Prophage induction by non-antibiotic compounds promotes transformation of released antibiotic resistance genes from cell lysis.

Prophages are prevalent among bacterial species, including strains carrying antibiotic resistance genes (ARGs). Prophage induction can be triggered by the SOS response to stressors, leading to cell lysis. In environments polluted by chemical stressors, ARGs and prophage co-harboring strains might pose an unknown risk of spreading ARGs through chemical pollutant-mediated prophage induction and subsequent cell lysis. In this study, we investigated the effects of common non-antibiotic water pollutants, triclosan and silver nanoparticles, on triggering prophage induction in clinical isolates carrying ARGs and the subsequent uptake of released ARGs by the naturally competent bacterium Acinetobacter baylyi. Our results demonstrate that both triclosan and silver nanoparticles, at environmentally relevant concentrations and those found in commercial products, significantly enhance prophage induction among various clinical isolates. Transmission electron microscopy imaging and plaque assays confirmed the production of infectious phage particles under non-antibiotic pollutants-mediated prophage induction. In addition, the rate of ARG transformation to A. baylyi significantly increased after the release of extracellular ARGs from prophage induction-mediated cell lysis. The mechanism of non-antibiotic pollutants-mediated prophage induction is primarily associated with excessive oxidative stress, which provokes the SOS response. Our findings offer insights into the role of non-antibiotic pollutants in promoting the dissemination of ARGs by triggering prophage induction.

Biologic and genomic characterization of a novel virulent Aeromonas hydrophila phage phiA051, with high homology to prophages.

Introduction: Aeromonas hydrophila is particularly harmful to freshwater aquaculture, and the search for phage is an effective biological control method, but reports of possible temperate phages and their mutants are rare in this field. In this study, a virulent phage highly homologous to prophage in the genomes of A. hydrophila was collected and preliminary biological characterization was carried out to understand its nature. Materials and methods: Water samples taken from eel ponds in Fujian, China were combined with the strain. Spot test method and double-layer agar plate assay was used for confirmation and purification. Phage virions were observed using transmission electron microscope. A total of 68 strains of Aeromonas spp. were used to determine the host range. MOI groups of 1,000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001 were prepared to detect the optimal MOI. The conditions of thermal stability assay were set as 30, 40, 50, 60, 70 and 80°C for 1 h, respectively, and conditions of acid and alkali stability assay were set as 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0 of pH. MOI of 0.01 and 0.1, respectively, are set to determine the inhibitory capacity of phage. Results: A novel virulent A. hydrophila phage designated phiA051 has been isolated from aquaculture water. Electron microscopic observation showed that the phage phiA051 was composed of an icosahedral capsid. The phage phiA051 possesses an optimal multiplicity of infection (MOI) of 0.01, and its burst size was 108 PFU/cell. The phage maintained a high viability at temperatures of 30-50°C or pH 6.0-10.0 for 1 h. Phage phiA051 has certain potentials in rapidly inhibiting the spread of pathogen early in the outbreak, and it has a linear dsDNA with GC content of 60.55% and a total length of 32,212 bp, including 46 ORFs. Discussion: The phage phiA051 behaved as a virulent phage. However, the BLASTN result showed that 23 of the top 25 hits were genomes of Aeromonas strains. It was suggested that phiA051 was probably derived from some prophage in the chromosome of Aeromonas. Further investigation of the mechanism how phage phiA051 transforms from a temperate phage to a virulent phage will provide a unique perspective and idea to explore the potential of prophages.

Gene content, phage cycle regulation model and prophage inactivation disclosed by prophage genomics in the Helicobacter pylori Genome Project.

