Gene duplication, gene loss, and recombination events with variola virus shaped the complex evolutionary path of historical American horsepox-based smallpox vaccines.

IMPORTANCE: Modern smallpox vaccines, such as those used against mpox, are made from vaccinia viruses, but it is still unknown whether cowpox, horsepox, or vaccinia viruses were used in the early 20th century or earlier. The mystery began to be solved when the genomes of six historical smallpox vaccines used in the United States from 1850 to 1902 were determined. Our work analyzed in detail the genomes of these six historical vaccines, revealing a complex genomic structure. Historical vaccines are highly similar to horsepox in the core of their genomes, but some are closer to the structure of vaccinia virus at the ends of the genome. One of the vaccines is a recombinant virus with parts of variola virus recombined into its genome. Our data add valuable information for understanding the evolutionary path of current smallpox vaccines and the genetic makeup of the potentially extinct group of horsepox viruses.

Description of optochin-resistant Streptococcus pneumoniae due to an uncommon mutation in the atpA gene and comparison with previously identified atpC mutants from Brazil.

Optochin susceptibility testing is a major assay used for presumptive identification of Streptococcus pneumoniae. Still, atypical optochin-resistant (Optr) pneumococci have been reported and this phenotype has been attributed to nucleotide substitutions in the genes coding for the F0F1ATPase. While substitutions in the atpC gene (c-subunit of ATPase) are more common and better characterized, data on mutations in the atpA (a-subunit) are still limited. We have characterized five Optr isolates presenting alterations in the atpA (Trp206Cys in four isolates and Trp206Ser in one isolate), constituting the first report of such mutations in Brazil. Most of the Optr isolates consisted of heterogeneous populations. Except for Opt MICs and the nucleotide changes in the atpA gene, Optr and Opts subpopulations originating from the same culture had identical characteristics. In addition, we compared phenotypic and genetic characteristics of these atpA mutants with those of atpC mutants previously identified in Brazil. No structural alterations were detected among predicted proteins, regardless of mutations in the coding gene, suggesting that, despite the occurrence of mutations, protein structures tend to be highly conserved, ensuring their functionalities. Phylogenetic analysis revealed that atypical Optr strains are true pneumococci and Opt resistance does not represent any apparent selective advantage for clinical isolates.

Characterization of Streptococcus pneumoniae serotype 19F-variants occurring in Brazil uncovers a predominant lineage that can lead to misinterpretation in capsular typing.

BACKGROUND: Streptococcus pneumoniae (S. pneumoniae) of serogroup 19 are mainly represented by serotypes 19A and 19F, which are associated with antimicrobial resistance and disease. The wzy gene, a component of the pneumococcal capsular locus, is the target to differentiate serotypes 19A and 19F by PCR-based capsular typing. In the last decade, allelic variants of the wzy19F gene have been described, leading to misinterpretation of capsular typing results. METHODS: A collection of 154 serotype 19F S. pneumoniae strains recovered from carriage and disease in Brazil was evaluated to identify and characterize wzy19F variant isolates. RESULTS: Eleven (7%) wzy19F variant isolates were detected and identified as belonging to ST810 (n = 10) or ST13673 (n = 1; single-locus variant of ST810). They were mostly recovered from diseased patients, susceptible to the antimicrobial agents tested (except for one multidrug-resistant strain) and did not harbor pili genes. Sequences of the wzy19F gene of these variants were identical to each other and to those previously described in Brazil, but slightly different from wzy19F variants identified in other countries. CONCLUSION: This study indicated that wzy19F variants present a geographically driven distribution and was the first to uncover phenotypic and genetic features of a wzy19F variant lineage occurring in Brazil since 1989.

Re-assembly of nineteenth-century smallpox vaccine genomes reveals the contemporaneous use of horsepox and horsepox-related viruses in the USA.

According to a recent article published in Genome Biology, Duggan and coworkers sequenced and partially assembled five genomes of smallpox vaccines from the nineteenth century. No information regarding the ends of genomes was presented, and they are important to understand the evolutionary relationship of the different smallpox vaccine genomes during the centuries. We re-assembled the genomes, which include the largest genomes in the vaccinia lineage and one true horsepox strain. Moreover, the assemblies reveal a diverse genetic structure in the genome ends. Our data emphasize the concurrent use of horsepox and horsepox-related viruses as the smallpox vaccine in the nineteenth century.

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.

