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

Olson, Adam B.; Sibley, Christopher D.; Schmidt, Lisa; Wilcox, Mark; Surette, Michael G.; Corbett, Cindi R.

doi:10.1128/jcm.02082-09

Cited by 28 publications

(27 citation statements)

References 61 publications

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“…The PCR targets were the lytA gene for S pneumoniae (21), the hpd gene for H influenzae (21), the nuc gene for S aureus (22), the spy gene for S pyogenes (23), and the 16S ribosomal RNA gene of both Streptococcus intermedius and Streptococcus constellatus (two of the three species in the S anginosus group) (24). …”

Section: Methodsmentioning

confidence: 99%

Real‐Time Polymerase Chain Reaction for Microbiological Diagnosis of Parapneumonic Effusions in Canadian Children

Pernica

Moldovan

Chan

et al. 2014

Canadian Journal of Infectious Diseases and Medical Microbiolog

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Community-acquired pneumonia with parapneumonic effusion/empyema is not uncommon in children and can cause serious illness; there -fore, the timely optimization of antimicrobial therapy is essential in this situation. The aim of this study was to determine whether using real-time polymerase chain reaction of pleural fluids to identify the causative organism improves the process of microbiological diagnosis in the context of community-acquired pneumonia with parapneumonic effusion/empyema. This technique was compared with traditional culture methods for microbiological diagnosis.

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Section: Methodsmentioning

confidence: 99%

Real‐Time Polymerase Chain Reaction for Microbiological Diagnosis of Parapneumonic Effusions in Canadian Children

Pernica

Moldovan

Chan

et al. 2014

Canadian Journal of Infectious Diseases and Medical Microbiolog

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“…This collection of SMG respiratory isolates was not recovered by conventional CF microbiology and enabled us to characterize the phenotypic properties of airway isolates and compare them to invasive strains (5). These results, in combination with analysis of the nucleotide sequence of the 16S rRNA gene of these strains, revealed clusters of isolates that included both CF and invasive isolates with indistinguishable phenotypic characteristics (5,9).…”

mentioning

confidence: 92%

The Streptococcus milleri Population of a Cystic Fibrosis Clinic Reveals Patient Specificity and Intraspecies Diversity

Sibley¹,

Sibley²,

Leong³

et al. 2010

J Clin Microbiol

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The genetic relatedness of Streptococcus milleri group isolates from the airways of cystic fibrosis patients was determined by using pulsed-field gel electrophoresis. This study reveals no evidence for patient-to-patient transmission in our patient population; however, within individual patients, complex inter-and intraspecies diversity and dynamics can be observed.In addition to their role in purulent infections (3,7,11), members of the Streptococcus milleri group (SMG), also known as the Streptococcus anginosus group, comprised of Streptococcus constellatus, Streptococcus intermedius, and Streptococcus anginosus, have emerged as clinically relevant in chronic airway infections in cystic fibrosis (CF) patients and have been implicated as etiologic agents of pulmonary exacerbation (6,10,13,14). We have recently described the isolation of a large number of SMG strains from a cohort of CF patients by using the semiselective medium McKay agar (12). This collection of SMG respiratory isolates was not recovered by conventional CF microbiology and enabled us to characterize the phenotypic properties of airway isolates and compare them to invasive strains (5). These results, in combination with analysis of the nucleotide sequence of the 16S rRNA gene of these strains, revealed clusters of isolates that included both CF and invasive isolates with indistinguishable phenotypic characteristics (5, 9).In this study, we evaluated whether patient-to-patient transmission was occurring in our CF patient cohort. The molecular epidemiological relationship of the SMG isolates was determined by using pulsed-field gel electrophoresis (PFGE) (4,8,15).PFGE was performed by modification of a protocol described by Bartie et al. (2). The isolates were cultured at 37°C for 48 h on brain heart infusion agar supplemented with colistin sulfate (10 g/ml) and oxolinic acid (5 g/ml) under anaerobic conditions. The cells were harvested and suspended to 20% transmittance (600 nm) in 100 mM Tris-HCl buffer (pH 7.6). Mutanolysin (100 U; Sigma-Aldrich, St. Louis, MO) was added to 500 l of cell suspension before an equal volume of molten 1% SeaKem Gold agarose (Lonza, Rockland, ME) was added. Plugs were cast at room temperature and then transferred to 1.5 ml of lysis solution (0.25 M EDTA [pH 9.0], 0.5% Brij 58, 2 g/liter sodium deoxycholate, 5 g/liter lauroyl sarcosine, 100 U/ml mutanolysin) and incubated at 37°C for 2 h. The lysis solution was replaced with 1.5 ml ESP solution (0.25 M EDTA [pH 9.5], 1% sodium lauroyl sarcosine, 0.5 mg/ml proteinase K) and incubated at 55°C for 2 h. The plugs were rinsed with 1 ml of distilled water and then washed for 10-min intervals, once with distilled water and three times with 1ϫ Tris-EDTA (TE; 10 mM Tris-Hcl, 1 mM EDTA [pH 8.0]) at room temperature.For restriction digestion, the plugs were preincubated in 300 l of 1ϫ reaction buffer (Invitrogen, Carlsbad, CA) at room temperature for 15 min and then replaced with fresh 1ϫ reaction buffer supplemented with 90 U of SmaI or ApaI (New England BioLabs, Beverly, MA)...

