Figure 1. Leveraging clinical microbiome data sets with computational analyses to identify communities and community members to model in vitro.
(A) Relative 16S rRNA gene abundance and prevalence of the top 10 cystic fibrosis (CF) lung pathogens in the 167 persons with CF (pwCF) data set used as the basis for developing the in vitro mixed community, as reported by Hampton et al., 2021. (B) Number of unique samples for which >70% of 16S rRNA reads are associated with the combined presence of Pseudomonas, Staphylococcus, Streptococcus, and Prevotella. #Indicates the number of samples (103) that meet this criterion from the total sample size of 167 pwCF. (C) Colony forming units (CFUs) counts of each microbial member grown as a monoculture and in a mixed community (Mix) for biofilm (B) and planktonic (P) fractions. CFUs were performed by plating on medium selective for the growth of each microorganism. Each data point presented in a column represents the average from at least three technical replicates performed at least on three different days (n=6). Statistical analysis was performed using ordinary one-way ANOVA and Tukey’s multiple comparisons posttest with *, p<0.05; ****, p<0.0001, ns = non-significant. Error bars represent SD. Pa = Pseudomonas aeruginosa, Sa = Staphylococcus aureus, Strep = Streptococcus sanguinis, Prev = Prevotella melaninogenica, and bd = below detection.
Figure 1—figure supplement 1. Leveraging clinical microbiome data sets with computational analyses to identify communities and community members to model in vitro.
(A) In silico prediction of metabolic flux between P. aeruginosa-dominated (Pa.D), Streptococcus-dominated (Strep.D) and the “mixed” Pa.M1 and Pa.M2 communities based on the abundance of P. aeruginosa, S. aureus, S. sanguinis, and P. melaninogenica analyzed by Hampton, O’Toole, and colleagues (Hampton et al., 2021). (B and C) Number of unique samples for which >70% of 16S rRNA reads are associated with the combined presence of Pseudomonas, Staphylococcus, Streptococcus, Prevotella with either Burkholderia (B), or Achromobacter (C). #Indicates the number of samples that meet these criteria from the total sample size is 167 persons with cystic fibrosis (pwCF). Compare the values in panels B,C with the 103 samples with >70% Pseudomonas, Staphylococcus, Streptococcus, and Prevotella shown in Figure 1B.
Figure 1—figure supplement 2. Prevotella melaninogenica does not grow as monoculture biofilms in artificial sputum medium (ASM) in anoxic conditions.
Colony forming units (CFUs) of P. melaninogenica grown in ASM or Prevotella growth medium (PGM) as planktonic or biofilm monospecies communities using an anoxic environmental chamber. Each data point presented in a column represents the average of three biological replicates each with three technical replicates (n=3). Prev = P. melaninogenica. Error bars represent SD. Statistical analyses were done using t-test.with **, p<0.01. ns = non-significant, bd = below detection.
Figure 1—figure supplement 3. Productivity of P. aeruginosa, S. aureus, S. sanguinis, and P. melaninogenica grown as monospecies or mixed planktonic/biofilm communities.
(A) Measures of productivity (or yield) represented as colony forming unit (CFU)/mL counts of P. aeruginosa, S. aureus, S. sanguinis, and P. melaninogenica grown in planktonic or biofilm and as pure or mixed communities. For theoretical productivity of planktonic and biofilm mixed communities, we assumed additivity of the four community members (denoted as ‘Mix (P/B) – Theoretical’). Observed yield of the mixed communities is based on actual CFU/mL yield counts of each of the four species shown as ‘Mix (P/B) – Observed’. (B) Productivity comparisons of P. aeruginosa, S. aureus, P. melaninogenica, and S. sanguinis grown as monospecies biofilms to their counterparts grown in a mixed biofilm community. Data points presented in (A) and (B) are tabulated from data shown in Figure 1C, each with three technical replicates performed on six different days (n=6). Raw data for the figure is presented in Source code 1. Pa = P. aeruginosa, Sa = S. aureus, Strep = S. sanguinis, and Prev = P. melaninogenica. Mix = mixed biofilm community. Y=yes, N=no. ns = non-significant. *, p<0.05; **, p<0.01; ***, p<0.001. Error bars represent SD. Statistical analyses were done using t-tests.
