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

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

Characteristics of NTM culture-positive samples.

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

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

Weight trends.

<p>(<b>A</b> and <b>B</b>) Mice inoculated with <i>M</i>. <i>abscessus</i> trended towards greater weight loss than mice inoculated with sterile thrombin/ fibrinogen. (N = 19–40 mice in each <i>M</i>. <i>abscessus</i> group, N = 6–16 mice in each sterile inoculum group). Mice receiving smooth morphotype inoculation that had some colony conversion to rough morphotype on either BALF, lung, or spleen cultures had trend towards greater weight loss and slower weight gain. (N = 8–10 for WT mice, 4–6 for CF mice). Data displayed as mean of group, pooled from 2–3 replicate experiments for each morphotype.</p

BALF neutrophil percentage.

<p>(<b>A</b>, <b>B</b>, <b>C</b> and <b>D</b>) Inoculation with rough morphotype causes greater BALF neutrophil percentage than smooth morphotype at 14 days post-inoculation in CF mice (<b>D</b>). Red circles indicate smooth morphotype inoculation with conversion to rough morphotype. Minimal BALF neutrophilia was seen after sterile thrombin/fibrinogen inoculation. (N = 9–20 mice in each <i>M</i>. <i>abscessus</i> group, N = 3–8 mice in each sterile group). * p< 0.05. Data displayed as mean ± SEM of group, pooled from 2–3 replicate experiments for each morphotype.</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

BALF neutrophils.

<p>(<b>A</b>, <b>B</b>, <b>C</b> and <b>D</b>) Inoculation with rough morphotype causes greater BALF neutrophilia than smooth morphotype at 14 days post-inoculation in both WT (<b>B</b>) and CF (<b>D</b>) mice. Red circles indicate smooth morphotype inoculation with conversion to rough morphotype. Minimal BALF neutrophilia was seen after sterile thrombin/fibrinogen inoculation. (N = 4–20 mice in each <i>M</i>. <i>abscessus</i> group, N = 3–8 mice in each sterile group). * p< 0.05. Data displayed as mean ± SEM of group, pooled from 2–3 replicate experiments for each morphotype.</p

Fibrin plug model.

<p>Thrombin and fibrinogen solutions, combined here on the bench top, form gelatinous fibrin plugs that retain the bacteria in the distal airways.</p