Supplementary Information s66

Supplementary Information

Sputum samples were collected in the UCSD Adult CF clinic, and microbial DNA was obtained following the procedure described (Lim et al 2012). The samples were sequenced using the Ion Torrent platform. Sequences were preprocessed according to Ion Torrent quality scores. High quality sequence was retained using Prinseq (Schmieder and Edwards 2011a) with the following command:

perl prinseq-lite-0.20.2/prinseq-lite.pl -verbose -trim_left 1 -trim_right 1 -trim_to_len 660 -derep 1245 -lc_method entropy -lc_threshold 50 -trim_qual_right 15 -trim_qual_left 15 -trim_qual_type mean -trim_qual_rule lt -trim_qual_window 2 -trim_qual_step 1 -trim_tail_left 5 -trim_tail_right 5 -min_len 60 -min_qual_mean 15 -ns_max_p 1 -rm_header -fastq file.fastq -log -out_bad null -out_good

Human sequences were removed from the data using Deconseq (Schmieder and Edwards 2011b) with the following command:

perl deconseq.pl -i 94 -c 90 -f filename -id deconseq_filename -dbs hsref,hs1,hs2,hs3,hs4,hsunique -dbs_retain vir,bact -z 3

Regression of Streptococcus species abundance vs 2,3-butanedione concentration was conducted in R with the following code and outputs:

linear model formula = Medianppt ~ Streptococcus

Residuals:

1 2 3 4 5 6 7

366.7 -480.2 313.2 -200.5 166.2 363.1 -528.4

Coefficients:

Estimate Std. Error t value Pr(>|t|)

(Intercept) -11.18 271.94 -0.041 0.9688

Streptococcus 53.93 13.58 3.971 0.0106 *

---

Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Residual standard error: 434.1 on 5 degrees of freedom

Multiple R-squared: 0.7592, Adjusted R-squared: 0.7111

F-statistic: 15.77 on 1 and 5 DF, p-value: 0.01063

Ferritin transcription and iron availability in CF sputum:

In oxygen depleted CF sputum, transcription of the iron storage protein ferritin provides evidence for the presence of an alternative electron acceptor. Abnormal iron and ferritin levels are an important part of the pathogenesis of many diseases, not just those specific to iron metabolism, including diseases characterized by infection and inflammation (Torti and Torti 2002). Increased iron and ferritin concentrations have been found in CF sputum in several studies (Ghio et al 2012, Gifford et al 2012, Reid et al 2007, Stites et al 1998). Iron may be liberated from ferritin by microbial proteases (Whitby et al 2006) or neutrophil elastase (Fischer et al 2009). In addition to the generation of destructive reactive oxygen species, iron can act as a final electron acceptor in anaerobic respiration, and could cycle the redox state of phenazines. Transcription of human ferritin is a marker of oxidative stress because it is induced via a defense mechanism that is activated by redox changes and other toxic molecules (Mackenzie et al 2008, Torti and Torti 2002, Tsuji et al 2000). Because these conditions are likely to exist in the CF lung environment, we searched metatranscriptome data from the San Diego CF cohort (Lim et al 2012) for human ferritin transcripts. Compared to control genes that are consistently transcribed across tissues, ferritin had one to two orders of magnitude more reads in the CF sputum metatranscriptome (Supplemental Figure 3). Thus, the transcription of ferritin, potentially in response to the redox state of the CF lung, supports the possibility that microbes in the CF lung have access to alternative electron acceptors for anaerobic respiration in the form of Fe3+, and phenazines that recycle their redox state through donation of electrons to Fe3+.

Supplementary Figure Legends

Supplementary Figure 1. Taxonomy of blast hits to genes involved in 2,3-butanedione metabolism (6278 hits) and phenazine synthesis (4109 hits) from 654,000 Roche 454 reads from 18 samples from 6 CF patients at between 2 and 4 time-points each (Lim et al 2012).

Supplementary Figure 2. Fraction of metagenomic reads assigned to several Streptoccocus spp. in three time-points from patient CF1.

Supplementary Figure 3. Ferritin transcript levels in CF metatranscriptomes (Lim et al. 2012) revealed a higher abundance of ferritin heavy chain (FTH1) and to a lesser extent ferritin light chain (FTL) mRNA in comparison to evenly expressed mRNA used as a marker in transcriptome studies (PGK1 and PPIA).

