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J Clin Microbiol. 2010 April; 48(4): 1150–1160.
Published online 2010 February 17. doi:  10.1128/JCM.02082-09
PMCID: PMC2849594

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[down-pointing small open triangle]

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 >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.

Cystic fibrosis (CF) is the most common fatal genetic disease affecting young Caucasians (13). It is a multiorgan disease that primarily affects the lungs and digestive system. Within the CF lungs, there is a buildup of thick mucus that is difficult to clear, leading to chronic bacterial colonization with high bacterial loads (34, 41, 49). However, it is not solely the presence of high bacterial loads in the lungs of CF patients but periods of pulmonary exacerbation, an overt immune response that leads to the majority of irreversible lung damage, that ultimately lead to pulmonary failure in 90% of afflicted individuals (34-36). Classically there are relatively few bacterial pathogens described in CF lung disease (15, 17, 18); however, CF should be considered a polymicrobial infectious disease, as the CF lungs are colonized by a diverse and dynamic consortium of bacteria, fungi, and viruses (1, 21-23, 44-46, 53).

Recently the Streptococcus anginosus group, herein referred to as the “Streptococcus milleri group” (SMG), which includes the three species S. anginosus, S. constellatus and S. intermedius, has been implicated in pulmonary exacerbations of CF patients (4, 37, 45). The SMG species have been identified as part of the microbiota of the respiratory tract, gastrointestinal tract, and genitourinary tract in 15 to 30% of healthy individuals (20, 38, 39, 43). However, each species within the SMG has the capacity to cause severe invasive infections throughout the body. SMG infection is the most common cause of brain and liver abscesses (8, 20, 32, 54) and is a major cause of empyema (30). Members of the SMG have been implicated in infection at all body sites, associated with skin and soft tissue (3, 56), abdomen (48), head and neck (16, 33, 48), pleuropulmonary (28, 48), cardiovascular and blood (25, 40), and genitourinary and musculoskeletal (7, 48) infections. There does appear to be species bias to some infections, as S. anginosus has been found to cause the majority of abdominal infections, while S. intermedius has been more often linked to liver and central nervous system (CNS) infections (10, 56). Members of the SMG have also been implicated as a common etiology of intra-abdominal abscesses developed by individuals who have received solid organ transplants and may have been underestimated as a cause of disease within this population (50).

SMG strains are phenotypically diverse, even within each species. However, most strains share some common characteristics such as slow growth rate, a distinctive “caramel smell,” their ability to hydrolyze arginine, acetoin production from glucose, and an inability to ferment sorbitol (9, 20, 38, 43). Microbiological differentiation of the three species within the SMG can be problematic. A few methods have been designed that allow for the differentiation of these three species; unfortunately, they are time-consuming, and results are variable (14, 31, 58). Recently a new medium that has been developed, McKay agar, that allows for the isolation of SMG from complex clinical samples; however, other organisms, including additional Streptococcus strains, can also be cultured on this medium (46a). Numerous molecular assays have been developed to differentiate SMG using cpn60 (53), rnpB (27, 52, 55), 16S rRNA genes (7, 10, 31), 16S-to-23S rRNA gene intergenic spacer (ITS) region (5, 11, 52, 57), and the penicillin-binding protein (51). These assays are limited by their need for nucleic acid sequence analysis or further PCR analysis required to differentiate SMG species.

The increased importance of SMG in human infections and the difficulty in microbial detection suggest a need for a rapid and reliable test to detect SMG from pure culture as well as complex polymicrobial diagnostic samples such as CF sputum samples (4, 37, 45). The development of a real-time PCR assay in combination with McKay agar isolation would reduce microbial identification time, thereby decreasing the period before the initiation of appropriate antibiotics, which in turn would resolve clinical symptoms more efficiently for all types of infections. This would also afford the opportunity for clinical intervention before the onset of pulmonary exacerbation preventing increased lung damage.

We have developed three real-time PCR assays. The first assay is based on cpn60, which detects S. constellatus and S. intermedius and many S. anginosus strains and allows for melting curve-based speciation. The second assay specifically detects S. anginosus, based on a conserved region of the 16S rRNA gene. Finally, the third assay specifically detects S. constellatus and S. intermedius based on a conserved region of the 16S rRNA gene. These assays provide a novel culture-independent strategy for the detection of this important group of emerging pathogens.

