Bacteriophage endolysins as a response to emerging foodborne pathogens

Continuous reports on foodborne outbreaks and increasing prevalence of antibiotic-resistant bacteria call for the development of novel preservation techniques that assure the safety of food products. Bacteriophage endolysins are highly active antibacterial peptidoglycan hydrolases that have evolved over millions of years to become the ultimate weapon against bacteria, with potential to be used as a food preservative. We here give an overview of all distinct endolysins encountered so far, we discuss their inherent qualities and review their role in preventing and controlling foodborne pathogens, divulging their potential for future applications.This work was supported by a grant from the Portuguese Foundation for Science and Technology in the scope of the Projects PTDC/AGR-ALI/100492/2008 and PTDC/AGR-ALI/121057/2010. Hugo Oliveira is paid by the FCT grant SFRH/BD/63734/2009

Hydrodynamic limit of multiscale viscoelastic models for rigid particle suspensions

We study the multiscale viscoelastic Doi model for suspensions of Brownian rigid rod-like particles, as well as its generalization by Saintillan and Shelley for self-propelled particles. We consider the regime of a small Weissenberg number, which corresponds to a fast rotational diffusion compared to the fluid velocity gradient, and we analyze the resulting hydrodynamic approximation. More precisely, we show the asymptotic validity of macroscopic nonlinear viscoelastic models, in form of so-called ordered fluid models, as an expansion in the Weissenberg number. The result holds for zero Reynolds number in 3D and for arbitrary Reynolds number in 2D. Along the way, we establish several new well-posedness and regularity results for nonlinear fluid models, which may be of independent interest.Comment: 64 page

The Role of Footwear, Foot Orthosis and Training-Related Strategies in the Prevention of Bone Stress Injuries: A Systematic Review and Meta-Analysis

International Journal of Exercise Science 16(3): 721-743, 2023. Objective: To evaluate the effectiveness of footwear, foot orthoses and training-related strategies to prevent lower extremity bone stress injury (BSI). Design: Systematic review and meta-analysis. Data sources: Four bibliographic databases (from inception until November 2021): Cochrane Central Register of Controlled Trials, MEDLINE, EMBASE and CINAHL. Eligibility criteria: Randomised controlled trials (RCTs) that assessed the risk of developing a BSI when using particular footwear, foot orthoses or training-related strategies such as muscle strengthening, stretching, and mechanical loading exercises. Results: Eleven studies were included in this systematic review. When wearing foot orthoses, the risk ratio of developing a BSI on any lower extremity bone is 0.47 (95% CI 0.26 to 0.87; p = 0.02). When doing pre-exercise dynamic stretching, the risk ratio of suffering a tibial BSI is 1.06 (95% CI 0.67 to 1.68; p = 0.79). No meta-analyses could be performed for footwear or training-related strategies. The quality of evidence for all these results is low considering the high risk of bias in each study, the low number of studies and the low number of cases in each study. Conclusion: This systematic review reveals the lack of high-quality studies in BSI prevention. Based on studies at high risk of bias, foot orthoses could potentially help prevent BSIs in the military setting. It is still unknown whether footwear and training-related strategies have any benefits. It is crucial to further investigate potential BSI prevention strategies in women and athletes. Research is also needed to assess the influence of running shoes and loading management on BSI incidence

Antibacterial activity on opportunistic Pseudomonas aeruginosa pathogen by a novel Salmonella phage endolysin

