High throughput platform technology for rapid target identification in personalized phage therapy.

As bacteriophages continue to gain regulatory approval for personalized human therapy against antibiotic-resistant infections, there is a need for transformative technologies for rapid target identification through multiple, large, decentralized therapeutic phages biobanks. Here, we design a high throughput phage screening platform comprised of a portable library of individual shelf-stable, ready-to-use phages, in all-inclusive solid tablets. Each tablet encapsulates one phage along with luciferin and luciferase enzyme stabilized in a sugar matrix comprised of pullulan and trehalose capable of directly detecting phage-mediated adenosine triphosphate (ATP) release through ATP bioluminescence reaction upon bacterial cell burst. The tablet composition also enhances desiccation tolerance of all components, which should allow easier and cheaper international transportation of phages and as a result, increased accessibility to therapeutic phages. We demonstrate high throughput screening by identifying target phages for select multidrug-resistant clinical isolates of Pseudomonas aeruginosa, Salmonella enterica, Escherichia coli, and Staphylococcus aureus with targets identified within 30-120 min.

Building pyramids against the evolutionary emergence of pathogens.

Mutations allowing pathogens to escape host immunity promote the spread of infectious diseases in heterogeneous host populations and can lead to major epidemics. Understanding the conditions that slow down this evolution is key for the development of durable control strategies against pathogens. Here, we use theory and experiments to compare the efficacy of three strategies for the deployment of resistance: (i) a mixing strategy where the host population contains two single-resistant genotypes, (ii) a pyramiding strategy where the host carries a double-resistant genotype, (iii) a combining strategy where the host population is a mix of a single-resistant genotype and a double-resistant genotype. First, we use evolutionary epidemiology theory to clarify the interplay between demographic stochasticity and evolutionary dynamics to show that the pyramiding strategy always yields lower probability of evolutionary emergence. Second, we test experimentally these predictions with the introduction of bacteriophages into bacterial populations where we manipulated the diversity and the depth of immunity using a Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated (CRISPR-Cas) system. These biological assays confirm that pyramiding multiple defences into the same host genotype and avoiding combination with single-defence genotypes is a robust way to reduce pathogen evolutionary emergence. The experimental validation of these theoretical recommendations has practical implications in various areas, including for the optimal deployment of resistance varieties in agriculture and for the design of durable vaccination strategies.

Complete genome of Escherichia phages Carena and JoYop.

Escherichia phages Carena and JoYop were isolated from water samples in Abidjan (Cote d'Ivoire). Their genomes comprise 39,283 and 169,193 bp, encoding 44 and 246 predicted genes, respectively. Carena shares 93.4% nucleotide identity with Escherichia podophage CarlSpitteler (Berlinvirus), and JoYop shows 95.6% identity with Escherichia myophage ADUt (Tequatrovirus).

Diversity of Salmonella enterica phages isolated from chicken farms in Kenya.

IMPORTANCE: Non-typhoidal Salmonella enterica infections are one of the leading causes of diarrhoeal diseases that spread to humans from animal sources such as poultry. Hence, keeping poultry farms free of Salmonella is essential for consumer safety and for a better yield of animal products. However, the emergence of antibiotic resistance due to over usage has sped up the search for alternative biocontrol methods such as the use of bacteriophages. Isolation and characterization of novel bacteriophages are key to adapt phage-based biocontrol applications. Here, we isolated and characterized Salmonella phages from samples collected at chicken farms and slaughterhouses in Kenya. The genomic characterization of these phage isolates revealed that they belong to four ICTV (International Committee on Taxonomy of Viruses) phage genera. All these phages are lytic and possibly suitable for biocontrol applications because no lysogenic genes or virulence factors were found in their genomes. Hence, we recommend further studies on these phages for their applications in Salmonella biocontrol.

Four additional natural 7-deazaguanine derivatives in phages and how to make them.

