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.

Predicting the fate of a microbial community and its member species relies on understanding the nature of their interactions. However, designing simple assays that distinguish between interaction types can be challenging. Here, we performed spent media assays based on the predictions of a mathematical model to decipher the interactions between four bacterial species: Agrobacterium tumefaciens (At), Comamonas testosteroni (Ct), Microbacterium saperdae (Ms) and Ochrobactrum anthropi (Oa). While most experimental results matched model predictions, the behavior of Ct did not: its lag phase was reduced in the pure spent media of At and Ms, but prolonged again when we replenished with our growth medium. Further experiments showed that the growth medium actually delayed the growth of Ct, leading us to suspect that At and Ms could alleviate this inhibitory effect. There was, however, no evidence supporting such "cross-detoxification" and instead, we identified metabolites secreted by At and Ms that were then consumed or "cross-fed" by Ct, shortening its lag phase. Our results highlight that even simple, defined growth media can have inhibitory effects on some species and that such negative effects need to be included in our models. Based on this, we present new guidelines to correctly distinguish between different interaction types, such as cross-detoxification and cross-feeding.

Bacterial viruses, or phage, play a key role in shaping natural 1 microbial communities. Yet much research on bacterial-phage 2 interactions has been conducted in liquid cultures involving sin-3 gle bacterial strains. Critically, phage often have a very narrow 4 host range meaning they can only ever target a subset of strains 5 in a community. Here we explore how strain diversity affects 6 the success of lytic phage in structured communities. In par-7 ticular, we infect a susceptible Pseudomonas aeruginosa strain 8 PAO1 with lytic phage Pseudomonas 352 in the presence versus 9 absence of an insensitive P. aeruginosa strain PA14, in liquid cul-10 ture versus colonies growing on agar. We find that competition 11 between the two bacterial strains reduces the likelihood of the 12 susceptible strain evolving resistance to the phage. This result 13 holds in liquid culture and in colonies. However, while in liq-14 uid the phage eliminate the whole sensitive population, colonies 15 contain refuges wherein bacteria can remain sensitive yet es-16 cape phage infection. These refuges form mainly due to reduced 17 growth in colony centers. We find little evidence that the pres-18 ence of the insensitive strain provides any additional protection 19 against phage. Our study reveals that living in a spatially struc-20 tured population can protect bacteria against phage infection, 21 while the presence of competing strains may instead reduce the 22 likelihood of evolving resistance to phage, if encountered. 23 Bacterial colony | resistance | evolution | microbial communities | population 24 dynamics | spatial structure | phage therapy 25 Correspondence: sara.mitri@unil.ch 26 82 survival rate compared to planktonic bacteria (25), particu-83 larly when exposed to antibiotics and importantly, also to 84 phage (26). More generally, phage population dynamics dif-85 fer radically between liquid bacterial cultures and bacteria 86 growing on solid surfaces (27). 87 Here we show that both of these factors -the presence of 88 other strains, and spatial structure -separately and combined 89 affect the outcome of phage predation on the pathogen Pseu-90 Testa et al. | bioRχiv | February 15, 2019 | 1-10In particular, we target P. aeruginosa strain PAO1 with Pseu-92 domonas phage 352 to which it is sensitive, in the presence 93 and absence of a second strain, P. aeruginosa PA14 that is in-94 sensitive to the phage. Since phage are so specific, we believe 95 the choice of a closely-related phage-insensitive strain to be 96 a realistic one. We compare the outcome for PAO1 in a well-97 mixed liquid environment and a structured biofilm (colony) 98 growing on a solid agar surface. 99 We find that in liquid, competition between the two strains 100 can reduce the population size of the target strain PAO1, giv-101 ing a competitive advantage to the phage and eliminating 102 PAO1 without the emergence of resistance. Indeed, evolv-103 ing resistance to the phage was the only way for PAO1 to 104 survive phage attack in liquid. In contrast, in a biofilm...

Communities of microbes colonize virtually every place on earth. Ultimately, we strive to predict and control how these communities behave, for example, if they reside in our guts and make us sick.