Prophages can have major clinical implications through their ability to change pathogenic bacterial traits. There is limited understanding of the prophage role in ecological, evolutionary, adaptive processes and pathogenicity of Helicobacter pylori, a widespread bacterium causally associated with gastric cancer. Inferring the exact prophage genomic location and completeness requires complete genomes. The international Helicobacter pylori Genome Project (HpGP) dataset comprises 1011 H. pylori complete clinical genomes enriched with epigenetic data. We thoroughly evaluated the H. pylori prophage genomic content in the HpGP dataset. We investigated population evolutionary dynamics through phylogenetic and pangenome analyses. Additionally, we identified genome rearrangements and assessed the impact of prophage presence on bacterial gene disruption and methylome. We found that 29.5% (298) of the HpGP genomes contain prophages, of which only 32.2% (96) were complete, minimizing the burden of prophage carriage. The prevalence of H. pylori prophage sequences was variable by geography and ancestry, but not by disease status of the human host. Prophage insertion occasionally results in gene disruption that can change the global bacterial epigenome. Gene function prediction allowed the development of the first model for lysogenic-lytic cycle regulation in H. pylori. We have disclosed new prophage inactivation mechanisms that appear to occur by genome rearrangement, merger with other mobile elements, and pseudogene accumulation. Our analysis provides a comprehensive framework for H. pylori prophage biological and genomics, offering insights into lysogeny regulation and bacterial adaptation to prophages.

Genomic analysis of "Candidatus Liberibacter africanus" strain from Zimbabwe reveals unique virulence and prophage characteristics compared to "Ca. Liberibacter asiaticus".

Citrus Huanglongbing (HLB) is caused by the phloem-limited α-proteobacterium "Candidatus Liberibacter spp.", among which "Ca. Liberibacter africanus" (CLaf) have posed a significant threat to citrus production in Africa near a century. CLaf is closely related to the globally prevalent "Ca. Liberibacter asiaticus" (CLas), whereas little is known about the virulence of CLaf, primarily due to limited genome resources. In this study, we completed the whole-genome assembly and annotation of CLaf strain Zim (from Zimbabwe). Compared to CLas, a total of 102 CLaf unique genes were identified, including 14 potential Sec-dependent effectors (SDEs) genes, 29 phage-associated genes, and 59 genes with hypothetical function. Among 14 SDEs, V9J15_03810 was able to induce a significant hypersensitive response (HR) in Nicotiana benthamiana, indicating its potential as a virulence factor for CLaf. Genome analysis showed that CLaf strain Zim genome harbored a complete prophage region (named P-Zim-1, 42,208 bp). P-Zim-1 retained two immunosuppressive peroxidase genes (V9J15_02125 and V9J15_02130) homologous to CLas prophage SC1/SC2, whereas the lysogen-associated genes encoding integrase (V9J15_01970) and repressor (V9J15_02080) were homologous to the prophage of "Ca. Liberibacter solanacearum", the causal agent of potato zebra chip disease. In addition, P-Zim-1 carried a novel CRISPR/Cas system, including a CRISPR array (located within V9J15_02040, ranging from 443,643 to 443,897) and five CRISPR-related Cas proteins (V9J15_02005, 02010, 02015, 02025 and 02035). This study first characterized the unique genomic feature of CLaf related to virulence and prophage, which will facilitate future research on CLaf biology and African HLB management.

Distinct prophage gene profiles of Staphylococcus aureus strains from atopic dermatitis patients and healthy individuals.

Staphylococcus aureus strains exhibit varying associations with atopic dermatitis (AD), but the genetic determinants underpinning the pathogenicity are yet to be fully characterized. To reveal the genetic differences between S. aureus strains from AD patients and healthy individuals (HE), we developed and employed a random forest classifier to identify potential marker genes responsible for their phenotypic variations. The classifier was able to effectively distinguish strains from AD and HE. We also uncovered strong links between certain marker genes and phage functionalities, with phage holin emerging as the most pivotal differentiating factor. Further examination of S. aureus gene content highlighted the genetic diversity and functional implications of prophages in driving differentiation between strains from AD and HE. The HE group exhibited greater gene content diversity, largely influenced by their prophages. While strains from both AD and HE universally housed prophages, those in the HE group were distinctively higher at the strain level. Moreover, although prophages in the HE group exhibited variously higher enrichment of differential functions, the AD group displayed a notable enrichment of virulence factors within their prophages, underscoring the important contribution of prophages to the pathogenesis of AD-associated strains. Overall, prophages significantly shape the genetic and functional profiles of S. aureus strains, shedding light on their pathogenic potential and elucidating the mechanisms behind the phenotypic variations in AD and HE environments. IMPORTANCE: Through a nuanced exploration of Staphylococcus aureus strains obtained from atopic dermatitis (AD) patients and healthy controls (HE), our research unveils pivotal genetic determinants influencing their pathogenic associations. Utilizing a random forest classifier, we illuminate distinct marker genes, with phage holin emerging as a critical differential factor, revealing the profound impact of prophages on genetic and pathogenic profiles. HE strains exhibited a diverse gene content, notably shaped by unique, heightened prophages. Conversely, AD strains emphasized a pronounced enrichment of virulence factors within prophages, signifying their key role in AD pathogenesis. This work crucially highlights prophages as central architects of the genetic and functional attributes of S. aureus strains, providing vital insights into pathogenic mechanisms and phenotypic variations, thereby paving the way for targeted AD therapeutic approaches and management strategies by demystifying specific genetic and pathogenic mechanisms.

