Competition quenching strategies reduce antibiotic tolerance in polymicrobial biofilms.

Bacteria typically live in dense communities where they are surrounded by other species and compete for a limited amount of resources. These competitive interactions can induce defensive responses that also protect against antimicrobials, potentially complicating the antimicrobial treatment of pathogens residing in polymicrobial consortia. Therefore, we evaluate the potential of alternative antivirulence strategies that quench this response to competition. We test three competition quenching approaches: (i) interference with the attack mechanism of surrounding competitors, (ii) inhibition of the stress response systems that detect competition, and (iii) reduction of the overall level of competition in the community by lowering the population density. We show that either strategy can prevent the induction of antimicrobial tolerance of Salmonella Typhimurium in response to competitors. Competition quenching strategies can thus reduce tolerance of pathogens residing in polymicrobial communities and could contribute to the improved eradication of these pathogens via traditional methods.

Competitive interactions facilitate resistance development against antimicrobials.

While the evolution of antimicrobial resistance is well studied in free-living bacteria, information on resistance development in dense and diverse biofilm communities is largely lacking. Therefore, we explored how the social interactions in a duo-species biofilm composed of the brewery isolates Pseudomonas rhodesiae and Raoultella terrigena influence the adaptation to the broad-spectrum antimicrobial sulfathiazole. Previously, we showed that the competition between these brewery isolates enhances the antimicrobial tolerance of P. rhodesiae. Here, we found that this enhanced tolerance in duo-species biofilms is associated with a strongly increased antimicrobial resistance development in P. rhodesiae. Whereas P. rhodesiae was not able to evolve resistance against sulfathiazole in monospecies conditions, it rapidly evolved resistance in the majority of the duo-species communities. Although the initial presence of R. terrigena was thus required for P. rhodesiae to acquire resistance, the resistance mechanisms did not depend on the presence of R. terrigena. Whole genome sequencing of resistant P. rhodesiae clones showed no clear mutational hot spots. This indicates that the acquired resistance phenotype depends on complex interactions between low-frequency mutations in the genetic background of the strains. We hypothesize that the increased tolerance in duo-species conditions promotes resistance by enhancing the selection of partially resistant mutants and opening up novel evolutionary trajectories that enable such genetic interactions. This hypothesis is reinforced by experimentally excluding potential effects of increased initial population size, enhanced mutation rate, and horizontal gene transfer. Altogether, our observations suggest that the community mode of life and the social interactions therein strongly affect the accessible evolutionary pathways toward antimicrobial resistance.IMPORTANCEAntimicrobial resistance is one of the most studied bacterial properties due to its enormous clinical and industrial relevance; however, most research focuses on resistance development of a single species in isolation. In the present study, we showed that resistance evolution of brewery isolates can differ greatly between single- and mixed-species conditions. Specifically, we observed that the development of antimicrobial resistance in certain species can be significantly enhanced in co-culture as compared to the single-species conditions. Overall, the current study emphasizes the need of considering the within bacterial interactions in microbial communities when evaluating antimicrobial treatments and resistance evolution.

Antibiotic Cycling Affects Resistance Evolution Independently of Collateral Sensitivity.

Antibiotic cycling has been proposed as a promising approach to slow down resistance evolution against currently employed antibiotics. It remains unclear, however, to which extent the decreased resistance evolution is the result of collateral sensitivity, an evolutionary trade-off where resistance to one antibiotic enhances the sensitivity to the second, or due to additional effects of the evolved genetic background, in which mutations accumulated during treatment with a first antibiotic alter the emergence and spread of resistance against a second antibiotic via other mechanisms. Also, the influence of antibiotic exposure patterns on the outcome of drug cycling is unknown. Here, we systematically assessed the effects of the evolved genetic background by focusing on the first switch between two antibiotics against Salmonella Typhimurium, with cefotaxime fixed as the first and a broad variety of other drugs as the second antibiotic. By normalizing the antibiotic concentrations to eliminate the effects of collateral sensitivity, we demonstrated a clear contribution of the evolved genetic background beyond collateral sensitivity, which either enhanced or reduced the adaptive potential depending on the specific drug combination. We further demonstrated that the gradient strength with which cefotaxime was applied affected both cefotaxime resistance evolution and adaptation to second antibiotics, an effect that was associated with higher levels of clonal interference and reduced cost of resistance in populations evolved under weaker cefotaxime gradients. Overall, our work highlights that drug cycling can affect resistance evolution independently of collateral sensitivity, in a manner that is contingent on the antibiotic exposure pattern.

