Phage-bacteria Interactions Research Articles

Characteristics of a novel temperate bacteriophage against Staphylococcus arlettae (vB_SarS_BM31).

Staphylococcus arlettae is a rarely reported coagulase-negative staphylococcus (CoNS) isolated from infected humans and livestock. Observing phage-bacteria interaction could improve the understanding of bacterial pathogenetic mechanisms, providing foundational evidence for phage therapy or phage detection. Herein, we aimed to characterise and annotate a novel bacteriophage, vB_SarS_BM31 (BM31), specific to S. arlettae. This bacteriophage was isolated from a milk sample associated with bovine mastitis and collected in the Sichuan Province, China. The BM31 genome comprised a linear double-stranded DNA of 42,271 base pair in length with a G + C content of 34.59%. A total of 65 open reading frames (ORFs) were assembled from phage DNA, of which 29 were functionally annotated. These functional genes were divided into four modules: the structural, DNA packing and replication, lysis, and lysogeny modules. Holin (ORF25), lysin (ORF26), and integrase (ORF28) were located closely in the entire BM31 genome and were important for lyse or lysogeny cycle of BM31. The phage was identified as a temperate phage according to whole genome analysis and life cycle assay, with basic biological characteristics such as small burst size, short latency period, and narrow host range, consistent with the characteristics of the family Siphoviridae, subcluster B14 of the Staphylococcus bacteriophage. The present isolation and characterisation of BM31 contributes to the Staphylococcus bacteriophage database and provides a theoretical foundation for its potential applications. To the best of our knowledge, BM31 is the only shared and completely reported phage against S. arlettae in the entire public database.

Phage Predation Promotes Filamentous Bacterium Piscinibacter Colonization and Improves Structural and Hydraulic Stability of Microbial Aggregates.

Although bacteria-phage interactions have broad environmental applications and ecological implications, the influence of phage predation on bacterial aggregation and structural stability remains largely unexplored. Herein, we demonstrate that inefficient lytic phage predation can promote host filamentous bacterium Piscinibacter colonization onto non-host Thauera aggregates, improving the structural and hydraulic stability of the dual-species aggregates. Specifically, phage predation at 103-104 PFU/mL (i.e., multiplication of infection at 0.01-0.1) promoted initial Piscinibacter colonization by 10-15 folds and resulted in 29-31% higher abundance of Piscinibacter in the stabilized aggregates than that in the control aggregates without phage predation. Transcriptomic analysis revealed upregulated genes related to quorum sensing (by 15-92 folds) and polysaccharide secretion (by 10-90 folds) within the treated aggregates, which was consistent with 120-172% higher content of polysaccharides for the treated dual-species aggregates. Confocal laser scanning microscopic images further confirmed the increase of filamentous bacteria and polysaccharides (both with wider distribution) within the dual-species aggregates. Accordlingly, the aggregates' structural strength (via atomic force microscopes) and shear resistance (via hydraulic stress tests) increased by 77 and 42%, respectively, relative to the control group. In the long-term experiments, the enhanced hydraulic stability of the treated aggregates could facilitate dwelling bacteria propagation in flow-through conditions. Overall, our study demonstrates that phage predation can promote bacterial aggregation and enhance aggregate structural stability, revealing the beneficial role of lytic phage predation on bacterial symbiosis and environmental adaptivity.

Revealing bacteria-phage interactions in human microbiome through the CRISPR-Cas immune systems.

