Impact of lysis method on community structure.

<p>Bray-Curtis-based nonmetric multidimensional scaling (NMDS) plot showing pairwise comparison of samples processed with the standard (blue symbols) or modified (red symbols) lysis protocols. Solid symbols represent paired samples with greater separation.</p

Characteristics of NTM culture-positive samples.

<p>Characteristics of NTM culture-positive samples.</p

Differences in alpha diversity between lysis protocols.

<p>Samples processed with the modified protocol had higher levels of (A) richness (p = 0.04, paired t-test), and higher (C) Shannon diversity (p = 0.004, paired t-test) than samples processed with the standard protocol. (B) Evenness did not differ between the lysis protocols (p = 0.06, paired t-test). Blue and red symbols represent paired samples with greater separation on NMDS. Error bars indicate mean and SD.</p

Improvement in NTM DNA extraction from spiked sputum samples with the modified as compared to the standard lysis protocol.

<p>Log₁₀ <i>atpE</i> gene copies/mL in DNA extracted from sputum spiked with either (A) MABSC or (B) MAC using the standard (blue circles) or the modified (red squares) lysis protocols. Error bars indicate mean and SD.</p

Culture-Independent Identification of Nontuberculous Mycobacteria in Cystic Fibrosis Respiratory Samples - Fig 2

<p>(A) Relative abundances of NTM OTUs and (B) total bacterial load in NTM culture-positive samples. (A) The mean relative abundance of NTM OTUs in the samples processed with the standard protocol was not significantly different from that observed when these samples were processed with the modified protocol (mean 0.098% and 1.21%, respectively; p = 0.08, paired t-test). (B) Total bacterial load in NTM culture-positive samples as measured by 16S rRNA gene qPCR did not significantly differ between lysis protocols. (p = 0.91, paired t-test). Error bars indicate mean and SD.</p

Fluctuations in airway bacterial communities associated with clinical states and disease stages in cystic fibrosis

<div><p>Bacteria that infect the airways of persons with cystic fibrosis (CF) include a group of well-described opportunistic pathogens as well as numerous, mainly obligate or facultative anaerobic species typically not reported by standard sputum culture. We sequenced the V3-V5 hypervariable region of the bacterial 16S rRNA gene in DNA derived from 631 sputum specimens collected from 111 CF patients over 10 years. We describe fluctuations in the relative abundances of typical CF pathogens, as well as anaerobic species, in relation to changes in patients’ clinical state and lung disease stage. Both bacterial community diversity and the relative abundance of anaerobes increased during exacerbation of symptoms (prior to antibiotic treatment), although this trend was not observed uniformly across disease stages. Community diversity and the relative abundance of anaerobic species decreased during antibiotic treatment. These results support current hypotheses regarding the role of anaerobes in CF pulmonary exacerbations and lung disease progression.</p></div

Subject demographics and sample characteristics.

<p>Subject demographics and sample characteristics.</p

Impact of DNA extraction method on measures of <i>Staphylococcus</i> relative abundance alpha diversities.

<p>(<b>A</b>) Relative abundance of <i>Staphylococcus</i> and (<b>B</b>) community diversity (Shannon index), (<b>C</b>) richness (number of observed OTUs) and (<b>D</b>) evenness (Shannon evenness) in <i>Staphylococcus</i>-rich and <i>Staphylococcus</i>-poor samples revealed by the standard lysis method (−LY, black bars) and the lysostaphin-lysozyme (+LY, white bars) method.</p

Relative abundance of anaerobic genera and Shannon diversity in bacterial communities based on patient clinical state and disease stage.

<p>The cumulative relative abundances of anaerobic genera (<i>Actinomyces</i>, <i>Fusobacterium</i>, <i>Gemella</i>, <i>Granulicatella</i>, <i>Porphyromonas</i>, <i>Prevotella</i>, <i>Rothia</i>, <i>Streptococcus</i> and <i>Veillonella</i> spp) are depicted by (A) clinical state and (B) disease stage. Bacterial community Shannon diversity is depicted by (C) clinical state and (D) disease stage. ***<i>P</i> ≤ 0.001 (GEE).</p

Culture and pyrosequencing based measures of <i>Staphylococcus</i> abundance.

1<p>Abundance of <i>S. aureus</i> in culture (reported by clinical microbiology laboratory).</p>2<p>Relative abundance by pyrosequencing without (−LY) and with (+LY) lysostaphin-lysozyme; no. <i>Staphylococcus</i> sequence reads/no. total sequence reads.</p>3<p>Total number of OTUs observed after normalization of sequence reads to 498, the smallest number of sequences obtained among the 34 samples.</p>4<p>The rank order of the relative abundance of <i>Staphylococcus</i>.</p>5<p>With the exception of sample 16, samples with “numerous <i>S. aureus</i>” detected in culture are referred to as <i>Staphylococcus</i>-rich samples. Sample 16 and all other samples are referred to as S<i>taphylococcus</i>-poor samples.</p><p>ND: not detected.</p