The Gram-negative pathogen Pseudomonas aeruginosa can cause severe infections of burn wound or cystic fibrosis on patients. Bacteriophage endolysin based strategy can offer a new alternative antimicrobial therapy. Endolysins are lytic enzymes that break down the peptidoglycan of bacterial cell wall at the late phage lytic cycle, however they are inactive on their own against Gram-negative bacteria when applied exogenously as recombinant proteins due to the peptidoglycan (endolysin substrate) protective outer membrane.We propose an innovative strategy to target Gram-negative Ps. aeruginosa based on the combination of endolysin enzymes and an outer membrane permeabilizing agent - ethylenediamine tetraacetic acid (EDTA).To validate this approach, we have isolated a novel Salmonella phage endolysin (68gpLys). Cloning this gene into E. coli expression system and subsequent large scale protein expression led to a high soluble yield of 14,3 mg/L of expression culture. In order to characterized it, muralytic assays on chloroform/Tris-HCl pretreated Ps. aeruginosa strain PAO1k (to remove the outer membrane) were made to check activity levels on substrate (398.05 Units/mM). The pH range was also determined with pH 7 being the optimum for the endolysin activity. For antimicrobial test, in vitro assays showed that incubation of 106 Ps. aeruginosa cells/mL with 0.5 mM EDTA and 5000 nM of 68gpLys, led to a strain inactivation of 3.42 ± 0.02 logarithmic reduction units in a time-frame of 30 min.Here we prove that the synergistic effect of endolysin 68gpLys with EDTA can significantly reduce Ps. aeruginosa contamination. These results suggests, the great potential of this strategy for prevention and/or control of other Gram-negative pathogens.Current work has been also development to engineer new endolysins with incorporated cell penetrating peptides (CPP), employing sited- and random-mutagenesis molecular techniques, to further enhance outer membrane permeabilization

Multicenter assessment of quantitative sensory testing (QST) for the detection of neuropathic-like pain responses using the topical capsaicin model

Background: The use of quantitative sensory testing (QST) in multicenter studies has been quite limited, due in part to lack of standardized procedures among centers. Aim: The aim of this study was to assess the application of the capsaicin pain model as a surrogate experimental human model of neuropathic pain in different centers and verify the variation in reports of QST measures across centers. Methods: A multicenter study conducted by the Quebec Pain Research Network in six laboratories allowed the evaluation of nine QST parameters in 60 healthy subjects treated with topical capsaicin to model unilateral pain and allodynia. The same measurements (without capsaicin) were taken in 20 patients with chronic neuropathic pain recruited from an independent pain clinic. Results: Results revealed that six parameters detected a significant difference between the capsaicin-treated and the control skin areas: (1) cold detection threshold (CDT) and (2) cold pain threshold (CPT) are lower on the capsaicin-treated side, indicating a decreased in cold sensitivity; (3) heat pain threshold (HPT) was lower on the capsaicin-treated side in healthy subjects, suggesting an increased heat pain sensitivity; (4) dynamic mechanical allodynia (DMA); (5) mechanical pain after two stimulations (MPS2); and (6) mechanical pain summation after ten stimulations (MPS10), are increased on the capsaicin-treated side, suggesting an increased in mechanical pain (P < 0.002). CDT, CPT and HPT showed comparable effects across all six centers, with CPT and HPT demonstrating the best sensitivity. Data from the patients showed significant difference between affected and unaffected body side but only with CDT. Conclusion: These results provide further support for the application of QST in multicenter studies examining normal and pathological pain responses

Investigating the biocontrol and anti-biofilm potential of a three phage cocktail against Cronobacter sakazakii in different brands of infant formula

Supplementary data to this article can be found online at: http://dx. doi.org/10.1016/j.ijfoodmicro.2017.04.009.In recent years, the microbiological safety of powdered infant formula has gained increasing attention due to the identification of contaminating C. sakazakii and its epidemiological link with life-threatening neonatal infections. Current intervention strategies have fallen short of ensuring the production of infant formula that is free from C. sakazakii. In this study, we describe the isolation and characterisation of three bacteriophages (phages) and their application as a phage cocktail to inhibit the growth of C. sakazakii in different brands of infant formula, while also assessing the phages ability to prevent biofilm formation. All three phages, isolated from slurry, possess a relatively broad host range, verified by their ability to infect across genera and species. When all three phages were combined and used as part of a phage cocktail, 73% coverage was obtained across all Cronobacter strains tested. Optimum thermo-tolerance and pH stability were determined between 4 °C37 °C, and pH 68, respectively, well within the normal range of application of infant formula. Genome sequencing and analysis revealed all the phages to be free from lysogenic properties, a trait which renders each favourable for phage therapy applications. As such, the combined-phage preparation (3 × 108 pfu/mL) was found to possess a strong bactericidal effect on C. sakazakii/C. sakazakii LUX cells ( 104 cfu/mL), resulting in a significant reduction in cell numbers, to below the limit of detection (< 10 cfu/mL). This was observed following a 20 h challenge in different brands of infant formula, where samples in the absence of the phage cocktail reached concentrations of ~ 109 cfu/mL. The phage cocktail also demonstrated promise in preventing the establishment of biofilm, as biofilm formation could not be detected for up to 48 h post treatment. These results highlight the potential application of this phage preparation for biocontrol of C. sakazakii contamination in reconstituted infant formula and also as a preventative agent against biofilm formation.This work was funded by Technological Sector Research Strand III ref. CRS/07/CR03. Angela Back from MRI Kiel is acknowledged for technical assistance in preparations for electron microscopy. Hugo Oliveira and Rob Lavigne contributed to the genome sequencing analysis, supported by the KULeuven GOA (GOA/15/006) Grant Phagebiosystems.info:eu-repo/semantics/publishedVersio