The human gut microbiome is composed of a diverse consortium of microorganisms. Relatively little is known about the diversity of the bacteriophage population and their interactions with microbial organisms in the human microbiome. Due to the persistent rivalry between microbial organisms (hosts) and phages (invaders), genetic traces of phages are found in the hosts’ CRISPR-Cas adaptive immune system. Mobile genetic elements (MGEs) found in bacteria include genetic material from phage and plasmids, often resultant from invasion events. We developed a computational pipeline (BacMGEnet), which can be used for inference and exploratory analysis of putative interactions between microbial organisms and MGEs (phages and plasmids) and their interaction network. Given a collection of genomes as the input, BacMGEnet utilizes computational tools we have previously developed to characterize CRISPR-Cas systems in the genomes, which are then used to identify putative invaders from publicly available collections of phage/prophage sequences. In addition, BacMGEnet uses a greedy algorithm to summarize identified putative interactions to produce a bacteria-MGE network in a standard network format. Inferred networks can be utilized to assist further examination of the putative interactions and for discovery of interaction patterns. Here we apply the BacMGEnet pipeline to a few collections of genomic/metagenomic datasets to demonstrate its utilities. BacMGEnet revealed a complex interaction network of the Phocaeicola vulgatus pangenome with its phage invaders, and the modularity analysis of the resulted network suggested differential activities of the different P. vulgatus’ CRISPR-Cas systems (Type I-C and Type II-C) against some phages. Analysis of the phage-bacteria interaction network of human gut microbiome revealed a mixture of phages with a broad host range (resulting in large modules with many bacteria and phages), and phages with narrow host range. We also showed that BacMGEnet can be used to infer phages that invade bacteria and their interactions in wound microbiome. We anticipate that BacMGEnet will become an important tool for studying the interactions between bacteria and their invaders for microbiome research.

Forty Years without Family: Three Novel Bacteriophages with High Similarity to SPP1 Reveal Decades of Evolutionary Stasis since the Isolation of Their Famous Relative.

SPP1, an extensively studied bacteriophage of the Gram-positive Bacillus subtilis, is a model system for the study of phage–host interactions. Despite progress in the isolation and characterization of Bacillus phages, no previously fully sequenced phages have shared more than passing genetic similarity to SPP1. Here, we describe three virulent phages very similar to SPP1; SPP1 has greater than 80% nucleotide sequence identity and shares more that 85% of its protein coding genes with these phages. This is remarkable, given more than 40 years between the isolation of SPP1 and these phages. All three phages have somewhat larger genomes and more genes than SPP1. We identified a new putative gene in SPP1 based on a conserved sequence found in all phages. Gene conservation connotes purifying selection and is observed in structural genes and genes involved in DNA metabolism, but also in genes of unknown function, suggesting an important role in phage survival independent of the environment. Patterns of divergence point to genes or gene domains likely involved in adaptation to diverse hosts or different environments. Ultimately, comparative genomics of related phages provides insight into the long-term selective pressures that affect phage–bacteria interactions and alter phage genome content.

Mucosal Environment Induces Phage Susceptibility in Streptococcus mutans.

Pathogenic bacteria are attracted toward mucosa, as it is their way of entry into the body. However, we know surprisingly little about the phage-bacterium interactions in the mucosal environment. Here, we explored the effect of the mucosal environment on growth characteristics and phage-bacterium interactions in Streptococcus mutans, a causative agent of dental caries. We found that although mucin supplementation increased bacterial growth and survival, it decreased S. mutans biofilm formation. More importantly, the presence of mucin had a significant effect on S. mutans phage susceptibility. In two experiments done in Brain Heart Infusion Broth, phage M102 replication was detected only with 0.2% mucin supplementation. In 0.1 × Tryptic Soy Broth, 0.5% mucin supplementation led to a 4-log increase in phage titers compared with the control. These results suggest that the mucosal environment can have a major role in the growth, phage sensitivity, and phage resistance of S. mutans, and underline the importance of understanding the effect of mucosal environment on phage-bacterium interactions.

RNA Management During T7 Infection.

Post-transcriptional regulation (PTR) determines the fate of RNA in the cell and represents an important control point in the flow of genetic information and thus underpins many, if not all, aspects of cell function. Host takeover by phages through misappropriation of the bacterial transcription machinery is a relatively advanced area of research. However, several phages encode small regulatory RNAs, which are major mediators of PTR, and produce specific proteins to manipulate bacterial enzymes involved in RNA degradation.1-4 However, PTR during phage development still represents an understudied area of phage-bacteria interaction biology. In this study, we discuss the potential role PTR could play in determining the fate of RNA during the lifecycle of the prototypic phage T7 in Escherichia coli.

Hypoxia Increases the Tempo of Phage Resistance and Mutational Bottlenecking of Pseudomonas aeruginosa.

