The Baseplate of Lactobacillus delbrueckii Bacteriophage Ld17 Harbors a Glycerophosphodiesterase.

Glycerophosphodiester phosphodiesterases (GDPDs; EC 3.1.4.46) typically hydrolyze glycerophosphodiesters to sn-glycerol 3-phosphate (Gro3P) and their corresponding alcohol during patho/physiological processes in bacteria and eukaryotes. GDPD(-like) domains were identified in the structural particle of bacterial viruses (bacteriophages) specifically infecting Gram-positive bacteria. The GDPD of phage 17 (Ld17; GDPDLd17), representative of the group b Lactobacillus delbrueckii subsp. bulgaricus (Ldb)-infecting bacteriophages, was shown to hydrolyze, besides the simple glycerophosphodiester, two complex surface-associated carbohydrates of the Ldb17 cell envelope: the Gro3P decoration of the major surface polysaccharide d-galactan and the oligo(glycerol phosphate) backbone of the partially glycosylated cell wall teichoic acid, a minor Ldb17 cell envelope component. Degradation of cell wall teichoic acid occurs according to an exolytic mechanism, and Gro3P substitution is presumed to be inhibitory for GDPDLd17 activity. The presence of the GDPDLd17 homotrimer in the viral baseplate structure involved in phage-host interaction together with the dependence of native GDPD activity, adsorption, and efficiency of plating of Ca(2+) ions supports a role for GDPDLd17 activity during phage adsorption and/or phage genome injection. In contrast to GDPDLd17, we could not identify any enzymatic activity for the GDPD-like domain in the neck passage structure of phage 340, a 936-type Lactococcus lactis subsp. lactis bacteriophage.

Discovery of a conjugative megaplasmid in Bifidobacterium breve.

Bifidobacterium breve is a common and sometimes very abundant inhabitant of the human gut. Genome sequencing of B. breve JCM 7017 revealed the presence of an extrachromosomal element, designated pMP7017 consisting of >190 kb, thus representing the first reported bifidobacterial megaplasmid. In silico characterization of this element revealed several genomic features supporting a stable establishment of the megaplasmid in its host, illustrated by predicted CRISPR-Cas functions that are known to protect the host against intrusion of foreign DNA. Interestingly, pMP7017 is also predicted to encode a conjugative DNA transfer apparatus and, consistent with this notion, we demonstrate here the conjugal transfer of pMP7017 to representative strains of B. breve and B. longum subsp. longum. We also demonstrate the presence of a megaplasmid with homology to pMP7017 in three B. longum subsp. longum strains.

Novel phage group infecting Lactobacillus delbrueckii subsp. lactis, as revealed by genomic and proteomic analysis of bacteriophage Ldl1.

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 +/- 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

Molecular characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages.

In this study, three phages infecting Lactobacillus delbrueckii subsp. bulgaricus, named Ld3, Ld17, and Ld25A, were isolated from whey samples obtained from various industrial fermentations. These phages were further characterized in a multifaceted approach: (i) biological and physical characterization through host range analysis and electron microscopy; (ii) genetic assessment through genome analysis; (iii) mass spectrometry analysis of the structural components of the phages; and (iv), for one phage, transcriptional analysis by Northern hybridization, reverse transcription-PCR, and primer extension. The three obtained phage genomes display high levels of sequence identity to each other and to genomes of the so-called group b L. delbrueckii phages c5, LL-Ku, and phiLdb, where some of the observed differences are believed to be responsible for host range variations.

Ribosome profiling reveals downregulation of UMP biosynthesis as the major early response to phage infection.