MATERIALS AND METHODS

Bacterial strains.

For real-time assay development 98 clinical SMG strains originally isolated from CF patients using McKay agar were utilized (46a). The 98 clinical strains were comprised of 40 S. anginosus, 32 S. intermedius, and 26 S. constellatus strains. The control bacterial strains used to test the specificity of the real-time PCR assays are listed in Table Table1,1, and these include isolates of all Streptococcus spp. that also grew on McKay agar, in addition to the SMG strains mentioned above, as well as American Type Culture Collection (ATCC) strains (Cederlane, Labs, Ontario, Canada) used as controls. The abbreviation “M#” represents a strain cultivated from CF sputum on McKay agar and the number of the isolate.

TABLE 1.
Strains used in this study

Chromosomal DNA isolation. (i) Physical disruption.

Strains were streaked for purity on Columbia blood agar (CBA) plates or brain heart infusion agar (BHIA) (Becton, Dickinson and Company [BD], Oakville, Ontario, Canada) and incubated for 24 h at 37°C plus 5% CO2. A single colony was inoculated into 20 ml of BHI broth (BD) and incubated at 37°C plus 5% CO2 for 24 h. One milliliter of culture was pelleted by centrifugation, followed by resuspension in 1 ml TN150 (10 mM Tris-HCl, 150 mM NaCl, pH 8.0; Sigma-Aldrich, Oakville, Ontario, Canada). The wash step was repeated twice, and the resuspended culture was added to a 2.0-ml tube with a screw cap and O-ring with 0.2 g of 0.1-mm-diameter glass beads and placed in the MiniBeadbeater-8 (BioSpec Products, Inc., Bartlesville, OK), which was run on the setting “homogenize” for 3 min. After cells were disrupted, a standard phenol-chloroform preparation was done (43a). The DNA was resuspended in 100 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0; Sigma-Aldrich) and stored at −20°C.

(ii) Boiling preparation.

Cultures were grown as described above, and the pellet was resuspended in 500 μl of TE buffer (Sigma-Aldrich) and lysed by boiling for 15 min. Boiled cells were pelleted, and the supernatant was removed to a new tube and stored at −20°C.

Sputum sample DNA isolation.

Sputum samples were collected in accordance with ethical guidelines established by the Office of Medical Bioethics, University of Calgary, and stored at −80°C (46a). Sputum was processed as previously described (42), with the exception that a 50-μl aliquot of each sputum preparation was further processed using the LOOXSTER prokaryotic DNA protocol (SIRS Labs, Germany) as per the manufacturer's protocol. This protocol is designed to decrease the concentration of host genomic DNA in complex samples by up to 90%.

CF sputum samples spiked with SMG were prepared as described above, except 1 ml of appropriate culture grown in BHI broth (BD) for 24 h at 37°C with 5% CO2 was pelleted and resuspended in 900 μl of 200 mM sodium phosphate buffer, followed by the addition of 300 μl of sputum. Spiked sputum samples were then processed as described above. The numbers of CFU spiked into sputum were determined by plating culture dilutions onto CBA or BHIA (BD) plates and incubating them for 48 h at 37°C with 5% CO2.

Conventional PCR.

Previously published primer sets were synthesized in house (DNA core facility, PHAC, Manitoba, Canada) and used to amplify portions of the cpn60 gene (2, 19) and the 16S rRNA gene (12). The cpn60 PCR was performed with high-fidelity Platinum Taq (Invitrogen, Burlington, Ontario, Canada), following the manufacturer's directions with the addition of 1 μM each primer, 0.2 mM each deoxynucleoside triphosphate (dNTP), and 3 mM MgSO4, while the 16S rRNA PCR was done with FastStart Taq (Roche Diagnostics, Laval, Quebec, Canada) as per the manufacturer's directions with 1 μM each primer and 0.2 mM each dNTP and 1.5 mM MgCl2. The thermocycling parameters for all PCRs included an initial denaturing step of 95°C for 5 min and a final extension at 68°C for 5 min. For cpn60, there were 40 cycles of 95°C for 30 s, 42°C for 30 s, and 68°C for 30 s. For 16S rRNA PCR, there were 32 cycles of 95°C for 30 s, 50°C for 45 s, and 68°C for 1 min.