Supplementary Figure 4. Linear model of Streptococcus abundance vs 2,3-butanedione concentration (ppt) from the seven CF patients in the cross-sectional study shows that a significant amount of variation in 2,3-butanedione production is explained by differences in Streptococcus abundance between patients. A) 2,3-butanedione vs Streptococcus abundance plot; B) Residuals vs fitted values from the linear model in R; and C) the standardized residuals vs theoretical quantiles (Normal Q-Q plot) from the linear model.

Supplementary Tables

Supplementary Table 1. BLASTn hits (minimum 60bp and 90% identity and E value of 1´10-10) to genes encoding enzymes for acetoin anabolism (budABC) and catabolism (acoA and acoB) in CF metagenomes from (Lim et al 2012).

budB
large / budB
small / budA / budC / acoA / acoB / Total
Streptococcus spp. / 102 / 47 / 33 / 32 / 61 / 60 / 335
Other / 15 / 6 / 0 / 0 / 0 / 1 / 22
E. coli / 44 / 3 / 0 / 0 / 0 / 0 / 47
P. aeruginosa / 18 / 7 / 0 / 0 / 7 / 7 / 39
S. maltophila / 23 / 0 / 0 / 0 / 0 / 0 / 23
Total / 202 / 63 / 33 / 32 / 68 / 68 / 466

Supplementary Table 2: Percentage of CF sputum samples containing Streptococcus spp. and Pseudomonas spp. in several recent culture-independent 16S rRNA sequence studies of CF microbial communities

Study / Hunter 2012 (Hunter et al 2012b) / Zhao 2012 (Zhao et al 2012) / Delhaes
2012 (Delhaes et al 2012) / Fodor 2012 (Fodor et al 2012) / Goddard 2012 (Goddard et al 2012) i / Filkins 2012 (Filkins et al 2012) / Lim 2012 (Lim et al 2012)
Streptococcus spp. (%) / 86 / >70 / 87 / 95 / ~44 / 79 / 89
Pseudomonas spp. (%) / 97 / >97 / 62 / >78 / >90 / 94 / 56
# CF Patients / 36 / 6 / 4 / 23 / 15 / 35 / 6
Samples / 36 / 126 / 8 / 63 / 43 / 35 / 18

i Lung ex-plant, sputum and mouthwash samples

Supplementary Table 3. 2,3-butanedione concentrations from CF patient, healthy control, and room samples during 7 time-points (shown in Figure 2).

2,3-butanedione (ppt) for patient shown in each column
Timepoint / Day / CF1 / CF1 / CF1 / Healthy (H5) / Room
A / 1 / 4336 / 4437 / 1482 / 261 / 248
B / 36 / 2850 / 2537 / 1861 / 495 / 3
C / 71 / 264 / 333 / 284 / 342 / 596
D / 113 / 5631 / 4851 / 4032 / 388 / 262
E / 142 / 12544 / 5335 / 17270 / 2287 / 485
F / 162 / 2235 / 11802 / 10867 / 945 / 178
G / 169 / 22234 / 26751 / 14445 / 701 / 241

Supplementary Table 4. 2,3-butanedione concentrations from cross-sectional study of seven CF patients, four healthy controls and room samples.

