Biophysical mechanisms of antimicrobial resistance in swarming B. subtilis

Swarms and biofilms are the two major modes of bacterial collectives and confer cells with emergent properties that lack as individuals, such as an increase in antibiotic tolerance. Swarming is a rapid type of surface colonization, and therefore its ability to withstand high antibiotic concentrations could lead to the subsequent establishment of high lyre- silient biofilms orgenetically resistant bacteria in regions that could not otherwise have been colonized. However, whether the development of biofilms or resistant microcolonies by swarms is possible is unknown. Using swarming Bacillus subtilis, we reveal that a biophysical mechanism, reminiscent of motility-induced phase separation (MIPS),under- pins a swarming-to-biofilm transition through a localized dynamic phase transition. This transition, triggered by an external stressor, is underpinned by a localized multilayer formation. Inspired by the thermodynamic properties of active matter, we demonstrated that such multilayer formation forms through a nucleation and growth process near an antibiotic gradient, and through spinodal decomposition in absence of stress. The nucle- ation and growth of multiple layers near the antibiotic, triggers waves of bacteria that move towards the antibiotic source, suggesting a novel mechanism of bacterial transport. When swarming to biofilm transition was prohibited by the environmental conditions, the swarm uses an alternative strategy to cope with the antibiotic gradient by developing re- sistant microcolonies. Quantification of this resistance displayed that the higher resistance to kanamycinis acquired together with resistance to other antibiotics targeting the same process. Inspired by the medical motivation of this project, we suggested solutions to both the emergence of the biofilm and the development of resistant bacteria by using the insight that we gained throughout the study. In particular, we proved that the biofilm formation can be reduced when splitting the total dose of antibiotics in two different time steps: the first triggers the multi layer formation and the second targets this key region in the swarm. Alternatively, when the swarm develops resistant colonies, we observed that these become more vulnerable to other drugs, so a tratement using certain sequence of antibiotics could be highly effective to kill multi drug resistant bacteria. These could lead to new strategies to tackle antimicrobial resistance

Swarming bacteria undergo localized dynamic phase transition to form stress-induced biofilms

Self-organized multicellular behaviors enable cells to adapt and tolerate stressors to a greater degree than isolated cells. However, whether and how cellular communities alter their collective behaviors adaptively upon exposure to stress is largely unclear. Here, we investigate this question using Bacillus subtilis, a model system for bacterial multicellularity. We discover that, upon exposure to a spatial gradient of kanamycin, swarming bacteria activate matrix genes and transit to biofilms. The initial stage of this transition is underpinned by a stress-induced multilayer formation, emerging from a biophysical mechanism reminiscent of motility-induced phase separation (MIPS). The physical nature of the process suggests that stressors which suppress the expansion of swarms would induce biofilm formation. Indeed, a simple physical barrier also induces a swarm-to-biofilm transition. Based on the gained insight, we propose a strategy of antibiotic treatment to inhibit the transition from swarms to biofilms by targeting the localized phase transition

The dynamics of single-to-multi layer transition in bacterial swarms

Wet self-propelled rods at high densities can exhibit a state of mesoscale turbulence: a disordered lattice of vortices with chaotic dynamics and a characteristic length scale. Such a state is commonly studied by a two-dimensional continuum model. However, less is known about the dynamic behaviour of self-propelled rods in three- or quasi-two- dimensions, which can be found in biological systems, for example, during the formation of bacterial aggregates and biolms. In this study, we characterised the formation of multi-layered islands in a monolayer of swarming cells using the rod-shaped bacteria B. subtilis as a model system. We focused on understanding how bacteria form multiple layers and how the presence of stress aects the multiple layer formation. Following our previous study where we reported that the initiation of the multilayer formation can be accounted by the framework of motility-induced phase separation (MIPS), this study analysed how this phase separation is impacted by the presence of stress, specially under the exposure to a gradient of antibiotic. The analyses show that in the presence of an antibiotic gradient, the multi-layer formation happens by a nucleation and growth of well-defined multilayered clusters instead of by the uncontrolled emergence of the multilayer, resembling the traditional thermodynamic processes of binodal and spinodal decomposition respectively. Finally, the multilayer gives place to waves of bacteria that can travel towards high concentrations of antibiotics and that resemble travelling waves predicted by simulations of mixtures of passive and active particles

