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Bacteria sense population density via the cell–cell communication system called quorum sensing (QS). The evolution of QS and its maintenance or loss in mixed bacterial communities is highly relevant to understanding how cell–cell signaling impacts bacterial fitness and competition, particularly under varying environmental conditions such as nutrient availability. We uncovered a phenomenon in whichVibriocells grown in minimal medium optimize expression of the methionine and tetrahydrofolate (THF) synthesis genes via QS. Strains that are genetically “locked” at high cell density grow slowly in minimal glucose media and suppressor mutants accumulate via inactivating mutations inmetF(methylenetetrahydrofolate reductase) andluxR(the master QS transcriptional regulator). In mixed cultures, QS mutant strains initially coexist with wild-type, but as glucose is depleted, wild-type outcompetes the QS mutants. Thus, QS regulation of methionine/THF synthesis is a fitness benefit that links nutrient availability and cell density, preventing accumulation of QS-defective mutants. 

ABSTRACT Chronic polymicrobial infections involvingPseudomonas aeruginosaandStaphylococcus aureusare prevalent, difficult to eradicate, and associated with poor health outcomes. Therefore, understanding interactions between these pathogens is important to inform improved treatment development. We previously demonstrated thatP. aeruginosais attracted toS. aureususing type IV pili (TFP)-mediated chemotaxis, but the impact of attraction onS. aureusgrowth and physiology remained unknown. Using live single-cell confocal imaging to visualize microcolony structure, spatial organization, and survival ofS. aureusduring coculture, we found that interspecies chemotaxis providesP. aeruginosaa competitive advantage by promoting invasion into and disruption ofS. aureusmicrocolonies. This behavior rendersS. aureussusceptible toP. aeruginosaantimicrobials. Conversely, in the absence of TFP motility,P. aeruginosacells exhibit reduced invasion ofS. aureuscolonies. Instead,P. aeruginosabuilds a cellular barrier adjacent toS. aureusand secretes diffusible, bacteriostatic antimicrobials like 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) into theS. aureuscolonies. Reduced invasion leads to the formation of denser and thickerS. aureuscolonies with increased HQNO-mediated lactic acid fermentation, a physiological change that could complicate treatment strategies. Finally, we show thatP. aeruginosamotility modifications of spatial structure enhance competition againstS. aureus. Overall, these studies expand our understanding of howP. aeruginosaTFP-mediated interspecies chemotaxis facilitates polymicrobial interactions, highlighting the importance of spatial positioning in mixed-species communities. IMPORTANCEThe polymicrobial nature of many chronic infections makes their eradication challenging. Particularly, coisolation ofPseudomonas aeruginosaandStaphylococcus aureusfrom airways of people with cystic fibrosis and chronic wound infections is common and associated with severe clinical outcomes. The complex interplay between these pathogens is not fully understood, highlighting the need for continued research to improve management of chronic infections. Our study unveils thatP. aeruginosais attracted toS. aureus, invades into neighboring colonies, and secretes anti-staphylococcal factors into the interior of the colony. Upon inhibition ofP. aeruginosamotility and thus invasion,S. aureuscolony architecture changes dramatically, wherebyS. aureusis protected fromP. aeruginosaantagonism and responds through physiological alterations that may further hamper treatment. These studies reinforce accumulating evidence that spatial structuring can dictate community resilience and reveal that motility and chemotaxis are critical drivers of interspecies competition. 

ABSTRACT Predators play a central role in shaping community structure, function, and stability. The degree to which bacteriophage predators (viruses that infect bacteria) evolve to be specialists with a single bacterial prey species versus generalists able to consume multiple types of prey has implications for their effect on microbial communities. The presence and abundance of multiple bacterial prey types can alter selection for phage generalists, but less is known about how interactions between prey shape predator specificity in microbial systems. Using a phenomenological mathematical model of phage and bacterial populations, we find that the dominant phage strategy depends on prey ecology. Given a fitness cost for generalism, generalist predators maintain an advantage when prey species compete, while specialists dominate when prey are obligately engaged in cross-feeding interactions. We test these predictions in a synthetic microbial community with interacting strains ofEscherichia coliandSalmonella entericaby competing a generalist T5-like phage able to infect both prey against P22vir, anS. enterica-specific phage. Our experimental data conform to our modeling expectations when prey species are competing or obligately mutualistic, although our results suggest that thein vitrocost of generalism is caused by a combination of biological mechanisms not anticipated in our model. Our work demonstrates that interactions between bacteria play a role in shaping ecological selection on predator specificity in obligately lytic bacteriophages and emphasizes the diversity of ways in which fitness trade-offs can manifest. IMPORTANCEThere is significant natural diversity in how many different types of bacteria a bacteriophage can infect, but the mechanisms driving this diversity are unclear. This study uses a combination of mathematical modeling and anin vitrosystem consisting ofEscherichia coli,Salmonella enterica, a T5-like generalist phage, and the specialist phage P22virto highlight the connection between bacteriophage specificity and interactions between their potential microbial prey. Mathematical modeling suggests that competing bacteria tend to favor generalist bacteriophage, while bacteria that benefit each other tend to favor specialist bacteriophage. Experimental results support this general finding. The experiments also show that the optimal phage strategy is impacted by phage degradation and bacterial physiology. These findings enhance our understanding of how complex microbial communities shape selection on bacteriophage specificity, which may improve our ability to use phage to manage antibiotic-resistant microbial infections. 

