Pseudomonas aeruginosa is an opportunistic pathogen that forms antibiotic-resistant biofilms, which facilitate chronic infections in immunocompromised hosts. We have previously shown that P. aeruginosa secretes outer-membrane vesicles that deliver a small RNA to human airway epithelial cells (AECs), in which it suppresses the innate immune response. Here, we demonstrate that interdomain communication through small RNA–containing membrane vesicles is bidirectional and that microRNAs (miRNAs) in extracellular vesicles (EVs) secreted by human AECs regulate protein expression, antibiotic sensitivity, and biofilm formation by P. aeruginosa. Specifically, human EVs deliver miRNA let-7b-5p to P. aeruginosa, which systematically decreases the abundance of proteins essential for biofilm formation, including PpkA and ClpV1-3, and increases the ability of beta-lactam antibiotics to reduce biofilm formation by targeting the beta-lactamase AmpC. Let-7b-5p is bioinformatically predicted to target not only PpkA, ClpV1, and AmpC in P. aeruginosa but also the corresponding orthologs in Burkholderia cenocepacia, another notorious opportunistic lung pathogen, suggesting that the ability of let-7b-5p to reduce biofilm formation and increase beta-lactam sensitivity is not limited to P. aeruginosa. Here, we provide direct evidence for transfer of miRNAs in EVs secreted by eukaryotic cells to a prokaryote, resulting in subsequent phenotypic alterations in the prokaryote as a result of this interdomain communication. Since let-7–family miRNAs are in clinical trials to reduce inflammation and because chronic P. aeruginosa lung infections are associated with a hyperinflammatory state, treatment with let-7b-5p and a beta-lactam antibiotic in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

There is an abundance of work on both P. aeruginosa and C. albicans in isolation, and quite some work as well on the way these two microbes interact. These studies do not, however, consider biofilm environments under flow, and our results here show that the expected outcome of interaction between these two pathogens can actually be reversed under flow, from pure antagonism to an increase in biomass on the part of both.

Pseudomonas aeruginosa strains PA14 and PAO1 are among the two best characterized model organisms used to study the mechanisms of biofilm formation, while also representing two distinct lineages of P. aeruginosa . Previous work has shown that PA14 and PAO1 use different strategies for surface colonization; they also have different extracellular matrix composition and different propensities to disperse from biofilms back into the planktonic phase surrounding them. We expand on this work here by exploring the consequences of these different biofilm production strategies during direct competition. Using differentially labeled strains and microfluidic culture methods, we show that PAO1 can outcompete PA14 in direct competition during early colonization and subsequent biofilm growth, that they can do so in constant and perturbed environments, and that this advantage is specific to biofilm growth and requires production of the Psl polysaccharide. In contrast, the P. aeruginosa PA14 is better able to invade pre-formed biofilms and is more inclined to remain surface-associated under starvation conditions. These data together suggest that while P. aeruginosa PAO1 and PA14 are both able to effectively colonize surfaces, they do so in different ways that are advantageous under different environmental settings. Importance Recent studies indicate that P. aeruginosa PAO1 and PA14 use distinct strategies to initiate biofilm formation. We investigated whether their respective colonization and matrix secretion strategies impact their ability to compete under different biofilm-forming regimes. Our work shows that these different strategies do indeed impact how these strains fair in direct competition: PAO1 dominates during colonization of a naïve surface, while PA14 is more effective in colonizing a pre-formed biofilm. These data suggest that even for very similar microbes there can be distinct strategies to successfully colonize and persist on surfaces during the biofilm life cycle.

Spatially structured communities of microbes - biofilms - are widespread in nature, and biofilm-dwelling microbes often respond to their environments in ways that are different from their planktonic counterparts. Further, most natural biofilms are multi-species mixtures of microorganisms; the ecology of intra- and inter-species interactions in these consortia, and the resulting effects on total community properties, are often not well understood. A common site of polymicrobial biofilm infections is the lungs of patients with cystic fibrosis (CF). CF is a genetic disorder in humans that leads to colonization of the lungs by a variety of microorganisms, including Pseudomonas aeruginosa and Candida albicans. These opportunistic pathogens are frequently co-isolated from infected lungs, in addition to other infection sites including urinary and intravenous catheters. To study how these microbes behave together in biofilms, we developed a modified artificial sputum medium that is optically clear for use with microfluidic culture. In addition, we engineered strains with optimized fluorescent protein expression constructs allowing for single-cell resolution confocal microscopy. Using these tools and recently developed methods for spatial analysis of 3-D image data, we found that both P. aeruginosa and C. albicans display increased biovolume accumulation in multi-species biofilms relative to single-species biofilms. This pattern did not occur in planktonic co-culture and was thus specific to the biofilm environment. Interestingly, introduction of P. aeruginosa supernatants over dual-species biofilms strongly reduced C. albicans biovolume. This suggests that products that accumulate in batch culture were still inhibitory to C. albicans under a flow regime, but that they their de novo production in mixed species biofilms was not sufficient to inhibit C. albicans biofilm accumulation. Altogether our results indicate a critical impact of flow environment for the outcome of polymicrobial interactions and the need for high-resolution analysis of such communities in future work.

Pseudomonas aeruginosa strains PA14 and PAO1 are among the two best characterized model organisms used to study the mechanisms of biofilm formation, while also representing two distinct lineages of P. aeruginosa. Our previous work showed that P. aeruginosa PA14 and PAO1 use distinct strategies to initiate biofilm growth. Using differentially-labeled strains and microfluidic devices, we show that PAO1 can outcompete PA14 in a head-to-head competition during early colonization of a surface, can do so in constant and perturbed environments, that this advantage is specific to biofilm growth and requires production of the Psl polysaccharide. In contrast, the P. aeruginosa PA14 exhibits a competitive fitness advantage when invading a pre-formed biofilm and is better able to tolerate starvation than PAO1 in the biofilm context. These data support the model that while P. aeruginosa PAO1 and PA14 are both able to effectively colonize surfaces, these strains use distinct strategies that are advantageous under different environmental settings.