule is antigenically diverse allowing the recognition of more than 90 serotypes (Bentley et al., 2006; Mostowy et al., 2017). In addition, the polysaccharide capsule is the basis of licensed vaccine formulations against pneumococcal disease, including the 7-valent pneumococcal conjugate vaccine (PCV7), the 10-valent PCV (PCV10), and the 13-valent PCV (PCV13) (WHO, 2012).

Penicillin non-susceptible pneumococci (PNSP) were recently listed as one of the most important antimicrobial-resistant threats worldwide (CDC, 2013; WHO, 2017). Increasing occurrence of PNSP has been detected since the first report in 1967 in Australia (Hansmann and Bullen, 1967; Castañeda et al., 1998; Appelbaum, 2002; Sadowy et al., 2010; Hackel et al., 2013; Kim et al., 2016). This characteristic seems to be more commonly associated with certain serotypes, such as serotype 14 and those included in serogroups 6, 19, and 23 (McGee et al., 2001; Lee et al., 2014). In Brazil, the emergence of PNSP was initially documented in the mid 1990s and it was initially attributed to the introduction of an internationally disseminated clone (namely ST156) expressing the capsular type 14 (Brandileone et al., 2006; Pinto et al., 2016).

Different measures can affect the epidemiology and evolution of PNSP isolates, including antibiotic therapy policies and the implementation of vaccines. However, such interventions may vary according to the geographical region (Guillemot et al., 1998; McCormick et al., 2003; Kim et al., 2016). Brazil is one of the 32 countries that have introduced PCV10 into the national immunization program, starting in 2010 (Brazil Ministry of Health, 2010). In turn, PCV13 has simultaneously replaced PCV7 in private immunization clinics. Thus, the aim of the present study was to investigate the distribution of capsular types and penicillin susceptibility profiles among pneumococcal isolates recovered from asymptomatic carriage and IPD over a period of 25 years in Brazil, comprising the periods before and after PCV introduction.

Differences in the distribution of pneumococcal serotypes between carriage and IPD isolates were observed. Some serotypes, including 3, 4, and 5, were exclusively detected among IPD cases. Previous studies have shown that certain capsular types are more prone to cause IPD while others are well-adapted to nasopharynx colonization (Bender et al., 2008; Weiser, 2010; Weinberger et al., 2011). Pneumococcal strains lacking the polysaccharide capsule (NT isolates), for example, are believed to be less virulent (Sharma et al., 2013). Accordingly, NT isolates were only identified among pneumococcal isolates recovered from asymptomatic carriage. On the other hand, a group of serotypes seems to be highly versatile, being frequently found in both carriage and IPD. In this study, three capsular types were frequently found regardless of clinical source, including 6B, 14, and 19F. Indeed, these serotypes are known to be common among carriage and IPD worldwide before PCV introduction (Hausdorff, 2007; Weinberger et al., 2011; Song et al., 2013).

Nearly 20% of all the isolates were PNSP, which is in accordance with previous data from Brazilian studies (Neves et al., 2013; Mott et al., 2014; dos Santos et al., 2015). However, differences on the distribution of penicillin resistance were also noted when carriage and IPD isolates were compared. PNSP occurrence, as well as penicillin MIC levels, were higher among carriage isolates. Indeed, certain serotypes almost exclusively found in IPD, such as serotype 3, were fully susceptible to penicillin. Several studies have shown that pneumococcal serotypes commonly found in carriage are more frequently associated with antimicrobial resistance than those exclusively found in IPD isolates (Weiser, 2010; Song et al., 2013; Zhou et al., 2015; Kim et al., 2016; Neves et al., 2017). This observation may be due, at least in part, to the fact that the human nasopharynx, in contrast to blood or cerebrospinal fluid, is a highly populated niche where genetic exchange among bacteria occurs and, thus, emergence of antimicrobial resistance traits can be favored (Andam and Hanage, 2015; Kim et al., 2016).

Although fluctuations on the occurrence of serotypes over time can happen naturally and should be carefully evaluated, our results suggest that the introduction of PCV7 and PCV13 in 2001 and 2010, respectively, did not seem to have affected pneumococcal epidemiology regarding serotype and PNSP distribution in our setting. This might be due, at least in part, to the fact that these PCVs were made available only in private clinics in Brazil. Indeed, usage of PCV7 and PCV13 in Brazil is very low due to their high cost (Brazil Ministry of Health, 2006; Medeiros et al., 2017; Neves et al., 2017). On the other hand, according to previous studies conducted in Brazil (dos Santos et al., 2013; Medeiros et al., 2017; Neves et al., 2018), our results suggest an important impact on serotype replacement after the implementation of PCV10. PCV10 serotypes showed a decreasing trend over time, especially in the late study period (2011–2014). In parallel, occurrence of non-PCV10 serotypes increased over time, surpassing the numbers of PCV10 serotypes in both carriage and IPD between 2011 and 2014. Similar observations have been made in other countries where PCV10 was routinely adopted, such as the Netherlands, Mozambique and Finland (Knol et al., 2015; Nhantumbo et al., 2017; Sihvonen et al., 2017).

