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. 2021 Apr 3:3:100047.
doi: 10.1016/j.bioflm.2021.100047. eCollection 2021 Dec.

Milieu matters: An in vitro wound milieu to recapitulate key features of, and probe new insights into, mixed-species bacterial biofilms

Snehal Kadam  1 , Vandana Madhusoodhanan  1 , Radhika Dhekane  1 , Devyani Bhide  2 , Rutuja Ugale  1 , Utkarsha Tikhole  1 , Karishma S Kaushik  1
Affiliations

Milieu matters: An in vitro wound milieu to recapitulate key features of, and probe new insights into, mixed-species bacterial biofilms

Snehal Kadam et al. Biofilm. .

Abstract

Bacterial biofilms are a major cause of delayed wound healing. Consequently, the study of wound biofilms, particularly in host-relevant conditions, has gained importance. Most in vitro studies employ refined laboratory media to study biofilms, representing conditions that are not relevant to the infection state. To mimic the wound milieu, in vitro biofilm studies often incorporate serum or plasma in growth conditions, or employ clot or matrix-based biofilm models. While incorporating serum or plasma alone is a minimalistic approach, the more complex in vitro wound models are technically demanding, and poorly compatible with standard biofilm assays. Based on previous reports of clinical wound fluid composition, we have developed an in vitro wound milieu (IVWM) that includes, in addition to serum (to recapitulate wound fluid), matrix elements and biochemical factors. With Luria-Bertani broth and Fetal Bovine Serum (FBS) for comparison, the IVWM was used to study planktonic growth, biofilm features, and interspecies interactions, of common wound pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. We demonstrate that the IVWM recapitulates widely reported in vivo biofilm features such as biomass formation, metabolic activity, increased antibiotic tolerance, 3D structure, and interspecies interactions for monospecies and mixed-species biofilms. Further, the IVWM is simple to formulate, uses laboratory-grade components, and is compatible with standard biofilm assays. Given this, it holds potential as a tractable approach to study wound biofilms under host-relevant conditions.

Keywords: Biofilms; Interspecies interactions; Milieu; Mixed-species; Pseudomonas aeruginosa; Staphylococcus aureus; Wounds.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
IVWM supports the planktonic growth of P. aeruginosa, but not of S. aureus. Planktonic growth curves of Pseudomonas aeruginosa (PAO1) and Staphylococcus aureus (AH133) were performed in Luria-Bertani broth (LB), Fetal Bovine Serum (FBS) and the in vitro wound milieu (IVWM). Optical density (OD600) was measured at intervals of 30 ​min for 12 ​h. Error bars represent SEM, n ​= ​3 (biological replicates).
Fig. 2
Fig. 2
In the IVWM, interspecies interactions between planktonic P. aeruginosa and S. aureus result in P. aeruginosa significantly outcompeting S. aureus. Viable counts of planktonic P. aeruginosa and S. aureus grown under mixed-species conditions in LB, FBS and IVWM were quantified by measuring Colony Forming Units (CFU). (A) CFUs of planktonic monospecies and mixed-species cultures of Pseudomonas aeruginosa and Staphylococcus aureus in LB, FBS and IVWM (B) Ratio of CFU/mL of the colony counts in monospecies to that in mixed-species planktonic cultures of Pseudomonas aeruginosa and Staphylococcus aureus in LB, FBS and IVWM. Error bars represent SEM, n ​= ​4 (biological replicates). A p-value of <0.05 was considered significant (∗).
Fig. 3
Fig. 3
IVWM supports the formation of metabolically-active biofilms of P. aeruginosa and S. aureus, and indicates the coexistence of both species under mixed-species conditions. Pre-formed 24-h biofilms of Pseudomonas aeruginosa and Staphylococcus aureus were quantified for metabolic activity by the XTT assay and for viability using the CFU technique. Assays were performed under monospecies and mixed-species conditions in LB, FBS and IVWM. (A) Metabolic activity normalized to log10 (CFU) of the biofilm for P. aeruginosa and S. aureus biofilms under monospecies and mixed-species states (B) Log10 (CFU) of viable biofilm cells of P. aeruginosa and S. aureus biofilms under monospecies and mixed-species states in LB, FBS and IVWM. Error bars represent SEM, n ​= ​3 (biological replicates). A p-value of <0.05 was considered significant (∗).
Fig. 4
Fig. 4
3D biofilm structure of P. aeruginosa and S. aureus single-species biofilms in IVWM are distinct from that in LB and FBS. Tile-scan confocal microscopy showing 3D structure and thickness of (A) P. aeruginosa (PAO1-mCherry) and (B) S. aureus (AH133-GFP) biofilms in LB, FBS and IVWM. To reduce the role of surface attachment, and better explore the role of the different media conditions, biofilms were grown in tissue-culture treated microtiter plates. The poor biomass formed by P. aeruginosa in LB, is likely due to the reduced surface attachment to the tissue-culture treated surfaces, and absence of host and matrix proteins in the media. For all images, given that the wells were not rinsed prior to imaging, the observed biomass represents not only biofilm, but also attached or unattached single bacterial cells within, and on top of, the dense bacterial mats (visible in the side view images). The grids are a result from stitching of the tiles in the tile-scan processing. Error bars represent SEM, n ​= ​3 (biological replicates). A p-value of <0.05 was considered significant (∗).
Fig. 5
Fig. 5
3D visualization of biofilm structure of mixed-species P. aeruginosa and S. aureus biofilms in IVWM shows distinct predominance of P. aeruginosa. (A) Tile scan confocal microscopy of P. aeruginosa (PAO1-mCherry) and S. aureus (AH133-GFP) mixed-species biofilms in LB, FBS and IVWM grown in tissue-culture treated microtiter plates. (B) Mean intensity of fluorescence (representing PAO1-mCherry and SA-GFP) across the Z-height of the biofilm (with the bottom as Z ​= ​0). Mean intensities were calculated using the LAS software as the average fluorescence intensity of each channel in each Z-plane and plotted as the mean intensity versus Z-position. This was averaged across 3 biological replicates, and therefore represents variation seen across biological replicates, as well as variation across the Z-plane. Given that the wells were not rinsed prior to imaging, the observed biomass represents not only biofilm, but also attached or unattached single bacterial cells within, and on top of, the dense bacterial mats (visible in the side view images). The inset images are zoomed in ~8 times. The grids are a result from stitching of the tiles in the tile-scan processing. Error bars represent SEM, n ​= ​3 (biological replicates).
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