Biology subjects

Domingo-Calap, P.

Publications and source records attributed to Domingo-Calap, P..

14 recordsLinked to original sources

bioRxiv2026

Lipid metabolic pathways determine phage infectivity in Mycobacterium abscessus

Mycobacterium abscessus is a rapidly growing non-tuberculous mycobacterium with high intrinsic antibiotic resistance, requiring innovative therapeutics. During infection, smooth and rough colony morphotypes can coexist in the human body, participating in the pathogenesis. Despite its increasing clinical importance and phage therapy being a last resort treatment recently, the genetic basis of phage interaction in M. abscessus remains poorly understood. Previous work has focused largely on rough strains or the surrogate host Mycobacterium smegmatis. Here, we isolated novel phages that efficiently infect both morphotypes, allowing us to characterize phage-resistant mutants from paired smooth and rough clinical isolates to determine the genetic basis of the infectivity. Integrating whole-genome sequencing, transcriptomics, and phage susceptibility and adsorption assays, we deeply analyzed 30 phage-resistant variants and found that resistance trajectories were shaped primarily by host morphotype rather than by the selecting phage. We confirmed the TPP locus as a conserved determinant of phage infection in both morphotypes and identified previously undescribed hotspot mutations in furB and nrnA, together with a multi-gene deletion, in smooth-derived resistant variants. Whereas the TPP locus and the multi-gene deletion represent stable genomic changes affecting lipid-associated loci, frameshift mutations in furB and nrnA, not previously linked to lipid metabolism, were accompanied by broad transcriptional rewiring of lipid-related genes. Resistance was mutation-dependent and consistently associated with impaired phage adsorption, indicating an early block in infection. Together, these findings show that phage recognition in M. abscessus is shaped by mycomembrane lipid architecture rather than a single dedicated receptor and uncover regulatory and metabolic pathways with implications for more durable phage-based therapies.

microbiology↗

bioRxiv2026

Phage-meropenem synergy against OXA-48-producing Klebsiella pneumoniae clinical isolates

Antimicrobial resistance is a growing global healthcare crisis, driven by the rapid spread of resistant pathogens that compromise existing treatments. Carbapenem-resistant Klebsiella pneumoniae is a major public health threat, requiring novel therapeutic strategies. Phage therapy, which employs phages to target bacterial pathogens, is a promising approach, particularly when combined with antibiotics to enhance efficacy through synergistic interactions. In this study, time-kill curve assays were used to evaluate the synergy between the lytic phage vB_Kpn_2-P4 and meropenem against twelve K. pneumoniae clinical isolates from Spanish hospitals that carried diverse carbapenemases. Notably, in OXA-48-producing isolates, this combination prevented the emergence of resistant mutants, highlighting the therapeutic potential of phage-antibiotic synergy. The observed effect, linked to the presence of the pOXA-48 plasmid, suggests a promising strategy for combating multidrug-resistant bacteria.

microbiology↗

bioRxiv2025

Quasineutral dynamics shape coexistence and clearance in a model ofin vitro phage-bacteria interactions

