Phage efficacy in infecting dual-strain biofilms ofPseudomonas aeruginosa

Abstract

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...

Adaptive lifestyle of bacteria determines phage-bacteria interaction

“…AEP1.3 in liquid culture was unexpected (Figure 1E). While it has been recorded that community composition affected phage susceptibility (Blazanin and Turner, 2021) literature has indicated that solid bacterial aggregates hindered phage propagation due to perturbed diffusion, fibrous barriers, and increased nutrient availability (Lourenço et al, 2018;Sousa and Rocha, 2019) and that contrary to our findings, bacteria were found to be more susceptible to phages in liquid culture (Testa et al, 2019). Since evidence of a phage that only infects bacteria on solid medium and not in liquid culture is rare, we chose to investigate this phenomenon in greater detail.…”

Phage cocktail strategies for the suppression of a pathogen in a cross‐feeding coculture

“…While we are not the first to consider using multispecies‐targeting cocktails, others have used them with a different goal – to target co‐occurring pathogens (Carson et al ., 2010; Lehman and Donlan, 2015; Oliveira et al ., 2018; Milho et al ., 2019; Zhao et al ., 2019). Additionally, others have explored phage treatment of pathogens in competitive ecological contexts, but limited their analysis to single phage treatments that targeted the focal bacterial species only (Harcombe and Bull, 2005; Brockhurst et al ., 2006; Wang et al ., 2017; Yu et al ., 2017; Testa et al ., 2019). Our research extended these foundational studies by including both pathogen‐targeting and multispecies‐targeting cocktails, and by addressing the role of cooperative cross‐feeding between a pathogen and another coculture member.…”

“…Experiments using cocultures with well‐defined interactions have helped elucidate a range of responses to phage infection which may be leveraged for phage therapy. For example, adding non‐host bacteria that compete with phage hosts for nutrients limits phage resistance evolution, thereby magnifying the efficacy of the phage (Harcombe and Bull, 2005; Brockhurst et al ., 2006; Wang et al ., 2017; Yu et al ., 2017; Testa et al ., 2019). Microbes can also engage in cooperative mutualistic interactions, where bacteria depend on others to cross‐feed nutrients (Schink, 2002; Mee et al ., 2014; Zelezniak et al ., 2015; D'Souza and Kost, 2016; Adamowicz et al ., 2018).…”