PhageLeads: Rapid Assessment of Phage Therapeutic Suitability Using an Ensemble Machine Learning Approach.

The characterization of therapeutic phage genomes plays a crucial role in the success rate of phage therapies. There are three checkpoints that need to be examined for the selection of phage candidates, namely, the presence of temperate markers, antimicrobial resistance (AMR) genes, and virulence genes. However, currently, no single-step tools are available for this purpose. Hence, we have developed a tool capable of checking all three conditions required for the selection of suitable therapeutic phage candidates. This tool consists of an ensemble of machine-learning-based predictors for determining the presence of temperate markers (integrase, Cro/CI repressor, immunity repressor, DNA partitioning protein A, and antirepressor) along with the integration of the ABRicate tool to determine the presence of antibiotic resistance genes and virulence genes. Using the biological features of the temperate markers, we were able to predict the presence of the temperate markers with high MCC scores (>0.70), corresponding to the lifestyle of the phages with an accuracy of 96.5%. Additionally, the screening of 183 lytic phage genomes revealed that six phages were found to contain AMR or virulence genes, showing that not all lytic phages are suitable to be used for therapy. The suite of predictors, PhageLeads, along with the integrated ABRicate tool, can be accessed online for in silico selection of suitable therapeutic phage candidates from single genome or metagenomic contigs.

Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types.

To provide protection against viral infection and limit the uptake of mobile genetic elements, bacteria and archaea have evolved many diverse defence systems. The discovery and application of CRISPR-Cas adaptive immune systems has spurred recent interest in the identification and classification of new types of defence systems. Many new defence systems have recently been reported but there is a lack of accessible tools available to identify homologs of these systems in different genomes. Here, we report the Prokaryotic Antiviral Defence LOCator (PADLOC), a flexible and scalable open-source tool for defence system identification. With PADLOC, defence system genes are identified using HMM-based homologue searches, followed by validation of system completeness using gene presence/absence and synteny criteria specified by customisable system classifications. We show that PADLOC identifies defence systems with high accuracy and sensitivity. Our modular approach to organising the HMMs and system classifications allows additional defence systems to be easily integrated into the PADLOC database. To demonstrate application of PADLOC to biological questions, we used PADLOC to identify six new subtypes of known defence systems and a putative novel defence system comprised of a helicase, methylase and ATPase. PADLOC is available as a standalone package (https://github.com/padlocbio/padloc) and as a webserver (https://padloc.otago.ac.nz).

Modeling Phage-Bacteria Dynamics.

Phage-bacteria interaction is a classic example of competitive coevolution in nature. Mathematical modeling of such interactions furnishes new insight into the dynamics of phage and bacteria. Besides its intrinsic value, a somewhat underutilized aspect of such insight is that it can provide beneficial inputs toward better experimental design. In this chapter, we discuss several modeling techniques that can be used to study the dynamics between phages and their host bacteria. Monte Carlo simulations and differential equations (both ordinary and delay differential equations) can be used to successfully model phage-bacteria dynamics in well-mixed populations. The presence of spatial restrictions in the interaction media significantly affects the dynamics of phage-bacteria interactions. For such cases, techniques like cellular automata and reaction-diffusion equations can be used to capture these effects adequately. We discuss details of the modeling techniques with specific examples.

Visualization and prediction of CRISPR incidence in microbial trait-space to identify drivers of antiviral immune strategy.

Bacteria and archaea are locked in a near-constant battle with their viral pathogens. Despite previous mechanistic characterization of numerous prokaryotic defense strategies, the underlying ecological drivers of different strategies remain largely unknown and predicting which species will take which strategies remains a challenge. Here, we focus on the CRISPR immune strategy and develop a phylogenetically-corrected machine learning approach to build a predictive model of CRISPR incidence using data on over 100 traits across over 2600 species. We discover a strong but hitherto-unknown negative interaction between CRISPR and aerobicity, which we hypothesize may result from interference between CRISPR-associated proteins and non-homologous end-joining DNA repair due to oxidative stress. Our predictive model also quantitatively confirms previous observations of an association between CRISPR and temperature. Finally, we contrast the environmental associations of different CRISPR system types (I, II, III) and restriction modification systems, all of which act as intracellular immune systems.

Filamentous phages: masters of a microbial sharing economy.