Sequence analysis.

PCR products were purified using the QIAquick PCR purification kit (Qiagen, Mississauga, Ontario, Canada) as per the manufacturer's protocols. For the 16S rRNA gene, the products were sequenced using the initial amplification primers and four additional oligonucleotides targeting internal segments to provide complete coverage for sequence analysis (12). The cpn60 gene products were sequenced with M13 primers (2). Sequencing was performed with an ABI3730 capillary electrophoresis instrument (Applied Biosystems, Foster City, CA). Sequence was analyzed using Lasergene (DNAstar). Multiple sequence alignments were performed using ClustalW (http://www.ebi.ac.uk/clustalw/) and organized for display using Boxshade (http://www.ch.embnet.org), and these data were deposited in GenBank with accession numbers as listed below.

Real-time PCR.

LightCycler Hybprobe primers and hybridization probes were designed using the LightCycler probe design software version 2.0. Primers and probes were designed based on multiple sequence alignments of the cpn60 gene and the 16S rRNA gene and synthesized by Tib-Molbiol (Berlin, Germany). The sequences and product sizes are listed in Table Table2.2. Sequence alignments of S. anginosus ATCC 33397, S. constellatus ATCC 27823, and S. intermedius ATCC 27335, along with representatives from 37 other Streptococcus spp., 4 Gemella spp., Staphylococcus spp., Pseudomonas aeruginosa, and Burkholderia cenocepacia, were prepared and analyzed to detect conserved regions within the SMG. Real-time PCR primers or probes were designed to bind to conserved regions that allow for detection of SMG.

TABLE 2.
SMG real-time PCR primers and probes

The LightCycler FastStart DNA Master HybProbe (Roche Diagnostic) was used for all real-time PCR assays. All reactions were carried out in the Lightcycler (Roche Diagnostic) 2.0 using 20-μl capillaries (Roche Diagnostic). The PCR thermocycler conditions for cpn60 (SMG_cpn60) included an initial denaturation of 95°C for 10 min, followed by amplification using 30 cycles of 95°C for 10 s, 55°C for 15 s, and 72°C for 15 s followed by melting curve analysis of 1 cycle of 95°C for 0 s, 40°C for 30 s, and 70°C for <1 s with a ramp rate of 0.05°C/s followed by a final cooling step of 40°C for 30 s. For the Streptococcus anginosus (16s_SA)- and Streptococcus intermedius/S. constellatus (16s_SCI)-specific 16S rRNA real-time PCR, all steps were the same as those above, except during amplification the annealing temperatures were 62°C and 60°C, respectively.

Nucleotide sequence accession numbers.

Each unique sequence for the cpn60 allele, including those from SMG and other Streptococcus spp. isolated on McKay agar (n = 135), has been uploaded to GenBank database (http://www.ncbi.nlm.nih.gov) under accession no. GQ251384 to GQ251518. The 16S rRNA sequence accession numbers have been submitted through the McKay agar study (46a).

RESULTS

Sequence analysis of ATCC and clinical isolates.

Sequence analysis for the partial segments of the 16S rRNA gene was completed for all Streptococcus spp., including ATCC type strains and clinical isolates listed in Table Table11 and all clinical SMG strains. After 16S rRNA analysis, there were 99 SMG isolates, including 40 S. anginosus, 33 S. intermedius, and 26 S. constellatus isolates. We also utilized as many sequences from ATCC type strains deposited in GenBank as possible to allow for proper identification (Fig. (Fig.1).1). There were a total of 20 16S rRNA alleles among the 99 SMG clinical isolates, with 12 alleles, 7 alleles, and 1 allele for S. anginosus, S. constellatus, and S. intermedius, respectively. Phylogenetic analysis of 16S rRNA gene showed SMG isolates grouped together and separate from other phylogenetically closely related species, with S. constellatus and S. intermedius more closely related to each other than to S. anginosus (Fig. (Fig.11).

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. ...

Sequence analysis for the cpn60 gene.