Analysis of the cell wall binding domain in bacteriocin-like lysin LysL from Lactococcus lactis LAC460.

Wild-type Lactococcus lactis strain LAC460 secretes prophage-encoded bacteriocin-like lysin LysL, which kills some Lactococcus strains, but has no lytic effect on the producer. LysL carries two N-terminal enzymatic active domains (EAD), and an unknown C-terminus without homology to known domains. This study aimed to determine whether the C-terminus of LysL carries a cell wall binding domain (CBD) for target specificity of LysL. The C-terminal putative CBD region of LysL was fused with His-tagged green fluorescent protein (HGFPuv). The HGFPuv_CBDlysL gene fusion was ligated into the pASG-IBA4 vector, and introduced into Escherichia coli. The fusion protein was produced and purified with affinity chromatography. To analyse the binding of HGFPuv_CBDLysL to Lactococcus cells, the protein was mixed with LysL-sensitive and LysL-resistant strains, including the LysL-producer LAC460, and the fluorescence of the cells was analysed. As seen in fluorescence microscope, HGFPuv_CBDLysL decorated the cell surface of LysL-sensitive L. cremoris MG1614 with green fluorescence, whereas the resistant L. lactis strains LM0230 and LAC460 remained unfluorescent. The fluorescence plate reader confirmed the microscopy results detecting fluorescence only from four tested LysL-sensitive strains but not from 11 tested LysL-resistant strains. Specific binding of HGFPuv_CBDLysL onto the LysL-sensitive cells but not onto the LysL-resistant strains indicates that the C-terminus of LysL contains specific CBD. In conclusion, this report presents experimental evidence of the presence of a CBD in a lactococcal phage lysin. Moreover, the inability of HGFPuv_CBDLysL to bind to the LysL producer LAC460 may partly explain the host's resistance to its own prophage lysin.

Isolation and characterization of novel acetogenic strains of the genera Terrisporobacter and Acetoanaerobium.

Due to their metabolic versatility in substrate utilization, acetogenic bacteria represent industrially significant production platforms for biotechnological applications such as syngas fermentation, microbial electrosynthesis or transformation of one-carbon substrates. However, acetogenic strains from the genera Terrisporobacter and Acetoanaerobium remained poorly investigated for biotechnological applications. We report the isolation and characterization of four acetogenic Terrisporobacter strains and one Acetoanaerobium strain. All Terrisporobacter isolates showed a characteristic growth pattern under a H2 + CO2 atmosphere. An initial heterotrophic growth phase was followed by a stationary growth phase, where continuous acetate production was indicative of H2-dependent acetogenesis. One of the novel Terrisporobacter isolates obtained from compost (strain COMT) additionally produced ethanol besides acetate in the stationary growth phase in H2-supplemented cultures. Genomic and physiological characterizations showed that strain COMT represented a novel Terrisporobacter species and the name Terrisporobacter vanillatitrophus is proposed (=DSM 116160T = CCOS 2104T). Phylogenomic analysis of the novel isolates and reference strains implied the reclassification of the T. petrolearius/T. hibernicus phylogenomic cluster to the species T. petrolearius and of the A. noterae/A. sticklandii phylogenomic cluster to the species A. sticklandii. Furthermore, we provide first insights into active prophages of acetogens from the genera Terrisporobacter and Acetoanaerobium.