patient / 2,3-butanedione (ppt) / Type / Status
CF1 / 2201 / CF / Suppressive
CF1 / 1370 / CF / Suppressive
CF1 / 3133 / CF / Suppressive
CF1 / 2781 / CF / Suppressive
CF2 / 1008 / CF / Suppressive
CF2 / 37 / CF / Suppressive
CF2 / 774 / CF / Suppressive
CF2 / 562 / CF / Suppressive
CF3 / 749 / CF / Suppressive
CF3 / 599 / CF / Suppressive
CF3 / 1612 / CF / Suppressive
CF4 / 1124 / CF / Suppressive
CF4 / 953 / CF / Suppressive
CF4 / 743 / CF / Suppressive
CF4 / 87 / CF / Suppressive
CF5 / 1681 / CF / Acute
CF5 / 1007 / CF / Acute
CF5 / 666 / CF / Acute
CF6 / 81 / CF / Acute
CF6 / 167 / CF / Acute
CF6 / 143 / CF / Acute
CF6 / 273 / CF / Acute
CF7 / 23 / CF / Acute
CF7 / 30 / CF / Acute
CF7 / 34 / CF / Acute
CF7 / 609 / CF / Acute
H1 / 43 / Control / Control
H1 / 347 / Control / Control
H2 / 132 / Control / Control
H2 / 357 / Control / Control
H2 / 1905 / Control / Control
H2 / 291 / Control / Control
H2 / 201 / Control / Control
H2 / 138 / Control / Control
H2 / 539 / Control / Control
H2 / 716 / Control / Control
H3 / 97 / Control / Control
H3 / 496 / Control / Control
H4 / 3750 / Control / Control
H4 / 4216 / Control / Control
Room / 326 / CF1 Room
Room / 16 / CF2 Room
Room / 10 / CF3 Room
Room / 11 / CF5 Room
Room / 10 / CF6 Room
Room / 30 / CF7 Room

Supplementary Table 5. Consequences of 2,3-butanedione (diacetyl) exposure in a wide variety of systems.

Location / Source / Concentration / Consequence / Reference
Human lung / Popcorn flavoring or diacetyl factory / Lung destruction (Obliterative Bronchiolitis) / (Egilman and Schilling 2012)
Sprague-Dawley rat lungs / Purified dose / 203–371 ppm diacetyl for 6 hours (comparable to factory levels) / Nasal and lung airway epithelia destruction and necrosis / (Hubbs et al 2002)
C57Bl/6 mice / Purified dose / 100 ppm diacetyl 6 h/day 5 days/week for 12 weeks / Injury to the epithelium of the nasal cavity and upper airways / (Morgan et al 2008)
Food additive as preservative / Harvested from Lactobacillus / Kill gram negatives / (Jay 1982)
Wine, butter, popcorn / Lactobacillus / Flavor / Multiple sources 1900-present (Bartowsky and Henschke 2004)
Mosquito nose / Purified dose / Firing of CO2 detection neurons, lose ability to use [CO2] to detect prey / (Turner et al 2011)
Microbial culture / Co-cultured B. subtilis with E. coli, and purified dose / Nanomolar to micromolar / Changes in global gene expression related to motility and antibiotic resistance / (Kim et al 2013)

Supplementary Table 6. Consequences of 2,3-butanediol exposure in a wide variety of systems.

Location / Source / Concentration / Consequence / Reference
Bioreactor with solid-state electrodes as electron acceptors / E. aerogenes / Stimulation of pyocyanin production in P. aeruginosa, anaerobic (microaerobic) respiration / (Venkataraman et al 2011)
Epithelia cells / Vibrio cholera / Suppression of inflammatory signals / (Bari et al 2011)
Rats / Purified dose / Inhibit neutrophils / (Hsieh et al 2007)
Arabidopsis / Root bacterial community, purified dose or B. subtilis / Trigger Induced Systemic Resistance (ISR) and protection from root rot / (Han et al 2006)
Tobacco plant / Pseudomonas chlororaphis and purified dose / Identified 2,3-butanediol as the molecular trigger of ISR, and tried with purified R-R-2,3-bd / Enhances aerial growth, trigger plant defense system through GacS sensor kinase / (Ryu et al 2004)
Potato and chicory / Dickeya and Pectobacteria / 40umol/g in infection, ND in healthy plant tissue / Massively produced 2,3-bd in soft rot infection, bd metabolism mutants not as destructive / (Effantin et al 2011)

Supplementary Table 7. Ion Torrent sequence quality filtering. Samples are labeled with an anonymous patient id (i.e. CF1) followed by a letter indicating the time-point and an abbreviation for their disease state (St = Stable, Tr = Treatment and Pt = Post Treatment).

