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Staphylococcus epidermidis has emerged as the most important pathogen in infections related to indwelling medical devices, and although these infections are not life-threatening, their frequency and the fact that they are extremely difficult to treat represent a serious burden on the public health system. Treatment is complicated by specific antibiotic resistance genes and the formation of biofilms. Hence, novel therapeutic strategies are needed to fight these infections. A novel bacteriophage CUB-EPI_14 specific to the bacterial species S. epidermidis was isolated from sewage and characterized genomically and phenotypically. Its genome contains a total of 46,098 bp and 63 predicted genes, among which some have been associated with packaging and lysis-associated proteins, structural proteins, or DNA- and metabolism-associated proteins. No lysogeny-associated proteins or known virulence proteins were identified in the phage genome. CUB-EPI_14 showed stability over a wide range of temperatures (from -20 °C to 50 °C) and pH values (pH 3-pH 12) and a narrow host range against S. epidermidis. Potent antimicrobial and antibiofilm activities were observed when the phage was tested against a highly susceptible bacterial isolate. These encouraging results open the door to new therapeutic opportunities in the fight against resilient biofilm-associated infections caused by S. epidermidis. ispartof: VIRUSES-BASEL vol:14 issue:6 ispartof: location:Switzerland status: published

La invención se refiere a una proteína quimérica con una actividad lítica mejorada contraStaphylococcus sp. y una mayor estabilidad que las endolisinas precursoras de la técnica actual. Por tanto, la invención se refiere a esta proteína quimérica y su uso como antimicrobiano, siendo útil para tratar y/o prevenir una infección porStaphylococcus sp., para eliminar biopelículas deStaphylococcus sp., para controlar la contaminación porStaphylococcus sp. en la industria alimentaria y para descontaminar superficies inanimadas sospechosas de contenerStaphylococcus sp. Por tanto, la presente invención pertenece al campo del tratamiento de infecciones o contaminaciones bacterianas., Consejo Superior de Investigaciones Científicas (CSIC), T3 Traducción de patente europea

Staphylococcus epidermidis has emerged as the most important pathogen in infections related to indwelling medical devices, and although these infections are not life-threatening, their frequency and the fact that they are extremely difficult to treat represent a serious burden on the public health system. Treatment is complicated by specific antibiotic resistance genes and the formation of biofilms. Hence, novel therapeutic strategies are needed to fight these infections. A novel bacteriophage CUB-EPI_14 specific to the bacterial species S. epidermidis was isolated from sewage and characterized genomically and phenotypically. Its genome contains a total of 46,098 bp and 63 predicted genes, among which some have been associated with packaging and lysis-associated proteins, structural proteins, or DNA- and metabolism-associated proteins. No lysogeny-associated proteins or known virulence proteins were identified in the phage genome. CUB-EPI_14 showed stability over a wide range of temperatures (from -20 degrees C to 50 degrees C) and pH values (pH 3-pH 12) and a narrow host range against S. epidermidis. Potent antimicrobial and antibiofilm activities were observed when the phage was tested against a highly susceptible bacterial isolate. These encouraging results open the door to new therapeutic opportunities in the fight against resilient biofilm-associated infections caused by S. epidermidis.

