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Understanding bacteriophage propagation on bacteria in different physiological conditions is imperative for predicting phage therapy efficacy on various bacterial infections, especially chronic ones. We investigated phage T4 propagation on bacteria E.coli grown in a chemostat at very low dilution rates extending down to 0.027 h −1 and bacteria exposed to nutrient deprivation. An increase in adsorption constant and latent period with dilution rate D decrease and burst size being proportional to dilution rate (D) was confirmed, consistent with previously published results, extending validity of previous findings. Additional bacterial exposure to starvation, either through nutrient cessation or transferring bacteria into SM buffer, sustained phage propagation during first hours of starvation and diminished to formation of a single phage per infected cell after 24 h. Nutrient deprivation effects were investigated on fast growing bacteria and bacteria in a death phase. While no phage generation was observed within bacteria in death phase, fast growing bacteria transferred into SM buffer generated a single phage within 48 h without lysis, indicating that bacterial exposure to nutrient depleted conditions triggers a so called “scavenger response” whose intensity depends on starvation exposure time. Graphical abstract Highlights Long term bacteria nutrient limitation still allows phage propagation Omitting nutrient supply diminish phage propagation over time till scavenger response occurs Sudden exposure of fast growing bacteria to environment without nutrient induces scavenger response

The world is on the cusp of a post-antibiotic era, but researchers and medical doctors have found a way forward—by looking back at how infections were treated before the advent of antibiotics, namely using phage therapy. Although bacteriophages (phages) continue to lack drug approval in Western medicine, an increasing number of patients are being treated on an expanded-access emergency investigational new drug basis. To streamline the production of high-quality and clinically safe phage preparations, we developed a systematic procedure for medicinal phage isolation, liter-scale cultivation, concentration and purification. The 16- to 21-day procedure described in this protocol uses a combination of modified classic techniques, modern membrane filtration processes and no organic solvents to yield on average 23 mL of 10 11 plaque-forming units (PFUs) per milliliter for Pseudomonas , Klebsiella , and Serratia phages tested. Thus, a single production run can produce up to 64,000 treatment doses at 10 9 PFUs, which would be sufficient for most expanded-access phage therapy cases and potentially for clinical phase I/II applications. The protocol focuses on removing endotoxins early by conducting multiple low-speed centrifugations, microfiltration, and cross-flow ultrafiltration, which reduced endotoxins by up to 10 6 -fold in phage preparations. Implementation of a standardized phage cultivation and purification across research laboratories participating in phage production for expanded-access phage therapy might be pivotal to reintroduce phage therapy to Western medicine. This protocol provides standardized laboratory manufacturing practices to select, cultivate and purify bacteriophages for human clinical applications. The procedure covers all stages from phage isolation and characterization to quality control.

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci in prokaryotes are composed of 30–40-base-pair repeats separated by equally short sequences of plasmid and bacteriophage origin known as spacers 1 – 3 . These loci are transcribed and processed into short CRISPR RNAs (crRNAs) that are used as guides by CRISPR-associated (Cas) nucleases to recognize and destroy complementary sequences (known as protospacers) in foreign nucleic acids 4 , 5 . In contrast to most Cas nucleases, which destroy invader DNA 4 – 7 , the type VI effector nuclease Cas13 uses RNA guides to locate complementary transcripts and catalyse both sequence-specific cis - and non-specific trans -RNA cleavage 8 . Although it has been hypothesized that Cas13 naturally defends against RNA phages 8 , type VI spacer sequences have exclusively been found to match the genomes of double-stranded DNA phages 9 , 10 , suggesting that Cas13 can provide immunity against these invaders. However, whether and how Cas13 uses its cis - and/or trans -RNA cleavage activities to defend against double-stranded DNA phages is not understood. Here we show that trans -cleavage of transcripts halts the growth of the host cell and is sufficient to abort the infectious cycle. This depletes the phage population and provides herd immunity to uninfected bacteria. Phages that harbour target mutations, which easily evade DNA-targeting CRISPR systems 11 – 13 , are also neutralized when Cas13 is activated by wild-type phages. Thus, by acting on the host rather than directly targeting the virus, type VI CRISPR systems not only provide robust defence against DNA phages but also prevent outbreaks of CRISPR-resistant phage. The RNA-cleaving Cas13 degrades both host and bacteriophage transcripts, thereby rendering infected cells dormant and broadly resistant to phage-mediated lysis.