Bacteriophages and bacteria are engaged in a constant arms race, continually evolving new molecular tools to survive one another. To protect their genomic DNA from restriction enzymes, the most common bacterial defence systems, double-stranded DNA phages have evolved complex modifications that affect all four bases. This study focuses on modifications at position 7 of guanines. Eight derivatives of 7-deazaguanines were identified, including four previously unknown ones: 2'-deoxy-7-(methylamino)methyl-7-deazaguanine (mdPreQ1), 2'-deoxy-7-(formylamino)methyl-7-deazaguanine (fdPreQ1), 2'-deoxy-7-deazaguanine (dDG) and 2'-deoxy-7-carboxy-7-deazaguanine (dCDG). These modifications are inserted in DNA by a guanine transglycosylase named DpdA. Three subfamilies of DpdA had been previously characterized: bDpdA, DpdA1, and DpdA2. Two additional subfamilies were identified in this work: DpdA3, which allows for complete replacement of the guanines, and DpdA4, which is specific to archaeal viruses. Transglycosylases have now been identified in all phages and viruses carrying 7-deazaguanine modifications, indicating that the insertion of these modifications is a post-replication event. Three enzymes were predicted to be involved in the biosynthesis of these newly identified DNA modifications: 7-carboxy-7-deazaguanine decarboxylase (DpdL), dPreQ1 formyltransferase (DpdN) and dPreQ1 methyltransferase (DpdM), which was experimentally validated and harbors a unique fold not previously observed for nucleic acid methylases.

Insight into the Binding Mechanisms of Quartz-Selective Peptides: Toward Greener Flotation Processes.

Mining practices, chiefly froth flotation, are being critically reassessed to replace their use of biohazardous chemical reagents in favor of biofriendly alternatives as a path toward green processes. In this regard, this study aimed at evaluating the interactions of peptides, as potential floatation collectors, with quartz using phage display and molecular dynamics (MD) simulations. Quartz-selective peptide sequences were initially identified by phage display at pH = 9 and further modeled by a robust simulation scheme combining classical MD, replica exchange MD, and steered MD calculations. Our residue-specific analyses of the peptides revealed that positively charged arginine and lysine residues were favorably attracted by the quartz surface at basic pH. The negatively charged residues at pH 9 (i.e., aspartic acid and glutamic acid) further showed affinity toward the quartz surface through electrostatic interactions with the positively charged surface-bound Na+ ions. The best-binding heptapeptide combinations, however, contained both positively and negatively charged residues in their composition. The flexibility of peptide chains was also shown to directly affect the adsorption behavior of the peptide. While attractive intrapeptide interactions were dominated by a weak peptide-quartz binding, the repulsive self-interactions in the peptides improved the binding propensity to the quartz surface. Our results showed that MD simulations are fully capable of revealing mechanistic details of peptide adsorption to inorganic surfaces and are an invaluable tool to accelerate the rational design of peptide sequences for mineral processing applications.

Longitudinal Study of Lactococcus Phages in a Canadian Cheese Factory.

The presence of virulent phages is closely monitored during cheese manufacturing, as these bacterial viruses can significantly slow down the milk fermentation process and lead to low-quality cheeses. From 2001 to 2020, whey samples from cheddar cheese production in a Canadian factory were monitored for the presence of virulent phages capable of infecting proprietary strains of Lactococcus cremoris and Lactococcus lactis used in starter cultures. Phages were successfully isolated from 932 whey samples using standard plaque assays and several industrial Lactococcus strains as hosts. A multiplex PCR assay assigned 97% of these phage isolates to the Skunavirus genus, 2% to the P335 group, and 1% to the Ceduovirus genus. DNA restriction profiles and a multilocus sequence typing (MLST) scheme distinguished at least 241 unique lactococcal phages from these isolates. While most phages were isolated only once, 93 of them (out of 241, 39%) were isolated multiple times. Phage GL7 was isolated 132 times from 2006 to 2020, demonstrating that phages can persist in a cheese factory for long periods of time. Phylogenetic analysis of MLST sequences showed that phages could be clustered based on their bacterial hosts rather than their year of isolation. Host range analysis showed that Skunavirus phages exhibited a very narrow host range, whereas some Ceduovirus and P335 phages had a broader host range. Overall, the host range information was useful in improving the starter culture rotation by identifying phage-unrelated strains and helped mitigating the risk of fermentation failure due to virulent phages. IMPORTANCE Although lactococcal phages have been observed in cheese production settings for almost a century, few longitudinal studies have been performed. This 20-year study describes the close monitoring of dairy lactococcal phages in a cheddar cheese factory. Routine monitoring was conducted by factory staff, and when whey samples were found to inhibit industrial starter cultures under laboratory conditions, they were sent to an academic research laboratory for phage isolation and characterization. This led to a collection of at least 241 unique lactococcal phages, which were characterized through PCR typing and MLST profiling. Phages of the Skunavirus genus were by far the most dominant. Most phages lysed a small subset of the Lactococcus strains. These findings guided the industrial partner in adapting the starter culture schedule by using phage-unrelated strains in starter cultures and removing some strains from the starter rotation. This phage control strategy could be adapted for other large-scale bacterial fermentation processes.

