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

Abstract Background Azithromycin is commonly prescribed drug for individuals with cystic fibrosis (CF), with demonstrated benefits in reducing lung function decline, exacerbation occurrence and improving nutrition. As azithromycin has antimicrobial activity against components of the uncultured microbiome and increasingly the CF microbiome is implicated in disease pathogenesis – we postulated azithromycin may act through its manipulation. Herein we sought to determine if the CF microbiome changed following azithromycin use and if clinical benefit observed during azithromycin use associated with baseline community structure. Results Drawing from a prospectively collected biobank we identified patients with sputum samples prior to, during and after initiating azithromycin and determined the composition of the CF microbial community by sequencing the V3-V4 region of the 16S rRNA gene. We categorized patients as responders if their rate of lung function decline improved after azithromycin initiation. Thirty-eight adults comprised our cohort, nine who had not utilized azithromycin in at least 3 years, and 29 who were completely naïve. We did not observe a major impact in the microbial community structure of CF sputum in the 2 years following azithromycin usage in either alpha or beta-diversity metrics. Seventeen patients (45%) were classified as Responders – demonstrating reduced lung function decline after azithromycin. Responders who were naïve to azithromycin had a modest clustering effect distinguishing them from those who were non-Responders, and had communities enriched with several organisms including Stenotrophomonas, but not Pseudomonas. Conclusions Azithromycin treatment did not associate with subsequent large changes in the CF microbiome structure. However, we found that baseline community structure associated with subsequent azithromycin response in CF adults.

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SERUM DERIVED PROTEINS AND ANTIBACTERIAL PROTEINS IN CYSTIC FIBROSIS SPUTUM

Proceedings of the Second Symposium Lyon, France, June 27–30, 1983 ◽

10.1515/9783112328262-028 ◽

1984 ◽

pp. 239-242

Keyword(s):

Cystic Fibrosis ◽

Cystic Fibrosis Sputum

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Release of the antimicrobial peptide LL-37 from DNA/F-actin bundles in cystic fibrosis sputum

European Respiratory Journal ◽

10.1183/09031936.00080806 ◽

2007 ◽

Vol 29 (4) ◽

pp. 624-632 ◽

Cited By ~ 68

Author(s):

R. Bucki ◽

F. J. Byfield ◽

P. A. Janmey

Keyword(s):

Cystic Fibrosis ◽

Antimicrobial Peptide ◽

Actin Bundles ◽

Cystic Fibrosis Sputum

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Cystic Fibrosis Sputum Impairs the Ability of Neutrophils to Kill Staphylococcus aureus

Pathogens ◽

10.3390/pathogens10060703 ◽

2021 ◽

Vol 10 (6) ◽

pp. 703

Author(s):

Kayla Fantone ◽

Samantha L. Tucker ◽

Arthur Miller ◽

Ruchi Yadav ◽

Eryn E. Bernardy ◽

...

Keyword(s):

Cystic Fibrosis ◽

Staphylococcus Aureus ◽

Healthy Volunteers ◽

Airway Disease ◽

Neutrophil Extracellular Trap ◽

Lung Function Decline ◽

Bacterial Killing ◽

Cystic Fibrosis Sputum ◽

Microbial Infections

Cystic fibrosis (CF) airway disease is characterized by chronic microbial infections and infiltration of inflammatory polymorphonuclear (PMN) granulocytes. Staphylococcus aureus (S. aureus) is a major lung pathogen in CF that persists despite the presence of PMNs and has been associated with CF lung function decline. While PMNs represent the main mechanism of the immune system to kill S. aureus, it remains largely unknown why PMNs fail to eliminate S. aureus in CF. The goal of this study was to observe how the CF airway environment affects S. aureus killing by PMNs. PMNs were isolated from the blood of healthy volunteers and CF patients. Clinical isolates of S. aureus were obtained from the airways of CF patients. The results show that PMNs from healthy volunteers were able to kill all CF isolates and laboratory strains of S. aureus tested in vitro. The extent of killing varied among strains. When PMNs were pretreated with supernatants of CF sputum, S. aureus killing was significantly inhibited suggesting that the CF airway environment compromises PMN antibacterial functions. CF blood PMNs were capable of killing S. aureus. Although bacterial killing was inhibited with CF sputum, PMN binding and phagocytosis of S. aureus was not diminished. The S. aureus-induced respiratory burst and neutrophil extracellular trap release from PMNs also remained uninhibited by CF sputum. In summary, our data demonstrate that the CF airway environment limits killing of S. aureus by PMNs and provides a new in vitro experimental model to study this phenomenon and its mechanism.

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Validating DNA Extraction Protocols for Bentonite Clay

mSphere ◽

10.1128/msphere.00334-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 3

Author(s):

Katja Engel ◽

Sara Coyotzi ◽

Melody A. Vachon ◽

Jennifer R. McKelvie ◽

Josh D. Neufeld

Keyword(s):

Microbial Community ◽

Nucleic Acid ◽

Nucleic Acids ◽

Dna Extraction ◽

Genomic Dna ◽

Bentonite Clay ◽

Dna Recovery ◽

Blocking Agents ◽

Clay Matrix ◽

Microbial Dna

ABSTRACT Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material “spiked” with Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination. IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.

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