CRISPR/Cas systems are bacterial adaptive immune systems that provide sequence-specific protection from invading nucleic acids, including from bacteriophages; in a notable reverse a vibriophage-encoded CRISPR/Cas system, used to disable a bacteriophage inhibitory chromosomal island in Vibrio cholerae , is identified. Vibriophage hijacks cholera pathogen defences The CRISPR/Cas bacterial adaptive immune systems provide sequence-specific protection from invading nucleic acids, including those of bacteriophage. As such, they are key weapons in an ongoing co-evolutionary arms race. Here Andrew Camilli and colleagues reveal a remarkable case in which these weapons have been turned on their bacterial bearers. The authors identify a vibriophage-encoded CRISPR/Cas system that is used to disarm a bacteriophage inhibitory chromosomal island in the bacterium Vibrio cholerae . Bacteriophages (or phages) are the most abundant biological entities on earth, and are estimated to outnumber their bacterial prey by tenfold 1 . The constant threat of phage predation has led to the evolution of a broad range of bacterial immunity mechanisms that in turn result in the evolution of diverse phage immune evasion strategies, leading to a dynamic co-evolutionary arms race 2 , 3 . Although bacterial innate immune mechanisms against phage abound, the only documented bacterial adaptive immune system is the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system, which provides sequence-specific protection from invading nucleic acids, including phage 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 . Here we show a remarkable turn of events, in which a phage-encoded CRISPR/Cas system is used to counteract a phage inhibitory chromosomal island of the bacterial host. A successful lytic infection by the phage is dependent on sequence identity between CRISPR spacers and the target chromosomal island. In the absence of such targeting, the phage-encoded CRISPR/Cas system can acquire new spacers to evolve rapidly and ensure effective targeting of the chromosomal island to restore phage replication.

The therapeutic use of bacteriophages has seen a renewal of interest blossom in the last few years. This reversion is due to increased difficulties in the treatment of antibiotic-resistant strains of bacteria. Bacterial resistance to antibiotics, a serious problem in contemporary medicine, does not implicate resistance to phage lysis mechanisms. Lytic bacteriophages are able to kill antibiotic-resistant bacteria at the end of the phage infection cycle. Thus, the development of phage therapy is potentially a way to improve the treatment of bacterial infections. However, there are antibacterial phage therapy difficulties specified by broadening the knowledge of the phage nature and influence on the host. It has been shown during experiments that both innate and adaptive immunity are involved in the clearance of phages from the body. Immunological reactions against phages are related to the route of administration and may vary depending on the type of bacterial viruses. For that reason, it is very important to test the immunological response of every single phage, particularly if intravenous therapy is being considered. The lack of these data in previous years was one of the reasons for phage therapy abandonment despite its century-long study. Promising results of recent research led us to look forward to a phage therapy that can be applied on a larger scale and subsequently put it into practice.