Simple Genomic Traits Predict Rates of Polysaccharide Biodegradation.

Natural and chemically modified polysaccharides are extensively employed across a wide array of industries, leading to their prevalence in the waste streams of industrialized societies. With projected increasing demand, a pressing challenge is to swiftly assess and predict their biodegradability to inform the development of new sustainable materials. In this study, we developed a scalable method to evaluate polysaccharide breakdown by measuring microbial growth and analyzing microbial genomes. Our approach, applied to polysaccharides with various structures, correlates strongly with well-established regulatory methods based on oxygen demand. We show that modifications to the polysaccharide structure decreased degradability and favored the growth of microbes adapted to break down chemically modified sugars. More broadly, we discovered two main types of microbial communities associated with different polysaccharide structures─one dominated by fast-growing microbes and another by specialized degraders. Surprisingly, we were able to predict biodegradation rates based only on two genomic features that define these communities: the abundance of genes related to rRNA (indicating fast growth) and the abundance of glycoside hydrolases (enzymes that break down polysaccharides), which together predict nearly 70% of the variation in polysaccharide breakdown. This suggests a trade-off, whereby microbes are either adapted for fast growth or for degrading complex polysaccharide chains, but not both. Finally, we observe that viral elements (prophages) encoded in the genomes of degrading microbes are induced in easily degradable polysaccharides, leading to complex dynamics in biomass accumulation during degradation. In summary, our work provides a practical approach for efficiently assessing polymer degradability and offers genomic insights into how microbes break down polysaccharides.

Removal of mobile genetic elements from the genome of Clostridioides difficile and the implications for the organism's biology.

Clostridioides difficile is an emerging pathogen of One Health significance. Its highly variable genome contains mobile genetic elements (MGEs) such as transposons and prophages that influence its biology. Systematic deletion of each genetic element is required to determine their precise role in C. difficile biology and contribution to the wider mobilome. Here, Tn5397 (21 kb) and ϕ027 (56 kb) were deleted from C. difficile 630 and R20291, respectively, using allele replacement facilitated by CRISPR-Cas9. The 630 Tn5397 deletant transferred PaLoc at the same frequency (1 × 10-7) as 630 harboring Tn5397, indicating that Tn5397 alone did not mediate conjugative transfer of PaLoc. The R20291 ϕ027 deletant was sensitive to ϕ027 infection, and contained two unexpected features, a 2.7 kb remnant of the mutagenesis plasmid, and a putative catalase gene adjacent to the deleted prophage was also deleted. Growth kinetics of R20291 ϕ027 deletant was similar to wild type (WT) in rich medium but marginally reduced compared with WT in minimal medium. This work indicates the commonly used pMTL8000 plasmid series works well for CRISPR-Cas9-mediated gene deletion, resulting in the largest deleted locus (56.8 kb) described in C. difficile. Removal of MGEs was achieved by targeting conjugative/integrative regions to promote excision and permanent loss. The deletants created will be useful strains for investigating Tn5397 or ϕ027 prophage contribution to host virulence, fitness, and physiology, and a platform for other mutagenesis studies aimed at functional gene analysis without native transposon or phage interference in C. difficile 630 and R20291.

Identification and Characterization of a Novel Prophage Lysin against Streptococcus dysgalactiae.

Streptococcus dysgalactiae infection can cause bovine mastitis and lead to huge economic losses for the dairy industry. The abuse of antibiotics has resulted in growing drug resistance of S. dysgalactiae, which causes hard-to-treat infections. Bacteriophage lysin, as a novel antibacterial agent, has great potential for application against drug-resistant gram-positive bacteria. However, few studies have been conducted on the prophage lysin of S. dysgalactiae. In this study, we mined a novel prophage lysin, named Lys1644, from a clinical S. dysgalactiae isolate by genome sequencing and bioinformatic analysis. Lys1644 was expressed and purified, and the lytic activity, antibacterial spectrum, optimal pH and temperature, lytic activity in milk in vitro, and synergistic bacteriostasis with antibiotics were assessed. The Lys1644 prophage lysin showed high bacteriolysis activity specifically on S. dysgalactiae, which resulted in CFU 100-fold reduction in milk. Moreover, Lys1644 maintained high activity over a wide pH range (pH 5-10) and a wide temperature range (4-42 °C). Synergistic bacteriostatic experiments showed that the combination of low-dose Lys1644 (50 µg/mL) with a subinhibitory concentration of aminoglycoside antibiotics (kanamycin or spectinomycin) can completely inhibit bacterial growth, suggesting that the combination of Lys1644 and antibiotics could be an effective therapeutic strategy against S. dysgalactiae infection.