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“…This work has raised the possibility that previously unrecognized species may be present in the lower airways and contribute to the devastating clinical manifestations of CF. Indeed, these findings have begun to influence clinical practice recommendations and fundamentally alter understanding of the disease (12)(13)(14).…”

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confidence: 99%

“…In addition, DNA-based measurements may be more sensitive than culture (17,18) and, unlike cultures performed by clinical laboratories, results are not filtered to disregard organisms that could be contaminants (19,20). Finally, DNA-based methods are primarily used to identify organisms not typically considered pathogens in CF (12,21). Many of these species are normal inhabitants of upper airways (22).…”

mentioning

confidence: 99%

Direct sampling of cystic fibrosis lungs indicates that DNA-based analyses of upper-airway specimens can misrepresent lung microbiota

Goddard

Staudinger²,

Dowd³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

238

222

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Recent work using culture-independent methods suggests that the lungs of cystic fibrosis (CF) patients harbor a vast array of bacteria not conventionally implicated in CF lung disease. However, sampling lung secretions in living subjects requires that expectorated specimens or collection devices pass through the oropharynx. Thus, contamination could confound results. Here, we compared culture-independent analyses of throat and sputum specimens to samples directly obtained from the lungs at the time of transplantation. We found that CF lungs with advanced disease contained relatively homogenous populations of typical CF pathogens. In contrast, upper-airway specimens from the same subjects contained higher levels of microbial diversity and organisms not typically considered CF pathogens. Furthermore, sputum exhibited day-to-day variation in the abundance of nontypical organisms, even in the absence of clinical changes. These findings suggest that oropharyngeal contamination could limit the accuracy of DNA-based measurements on upper-airway specimens. This work highlights the importance of sampling procedures for microbiome studies and suggests that methods that account for contamination are needed when DNA-based methods are used on clinical specimens.

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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

Cited by 28 publications

References 61 publications

Real‐Time Polymerase Chain Reaction for Microbiological Diagnosis of Parapneumonic Effusions in Canadian Children

Real‐Time Polymerase Chain Reaction for Microbiological Diagnosis of Parapneumonic Effusions in Canadian Children

The Streptococcus milleri Population of a Cystic Fibrosis Clinic Reveals Patient Specificity and Intraspecies Diversity

Direct sampling of cystic fibrosis lungs indicates that DNA-based analyses of upper-airway specimens can misrepresent lung microbiota

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