Bacteria have evolved diverse defense mechanisms to counter bacteriophage attacks. Genetic programs activated upon infection characterize phage-host molecular interactions and ultimately determine the outcome of the infection. In this study, we applied ribosome profiling to monitor protein synthesis during the early stages of sk1 bacteriophage infection in Lactococcus cremoris. Our analysis revealed major changes in gene expression within 5 minutes of sk1 infection. Notably, we observed a specific and severe downregulation of several pyr operons which encode enzymes required for uridine monophosphate biosynthesis. Consistent with previous findings, this is likely an attempt of the host to starve the phage of nucleotides it requires for propagation. We also observed a gene expression response that we expect to benefit the phage. This included the upregulation of 40 ribosome proteins that likely increased the host's translational capacity, concurrent with a downregulation of genes that promote translational fidelity (lepA and raiA). In addition to the characterization of host-phage gene expression responses, the obtained ribosome profiling data enabled us to identify two putative recoding events as well as dozens of loci currently annotated as pseudogenes that are actively translated. Furthermore, our study elucidated alterations in the dynamics of the translation process, as indicated by time-dependent changes in the metagene profile, suggesting global shifts in translation rates upon infection. Additionally, we observed consistent modifications in the ribosome profiles of individual genes, which were apparent as early as 2 minutes post-infection. The study emphasizes our ability to capture rapid alterations of gene expression during phage infection through ribosome profiling. IMPORTANCE: The ribosome profiling technology has provided invaluable insights for understanding cellular translation and eukaryotic viral infections. However, its potential for investigating host-phage interactions remains largely untapped. Here, we applied ribosome profiling to Lactococcus cremoris cultures infected with sk1, a major infectious agent in dairy fermentation processes. This revealed a profound downregulation of genes involved in pyrimidine nucleotide synthesis at an early stage of phage infection, suggesting an anti-phage program aimed at restricting nucleotide availability and, consequently, phage propagation. This is consistent with recent findings and contributes to our growing appreciation for the role of nucleotide limitation as an anti-viral strategy. In addition to capturing rapid alterations in gene expression levels, we identified translation occurring outside annotated regions, as well as signatures of non-standard translation mechanisms. The gene profiles revealed specific changes in ribosomal densities upon infection, reflecting alterations in the dynamics of the translation process.

Crowdsourcing forensics: Creating a curated catalog of digital forensic artifacts.

The increasing volume, variety, velocity, distribution, structural intricacy, and complexity of use of digital evidence can make it difficult for practitioners to find and understand the most forensically useful information (Casey E. Digital evidence and computer crime: Forensic science, computers, and the Internet. Academic Press; 2011. p. 31; Pollitt M. The hermeneutics of the hard drive: Using narratology, natural language processing, and knowledge management to improve the effectiveness of the digital forensic process [PhD dissertation]. University of Central Florida; 2011). Digital forensic practitioners currently search for information and solutions in an ad hoc manner, leading to results that are unstructured, unverified, and sometimes incomplete. As a result, certain digital evidence is being missed or misinterpreted. To mitigate risks of knowledge gaps, there is a pressing need for a systematic mechanism that practitioners can use to codify and combine their collective knowledge. This work presents the design and development of a solution that catalogs crowdsourced knowledge of digital forensic artifacts in a well-structured, easily searchable form to support efficient and automated extraction of pertinent information, improving availability and reliability of interpretation of artifacts (general acceptance). Technical implementation and artifact curation are discussed with illustrative examples and recommendations for future work.

Novel Siphoviridae phage PMBT4 belonging to the group b Lactobacillus delbrueckii subsp. bulgaricus phages.

A novel Lactobacillus delbrueckii bacteriophage PMBT4 was isolated from the Nigerian fermented milk product nono. The phage possesses a long and thin, non-contractile tail and an isometric head, indicating that it belongs to the Siphoviridae family. A neck passage structure (`collar`), previously hypothesized to be encoded by two genes located in the Lactobacillus delbrueckii phage LL-K insertion sequence (KIS) element, as well as in two additional Lb. delbrueckii phages Ld17 and Ld25A, could also be observed on an estimated 1-5% of phage particles by transmission electron microscopy. However, neither mapping of high throughput sequencing data to KIS element genes from Lb. delbrueckii phages LL-K, Ld17 and Ld25A nor PCR amplification of the KIS element genes could corroborate the presence of these genes in the PMBT4 genome. The PMBT4 genome consists of 31,399 bp with a mol% GC content of 41.6 and exhibits high (95-96%) sequence homologies to Lb. delbrueckii phages c5, Ld3, Ld25A and Ld17, which assigned PMBT4 as a new member of this genus, i.e. the Cequinquevirus genus.

Needle in a Whey-Stack: PhRACS as a Discovery Tool for Unknown Phage-Host Combinations.