Bacteriophage ("bacteria eaters") or phage is the collective term for viruses that infect bacteria. While most phages are pathogens that kill their bacterial hosts, the filamentous phages of the sub-class Inoviridae live in cooperative relationships with their bacterial hosts, akin to the principal behaviours found in the modern-day sharing economy: peer-to-peer support, to offset any burden. Filamentous phages impose very little burden on bacteria and offset this by providing service to help build better biofilms, or provision of toxins and other factors that increase virulence, or modified behaviours that provide novel motile activity to their bacterial hosts. Past, present and future biotechnology applications have been built on this phage-host cooperativity, including DNA sequencing technology, tools for genetic engineering and molecular analysis of gene expression and protein production, and phage-display technologies for screening protein-ligand and protein-protein interactions. With the explosion of genome and metagenome sequencing surveys around the world, we are coming to realize that our knowledge of filamentous phage diversity remains at a tip-of-the-iceberg stage, promising that new biology and biotechnology are soon to come.

Bacteriophage production processes.

High quantities of bacteriophages are currently used in the food industry and agriculture. However, growing antibiotic resistance of bacteria has recently awakened the interest to use bacteriophages for the treatment of bacterial infections in humans indicating that even higher quantities will be required in the future. High demand combined with a wide range of applications requires also efficient bacteriophage production processes operating at low production costs and with high productivity. To achieve this goal, different approaches were introduced and extensive studies of various parameters affecting bacteriophage formation were investigated. In this mini-review, we provide a short overview about different operation modes of bacteriophage production such as batch, semi-continuous and especially continuous with the pros and cons of each. We present factors affecting bacterial physiological state, its effect on phage formation and provide a description of methods for determination of bacteriophage growth parameters, through which bacteriophage formation is obtained. Understanding of described phenomena and inclusion of potential occurrence of mutations and selection in continuous systems enables evaluation of continuous process productivity and its optimization.

Responses of intestinal virome to silver nanoparticles: safety assessment by classical virology, whole-genome sequencing and bioinformatics approaches.

BACKGROUND: Effects of silver nanoparticles (AgNP) on the intestinal virome/phage community are mostly unknown. The working hypothesis of this study was that the exposure of pharmaceutical/nanomedicine and other consumer-use material containing silver ions and nanoparticles to the gastrointestinal tract may result in disturbance of the beneficial gut viruses/phages. METHODS: This study assesses the impact of AgNP on the survival of individual bacteriophages using classical virology cultivation and electron microscopic techniques. Moreover, how the ingested AgNP may affect the intestinal virus/phages was investigated by conducting whole-genome sequencing (WGS). RESULTS: The viral cultivation methods showed minimal effect on selected viruses during short-term exposure (24 h) to 10 nm AgNP. However, long-term exposure (7 days) resulted in significant reduction in the viral/phage population. Data obtained from WGS were filtered and compared with a nonredundant viral database composed of the complete viral genomes from NCBI using KRAKEN (confidence scoring threshold of 0.5). To compare the relative differential changes, the sequence counts in each treatment group were normalized to account for differences in DNA sequencing library sizes. Bioinformatics techniques were developed to visualize the virome comparative changes in a phylogenic tree graph. The computed data revealed that AgNP had an impact on several intestinal bacteriophages that prey on bacterial genus Enterobacteria, Yersinia and Staphylococcus as host species. Moreover, there was an independent effect of nanoparticles and released ions. CONCLUSION: Overall, this study reveals that the small-size AgNP could lead to perturbations of the gut microbial ecosystem, leading to the inactivation of resident phages that play an important role in influencing gastrointestinal health.

Bacteriophage Clinical Use as Antibacterial "Drugs": Utility and Precedent.

For phage therapy-the treatment of bacterial infections using bacterial viruses-a key issue is the conflict between apparent ease of clinical application, on the one hand, and on the other hand, numerous difficulties that can be associated with undertaking preclinical development. These conflicts between achieving efficacy in the real world versus rigorously understanding that efficacy should not be surprising because equivalent conflicts have been observed in applied biology for millennia: exploiting the inherent, holistic tendencies of useful systems, e.g., of dairy cows, inevitably is easier than modeling those systems or maintaining effectiveness while reducing such systems to isolated parts. Trial and error alone, in other words, can be a powerful means toward technological development. Undertaking trial and error-based programs, especially in the clinic, nonetheless is highly dependent on those technologies possessing both inherent safety and intrinsic tendencies toward effectiveness, but in this modern era we tend to forget that ideally there would exist antibacterials which could be thus developed, that is, with tendencies toward both safety and effectiveness, and which are even relatively inexpensive. Consequently, we tend to demand rigor as well as expense of development even to the point of potentially squandering such utility, were it to exist. In this review I lay out evidence that in phage therapy such potential, in fact, does exist. Advancement of phage therapy unquestionably requires effective regulation as well as rigorous demonstration of efficacy, but after nearly 100 years of clinical practice, perhaps not as much emphasis on strictly laboratory-based proof of principle.