Streptococcus pneumoniae remains a major agent of invasive diseases, especially in children and the elderly. The presence of pneumococcal capsule, pneumococcal surface protein A (PspA), and pilus type 1 (PI-1) and the ability of colony phase variation are assumed to play important roles in the virulence potential of this microorganism. Differences in the capsular polysaccharide allow the characterization of more than 90 pneumococcal serotypes; among them, serotype 14 and serogroup 9 stand out due to their prevalence in the pre- pneumococcal conjugate vaccine era and frequent association with penicillin non-susceptibility. Here we investigated the distribution of PI-1 and pspA genes and colony phase variants among 315 S. pneumoniae isolates belonging to serotype 14 and serogroup 9, recovered over 20 years in Brazil, and correlated these characteristics with penicillin susceptibility and genotype as determined by multilocus sequence typing. All strains were shown to carry pspA genes, with those of family 2 (pspA2) being the most common, and nearly half of the strains harbored P1-1 genes. The pspA gene family and the presence of PI-1 genes were conserved features among strains belonging to a given clone. A trend for increasing the occurrence of pspA2 and PI-1 genes over the period of investigation was observed, and it coincided with the dissemination of CC156 (Spain9V -3) clone in Brazil, suggesting a role for these virulence attributes in the establishment and the persistence of this successful clone. Opaque variant was the colony phenotype most frequently observed, regardless of clonal type. On the other hand, the transparent variant was more commonly associated with penicillin-non-susceptible pneumococci and with strains presenting evidence of recombination events involving the genes coding for polysaccharide capsule and PspA, suggesting that pneumococcal transparent variants may present a higher ability to acquire exogenous DNA. The results bring to light new information about the virulence potentials of serotype 14 and serogroup 9 S. pneumoniae isolates representing the major clones that have been associated with the emergence and the dissemination of antimicrobial resistance in our setting since the late 1980s.

Population structure of Streptococcus pneumoniae colonizing children before and after universal use of pneumococcal conjugate vaccines in Brazil: emergence and expansion of the MDR serotype 6C-CC386 lineage.

Objectives: To determine the population structure and change in drug resistance of pneumococci colonizing children before and after the introduction of the 10-valent and 13-valent pneumococcal conjugate vaccines (PCV10/13) in Brazil. Methods: We used MLST to analyse 256 pneumococcal isolates obtained from children aged <6 years before (2009-10; n = 125) and after (2014; n = 131) the introduction of the PCV10 and PCV13. Antimicrobial susceptibility and capsular types were previously determined. Results: We identified 97 different STs. Ninety (35.2%) isolates were related to international clones. The most frequent lineages were serogroup 6-CC724 (where CC stands for clonal complex) and the MDR serotype 6C-CC386 in the pre- and post-PCV10/13 periods, respectively. Penicillin-non-susceptible pneumococci (PNSP) formed 24% and 38.9% of the pre- and post-PCV10/13 isolates, respectively (P = 0.01). In the pre-PCV10/13 period, serotype 14-ST156 was the predominant penicillin-non-susceptible lineage, but it was not detected in the post-PCV10/13 period. Serotype 14-ST156 and serotype 19A-ST320 complex isolates had the highest penicillin and ceftriaxone MICs in the pre- and post-PCV10/13 periods, respectively. In turn, serotype 6C-CC386 comprised almost 30% of the PNSP and over 40% of the erythromycin-resistant isolates (MIC >256 mg/L) in the post-PCV10/13 period. Conclusions: Although PNSP strains were polyclonal, most resistant isolates belonged to a single genotype from each period. Higher erythromycin resistance prevalence (42%) in the post-PCV10/13 period was mainly attributed to MDR serotype 6C-CC386. Ongoing surveillance of pneumococcal clonal composition is important to evaluate PCV use outcomes and to identify factors other than PCVs that drive pneumococcal drug resistance evolution.

Phenotypic and molecular characterization of optochin-resistant Streptococcus pneumoniae isolates from Brazil, with description of five novel mutations in the ATPC gene.

Optochin (Opt) susceptibility is used largely for the identification of Streptococcus pneumoniae in diagnostic laboratories. Opt-resistant (Opt(r)) S. pneumoniae isolates have been reported, however, indicating the potential for misidentification of this important pathogen. Point mutations in the atpC gene have been associated with the emergence of Opt(r) S. pneumoniae, but data on the characterization of such atypical variants of S. pneumoniae are still limited. The present report describes the results of a polyphasic approach to identifying and characterizing 26 Opt(r) S. pneumoniae isolates recovered from patients or carriers living in Brazil. Sixteen isolates consisted of heterogeneous populations, and 10 isolates were homogeneously Opt(r). The isolates had different serotypes and antimicrobial susceptibility profiles. They also presented diverse genetic characteristics, as indicated by pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem-repeat analysis (MLVA), and pspA gene typing. Except for Opt MICs (4- to 64-fold higher among Opt(r) variants), Opt(r) and Opt-susceptible (Opt(s)) subpopulations originating from the same culture had identical characteristics. Sequencing of the atpC gene of the Opt(r) variants revealed 13 different nucleotide changes distributed among eight different codons. Changes in codon 49 were the most frequent, suggesting that this might be a hot spot for optochin resistance-conferring mutations. On the other hand, five novel types of mutations in the atpC gene (Met13Ile, Gly18Ser, Gly20Ala, Ala31Val, and Ala49Gly) were identified. In silico prediction modeling indicated that the atpC gene mutations corresponded to alterations in the transmembrane region of the ATPase, leading to a higher hydrophobicity profile in α-helix 1 and to a lower hydrophobicity profile in α-helix 2.