Characterization of Streptococcus milleri Group Isolates from Expectorated Sputum of Adult Patients with Cystic Fibrosis

With the recent insights into the Streptococcus milleri group (SMG) as pulmonary pathogens in patients with cystic fibrosis (CF), we sought to characterize 128 isolates from the sputum of adults with CF, along with 45 isolates from patients with invasive diseases for comparison. The tests performed included Lancefield grouping; tests for hemolysis; tests for the production of hyaluronidase, chondroitin sulfatase, DNase, proteases, and hydrogen peroxide; and PCR for the detection of the intermedilysin gene (ily). We also generated biochemical profiles with the Rapid ID Strep 32 API system and tested cell-free supernatants for the presence of the signal molecule autoinducer-2 (AI-2) using a Vibrio harveyi bioassay with a subset of CF strains. The S. intermedius isolates from both strain collections were similar, while the S. constellatus and S. anginosus isolates yielded several biotypes that differed in prevalence between the two strain collections. Beta-hemolytic, Lancefield group C S. constellatus comprised 74.4% of the S. constellatus isolates from patients with CF but only 13.3% of the corresponding isolates from patients with invasive infections. This was the only S. constellatus biotype associated with pulmonary exacerbations. Hyaluronidase-positive S. anginosus was detected only among the isolates from patients with CF. Strain-to-strain variability in AI-2 expression was evident, with the mean values being the highest for S. anginosus, followed by S. constellatus and then S. intermedius. Cluster analysis and 16S rRNA sequencing revealed that the species of SMG could be accurately determined with a minimum of three phenotypic tests: tests for the Lancefield group, hyaluronidase production, and chondroitin sulfatase production. Furthermore, isolates from patients with invasive infections clustered with isolates from the sputum of patients with CF, suggesting that the respiratory tract isolates were equally pathogenic.