The field of metagenomics has rapidly expanded to become the go-to method for complex microbial community analyses. However, there is currently no straightforward route from metagenomics to traditional culture-based methods of strain isolation, particularly in (bacterio)phage biology, leading to an investigative bottleneck. Here, we describe a method that exploits specific phage receptor binding protein (RBP)-host cell surface receptor interaction enabling isolation of phage-host combinations from an environmental sample. The method was successfully applied to two complex sample types-a dairy-derived whey sample and an infant fecal sample, enabling retrieval of specific and culturable phage hosts. IMPORTANCE PhRACS aims to bridge the current divide between in silico genetic analyses (i.e., phageomic studies) and traditional culture-based methodology. Through the labeling of specific bacterial hosts with fluorescently tagged recombinant phage receptor binding proteins and the isolation of tagged cells using flow cytometry, PhRACS allows the full potential of phageomic data to be realized in the wet laboratory.

Inter-regional digital forensic knowledge management: needs, challenges, and solutions.

Increasing demand for digital evidence in criminal investigations is driving decentralization of forensic capabilities closer to the crime scene. Law enforcement agencies are struggling to keep pace with technological developments, cybercrime growth, and scientific advances. In federated environments, digital forensic knowledge and practices vary widely across regions. To reduce delays, wasted resources, missed opportunities, mistakes, and misinterpretations, there is a pressing need to balance the democratization of digital forensic capabilities with knowledge management and sharing between decentralized regions. There are multiple forms of knowledge to be managed, including procedural, technical, investigative, scientific, behavioral, crime analysis, and forensic intelligence. In addition, there are multiple knowledge producers and consumers, including police investigators, digital forensic practitioners, criminal intelligence analysts, attorneys, and judges. Knowledge management becomes even more challenging when multiple interdependent regions are involved, speaking different languages. Taking all of these factors into consideration, this work presents an inter-regional knowledge management solution for improving the quality, consistency, reliability, efficiency, cost-effectiveness, and return on investment of digital forensic capabilities. The basis of this work is a community-driven initiative of Swiss regional police authorities. Interviews were conducted with 15 digital forensic units to determine their current knowledge management practices and needs. The results were then generalized into a prioritized set of requirements for inter-regional digital forensic knowledge management that may be applicable in other countries. These requirements were used to evaluate knowledge management platforms, and one was selected. Implementation, operations, and maintenance challenges of an inter-regional digital forensic knowledge management platform are discussed.

A formalized model of the Trace.

This work proposes a formalized model, grounded in forensic science, to support a unified understanding of the Trace across scientific disciplines. The model is precisely defined in mathematical terms that reflect the dynamics of an offense as expressed in Locard's Exchange principle. Specifically, this mathematical approach represents the Trace as the modification of a Scene, subsequently perceptible, resulting from the Event under investigation. Examples are provided to illustrate how this conceptualization applies to forensic science, including DNA and digital evidence. Broader implications of this model are presented in the context of COVID-19, emphasizing the value of cohesive scientific study of the Trace. The aim of this work is to stimulate more formalized study of the Trace, both from tangible and abstract perspectives, and to strengthen forensic science as a whole.

Phageome Analysis of Bifidobacteria-Rich Samples.

Bifidobacteria are important early colonizers of the human intestinal tract. The relative abundance of bifidobacterial species may be modulated, in part, by bacteriophage activity. Metagenomic studies of these populations is a crucial step in understanding this important interaction. This chapter outlines the technical instructions required to analyze the virome of a bifidobacteria-rich sample, for example, an infant fecal sample.

In Vitro and In Vivo Assessment of the Potential of Escherichia coli Phages to Treat Infections and Survive Gastric Conditions.

Enterotoxigenic Escherichia coli (ETEC) and Shigella ssp. infections are associated with high rates of mortality, especially in infants in developing countries. Due to increasing levels of global antibiotic resistance exhibited by many pathogenic organisms, alternative strategies to combat such infections are urgently required. In this study, we evaluated the stability of five coliphages (four Myoviridae and one Siphoviridae phage) over a range of pH conditions and in simulated gastric conditions. The Myoviridae phages were stable across the range of pH 2 to 7, while the Siphoviridae phage, JK16, exhibited higher sensitivity to low pH. A composite mixture of these five phages was tested in vivo in a Galleria mellonella model. The obtained data clearly shows potential in treating E. coli infections prophylactically.