Among the non-PCV10 serotypes emerging after PCV10 introduction, serotype 19A was an important serotype associated with both carriage and IPD. Although emergence of this serotype after PCV7 introduction in certain high-income countries is a well-established fact (Isaacman et al., 2010; Isturiz et al., 2017), serotype 19A emergence after PCV10 introduction in Brazil still seems to be a contradictory issue. While certain studies reveal that occurrence of this serotype has not significantly changed (Medeiros et al., 2017; Neves et al., 2018), others report an increasing rate (Cassiolato et al., 2018; Christophe et al., 2018). We also observed that serotypes 3, 8, 20, and 23A emerged among isolates from IPD cases, whereas serotypes 6C, 35B, and NT isolates were more commonly associated with asymptomatic carriage. Emergence of serotype 6C in carriage and of serotypes 3 and 8 in IPD after PCV10 implementation in Brazil has been recently described (Medeiros et al., 2017; Christophe et al., 2018; Neves et al., 2018). Of note, all these emerging non-PCV10 serotypes have been circulating in our setting since the 1990s, reinforcing the possibility of serotype replacement phenomenon.

Moreover, while PNSP numbers and levels decreased significantly in the late period of the present study (2011–2014) among IPD isolates, they kept increasing among isolates from carriage. Accordingly, many studies have reported lower frequencies and levels of PNSP among IPD isolates after PCV10 introduction in Brazil (dos Santos et al., 2013; Medeiros et al., 2017). In turn, antimicrobial resistance levels among pneumococcal isolates from asymptomatic carriage have been increasing despite of vaccination. Recently, Neves et al. (2018) have suggested that this is probably due to the emergence of multidrug resistant lineages belonging to non-PCV10 serotypes, such as the serotype 6C-CC386, among carriage isolates. On the other hand, serotypes emerging among IPD isolates after PCV10 introduction, such as 3, 8 and 20, were shown to be fully susceptible to penicillin. These observations suggest that the PCV10 impact on the reduction of PNSP occurrence and level might be more relevant for IPD than for carriage. This suggestion can also be supported by the observation that, before PCV10 introduction, PNSP isolates recovered from IPD were almost completely represented by PCV10 serotypes (nearly 90%), while only half of PNSP strains recovered from asymptomatic carriage comprised PCV10 serotypes.

Penicillin non-susceptible pneumococci have been listed as one of the major antimicrobial resistance threats among bacterial pathogens (CDC, 2013; WHO, 2017). Although they represent a global public health threat, occurrence and epidemiology of PNSP vary according to the geographic region. Taken our results into consideration, from the mid 1990s until 2010, serotype 14 played a major role in the dispersion of penicillin non-susceptibility, especially among IPD isolates. Indeed, it was previously shown that an internationally disseminated clone belonging to this serotype (namely ST156), which was also frequently associated with IPD worldwide, was the main reason for PNSP emergence in Brazil in the pre-vaccination era (Barroso et al., 2012; Pinto et al., 2016). After 2010, however, this scenario has changed and a more diversified panel of serotypes has been associated with penicillin non-susceptibility, regardless of clinical source. Among IPD isolates specifically, serotype 19A PNSP emerged significantly, surpassing the previous number of serotype 14 PNSP isolates.

Major limitations of this study are related to the characteristics of the population included. It is known that age of individuals is an important feature and may have an influence on serotype distribution. However, we were not able to assess this issue in detail since information was not available for a large proportion of the isolates analyzed, although we estimate from available data that most of strains were recovered from children. In addition, although Brazil is a country with continental dimensions and, thus, might present discrepancies between regions, the Southeastern region, represented here by five different cities, is the most populated one. According to the last official demographic survey conducted in Brazil (Instituto Brasileiro de Geografia e Estatística [IBGE], 2010), population living in the Southeastern region accounted for nearly half of the whole Brazilian population. Moreover, this region can be considered as representative of the ethnic, social, and economic diversity of the Brazilian population due to the historic high flow of domestic in-migration.

Penicillin non-susceptible pneumococci evolution can be driven by different interventions such as antibiotic therapy policies and vaccine implementation (Guillemot et al., 1998; McCormick et al., 2003; Kim et al., 2016). These aspects usually differ by country; for example, Brazil is one of the 32 countries that have adopted PCV10 in the national immunization program instead of PCV7/PCV13, adopted by other 98 countries (Brazil Ministry of Health, 2010). Therefore, gathering information on PNSP epidemiology over a long period of time can contribute to a better understanding of their evolution and the impact of different vaccination strategies. Overall, our results show the emergence of non-PCV10 serotypes after 2010 in Brazil and the emergence and spread of PNSP associated with carriage. On the other hand, PCV10 has been effective in decreasing PNSP rates and levels among IPD isolates, but it has not avoided serotype replacement. These results reinforce the need of continuous surveillance of PNSP in the post-vaccine introduction era and may contribute to the development of more effective measures to control the spread of drug-resistant pneumococci.

JP and LT coordinated the study. TP, LT, and JP contributed to the conception and design of the work. TP, FN, AS, LO, NC, CM-S, and LC performed the experiments and analyzed the data. LO performed the statistical analyses. TP, LO, JP, and LT wrote the manuscript. All authors revised and approved the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.