Impact of mixed Staphylococcus aureus-Pseudomonas aeruginosa biofilm on susceptibility to antimicrobial treatments in a 3D in vitro model

Abstract

Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacteria co-isolated from chronic infected wounds. Their interactions remain unclear but this coexistence is beneficial for both bacteria and may lead to resistance to antimicrobial treatments. Besides, developing an in vitro model where this coexistence is recreated remains challenging, making difficult their study. The aim of this work was to develop a reliable polymicrobial in vitro model of both species to further understand their interrelationships and the effects of different antimicrobials in coculture. In this work, bioluminescent and fluorescent bacteria were used to evaluate the activity of two antiseptics (chlorhexidine and thymol) against these bacteria planktonically grown, or when forming single and mixed biofilms. At the doses tested (0.4-1,000 mg/L), thymol showed selective antimicrobial action against S. aureus in planktonic and biofilm states, in contrast with chlorhexidine which exerted antimicrobial effects against both bacteria. Furthermore, the initial conditions for both bacteria in the co-culture determined the antimicrobial outcome, showing that P. aeruginosa impaired the proliferation and metabolism of S. aureus. Moreover, S. aureus showed an increased tolerance against antiseptic treatments when co-cultured, attributed to the formation of a thicker mixed biofilm compared to those obtained when monocultured, and also, by the reduction of S. aureus metabolic activity induced by diffusible molecules produced by P. aeruginosa. This work underlines the relevance of polymicrobial populations and their crosstalk and microenvironment in the search of disruptive and effective treatments for polymicrobial biofilms.

Keywords: Pseudomonas aeruginosa; Staphylococcus aureus; Antiseptics; Chlorhexidine; Polymicrobial biofilm; Thymol.

Fig. 1

Bioluminescence signals from (a) Planktonic S. aureus Xen36 (10⁷ CFU/mL) co-cultured with increasing amounts of planktonic P. aeruginosa PAO1 (10¹-10⁸ CFU/mL) and (b) Planktonic P. aeruginosa Lux (10¹-10⁸ CFU/mL) co-cultured with a fixed amount of S. aureus ATCC 29213 (10⁷ CFU/mL). The acronym NSI stands for “Normalized Signal Intensity”. Control samples (Ctrl) represent the normal growth of cultures of S. aureus without the addition of P. aeruginosa. (c) Bacterial counts of both species in the cultures after 48 h (data obtained with conditions (a) and (b) were similar). The data are represented as Mean ± SD of three independent experiments.

Fig. 2

Bioluminescence signals from (a) 48-hours old S. aureus Xen36 biofilm supplemented with increasing amounts of planktonic P. aeruginosa PAO1 (10¹-10⁸ CFU/mL) or (b) 48-hours old S. aureus ATCC 29,213 supplemented with increasing amounts of planktonic bioluminescent P. aeruginosa lux (10¹-10⁸ CFU/mL). (c) Mean bacterial counts of both species in the cultures after 48 h (data obtained with conditions (a) and (b) were similar). The data are represented as Mean ± SD of three independent experiments.

Fig. 3

Interaction between both species in mixed biofilm: (a) Schematic overview of the methodology developed to evaluate the interaction between bacterial species: i.S. aureus Xen36 single biofilm culture without insert. ii.S. aureus Xen36 and P. aeruginosa PAO1 mixed biofilm without insert. iii.S. aureus Xen36 and P. aeruginosa single biofilm in the insert. iv.S. aureus Xen36 single biofilm, and S. aureus Xen36 and P. aeruginosa PAO1 mixed biofilm in the insert. (b) Bioluminescence signal of S. aureus Xen36 over 48 h. (c)S. aureus Xen36 counting measured after 48 h.

Fig. 4

S. aureus GFP fluorescence kinetics measured over 70 h after the addition of different percentages of conditioned medium (CM) obtained from an isolated culture of P. aeruginosa PAO1.

Fig. 5

Bactericidal activity of CHXD (a) and THY (b) against S. aureus Xen36 in planktonic, single-species biofilm and mixed biofilm formed with P. aeruginosa PAO1 after 24 h. Bactericidal activity of CHXD (c) and THY (d) against P. aeruginosa Lux in planktonic, single-species biofilm, and mixed biofilm formed with S. aureus 29213 after 24 h.

Fig. 6

CLSM biofilm visualization: (a) Single S. aureus GFP biofilm and (b) Mixed S. aureus GFP / P. aeruginosa BFP biofilm. Biofilms were visualized on days 2, 4 and 5 after treatment with 10 mg/L or 40 mg/L of CHXD. S. aureus GFP is emitting in green, P. aeruginosa BFP in blue, and dead bacteria in red, regardless of the species. Images were constructed from the z-stack acquisition.

Fig. 7

CLSM images of mixed biofilms observed on day 5: (a) Before the addition of SYTO-9, where P. aeruginosa is stained in blue, dead bacteria in red, and the signal of S. aureus (green) is not appreciable. (b) The same biofilm after the addition of SYTO-9, which labels all bacteria both live and dead in green, including S. aureus. Images below were constructed from the z-stack acquisition.

Fig. 8

Schematic overview of the strategies used to obtain polymicrobial biofilms: (a) and (b) schemes represent the first methodology, where the co-culture was established by adding the bacterial species in their planktonic state. (c) and (d) schemes depict the second methodology used, involving the addition of P. aeruginosa in the planktonic state to a pre-formed 48 h old S. aureus biofilm.