Sequence analysis for the cpn60 gene showed that there were a total of 30 alleles for the 99 SMG clinical isolates, with 12, 8, and 11 alleles for S. anginosus, S. constellatus, and S. intermedius, respectively. Phylogenetically, the same trend that was seen for the 16S rRNA gene was also seen for the cpn60 gene, except there was increased sequence diversity within species of SMG (Fig. (Fig.2A).2A). Streptococcus intermedius showed the greatest differences with intraspecies diversity between the 16S rRNA and cpn60 phylogeny, with only a single 16S rRNA allele and 11 cpn60 alleles. S. anginosus showed the most intraspecies variability among the cpn60 alleles, with 93.4 to 100% nucleotide identity, followed by S. constellatus with 94.6 to 99.8% nucleotide identity and S. intermedius with 96.5 to 99.5% nucleotide identity (Fig. (Fig.2B).2B). Based on the sequence diversity, we were able to develop three real-time PCR assays that can be used to specifically identify members of the SMG.

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 ...

Development of real-time PCR assays.

The sensitivity of the SMG_cpn60 real-time PCR assays was tested using 40 amplification cycles with a dilution series from 108 CFU to <1 CFU for S. anginosus ATCC 33397, S. constellatus ATCC 27823, and S. intermedius ATCC 27335 (Fig. (Fig.3A).3A). The detection limit was <10 CFU for S. constellatus and S. intermedius, with a crossing point (CP) of >35 cycles for the lowest detectable dilution. Melting curve analysis of 21 replicates showed melting temperatures (Tms) of 59.02°C ± 0.42°C for S. intermedius and 65.00°C ± 0.22°C for S. constellatus. The detection limit for S. anginosus using crossing points was only 107 CFU. However, a melting curve was produced for 105 CFU, with a melting temperature of 53.25°C ± 0.33°C. Figure Figure44 demonstrates sequence variation and related melting curve temperatures produced for SMG at the cpn60 loci in the region covered by the primers and probes for the real-time PCR assay. These sequence differences in the cpn60 gene allow for speciation of SMG based on the Tms of the amplification products. Crossing points from closely related Streptococcus spp. were only observed using 40 or more amplification cycles and high concentrations of genomic DNA (>106 CFU). Thus, we defined the parameters of the assay at 30 cycles. Utilizing 30 cycles decreased the lower level of detection to 103 CFU from <10 CFU for S. intermedius and S. constellatus and 107 CFU for S. anginosus, with only three non-SMG strains producing any amplification product. Streptococcus bovis had an amplification product with a mean Tm (using results from 15 different runs) of 55.15°C ± 0.13°C; S. infantis and S. gordonii did not have CPs, but they had Tms of 52.04 ± 0.23°C (22 runs) and 51.24 ± 0.14°C (14 runs), respectively (Fig. (Fig.44).

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 ...
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 ...

For the 16s_SA assay, a conserved region within the 16S rRNA sequence was used to design primers specific to S. anginosus ATCC 33397, while the 16s_SCI assay was designed based on conserved sequence for S. intermedius ATCC 27335 and S. constellatus ATCC 27823. Sensitivities of the 16S rRNA real-time PCR assays were tested using 40 amplification cycles with a dilution series from 108 CFU to <1 CFU for S. anginosus, S. constellatus, and S. intermedius with a detection limit of <10 CFU (Fig. (Fig.3B).3B). For the 16s_SCI assay, S. constellatus and S. intermedius at a concentration of 108 CFU had crossing points similar to 102 CFU for S. anginosus (Fig. (Fig.3C).3C). The 16s_SCI assay produced the same results as the 16s_SA assay, with the exception that S. intermedius and S. constellatus were both detected with a sensitivity of <10 CFU, while high concentrations of S. anginosus were detected at a CP similar to 102 CFU. The melting curves are the same for all three species, and when the number of amplification cycles was reduced to 30, only S. intermedius and S. constellatus or S. anginosus produced a product for the 16s_SCI assay or 16s_SA assay, respectively, with a detection limit of 103 CFU. Thus, using the protocol with 30 amplification cycles only targeted species were positive, without false positives from any other Streptococcus spp. tested.

Real-time PCR assays using spiked CF sputum samples.