Adaptation to Parasites and Costs of Parasite Resistance in Mutator and Nonmutator Bacteria.

Parasitism creates selection for resistance mechanisms in host populations and is hypothesized to promote increased host evolvability. However, the influence of these traits on host evolution when parasites are no longer present is unclear. We used experimental evolution and whole-genome sequencing of Escherichia coli to determine the effects of past and present exposure to parasitic viruses (phages) on the spread of mutator alleles, resistance, and bacterial competitive fitness. We found that mutator alleles spread rapidly during adaptation to any of four different phage species, and this pattern was even more pronounced with multiple phages present simultaneously. However, hypermutability did not detectably accelerate adaptation in the absence of phages and recovery of fitness costs associated with resistance. Several lineages evolved phage resistance through elevated mucoidy, and during subsequent evolution in phage-free conditions they rapidly reverted to nonmucoid, phage-susceptible phenotypes. Genome sequencing revealed that this phenotypic reversion was achieved by additional genetic changes rather than by genotypic reversion of the initial resistance mutations. Insertion sequence (IS) elements played a key role in both the acquisition of resistance and adaptation in the absence of parasites; unlike single nucleotide polymorphisms, IS insertions were not more frequent in mutator lineages. Our results provide a genetic explanation for rapid reversion of mucoidy, a phenotype observed in other bacterial species including human pathogens. Moreover, this demonstrates that the types of genetic change underlying adaptation to fitness costs, and consequently the impact of evolvability mechanisms such as increased point-mutation rates, depend critically on the mechanism of resistance.

Spatial structure mitigates fitness costs in host-parasite coevolution.

The extent of population mixing is known to influence the coevolutionary outcomes of many host and parasite traits, including the evolution of generalism (the ability to resist or infect a broad range of genotypes). While the segregation of populations into interconnected demes has been shown to influence the evolution of generalism, the role of local interactions between individuals is unclear. Here, we combine an individual-based model of microbial communities with a well-established framework of genetic specificity that matches empirical observations of bacterium-phage interactions. We find the evolution of generalism in well-mixed populations to be highly sensitive to the severity of associated fitness costs, but the constraining effect of costs on the evolution of generalism is lessened in spatially structured populations. The contrasting outcomes between the two environments can be explained by different scales of competition (i.e., global vs. local). These findings suggest that local interactions may have important effects on the evolution of generalism in host-parasite interactions, particularly in the presence of high fitness costs.

Nasty viruses, costly plasmids, population dynamics, and the conditions for establishing and maintaining CRISPR-mediated adaptive immunity in bacteria.

Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR) abound in the genomes of almost all archaebacteria and nearly half the eubacteria sequenced. Through a genetic interference mechanism, bacteria with CRISPR regions carrying copies of the DNA of previously encountered phage and plasmids abort the replication of phage and plasmids with these sequences. Thus it would seem that protection against infecting phage and plasmids is the selection pressure responsible for establishing and maintaining CRISPR in bacterial populations. But is it? To address this question and provide a framework and hypotheses for the experimental study of the ecology and evolution of CRISPR, I use mathematical models of the population dynamics of CRISPR-encoding bacteria with lytic phage and conjugative plasmids. The results of the numerical (computer simulation) analysis of the properties of these models with parameters in the ranges estimated for Escherichia coli and its phage and conjugative plasmids indicate: (1) In the presence of lytic phage there are broad conditions where bacteria with CRISPR-mediated immunity will have an advantage in competition with non-CRISPR bacteria with otherwise higher Malthusian fitness. (2) These conditions for the existence of CRISPR are narrower when there is envelope resistance to the phage. (3) While there are situations where CRISPR-mediated immunity can provide bacteria an advantage in competition with higher Malthusian fitness bacteria bearing deleterious conjugative plasmids, the conditions for this to obtain are relatively narrow and the intensity of selection favoring CRISPR weak. The parameters of these models can be independently estimated, the assumption behind their construction validated, and the hypotheses generated from the analysis of their properties tested in experimental populations of bacteria with lytic phage and conjugative plasmids. I suggest protocols for estimating these parameters and outline the design of experiments to evaluate the validity of these models and test these hypotheses.

Cultivation-based assessment of lysogeny among soil bacteria.