Sample / Input sequences / After Prinseq / % / length / duplicates / After Deconseq
1 / CF1-L-St / 1,780,819 / 1,494,421 / 84% / 133 / 57,857 / 1317296
2 / CF2-E-St / 1,973,912 / 1,593,530 / 81% / 150 / 158,584 / 925360*
3 / CF3-A-St / 1,780,474 / 1,436,594 / 81% / 144 / 154,059 / 1,417,311
4 / CF4-A-St / 5,877,520 / 4,334,671 / 74% / 139 / 987,405 / 4,762,081
5 / CF5-A-Tr / 1,162,089 / 964,098 / 83% / 144 / 59,996 / 549831
6 / CF6-A-St / 1,836,488 / 1,471,073 / 80% / 136 / 171,483 / 1356155
7 / CF7-A-Pt / 1,428,012 / 1,593,530 / 81% / 150 / 580,864 / 1139292
8 / H2-A / 3,956,658 / 2,992,662 / 76% / 145 / 476112 / 2096847
TOTAL / 19,795,972 / 15,880,579 / 2,646,360 / 13,564,173

*half of total available data processed

Supplementary Table 8. Summary of clinical information, 2,3 butanedione concentration (ppt) and abundance of three most prevalent bacteria for CF patients

patient / CF1 / CF2 / CF3 / CF4 / CF5 / CF6 / CF7
age / 40 / 39 / 27 / 52 / 31 / 21 / 44
Rothia / 16.6 / 68.9 / 79 / 4.6 / 73.7 / 0.0 / 15.5
Pseudomonas / 11.8 / 1.1 / 0 / 77.6 / 0.0 / 31 / 0.0
Streptococcus / 39.6 / 21.5 / 8 / 9.2 / 22.6 / 0.0 / 10.6
Acute antibiotic treatment / 0 / 0 / 0 / 0 / 1 / 1 / 1
Days since acute
abx treatment / 62 / 108 / 368 / 315 / 0 / 0 / 5
Medianppt / 2491 / 668 / 749 / 848 / 1007 / 155 / 32

Supplementary Table 9. Pearson correlations between data from Supplementary Table 8, the clinical information, 2,3 butanedione concentration (ppt) and abundance of three most prevalent bacteria for CF patients

pearson correlations with cor() in R
age / Rothia / Pseudo
monas / Strepto
coccus / Acute
abx / Days
since
abx / 2,3bd
(ppt)
median
age / 1 / -0.3 / 0.43 / 0.28 / -0.38 / 0.21 / 0.19
Rothia / 1.00 / -0.63 / 0.20 / -0.19 / 0.23 / 0.03
Pseudomonas / 1.00 / -0.32 / -0.23 / 0.39 / -0.02
Streptococcus / 1.00 / -0.35 / -0.24 / 0.87
Acute antibiotic treatment / 1.00 / -0.73 / -0.52
Days since acute abx treatment / 1.00 / 0.05
Median 2,3bd (ppt) / 1.00

Supplementary Table 10. Taxonomic abundance of each sample from cross-sectional study determined by Metaphlan (results shown in Figure 4).

genus / 1 Stable / 2 Stable / 3 Stable / 4 Stable / 5 Stable / 6 Tr. / 7 Post-Tr / CF average / Healthy / Pooled CF
Rothia / 16.6 / 68.9 / 78.9 / 4.6 / 73.7 / 15.5 / 36.9 / 11.3 / 30.4
Pseudomonas / 11.8 / 1.1 / 77.6 / 30.6 / 17.3 / 16.1 / 5.0
Streptococcus / 39.6 / 21.5 / 8.3 / 9.2 / 22.6 / 10.6 / 16.0 / 40.3 / 34.0
Staphylococcus / 63.0 / 9.0
Bordetella / 30.4 / 4.3
Achromobacter / 26.7 / 3.8
Veillonella / 17.8 / 2.3 / 1.8 / 3.1 / 2.0
Actinomyces / 1.3 / 3.1 / 3.6 / 1.1 / 1.0 / 3.2
Neisseria / 0.0 / 9.2
Prevotella / 5.0 / 1.3 / 0.9 / 3.9
Stenotrophomonas / 1.1 / 0.2 / 7.7
Gemella / 1.2 / 0.2 / 4.6
Escherichia / 1.1 / 0.2 / 4.6
Burkholderia / 5.0 / 0.7
Haemophilus / 0.0 / 3.7
Oribact-erium / 3.6 / 0.5

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