Viruses that infect bacteria (i.e., phages) are abundant and widespread in the human body, and new anti-infective approaches such as phage therapy are essential for the future of effective medicine. Our understanding of microenvironmental factors such as tissue oxygen availability at the site of phage–bacteria interaction remains limited, and it is unknown whether evolved resistance is sculpted differentially under normoxia vs. hypoxia. We, therefore, analyzed the phage–bacteria interaction landscape via adsorption, one-step, time-kill dynamics, and genetic evolution under both normoxia and hypoxia. This revealed that adsorption of phages to Pseudomonas aeruginosa decreased under 14% environmental oxygen (i.e., hypoxia), but phage time-kill and one-step growth kinetics were not further influenced. Tracking the adaptation of P. aeruginosa to phages uncovered a higher frequency of phage resistance and constrained types of spontaneous mutation under hypoxia. Given the interest in developing phage therapies, developing our understanding of the phage–pathogen interaction under microenvironmental conditions resembling those in the body offers insight into possible strategies to overcome multidrug-resistant (MDR) bacteria.

Mutualistic interplay between bacteriophages and bacteria in the human gut.

Bacteriophages (phages) are often described as obligate predators of their bacterial hosts, and phage predation is one of the leading forces controlling the density and distribution of bacterial populations. Every 48 h half of all bacteria on Earth are killed by phages. Efficient killing also forms the basis of phage therapy in humans and animals and the use of phages as food preservatives. In turn, bacteria have a plethora of resistance systems against phage attack, but very few bacterial species, if any, have entirely escaped phage predation. However, in complex communities and environments such as the human gut, this antagonistic model of attack and counter-defence does not fully describe the scope of phage-bacterium interactions. In this Review, we explore some of the more mutualistic aspects of phage-bacterium interactions in the human gut, and we suggest that the relationship between phages and their bacterial hosts in the gut is best characterized not as a fight to the death between enemies but rather as a mutualistic relationship between partners.

Mucin induces CRISPR-Cas defense in an opportunistic pathogen

Parasitism by bacteriophages has led to the evolution of a variety of defense mechanisms in their host bacteria. However, it is unclear what factors lead to specific defenses being deployed upon phage infection. To explore this question, we co-evolved the bacterial fish pathogen Flavobacterium columnare and its virulent phage V156 in presence and absence of a eukaryotic host signal (mucin) for sixteen weeks. The presence of mucin leads to a dramatic increase in CRISPR spacer acquisition, especially in low nutrient conditions where over 60% of colonies obtain at least one new spacer. Additionally, we show that the presence of a competitor bacterium further increases CRISPR spacer acquisition in F. columnare. These results suggest that ecological factors are important in determining defense strategies against phages, and that the phage-bacterium interactions on mucosal surfaces may select for the diversification of bacterial immune systems.

Individual-based model highlights the importance of trade-offs for virus-host population dynamics and long-term co-existence.

Viruses play diverse and important roles in ecosystems. In recent years, trade-offs between host and virus traits have gained increasing attention in viral ecology and evolution. However, microbial organism traits, and viral population parameters in particular, are challenging to monitor. Mathematical and individual-based models are useful tools for predicting virus-host dynamics. We have developed an individual-based evolutionary model to study ecological interactions and evolution between bacteria and viruses, with emphasis on the impacts of trade-offs between competitive and defensive host traits on bacteria-phage population dynamics and trait diversification. Host dynamics are validated with lab results for different initial virus to host ratios (VHR). We show that trade-off based, as opposed to random bacteria-virus interactions, result in biologically plausible evolutionary outcomes, thus highlighting the importance of trade-offs in shaping biodiversity. The effects of nutrient concentration and other environmental and organismal parameters on the virus-host dynamics are also investigated. Despite its simplicity, our model serves as a powerful tool to study bacteria-phage interactions and mechanisms for evolutionary diversification under various environmental conditions.

Understanding the Impacts of Bacteriophage Viruses: From Laboratory Evolution to Natural Ecosystems.

