A summary of the main themes and findings presented at the ASM Intermountain Branch meeting (2024).

The annual meeting for the Intermountain Branch was held in April 2024 on the campus of Brigham Young University. There were 127 branch members from Utah, Idaho, and Nevada who attended the meeting and were composed of undergraduate students, graduate or medical students, and faculty. This report highlights the diversity of, and the emerging trends in, the research conducted by American Society for Microbiology members in the Intermountain Branch.

A novel genus of Pectobacterium bacteriophages display broad host range by targeting several species of Danish soft rot isolates.

The bacterial diseases black leg and soft rot in potatoes cause heavy losses of potatoes worldwide. Bacteria within the genus Pectobacteriaceae are the causative agents of black leg and soft rot. The use of antibiotics in agriculture is heavily regulated and no other effective treatment currently exists, but bacteriophages (phages) have shown promise as potential biocontrol agents. In this study we isolated soft rot bacteria from potato tubers and plant tissue displaying soft rot or black leg symptoms collected in Danish fields. We then used the isolated bacterial strains as hosts for phage isolation. Using organic waste, we isolated phages targeting different species within Pectobacterium. Here we focus on seven of these phages representing a new genus primarily targeting P. brasiliense; phage Ymer, Amona, Sabo, Abuela, Koroua, Taid and Pappous. TEM image of phage Ymer showed siphovirus morphotype, and the proposed Ymer genus belongs to the class Caudoviricetes, with double-stranded DNA genomes varying from 39kb to 43kb. In silico host range prediction using a CRISPR-Cas spacer database suggested both P. brasiliense, P. polaris and P. versatile as natural hosts for phages within the proposed Ymer genus. A following host range experiment, using 47 bacterial isolates from Danish tubers and plants symptomatic with soft rot or black leg disease verified the in silico host range prediction, as the genus as a group were able to infect all three Pectobacterium species. Phages did, however, primarily target P. brasiliense isolates and displayed differences in host range even within the species level. Two of the phages were able to infect two or more Pectobacterium species. Despite no nucleotide similarity with any phages in the NCBI database, the proposed Ymer genus did share some similarity at the protein level, as well as gene synteny, with currently known phages. None of the phages encoded integrases or other genes typically associated with lysogeny. Similarly, no virulence factors nor antimicrobial resistance genes were found, and combined with their ability to infect several soft rot-causing Pectobacterium species from Danish fields, demonstrates their potential as biocontrol agents against soft rot and black leg diseases in potatoes.

Exploring the historical roots, advantages and efficacy of phage therapy in plant diseases management.

In the drug-resistance era, phage therapy has received considerable attention from worldwide researchers. Phage therapy has been given much attention in public health but is rarely applied to control plant diseases. Herein, we discuss phage therapy as a biocontrol approach against several plant diseases. The emergence of antibiotic resistance in agriculturally important pathogenic bacteria and the toxic nature of different synthetic compounds used to control microbes has driven researchers to rethink the century-old strategy of phage therapy''. Compared to other treatment strategies, phage therapy offers remarkable advantages such as high specificity, less chances of drug resistance, non-harmful nature, and benefit to soil microbial flora. The optimizations and protective formulations of phages are significant accomplishments; however, steps towards a better understanding of the physiologic characteristics of phages need to be preceded to commercialize their use. The future of phage therapy in the context of plant disease management is promising and could play a significant role in sustainable agriculture. Ongoing research will likely affirm the safety of phage therapy, ensuring that it does not harm non-target organisms, including beneficial soil microbes. Phage therapy could become vital in addressing global food security challenges, particularly in regions heavily impacted by plant bacterial diseases. Efforts to create formulations that enhance the stability and shelf-life of phages will be crucial, especially for their use in varied environmental conditions.

