Table 1.
Description and serotype of the L1, L2, and L3 L. monocytogenes strains and their capacity to form biofilms.
Table 2.
Composition of the different cultures and co-cultures of L. monocytogenes and Veillonella.
Fig 1.
Static Biofilms Assay Plates plan.
The wells of 96-well plates were inoculated with a mix of L1 or L2 L. monocytogenes strain cultures, TSB broth, and Veillonella (V1 or V2) or L. monocytogenes (L1 or L2) strain supernatants (T2, T3, or T4). Created with BioRender.com.
Fig 2.
Absorbance measurements of mono L. monocytogenes cultures and mixed cultures of L. monocytogenes and Veillonella at 37°C.
a) Mono- and co-cultures involving L1; b) Mono- and co-cultures involving L2; c) Mono- and co-cultures involving L3. In red: monoculture of V1; in orange: monoculture of V2; in purple: monoculture L1, L2, or L3; in green: co-culture of L1, L2, or L3 with V1; in blue: co-culture of L. monocytogenes L1, L2, or L3 with V2. *Indicates a statistically significant result.
Fig 3.
Viable counts of L. monocytogenes in monocultures and mixed cultures with Veillonella at 37°C.
a) mono- and co-cultures involving L1; b) mono- and co-cultures involving L2 and c) mono- and co-cultures involving L3. In purple: monoculture of L. monocytogenes (L1, L2, or L3); in green: co-culture of L. monocytogenes (L1, L2, or L3) with Veillonella strain 17744 (V1); in blue: co-culture of L. monocytogenes (L1, L2, or L3) with Veillonella strain 17748 (V2). *Indicates a statistically significant result.
Fig 4.
L. monocytogenes cultures viable cell counts when exposed to different supernatants at 12°C.
a) Mean viable counts (all harvesting times combined) of the three L. monocytogenes cultures (L1, L2, and L3) exposed for 24h to their own supernatants (SL1, SL2, or SL3) or to the supernatant of Veillonella (SV1 or SV2); b) Impact of supernatant harvesting times (T0, T1, T2, T3, T4, or T5) on mean viable counts of L. monocytogenes cultures (L1, L2 and L3) after 24 hours of contact; c) Impact of the contact time (24 or 48 hours) between the L. monocytogenes cultures (L1, L2 and L3) and the different supernatants.
Fig 5.
Listeria monocytogenes biofilm biovolume (crystal violet) at 12°C under static conditions.
a) Absorbance measurements of L1 exposed to different T2 supernatants; b) Absorbance measurements of L1 exposed to different T3 supernatants; c) Absorbance measurements of L1 exposed to different T4 supernatants; d) Absorbance measurements of L2 exposed to different T2 supernatants; e) Absorbance measurements of L2 exposed to different T3 supernatants; f) Absorbance measurements of L2 exposed to different T4 supernatants. L1+SV1 in light blue; L1+SV2 in medium blue; L2+SV1 in light green; L2+SV2 in medium green; L1+SV1 in dark blue; and L2+SL2 in dark green. *Indicates a statistically significant result.
Fig 6.
Listeria monocytogenes biofilm growth under dynamic conditions subjected to the contact of different supernatants.
A – Exposure to the 48-hour L2 (SL2) supernatant during the adhesion and flow phases; B – Exposure to the 48-hour V2 (SV2) supernatant during the adhesion and flow phases; C – Exposure to the 48-hour L2 (SL2) supernatant during the adhesion phase (4 hours) followed by exposure to the 48-hour V2 supernatant (SV2) during the flow phase (20 hours). T0 corresponds to the time at which the cells were put in the growth chamber; T4 is the time following the four hours of adhesion; and T20 is the time following the 20-hour flow phase. Created with BioRender.com.
Fig 7.
Composition of dead and live cells in the biofilms of L. monocytogenes strain L2 exposed to the contact of different supernatants.
SYTO 9 – Individual visualization of live population; PI – Individual visualization of the dead population; Merge – Merger of the live and dead populations (total biomass); 3D – Three dimensional images constructed using 13 to 25 image layers separated from each other by 1.47 µm from the bottom to the surface of the biofilms. A – Exposure to the 48-hour L2 (SL2) supernatant during the adhesion and flow phases; B – Exposure to the 48-hour V2 (SV2) supernatant after 48 hours during the adhesion and flow phases; C – Exposure to the 48-hour L2 (SL2) supernatant during the adhesion phase (4 hours) followed by exposure to the 48-hour V2 supernatant (SV2) during the flow phase (20 hours). The 3D images were performed using the Image-Pro 3D Suite, version 6.1. Created with BioRender.com.
Fig 8.
Biovolume calculations of biofilms formed in microfluidic conditions by Listeria monocytogenes L2 exposed to the contact of different supernatants.
a) Total biovolumes of biofilms; b) Biovolumes of live and dead biomass in each biofilm type. SL2 – Exposure to the 48-hour L2 L. monocytogenes strain supernatant; SV2 – Exposure to the 48-hour Veillonella V2 strain supernatant; SL2 + SV2 – Exposure to the 48-hour L. monocytogenes L2 supernatant followed by exposure to the 48-hour Veillonella V2. supernatant. *р < 0.05.
Fig 9.
CLSM images of the organization of biofilms of Listeria monocytogenes L2 and Veillonella V2 after 48 hours at 37°C under anaerobic conditions.
L – Listeria monocytogenes L2 mono-species biofilm; V – Veillonella V2 mono-species biofilm; M – dual-species biofilm of Listeria monocytogenes L2 and Veillonella V2. Blue indicates live bacteria, green indicates dead bacteria, and red indicates Listeria monocytogenes (Gram+). The images were performed using the Image-Pro 3D Suite, version 6.1. Created with BioRender.com.