Viruses of bacteria (bacteriophages or phage) have broad effects on bacterial ecology and evolution in nature that mediate microbial interactions, shape bacterial diversity, and influence nutrient cycling and ecosystem function. The unrelenting impact of phages within the microbial realm is the result, in large part, of their ability to rapidly evolve in response to bacterial host dynamics. The knowledge gained from laboratory systems, typically using pairwise interactions between single-host and single-phage systems, has made clear that phages coevolve with their bacterial hosts rapidly, somewhat predictably, and primarily by counteradapting to host resistance. Recent advancement in metagenomics approaches, as well as a shifting focus toward natural microbial communities and host-associated microbiomes, is beginning to uncover the full picture of phage evolution and ecology within more complex settings. As these data reach their full potential, it will be critical to ask when and how insights gained from studies of phage evolution in vitro can be meaningfully applied to understanding bacteria-phage interactions in nature. In this review, we explore the myriad ways that phagesshape and are themselves shaped by bacterial host populations and communities, with a particular focus on observed and predicted differences between the laboratory and complex microbial communities.

Parallel phage resistance - virulence trade - offs during clinical phage therapy and in vitro

With rising antibiotic resistance, modern medicine needs new approaches for tackling bacterial infections. Phage therapy uses the viruses of pathogenic bacteria to clear the infection. Unlike antibiotics, phage can evolve if bacteria become resistant to maintain or even increase their infectivity (coevolution). While laboratory studies can give insight into complex bacteria-phage interactions, whether they act as a true representation of phage therapy in patients is unknown. Here, we compared phage therapy in a patient to that of in vitro experiments. The patient had been admitted with aPseudomonas aeruginosainfection and was successfully treated with a phage cocktail. Bacteria were isolated before and during phage therapy, allowing us to follow bacteria-phage coevolution in the patient while doing experiments on the same clones in vitro. In vivo and in vitro, bacteria rapidly evolved resistance with little or no evidence of bacteria - phage coevolution. Although resistance mechanisms differed, parallel resistance - virulence trade-offs were found in vivo and in vitro. Therefore, phage resistance could increase treatment success and our results indicate to what extent bacteria - phage evolutionary dynamics can be predicted from in vitro experiments.

The Gut Microbiota of Healthy and Flavobacterium psychrophilum-Infected Rainbow Trout Fry Is Shaped by Antibiotics and Phage Therapies.

In the aquaculture sector, there is an increased interest in developing environmentally friendly alternatives to antibiotics in the treatment and prevention of bacterial infections. This requires an understanding of the effects of different treatments on the fish microbiota as a measure for improving the fish health status. In this study, we focused on the freshwater pathogen Flavobacterium psychrophilum and investigated the effects of antibiotics (florfenicol) and phage therapies on the gut microbiota of healthy and infected rainbow trout fry (1–2 g). Florfenicol-coated feed was administered for 10 days, starting two days after the infection procedure. A two-component mix of phage targeting F. psychrophilum (FpV4 and FPSV-D22) was continuously delivered by feed with a prophylactic period of 12 days. Samples of the distal intestine were collected over time (day -1 and 1, 8, and 33 days post-infection) and analyzed by community analysis targeting the 16S rRNA gene (V3–V4 region). Results showed the dysbiosis effect caused both by the infection and by florfenicol administration. Shifts in the overall composition were detected by β-diversity analysis, and changes in specific populations were observed during taxonomic mapping. Measures of α-diversity were only affected in infected fish (large variation observed 1 and 8 dpi). These community alterations disappeared again when fish recovered from the infection and the antibiotic treatment was terminated (33 dpi). Interestingly, phage addition altered the microbiota of the fish independently of the presence of their target bacterium. The overall gut bacterial community in fish fed phage-treated feed was different from the controls at each time point as revealed by β-diversity analysis. However, it was not possible to identify specific bacterial populations responsible for these changes except for an increase of lactic acid bacteria 33 dpi. Overall, the results indicate that the administered phages might affect the complex network of phage-bacteria interactions in the fish gut. Nevertheless, we did not observe negative effects on fish health or growth, and further studies should be directed in understanding if these changes are beneficial or not for the fish health with an additional focus on the host immune response.

Combination of invivo phage therapy data with in silico model highlights key parameters for pneumonia treatment efficacy.