Phage therapy, which uses viruses that infect bacteria to target and lyse specific bacterial pathogens, has re-emerged as a promising strategy to combat antibiotic-multiresistant bacteria. Advances in metagenomics and synthetic biology, together with systems biology approaches combining mathematical modeling with experimental data, provide excellent opportunities to understand phage-bacteria dynamics. Here we analyze a mathematical model successfully calibrated using in vitro data on the multidrug-resistant bacterium Klebsiella pneumoniae in the presence of the phage vB Kpn 2-P4. The model describes a system with a susceptible bacterial population that can generate phage-resistant mutants. By analyzing the equilibria and bifurcations of the model, we identify a coexistence scenario between phage-resistant bacteria and phages governed by a quasineutral line of equilibria with both stable and unstable segments. Biologically, this quasineutral structure implies that phage-resistant bacteria can persist across a wide range of phage densities without selective pressure favoring a unique outcome, making clearance highly sensitive to additional mortality mechanisms. The clearance of phage-resistant bacteria can be achieved by combining phage activity with an increased death rate of the resistant strains. This process is governed by a global transcritical bifurcation of the quasineutral line. Our model offers mechanistic insight into potential scenarios leading to the complete elimination of phage-resistant bacteria. Author summaryBacteriophages--viruses that infect bacteria--are being reconsidered as alternatives to antibiotics, but bacterial resistance to phages often emerges rapidly. Understanding when phages and bacteria coexist and when resistant bacteria can be eliminated remains a major challenge. In this study, we analyze a mathematical model calibrated with in vitro data describing interactions between bacteria, bacteriophages, and phage-resistant mutants. Using tools from dynamical systems theory, we show that coexistence between phages and resistant bacteria is organized by a quasineutral line of equilibrium states rather than a single stable outcome. This structure explains why long-term dynamics can be highly sensitive to initial conditions without being chaotic. We further identify a bifurcation that leads to the extinction of resistant bacteria when their effective mortality exceeds a critical threshold. Our results provide a mechanistic framework for interpreting resistance-driven outcomes in phage-bacteria systems and highlight how mathematical structure can constrain therapeutic strategies, even in simple experimental settings.

systems biology↗

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

Isolation and characterization of broad-range Staphylococcus epidermidis sepunaviruses

Staphylococcus epidermidis is a major contributor to biofilm-associated infections, bacteraemia, and sepsis in humans. Additionally, it is an important veterinary pathogen. The spread of multi-drug resistant S. epidermidis (MRSE) poses an even greater challenge, requiring the development of antibiotic-alternative approaches. Here, we isolated four new broad-range phages capable of infecting a large panel of 65 clinical and veterinary isolates of S. epidermidis. Phages with the broadest host range produced plaques on 37% of the hosts tested and halos on an additional 40%. These phages belonged to the Sepunavirus genus, as supported by their morphology and genome analysis. A genome-wide association study identified a significant correlation between resistance to phage lysis and the presence of the sbcCD complex and the ica operon, highlighting the protective role of biofilms against the phages isolated in this study. Furthermore, distinct phage-encoded methyltransferases identified in the phage genomes may contribute to differences in host ranges. This study advances our knowledge on the diversity and characteristics of S. epidermidis phages, which could be considered as interesting tools for phage therapeutics. RepositoriesPhage genomes are available under Genbank accession numbers PV426907-PV426910. Bacterial genome sequences determined as part of this study, and all Bakta annotations, are available via Figshare (doi:10.6084/m9.figshare.29066417 and doi:10.6084/m9.figshare.29079968 respectively).

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↗

bioRxiv2024

Evolutionary strategies of Klebsiella phages in a host-diverse environment

Phage-host interactions have been studied using one or a few phages and hosts in simple, controlled systems. Here, we implemented an experimental design to evolve a 12-phage cocktail in a highly diverse environment, including a combination of 39 Klebsiella spp. capsular types. Our results showed how phages modulate their host range through different strategies, adopting a more generalist lifestyle or maintaining their host specificity, depending on the versatility of receptor binding proteins. Some of these proteins were mutational hotspots during the evolution experiment, mainly due to their intrinsic versatility, allowing a broader or narrower host range, and host diversity in the environment. Other mutations found in evolved phages were associated with evading bacterial defense systems or improving fitness on current hosts. Ecological dynamics between phages and hosts, including prophage activation and recombination events, also determined the fate of phage populations. In addition, single phage evolution experiments were performed to validate the results, allowing a model of receptor binding protein evolution and host range modulation to be proposed. This work is a step forward in the understanding of phage-host interactions and the appropriate implementation of phages as biomedical tools, providing insights into their impact in complex environments such as the human microbiota.

evolutionary biology↗

bioRxiv2024

Unlocking data in Klebsiella lysogens to predict capsular type-specificity of phage depolymerases

