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

Abstract

Cystic fibrosis (CF) is a multiorgan disease, with the majority of mortalities resulting from pulmonary failure due to repeated pulmonary exacerbations. Recently, members of the Streptococcus anginosus group (S. anginosus, S. constellatus, and S. 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. The cpn60 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 the S. constellatus (96%), S. intermedius (94%), and S. anginosus (60%) strains to be correctly identified. To increase specificity for S. anginosus, two 16S rRNA real-time PCR assays were developed targeting the 16S rRNA gene. The 16s_SA assay is specific for S. anginosus (100%), while the 16s_SCI assay is specific for S. constellatus and S. intermedius (100%). These assays can detect <10 genome equivalents in pure culture and >10(4) 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.

FIG. 1.

Dendrogram analysis of a variety of Streptococcus spp. using a 1,515-bp portion of the 16S rRNA gene sequence corresponding to positions 33 to 1547 of S. anginosus AF104678. The dendrogram was generated using Splitstree4 version 4.10 (26). The SMG spp. are highlighted with a black box. The accession numbers for the nucleotide sequences used in this analysis are as follows: S. agalactiae, AE009948; S. alactolyticus, AF201899; S. anginosus, AF104678; S. australis, AY485604; S. bovis, AB002482; S. canis, DQ303184; S. constellatus, AF104676; S. cristatus, AY584476; S. didelphis, DQ303185; S. downei, AY188350; S. dysgalactiae, AY584478; S. equinus, AB362710; S. equi subsp. equi, EF406032; S. gordonii, CP000725; S. hyointestinalis, AF201898; S. infantarius, AF429762; S. iniae, DQ303187; S. intermedius, AF104671; S. mitis, AF003929; S. mutans, DQ303189; S. oralis, AY485602; S. orisratti, AF124350; S. parasanguinis, DQ303191; S. phocae, AF235052; S. pneumoniae, AY485600; S. porcinus, AB002523; S. pyogenes, FJ662846; S. salivarius, AY188352; S. sanguinis, AY691542; S. sinensis, AF432856; S. thermophilus, AY687382; S. thoraltensis, Y09007; S. uberis, AM946015; and S. vestibularis, AY188353.

FIG. 2.

(A) Dendrogram analysis of DNA sequences for a 552-bp portion of the cpn60 gene. For panel A, a dendrogram of different Streptococcus species was constructed using the same sequences previously published (1), along with the following species (with accession numbers shown in parentheses): S. cristatus (AF352802), S. didelphis (DQ303185), S. downei (AY188350), S. hyointestinalis (AF201898), S. infantarius (AF429762), S. orisratti (AY123729), S. sinensis (AY360223), S. thoraltensis (AY123725), and S. uberis (AY123724). The SMG spp. are highlighted with a black box. (B) Phylogenetic relationship of the SMG ATCC type strains and the strains isolated from McKay agar utilized in this study across a 552-bp portion of cpn60. All isolates with the same sequence have been grouped. SA; Streptococcus anginosus, SI; Streptococcus intermedius; and SC, Streptococcus constellatus. “M#” represents “McKay agar isolate.”

FIG. 3.

Detection limits for the SMG-specific real-time PCR assays showing the relationship between CFU and CP for each assay. (A) SMG_cpn60; (B) 16s_SA; and (C) 16s_SCI. In each graph, S. anginosus (S. ang) is represented by a circle, S. constellatus (S. const) by a square, and S. intermedius (S. inter) by a triangle. The gray vertical line represents the current detection limit for SMG using McKay agar (10⁴ CFU/ml) to isolate SMG from sputum samples.

FIG. 4.

Alignment of partial sequence of the cpn60 gene-binding region for the SMG_cpn60 real-time PCR assay. The primers and probes were designed to match S. constellatus ATCC 27823 (no. 1). All S. intermedius strains have an SNP in the forward primer, and S. intermedius ATCC 27335 (no. 2) has 3 SNPs in the LCRed640-labeled probe, while the S. intermedius T_m 61.5 strain (no. 3) has two SNPs in the LCRed640 probe and the S. intermedius T_m 60.3 strain (no. 4) has 2 SNPs in the LCRed640-labeled probe and one SNP in the fluorescein-labeled probe. S. anginosus ATCC 33397 (no. 5) has three SNPs in the fluorescein-labeled probe and four SNPs in the LCRed640-labeled probe, as well as four SNPs in the reverse primer. Streptococcus bovis (no. 6), S. gordonii (no. 7), and S. infantis (no. 8) are the only non-SMG spp. tested that produce a signal using this assay, with T_ms of 54.67°C ± 0.17°C, 51.24°C ± 0.14°C, and 52.04°C ± 0.23°C, respectively.