Diversity and phage sensitivity to phages of porcine enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a diverse and poorly characterized E. coli pathotype that causes diarrhea in humans and animals. Phages have been proposed for the veterinary biocontrol of ETEC, but effective solutions require understanding of porcine ETEC diversity that affects phage infection. Here, we sequenced and analyzed the genomes of the PHAGEBio ETEC collection, gathering 79 diverse ETEC strains isolated from European pigs with post-weaning diarrhea (PWD). We identified the virulence factors characterizing the pathotype and several antibiotic resistance genes on plasmids, while phage resistance genes and other virulence factors were mostly chromosome encoded. We experienced that ETEC strains were highly resistant to Enterobacteriaceae phage infection. It was only by enrichment of numerous diverse samples with different media and conditions, using the 41 ETEC strains of our collection as hosts, that we could isolate two lytic phages that could infect a large part of our diverse ETEC collection: vB_EcoP_ETEP21B and vB_EcoS_ETEP102. Based on genome and host range analyses, we discussed the infection strategies of the two phages and identified components of lipopolysaccharides ( LPS) as receptors for the two phages. Our detailed computational structural analysis highlights several loops and pockets in the tail fibers that may allow recognition and binding of ETEC strains, also in the presence of O-antigens. Despite the importance of receptor recognition, the diversity of the ETEC strains remains a significant challenge for isolating ETEC phages and developing sustainable phage-based products to address ETEC-induced PWD.IMPORTANCEEnterotoxigenic Escherichia coli (ETEC)-induced post-weaning diarrhea is a severe disease in piglets that leads to weight loss and potentially death, with high economic and animal welfare costs worldwide. Phage-based approaches have been proposed, but available data are insufficient to ensure efficacy. Genome analysis of an extensive collection of ETEC strains revealed that phage defense mechanisms were mostly chromosome encoded, suggesting a lower chance of spread and selection by phage exposure. The difficulty in isolating lytic phages and the molecular and structural analyses of two ETEC phages point toward a multifactorial resistance of ETEC to phage infection and the importance of extensive phage screenings specifically against clinically relevant strains. The PHAGEBio ETEC collection and these two phages are valuable tools for the scientific community to expand our knowledge on the most studied, but still enigmatic, bacterial species-E. coli.

Artificial Phages with Biocatalytic Spikes for Synergistically Eradicating Antibiotic-Resistant Biofilms.

Antibiotic-resistant pathogens have become a global public health crisis, especially biofilm-induced refractory infections. Efficient, safe, and biofilm microenvironment (BME)-adaptive therapeutic strategies are urgently demanded to combat antibiotic-resistant biofilms. Here, inspired by the fascinating biological structures and functions of phages, the de novo design of a spiky Ir@Co3O4 particle is proposed to serve as an artificial phage for synergistically eradicating antibiotic-resistant Staphylococcus aureus biofilms. Benefiting from the abundant nanospikes and highly active Ir sites, the synthesized artificial phage can simultaneously achieve efficient biofilm accumulation, extracellular polymeric substance (EPS) penetration, and superior BME-adaptive reactive oxygen species (ROS) generation, thus facilitating the in situ ROS delivery and enhancing the biofilm eradication. Moreover, metabolomics found that the artificial phage obstructs the bacterial attachment to EPS, disrupts the maintenance of the BME, and fosters the dispersion and eradication of biofilms by down-regulating the associated genes for the biosynthesis and preservation of both intra- and extracellular environments. The in vivo results demonstrate that the artificial phage can treat the biofilm-induced recalcitrant infected wounds equivalent to vancomycin. It is suggested that the design of this spiky artificial phage with synergistic "penetrate and eradicate" capability to treat antibiotic-resistant biofilms offers a new pathway for bionic and nonantibiotic disinfection.

Acinetobacter baumannii satellite phage Aci01-2-Phanie depends on a helper myophage for its multiplication.