Viral entry is a critical step in the infection process. Klebsiella spp. and other clinically relevant bacteria often express a complex polysaccharide capsule that acts as a barrier to phage entry. In turn, most Klebsiella phages encode depolymerases for capsule removal. This virus-host arms race has led to extensive genetic diversity in both capsules and depolymerases, complicating our ability to understand their interaction. This study exploits the information encoded in Klebsiella prophages to model the interplay between the bacteria, the prophages, and their depolymerases, using a graph neural network and a sequence clustering-based method. Both approaches showed significant predictive ability for prophages capsular tropism and, importantly, were transferrable to lytic phages. In addition to creating a comprehensive database linking depolymerase sequences to their specific targets, this study demonstrates the predictability of phage-host interactions at the subspecies level, providing new insights for improving the therapeutic and industrial applicability of phages.

bioinformatics↗

bioRxiv2024

Mobile Integrons Encode Phage Defence Systems

Integrons are bacterial genetic elements that capture, stockpile and modulate the expression of genes encoded in integron cassettes. Mobile Integrons (MI) are borne on plasmids, acting as a vehicle for hundreds of antimicrobial resistance genes among key pathogens. These elements also carry gene cassettes of unknown function (gcus) whose role and adaptive value remains unexplored. Here we show that gcus encode phage resistance systems, many of which are novel. Bacteriophage resistance integron cassettes (BRiCs) can be combined and mixed with resistance cassettes to produce multiphage or drug/phage-resistance. The fitness costs of BRiCs are variable, dependent on the genetic context, and can be modulated by changing the order of cassettes in the array. Hence, MIs act as highly mobile, low-cost defense islands. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/601719v4_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@965766org.highwire.dtl.DTLVardef@442763org.highwire.dtl.DTLVardef@7600d4org.highwire.dtl.DTLVardef@13068ca_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOSummary FigureC_FLOATNO Novel phage defense systems identified in Mobile Integrons. We confronted genes of unknown function from mobile integrons against a panel of phage. We characterized 13 Bacteriophage Resistance integron Cassettes (BRiCs) and confirmed their function in Klebsiella pneumoniae and Pseudomonas aeruginosa. Combined with other cassettes, BRiCs produce multi-phage/antibiotic resistance. Additionally, their cost can be reduced in an array. C_FIG

microbiology↗

bioRxiv2024

DepoScope: accurate phage depolymerase annotation and domain delineation using large language models

Bacteriophages (phages) are viruses that infect bacteria. Many of them produce specific enzymes called depolymerases to break down external polysaccharide structures. Accurate annotation and domain identification of these depolymerases are challenging due to their inherent sequence diversity. Hence, we present DepoScope, a machine learning tool that combines a fine-tuned ESM-2 model with a convolutional neural network to precisely identify depolymerase sequences and their enzymatic domains. To accomplish this, we curated a dataset from the INPHARED phage genome database, created a polysaccharide-degrading domain database, and applied sequential filters to construct a high-quality dataset, which are subsequently used to train DepoScope. Our work is the first approach that combines sequence-level predictions with amino-acid-level predictions for an accurate depolymerase detection and functional domain identification. In that way, we believe that DepoScope can enhance our understanding of phage-host interactions at the level of depolymerases. Summary with Key MessagesO_LIPhage depolymerases are proteins that play a crucial role in the first step of a phage replication cycle. As a result, they are both important from a biological perspective and a therapeutical perspective. C_LIO_LICurrent methods to accurately annotate phage depolymerases and their associated enzymatic domains remains challenging due to their inherent high sequence diversity. C_LIO_LIWe have developed DepoScope, a language-based artificial intelligence model that can accurately identify phage depolymerases and their specific enzymatic domains. C_LIO_LIWe provide full public access to the DepoScope code and database to give broad access to the research community and promote further research. C_LI

bioinformatics↗

bioRxiv2024

Targeted phage hunting to specific Klebsiella pneumoniae clinical isolates is an efficient antibiotic resistance and infection control strategy