Interspecies ecological competition rejuvenates decayed Geobacter electroactive biofilm.

Bioelectrochemical systems (BESs) exploit electroactive biofilms (EABs) for promising applications in biosensing, wastewater treatment, energy production, and chemical biosynthesis. However, during the operation of BESs, EABs inevitably decay. Seeking approaches to rejuvenate decayed EABs is critical for the sustainability and practical application of BESs. Prophage induction has been recognized as the primary reason for EAB decay. Herein, we report that introducing a competitive species of Geobacter uraniireducens suspended prophage induction in Geobacter sulfurreducens and thereby rejuvenated the decayed G. sulfurreducens EAB. The transcriptomic profile of G. sulfurreducens demonstrated that the addition of G. uraniireducens significantly affected the expression of metabolism- and stress response system-related genes and in particular suppressed the induction of phage-related genes. Mechanistic analyses revealed that interspecies ecological competition exerted by G. uraniireducens suppressed prophage induction. Our findings not only reveal a novel strategy to rejuvenate decayed EABs, which is significant for the sustainability of BESs, but also provide new knowledge for understanding phage-host interactions from an ecological perspective, with implications for developing therapies to defend against phage attack.

Discovery of Antimicrobial Lysins from the "Dark Matter" of Uncharacterized Phages Using Artificial Intelligence.

The rapid rise of antibiotic resistance and slow discovery of new antibiotics have threatened global health. While novel phage lysins have emerged as potential antibacterial agents, experimental screening methods for novel lysins pose significant challenges due to the enormous workload. Here, the first unified software package, namely DeepLysin, is developed to employ artificial intelligence for mining the vast genome reservoirs ("dark matter") for novel antibacterial phage lysins. Putative lysins are computationally screened from uncharacterized Staphylococcus aureus phages and 17 novel lysins are randomly selected for experimental validation. Seven candidates exhibit excellent in vitro antibacterial activity, with LLysSA9 exceeding that of the best-in-class alternative. The efficacy of LLysSA9 is further demonstrated in mouse bloodstream and wound infection models. Therefore, this study demonstrates the potential of integrating computational and experimental approaches to expedite the discovery of new antibacterial proteins for combating increasing antimicrobial resistance.

Unlocking the genomic potential of Red Sea coral probiotics.

The application of beneficial microorganisms for corals (BMC) decreases the bleaching susceptibility and mortality rate of corals. BMC selection is typically performed via molecular and biochemical assays, followed by genomic screening for BMC traits. Herein, we present a comprehensive in silico framework to explore a set of six putative BMC strains. We extracted high-quality DNA from coral samples collected from the Red Sea and performed PacBio sequencing. We identified BMC traits and mechanisms associated with each strain as well as proposed new traits and mechanisms, such as chemotaxis and the presence of phages and bioactive secondary metabolites. The presence of prophages in two of the six studied BMC strains suggests their possible distribution within beneficial bacteria. We also detected various secondary metabolites, such as terpenes, ectoines, lanthipeptides, and lasso peptides. These metabolites possess antimicrobial, antifungal, antiviral, anti-inflammatory, and antioxidant activities and play key roles in coral health by reducing the effects of heat stress, high salinity, reactive oxygen species, and radiation. Corals are currently facing unprecedented challenges, and our revised framework can help select more efficient BMC for use in studies on coral microbiome rehabilitation, coral resilience, and coral restoration.

Uncovering Endolysins against Methicillin-Resistant Staphylococcus aureus Using a Microbial Single-Cell Genome Database.