Brieuc Van Nieuwenhuyse1, Dimitri Van der Linden1,2, Olga Chatzis2, Cédric Lood3,4, Jeroen Wagemans3, Rob Lavigne4, Catherine de Magnée5, Étienne Sokal1,6, Hector Rodriguez-Villalobos7, Sarah Djebara8, Maya Merabishvili9, Patrick Soentjens8, Jean-Paul Pirnay9. 1Institute of Experimental and Clinical Research's Pediatric department, UCLouvain, Brussels, Belgium; 2Pediatric Infectious Diseases, General Pediatrics Department, Cliniques universitaires Saint-Luc, Brussels, Belgium; 3Department of Biosystems, Laboratory of Gene Technology, KULeuven, Leuven, Belgium; 4Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, KULeuven, Leuven, Belgium; 5Pediatric and Transplantation Surgery, Cliniques universitaires Saint-Luc, Brussels, Belgium; 6Pediatric Hepatology and Gastro-enterology, Cliniques universitaires Saint-Luc, Brussels, Belgium; 7Department of Microbiology, Cliniques universitaires Saint-Luc, Brussels, Belgium; 8Center for Infectious Diseases, Queen Astrid Military Hospital, Brussels, Belgium; 9Laboratory for Molecular and Cellular Technology, Queen Astrid Military Hospital, Brussels, Belgium A 14-month old boy undergoes a first liver transplantation (LT) (Day 0), from an ABO-incompatible living donor. On D+20, we detect a fecal carriage of an extensively drug-resistant (XDR) Pseudomonas aeruginosa (Pa) strain. Besides intermediate susceptibility to aztreonam and colistin and susceptibility to gentamycin, the strain is resistant to all other antibiotics. On D+53, the child enters a severe septic state due to a bacteremia with the same Pa strain. New antibiogram suggests a resistance to colistin. Liver bilomas' drainage material is cultured and grows the same Pa strain. Admission to the pediatric intensive care unit and adjunction of intravenous (IV) aztreonam, gentamycin, and colistin led to no improvement on the microbiological or clinical levels during the next four days. By collaborating with Queen Astrid Military Hospital (Brussels, Belgium), we initiated phage therapy (PT) on D+57 in accordance to the Article 37 of the Declaration of Helsinki and with the patient's parents' consent. PT is the use of lytic bacteriophage viruses to achieve antibacterial effect. Phage cocktail BFC1 contains two anti-Pa phages (PNM and 14/1) and one anti-Staphylococcus aureus phage (ISP). BFC1 was administered in situ by instillations through biliary catheter during six days, and in IV for 86 days (72 days until 2nd LT, 14 days afterwards), the longest described duration for IV PT in a child. Previous antibiotic therapy was pursued all along. Intraoperative PT was performed during 2nd LT by bathing the peritoneal cavity in phage solution during the anhepatic phase. To further our understanding of the case, seven Pa isolates, both bloodborne and liver-borne, were sequenced. Serum samples obtained before, during, and after phage therapy were analyzed through double agar overlay method to search for phage immune neutralization (PIN). Phage-induced virulence tradeoffs (PIVT) assays were performed in a Galleria mellonella model. In vitro phage-antibiotic interactions were evaluated with OmniLog® system. PT initiation was followed by immediate (

Objectives: Endolysins, bacteriophage-derived enzymes that degrade the bacterial cell wall, are a novel class of antimicrobials that are breaking through in human and veterinary medicine. In this work PlySs2 & -9, two endolysins derived from Streptococcus suis bacteriophages, have been characterized for their lytic activity towards Streptococcus uberis. This latter mentioned pathogen is among the top causative agents of bovine mastitis, defined as an inflammation of the cow’s udder, with high economic impact on the dairy industry. S. uberis can circumvent standard antibiotic therapy due to biofilm formation, intracellular persistence and antimicrobial resistance, which subsequently leads to chronic infections that remain difficult to eradicate. Methods: PlySs2 & -9 were synthesized after their discovery in S. suis prophages and standard cloning techniques were used to clone out individual subdomains. Polyhistidine-tagged proteins were expressed in Escherichia coli BL21 (DE3) and chromatographically purified. A combination of qualitative (spot-on-plate) and quantitative (turbidity reduction, logarithmic reduction & MIC) assays evaluated the lytic activity on reference strains and (sub)clinical isolates, whilst fluorescence microscopy evaluated binding of the AlexaFluor-conjugated cell-wall binding subdomains. Results & Conclusions: PlySs2 and PlySs9 both have in vitro muralytic activity against a broad range of (sub)clinical bovine mastitis S. uberis strains. In depth characterization further substantiated PlySs9 as the more potent endolysin, killing one additional log and showing a lower MIC than PlySs2. Additional molecular dissection of both endolysins showed that the individual subdomains have deficient killing and binding activity compared to their respective parental endolysin.

The deeply intertwined evolutionary history between bacteriophages and bacteria has endowed phages with highly specific mechanisms to hijack bacterial cell metabolism for their propagation. Here, we present a comprehensive, phage-driven strategy to reveal novel antibacterial targets by the exploitation of phage-bacteria interactions. This strategy will enable the design of small molecules, which mimic the inhibitory phage proteins, and allow the subsequent hit-to-lead development of these antimicrobial compounds. This proposed small molecule approach is distinct from phage therapy and phage enzyme-based antimicrobials and may produce a more sustainable generation of new antibiotics that exploit novel bacterial targets and act in a pathogen-specific manner.

The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell. IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.