Background Autism spectrum disorders (ASD) are complex neurobiological disorders that impair social interactions and communication and lead to restricted, repetitive, and stereotyped patterns of behavior, interests, and activities. The causes of these disorders remain poorly understood, but gut microbiota, the 10 13 bacteria in the human intestines, have been implicated because children with ASD often suffer gastrointestinal (GI) problems that correlate with ASD severity. Several previous studies have reported abnormal gut bacteria in children with ASD. The gut microbiome-ASD connection has been tested in a mouse model of ASD, where the microbiome was mechanistically linked to abnormal metabolites and behavior. Similarly, a study of children with ASD found that oral non-absorbable antibiotic treatment improved GI and ASD symptoms, albeit temporarily. Here, a small open-label clinical trial evaluated the impact of Microbiota Transfer Therapy (MTT) on gut microbiota composition and GI and ASD symptoms of 18 ASD-diagnosed children. Results MTT involved a 2-week antibiotic treatment, a bowel cleanse, and then an extended fecal microbiota transplant (FMT) using a high initial dose followed by daily and lower maintenance doses for 7–8 weeks. The Gastrointestinal Symptom Rating Scale revealed an approximately 80% reduction of GI symptoms at the end of treatment, including significant improvements in symptoms of constipation, diarrhea, indigestion, and abdominal pain. Improvements persisted 8 weeks after treatment. Similarly, clinical assessments showed that behavioral ASD symptoms improved significantly and remained improved 8 weeks after treatment ended. Bacterial and phagedeep sequencing analyses revealed successful partial engraftment of donor microbiota and beneficial changes in the gut environment. Specifically, overall bacterial diversity and the abundance of Bifidobacterium , Prevotella , and Desulfovibrio among other taxa increased following MTT, and these changes persisted after treatment stopped (followed for 8 weeks). Conclusions This exploratory, extended-duration treatment protocol thus appears to be a promising approach to alter the gut microbiome and virome and improve GI and behavioral symptoms of ASD. Improvements in GI symptoms, ASD symptoms, and the microbiome all persisted for at least 8 weeks after treatment ended, suggesting a long-term impact. Trial registration This trial was registered on the ClinicalTrials.gov, with the registration number  NCT02504554

Foodborne illnesses remain a major cause of hospitalization and death worldwide despite many advances in food sanitation techniques and pathogen surveillance. Traditional antimicrobial methods, such as pasteurization, high pressure processing, irradiation, and chemical disinfectants are capable of reducing microbial populations in foods to varying degrees, but they also have considerable drawbacks, such as a large initial investment, potential damage to processing equipment due to their corrosive nature, and a deleterious impact on organoleptic qualities (and possibly the nutritional value) of foods. Perhaps most importantly, these decontamination strategies kill indiscriminately, including many—often beneficial—bacteria that are naturally present in foods. One promising technique that addresses several of these shortcomings is bacteriophage biocontrol, a green and natural method that uses lytic bacteriophages isolated from the environment to specifically target pathogenic bacteria and eliminate them from (or significantly reduce their levels in) foods. Since the initial conception of using bacteriophages on foods, a substantial number of research reports have described the use of bacteriophage biocontrol to target a variety of bacterial pathogens in various foods, ranging from ready-to-eat deli meats to fresh fruits and vegetables, and the number of commercially available products containing bacteriophages approved for use in food safety applications has also been steadily increasing. Though some challenges remain, bacteriophage biocontrol is increasingly recognized as an attractive modality in our arsenal of tools for safely and naturally eliminating pathogenic bacteria from foods.

Many bacterial and archaeal organisms use clustered regularly interspaced short palindromic repeats–CRISPR associated (CRISPR–Cas) systems to defend themselves from mobile genetic elements. These CRISPR–Cas systems are classified into six types based on their composition and mechanism. CRISPR–Cas enzymes are widely used for genome editing and offer immense therapeutic opportunity to treat genetic diseases. To realize their full potential, it is important to control the timing, duration, efficiency and specificity of CRISPR–Cas enzyme activities. In this Review we discuss the mechanisms of natural CRISPR–Cas regulatory biomolecules and engineering strategies that enhance or inhibit CRISPR–Cas immunity by altering enzyme function. We also discuss the potential applications of these CRISPR regulators and highlight unanswered questions about their evolution and purpose in nature. This Review summarizes recent advances in CRISPR–Cas regulation mechanisms by natural biomolecules that enhance or inhibit CRIPSR–Cas immunity, as well as their applications in CRISPR biology and technologies.

