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International audience

Phages are complex biomolecular machineries that have to survive in a bacterial world. Phage genomes show many adaptations to their lifestyle such as shorter genes, reduced capacity for redundant DNA sequences, and the inclusion of tRNAs in their genomes. In addition, phages are not free-living, they require a host for replication and survival. These unique adaptations provide challenges for the bioinformatics analysis of phage genomes. In particular, ORF calling, genome annotation, noncoding RNA (ncRNA) identification, and the identification of transposons and insertions are all complicated in phage genome analysis. We provide a road map through the phage genome annotation pipeline, and discuss the challenges and solutions for phage genome annotation as we have implemented in the rapid annotation using subsystems (RAST) pipeline.

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.

Resistance of bacteria to antibiotics just keeps growing. Industry’s antibiotic pipeline is running dry. Before the pharmaceutical development of antibiotics, natural bacteriophages (=bacterial viruses) were commercialised and used to kill pathogenic bacteria. This therapeutic application of natural bacteriophages was called “bacteriophage therapy”. Today, countries like Poland, Georgia and Russia are still practising bacteriophage therapy. The European Union and “modern” medicine as a whole needs an urgent return of bacteriophage therapy as part of its armamentarium to fight bacterial resistance to antibiotics. This paper reflects on the issue and proposes a European regulatory frame that could fit the re-introduction of bacteriophage therapy without losing sight of safety, quality and efficacy aspects.

Many bacteria are able to assume a transient cell wall-deficient (or L-form) state under favorable osmotic conditions. Cell wall stress such as exposure to β-lactam antibiotics can enforce the transition to and maintenance of this state. L-forms actively proliferate and can return to the walled state upon removal of the inducing agent. We have adopted Escherichia coli as a model system for the controlled transition to and reversion from the L-form state, and have studied these dynamics with genetics, cell biology and ‘omics’ technologies. As such, a transposon mutagenesis screen underscored the requirement for the Rcs phosphorelay and colanic acid synthesis, while proteomics show only little differences between rods and L-forms. In contrast, metabolome comparison reveals the high abundance of lysophospholipids and phospholipids with unsaturated or cyclopropanized fatty acids in E. coli L-forms. This increase of membrane lipids associated with increased membrane fluidity may facilitate proliferation through bud formation. Visualization of the residual peptidoglycan with a fluorescently labeled peptidoglycan binding protein indicates de novo cell wall synthesis and a role for septal peptidoglycan synthesis during bud constriction. The DD-carboxypeptidases PBP5 and PBP6 are three- and four-fold upregulated in L-forms, indicating a specific role for regulation of crosslinking during L-form proliferation.

International audience

For practitioners at hospitals seeking to use natural (not genetically modified, as appearing in nature) bacteriophages for treatment of antibiotic-resistant bacterial infections (bacteriophage therapy), Europe's current regulatory framework for medicinal products hinders more than it facilitates. Although many experts consider bacteriophage therapy to be a promising complementary (or alternative) treatment to antibiotic therapy, no bacteriophage-specific framework for documentation exists to date. Decades worth of historical clinical data on bacteriophage therapy (from Eastern Europe, particularly Poland, and the former Soviet republics, particularly Georgia and Russia, as well as from today's 27 EU member states and the US) have not been taken into account by European regulators because these data have not been validated under current Western regulatory standards. Consequently, applicants carrying out standard clinical trials on bacteriophages in Europe are obliged to initiate clinical work from scratch. This paper argues for a reduced documentation threshold for Phase 1 clinical trials of bacteriophages and maintains that bacteriophages should not be categorized as classical medicinal products for at least two reasons: (1) such a categorization is scientifically inappropriate for this specific therapy and (2) such a categorization limits the marketing authorization process to industry, the only stakeholder with sufficient financial resources to prepare a complete dossier for the competent authorities. This paper reflects on the current regulatory framework for medicines in Europe and assesses possible regulatory pathways for the (re-)introduction of bacteriophage therapy in a way that maintains its effectiveness and safety as well as its inherent characteristics of sustainability and in situ self-amplification and limitation.

Childhood absence epilepsy is a prototypic form of generalized nonconvulsive epilepsy characterized by short impairments of consciousness concomitant with synchronous and bilateral spike-and-wave discharges in the electroencephalogram. For scientists in this field, the BS/Orl and BR/Orl mouse lines, derived from a genetic selection, constitute an original mouse model "in mirror" of absence epilepsy. The potential of MALDI imaging mass spectrometry (IMS) for the discovery of potential biomarkers is increasingly recognized. Interestingly, statistical analysis tools specifically adapted to IMS data sets and methods for the identification of detected proteins play an essential role. In this study, a new cross-classification comparative design using a combined discrete wavelet transformation-support vector machine classification was developed to discriminate spectra of brain sections of BS/Orl and BR/Orl mice. Nineteen m/z ratios were thus highlighted as potential markers with very high recognition rates (87-99%). Seven of these potential markers were identified using a top-down approach, in particular a fragment of Synapsin-I. This protein is yet suspected to be involved in epilepsy. Immunohistochemistry and Western Blot experiments confirmed the differential expression of Synapsin-I observed by IMS, thus tending to validate our approach. Functional assays are being performed to confirm the involvement of Synapsin-I in the mechanisms underlying childhood absence epilepsy.

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We describe the small-scale, laboratory-based, production and quality control of a cocktail, consisting of exclusively lytic bacteriophages, designed for the treatment of Pseudomonas aeruginosa and Staphylococcus aureus infections in burn wound patients. Based on successive selection rounds three bacteriophages were retained from an initial pool of 82 P. aeruginosa and 8 S. aureus bacteriophages, specific for prevalent P. aeruginosa and S. aureus strains in the Burn Centre of the Queen Astrid Military Hospital in Brussels, Belgium. This cocktail, consisting of P. aeruginosa phages 14/1 (Myoviridae) and PNM (Podoviridae) and S. aureus phage ISP (Myoviridae) was produced and purified of endotoxin. Quality control included Stability (shelf life), determination of pyrogenicity, sterility and cytotoxicity, confirmation of the absence of temperate bacteriophages and transmission electron microscopy-based confirmation of the presence of the expected virion morphologic particles as well as of their specific interaction with the target bacteria. Bacteriophage genome and proteome analysis confirmed the lytic nature of the bacteriophages, the absence of toxin-coding genes and showed that the selected phages 14/1, PNM and ISP are close relatives of respectively F8, phiKMV and phage G1. The bacteriophage cocktail is currently being evaluated in a pilot clinical study cleared by a leading Medical Ethical Committee.

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