We report the discovery of a satellite-helper phage system with a novel type of dependence on a tail donor. The Acinetobacter baumannii satellite podovirus Aci01-2-Phanie (short name Phanie) uses a phage phi29-like DNA replication and packaging mode. Its linear 11,885 bp dsDNA genome bears 171 bp inverted terminal repeats (ITR). Phanie is related to phage DU-PP-III from Pectobacterium and to members of the Astrithrvirus from Salmonella enterica. Together, they form a new clade of phages with 27% to 30% identity over the whole genome. Detailed 3D protein structure prediction and mass spectrometry analyses demonstrate that Phanie encodes its capsid structural genes and genes necessary to form a short tail. However, our study reveals that Phanie virions are non-infectious unless they associate with the contractile tail of an unrelated phage, Aci01-1, to produce chimeric myoviruses. Following the coinfection of Phanie with myovirus Aci01-1, hybrid viral particles composed of Phanie capsids and Aci01-1 contractile tails are assembled together with Phanie and Aci01-1 particles.IMPORTANCEThere are few reported cases of satellite-helper phage interactions but many more may be yet undiscovered. Here we describe a new mode of satellite phage dependence on a helper phage. Phanie, like phage phi29, replicates its linear dsDNA by a protein primed-mechanism and protects it inside podovirus-like particles. However, these particles are defective, requiring the acquisition of the tail from a myovirus helper for production of infectious virions. The formation of chimeras between a phi29-like podovirus and a helper contractile tail reveals an unexpected association between very different bacterial viruses.

Genomic and taxonomic evaluation of 38 Treponema prophage sequences.

BACKGROUND: Despite Spirochetales being a ubiquitous and medically important order of bacteria infecting both humans and animals, there is extremely limited information regarding their bacteriophages. Of the genus Treponema, there is just a single reported characterised prophage. RESULTS: We applied a bioinformatic approach on 24 previously published Treponema genomes to identify and characterise putative treponemal prophages. Thirteen of the genomes did not contain any detectable prophage regions. The remaining eleven contained 38 prophage sequences, with between one and eight putative prophages in each bacterial genome. The prophage regions ranged from 12.4 to 75.1 kb, with between 27 and 171 protein coding sequences. Phylogenetic analysis revealed that 24 of the prophages formed three distinct sequence clusters, identifying putative myoviral and siphoviral morphology. ViPTree analysis demonstrated that the identified sequences were novel when compared to known double stranded DNA bacteriophage genomes. CONCLUSIONS: In this study, we have started to address the knowledge gap on treponeme bacteriophages by characterising 38 prophage sequences in 24 treponeme genomes. Using bioinformatic approaches, we have been able to identify and compare the prophage-like elements with respect to other bacteriophages, their gene content, and their potential to be a functional and inducible bacteriophage, which in turn can help focus our attention on specific prophages to investigate further.

Data-Driven Engineering of Phages with Tunable Capsule Tropism for Klebsiella pneumoniae.

Klebsiella pneumoniae, a major clinical pathogen known for causing severe infections, is attracting heightened attention due to its escalating antibiotic resistance. Phages are emerging as a promising alternative to antibiotics; however, their specificity to particular hosts often restricts their use. In this study, a collection of 114 phages is obtained and subjected to analysis against 238 clinical K. pneumoniae strains, revealing a spectrum of lytic behaviors. A correlation between putative tail protein clusters and lysis patterns leads to the discovery of six receptor-binding protein (RBP) clusters that determine host capsule tropism. Significantly, RBPs with cross-capsular lysis capabilities are identified. The newly-identified RBPs provide a toolbox for customizing phages to target diverse capsular types. Building on the toolbox, the engineered phages with altered RBPs successfully shifted and broadened their host capsule tropism, setting the stage for tunable phage that offer a precise and flexible solution to combat K. pneumoniae infections.

Novel phage infecting the Roseobacter CHUG lineage reveals a diverse and globally distributed phage family.

