Impact of Enhanced Staphylococcus DNA Extraction on Microbial Community Measures in Cystic Fibrosis Sputum

Jiangchao Zhao,John J Lipuma,Joseph F Petrosino,Linda M Kalikin,Jun Li,Lisa A Carmody,Vincent B Young,Patrick D Schloss,John R Battista

doi:10.1371/journal.pone.0033127

Jiangchao Zhao, John J Lipuma + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0033127

Copy DOI

Abstract

Staphylococcus aureus is a common constituent of the bacterial community inhabiting the airways of persons with cystic fibrosis (CF). Culture-independent studies have shown that this species is often present in relatively high abundance and would therefore be expected to exert a pronounced effect on measures of CF airway bacterial community structure. We investigated the impact of DNA extraction method on pyrosequencing-based measures of Staphylococcus abundance and bacterial community structure in 17 sputum samples from five CF patients. Staphylococcus was detected in fewer samples when DNA was extracted using a standard bacterial lysis method compared to when DNA was extracted using a lysis buffer amended with lysostaphin and lysozyme. The standard lysis method resulted in significantly lower measures of Staphylococcus relative abundance and higher levels of community diversity, richness, and evenness compared to the lysostaphin-lysozyme modified method. Measures of community dynamics in serial sputum samples from the same individual were nevertheless highly concordant between the two DNA extraction methods. These results illustrate the impact of DNA preparation method on measures of Staphylococcus abundance and bacterial community structures in studies of the airways microbiota in CF.

Highlights

Cystic fibrosis (CF), an autosomal recessive genetic disease affecting more than 70,000 people worldwide, is caused by mutations in the CF transmembrane conductance regulator gene
Conventional culturebased microbiology has identified a number of bacterial species that commonly infect the airways of persons with CF, including Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia complex, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans [2]
In preliminary experiments assessing the airways microbiome in CF, we found that the combination of lysostaphin and lysozyme increased the yield of Staphylococcus DNA from sputum samples compared with a standard bacterial cell lysis buffer

Summary

Introduction

Cystic fibrosis (CF), an autosomal recessive genetic disease affecting more than 70,000 people worldwide, is caused by mutations in the CF transmembrane conductance regulator gene. Conventional culturebased microbiology has identified a number of bacterial species that commonly infect the airways of persons with CF, including Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia complex, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans [2]. Among these species, S. aureus typically infects patients at an early age, being most frequently isolated from infants and children with CF [2,3]. We wished to further examine the impact of DNA preparation method on pyrosequencing-based measures of Staphylococcus abundance and overall bacterial community structure in sputum specimens from patients culturepositive for S. aureus

Methods

Results

Conclusion