Oxygen spatio-temporal distribution in a 4-species adherent community of bacteria
Authors: Ben Youssef Wafa , Monmeyran Amaury , Sureau Franck , Panier Thomas , Henry Nelly 
Published: July 2020

&lt;p&gt;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160; More than 30 years have passed now since the pioneering work of Costerton and co-workers&lt;sup&gt;e.g.,1&lt;/sup&gt;. We have learned that the biological functions of the cells embedded in the complex, self-produced polymeric extracellular matrix, differ radically from the ones of the planktonic cells. Emergent properties such as enhanced antimicrobial resistance appear.&amp;#160; Biofilms are widely spread in different habitats, both in the environment and the living organisms. Mostly, the characterization of this bacterial specific phenotype has been carried out using mono-species lab models. Yet, these systems are in marked contrast to the biofilms found in the environment. Those are usually complex and contain multiple bacterial species and, in many cases, also fungi, algae, and protozoa&lt;sup&gt;2&lt;/sup&gt;. To take this into account, researches have recently turned to multispecies communities, aiming at describing the interspecies interactions in order to decipher the mechanisms underlying the properties of these complex consortia.&lt;/p&gt;
&lt;p&gt;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160; We present here a simplified model community consisting of 4 species &amp;#8212; &lt;em&gt;Bacillus thuringiensis, Kocuria salsicia, Pseudomonas fluorescens, Rhodocyclus sp.&lt;/em&gt; &amp;#8212; elaborated from a natural environment to investigate the mechanisms supporting the formation of a multispecies consortium. We have been able to grow the 4-species biofilm under flow in a millimetric channel made of PDMS, which enabled to monitor the biofilm settlement and development using video-microscopy&lt;sup&gt;3&lt;/sup&gt;. We found a deterministic development which follows defined kinetics and spatial distribution, suggesting that the formation of this adherent community is dominated by the self-induced modulation of the environmental parameters. To clarify this hypothesis, we focused our attention on the spatio-temporal distribution of oxygen and we devised an original experiment to map &lt;em&gt;in situ&lt;/em&gt; and in real-time the evolution of oxygen level within the 4-species biofilm.&lt;/p&gt;
&lt;p&gt;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160; We used an O&lt;sub&gt;2&lt;/sub&gt; fluorescent probe made of a Ruthenium complex encapsulated in lipidic micelles to overcome the metal toxicity. We derived local oxygen concentration in the biofilm from fluorescent-lifetime imaging microscopy (FLIM) measurements of the probe &lt;em&gt;in situ&lt;/em&gt;. The setup was equipped with a light sheet to ensure the optical sectioning for a 3D mapping. We will show here the spatial and temporal characteristics of the method and the first O&lt;sub&gt;2&lt;/sub&gt; map obtained on a growing biofilm.&lt;/p&gt;
&lt;p&gt;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160; To conclude, we will discuss how the monitoring of oxygen spatio-temporal distribution in a model community can help to elucidate basic interspecies interactions and reveal general mechanisms likely to govern number of more complex natural systems.&lt;/p&gt;
&lt;p&gt;&amp;#160;&lt;/p&gt;
&lt;ol&gt;
&lt;li&gt;Costerton, J. W., Stewart, P. S. &amp; Greenberg, E. P. Bacterial biofilms: a common cause of persistent infections. Science &lt;strong&gt;284&lt;/strong&gt;, 1318&amp;#8211;1322 (1999).&lt;/li&gt;
&lt;li&gt;Hall-Stoodley, L., Costerton, J. W. &amp; Stoodley, P. Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol &lt;strong&gt;2&lt;/strong&gt;, 95&amp;#8211;108 (2004).&lt;/li&gt;
&lt;li&gt;Thomen, P. et al. Bacterial biofilm under flow: First a physical struggle to stay, then a matter of breathing. PLoS One &lt;strong&gt;12&lt;/strong&gt;, e0175197 (2017).&lt;/li&gt;
&lt;/ol&gt;
&lt;p&gt;&amp;#160;&lt;/p&gt;