Lysogeny has long been proposed as an important long-term maintenance strategy for autochthonous soil bacteriophages (phages). Whole genome sequence data indicate that prophage-derived sequences pervade prokaryotic genomes, but the connection between inferred prophage sequence and an active temperate phage is tenuous. Thus, definitive evidence of phage production from lysogenic prokaryotes will be critical in determining the presence and extent of temperate phage diversity existing as prophage within bacterial genomes and within environmental contexts such as soils. This study optimized methods for systematic and definitive determination of lysogeny within a collection of autochthonous soil bacteria. Twenty bacterial isolates from a range of Delaware soil environments (five from each soil) were treated with the inducing agents mitomycin C (MC) or UV light. Six isolates (30%) carried inducible temperate phages as evidenced by an increase in virus direct counts. The magnitude of induction response was highly dependent upon specific induction conditions, and corresponding burst sizes ranged from 1 to 176. Treatment with MC for 30 min yielded the largest induction responses for three of the six lysogens. Morphological analysis revealed that four of the lysogens produced lambda-like Siphoviridae particles, whereas two produced Myoviridae particles. Additionally, pulsed-field gel electrophoresis data indicated that two of the six lysogens were polylysogens, producing more than one distinct type of phage particle. These results suggest that lysogeny is relatively common among soil bacteria.

Bacteriophages for plant disease control.

The use of phages for disease control is a fast expanding area of plant protection with great potential to replace the chemical control measures now prevalent. Phages can be used effectively as part of integrated disease management strategies. The relative ease of preparing phage treatments and low cost of production of these agents make them good candidates for widespread use in developing countries as well. However, the efficacy of phages, as is true of many biological control agents, depends greatly on prevailing environmental factors as well as on susceptibility of the target organism. Great care is necessary during development, production and application of phage treatments. In addition, constant monitoring for the emergence of resistant bacterial strains is essential. Phage-based disease control management is a dynamic process with a need for continuous adjustment of the phage preparation in order to effectively fight potentially adapting pathogenic bacteria.

Coevolutionary arms races between bacteria and bacteriophage.

We propose a computational and theoretical framework for analyzing rapid coevolutionary dynamics of bacteriophage and bacteria in their ecological context. Bacteriophage enter host cells via membrane-bound surface receptors often responsible for nutrient uptake. As such, a selective pressure will exist for the bacteria to modify its receptor configuration and, in turn, for the phage to modify its tail fiber. A mathematical model of these trait adaptations is developed by using the framework of adaptive dynamics. Host strains differ in their efficiency of resource uptake and resistance to phage, whereas phage strains differ in their host preference for adsorption. We solve the evolutionary ecology model and find the conditions for coevolutionary branching and relevant dimensionless parameters leading to distinct quasispecies. We confirm these calculations using stochastic Monte Carlo simulations of populations evolving in a chemostat with fixed washout rate and inflow resource density. We find that multiple quasispecies of bacteria and phage can coexist in a homogeneous medium with a single resource. When diversification occurs, quasispecies of phage adsorb effectively to only a limited subset of the total number of quasispecies of bacteria, i.e., functional differences between quasispecies arise endogenously within the evolutionary ecology framework. Finally, we discuss means to relate predictions of this model to experimental studies in the chemostat, using the model organisms Escherichia coli and the virulent strain of lambda phage.

The impact of bacteriophage genomics.

The discovery of (bacterio)phages revolutionised microbiology and genetics, while phage research has been integral to answering some of the most fundamental biological questions of the twentieth century. The susceptibility of bacteria to bacteriophage attack can be undesirable in some cases, especially in the dairy industry, but can be desirable in others, for example, the use of bacteriophage therapy to eliminate pathogenic bacteria. The relative ease with which entire bacteriophage genome sequences can now be elucidated has had a profound impact on the study of these bacterial parasites.

Evolutionary role of restriction/modification systems as revealed by comparative genome analysis.

Type II restriction modification systems (RMSs) have been regarded either as defense tools or as molecular parasites of bacteria. We extensively analyzed their evolutionary role from the study of their impact in the complete genomes of 26 bacteria and 35 phages in terms of palindrome avoidance. This analysis reveals that palindrome avoidance is not universally spread among bacterial species and that it does not correlate with taxonomic proximity. Palindrome avoidance is also not universal among bacteriophage, even when their hosts code for RMSs, and depends strongly on the genetic material of the phage. Interestingly, palindrome avoidance is intimately correlated with the infective behavior of the phage. We observe that the degree of palindrome and restriction site avoidance is significantly and consistently less important in phages than in their bacterial hosts. This result brings to the fore a larger selective load for palindrome and restriction site avoidance on the bacterial hosts than on their infecting phages. It is then consistent with a view where type II RMSs are considered as parasites possibly at the verge of mutualism. As a consequence, RMSs constitute a nontrivial third player in the host-parasite relationship between bacteria and phages.