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Dual species biofilm culture of Staphylococcus aureus and a bioluminescent strain of Pseudomonas aeruginosa

Steven, Joshua; Robinson, Gareth; Nelson, Shona


Joshua Steven

Gareth Robinson
Senior Lecturer in Science Communication


Poster presentation.

Pseudomonas aeruginosa and Staphylococcus aureus are significant pathogens of wounds and the cystic fibrosis lung. There are likely to be complex interactions between the two species, with biofilm co-culture affording them increased tolerance to antimicrobials and enhanced mutual survival. This present study used a simple in vitro biofilm model to co-culture S. aureus and a bioluminescent strain of P. aeruginosa to assess their relative competitive fitness and to use light output as a real-time indicator of P. aeruginosa’s metabolism on antibiotic challenge.

Materials and Methods
Biofilm model
S. aureus ATCC 6358 and P. aeruginosa PAO1 pBBR1 MCS5-lite biofilms were grown on a foam substratum in simulated serum (SS), at 37˚C.
Mono-species biofilms (48 h) were challenged with the second species. Starting inocula were ca. 1 x 108 cfu.
After a further 24 h, biofilms were disaggregated3 and viable cells enumerated on mannitol salts agar (S. aureus) and nutrient agar + gentamicin (10 µg/mL) (P. aeruginosa).
Antibiotic challenge
Biofilms (48 h) were exposed to ciprofloxacin in SS at the minimum inhibitory concentration (MIC) for both species (0.5 µg/mL) and at 5 x MIC. Bioluminescence was monitored over 24 h.

Biofilm population dynamics
Both P. aeruginosa and S. aureus were able to colonise the substratum and increase in population density over 48 h.
When challenged with S. aureus, P. aeruginosa populations were not significantly affected but the S. aureus population, by 72 h, was over 1 log10 lower than its initial inoculum size.
When challenged with P. aeruginosa, S. aureus populations by 72 h were 0.69 log10 lower than when unchallenged and the P. aeruginosa population increased 1.3 log10.
Real-time imaging of antibiotic challenge
Light output from ciprofloxacin-challenged biofilms demonstrated that P. aeruginosa was metabolically active, even at 5 x MIC.

P. aeruginosa was dominant over S. aureus, particularly when already established as a biofilm. P. aeruginosa was not affected, either numerically or metabolically, by the presence of S. aureus and biofilm cells were able to maintain viability in inhibitory concentrations of ciprofloxacin. Interestingly, light output in ciprofloxacin-challenged populations was detected even when control biofilms had ceased to bioluminesce. This model, used with bioluminescent reporters, can generate real-time information about biofilm cell activity and may aid our understanding of the physiology of multispecies biofilm interactions.


Steven, J., Robinson, G., & Nelson, S. (2016, October). Dual species biofilm culture of Staphylococcus aureus and a bioluminescent strain of Pseudomonas aeruginosa

Presentation Conference Type Poster
Start Date Oct 10, 2016
End Date Oct 10, 2016
Acceptance Date Sep 5, 2016
Publication Date Oct 10, 2016
Peer Reviewed Peer Reviewed
Additional Information Title of Conference or Conference Proceedings : Society for Applied Microbiology Early Career Scientist Conference