API480: features towards therapy in honeybee hives

American foulbrood disease (AFB) is a devastating bacterial disease affecting honeybees. It is caused by Paenibacillus larvae, a worldwide-distributed spore forming Gram-positive bacterium which spread easily across apiaries producing highly resistant spores. When AFB symptoms are found the burning of contaminated hives is mandatory causing serious economic losses [1]. Bacteriophages (phages) are being considered valuable solutions to the control of this infection [2-5]. So far, 48 Siphoviridae P. larvae phages sequences are known and most encode integration genes suggesting a temperate lifestyle. All of these 48 phages seem to have a common evolutionary ancestor showing an overall common structure. Their genomes were grouped into four clusters (with Fern, Harrison, Vegas and Halcyone as representative phages) and one singleton (phage Lily) [6].Project APILYSE,PTDC/CVT-EPI/4008/2014-POCI-01-0145-FEDER-016598,-funded by FEDER through COMPETE2020-Programa Operacional Competitividade e Internacionalização(POCI) and the Portuguese Foundation for Science and Technology(FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTec Norte operation(NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte.The work was also supported by CEB-UM that provided the laboratorial facilities to perform this research. HR was supported by FCT through the grant SFRH/BD/128859/2017. MB and RL were supported by KULeuven through a GOAgrant[3E140356]info:eu-repo/semantics/publishedVersio

Biochemical characterization of highly stable endolysins with a powerful and broad anti-Gram-negative lytic activity in the presence of weak acids

EMBO Conference on Viruses of Microbes III: Structure and Function - from Molecules to Communities (Programme and Abstract Book)info:eu-repo/semantics/publishedVersio

Erratum for Oliveira et al., "Functional Analysis and Antivirulence Properties of a New Depolymerase from a Myovirus That Infects Acinetobacter baumannii Capsule K45"

Volume 93, no. 4, e01163-18, 2019, https://doi.org/10.1128/JVI.01163-18. Page 14, Acknowledgments, line 4: (POCI-01-0145-FEDER-016678) should read (POCI-01-0145-FEDER-016643).info:eu-repo/semantics/publishedVersio

CkP1 bacteriophage, a S16-like myovirus that recognizes Citrobacter koseri lipopolysaccharide through its long tail fibers

The online version contains supplementary material available at https://doi.org/10.1007/s00253-023-12547-8.Citrobacter koseri is an emerging Gram-negative bacterial pathogen, which causes urinary tract infections. We isolated and characterized a novel S16-like myovirus CKP1 (vB\_CkoM\\_CkP1), infecting C. koseri. CkP1 has a host range covering the whole C. koseri species, i.e., all strains that were tested, but does not infect other species. Its linear 168,463-bp genome contains 291 coding sequences, sharing sequence similarity with the Salmonella phage S16. Based on surface plasmon resonance and recombinant green florescence protein fusions, the tail fiber (gp267) was shown to decorate C. koseri cells, binding with a nanomolar affinity, without the need of accessory proteins. Both phage and the tail fiber specifically bind to bacterial cells by the lipopolysaccharide polymer. We further demonstrate that CkP1 is highly stable towards different environmental conditions of pH and temperatures and is able to control C. koseri cells in urine samples. Altogether, CkP1 features optimal in vitro characteristics to be used both as a control and detection agent towards drug-resistant C. koseri infections.Open access funding provided by FCT|FCCN (b-on). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit. DB is supported by the Research Foundation – Flanders (FWO), grant number 1S69520N. JO received a predoctoral fellowship from the UAB and a FEMS research and training grant.info:eu-repo/semantics/publishedVersio