Infant-Associated Bifidobacterial ß-Galactosidases and Their Ability to Synthesize Galacto-Oligosaccharides.

Galacto-oligosaccharides (GOS) represent non-digestible glycans that are commercially produced by transgalactosylation of lactose, and that are widely used as functional food ingredients in prebiotic formulations, in particular in infant nutrition. GOS consumption has been reported to enhance growth of specific bacteria in the gut, in particular bifidobacteria, thereby supporting a balanced gut microbiota. In a previous study, we assessed the hydrolytic activity and substrate specificity of seventeen predicted ß-galactosidases encoded by various species and strains of infant-associated bifidobacteria. In the current study, we further characterized seven out of these seventeen bifidobacterial ß-galactosidases in terms of their kinetics, enzyme stability and oligomeric state. Accordingly, we established whether these ß-galactosidases are capable of synthesizing GOS via enzymatic transgalactosylation employing lactose as the feed substrate. Our findings show that the seven selected enzymes all possess such transgalactosylation activity, though they appear to differ in their efficiency by which they perform this reaction. From chromatography analysis, it seems that these enzymes generate two distinct GOS mixtures: GOS with a relatively short or long degree of polymerization profile. These findings may be the stepping stone for further studies aimed at synthesizing new GOS variants with novel and/or enhanced prebiotic activities and potential for industrial applications.

Digital transformation risk management in forensic science laboratories.

Technological advances are changing how forensic laboratories operate in all forensic disciplines, not only digital. Computers support workflow management, enable evidence analysis (physical and digital), and new technology enables previously unavailable forensic capabilities. Used properly, the integration of digital systems supports greater efficiency and reproducibility, and drives digital transformation of forensic laboratories. However, without the necessary preparations, these digital transformations can undermine the core principles and processes of forensic laboratories. Pertinent examples of problems involving technology that have occurred in laboratories are provided, along with opportunities and risk mitigation strategies, based on the authors' experiences. Forensic preparedness concentrating on digital data reduces the cost and operational disruption of responding to various kinds of problems, including misplaced exhibits, allegations of employee misconduct, disclosure requirements, and information security breaches. This work presents recommendations to help forensic laboratories prepare for and manage these risks, to use technology effectively, and ultimately strengthen forensic science. The importance of involving digital forensic expertise in risk management of digital transformations in laboratories is emphasized. Forensic laboratories that do not adopt forensic digital preparedness will produce results based on digital data and processes that cannot be verified independently, leaving them vulnerable to challenge. The recommendations in this work could enhance international standards such as ISO/IEC 17025 used to assess and accredit laboratories.

The Impact and Applications of Phages in the Food Industry and Agriculture.

Food security is currently a global socio-political pressure point that is exacerbated by the everincreasing world population[...].

The Kodak Syndrome: Risks and Opportunities Created by Decentralization of Forensic Capabilities.

Forensic science laboratories are being challenged by the expanding decentralization of forensic capabilities, particularly for digital traces. This study recommends laboratories undertake digital transformations to capitalize on the decentralization movement, develop a more comprehensive understanding of crime and security-relevant problems, and play a more central role in problem-solving collaboratively with law enforcement organizations and other stakeholders. A framework for the bilateral transfer of information and knowledge is proposed to magnify the impact of forensic science laboratories on abating crime, strengthening security, and reinforcing the criminal justice system. To accomplish digital transformations, laboratories require personnel with different expertise, including investigative reasoning, knowledge codification, data analytics, and forensic intelligence. Ultimately, this study encourages managers, educators, researchers, and policymakers to look beyond the usefulness of forensic results for solving individual investigations, and to realize the value of combined forensic knowledge and intelligence for developing broader strategies to deal with crime in digitalized society.

A Quest of Great Importance-Developing a Broad Spectrum Escherichiacoli Phage Collection.