Endolysins, peptidoglycan hydrolases derived from bacteriophages (phages), are being developed as a promising alternative to conventional antibiotics. To obtain highly active endolysins, a diverse library of these endolysins is vital. We propose here microbial single-cell genome sequencing as an efficient tool to discover dozens of previously unknown endolysins, owing to its culture-independent sequencing method. As a proof of concept, we analyzed and recovered endolysin genes within prophage regions of Staphylococcus single-amplified genomes in human skin microbiome samples. We constructed a library of chimeric endolysins by shuffling domains of the natural endolysins and performed high-throughput screening against Staphylococcus aureus. One of the lead endolysins, bbst1027, exhibited desirable antimicrobial properties, such as rapid bactericidal activity, no detectable resistance development, and in vivo efficacy. We foresee that this endolysin discovery pipeline is in principle applicable to any bacterial target and boost the development of novel antimicrobial agents.

Identifying components of the Shewanella phage LambdaSo lysis system.

Phage-induced lysis of Gram-negative bacterial hosts usually requires a set of phage lysis proteins, a holin, an endopeptidase, and a spanin system, to disrupt each of the three cell envelope layers. Genome annotations and previous studies identified a gene region in the Shewanella oneidensis prophage LambdaSo, which comprises potential holin- and endolysin-encoding genes but lacks an obvious spanin system. By a combination of candidate approaches, mutant screening, characterization, and microscopy, we found that LambdaSo uses a pinholin/signal-anchor-release (SAR) endolysin system to induce proton leakage and degradation of the cell wall. Between the corresponding genes, we found that two extensively nested open-reading frames encode a two-component spanin module Rz/Rz1. Unexpectedly, we identified another factor strictly required for LambdaSo-induced cell lysis, the phage protein Lcc6. Lcc6 is a transmembrane protein of 65 amino acid residues with hitherto unknown function, which acts at the level of holin in the cytoplasmic membrane to allow endolysin release. Thus, LambdaSo-mediated cell lysis requires at least four protein factors (pinholin, SAR endolysin, spanin, and Lcc6). The findings further extend the known repertoire of phage proteins involved in host lysis and phage egress. IMPORTANCE: Lysis of bacteria can have multiple consequences, such as the release of host DNA to foster robust biofilm. Phage-induced lysis of Gram-negative cells requires the disruption of three layers, the outer and inner membranes and the cell wall. In most cases, the lysis systems of phages infecting Gram-negative cells comprise holins to disrupt or depolarize the membrane, thereby releasing or activating endolysins, which then degrade the cell wall. This, in turn, allows the spanins to become active and fuse outer and inner membranes, completing cell envelope disruption and allowing phage egress. Here, we show that the presence of these three components may not be sufficient to allow cell lysis, implicating that also in known phages, further factors may be required.

What's on a prophage: analysis of Salmonella spp. prophages identifies a diverse range of cargo with multiple virulence- and metabolism-associated functions.

The gain of mobile elements, such as prophages, can introduce cargo to the recipient bacterium that could facilitate its persistence in or expansion to a new environment, such as a host. While previous studies have focused on identifying and characterizing the genetic diversity of prophages, analyses characterizing the cargo that prophages carry have not been extensively explored. We characterized prophage regions from 303 Salmonella spp. genomes (representing 254 unique serovars) to assess the distribution of prophages in diverse Salmonella. On average, prophages accounted for 3.7% (0.1%-8.8%) of the total genomic content of each isolate. Prophage regions annotated as Gifsy 1 and Salmon Fels 1 were the most commonly identified intact prophages, suggesting that they are common throughout the Salmonella genus. Among 21,687 total coding sequences (CDSs) from intact prophage regions in subsp. enterica genomes, 7.5% (median; range: 1.1%-47.6%) were categorized as having a function not related to prophage integration or phage structure, some of which could potentially provide a functional attribute to the host Salmonella cell. These predicted functions could be broadly categorized into CDSs involved in: (i) modification of cell surface structures (i.e., glycosyltransferases); (ii) modulation of host responses (e.g., SodC/SodA, SopE, ArtAB, and typhoid toxin); (iii) conferring resistance to heavy metals and antimicrobials; (iv) metabolism of carbohydrates, amino acids, and nucleotides; and (v) DNA replication, repair, and regulation. Overall, our systematic analysis of prophage cargo highlights a broader role for prophage cargo in influencing the metabolic, virulence, and resistance characteristics of Salmonella. IMPORTANCE: Lysogenic bacteriophages (phages) can integrate their genome into a bacterial host's genome, potentially introducing genetic elements that can affect the fitness of the host bacterium. The functions of prophage-encoded genes are important to understand as these genes could be mobilized and transferred to a new host. Using a large genomic dataset representing >300 isolates from all known subspecies and species of Salmonella, our study contributes important new findings on the distribution of prophages and the types of cargo that diverse Salmonella prophages carry. We identified a number of coding sequences (CDSs) annotated as having cell surface-modifying attributes, suggesting that prophages may have played an important role in shaping Salmonella's diverse surface antigen repertoire. Furthermore, our characterization of prophages suggests that they play a broader role in facilitating the acquisition and transfer of CDSs associated with metabolism, DNA replication and repair, virulence factors, and to a lesser extent, antimicrobial resistance.

