MLVA Typing of Streptococcus pneumoniae Isolates with Emphasis on Serotypes 14, 9N and 9V: Comparison of Previously Described Panels and Proposal of a Novel 7 VNTR Loci-Based Simplified Scheme

Abstract

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.

Virulence-Associated Characteristics of Serotype 14 and Serogroup 9 Streptococcus pneumoniae Clones Circulating in Brazil: Association of Penicillin Non-susceptibility With Transparent Colony Phenotype Variants

“…The opaque phenotype was the most common overall and also within each one of all the clones investigated in this study, except for ST199 isolates, which we have shown to be a probable result of capsular switching events ( Costa et al, 2016 ). Among these three serotype 14 ST199 variants, two were exclusively composed of transparent variants, while the third was composed of a mixed phenotype, indicating that the genotype in which capsular recombination events were assumed to occur was also the only genotype where the transparent phenotype was more frequent.…”

“…However, ST199 isolates usually belong to serogroups 15 or 19 2 , and the ones included in the present study were of serotype 14, representing the first serotype 14 variants of this clone to be reported ( Pinto et al, 2016 ). We have also previously shown that these serotype 14 ST199 variants might have been generated by capsular switching events since they were genetically more closely related to strains belonging to serotype 15B than to other isolates of serotype 14 ( Costa et al, 2016 ). Previous studies have indicated that capsular switching events in pneumococci can lead to other simultaneous recombination events spanning a genome region of approximately 750 kb, which includes the capsular locus and the pilus type 1 operon ( Sjöström et al, 2007 ; Croucher et al, 2014 ; Metcalf et al, 2016 ); thus, in these serotype 14 ST199 variants, PI-1 coding genes could have been lost during recombination events in the capsular locus that led to serotype switching.…”

“…In addition, genetic analyses have demonstrated that BOX elements (repeat sequences in the pneumococcal genome) can have a role in the frequency of phase variation ( Saluja and Weiser, 1995 ; Li et al, 2016 ). Many of the 315 pneumococcal isolates included in this study were previously analyzed by MLVA ( Costa et al, 2016 ), a methodology that targets BOX elements. This previous study showed interesting differences in the number of repeat sequences in certain BOX elements between ST156 and ST162 isolates.…”

“…This previous study showed interesting differences in the number of repeat sequences in certain BOX elements between ST156 and ST162 isolates. In the BOX element represented by locus Spneu40, for example, two or three repeats were found in ST162 isolates, while a number of eight repeats were observed in ST156 isolates ( Costa et al, 2016 ).…”

“…All the isolates were previously characterized regarding capsular type, antimicrobial susceptibility profile, and multilocus sequence typing ( Pinto et al, 2016 ). A number of them (89 isolates) were also previously characterized by multiple locus variable-number tandem repeat (VNTR) analysis (MLVA; Costa et al, 2016 ).…”