Bacteriophages play an essential role in shaping the diversity and metabolism of bacterial communities. Marine Roseobacter group is an abundant heterotrophic bacterial group that is involved in many major element cycles, especially carbon and sulfur. Members of the Roseobacter CHUG (Clade Hidden and Underappreciated Globally) lineage are globally distributed and are activated in pelagic marine environments. In this study, we isolated and characterized a phage, CRP-810, that infects the CHUG strain FZCC0198. The genome of CRP-810 was dissimilar to those of other known phages. Additionally, 251 uncultured viral genomes (UViGs) closely related to CRP-810 were obtained from the uncultivated marine viral contig databases. Comparative genomic and phylogenetic analyses revealed that CRP-810 and these related UViGs exhibited conserved genome synteny, representing a new phage family with at least eight subgroups. Most of the CRP-810-type phages contain an integrase gene, and CRP-810 can be integrated into the host genome. Further analysis revealed that three CRP-810-type members were prophages found in the genomes of marine SAR11, Poseidonocella, and Sphingomonadaceae. Finally, viromic read-mapping analysis showed that CRP-810-type phages were globally distributed and displayed distinct biogeographic patterns related to temperature and latitude. Many members with a lower G + C content were mainly distributed in the trade station, whereas members with a higher G + C content were mainly distributed in polar and westerlies station, indicating that the niche differentiation of phages was subject to host adaptation. Collectively, these findings identify a novel phage family and expand our understanding of phylogenetic diversity, evolution, and biogeography of marine phages. IMPORTANCE: The Roseobacter CHUG lineage, affiliated with the Pelagic Roseobacter Cluster (PRC), is widely distributed in the global oceans and is active in oligotrophic seawater. However, knowledge of the bacteriophages that infect CHUG members is limited. In this study, a CHUG phage, CRP-810, that infects the CHUG strain FZCC0198, was isolated and shown to have a novel genomic architecture. In addition, 251 uncultured viral genomes closely related to CRP-810 were recovered and included in the analyses. Phylogenomic analyses revealed that the CRP-810-type phages represent a new phage family containing at least eight genus-level subgroups. Members of this family were predicted to infect various marine bacteria. We also demonstrated that the CRP-810-type phages are widely distributed in global oceans and display distinct biogeographic patterns related to latitude. Collectively, this study provides important insights into the genomic organization, diversity, and ecology of a novel phage family that infect ecologically important bacteria in the global ocean.

Mixed waste contamination selects for a mobile genetic element population enriched in multiple heavy metal resistance genes.

Mobile genetic elements (MGEs) like plasmids, viruses, and transposable elements can provide fitness benefits to their hosts for survival in the presence of environmental stressors. Heavy metal resistance genes (HMRGs) are frequently observed on MGEs, suggesting that MGEs may be an important driver of adaptive evolution in environments contaminated with heavy metals. Here, we report the meta-mobilome of the heavy metal-contaminated regions of the Oak Ridge Reservation subsurface. This meta-mobilome was compared with one derived from samples collected from unimpacted regions of the Oak Ridge Reservation subsurface. We assembled 1615 unique circularized DNA elements that we propose to be MGEs. The circular elements from the highly contaminated subsurface were enriched in HMRG clusters relative to those from the nearby unimpacted regions. Additionally, we found that these HMRGs were associated with Gamma and Betaproteobacteria hosts in the contaminated subsurface and potentially facilitate the persistence and dominance of these taxa in this region. Finally, the HMRGs were associated with conjugative elements, suggesting their potential for future lateral transfer. We demonstrate how our understanding of MGE ecology, evolution, and function can be enhanced through the genomic context provided by completed MGE assemblies.

Phage-plasmids: missed links between mobile genetic elements.

Phages and plasmids are discrete mobile genetic elements (MGEs) with critical roles in gene dissemination across bacteria but limited scope for exchanging DNA between them. By investigating recent gene-sharing events, Pfeifer and Rocha describe how the hybrid elements phage-plasmids (P-Ps) promote gene flow between MGE types and evolve into new ones.

Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans.

Plasmid-encoded type IV-A CRISPR-Cas systems lack an acquisition module, feature a DinG helicase instead of a nuclease, and form ribonucleoprotein complexes of unknown biological functions. Type IV-A3 systems are carried by conjugative plasmids that often harbor antibiotic-resistance genes and their CRISPR array contents suggest a role in mediating inter-plasmid conflicts, but this function remains unexplored. Here, we demonstrate that a plasmid-encoded type IV-A3 system co-opts the type I-E adaptation machinery from its host, Klebsiella pneumoniae (K. pneumoniae), to update its CRISPR array. Furthermore, we reveal that robust interference of conjugative plasmids and phages is elicited through CRISPR RNA-dependent transcriptional repression. By silencing plasmid core functions, type IV-A3 impacts the horizontal transfer and stability of targeted plasmids, supporting its role in plasmid competition. Our findings shed light on the mechanisms and ecological function of type IV-A3 systems and demonstrate their practical efficacy for countering antibiotic resistance in clinically relevant strains.