CF sputum samples that were SMG culture negative (46a) were split into aliquots, each aliquot was spiked with either S. anginosus, S. intermedius, or S. constellatus, and serial dilutions were done. The 16s_SCI real-time PCR assay was able to detect S. intermedius or S. constellatus at concentrations as low as 104 CFU, while S. anginosus was negative for all dilutions. The detection limit for the 16s_SA assay was 104 CFU for S. anginosus, while there was no amplification for either S. constellatus or S. intermedius spiked samples. For the SMG_cpn60 assay, the detection limit was 104 CFU for S. constellatus and S. intermedius, while S. anginosus was detected only at levels of >107 CFU. The detection limit was consistently 1 order of magnitude higher for samples that had been treated with the LOOXSTER kit, although highly concentrated samples (108 CFU) had decreased CP after treatment with the LOOXSTER kit (Table (Table33).

TABLE 3.
SMG detection limits in sputum samples with the developed real-time PCR assays

Specificity of the SMG real-time PCR assays.

After development and optimization of the real-time PCR assays using ATCC type strains, bacterial isolates from the McKay agar study (46a) were used to test the specificity of the assays. The SMG_cpn60 real-time PCR assay had crossing points for all S. intermedius (n = 32) strains, although the Tms for the McKay agar isolates were different from those for S. intermedius strain ATCC 27335, which was used to optimize the assay. The majority of S. intermedius strains had one of two predominant Tms; 59.84°C ± 0.17°C for 16 strains and 61.16°C ± 0.16°C for 14 strains, while 2 had Tms of 64.75°C ± 0.01°C. For the S. constellatus (n = 26) clinical isolates, 25 were positive using the SMG_cpn60 assay. Of these, 19 had Tms the same as S. constellatus ATCC 27823 (64.82°C ± 0.13°C), while five strains had Tms of 58.44°C ± 0.05°C, similar to S. intermedius ATCC 27335. For S. anginosus, the majority of strains (24/40) had Tms similar to those of the type strain S. anginosus ATCC 33397 (53.23°C ± 0.38°C). Of the 16 strains that had different Tms, six had Tms of 64.8°C ± 0.16°C, similar to S. constellatus ATCC 27823, and two had Tms of 61.06°C ± 0.02°C, similar to S. intermedius with eight negative strains.

The 16s_SCI real-time PCR assay was able to detect all of the McKay agar study S. intermedius (n = 33) and S. constellatus (n = 26) strains while being negative for all S. anginosus (n = 40) strains. The mean Tm for all strains was 57.36°C ± 0.25°C. The 16s_SA assay was positive for all S. anginosus (n = 40) strains and negative for all non-S. anginosus SMG (n = 59) strains tested. The Tms for the S. anginosus assay were 60.54°C ± 0.30°C for 35 S. anginosus clinical strains and 57.3°C ± 0.53°C for five other S. anginosus clinical isolates. This was due to a single nucleotide polymorphism (SNP) in the forward and reverse primers preventing optimal amplification for these isolates. Both assays were also negative for all non-SMG isolates tested (n = 45), including sample strains from all non-SMG Streptococcus (n = 34) and non-Streptococcus (n = 11) spp. that were recovered from CF sputum on McKay agar in a previous study (46a).

Real-time PCR detection of SMG in CF sputa.

The 16s_SCI real-time PCR assay was able to detect S. intermedius or S. constellatus in five sputum samples with known culture concentrations of SMG, as well as two sputum samples with mixed-infection culture data from S. anginosus and either S. intermedius or S. constellatus, with a detection limit of 105 CFU (Table (Table4).4). In samples treated with the LOOXSTER kit the 16S_SCI assay detected all five S. intermedius and S. constellatus culture-positive sputum samples, as well as two mixed infections. Two culture-negative samples (one of which was from a patient who was later confirmed as culture positive for SMG) were positive with late signals and weak curves for Tms (Table (Table44).

TABLE 4.
SMG detection in CF sputum via culture and real-time PCR assays

The 16s_SA real-time PCR assay detected one of the three sputum samples that were culture positive for S. anginosus. One sample that did not produce a signal was at a concentration of 104 CFU at the lower limit of detection for this assay, and the other was a mixed infection with S. intermedius. All 10 sputum samples that were culture negative for S. anginosus were also negative by this assay (Table (Table4).4). The 16s_SA assay had similar results to those described above for sputum samples that were treated with the LOOXSTER kit, although the mixed-infection sample was positive after LOOXSTER treatment (Table (Table44).

