MEndoB, a chimeric lysin featuring a novel domain architecture and superior activity for the treatment of staphylococcal infections.

Bacterial infections are a growing global healthcare concern, as an estimated annual 4.95 million deaths are associated with antimicrobial resistance (AMR). Methicillin-resistant Staphylococcus aureus is one of the deadliest pathogens and a high-priority pathogen according to the World Health Organization. Peptidoglycan hydrolases (PGHs) of phage origin have been postulated as a new class of antimicrobials for the treatment of bacterial infections, with a novel mechanism of action and no known resistances. The modular architecture of PGHs permits the creation of chimeric PGH libraries. In this study, the chimeric enzyme MEndoB was selected from a library of staphylococcal PGHs based on its rapid and sustained activity against staphylococci in human serum. The benefit of the presented screening approach was illustrated by the superiority of MEndoB in a head-to-head comparison with other PGHs intended for use against staphylococcal bacteremia. MEndoB displayed synergy with antibiotics and rapid killing in human whole blood with complete inhibition of re-growth over 24 h at low doses. Successful treatment of S. aureus-infected zebrafish larvae with MEndoB provided evidence for its in vivo effectiveness. This was further confirmed in a lethal systemic mouse infection model in which MEndoB significantly reduced S. aureus loads and tumor necrosis factor alpha levels in blood in a dose-dependent manner, which led to increased survival of the animals. Thus, the thorough lead candidate selection of MEndoB resulted in an outstanding second-generation PGH with in vitro, ex vivo, and in vivo results supporting further development.IMPORTANCEOne of the most pressing challenges of our era is the rising occurrence of bacteria that are resistant to antibiotics. Staphylococci are prominent pathogens in humans, which have developed multiple strategies to evade the effects of antibiotics. Infections caused by these bacteria have resulted in a high burden on the health care system and a significant loss of lives. In this study, we have successfully engineered lytic enzymes that exhibit an extraordinary ability to eradicate staphylococci. Our findings substantiate the importance of meticulous lead candidate selection to identify therapeutically promising peptidoglycan hydrolases with unprecedented activity. Hence, they offer a promising new avenue for treating staphylococcal infections.

Analysis of circulating ceramides and hexosylceramides in patients with coronary artery disease and type II diabetes mellitus.

BACKGROUND: Cardiovascular disease (CVD) remains the leading cause of death worldwide. The main driving force behind this association is coronary artery disease (CAD), the manifestation of atherosclerosis in the coronary circulation. Cornerstones in the development of CAD are pathologies in lipid metabolism. In recent years, ongoing research has identified ceramides, a subclass of sphingolipids to be mediators of CVD. The aim of this study is to investigate the influence of type II diabetes mellitus (DM) on circulating ceramides and hexosylceramides (HexCers) in CAD patients. METHODS: 24 patients aged 40-90 years with CAD confirmed by angiography were included into a pilot study. Patients with DM were identified by analysis of discharge letters or other medical documents available at the study center. During coronary angiography, arterial blood samples were collected and quantification of sphingolipids in patient serum was performed by mass spectrometry. RESULTS: Statistical analysis showed nine significantly different HexCers in CAD patients with DM compared to patients without DM. Among the nine significantly regulated HexCers, we identified seven d18:1 HexCers. This group contributes to the fourth most abundant subgroup of total ceramides and HexCers in this dataset. HexCer-d18:1-23:1(2-OH) showed the strongest downregulation in the patient group with DM. CONCLUSION: This study suggests that levels of circulating HexCers are downregulated in patients with CAD and concomitant DM compared to patients without DM. Further research is needed to investigate the underlying mechanisms and the suitability of HexCers as possible mediators and/or prognostic markers in CAD.

Bacteriophage S6 requires bacterial cellulose for Erwinia amylovora infection.

Bacteriophages are highly selective in targeting bacteria. This selectivity relies on the specific adsorption of phages to the host cell surface. In this study, a Tn5 transposon mutant library of Erwinia amylovora, the causative agent of fire blight, was screened to identify bacterial receptors required for infection by the podovirus S6. Phage S6 was unable to infect mutants with defects in the bacterial cellulose synthase operon (bcs). The Bcs complex produces and secretes bacterial cellulose, an extracellular polysaccharide associated with bacterial biofilms. Deletion of the bcs operon or associated genes (bcsA, bcsC and bcsZ) verified the crucial role of bacterial cellulose for S6 infection. Application of the cellulose binding dye Congo Red blocked infection by S6. We demonstrate that infective S6 virions degraded cellulose and that Gp95, a phage-encoded cellulase, is involved to catalyse the reaction. In planta S6 did not significantly inhibit fire blight symptom development. Moreover, deletion of bcs genes in E. amylovora did not affect bacterial virulence in blossom infections, indicating that sole application of cellulose targeting phages is less appropriate to biologically control E. amylovora. The interplay between cellulose synthesis, host cell infection and maintenance of the host cell population is discussed.

Ultrasensitive Detection of Salmonella and Listeria monocytogenes by Small-Molecule Chemiluminescence Probes.

Detection of Salmonella and L. monocytogenes in food samples by current diagnostic methods requires relatively long time to results (2-6 days). Furthermore, the ability to perform environmental monitoring at the factory site for these pathogens is limited due to the need for laboratory facilities. Herein, we report new chemiluminescence probes for the ultrasensitive direct detection of viable pathogenic bacteria. The probes are composed of a bright phenoxy-dioxetane luminophore masked by triggering group, which is activated by a specific bacterial enzyme, and could detect their corresponding bacteria with an LOD value of about 600-fold lower than that of fluorescent probes. Moreover, we were able to detect a minimum of 10 Salmonella cells within 6 h incubation. The assay allows for bacterial enrichment and detection in one test tube without further sample preparation. We anticipate that this design strategy will be used to prepare analogous chemiluminescence probes for other enzymes relevant to specific bacteria detection and point-of-care diagnostics.