Klebsiella pneumoniae is one of the most threatening multi-drug resistant pathogens today, with phage therapy being a promising alternative for personalized treatments. However, the intrinsic capsule diversity in Klebsiella spp. poses a substantial barrier to phage host range, complicating the development of broad-spectrum phage-based treatments. Here, we have isolated and genomically characterized phages capable of infecting each of the acquired 77 reference serotypes of Klebsiella spp., including capsular types widespread among high-risk K. pneumoniae clones causing nosocomial infections. We demonstrated the possibility of isolating phages for all capsular types in the collection, revealing high capsular specificity among taxonomically related phages, in contrast to a few phages that exhibited broad-spectrum infection capabilities. To decipher the determinants of the specificity of these phages, we focused on their receptor-binding proteins, with particular attention to depolymerase domains. We also explored the possibility of designing a broad-spectrum phage cocktail based on phages isolated in reference capsular type strains, and determining the ability to lysate relevant clinical isolates. Interestingly, a combination of 12 phages capable of infecting 60% of the reference Klebsiella spp. serotypes was tested on a panel of carbapenem-resistant K. pneumoniae clinical isolates. Our results suggest that in a highly variable encapsulated bacterial host, phage hunting must be directed to the specific Klebsiella isolates. This work is a step forward in the understanding of the complexity of phage-host interactions, and highlights the importance of implementing precise and phage-specific strategies to treat K. pneumoniae infections worldwide.

microbiology↗

bioRxiv2023

Capsules and their traits shape phage susceptibility and plasmid conjugation efficiency

Bacterial evolution is affected by mobile genetic elements such as phages and conjugative plasmids, which may provide novel adaptive traits but also incur in fitness costs. Infection by these elements is affected by the bacterial capsule. Yet, its importance has been difficult to quantify and characterise because of the high diversity of bacterial genomes regarding confounding mechanisms such as anti-viral systems. We swapped capsule loci between Klebsiella pneumoniae strains to quantify their effect on transfer of conjugative plasmids and phages independently of the genetic background. Capsule swaps systematically invert phage susceptibility, demonstrating that serotypes are key determinants of phage infection. Capsule types also affect conjugation efficiency in both donor and recipient cells depending on the serotype, a mechanism shaped by the capsule volume and depending on the structure of the conjugative pilus. Comparative genomics confirmed that more permissive serotypes in the lab correspond to the strains acquiring more conjugative plasmids in nature. The pili least sensitive to capsules (F-like) are also the most frequent in the species plasmids, and are the only ones associated with both antibiotic resistance and virulence factors, driving the convergence between virulence and antibiotics resistance in the population. These results show how the traits of cellular envelopes define slow and fast lanes of infection by mobile genetic elements, with implications for population dynamics and horizontal gene transfer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/536574v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@157bc4eorg.highwire.dtl.DTLVardef@10bc979org.highwire.dtl.DTLVardef@d85080org.highwire.dtl.DTLVardef@18d331e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Genetic determinants of host tropism in Klebsiella phages

Bacteriophages play key roles in bacterial ecology and evolution and are potential antimicrobials. However, the determinants of phage-host specificity remain elusive. Here, we used 46 newly-isolated phages to challenge 138 representative clinical isolates of Klebsiella pneumoniae, a widespread opportunistic pathogen. Spot tests revealed a narrow host range for most phages, with <2% of 6319 phage-host combinations tested yielding detectable interactions. Bacterial capsule diversity was the main factor restricting phage host range. Consequently, phage-encoded depolymerases were key determinants of host tropism, and we identified depolymerase sequence types associated with the ability to infect specific capsular types across phage families. Phages showing a capsule-independent mode of entry exhibited a much broader host range, but their infectivity was still restricted by complex intracellular defense mechanisms. These findings expand our knowledge of the complex interactions between bacteria and their viruses, and have implications for the biomedical and biotechnological use of phages.

microbiology↗