Streptococcus pneumoniae can be classified in more than 90 capsular types, as traditionally determined by serological methods and more recently by PCR-based techniques. Such methods, however, can be expensive, laborious or unable to accurately discriminate among certain serotypes. Therefore, determination of capsular types, although extremely important for epidemiological purposes and for estimating the impact of pneumococcal conjugate vaccines, is mainly restricted to research laboratories, being rarely performed in the clinical setting. In the present study, MALDI-TOF MS was evaluated as an alternative tool to characterize 416 pneumococcal isolates belonging to serotypes 6A, 6B, 6C, 9N, 9V or 14. For MALDI-TOF MS analysis, each isolate was submitted to an extraction protocol using formic acid and acetonitrile. Measurements were performed with a Bruker Microflex LT mass spectrometer using default parameters and generating spectra in the range of 2,000–20,000 m/z. Spectra were analyzed with the BioNumerics software v7.6. Isolates were mainly distributed according to the capsular type in a Neighbor Joining tree and serotypes investigated were successfully discriminated by the presence/absence of 14 selected biomarkers. The results suggest that MALDI-TOF MS is a promising alternative for typing pneumococcal strains, highlighting its usefulness for rapid and cost-effective routine application in clinical laboratories.

Streptococcus pneumoniae is a major cause of community-acquired pneumonia and meningitis, and it is also found as a commensal, colonizing the human upper respiratory tract of a portion of the human population. Its polysaccharide capsule allows the recognition of more than 90 capsular types and represents the target of the currently available pneumococcal conjugate vaccines (PCVs), such as the 10-valent (PCV10) and the 13-valent (PCV13). Penicillin non-susceptible pneumococci (PNSP) have been listed as one of the current major antimicrobial-resistant pathogen threats. In Brazil, the emergence of PNSP was initially detected in the mid 1990s and PCV10 has been part of the National Immunization Program since 2010. Here, we investigated the distribution of capsular types and penicillin susceptibility profiles of 783 pneumococcal strains isolated in Brazil between 1990 and 2014 to assess the evolution of penicillin non-susceptibility among pneumococci associated with asymptomatic carriage and invasive pneumococcal disease (IPD). The most common serotypes among carriage isolates were 19F, 6B, 6C, 23F, and 14. Among IPD isolates, the most frequent types were 14, 3, 6B, 5, 19F, and 4. We detected 21 types exclusively associated with IPD isolates, whereas non-typeable (NT) isolates were only detected in carriage. Nearly half of the isolates belonged to PCV10 serotypes, which remarkably decreased in occurrence (by nearly 50%) after PCV10 introduction (2011–2014), while non-PCV10 serotypes increased. PNSP frequency and levels were much higher among carriage isolates, but PNSP belonging to PCV10 serotypes were more common in IPD. While the occurrence of PNSP has decreased significantly among IPD isolates since 2011, it kept increasing among carriage strains. Such a difference can be attributed to the serotypes that emerged in each clinical source after PCV10 usage. PNSP with multidrug resistance profiles that emerged within carriage isolates comprised mostly serotypes 6C and 35B, as well as NT isolates. In turn, penicillin-susceptible capsular types 3, 20, and 8 have risen among IPD. Overall, our results reinforce the relevance of PNSP surveillance over a long period of time to better understand the dynamics of antimicrobial resistance in response to PCV introduction and may also contribute to improve control measures toward drug-resistant pneumococci.

Streptococcus pneumoniae remains as an important cause of community-acquired bacterial infections, and the nasopharynx of asymptomatic carriers is the major reservoir of this microorganism. Pneumococcal strains of serotype 14 and serogroup 9 are among the most frequently isolated from both asymptomatic carriers and patients with invasive disease living in Brazil. Internationally disseminated clones belonging to such serotypes have been associated with the emergence and spread of antimicrobial resistance in our setting, highlighting the need for epidemiological tracking of these isolates. In this scenario, Multiple Loci VNTR Analysis (MLVA) has emerged as an alternative tool for the molecular characterization of pneumococci, in addition to more traditional techniques such as Multi-Locus Sequence Typing (MLST) and Pulsed-Field Gel Electrophoresis (PFGE). In the present study, 18 VNTR loci, as well as other previously described reduced MLVA panels (7 VNTR loci), were evaluated as tools to characterize pneumococcal strains of serotypes 14, 9N and 9V belonging to international and regional clones isolated in Brazil. The 18 VNTR loci panel was highly congruent with MLST and PFGE, being also useful for indicating the genetic relationship with international clones and for discriminating among strains with indistinguishable STs and PFGE profiles. Analysis of the results also allowed deducing a novel shorter 7 VNTR loci panel, keeping a high discriminatory power for isolates of the serotypes investigated and a high congruence level with MLST and PFGE. The newly proposed simplified panel was then evaluated for typing pneumococcal strains of other commonly isolated serotypes. The results indicate that MLVA is a faster and easier to perform, reliable approach for the molecular characterization of S. pneumoniae isolates, with potential for cost-effective application, especially in resource-limited countries.