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.