Numerous ecological interactions among microbes—for example, competition for space and resources, or interaction among phages and their bacterial hosts—are likely to occur simultaneously in multispecies biofilm communities. While biofilms formed by just a single species occur, multispecies biofilms are thought to be more typical of microbial communities in the natural environment. Previous work has shown that multispecies biofilms can increase, decrease, or have no measurable impact on phage exposure of a host bacterium living alongside another species that the phages cannot target. The reasons underlying this variability are not well understood, and how phage–host encounters change within multispecies biofilms remains mostly unexplored at the cellular spatial scale. Here, we study how the cellular scale architecture of model 2-species biofilms impacts cell–cell and cell–phage interactions controlling larger scale population and community dynamics. Our system consists of dual culture biofilms ofEscherichia coliandVibrio choleraeunder exposure to T7 phages, which we study using microfluidic culture, high-resolution confocal microscopy imaging, and detailed image analysis. As shown previously, sufficiently mature biofilms ofE.colican protect themselves from phage exposure via their curli matrix. Before this stage of biofilm structural maturity,E.coliis highly susceptible to phages; however, we show that these bacteria can gain lasting protection against phage exposure if they have become embedded in the bottom layers of highly packed groups ofV.choleraein co-culture. This protection, in turn, is dependent on the cell packing architecture controlled byV.choleraebiofilm matrix secretion. In this manner,E.colicells that are otherwise susceptible to phage-mediated killing can survive phage exposure in the absence of de novo resistance evolution. While co-culture biofilm formation withV.choleraecan confer phage protection toE.coli, it comes at the cost of competing withV.choleraeand a disruption of normal curli-mediated protection forE.colieven in dual species biofilms grown over long time scales. This work highlights the critical importance of studying multispecies biofilm architecture and its influence on the community dynamics of bacteria and phages. 

Biofilm formation, including adherence to surfaces and secretion of extracellular matrix, is common in the microbial world, but we often do not know how interaction at the cellular spatial scale translates to higher-order biofilm community ecology. Here we explore an especially understudied element of biofilm ecology, namely predation by the bacteriumBdellovibrio bacteriovorus. This predator can kill and consume many different Gram-negative bacteria, includingVibrio choleraeandEscherichia coli.V. choleraecan protect itself from predation within densely packed biofilm structures that it creates, whereasE. colibiofilms are highly susceptible toB. bacteriovorus. We explore how predator–prey dynamics change whenV. choleraeandE. coliare growing in biofilms together. We find that in dual-species prey biofilms,E. colisurvival underB. bacteriovoruspredation increases, whereasV. choleraesurvival decreases.E. colibenefits from predator protection when it becomes embedded within expanding groups of highly packedV. cholerae. But we also find that the ordered, highly packed, and clonal biofilm structure ofV. choleraecan be disrupted ifV. choleraecells are directly adjacent toE. colicells at the start of biofilm growth. When this occurs, the two species become intermixed, and the resulting disordered cell groups do not block predator entry. Because biofilm cell group structure depends on initial cell distributions at the start of prey biofilm growth, the surface colonization dynamics have a dramatic impact on the eventual multispecies biofilm architecture, which in turn determines to what extent both species survive exposure toB. bacteriovorus. 

ABSTRACT 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, P. aeruginosa PA14 is better able to invade preformed 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 naive surface, while PA14 is more effective in colonizing a preformed 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. 

ABSTRACT Microbe-microbe interactions can strongly influence growth and biofilm formation kinetics. For Pseudomonas aeruginosa and Candida albicans , which are found together in diverse clinical sites, including urinary and intravenous catheters and the lungs of individuals with cystic fibrosis (CF), we compared the kinetics of biofilm formation by each species in dual-species and single-species biofilms. We engineered fluorescent protein constructs for P. aeruginosa (producing mKO-κ ) and C. albicans (producing mKate2 ) that did not alter growth and enabled single-cell resolution imaging by live-sample microscopy. Using these strains in an optically clear derivative of synthetic CF sputum medium, we found that both P. aeruginosa and C. albicans displayed increased biovolume accumulation—by three- and sixfold, respectively—in dual-species biofilms relative to single-species biofilms. This result was specific to the biofilm environment, as enhanced growth was not observed in planktonic cocultures. Stimulation of C. albicans biofilm formation occurred regardless of whether P. aeruginosa was added at the time of fungal inoculation or 24 h after the initiation of biofilm development. P. aeruginosa biofilm increases in cocultures did not require the Pel extracellular polysaccharide, phenazines, and siderophores known to influence C. albicans . P. aeruginosa mutants lacking Anr, LasR, and BapA were not significantly stimulated by C. albicans , but they still promoted a significant enhancement of biofilm development of the fungus, suggesting a fungal response to the presence of bacteria. Last, we showed that a set of P. aeruginosa clinical isolates also prompted an increase of biovolume by C. albicans in coculture. IMPORTANCE 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. Our work also highlights the importance of cellular-scale spatial structure in biofilms for understanding multispecies population dynamics. 

Bacteriophages can be trapped in the matrix of bacterial biofilms, such that the cells inside them are protected. It is not known whether these phages are still infectious and whether they pose a threat to newly arriving bacteria. Here, we address these questions using Escherichia coli and its lytic phage T7. Prior work has demonstrated that T7 phages are bound in the outermost curli polymer layers of the E. coli biofilm matrix. We show that these phages do remain viable and can kill colonizing cells that are T7-susceptible. If cells colonize a resident biofilm before phages do, we find that they can still be killed by phage exposure if it occurs soon thereafter. However, if colonizing cells are present on the biofilm long enough before phage exposure, they gain phage protection via envelopment within curli-producing clusters of the resident biofilm cells.