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Cystic fibrosis (CF) is a multiorgan disease, with the majority of mortalities resulting from pulmonary failure due to repeated pulmonary exacerbations. Recently, members of theStreptococcus anginosus group (S. anginosus ,S. constellatus , andS. intermedius ), herein referred to as the “Streptococcus milleri group” (SMG) have been implicated as important etiological pathogens contributing to pulmonary exacerbations in CF patients. This is partly due to better microbiological detection of the SMG species through the development of a novel specific medium termed “McKay agar.” McKay agar demonstrated that SMG has been an underreported respiratory pathogen contributing to lung exacerbations. Our aim was to develop a real-time PCR assay to expedite the detection of SMG within diagnostic samples. Thecpn60 gene was chosen as a target, with all three members amplified using a single hybridization probe set. SMG strain analysis showed that speciation based on melting curve analysis allowed for the majority of theS. constellatus (96%),S. intermedius (94%), andS. anginosus (60%) strains to be correctly identified. To increase specificity forS. anginosus , two 16S rRNA real-time PCR assays were developed targeting the 16S rRNA gene. The 16s_SA assay is specific forS. anginosus (100%), while the 16s_SCI assay is specific forS. constellatus andS. intermedius (100%). These assays can detect  104 genome equivalents in sputum samples, making this a great tool for assessment of the presence of SMG in complex polymicrobial samples. Novel molecular methods were developed providing detection ability for SMG, an emerging opportunistic pathogen.

journal of clinical microbiology

Development of Real-Time PCR Assays for Detection of the <i>Streptococcus milleri</i> Group from Cystic Fibrosis Clinical Specimens by Targeting the <i>cpn60</i> and 16S rRNA Genes

Development of Real-Time PCR Assays for Detection of the Streptococcus milleri Group from Cystic Fibrosis Clinical Specimens by Targeting the cpn60 and 16S rRNA Genes