Table 1.
Bacterial strains.
Fig 1.
Population dynamics and virulence factor production in co-culture biofilms.
Bacterial strains—Pa, P. aeruginosa CF2004; Sm, S. mitis NCTC 12261T; Sg, S. gordonii NCTC 7865T; Ss, S. sanguinis NCTC 7863T; So, S. oralis ATCC 35037T and So spxB, S. oralis ATCC 35037T spxB mutant. Inoculation ratios for Pa:Sm, Pa:Sg, Pa:Ss, Pa:So and Pa:So spxB were 1:6, 1:18, 1:24, 1:6 and 1:5 respectively. (A) Biofilms formed on nitrocellulose filters after 48 h (37°C, 10% CO2). (B) Quantitative bacteriology (CFU / filter) of monoculture and co-culture biofilms sampled after 48 h (at 37°C, 10% CO2); shaded bars P. aeruginosa CF2004 population, unshaded bars streptococcal population; significant differences between cfu in monocultures and co-cultures (p<0.001***) using two-tailed t-test were observed for all streptococci with the exception of the S.oralis spxB mutant. (C) Pyocyanin and (D) elastase activity per P. aeruginosa cfu. Fold increase in co-culture biofilms compared with mono-culture control displayed. Biofilms sampled after 48 h (at 37°C); standard deviation of triplicate or quadruplicate cultures shown. Results significantly different from control using ANOVA with Holm Sidak post hoc test are denoted with an asterisk (*p<0.05, ***p<0.001). A significant fold-increase in pyocyanin was also observed between the Pa+Sm / Pa+Ss pairing (p<0.001) and for elastase production significant increases were observed for the following pairings; Pa+Sm / Pa+Ss, Pa+Sm / Pa+So spxB (p<0.001), Pa+Sm / Pa+Sg (p<0.01) and Pa+Sg / Pa+Ss (p<0.05).
Fig 2.
Catalase-mediated protection of the CF2004 population when grown in biofilm co-culture with S. oralis.
Bacterial strains—Pa, P. aeruginosa CF2004; So, S. oralis ATCC 35037T. A) Biofilms grown on nitrocellulose filters for 48 h (37°C, 10% CO2) in the presence and absence of catalase (500 units). (B) Quantitative bacteriology (CFU / filter) of samples taken from central regions of biofilms (+ or - catalase) after 24 and 48 h at 37°C (+10% CO2); shaded bars 24 h population, unshaded bars 48 h population. Detection limit; 1.0x103 CFU/ sample.
Fig 3.
Quantitative bacteriology and H2O2 production in streptococcal mono-culture biofilms.
A) Quantitative bacteriology and B) H2O2 production in monoculture biofilms sampled after 24 h incubation at 37°C in the indicated atmosphere. p values using ANOVA and Holm-Sidak post hoc test for pairwise comparisons of the quantitative bacteriology data and H2O2 production are shown in accompanying matrices (Aii, Aiii, Bii and Biii). C) Quantitative bacteriology of Streptococcus oralis monoculture biofilms with and without catalase. Significant difference (p<0.001***) using the two-tailed t-test shown.
Fig 4.
Antagonism of P. aeruginosa growth by pioneering streptococcal colonisers.
Bacterial strains—Pa, P. aeruginosa CF2004; Sm, S. mitis NCTC 12261T; Sg, S. gordonii NCTC 7865T; Ss, S. sanguinis NCTC 7863T; So, S. oralis ATCC 35037T, and So spxB, S. oralis ATCC 35037T spxB mutant. Streptococcal spp were inoculated onto the agar surface as pioneer colonisers and incubated for 24 h at 37°C h in an A and C) aerobic (+10% CO2) and B and D) anaerobic atmosphere before CF2004 cultures were adjacently inoculated as secondary colonisers and grown for a further 24 h at 37°C h in an aerobic atmosphere (+10% CO2).
Fig 5.
S. oralis also inhibits growth of other common P. aeruginosa CF phenotypes.
Bacterial strains—768, P. aeruginosa 768; H129, P. aeruginosa H129; DWW2, P. aeruginosa DWW2; So, S. oralis ATCC 35037T. (A) Biofilms formed on nitrocellulose filters after 48 h (37°C, 10% CO2). (B) Quantitative bacteriology (CFU / filter) of samples taken from central regions of monoculture and co-culture biofilms after 24 and 48 h at 37°C (+10% CO2); shaded bars 24 h population, unshaded bars 48 h population. Detection limit; 1.0x103 CFU/ Sample. Standard deviation shown for duplicate or triplicate samples.
Fig 6.
Enhancement of P. aeruginosa CF2004 virulence factor by S. oralis in biofilms incubated without added CO2.
Bacterial strains—Pa, P. aeruginosa CF2004; So, S. oralis ATCC 35037T. Biofilms were grown aerobically on nitrocellulose filters for 48 h at 37°C. A) photographs and (B) quantitative bacteriology (CFU / filter) of monoculture and co-culture biofilms; shaded bars P. aeruginosa CF2004 population, unshaded bars streptococcal population. (C) Pyocyanin and (D) elastase activity in biofilms. Standard deviation of triplicate or quadruplicate cultures shown. Results significantly different from control using the two-tailed t-test are denoted with asterisks (*p<0.05, ** p<0.01).