Genetic recombination-mediated evolutionary interactions between phages of potential industrial importance and prophages of their hosts within or across the domains of Escherichia, Listeria, Salmonella, Campylobacter, and Staphylococcus.

BACKGROUND: The in-depth understanding of the role of lateral genetic transfer (LGT) in phage-prophage interactions is essential to rationalizing phage applications for human and animal therapy, as well as for food and environmental safety. This in silico study aimed to detect LGT between phages of potential industrial importance and their hosts. METHODS: A large array of genetic recombination detection algorithms, implemented in SplitsTree and RDP4, was applied to detect LGT between various Escherichia, Listeria, Salmonella, Campylobacter, Staphylococcus, Pseudomonas, and Vibrio phages and their hosts. PHASTER and RAST were employed respectively to identify prophages across the host genome and to annotate LGT-affected genes with unknown functions. PhageAI was used to gain deeper insights into the life cycle history of recombined phages. RESULTS: The split decomposition inferences (bootstrap values: 91.3-100; fit: 91.433-100), coupled with the Phi (0.0-2.836E-12) and RDP4 (P being well below 0.05) statistics, provided strong evidence for LGT between certain Escherichia, Listeria, Salmonella, and Campylobacter virulent phages and prophages of their hosts. The LGT events entailed mainly the phage genes encoding for hypothetical proteins, while some of these genetic loci appeared to have been affected even by intergeneric recombination in specific E. coli and S. enterica virulent phages when interacting with their host prophages. Moreover, it is shown that certain L. monocytogenes virulent phages could serve at least as the donors of the gene loci, involved in encoding for the basal promoter specificity factor, for L. monocytogenes. In contrast, the large genetic clusters were determined to have been simultaneously exchanged by many S. aureus prophages and some Staphylococcus temperate phages proposed earlier as potential therapeutic candidates (in their native or modified state). The above genetic clusters were found to encompass multiple genes encoding for various proteins, such as e.g., phage tail proteins, the capsid and scaffold proteins, holins, and transcriptional terminator proteins. CONCLUSIONS: It is suggested that phage-prophage interactions, mediated by LGT (including intergeneric recombination), can have a far-reaching impact on the co-evolutionary trajectories of industrial phages and their hosts especially when excessively present across microbially rich environments.

Comparative Analyses of Bacteriophage Genomes.

Bacterial viruses (bacteriophages or phages) are the most abundant and diverse biological entities on Earth. There is a renewed worldwide interest in phage-centered research motivated by their enormous potential as antimicrobials to cope with multidrug-resistant pathogens. An ever-growing number of complete phage genomes are becoming available, derived either from newly isolated phages (cultivated phages) or recovered from metagenomic sequencing data (uncultivated phages). Robust comparative analysis is crucial for a comprehensive understanding of genotypic variations of phages and their related evolutionary processes, and to investigate the interaction mechanisms between phages and their hosts. In this chapter, we present a protocol for phage comparative genomics employing tools selected out of the many currently available, focusing on complete genomes of phages classified in the class Caudoviricetes. This protocol provides accurate identification of similarities, differences, and patterns among new and previously known complete phage genomes as well as phage clustering and taxonomic classification.

