Characterization of Nontypeable and Atypical Streptococcus pneumoniae Pediatric Isolates from 1994 to 2010

Streptococcus pneumoniae is a major cause of bacteremia, meningitis, pneumonia, sinusitis, and acute otitis media in children. Although optochin susceptibility, bile solubility, and Quellung testing are the standards for identifying and differentiating pneumococci, there are several reports of nontypeable pneumococci that give inconsistent results with one or more of these tests. We characterized 52 isolates previously labeled as nontypeable pneumococci. Microbiological methods included repeating the Quellung reaction using a new and expanded group of antisera, optochin susceptibility and bile solubility tests, and automated Vitek 2 identification. Molecular methods included PCR detection of ply and psaA genes, multilocus sequence typing (MLST), 16S rRNA gene sequencing, and pyrosequencing. Of the 52 isolates, 38 (73%) were optochin susceptible, were psaA and ply positive, and could be serotyped by the Quellung reaction. The remaining 14 isolates, isolated from patients with otitis media (n = 6), bacteremia (n = 6), meningitis (n = 1), and pneumonia (n = 1), underwent further analysis. Three of these 14 isolates were nontypeable due to autoagglutination but were pneumococci by all tests and represented pneumococcal sequence types previously recognized by MLST. The 11 remaining isolates were optochin resistant, and 6 of these were bile soluble. Three of 11 were both psaA and ply positive and clustered with pneumococci by MLST (2 were bile soluble); 8 lacked psaA (5 ply positive, 4 bile soluble) and likely belonged to other Streptococcus species. In conclusion, few isolates were truly nontypeable by Quellung reaction, and MLST and the presence of psaA proved useful in distinguishing between atypical pneumococci and other streptococcal species.