The clinical (re)development of bacteriophage (phage) therapy to treat antibiotic-resistant infections faces the challenge of understanding the dynamics of phage-bacteria interactions in the invivo context. Here, we develop a general strategy coupling invitro and invivo experiments with a mathematical model to characterize the interplay between phage and bacteria during pneumonia induced by a pathogenic strain of Escherichia coli. The model allows the estimation of several key parameters for phage therapeutic efficacy. In particular, it quantifies the impact of dose and route of phage administration as well as the synergism of phage and the innate immune response on bacterial clearance. Simulations predict a limited impact of the intrinsic phage characteristics in agreement with the current semi-empirical choices of phages for compassionate treatments. Model-based approaches will foster the deployment of future phage-therapy clinical trials.

Global Transcriptomic Analysis of Bacteriophage-Host Interactions between a Kayvirus Therapeutic Phage and Staphylococcus aureus.

ABSTRACTKayviruses are polyvalent broad host range staphylococcal phages with a potential to combat staphylococcal infections. However, the implementation of rational phage therapy in medicine requires a thorough understanding of the interactions between bacteriophages and pathogens at omics level. To evaluate the effect of a phage used in therapy on its host bacterium, we performed differential transcriptomic analysis by RNA-Seq from bacteriophage K of genus Kayvirus infecting two Staphylococcus aureus strains, prophage-less strain SH1000 and quadruple lysogenic strain Newman. The temporal transcriptional profile of phage K was comparable in both strains except for a few loci encoding hypothetical proteins. Stranded sequencing revealed transcription of phage noncoding RNAs that may play a role in the regulation of phage and host gene expression. The transcriptional response of S. aureus to phage K infection resembles a general stress response with differential expression of genes involved in a DNA damage response. The host transcriptional changes involved upregulation of nucleotide, amino acid and energy synthesis and transporter genes and downregulation of host transcription factors. The interaction of phage K with variable genetic elements of the host showed slight upregulation of gene expression of prophage integrases and antirepressors. The virulence genes involved in adhesion and immune evasion were only marginally affected, making phage K suitable for therapy.IMPORTANCE Bacterium Staphylococcus aureus is a common human and veterinary pathogen that causes mild to life-threatening infections. As strains of S. aureus are becoming increasingly resistant to multiple antibiotics, the need to search for new therapeutics is urgent. A promising alternative to antibiotic treatment of staphylococcal infections is a phage therapy using lytic phages from the genus Kayvirus. Here, we present a comprehensive view on the phage-bacterium interactions on transcriptomic level that improves the knowledge of molecular mechanisms underlying the Kayvirus lytic action. The results will ensure safer usage of the phage therapeutics and may also serve as a basis for the development of new antibacterial strategies.

Mechanisms of interactions between bacteria and bacteriophage mediate by quorum sensing systems.

Bacteriophage (phage) and their host bacteria coevolve with each other over time. Quorum sensing (QS) systems play an important role in the interaction between bacteria and phage. In this review paper, we summarized the function of QS systems in bacterial biofilm formation, phage adsorption, lysis-lysogeny conversion of phage, coevolution of bacteria and phage, and information exchanges in phage, which may provide reference to future research on alternative control strategies for antibiotic-resistant and biofilm-forming pathogens by phage. KEY POINTS: • Quorum sensing (QS) systems influence bacteria-phage interaction. • QS systems cause phage adsorption and evolution and lysis-lysogeny conversion. • QS systems participate in biofilm formation and co-evolution with phage of bacteria.

Identifying biomarkers of the gut bacteria, bacteriophages and serum metabolites associated with three weaning periods in piglets

BackgroundThe establishment of the piglet gut microbiome has a prolonged influence on host health, as it sets the stage for establishment of the adult swine microbiome. Substantial changes in host metabolism and immunity around the time of weaning may be accompanied by alterations in the gut microbiome. In this study, we systematically evaluated differences in the gut microbiome and host metabolites among three weaning periods using shotgun metagenomic sequencing and untargeted metabolomic profiling in piglets.ResultsWe identified that P. copri was the most significantly different species among three weaning periods, and was the key bacterial species for mitigating piglet adaptation during the weaning transition, while Bacillus_phage_BCD7, the only differential bacteriophages, was significantly and positively correlated with P. copri enriched in day 28 group. Additionally, P. copri and Bacillus_phage_BCD7 was significantly correlated with the shifts of functional capacities of the gut microbiome and several CAZymes in day 28 group. Furthermore, the altered metabolites we observed were enriched in pathways matched to the functional capacity of the gut microbiome e.g., aminoacyl-tRNA biosynthesis.ConclusionThe results from this study indicate that the bacteria-phage interactions and host-microbial interactions during the weaning transition impact host metabolism, leading to beneficial host changes among three weaning periods.