Four culture-positive sputum samples were positive for S. intermedius and S. constellatus using the SMG_cpn60 assay, but did not detect S. intermedius at a concentration of 104 CFU or S. intermedius in a mixed infection with S. anginosus. The assay also allowed speciation of the positive samples based on the Tms produced. There was also one sputum sample that was culture negative for SMG that produced a late signal (Table (Table4).4). The SMG_cpn60 assay detected all five SMG culture-positive sputum samples after they were processed with the LOOXSTER kit, as well as the two mixed-infection samples. Two culture-negative samples also produce a signal for the same two culture-negative sputum samples that were detected using the 16s_SCI assay, one of which did not have a Tm (Table (Table44).

DISCUSSION

Three real-time PCR assays have been designed that specifically detect members of the SMG based on sequence analysis of the 16S rRNA and the cpn60 gene. Phylogenetic analysis of 16S rRNA gene showed SMG members grouped together and separate from other closely related species, with S. constellatus and S. intermedius more closely related to each other than S. anginosus, as has been found in previous studies (7, 24, 53). This trend was also seen for the cpn60 gene, except that there was increased sequence diversity within each species of SMG. This is in accordance with previous studies that show there is an increase in sequence diversity within the cpn60 gene as compared to 16S rRNA gene when looking at closely related isolates (24, 53). We found that there is more diversity in the cpn60 gene for S. anginosus and S. constellatus than S. intermedius. This is slightly different from results found in a previous study that found cpn60 in S. constellatus to be highly conserved (53); however, this study used fewer isolates than our study, which may account for the increased variability found. Also, the overall amount of intraspecies variability, as measured by percent nucleotide identity (results not shown), was similar for our isolates to that seen in the study by Teng et al. (53). This suggests that the overall variability within the cpn60 gene is similar for all members of the SMG and provides a suitable target to differentiate SMG (53).

Each of the 16S rRNA real-time assays was specific for SMG, while the assay targeting the cpn60 gene had amplification products for Streptococcus bovis, S. infantis, and S. gordonii. Although the mean Tms produced for these strains were similar to those for S. anginosus, there was a difference of at least 2 standard deviations between them. Thus, when considering Tms, the SMG species can be identified based on the specific Tms produced. Increased diversity in the cpn60 gene compared to the 16S rRNA gene is likely the reason some Streptococcus spp. have amplification products for this assay. The results from our SMG_cpn60 real-time PCR assay are comparable to the one other real-time PCR assay developed to detect SMG which was developed based on a conserved region of the ITS of SMG (11). The previously published assay can also be used to identify the three members of the SMG based on Tms; however, it also produces Tms for a wide variety of other Streptococcus spp., including two that are indiscernible from Streptococcus constellatus based on Tm analysis alone. Thus, the assay developed by Desar et al. (11) is valuable for analysis of pure culture but would not be as effective when used to test complex samples (11). Given the diversity of the Streptococci in human airways, additional approaches would be required to rule out these species if SMG detection was attempted from nonsterile human specimens (such as abscesses or sputum), thus limiting the benefit of the current real-time assay. Recent findings show the increase in numbers of SMG can lead to exacerbations for CF patients (4, 37, 45) and are likely an underrepresented cause of respiratory illnesses (50, 59). Due to this underrepresentation, sensitive and specific assays are required that can identify SMG members within complex clinical samples. Compared to the ITS real-time PCR assay, our SMG_cpn60 assay offers more specificity for SMG and allows the assays to be used for analysis of more complex samples.

The SMG_cpn60 real-time PCR assay is very successful for identification of members of the SMG; however, due to members of SMG carrying similar cpn60 alleles, this assay may not discern between the three species of SMG. For example, >10% of clinical SMG isolates used had cpn60 alleles that would suggest another species when compared to the sequence results from 16S rRNA analysis. These strains would still be identified as members of SMG using the SMG_cpn60 real-time PCR assay; however, they could be speciated differently depending on which method of speciation was used. To alleviate this, we also developed two real-time PCR assays based on species-specific regions of the 16S rRNA gene. A 16s_SA and 16s_SCI assay were developed that allowed for specific identification of all S. anginosus or S. constellatus and S. intermedius isolates, respectively. Our 16s_SA assay is very specific and only amplifies S. anginosus, from both pure culture and from sputum samples, providing a method to identify S. anginosus and eliminating the worry of false-positive results for S. bovis, S. infantis, and S. gordonii for the SMG_cpn60 assay. The 16s_SCI assay was positive for all S. constellatus and S. intermedius strains and negative for all S. anginosus strains from our clinical isolates; however, this assay was not able to speciate S. intermedius and S. constellatus. When the 16s_SCI assay is used in combination with the SMG_cpn60 real-time assay, they provide a method for speciation for the majority of SMG strains. When testing complex clinical samples, it may be better to use the 16s_SA or 16s_SCI assays than the SMG_cpn60 assay since they have greater sensitivity when testing complex samples. If speciation were required, the SMG_cpn60 assay could be run subsequently to confirm species present.