Figure 1—figure supplement 3—source data 1. Raw data for productivity calculation of P. aeruginosa, S. aureus, S. sanguinis, and P. melaninogenica grown in various conditions.
Data presented in Figure 1—figure supplement 3 represents the average of six biological replicates, each with three technical replicates performed at least on three different days. For each microorganism grown in the mixed communities, the log10 geometric mean value of the three biological replicates was transformed to colony forming unit (CFU)/mL counts and divided by the total CFU/mL of the community (total community). Data visualization was done using GraphPad Prism 9 (version 9.2.0).
elife-81604-fig1-figsupp3-data1.xlsx (18.3KB, xlsx)
Figure 1—figure supplement 4. Microbial composition range of in vivo CF mixed communities compared with the in vitro model.
(A) Relative abundance ranges of 16S rRNA reads assigned to Pseudomonas, Staphylococcus, Streptococcus, and Prevotella detected in the M1/M2 community types identified previously (Hampton et al., 2021). These data were plotted against the relative abundance of P. aeruginosa, S. aureus, S. sanguinis, and P. melaninogenica grown in the in vitro mixed community shown in Figure 1C. (B) In vitro relative abundance of P. aeruginosa, S. aureus, S. sanguinis, and P. melaninogenica in mixed communities where P. aeruginosa was maintained at the same starting inoculum and other microbes grown 10× in excess or diluted from 1:10 to 1:1000 relative to P. aeruginosa. Data points presented in (B) represents the average from three biological replicates, each with three technical replicates performed on three different days (n=3). Raw data for (B) is presented in Source code 2. Pa = P. aeruginosa, others = S. aureus, S. sanguinis, and P. melaninogenica.
Figure 1—figure supplement 4—source data 1. Raw data for in vitro relative abundance of P. aeruginosa, S. aureus, S. sanguinis, and P. melaninogenica grown in various ratios in mixed communities.
Data presented in Figure 1—figure supplement 4 represents the average of three biological replicates, each with three technical replicates performed on three different days. For each microorganism grown in the mixed communities, the log10 geometric mean value of the three biological replicates was transformed to colony forming unit (CFU)/mL counts and divided by the total CFU/mL of the community (total community). Data visualization was done using GraphPad Prism 9 (version 9.2.0).
elife-81604-fig1-figsupp4-data1.xlsx (14.7KB, xlsx)
Figure 1—figure supplement 5. Testing additional laboratory and clinical strains in the in vitro polymicrobial community model.
Colony forming units (CFUs) of communities including different (A) S. aureus strains, (B) P. melaninogenica (P. mel), Prevotella intermedia (P. int), (C) Streptococcus spp., and (D) P. aeruginosa strains. Various clinical isolates/strains pairs shown in panels A–D were tested in the in vitro polymicrobial system to determine if the observations reported in Figure 1C were also applicable to these strains. Each data point presented in a column represents the average from at least three technical replicates performed at least on three different days (n=3). Statistical analysis was performed using ordinary one-way ANOVA and Tukey’s multiple comparisons posttest with *, p<0.05; **, p<0.01; ***, p<0.001, and ****, p<0.0001, ns = non-significant. Error bars represent SD. Pa = P. aeruginosa PA14, Sa = S. aureus Newman, Strep = S. sanguinis, Prev = P. melaninogenica, and bd = below detection.
Figure 1—figure supplement 6. Fourteen days co-culture experiment of community members grown in a planktonic and biofilm mixed communities.
Colony forming units (CFUs) of (A) P. aeruginosa, (B) S. aureus, (C) S. sanguinis, and (D) P. melaninogenica. Red lines represent biofilm cells and black lines the planktonic cells. Each data point presented in the curves represents the average from at least three technical replicates performed at least on three different days (n=3). Every 24 hr the medium was removed, and then fresh medium added. Statistical analysis was performed using ordinary one-way ANOVA and Dunnett’s multiple comparisons posttest with the 24 hr timepoint as control. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Error bars represent SD.