Biology subjects

Mora-Quilis, L.

Publications and source records attributed to Mora-Quilis, L..

3 recordsLinked to original sources

bioRxiv2025

Coevolution of a phage cocktail constrains reversible phenotypic resistance in K. pneumoniae

The therapeutic potential of phages is frequently compromised by rapid bacterial adaptation. Strategies such as phage cocktails and phage training are widely used to counteract bacterial resistance. Although phage receptor mutations are highly common, bacteria can also exploit flexible non-genetic strategies to evade phage recognition. Overcoming such phenotypic resistance remains particularly challenging due to its transient and reversible nature. Here, we investigated the coevolutionary dynamics between Klebsiella pneumoniae and a phage cocktail with complementary host tropisms: a capsule-dependent phage and an acapsular-targeting phage, coevolved with bacteria either individually or in combination. The coevolutionary phage training demonstrated that evolution of the acapsular-targeting phage was the main driver of the enhanced resistance delay observed. Notably, while bacteria could evade individual phages through non-mutational mechanisms, the combined selective pressure imposed by the phage cocktail constrained this reversible escape route, forcing bacteria to evolve mutation-based resistance mechanisms. Together, these findings highlight the importance of elucidating phage-bacteria coevolutionary dynamics to optimize phage-based therapeutics design and reshape bacterial evolutionary trajectories toward favourable outcomes. ImportancePhage therapy is re-emerging as a promising alternative to antibiotics, yet its long-term efficacy is often limited by the rapid evolution of bacterial resistance. While phage cocktails and experimental phage training have been widely proposed to mitigate this problem, most studies focus on genetically fixed resistance, overlooking reversible phenotypic resistance mechanisms. Here, we show that phenotypic resistance based on capsule regulation enables bacteria to evade single-phage treatments without acquiring mutations. In contrast, coevolutionary training of a two-phage cocktail targeting complementary bacterial phenotypes constrains this potentially low-cost escape route, forcing bacteria to adopt a mutation-based resistance, typically associated with higher fitness costs. These findings highlight the importance of phage cocktails not only as therapeutic tools, but also as ecological drivers that redirect bacterial evolutionary trajectories.

microbiology↗

bioRxiv2025

Phenotypic Heterogeneity Shapes Phage Resistance and Cocktail Efficacy in Klebsiella pneumoniae

The emergence of phage-resistant bacteria poses a significant challenge to the success of phage therapy. Although phage cocktails can delay resistance, their efficacy relies on the ability to target the full spectrum of resistant variants, which are often more diverse than that represented by clonal isolates. In this study, we investigated how phenotypic diversity within phage-resistant Klebsiella pneumoniae influences susceptibility to newly isolated phages and the effectiveness of phage cocktails. We isolated phages from three cultures resistant to a capsule-dependent phage: a heterogeneous acapsular population, an acapsular mutant with a stable phenotype, and a capsule-reverted isolate that regained capsule expression upon removal of phage pressure. These phages differed in host tropism and in their capacity to delay resistance emergence when combined with the capsule-dependent phage. Phages targeting acapsular variants, particularly those isolated from the heterogeneous population, were the most effective, exhibiting strong synergy. Single-cell analyses further revealed that sustained selective pressure from the capsule-dependent phage prevents capsule reversion and maintains cocktail efficacy. Overall, our results highlight the importance of accounting for phenotypic heterogeneity when designing phage therapies and support population-level approaches for optimizing phage cocktail composition. ImportancePhage therapy is a promising alternative to antibiotics, but its success is often limited by the rapid emergence of phage-resistant bacteria. These resistant populations can be highly heterogeneous, comprising both stable mutants and variants with reversible, non-genetic resistance. In this study, we explore how this phenotypic diversity influences the effectiveness of phage cocktails. By isolating new phages and testing them in combination, we demonstrate that the selective pressure exerted by specific phages can prevent the reversion in transiently resistant variants, thereby sustaining treatment efficacy. Our findings highlight the need to consider not only the range of bacterial targets but also how phage pressure shapes bacterial population dynamics. This work offers a more refined strategy for designing phage cocktails with improved clinical potential.

microbiology↗

bioRxiv2025

Phenotypic resistance to phage infection through capsule expression shutdown in Klebsiella pneumoniae

Capsule loss is a major mechanism by which bacteria evade phage infection. This process has traditionally been attributed to mutations in capsule biosynthesis genes. Here, we investigated phage resistance in Klebsiella pneumoniae, a medically relevant encapsulated bacterium. Phage infection rapidly selected for resistant acapsular cells. As expected, transcriptomic analysis revealed a marked downregulation of capsule biosynthesis genes. However, full genome sequencing showed that capsule loss occurred without evidence of mutations, and acapsular phage-resistant cells were able to rapidly restore their capsule once phage pressure was removed. These findings highlight that phage-driven selective pressure can act on non-heritable variation in gene expression, providing a faster and more flexible resistance mechanism compared to the traditional mutation-selection process. IMPORTANCEBacteriophages are increasingly considered as alternatives or complements to antibiotics, particularly against multidrug-resistant pathogens like Klebsiella pneumoniae. A key barrier to effective phage therapy is the rapid emergence of bacterial resistance. Capsule loss is a common resistance mechanism, traditionally linked to genetic mutations. Here, we show that K. pneumoniae can evade phage infection through reversible, non-mutational downregulation of capsule biosynthesis. This phenotypic adaptation allows rapid resistance development without compromising long-term fitness. Our findings reveal a flexible, non-genetic resistance strategy that may limit the durability of phage therapy and should be considered in future phage treatment designs.

microbiology↗