A Novel Phage Infecting the Marine Photoheterotrophic Bacterium Citromicrobium bathyomarinum.

This study isolated and characterized a new phage infecting the marine photoheterotrophic bacterium Citromicrobium bathyomarinum, which fills the gap in research on phages targeting this ecologically important species. The phage vB_CbaS-RXM (RXM) has a dsDNA genome with a length of 104,206 bp and G+C content of 61.64%. The taxonomic analysis found a close evolutionary relationship between RXM, Erythrobacter phage vB_EliS-L02, and Sphingobium phage Lacusarx, and we propose that RXM represents a new species of the Lacusarxvirus genus. A one-step growth curve revealed a burst size of 75 plaque-forming units (PFUs) per cell in a 3-hour infection cycle. The lysis profile of RXM showed an intraspecific lethal rate of 26.3% against 38 citromicrobial strains. RXM contains 15 auxiliary metabolic genes (AMGs) related to diverse cellular processes, such as putative metabolic innovation and hijacking of host nucleotide metabolism to enhance its biosynthetic capacity. An in-depth analysis showed that phage functional genes strongly rely on the host for translation, while the translation of unique phage genes with less host dependency may be complemented by phage tRNA. Overall, our study investigated the infection kinetics, genetic traits, taxonomy, and predicted roles of AMGs and tRNA genes of this new phage, which contributes to a better understanding of phage diversity and phage–bacterium interactions.

Campylobacter Bacteriophage Cocktail Design Based on an Advanced Selection Scheme.

Campylobacteriosis is a worldwide-occurring disease and has been the most commonly reported zoonotic gastrointestinal infection in the European Union in recent years. The development of successful phage-based intervention strategies will require a better understanding of phage–bacteria interactions to facilitate advances in phage cocktail design. Therefore, this study aimed to investigate the effects of newly isolated group II and group III phages and their combinations on current Campylobacter field strains. A continuous workflow for host range and efficiency of plating (EOP) value determination was combined with a qPCR-based phage group identification and a liquid-based planktonic killing assay (PKA). An advanced analysis scheme allowed us to evaluate phage cocktails by their efficacy in inhibiting bacterial population growth and the resulting phage concentrations. The results of this study indicate that data obtained from PKAs are more accurate than host range data based on plaque formation (EOP). Planktonic killing assays with Campylobacter appear to be a useful tool for a straightforward cocktail design. Results show that a group II phage vB_CcM-LmqsCP218-2c2 and group III phage vB_CjM-LmqsCP1-1 mixture would be most promising for practical applications against Campylobacter coli and Campylobacter jejuni.

Resolving the structure of phage\u2013bacteria interactions in the context of natural diversity

Microbial communities are shaped by viral predators. Yet, resolving which viruses (phages) and bacteria are interacting is a major challenge in the context of natural levels of microbial diversity. Thus, fundamental features of how phage-bacteria interactions are structured and evolve in the wild remain poorly resolved. Here we use large-scale isolation of environmental marine Vibrio bacteria and their phages to obtain estimates of strain-level phage predator loads, and use all-by-all host range assays to discover how phage and host genomic diversity shape interactions. We show that lytic interactions in environmental interaction networks (as observed in agar overlay) are sparse—with phage predator loads being low for most bacterial strains, and phages being host-strain-specific. Paradoxically, we also find that although overlap in killing is generally rare between tailed phages, recombination is common. Together, these results suggest that recombination during cryptic co-infections is an important mode of phage evolution in microbial communities. In the development of phages for bioengineering and therapeutics it is important to consider that nucleic acids of introduced phages may spread into local phage populations through recombination, and that the likelihood of transfer is not predictable based on lytic host range.