Genome Analysis of Epsilon CrAss-like Phages.

CrAss-like phages play an important role in maintaining ecological balance in the human intestinal microbiome. However, their genetic diversity and lifestyle are still insufficiently studied. In this study, a novel CrAssE-Sib phage genome belonging to the epsilon crAss-like phage genomes was found. Comparative analysis indicated that epsilon crAss-like phages are divided into two putative genera, which were proposed to be named Epsilonunovirus and Epsilonduovirus; CrAssE-Sib belongs to the former. The crAssE-Sib genome contains a diversity-generating retroelement (DGR) cassette with all essential elements, including the reverse transcriptase (RT) and receptor binding protein (RBP) genes. However, this RT contains the GxxxSP motif in its fourth domain instead of the usual GxxxSQ motif found in all known phage and bacterial DGRs. RBP encoded by CrAssE-Sib and other Epsilonunoviruses has an unusual structure, and no similar phage proteins were found. In addition, crAssE-Sib and other Epsilonunoviruses encode conserved prophage repressor and anti-repressors that could be involved in lysogenic-to-lytic cycle switches. Notably, DNA primase sequences of epsilon crAss-like phages are not included in the monophyletic group formed by the DNA primases of all other crAss-like phages. Therefore, epsilon crAss-like phage substantially differ from other crAss-like phages, indicating the need to classify these phages into a separate family.

Genomic analysis of a novel phage vB_SenS_ST1UNAM with lytic activity against Salmonella enterica serotypes.

In this study, we present the complete annotated genome of a novel Salmonella phage, vB_SenS_ST1UNAM. This phage exhibits lytic activity against several Salmonella enterica serotypes, such as S. Typhi, S. Enteritidis, and S. Typhimurium strains, which are major causes of foodborne illness worldwide. Its genome consists of a linear, double-stranded DNA of 47,877 bp with an average G+C content of 46.6%. A total of 85 coding regions (CDS) were predicted, of which only 43 CDS were functionally assigned. Neither genes involved in the regulation of lysogeny, nor antibiotic resistance genes were identified. This phage harbors a lytic cassette that encodes a type II-holin and a Rz/Rz1-like spanin complex, along with a restriction-modification evasion system and a depolymerase that degrades Salmonella exopolysaccharide. Moreover, the comparative analysis with closely related phage genomes revealed that vB_SenS_ST1UNAM represents a novel genus, for which the genus "Gomezvirus" within the subfamily "ST1UNAM-like" is proposed.

Genome sequences of the first Autographiviridae phages infecting marine Roseobacter.

The ubiquitous and abundant marine phages play critical roles in shaping the composition and function of bacterial communities, impacting biogeochemical cycling in marine ecosystems. Autographiviridae is among the most abundant and ubiquitous phage families in the ocean. However, studies on the diversity and ecology of Autographiviridae phages in marine environments are restricted to isolates that infect SAR11 bacteria and cyanobacteria. In this study, ten new roseophages that infect marine Roseobacter strains were isolated from coastal waters. These new roseophages have a genome size ranging from 38 917 to 42 634 bp and G+C content of 44.6-50 %. Comparative genomics showed that they are similar to known Autographiviridae phages regarding gene content and architecture, thus representing the first Autographiviridae roseophages. Phylogenomic analysis based on concatenated conserved genes showed that the ten roseophages form three distinct subgroups within the Autographiviridae, and sequence analysis revealed that they belong to eight new genera. Finally, viromic read-mapping showed that these new Autographiviridae phages are widely distributed in global oceans, mostly inhabiting polar and estuarine locations. This study has expanded the current understanding of the genomic diversity, evolution and ecology of Autographiviridae phages and roseophages. We suggest that Autographiviridae phages play important roles in the mortality and community structure of roseobacters, and have broad ecological applications.

Prophage maintenance is determined by environment-dependent selective sweeps rather than mutational availability.