Biofilm and swarming emergent behaviours controlled through the aid of biophysical understanding and tools

Bacteria can organise themselves into communities in the forms of biofilms and swarms. Through chemical and physical interactions between cells, these communities exhibit emergent properties that individual cells alone do not have. While bacterial communities have been mainly studied in the context of biochemistry and molecular biology, recent years have seen rapid advancements in the biophysical understanding of emergent phenomena through physical interactions in biofilms and swarms. Moreover, new technologies to control bacterial emergent behaviours by physical means are emerging in synthetic biology. Such technologies are particularly promising for developing engineered living materials (ELM) and devices and controlling contamination and biofouling. In this minireview, we overview recent studies unveiling physical and mechanical cues that trigger and affect swarming and biofilm development. In particular, we focus on cell shape, motion and density as the key parameters for mechanical cell–cell interactions within a community. We then showcase recent studies that use physical stimuli for patterning bacterial communities, altering collective behaviours and preventing biofilm formation. Finally, we discuss the future potential extension of biophysical and bioengineering research on microbial communities through computational modelling and deeper investigation of mechano-electrophysiological coupling

Membrane targeted Azobenzene drives optical modulation of bacterial membrane potential

Recent studies have shown that bacterial membrane potential is dynamic and plays signaling roles. Yet, little is still known about the mechanisms of membrane potential dynamics regulation—owing to a scarcity of appropriate research tools. Optical modulation of bacterial membrane potential could fill this gap and provide a new approach for studying and controlling bacterial physiology and electrical signaling. Here, the authors show that a membrane-targeted azobenzene (Ziapin2) can be used to photo-modulate the membrane potential in cells of the Gram-positive bacterium Bacillus subtilis. It is found that upon exposure to blue–green light (λ = 470 nm), isomerization of Ziapin2 in the bacteria membrane induces hyperpolarization of the potential. To investigate the origin of this phenomenon, ion-channel-deletion strains and ion channel blockers are examined. The authors found that in presence of the chloride channel blocker idanyloxyacetic acid-94 (IAA-94) or in absence of KtrAB potassium transporter, the hyperpolarization response is attenuated. These results reveal that the Ziapin2 isomerization can induce ion channel opening in the bacterial membrane and suggest that Ziapin2 can be used for studying and controlling bacterial electrical signaling. This new optical tool could contribute to better understand various microbial phenomena, such as biofilm electric signaling and antimicrobial resistance

Biophysical mechanisms of antimicrobial resistance in swarming B. subtilis

Swarming and biofilm formation are two modes of bacterial collective behavior that en- able cells to increase their tolerance to anti-microbial agents. Swarming is a rapid type of surface colonization, and therefore its ability to withstand high antibiotic concentrations could lead to the subsequent establishment of highly resilient biofilms in regions that could not otherwise have been colonized. However, whether such a transition can happen at all, and how, remains unclear. Using Bacillus subtilis, here we reveal that a biophysical mech- anism, reminiscent of motility-induced phase separation (MIPS), underpins a swarming- to-biofilm transition through a localized dynamic phase transition. Guided by the MIPS paradigm, we demonstrate that the transition is triggered by environmental stressors such as an antibiotic gradient and a simple physical barrier. Based on the biophysical insight, we propose a promising strategy of antibiotic treatment to effectively inhibit the emer- gence of biofilms from swarms. Alternatively to biofilm formation, swarms can develop genetically resistant colonies when exposed to a gradient of antibiotics. I demonstrate that those colonies have the same fitness than the Wild Type and that by developing resistance to a specific antibiotic they become weaker against other antimicrobials.Peer reviewe