Inhomogeneity in bacterial survival against environmental stresses as a community

Biofilm reduces the efficiency of antimicrobial agents, hindering treatment and recovery of patients worldwide. One of the reasons for the difficulty of treatment is the tight envelope of extracellular polymeric substances (EPS) around the biofilm. The EPS structure exerts constant osmotic pressure to the bacteria inside it, but the effect of osmotic pressure is not very well documented. Here, we report an effort to characterize and quantify the heterogeneity of bacterial survival within a community that mimics the in vivo environment, and tested how different osmolytes impact the cell wall integrity and metabolic activities of many different bacteria inside the aggregates. It shows that bacteria is very effective combating high concentrations of antibiotics as a community, and there is a significant heterogeneity in survival rate, with the bacteria on the surface of the aggregate more susceptible than the ones deeper in the aggregate. Surprisingly, this pattern is reversed if NaCl was introduced, with more cells dying in the middle of the aggregates, even though the outer rim of aggregates were directly exposed to the osmolytes. Yet another osmolyte - sucrose - saved the dying bacteria regardless of the position it is within the aggregates. Co-exposure to antibiotics and osmolytes also yielded different, sometimes opposite results depending on conditions. In general, the bacterial reactions to osmotic pressure in an aggregate should always be discussed in a case-by-case principle, since the species, the types of osmolytes and antibiotics, as well as the exposure combination all play some roles in the process. This work raised many questions about metabolic modifications of bacteria in biofilm, the interplay between osmotic pressure and antibiotics that caused the heterogeneity of individual bacteria in biofilm, whose underlying mechanisms still await further explorations.