Shigella ssp. and enterotoxigenic Escherichiacoli are the most common etiological agents of diarrheal diseases in malnourished children under five years of age in developing countries. The ever-growing issue of antibiotic resistance and the potential negative impact of antibiotic use on infant commensal microbiota are significant challenges to current therapeutic approaches. Bacteriophages (or phages) represent an alternative treatment that can be used to treat specific bacterial infections. In the present study, we screened water samples from both environmental and industrial sources for phages capable of infecting E. coli laboratory strains within our collection. Nineteen phages were isolatedand tested for their ability to infect strains within the ECOR collection and E. coli O157:H7 Δstx. Furthermore, since coliphages have been reported to cross-infect certain Shigella spp., we also evaluated the ability of the nineteen phages to infect a representative Shigella sonnei strain from our collection. Based on having distinct (although overlapping in some cases) host ranges, ten phage isolates were selected for genome sequence and morphological characterization. Together, these ten selected phages were shown to infect most of the ECOR library, with 61 of the 72 strains infected by at least one phage from our collection. Genome analysis of the ten phages allowed classification into five previously described genetic subgroups plus one previously underrepresented subgroup.

Digital Stratigraphy: Contextual Analysis of File System Traces in Forensic Science.

This work introduces novel methods for conducting forensic analysis of file allocation traces, collectively called digital stratigraphy. These in-depth forensic analysis methods can provide insight into the origin, composition, distribution, and time frame of strata within storage media. Using case examples and empirical studies, this paper illuminates the successes, challenges, and limitations of digital stratigraphy. This study also shows how understanding file allocation methods can provide insight into concealment activities and how real-world computer usage can complicate digital stratigraphy. Furthermore, this work explains how forensic analysts have misinterpreted traces of normal file system behavior as indications of concealment activities. This work raises awareness of the value of taking the overall context into account when analyzing file system traces. This work calls for further research in this area and for forensic tools to provide necessary information for such contextual analysis, such as highlighting mass deletion, mass copying, and potential backdating.

In Vitro Characteristics of Phages to Guide 'Real Life' Phage Therapy Suitability.

The increasing problem of antibiotic-resistant pathogens has put enormous pressure on healthcare providers to reduce the application of antibiotics and to identify alternative therapies. Phages represent such an alternative with significant application potential, either on their own or in combination with antibiotics to enhance the effectiveness of traditional therapies. However, while phage therapy may offer exciting therapeutic opportunities, its evaluation for safe and appropriate use in humans needs to be guided initially by reliable and appropriate assessment techniques at the laboratory level. Here, we review the process of phage isolation and the application of individual pathogens or reference collections for the development of specific or "off-the-shelf" preparations. Furthermore, we evaluate current characterization approaches to assess the in vitro therapeutic potential of a phage including its spectrum of activity, genome characteristics, storage and administration requirements and effectiveness against biofilms. Lytic characteristics and the ability to overcome anti-phage systems are also covered. These attributes direct phage selection for their ultimate application as antimicrobial agents. We also discuss current pitfalls in this research area and propose that priority should be given to unify current phage characterization approaches.

Tracing mother-infant transmission of bacteriophages by means of a novel analytical tool for shotgun metagenomic datasets: METAnnotatorX.

BACKGROUND: Despite the relevance of viral populations, our knowledge of (bacterio) phage populations, i.e., the phageome, suffers from the absence of a "gold standard" protocol for viral DNA extraction with associated in silico sequence processing analyses. To overcome this apparent hiatus, we present here a comprehensive performance evaluation of various protocols and propose an optimized pipeline that covers DNA extraction, sequencing, and bioinformatic analysis of phageome data. RESULTS: Five widely used protocols for viral DNA extraction from fecal samples were tested for their performance in removal of non-viral DNA. Moreover, we developed a novel bioinformatic platform, METAnnotatorX, for metagenomic dataset analysis. This in silico tool facilitates a range of read- and assembly-based analyses, including taxonomic profiling using an iterative multi-database pipeline, classification of contigs at genus and species level, as well as functional characterizations of reads and assembled data. Performances of METAnnotatorX were assessed through investigation of seven mother-newborn pairs, leading to the identification of shared phage genotypes, of which two were genomically decoded and characterized. METAnnotatorX was furthermore employed to evaluate a protocol for the identification of contaminant non-viral DNA in sequenced datasets and was exploited to determine the amount of metagenomic data needed for robust evaluation of human adult-derived (fecal) phageomes. CONCLUSIONS: Results obtained in this study demonstrate that a comprehensive pipeline for analysis of phageomes will be pivotal for future explorations of the ecology of phages in the gut environment as well as for understanding their impact on the physiology and bacterial community kinetics as players of dysbiosis and homeostasis in the gut microbiota.