Competition and coevolution drive the evolution and the diversification of CRISPR immunity

The diversity of resistance fuels host adaptation to infectious diseases and challenges the ability of pathogens to exploit host populations. Yet, how this host diversity evolves over time remains unclear because it depends on the interplay between intraspecific competition and coevolution with pathogens. Here we study the effect of a coevolving phage population on the diversification of bacterial CRISPR immunity across space and time. We demonstrate that the negative-frequency-dependent selection generated by coevolution is a powerful force that maintains host resistance diversity and selects for new resistance mutations in the host. We also find that host evolution is driven by asymmetries in competitive abilities among different host genotypes. Even if the fittest host genotypes are targeted preferentially by the evolving phages they often escape extinctions through the acquisition of new CRISPR immunity. Together, these fluctuating selective pressures maintain diversity, but not by preserving the pre-existing host composition. Instead, we repeatedly observe the introduction of new resistance genotypes stemming from the fittest hosts in each population. These results highlight the importance of competition on the transient dynamics of host-pathogen coevolution.

Effects of the Newly Isolated T4-Like Phage on Transmission of Plasmid-Borne Antibiotic Resistance Genes via Generalized Transduction

Bacteriophages are the most abundant biological entities on earth and may play an important role in the transmission of antibiotic resistance genes (ARG) from host bacteria. Although the specialized transduction mediated by the temperate phage targeting a specific insertion site is widely explored, the carrying characteristics of “transducing particles” for different ARG subtypes in the process of generalized transduction remains largely unclear. Here, we isolated a new T4-like lytic phage targeting transconjugant Escherichia coli C600 that contained plasmid pHNAH67 (KX246266) and encoded 11 different ARG subtypes. We found that phage AH67C600_Q9 can misload plasmid-borne ARGs and package host DNA randomly. Moreover, for any specific ARG subtype, the carrying frequency was negatively correlated with the multiplicity of infection (MOI). Further, whole genome sequencing (WGS) identified that only 0.338% (4/1183) of the contigs of an entire purified phage population contained ARG sequences; these were floR, sul2, aph(4)-Ia, and fosA. The low coverage indicated that long-read sequencing methods are needed to explore the mechanism of ARG transmission during generalized transduction.

Standard Bacteriophage Purification Procedures Cause Loss in Numbers and Activity

For decades, bacteriophage purification has followed structured protocols focused on generating high concentrations of phage in manageable volumes. As research moves toward understanding complex phage populations, purification needs have shifted to maximize the amount of phage while maintaining diversity and activity. The effects of standard phage purification procedures such as polyethylene glycol (PEG) precipitation and cesium chloride (CsCl) density gradients on both diversity and activity of a phage population are not known. We have examined the effects of PEG precipitation and CsCl density gradients on a number of known phage (M13, T4, and ΦX 174) of varying structure and size, individually and as mixed sample. Measurement of phage numbers and activity throughout the purification process was performed. We demonstrate that these methods, used routinely to generate “pure” phage samples, are in fact detrimental to retention of phage number and activity; even more so in mixed phage samples. As such, minimal amounts of processing are recommended to introduce less bias and maintain more of a phage population.

Bacteria-Bacteriophage Cycles Facilitate Cholera Outbreak Cycles: An Indirect Susceptible - Infected - Bacteria - Phage (Isibp) Model-Based Mathematical Study

Cholera is an acute enteric infectious disease caused by the Gram-negative bacterium Vibrio Cholerae. Despite a huge body of research, the precise nature of its transmission dynamics has yet to be fully elucidated. Mathematical models can be useful to better understand how an infectious agent can spread and be properly controlled. We develop a compartmental model describing a Human population, a bacterial population as well as a phage population. We show that there might be eight equilibrium points; one of which is a disease free equilibrium point. We carry out numerical simulations and sensitivity analyses and we show that the presence of phage can reduce the number of infectious individuals. Moreover, we discuss the main implications in terms of public health management and control strategies.

Enteric Phageome Alterations in Patients With Type 2 Diabetes

Type 2 diabetes is a complex metabolic disease and has been shown to involve alteration of the gut microbiota. Previous studies have primarily focused on changes in the bacterial microbiome, while ignoring the phage community composition. Extracellular phages can lyse host bacteria and thus influence the microbiota through positive or negative interactions with bacteria. We investigated changes in the extracellular phageome and discussed its role in T2D pathogenesis. We used a sequencing-based approach to identify bacteriophage after isolation of VLPs (virus like particles) from fecal samples. We identified 330 species of phages according to the predicted host bacteria from T2D patients (N=17) and nondiabetic controls (N=29). The phageome characteristics were highly diverse among individuals. In the T2D group, the intestinal phage population was altered, and the abundance of phages specific to Enterobacteriaceae hosts increased markedly. Meanwhile, the abundance of Enterobacteriaceae in the gut was significantly increased, and systemic LPS content elevation was observed in the T2D group. Additionally, a consortia of eight phages was found to distinguish T2D patients from nondiabetic controls with good performance (AUC>0.99).