Nutrient Availability and Biofilm Polysaccharide Shape the Bacillaene-Dependent Antagonism of Bacillus subtilis against Salmonella Typhimurium.

Salmonella enterica is one of the most common foodborne pathogens and, due to the spread of antibiotic resistance, new antimicrobial strategies are urgently needed to control it. In this study, we explored the probiotic potential of Bacillus subtilis PS-216 and elucidated the mechanisms that underlie the interactions between this soil isolate and the model pathogenic strain S. Typhimurium SL1344. The results reveal that B. subtilis PS-216 inhibits the growth and biofilm formation of S. Typhimurium through the production of the pks cluster-dependent polyketide bacillaene. The presence of S. Typhimurium enhanced the activity of the PpksC promoter that controls bacillaene production, suggesting that B. subtilis senses and responds to Salmonella. The level of Salmonella inhibition, overall PpksC activity, and PpksC induction by Salmonella were all higher in nutrient-rich conditions than in nutrient-depleted conditions. Although eliminating the extracellular polysaccharide production of B. subtilis via deletion of the epsA-O operon had no significant effect on inhibitory activity against Salmonella in nutrient-rich conditions, this deletion mutant showed an enhanced antagonism against Salmonella in nutrient-depleted conditions, revealing an intricate relationship between exopolysaccharide production, nutrient availability, and bacillaene synthesis. Overall, this work provides evidence on the regulatory role of nutrient availability, sensing of the competitor, and EpsA-O polysaccharide in the social outcome of bacillaene-dependent competition between B. subtilis and S. Typhimurium. IMPORTANCE Probiotic bacteria represent an alternative for controlling foodborne disease caused by Salmonella enterica, which constitutes a serious concern during food production due to its antibiotic resistance and resilience to environmental stress. Bacillus subtilis is gaining popularity as a probiotic, but its behavior in biofilms with pathogens such as Salmonella remains to be elucidated. Here, we show that the antagonism of B. subtilis is mediated by the polyketide bacillaene and that the production of bacillaene is a highly dynamic trait which depends on environmental factors such as nutrient availability and the presence of competitors. Moreover, the production of extracellular polysaccharides by B. subtilis further alters the influence of these factors. Hence, this work highlights the inhibitory effect of B. subtilis, which is condition-dependent, and the importance of evaluating probiotic strains under conditions relevant to the intended use.

Permissive aggregative group formation favors coexistence between cooperators and defectors in yeast.

In Saccharomyces cerevisiae, the FLO1 gene encodes flocculins that lead to formation of multicellular flocs, that offer protection to the constituent cells. Flo1p was found to preferentially bind to fellow cooperators compared to defectors lacking FLO1 expression, enriching cooperators within the flocs. Given this dual function in cooperation and kin recognition, FLO1 has been termed a "green beard gene". Because of the heterophilic nature of the Flo1p bond however, we hypothesize that kin recognition is permissive and depends on the relative stability of the FLO1+/flo1- versus FLO1+/FLO1+ detachment force F. We combine single-cell measurements of adhesion, individual cell-based simulations of cluster formation, and in vitro flocculation to study the impact of relative bond stability on the evolutionary stability of cooperation. We identify a trade-off between both aspects of the green beard mechanism, with reduced relative bond stability leading to increased kin recognition at the expense of cooperative benefits. We show that the fitness of FLO1 cooperators decreases as their frequency in the population increases, arising from the observed permissive character (F+- = 0.5 F++) of the Flo1p bond. Considering the costs associated with FLO1 expression, this asymmetric selection often results in a stable coexistence between cooperators and defectors.

Evolution-proof inhibitors of public good cooperation: a screening strategy inspired by social evolution theory.