Viruses that infect microbial hosts have traditionally been studied in laboratory settings with a focus on either obligate lysis or persistent lysogeny. In the environment, these infection archetypes are part of a continuum that spans antagonistic to beneficial modes. In this Review, we advance a framework to accommodate the context-dependent nature of virus–microorganism interactions in ecological communities by synthesizing knowledge from decades of virology research, eco-evolutionary theory and recent technological advances. We discuss that nuanced outcomes, rather than the extremes of the continuum, are particularly likely in natural communities given variability in abiotic factors, the availability of suboptimal hosts and the relevance of multitrophic partnerships. We revisit the ‘rules of life’ in terms of how long-term infections shape the fate of viruses and microbial cells, populations and ecosystems.In this Review, Correa and colleagues revisit the rules of life for viruses of microorganisms by advancing a conceptual framework that recognizes virus–host interactions across a continuum of infection modalities and by examining the influence of these modalities on viruses, their hosts and ecosystems.

A powerful contributor to prokaryotic evolution is horizontal gene transfer (HGT) through transformation, conjugation, and transduction, which can be advantageous, neutral, or detrimental to fitness. Bacteria and archaea control HGT and phage infection through CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) adaptive immunity. Although the benefits of resisting phage infection are evident, this can come at a cost of inhibiting the acquisition of other beneficial genes through HGT. Despite the ability of CRISPR-Cas to limit HGT through conjugation and transformation, its role in transduction is largely overlooked. Transduction is the phage-mediated transfer of bacterial DNA between cells and arguably has the greatest impact on HGT. We demonstrate that in Pectobacterium atrosepticum , CRISPR-Cas can inhibit the transduction of plasmids and chromosomal loci. In addition, we detected phage-mediated transfer of a large plant pathogenicity genomic island and show that CRISPR-Cas can inhibit its transduction. Despite these inhibitory effects of CRISPR-Cas on transduction, its more common role in phage resistance promotes rather than diminishes HGT via transduction by protecting bacteria from phage infection. This protective effect can also increase transduction of phage-sensitive members of mixed populations. CRISPR-Cas systems themselves display evidence of HGT, but little is known about their lateral dissemination between bacteria and whether transduction can contribute. We show that, through transduction, bacteria can acquire an entire chromosomal CRISPR-Cas system, including cas genes and phage-targeting spacers. We propose that the positive effect of CRISPR-Cas phage immunity on enhancing transduction surpasses the rarer cases where gene flow by transduction is restricted. IMPORTANCE The generation of genetic diversity through acquisition of DNA is a powerful contributor to microbial evolution and occurs through transformation, conjugation, and transduction. Of these, transduction, the phage-mediated transfer of bacterial DNA, is arguably the major route for genetic exchange. CRISPR-Cas adaptive immune systems control gene transfer by conjugation and transformation, but transduction has been mostly overlooked. Our results indicate that CRISPR-Cas can impede, but typically enhances the transduction of plasmids, chromosomal genes, and pathogenicity islands. By limiting wild-type phage replication, CRISPR-Cas immunity increases transduction in both phage-resistant and -sensitive members of mixed populations. Furthermore, we demonstrate mobilization of a chromosomal CRISPR-Cas system containing phage-targeting spacers by generalized transduction, which might partly account for the uneven distribution of these systems in nature. Overall, the ability of CRISPR-Cas to promote transduction reveals an unexpected impact of adaptive immunity on horizontal gene transfer, with broader implications for microbial evolution. The generation of genetic diversity through acquisition of DNA is a powerful contributor to microbial evolution and occurs through transformation, conjugation, and transduction. Of these, transduction, the phage-mediated transfer of bacterial DNA, is arguably the major route for genetic exchange. CRISPR-Cas adaptive immune systems control gene transfer by conjugation and transformation, but transduction has been mostly overlooked. Our results indicate that CRISPR-Cas can impede, but typically enhances the transduction of plasmids, chromosomal genes, and pathogenicity islands. By limiting wild-type phage replication, CRISPR-Cas immunity increases transduction in both phage-resistant and -sensitive members of mixed populations. Furthermore, we demonstrate mobilization of a chromosomal CRISPR-Cas system containing phage-targeting spacers by generalized transduction, which might partly account for the uneven distribution of these systems in nature. Overall, the ability of CRISPR-Cas to promote transduction reveals an unexpected impact of adaptive immunity on horizontal gene transfer, with broader implications for microbial evolution.