In through the Out Door: A Functional Virulence Factor Secretion System Is Necessary for Phage Infection in Ralstonia solanacearum.

Bacteriophages put intense selective pressure on microbes, which must evolve diverse resistance mechanisms to survive continuous phage attacks. We used a library of spontaneous Bacteriophage Insensitive Mutants (BIMs) to learn how the plant pathogen Ralstonia solanacearum resists the virulent lytic podophage phiAP1. Phenotypic and genetic characterization of many BIMs suggested that the R. solanacearum Type II Secretion System (T2SS) plays a key role in phiAP1 infection. Using precision engineered mutations that permit T2SS assembly but either inactivate the T2SS GspE ATPase or sterically block the secretion portal, we demonstrated that phiAP1 needs a functional T2SS to infect R. solanacearum. This distinction between the static presence of T2SS components, which is necessary but not sufficient for phage sensitivity, and the energized and functional T2SS, which is sufficient, implies that binding interactions alone cannot explain the role of the T2SS in phiAP1 infection. Rather, our results imply that some aspect of the resetting of the T2SS, such as disassembly of the pseudopilus, is required. Because R. solanacearum secretes multiple virulence factors via the T2SS, acquiring resistance to phiAP1 also dramatically reduced R. solanacearum virulence on tomato plants. This acute fitness trade-off suggests this group of phages may be a sustainable control strategy for an important crop disease. IMPORTANCE Ralstonia solanacearum is a destructive plant pathogen that causes lethal bacterial wilt disease in hundreds of diverse plant hosts, including many economically important crops. Phages that kill R. solanacearum could offer effective and environmentally friendly wilt disease control, but only if the bacterium cannot easily evolve resistance. Encouragingly, most R. solanacearum mutants resistant to the virulent lytic phage phiAP1 no longer secreted multiple virulence factors and had much reduced fitness and virulence on tomato plants. Further analysis revealed that phage phiAP1 needs a functional type II secretion system to infect R. solanacearum, suggesting this podophage uses a novel infection mechanism.

Genome Sequence of SN1, a Bacteriophage That Infects Sphaerotilus natans and Pseudomonas aeruginosa.

Phage SN1 infects Sphaerotilus natans and Pseudomonas aeruginosa strains. Its genome consists of 61,858 bp (64.3% GC) and 89 genes, including 32 with predicted functions. SN1 genome is very similar to Pseudomonas phage M6, which contains hypermodified thymidines. Genome analyses revealed similar base-modifying genes as those found in M6.

A truncated anti-CRISPR protein prevents spacer acquisition but not interference.

CRISPR-Cas systems in prokaryotic cells provide an adaptive immunity against invading nucleic acids. For example, phage infection leads to addition of new immunity (spacer acquisition) and DNA cleavage (interference) in the bacterial model species Streptococcus thermophilus, which primarily relies on Cas9-containing CRISPR-Cas systems. Phages can counteract this defense system through mutations in the targeted protospacers or by encoding anti-CRISPR proteins (ACRs) that block Cas9 interference activity. Here, we show that S. thermophilus can block ACR-containing phages when the CRISPR immunity specifically targets the acr gene. This in turn selects for phage mutants carrying a deletion within the acr gene. Remarkably, a truncated acrIIA allele, found in a wild-type virulent streptococcal phage, does not block the interference activity of Cas9 but still prevents the acquisition of new immunities, thereby providing an example of an ACR specifically inhibiting spacer acquisition.

Complete Genome Sequences of 10 Lactococcal Skunavirus Phages Isolated from Cheddar Cheese Whey Samples in Canada.

We report the complete genome sequences of 10 virulent phages of the Skunavirus genus (Siphoviridae) that infect Lactococcus lactis strains used for cheddar cheese production in Canada. Their linear genomes range from 28,969 bp to 31,042 bp with GC contents of 34.1 to 35.1% and 55 to 60 predicted open reading frames (ORFs).

