Competition between microbes is extremely common, with many investing in mechanisms to harm other strains and species. Yet positive interactions between species have also been documented. What makes species help or harm each other is currently unclear. Here, we studied the interactions between 4 bacterial species capable of degrading metal working fluids (MWF), an industrial coolant and lubricant, which contains growth substrates as well as toxic biocides. We were surprised to find only positive or neutral interactions between the 4 species. Using mathematical modeling and further experiments, we show that positive interactions in this community were likely due to the toxicity of MWF, whereby each species’ detoxification benefited the others by facilitating their survival, such that they could grow and degrade MWF better when together. The addition of nutrients, the reduction of toxicity, or the addition of more species instead resulted in competitive behavior. Our work provides support to the stress gradient hypothesis by showing how harsh, toxic environments can strongly favor facilitation between microbial species and mask underlying competitive interactions.

Background.Increasing antibiotic resistance warrants therapeutic alternatives. Here we investigated the efficacy of bacteriophage-therapy (phage) alone or combined with antibiotics against experimental endocarditis (EE) due to Pseudomonas aeruginosa, an archetype of difficult-to-treat infection.Methods.In vitro fibrin clots and rats with aortic EE were treated with an antipseudomonas phage cocktail alone or combined with ciprofloxacin. Phage pharmacology, therapeutic efficacy, and resistance were determined.Results.In vitro, single-dose phage therapy killed 7 log colony-forming units (CFUs)/g of fibrin clots in 6 hours. Phage-resistant mutants regrew after 24 hours but were prevented by combination with ciprofloxacin (2.5 × minimum inhibitory concentration). In vivo, single-dose phage therapy killed 2.5 log CFUs/g of vegetations in 6 hours (P < .001 vs untreated controls) and was comparable with ciprofloxacin monotherapy. Moreover, phage/ciprofloxacin combinations were highly synergistic, killing >6 log CFUs/g of vegetations in 6 hours and successfully treating 64% (n = 7/11) of rats. Phage-resistant mutants emerged in vitro but not in vivo, most likely because resistant mutations affected bacterial surface determinants important for infectivity (eg, the pilT and galU genes involved in pilus motility and LPS formation).Conclusions.Single-dose phage therapy was active against P. aeruginosa EE and highly synergistic with ciprofloxacin. Phage-resistant mutants had impaired infectivity. Phage-therapy alone or combined with antibiotics merits further clinical consideration.

Bacterial viruses, or phage, are key members of natural microbial communities. Yet much research on bacterial-phage interactions has been conducted in liquid cultures involving single bacterial strains. Here we explored how bacterial diversity affects the success of lytic phage in structured communities. We infected a sensitive Pseudomonas aeruginosa strain PAO1 with a lytic phage Pseudomonas 352 in the presence versus absence of an insensitive P. aeruginosa strain PA14, in liquid culture versus colonies on agar. We found that both in liquid and in colonies, inter-strain competition reduced resistance evolution in the susceptible strain and decreased phage population size. However, while all sensitive bacteria died in liquid, bacteria in colonies could remain sensitive yet escape phage infection, due mainly to reduced growth in colony centers. In sum, spatial structure can protect bacteria against phage infection, while the presence of competing strains reduces the evolution of resistance to phage.

1 Competition between microbes is extremely common, with 2 many investing in a wide range of mechanisms to harm 3 other strains and species. Yet positive interactions between 4 species have also been documented. What makes species 5 help or harm each other is currently unclear. Here, we stud-6 ied the interactions between four bacterial species capa-7 ble of degrading Metal-Working Fluids (MWF), an industrial 8 coolant and lubricant, which contains growth substrates as 9 well as toxic biocides. We were surprised to find only posi-10 tive or neutral interactions between the four species. Using 11 mathematical modeling and further experiments, we show 12 that positive interactions in this community are likely due 13 to the toxicity of MWF, whereby each species' detoxification 14 benefited the others by facilitating their survival, such that 15 they could grow and degrade MWF better when together. 16 The addition of nutrients, the reduction of toxicity or the 17 addition of more species instead resulted in competitive 18 behavior. Our work provides support to the stress gradi-19 ent hypothesis by showing how harsh, toxic environments 20 can strongly favor facilitation between microbial species and 21 mask underlying competitive interactions. 22 Cooperation | Competition | Mutualism | Metal Working Fluid | Stress Gradient 23 Hypothesis | Species diversity | Community function | Bacterial community 24 Correspondence: sara.mitri@unil.ch 25 82 resents a tractable model system for exploring how abiotic 83 and biotic interactions shape the ecological dynamics of mi-84 crobial communities. By quantifying MWF degradation effi-85 ciency and mapping it to species composition and their inter-86 actions, this model system can also help answer another key 87 question in microbial ecology: how do inter-species interac-88 tions affect ecosystem functioning? 89 Below, we show that when growing in MWF, facilitation 90 dominates interactions between these four species, and that 91 this is likely due to the toxicity of MWF. By making the 92 Piccardi et al. | bioRχiv | April 11, 2019 | 1-9 93 tions become competitive, in a pattern that is consistent with 94 the SGH. In turn, degradation efficiency only improves with 95 community size when the environment is toxic and interac-96 tions are positive. Our experiments shed light on how nutrient 97 and toxicity gradients modulate interactions between species 98 and community functioning. 99 Results 100 Facilitation dominates the community in MWF. We first 101 characterized the effect of each species in the MWF com-102 munity on the others. The four species were incubated alone 103 (mono-culture) or in combination with a second species (pair-104 wise co-culture) in shaken flasks containing MWF medium 105 over 12 days (see Methods). The inoculum volume for each 106 species was held constant across all conditions, i.e. the total 107 was higher in co-cultures. In mono-culture, C. testosteroni 108 was able to survive and grow in MWF, while A. tumefaciens 109 survived in some replicates, and M. sa...

Predicting whether microbial invaders will colonize an environment is critical for managing natural and engineered ecosystems, and controlling infectious disease. Invaders often face competition by resident microbes. But how invasions play out in communities dominated by facilitative interactions is less clear. We previously showed that growth medium toxicity can promote facilitation between four bacterial species, as species that cannot grow alone rely on others to survive. Following the same logic, here we allowed other bacterial species to invade the four-species community and found that invaders could more easily colonize a toxic medium when the community was present. In a more benign environment instead, invasive species that could survive alone colonized more successfully when the residents were absent. Next, we asked whether early colonists could exclude future ones through a priority effect, by inoculating the invaders into the resident community only after its members had co-evolved for 44 weeks. Compared to the ancestral community, the co-evolved resident community was more competitive toward invaders and less affected by them. Our experiments show how communities may assemble by facilitating one another in harsh, sterile environments, but that arriving after community members have co-evolved can limit invasion success.