Biodiversity Of Bacteriophages From Lysed Batch Culture Of Recombinant Escherichia Coli BL21(DE3) Examined By Anion Exchange Chromatography And TEM

It was found that the phage population assaulted a batch culture of an industrial recombinant derivative of E. coli BL21(DE3) was attenuated that manifested in producing pinprick-type plaques and their inability to propagation in subsequent passages. Because of this, the goals of the present research were to evaluate biodiversity and scrutinize the possible virion structural defects of the attenuated phage population prior to pure phage lines isolation. The anion exchange chromatography (AEC) was chosen as the principal method allowing to get a comparative phage population profile based on the virion net surface charge, as well as to treat the 5-liter virion-contained sample and collect high-quality concentrated and separated phage fractions to further analysis. The isolate consisted of a mix of two phages belonging to Myoviridae (A2-morphotype) and Siphoviridae (B1-morphotype) families. By restriction analysis, the main portion of this phage pool (about 99% of all virions) was identified as the primary population of myophage Lw1, which possessed its own intra-population biodiversity (heterogeneity). It consisted of a major and two minor subpopulations that differed by phage capsid size and shape. The subpopulation III consisted of aberrant tubby-phages with triprolate (expanded at both sides) capsids having low aspect ratio.

Enteric phageome alternations in Type 2 diabetes disease

Background Type 2 diabetes (T2D) is a complex metabolic disease and has been proved to involve in the alternation of the gut microbiota. The previous studies primarily focused on the changes in bacteriome while ignoring the phage community composition. The extracellular phages could lyse the host bacteria, and thus influence the microbiota through the positive or negative interactions with bacteria. We investigated the change of extracellular phageome and explored its role in T2D pathogenesis.Results We used a sequencing-based approach to identify the bacteriophage after isolation of VLPs from the fecal samples. We identified 330 phages according to the predicted host bacteria. The phageome characteristics were highly diverse among individuals. In the T2D group, the intestinal phage population is altered and the abundance of 7 identified phages specific to Enterobacteriaceae hosts were found increased markedly. Additionally, the abundance of Enterobacteriaceae bacteria in gut was significantly increased and the systemic LPS elevation was observed in T2D group. Several phage consortia were found to have significant correlations with T2D disease indicators.Conclusions The alteration of bacteriophages predicted to infect Enterobacteriaceae in the gut was observed in this study, which was expected to be a new source of systemic LPS in T2D patients, and may contribute to the pathogenesis of the disease. The data present in this study revealed the similar variation trend in enteric bacteriome and the correlated bacteriophages, which is likely to shed considerable light on overall understanding the interactions between microbiome and metabolic diseases.

Spatial structure affects phage efficacy in infecting dual-strain biofilms of Pseudomonas aeruginosa

Bacterial viruses, or phage, are key members of natural microbial communities. Yet much research on bacterial-phage interactions has been conducted in liquid cultures involving single bacterial strains. Here we explored how bacterial diversity affects the success of lytic phage in structured communities. We infected a sensitive Pseudomonas aeruginosa strain PAO1 with a lytic phage Pseudomonas 352 in the presence versus absence of an insensitive P. aeruginosa strain PA14, in liquid culture versus colonies on agar. We found that both in liquid and in colonies, inter-strain competition reduced resistance evolution in the susceptible strain and decreased phage population size. However, while all sensitive bacteria died in liquid, bacteria in colonies could remain sensitive yet escape phage infection, due mainly to reduced growth in colony centers. In sum, spatial structure can protect bacteria against phage infection, while the presence of competing strains reduces the evolution of resistance to phage.

Diversity in CRISPR-based immunity protects susceptible genotypes by restricting phage spread and evolution

Diversity in host resistance often associates with reduced pathogen spread. This may result from ecological and evolutionary processes, likely with feedback between them. Theory and experiments on bacteria-phage interactions have shown that genetic diversity of the bacterial adaptive immune system can limit phage evolution to overcome resistance. Using the CRISPR-Cas bacterial immune system and lytic phage, we engineered a host-pathogen system where each bacterial host genotype could be infected by only one phage genotype. With this model system, we explored how CRISPR diversity impacts the spread of phage when they can overcome a resistance allele, how immune diversity affects the evolution of the phage to increase its host range, and if there was feedback between these processes. We show that increasing CRISPR diversity benefits susceptible bacteria via a dilution effect, which limits the spread of the phage. We suggest that this ecological effect impacts the evolution of novel phage genotypes, which then feeds back into phage population dynamics.

A type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity

CRISPR-Cas systems provide sequence-specific immunity against phages and mobile genetic elements using CRISPR-associated nucleases guided by short CRISPR RNAs (crRNAs). Type III systems exhibit a robust immune response that can lead to the extinction of a phage population, a feat coordinated by a multi-subunit effector complex that destroys invading DNA and RNA. Here, we demonstrate that a model type III system in Staphylococcus epidermidis relies upon the activities of two degradosome-associated nucleases, PNPase and RNase J2, to mount a successful defense. Genetic, molecular, and biochemical analyses reveal that PNPase promotes crRNA maturation, and both nucleases are required for efficient clearance of phage-derived nucleic acids. Furthermore, functional assays show that RNase J2 is essential for immunity against diverse mobile genetic elements originating from plasmid and phage. Altogether, our observations reveal the evolution of a critical collaboration between two nucleic acid degrading machines which ensures cell survival when faced with phage attack.