Our lower limit of detection for each assay was >10 CFU for pure cultures, which is comparable to published real-time PCR studies. This level of detection would allow for specific and sensitive detection of SMG (6, 29, 60). The use of universally encoded gene products in our PCR may lead to the potential for false positives (47). When more than 30 amplification cycles were run, we observed a few false-positive amplification products with high CP values and indeterminate assay results. To decrease aberrant assay results, we reduced the number of amplification cycles, as similarly conducted for the previously published ITS real-time PCR assay (11). The lower level of detection when using complex polymicrobial samples was approximately 104 CFU for each assay, although samples at the lower limit of detection show some variability among repeats, and thus samples with detection of 105 CFU and above show less variability and more reliable results. Due to this decrease in sensitivity, speciation is harder to do in complex samples, although the combination of the three real-time PCR assays allows for speciation of the majority of SMG isolates when testing complex sputum samples. Our lower limit of detection allows us to detect SMG at levels below that required to cause an exacerbation in CF patients and may provide a valuable tool to clinicians when predicting SMG-induced exacerbations. The use of the LOOXSTER kit increases the sensitivity of our assays by reducing the total host genomic DNA, although the cost per sample is still prohibitive for use in a diagnostic setting. There were also two sputum samples that were positive for SMG by real-time PCR that were negative by culture method. Unfortunately, we cannot confirm if these sputa are negative or are carrying SMG at a level below the limit of detection by culture method, with approximately 104 CFU using the SMG-specific McKay agar. However, one of the patients tested culture positive for SMG in the next sputum sample analyzed (several months later), suggesting that the real-time PCR was accurate and more sensitive than the original culturing method due to overgrowth from other bacterial species. Crossing points for both these sputa were late (>27 cycles) and would suggest SMG levels below 104 CFU. Further work will have to be done to validate the lower levels of infection in sputum to provide accurate detection of SMG below the level of culturing.

With the increasing importance of SMG in clinical settings, the need for tools that allow quick identification as well as potential for quantification are required. We have developed three real-time PCR assays providing new tools to allow quick identification of SMG from pure culture or from complex polymicrobial samples that can be used in conjunction with the McKay agar culture method to decrease the time required to identify SMG isolates. These molecular assays may provide the ability to perform retrospective analysis of exacerbation sputum samples due to the culture-independent nature of the assays, which will provide more information as to the historical importance of SMG within CF cohorts, as well as identifying SMG carriers within the CF population. These assays will provide insight into the overall clinical importance of SMG by reducing the difficulty in detecting these pathogens in clinical samples and will give a more accurate perspective on the clinical burden of SMG. These assays combined with new culture methods will provide clinicians with an increased ability to detect SMG from diagnostic samples, including other respiratory specimens, such as those from chronic bronchiectasis patients, where SMG may currently go unrecognized.

Acknowledgments

This work was supported by the Public Health Agency of Canada, a Federal Intramural R&D Initiative project, and a Canadian Cystic Fibrosis Foundation grant (to M.G.S.). M.G.S. is supported as an Alberta Heritage Foundation for Medical Research Scientist and Canada Research Chair in Microbial Gene Expression. C.D.S. is supported by an Alberta Heritage Foundation for Medical Research studentship and a Canada Graduate Scholarship from the Canadian Institutes of Health Research.

The views and opinions expressed herein are those of the authors only and do not necessarily represent the views and opinions of the Public Health Agency of Canada or the government of Canada.

Footnotes

[down-pointing small open triangle]Published ahead of print on 17 February 2010.

The authors have paid a fee to allow immediate free access to this article.

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