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

Abstract

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)...

Clinical Significance of Microbial Infection and Adaptation in Cystic Fibrosis

“…Resolution of the symptoms of clinical exacerbation was associated with antistreptococcal therapy and reduction in the density of this bacterium in sputum, suggesting that these bacteria may be a cause of pulmonary exacerbations in CF (544). Genotyping did not show evidence of patient-to-patient transmission, suggesting that patients are infected by their own endogenous flora (546). Other streptococci, such as S. agalactiae, have also been associated with CF.…”

Polymicrobial Interactions: Impact on Pathogenesis and Human Disease

“…Furthermore, it was demonstrated that some of these same gene modulations could be mediated by the quorum-sensing molecule AI-2, which can accumulate to high levels in the sputum of CF patients, suggesting that intermicrobial communication between microbes of the "normal" flora may exacerbate CF disease caused by classical pathogens. A culture-independent analysis of a small cohort of CF patients revealed that SMG organisms not only were found during episodes of CF airway exacerbations but also were the numerically dominant species present (195). While the virulence mechanisms of SMG are unclear at this time, future studies should determine whether the increased infectious burden, the quorum-sensingenhanced virulence of other species present, or a combination of these effects results in enhanced CF disease.…”

Pharmacokinetics and Pharmacodynamics of Aerosolized Antibacterial Agents in Chronically Infected Cystic Fibrosis Patients

“…For example, it was demonstrated in experimental animals that the presence of avirulent, oropharyngeal flora significantly increased the pathogenicity of P. aeruginosa (345). Another example is the need for antibiotic therapy of the Streptococcus milleri group as a new pathogen in the CF lung in order to prevent a loss of lung function and reduce the frequency of exacerbations (367). The actual culture-based techniques to identify causative pathogens of CF lung disease are inadequate to identify microbial community richness.…”