The effect of interspecies interactions on Pseudomonas aeruginosa biofilm formation and drug susceptibility in an artificial cystic fibrosis lung microbiome Eva Vandeplassche1, Tom Coenye1, Aurélie Crabbé1 1Laboratory
of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium
[email protected]
Introduction Chronic lung infections are the main cause of morbidity and mortality in cystic fibrosis (CF) patients. The source of this continuing infection and inflammation is a polymicrobial community, known as the CF lung microbiome, which forms biofilms that are highly resistant to antimicrobial therapy. One of the most frequently isolated biofilm-forming bacteria in the CF lung is the opportunistic pathogen Pseudomonas aeruginosa. This bacterium poses a great challenge for treatment of CF patients since it is highly virulent and intrinsically resistant to many antimicrobials. However, the exact role of the CF microbiome in P. aeruginosa virulence and drug susceptibility remains unclear. In this study, P. aeruginosa biofilm formation and drug susceptibility in the presence of specific bacterial species of the CF lung microbiome (CF microbiome members; CFMM) was examined.
Methods CFMM composition
Experiment set-up
Selection of six bacterial species that are frequently isolated from the CF lung1,2. For Pseudomonas aeruginosa3, three different strains were selected: the laboratory strain PAO1, the early CF infection strain AA2, and the late CF infection strain AA444
Pseudomonas aeruginosa (PAO1, AA2, and AA44) Staphylococcus aureus Streptococcus anginosus Achromobacter xylosoxidans Rothia mucilaginosa Gemella haemolysans
24 h biofilm formation Bacterial suspensions of P. aeruginosa and CFMM in a 1:1 ratio Biofilm growth in a PVC 96well microtiter plate, incubated in microaerophilic conditions
Plating method
24 h treatment with tobramycin; or untreated Incubated in microaerophilic conditions
On a Pseudomonas aeruginosa selective agar LB agar + 1.25 mg/L triclosan
Results Biofilm formation of P. aeruginosa PAO1 + CFMM 9 7,79 7,42
7,47
7,75
7,37
7,61
8
7,50
7
7
6
6
LOG CFU/mL
5 4 3
*
7,63
7,75 6,92
6,92
6,70
7,27
8
7,46
5 4 3
1
0
0
0
AA2
P+all
2+R
6
6
2+G
Added CF microbiome member(s)
Biofilm of P. aeruginosa AA44 + CFMM, treated with tobramycin
*
*
*
* 6
4,60
4,52 3,60
5
3,26
3 2
5
4,36
3,90 3,43
4 3,22
3,56
3,11
3 2
4
1
0
0
0
AA2
2+R 2+A 2+SP 2+Str 2+G 2+all Added CF microbiome member(s)
3,53
4,04
3,38
2
1
P+R P+A P+SP P+Str P+G P+all Added CF microbiome member(s)
3,49
3,75
3
1
PAO1
4,77
4,09
3,74
3,93
LOG CFU/mL
5
6,93
AA44 44+R 44+A 44+SP 44+Str 44+G 44+all
2+all
4,91
4,90
4
2+SP 2+Str
Biofilm of P. aeruginosa AA2 + CFMM, treated with tobramycin
*
*
2+A
Added CF microbiome member(s)
Biofilm of P. aeruginosa PAO1 + CFMM, treated with tobramycin
7,02
3
1
P+G
7,21
4
1
P+SP P+Str
7,16
5
2
P+A
7,67
7
2
P+R
7,41
7,38
6
Added CF microbiome member(s)
LOG CFU/mL
9
2
PAO1
Susceptibility to tobramycin
*
LOG CFU/mL
LOG CFU/mL
Biofilm formation
8
*
LOG CFU/mL
9
Figure 1-3. Biofilm formation of Pseudomonas aeruginosa in the presence of CF microbiome members.
Biofilm formation of P. aeruginosa AA44 + CFMM
Biofilm formation of P. aeruginosa AA2 + CFMM
AA44 44+R 44+A 44+SP 44+Str 44+G 44+all Added CF microbiome member(s)
P = PAO1, 2 = AA2, 44 = AA44, R = Rothia mucilaginosa, A = Achromobacter xylosoxidans, SP = Staphylococcus aureus SP123, Str = Streptococcus anginosus, G = Gemella haemolysans, all = R + A + SP + Str + G. Mean CFU/mL (in log) with standard errors on the error bars is depicted for multi- and monospecies biofilms of P. aeruginosa PAO1 (left), AA2 (middle), and AA44 (right) with CFMM. n ≥ 3, * p < 0.05
Pseudomonas aeruginosa AA2 forms significantly less biofilm when in a mixed biofilm with either Rothia, Achromobacter or SP123, compared to a monospecies biofilm of AA2. PAO1 and AA44 did not show significant differences in biofilm formation. Figure 4-6. Biofilm of Pseudomonas aeruginosa after 24h treatment with tobramycin, in the presence of CF microbiome members. P = PAO1, 2 = AA2, 44 = AA44, R = Rothia mucilaginosa, A = Achromobacter xylosoxidans, SP = Staphylococcus aureus SP123, Str = Streptococcus anginosus, G = Gemella haemolysans, all = R + A + SP + Str + G. Mean CFU/mL (in log) with standard errors on the error bars is depicted for multi- and monospecies biofilms of P. aeruginosa PAO1 (left), AA2 (middle), and AA44 (right) with CFMM. n ≥ 3, * p < 0.05
Pseudomonas aeruginosa PAO1 is significantly more susceptible to tobramycin in a mixed biofilm with either Rothia or Streptococcus, compared to a monospecies biofilm of PAO1. AA2 is significantly more susceptible to tobramycin when in a mixed biofilm with either Rothia, Achromobacter, Gemella or the combined CFMM. AA44 did not show significant differences.
Conclusion
References 1 Surette,
We conclude that P. aeruginosa biofilm formation and susceptibility to tobramycin is influenced by members of the CF lung microbiome. Therefore, studying P. aeruginosa antibiotic susceptibility in the context of the microbiome could help design treatment strategies with improved efficacy in CF patients.
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(2014) 2 Zhao, J., et al., Proc Natl Acad Sci U S A,.109(15):5809-14 (2012) 3 De Soyza, A., et al., Microbiology Open, 2(6): 1010–1023 (2013) 4 Bragonzi, A., et al., Am J Respir Crit Care Med Vol 180. p 138–145 (2009)
Acknowledgements This research was funded by an FWO Odysseus grant (A.C.)