Structural Insights into Lactococcal Siphophage p2 Baseplate Activation Mechanism.

Virulent phages infecting L. lactis, an industry-relevant bacterium, pose a significant risk to the quality of the fermented milk products. Phages of the Skunavirus genus are by far the most isolated lactococcal phages in the cheese environments and phage p2 is the model siphophage for this viral genus. The baseplate of phage p2, which is used to recognize its host, was previously shown to display two conformations by X-ray crystallography, a rested state and an activated state ready to bind to the host. The baseplate became only activated and opened in the presence of Ca2+. However, such an activated state was not previously observed in the virion. Here, using nanobodies binding to the baseplate, we report on the negative staining electron microscopy structure of the activated form of the baseplate directly observed in the p2 virion, that is compatible with the activated baseplate crystal structure. Analyses of this new structure also established the presence of a second distal tail (Dit) hexamer as a component of the baseplate, the topology of which differs largely from the first one. We also observed an uncoupling between the baseplate activation and the tail tip protein (Tal) opening, suggesting an infection mechanism more complex than previously expected.

A Lactococcal Phage Protein Promotes Viral Propagation and Alters the Host Proteomic Response During Infection.

The lactococcal virulent phage p2 is a model for studying the Skunavirus genus, the most prevalent group of phages causing milk fermentation failures in cheese factories worldwide. This siphophage infects Lactococcus lactis MG1363, a model strain used to study Gram-positive lactic acid bacteria. The structural proteins of phage p2 have been thoroughly described, while most of its non-structural proteins remain uncharacterized. Here, we developed an integrative approach, making use of structural biology, genomics, physiology, and proteomics to provide insights into the function of ORF47, the most conserved non-structural protein of unknown function among the Skunavirus genus. This small phage protein, which is composed of three α-helices, was found to have a major impact on the bacterial proteome during phage infection and to significantly reduce the emergence of bacteriophage-insensitive mutants.

Source Tracking Based on Core Genome SNV and CRISPR Typing of Salmonella enterica Serovar Heidelberg Isolates Involved in Foodborne Outbreaks in Québec, 2012.

Whole-genome sequencing (WGS) is the method of choice for bacterial subtyping and it is rapidly replacing the more traditional methods such as pulsed-field gel electrophoresis (PFGE). Here we used the high-resolution core genome single nucleotide variant (cgSNV) typing method to characterize clinical and food from Salmonella enterica serovar Heidelberg isolates in the context of source attribution. Additionally, clustered regularly interspaced short palindromic repeats (CRISPR) analysis was included to further support this method. Our results revealed that cgSNV was highly discriminatory and separated the outbreak isolates into distinct clusters (0-4 SNVs). CRISPR analysis was also able to distinguish outbreak strains from epidemiologically unrelated isolates. Specifically, our data clearly demonstrated the strength of these two methods to determine the probable source(s) of a 2012 epidemiologically characterized outbreak of S. Heidelberg. Using molecular cut-off of 0-10 SNVs, the cgSNV analysis of 246 clinical and food isolates of S. Heidelberg collected in Québec, in the same year of the outbreak event, revealed that retail and abattoir chicken isolates likely represent an important source of human infection to S. Heidelberg. Interestingly, the isolates genetically related by cgSNV also harbored the same CRISPR as outbreak isolates and clusters. This indicates that CRISPR profiles can be useful as a complementary approach to determine source attribution in foodborne outbreaks. Use of the genomic analysis also allowed to identify a large number of cases that were missed by PFGE, indicating that most outbreaks are probably underestimated. Although epidemiological information must still support WGS-based results, cgSNV method is a highly discriminatory method for the resolution of outbreak events and the attribution of these events to their respective sources. CRISPR typing can serve as a complimentary tool to this analysis during source tracking.

DNA tandem repeats contribute to the genetic diversity of Brevibacterium aurantiacum phages.