Interference with public good cooperation provides a promising novel antimicrobial strategy since social evolution theory predicts that resistant mutants will be counter-selected if they share the public benefits of their resistance with sensitive cells in the population. Although this hypothesis is supported by a limited number of pioneering studies, an extensive body of more fundamental work on social evolution describes a multitude of mechanisms and conditions that can stabilize public behaviour, thus potentially allowing resistant mutants to thrive. In this paper we theorize on how these different mechanisms can influence the evolution of resistance against public good inhibitors. Based hereon, we propose an innovative 5-step screening strategy to identify novel evolution-proof public good inhibitors, which involves a systematic evaluation of the exploitability of public goods under the most relevant experimental conditions, as well as a careful assessment of the most optimal way to interfere with their action. Overall, this opinion paper is aimed to contribute to long-term solutions to fight bacterial infections.

Microbial Interspecies Interactions and Their Impact on the Emergence and Spread of Antimicrobial Resistance.

Bacteria are social organisms that commonly live in dense communities surrounded by a multitude of other species. The competitive and cooperative interactions between these species not only shape the bacterial communities but also influence their susceptibility to antimicrobials. While several studies have shown that mixed-species communities are more tolerant toward antimicrobials than their monospecies counterparts, only limited empirical data are currently available on how interspecies interactions influence resistance development. We here propose a theoretic framework outlining the potential impact of interspecies social behavior on different aspects of resistance development. We identify factors by which interspecies interactions might influence resistance evolution and distinguish between their effect on (a) the emergence of a resistant mutant and (b) the spread of this resistance throughout the population. Our analysis indicates that considering the social life of bacteria is imperative to the rational design of more effective antibiotic treatment strategies with a minimal hazard for resistance development.

2-Aminoimidazoles as potent inhibitors of contaminating brewery biofilms.

Cleaning and disinfection protocols are not always able to remove biofilm microbes present in breweries, indicating that novel anti-biofilm strategies are needed. The preventive activities of three in-house synthesized members of the 2-aminoimidazole class of anti-biofilm molecules were studied against 17 natural brewery biofilms and benchmarked against 18 known inhibitors. Two 2-aminoimidazoles belonged to the top six inhibitors, which were retested against 12 defined brewery biofilm models. For the three best inhibitors, tannic acid (n° 1), 2-aminoimidazole imi-AAC-5 (n° 2), and baicalein (n° 3), the effect on the microbial metabolic activity was evaluated. Here, the top three inhibitors showed similar effectiveness, with baicalein possessing a slightly higher efficacy. Even though the 2-aminoimidazole was the second-best inhibitor, it showed a lower biocidal activity than tannic acid, making it less prone to resistance evolution. Overall, this study supports the potential of 2-aminoimidazoles as a preventive anti-biofilm strategy.

Pseudomonas putida as a potential biocontrol agent against Salmonella Java biofilm formation in the drinking water system of broiler houses.

BACKGROUND: Environmental biofilms can induce attachment and protection of other microorganisms including pathogens, but can also prevent them from invasion and colonization. This opens the possibility for so-called biocontrol strategies, wherein microorganisms are applied to control the presence of other microbes. The potential for both positive and negative interactions between microbes, however, raises the need for in depth characterization of the sociobiology of candidate biocontrol agents (BCAs). The inside of the drinking water system (DWS) of broiler houses is an interesting niche to apply BCAs, because contamination of these systems with pathogens plays an important role in the infection of broiler chickens and consequently humans. In this study, Pseudomonas putida, which is part of the natural microbiota in the DWS of broiler houses, was evaluated as BCA against the broiler pathogen Salmonella Java. RESULTS: To study the interaction between these species, an in vitro model was developed simulating biofilm formation in the drinking water system of broilers. Dual-species biofilms of P. putida strains P1, P2, and P3 with S. Java were characterized by competitive interactions, independent of P. putida strain, S. Java inoculum density and application order. When equal inocula of S. Java and P. putida strains P1 or P3 were simultaneously applied, the interaction was characterized by mutual inhibition, whereas P. putida strain P2 showed an exploitation of S. Java. Lowering the inoculum density of S. Java changed the interaction with P. putida strain P3 also into an exploitation of S. Java. A further increase in S. Java inhibition was established by P. putida strain P3 forming a mature biofilm before applying S. Java. CONCLUSIONS: This study provides the first results showing the potential of P. putida as BCA against S. Java in the broiler environment. Future work should include more complex microbial communities residing in the DWS, additional Salmonella strains as well as chemicals typically used to clean and disinfect the system.

