Organisms belonging to the Streptococcus milleri group (SMG) are known for their role in pyogenic infections but have recently been implicated as etiological agents of pulmonary exacerbation in adult patients with cystic fibrosis (CF). The prolonged exposure of CF patients to antibiotics prompted us to investigate the susceptibility profiles of 118 SMG isolates from the airways of CF patients to 12 antibiotics compared to 43 SMG isolates from patients with invasive infections. We found that ϳ60% of all isolates failed to grow using the standard medium for disc diffusion, Mueller-Hinton blood agar (MHBA), so we explored the usefulness of brain heart infusion (BHI) agar for susceptibility testing. Zone-of-inhibition comparisons between BHI and MHBA showed strong correlations for six antibiotics, and interpretations were similar for both medium types. For ceftriaxone and cefepime, both groups of isolates were highly susceptible. Tetracycline resistance levels were comparable between the two groups (22% in CF isolates and 17.4% in invasive isolates). However, more than half of the CF isolates were not susceptible to azithromycin, erythromycin, and clindamycin, compared to 11%, 13%, and 6.5% of invasive isolates, respectively. There were 5-fold and 8-fold increased risks of azithromycin and clindamycin resistance, respectively, for the isolates from the airways of CF patients relative to the invasive isolates. Macrolide resistance was strongly linked to chronic azithromycin therapy in CF patients. This study shows that BHI agar is a suitable alternative for antimicrobial susceptibility testing for the SMG and that SMG isolates from the airways of CF patients are more resistant to macrolides and clindamycin than strains isolated from patients with invasive infections.The members of the Streptococcus milleri group (SMG; also known as the Streptococcus anginosus group), consisting of Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius, are considered commensal organisms notable for their tendency to cause pyogenic infections (33). In a recent population-based epidemiological study it was observed that members of the SMG were responsible for as many invasive streptococcal diseases as all other streptococci combined (25). The members of the SMG are also the most common cause of brain (10, 13) and liver (12, 28) abscesses. Furthermore, members of the SMG are recognized respiratory pathogens. The members of the SMG are commonly associated with pleural empyema (1, 24, 31), especially as a complication of community-acquired pneumonia (1,35,45,48), and there is evidence that the SMG members are a cause of "culturenegative" community-acquired pneumonia (21). Recent studies have shown that the SMG may be an intrinsic component of the cystic fibrosis (CF) airway microbiome, with a high carriage rate observed for the sputum of adult CF patients (20,36,37). Moreover, members of the SMG were implicated in a large proportion of pulmonary exacerbations requiring hospitalization of adults with CF (29,36,3...