This report presents the characterization of the first virulent phages infecting Brevibacterium aurantiacum, a bacterial species used during the manufacture of surface-ripened cheeses. These phages were also responsible for flavour and colour defects in surface-ripened cheeses. Sixteen phages (out of 62 isolates) were selected for genome sequencing and comparative analyses. These cos-type phages with a long non-contractile tail currently belong to the Siphoviridae family (Caudovirales order). Their genome sizes vary from 35,637 to 36,825 bp and, similar to their host, have a high GC content (~61%). Genes encoding for an immunity repressor, an excisionase and a truncated integrase were found, suggesting that these virulent phages may be derived from a prophage. Their genomic organization is highly conserved, with most of the diversity coming from the presence of long (198 bp) DNA tandem repeats (TRs) within an open reading frame coding for a protein of unknown function. We categorized these phages into seven genomic groups according to their number of TR, which ranged from two to eight. Moreover, we showed that TRs are widespread in phage genomes, found in more than 85% of the genomes available in public databases.

Novel Genus of Phages Infecting Streptococcus thermophilus: Genomic and Morphological Characterization.

Streptococcus thermophilus is a lactic acid bacterium commonly used for the manufacture of yogurt and specialty cheeses. Virulent phages represent a major risk for milk fermentation processes worldwide, as they can inactivate the added starter bacterial cells, leading to low-quality fermented dairy products. To date, four genetically distinct groups of phages infecting S. thermophilus have been described. Here, we describe a fifth group. Phages P738 and D4446 are virulent siphophages that infect a few industrial strains of S. thermophilus The genomes of phages P738 and D4446 were sequenced and found to contain 34,037 and 33,656 bp as well as 48 and 46 open reading frames, respectively. Comparative genomic analyses revealed that the two phages are closely related to each other but display very limited similarities to other S. thermophilus phages. In fact, these two novel S. thermophilus phages share similarities with streptococcal phages of nondairy origin, suggesting that they emerged recently in the dairy environment.IMPORTANCE Despite decades of research and adapted antiphage strategies such as CRISPR-Cas systems, virulent phages are still a persistent risk for the milk fermentation industry worldwide, as they can cause manufacturing failures and alter product quality. Phages P738 and D4446 are novel virulent phages that infect the food-grade Gram-positive bacterial species Streptococcus thermophilus These two related viruses represent a fifth group of S. thermophilus phages, as they are significantly distinct from other known S. thermophilus phages. Both phages share similarities with phages infecting nondairy streptococci, suggesting their recent emergence and probable coexistence in dairy environments. These findings highlight the necessity of phage surveillance programs as the phage population evolves in response to the application of antiphage strategies.

Author Correction: A mutation in the methionine aminopeptidase gene provides phage resistance in Streptococcus thermophilus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Virulent coliphages in 1-year-old children fecal samples are fewer, but more infectious than temperate coliphages.

Bacteriophages constitute an important part of the human gut microbiota, but their impact on this community is largely unknown. Here, we cultivate temperate phages produced by 900 E. coli strains isolated from 648 fecal samples from 1-year-old children and obtain coliphages directly from the viral fraction of the same fecal samples. We find that 63% of strains hosted phages, while 24% of the viromes contain phages targeting E. coli. 150 of these phages, half recovered from strain supernatants, half from virome (73% temperate and 27% virulent) were tested for their host range on 75 E. coli strains isolated from the same cohort. Temperate phages barely infected the gut strains, whereas virulent phages killed up to 68% of them. We conclude that in fecal samples from children, temperate coliphages dominate, while virulent ones have greater infectivity and broader host range, likely playing a role in gut microbiota dynamics.

7-Deazaguanine modifications protect phage DNA from host restriction systems.

Genome modifications are central components of the continuous arms race between viruses and their hosts. The archaeosine base (G+), which was thought to be found only in archaeal tRNAs, was recently detected in genomic DNA of Enterobacteria phage 9g and was proposed to protect phage DNA from a wide variety of restriction enzymes. In this study, we identify three additional 2'-deoxy-7-deazaguanine modifications, which are all intermediates of the same pathway, in viruses: 2'-deoxy-7-amido-7-deazaguanine (dADG), 2'-deoxy-7-cyano-7-deazaguanine (dPreQ0) and 2'-deoxy-7- aminomethyl-7-deazaguanine (dPreQ1). We identify 180 phages or archaeal viruses that encode at least one of the enzymes of this pathway with an overrepresentation (60%) of viruses potentially infecting pathogenic microbial hosts. Genetic studies with the Escherichia phage CAjan show that DpdA is essential to insert the 7-deazaguanine base in phage genomic DNA and that 2'-deoxy-7-deazaguanine modifications protect phage DNA from host restriction enzymes.