Biofilm Bacteria Use Stress Responses to Detect and Respond to Competitors.

Bacteria use complex regulatory networks to cope with stress, but the function of these networks in natural habitats is poorly understood. The competition sensing hypothesis states that bacterial stress response systems can serve to detect ecological competition, but studying regulatory responses in diverse communities is challenging. Here, we solve this problem by using differential fluorescence induction to screen the Salmonella Typhimurium genome for loci that respond, at the single-cell level, to life in biofilms with competing strains of S. Typhimurium and Escherichia coli. This screening reveals the presence of competing strains drives up the expression of genes associated with biofilm matrix production (CsgD pathway), epithelial invasion (SPI1 invasion system), and, finally, chemical efflux and antibiotic tolerance (TolC efflux pump and AadA aminoglycoside 3-adenyltransferase). We validate that these regulatory changes result in the predicted phenotypic changes in biofilm, mammalian cell invasion, and antibiotic tolerance. We further show that these responses arise via activation of major stress responses, providing direct support for the competition sensing hypothesis. Moreover, inactivation of the type VI secretion system (T6SS) of a competitor annuls the responses to competition, indicating that T6SS-derived cell damage activates these stress response systems. Our work shows that bacteria use stress responses to detect and respond to competition in a manner important for major phenotypes, including biofilm formation, virulence, and antibiotic tolerance.

Inhibiting bacterial cooperation is an evolutionarily robust anti-biofilm strategy.

Bacteria commonly form dense biofilms encased in extracellular polymeric substances (EPS). Biofilms are often extremely tolerant to antimicrobials but their reliance on shared EPS may also be a weakness as social evolution theory predicts that inhibiting shared traits can select against resistance. Here we show that EPS of Salmonella biofilms is a cooperative trait whose benefit is shared among cells, and that EPS inhibition reduces both cell attachment and antimicrobial tolerance. We then compare an EPS inhibitor to conventional antimicrobials in an evolutionary experiment. While resistance against conventional antimicrobials rapidly evolves, we see no evolution of resistance to EPS inhibition. We further show that a resistant strain is outcompeted by a susceptible strain under EPS inhibitor treatment, explaining why resistance does not evolve. Our work suggests that targeting cooperative traits is a viable solution to the problem of antimicrobial resistance.

Agaric acid reduces Salmonella biofilm formation by inhibiting flagellar motility.

Salmonella biofilms are a common cause of contaminations in the food or feed industry. In a screening for novel compounds to combat biofilm-associated foodborne outbreaks, we identified agaric acid as a Salmonella Typhimurium biofilm inhibitor that does not affect planktonic growth. Importantly, the remaining biofilm cells after preventive treatment with agaric acid were significantly more sensitive to the common disinfectant hydrogen peroxide. Screening of a GFP-promoter fusion library of biofilm related genes revealed that agaric acid downregulates the transcription of genes responsible for flagellar motility. Concurrently, swimming motility was completely abrogated in the presence of agaric acid, indicating that biofilm inhibition occurs via interference with the motility phenotype. Moreover, agaric acid also reduced biofilm formation of Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. Agaric acid thus shows potential as an anti-virulence compound that inhibits both motility and biofilm formation.

Meeting report on the ASM Conference on Mechanisms of Interbacterial Cooperation and Competition.

The ASM Conference on Mechanisms of Interbacterial Cooperation and Competition was held in Washington DC, from 1 to 4 March 2017. The conference provided an international forum for sociomicrobiologists from different disciplines to present and discuss new findings. The meeting covered a wide range of topics, spanning molecular mechanisms, ecology, evolution, computation and manipulation of interbacterial interactions, and encompassed social communities in medicine, the natural environment, and industry. This report summarizes the presentations and emerging themes.