Characterization of Streptococcus milleri group isolates from expectorated sputum of adult patients with cystic fibrosis

Abstract

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.

FIG. 1.

Detection of AI-2 in SMG isolates. As a measure of AI-2 expression, the maximum counts per second (CPS_max) were determined for three replicates of each supernatant and the negative control (sterile Todd-Hewitt-yeast broth) during a 24-h assay period. Black circles, ratio of the average maximum counts per second for each supernatant to the average maximum counts per second for the negative control; bars, mean for each species.

FIG. 2.

Cluster analysis of phenotypic test results for the SMG. Blue, S. anginosus; red, S. constellatus; green, S. intermedius. Abbreviations and symbols: APPA, alanyl-phenylalanyl-proline arylamidase; GTA, glycyl-tryptophan arylamidase; stars, strains from patients with invasive infections; circles with dots in the center, reference strains, which clustered accordingly. The cluster is divided into two main groups: the upper group consists of less active strains, which showed fewer positive results than the lower, more active group. Subcluster designations are indicated on the right.

FIG. 3.

Proposed scheme for determination of the SMG species. The assignment of subtypes, namely, S. constellatus subsp. constellatus, S. constellatus subsp. pharyngis, and DNA group 2 strains, was based on the strain descriptions provided by Whiley et al. (43).