Exploring the diversity and evolutionary strategies of prophages in Hyphomicrobiales, comparing animal-associated with non-animal-associated bacteria.

The Hyphomicrobiales bacterial order (previously Rhizobiales) exhibits a wide range of lifestyle characteristics, including free-living, plant-association, nitrogen-fixing, and association with animals (Bartonella and Brucella). This study explores the diversity and evolutionary strategies of bacteriophages within the Hyphomicrobiales order, comparing animal-associated (AAB) with non-animal-associated bacteria (NAAB). We curated 560 high-quality complete genomes of 58 genera from this order and used the PHASTER server for prophage annotation and classification. For 19 genera with representative genomes, we curated 96 genomes and used the Defense-Finder server to summarize the type of anti-phage systems (APS) found in this order. We analyzed the genetic repertoire and length distributions of prophages, estimating evolutionary rates and comparing intact, questionable, and incomplete prophages in both groups. Analyses of best-fit parameters and bootstrap sensitivity were used to understand the evolutionary processes driving prophage gene content. A total of 1860 prophages distributed in Hyphomicrobiales were found, 695 in AAB and 1165 in the NAAB genera. The results revealed a similar number of prophages per genome in AAB and NAAB and a similar length distribution, suggesting shared mechanisms of genetic acquisition of prophage genes. Changes in the frequency of specific gene classes were observed between incomplete and intact prophages, indicating preferential loss or enrichment in both groups. The analysis of best-fit parameters and bootstrap sensitivity tests indicated a higher selection coefficient, induction rate, and turnover in NAAB genomes. We found 68 types of APS in Hyphomicrobiales; restriction modification (RM) and abortive infection (Abi) were the most frequent APS found for all Hyphomicrobiales, and within the AAB group. This classification of APS showed that NAAB genomes have a greater diversity of defense systems compared to AAB, which could be related to the higher rates of prophage induction and turnover in the latter group. Our study provides insights into the distributions of both prophages and APS in Hyphomicrobiales genomes, demonstrating that NAAB carry more defense systems against phages, while AAB show increased prophage stability and an increased number of incomplete prophages. These results suggest a greater role for domesticated prophages within animal-associated bacteria in Hyphomicrobiales.

Integrative and Conjugative Elements and Prophage DNA as Carriers of Resistance Genes in Erysipelothrix rhusiopathiae Strains from Domestic Geese in Poland.

Goose erysipelas is a serious problem in waterfowl breeding in Poland. However, knowledge of the characteristics of Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, the antimicrobial susceptibility and serotypes of four E. rhusiopathiae strains from domestic geese were determined, and their whole-genome sequences (WGSs) were analyzed to detect resistance genes, integrative and conjugative elements (ICEs), and prophage DNA. Sequence type and the presence of resistance genes and transposons were compared with 363 publicly available E. rhusiopathiae strains, as well as 13 strains of other Erysipelothrix species. Four strains tested represented serotypes 2 and 5 and the MLST groups ST 4, 32, 242, and 243. Their assembled circular genomes ranged from 1.8 to 1.9 kb with a GC content of 36-37%; a small plasmid was detected in strain 1023. Strains 1023 and 267 were multidrug-resistant. The resistance genes detected in the genome of strain 1023 were erm47, tetM, and lsaE-lnuB-ant(6)-Ia-spw cluster, while strain 267 contained the tetM and ermB genes. Mutations in the gyrA gene were detected in both strains. The tetM gene was embedded in a Tn916-like transposon, which in strain 1023, together with the other resistance genes, was located on a large integrative and conjugative-like element of 130 kb designated as ICEEr1023. A minor integrative element of 74 kb was identified in strain 1012 (ICEEr1012). This work contributes to knowledge about the characteristics of E. rhusiopathiae bacteria and, for the first time, reveals the occurrence of erm47 and ermB resistance genes in strains of this species. Phage infection appears to be responsible for the introduction of the ermB gene into the genome of strain 267, while ICEs most likely play a key role in the spread of the other resistance genes identified in E. rhusiopathiae.