Prophages, viral sequences integrated into bacterial genomes, can be beneficial and costly. Despite the risk of prophage activation and subsequent bacterial death, active prophages are present in most bacterial genomes. However, our understanding of the selective forces that maintain prophages in bacterial populations is limited. Combining experimental evolution with stochastic modeling, we show that prophage maintenance and loss are primarily determined by environmental conditions that alter the net fitness effect of a prophage on its bacterial host. When prophages are too costly, they are rapidly lost through environment-specific sequences of selective sweeps. Conflicting selection pressures that select against the prophage but for a prophage-encoded accessory gene can maintain prophages. The dynamics of prophage maintenance additionally depend on the sociality of this accessory gene. Prophage-encoded genes that exclusively benefit the lysogen maintain prophages at higher frequencies compared with genes that benefit the entire population. That is because the latter can protect phage-free "cheaters," reducing the benefit of maintaining the prophage. Our simulations suggest that environmental variation plays a larger role than mutation rates in determining prophage maintenance. These findings highlight the complexity of selection pressures that act on mobile genetic elements and challenge our understanding of the role of environmental factors relative to random chance events in shaping the evolutionary trajectory of bacterial populations. By shedding light on the key factors that shape microbial populations in the face of environmental changes, our study significantly advances our understanding of the complex dynamics of microbial evolution and diversification.

The role of rhizosphere phages in soil health.

While the One Health framework has emphasized the importance of soil microbiomes for plant and human health, one of the most diverse and abundant groups-bacterial viruses, i.e. phages-has been mostly neglected. This perspective reviews the significance of phages for plant health in rhizosphere and explores their ecological and evolutionary impacts on soil ecosystems. We first summarize our current understanding of the diversity and ecological roles of phages in soil microbiomes in terms of nutrient cycling, top-down density regulation, and pathogen suppression. We then consider how phages drive bacterial evolution in soils by promoting horizontal gene transfer, encoding auxiliary metabolic genes that increase host bacterial fitness, and selecting for phage-resistant mutants with altered ecology due to trade-offs with pathogen competitiveness and virulence. Finally, we consider challenges and avenues for phage research in soil ecosystems and how to elucidate the significance of phages for microbial ecology and evolution and soil ecosystem functioning in the future. We conclude that similar to bacteria, phages likely play important roles in connecting different One Health compartments, affecting microbiome diversity and functions in soils. From the applied perspective, phages could offer novel approaches to modulate and optimize microbial and microbe-plant interactions to enhance soil health.

Advances in Therapeutic Strategies for the Management of Clostridioides difficile Infection.

The infection caused by Clostridioides difficile represents one of the bacterial infections with the greatest increase in incidence among nosocomial infections in recent years. C. difficile is a Gram-positive bacterium able to produce toxins and spores. In some cases, infection results in severe diarrhoea and fulminant colitis, which cause prolonged hospitalisation and can be fatal, with repercussions also in terms of health economics. C. difficile is the most common cause of antibiotic-associated diarrhoea in the healthcare setting. The problem of bacterial forms that are increasingly resistant to common antibiotic treatments is also reflected in C. difficile infection (CDI). One of the causes of CDI is intestinal dysmicrobialism induced by prolonged antibiotic therapy. Moreover, in recent years, the emergence of increasingly virulent strains resistant to antibiotic treatment has made the picture even more complex. Evidence on preventive treatments to avoid recurrence is unclear. Current guidelines indicate the following antibiotics for the treatment of CDI: metronidazole, vancomycin, and fidaxomycin. This short narrative review provides an overview of CDI, antibiotic resistance, and emerging treatments.

Gut phageome: challenges in research and impact on human microbiota.

The human gut microbiome plays a critical role in maintaining our health. Fluctuations in the diversity and structure of the gut microbiota have been implicated in the pathogenesis of several metabolic and inflammatory conditions. Dietary patterns, medication, smoking, alcohol consumption, and physical activity can all influence the abundance of different types of microbiota in the gut, which in turn can affect the health of individuals. Intestinal phages are an essential component of the gut microbiome, but most studies predominantly focus on the structure and dynamics of gut bacteria while neglecting the role of phages in shaping the gut microbiome. As bacteria-killing viruses, the distribution of bacteriophages in the intestine, their role in influencing the intestinal microbiota, and their mechanisms of action remain elusive. Herein, we present an overview of the current knowledge of gut phages, their lifestyles, identification, and potential impact on the gut microbiota.