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Human norovirus (HuNoV) is an enteric infectious pathogen belonging to the Caliciviridae family that causes occasional epidemics. Circulating alcohol-tolerant viral particles that are readily transmitted via food-borne routes significantly contribute to the global burden of HuNoV-induced gastroenteritis. Moreover, contact with enzymes secreted by other microorganisms in the environment can impact the infectivity of viruses. Hence, understanding the circulation dynamics of Caliciviridae is critical to mitigating epidemics. Accordingly, in this study, we screened whether environmentally abundant secretase components, particularly proteases, affect Caliciviridae infectivity. Results showed that combining Bacillaceae serine proteases with epsilon-poly-l-lysine (EPL) produced by Streptomyces—a natural antimicrobial—elicited anti-Caliciviridae properties, including against the epidemic HuNoV GII.4_Sydney_2012 strain. In vitro and in vivo biochemical and virological analyses revealed that EPL has two unique synergistic viral inactivation functions. First, it maintains an optimal pH to promote viral surface conformational changes to the protease-sensitive structure. Subsequently, it inhibits viral RNA genome release via partial protease digestion at the P2 and S domains in the VP1 capsid. This study provides new insights regarding the high-dimensional environmental interactions between bacteria and Caliciviridae, while promoting the development of protease-based anti-viral disinfectants. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa and Klebsiella pneumoniae are notorious for their resistance to antibiotics and propensity for biofilm formation, posing significant threats to human health. Epsilon-poly-L-lysine (ε-PL) emerges as a naturally occurring antimicrobial poly(amino acid), which positions it as a prospective agent for addressing challenges linked to multidrug resistance. ε-PL symbolizes a promising avenue in the pursuit of efficacious therapeutic strategies and warrants earnest consideration within the realm of clinical treatment. Thus, our objective was to determine the antibiotic susceptibility profiles of 38 selected P. aeruginosa and ESBL-producing K. pneumoniae clinical isolates and determine the ability of ε-PL to inhibit biofilm formation. After PCR analysis, detection of genes related to β-lactamases was observed among the selected isolates of P. aeruginosa [blaSPM (35.7%), blaKPC (35.7%), blaSHV (14.3%), blaCTX-M (14.3%), blaOXA (14.3%), blaTEM (7.1%), blaPER (7.1%), blaVIM (7.1%), and blaVIM-2 (7.1%)] and K. pneumoniae [blaCTX-M (91.7%), blaTEM (83.3%), blaKPC (16.7%), blaNDM (12.5%), and blaOXA (4.2%)]. The results of testing the activity of ε-PL against the clinical isolates showed relatively high minimum inhibitory concentrations (MICs) for the P. aeruginosa (range: 8–64 µg/mL) and K. pneumoniae isolates (range: 16–32 µg/mL). These results suggest the need for prior optimization of ε-PL concerning its viability as an alternative to antibiotics for treating infections caused by P. aeruginosa and K. pneumoniae of clinical origin. It is noteworthy that, in the context of a low antibiotic discovery rate, ε-PL could play a significant role in this quest, considering its low toxicity and the unlikely development of resistance. Upon exposure to ε-PL, P. aeruginosa and K. pneumoniae isolates exhibited a reduction in biofilm production, with ε-PL concentration showing an inverse relationship, particularly in isolates initially characterized as strong or moderate producers, indicating its potential as a natural antimicrobial agent with further research needed to elucidate optimal concentrations and application methods across different bacterial species. Further research is needed to optimize its use and explore its potential in various applications. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance has become a major problem over the years and threatens to remain in the future, at least until a solution is found. Silver nanoparticles (Ag-NPs) and antimicrobial polymers (APs) are known for their antimicrobial properties and can be considered an alternative approach to fighting resistant microorganisms. Hence, the main goal of this research is to shed some light on the antimicrobial properties of Ag-NPs and APs (chitosan (CH), poly-L-lysine (PLL), ε-poly-L-lysine (ε-PLL), and dopamine (DA)) when used alone and complexed to explore the potential enhancement of the antimicrobial effect of the combination Ag-NPs + Aps. The resultant nanocomplexes were chemically and morphologically characterized by UV-visible spectra, zeta potential, transmission electron microscopy, and Fourier-transform infrared spectroscopy. Moreover, the Ag-NPs, APs, and Ag-NPs + APs nanocomplexes were tested against Gram-positive Staphylococcus aureus (S. aureus) and the Gram-negative Escherichia coli (E. coli) bacteria, as well as the fungi Candida albicans (C. albicans). Overall, the antimicrobial results showed potentiation of the activity of the nanocomplexes with a focus on C. albicans. For the biofilm eradication ability, Ag-NPs and Ag-NPs + DA were able to significantly remove S. aureus preformed biofilm, and Ag-NPs + CH were able to significantly destroy C. albicans biofilm, with both performing better than Ag-NPs alone. Overall, we have proven the successful conjugation of Ag-NPs and APs, with some of these formulations showing potential to be further investigated for the treatment of microbial infections. [ABSTRACT FROM AUTHOR]

Bacterial infections are the major inhibitory factors for natural wound healing. 3D bioprinted nanocellulose-based composite scaffold with a desirable porous and shape specificity is highly effective for antibacterial applications. However, the slow self-healing and lack of antibacterial activities of cellulose cause bacterial infection. In this work, Epsilon-poly-L-lysine (EPL) bioconjugated cellulose nanofibrils (TCNFs) were subjected to bioprinting with a customizable 3D scaffold with biocompatible and antibacterial activities. Results show that the 3D composite scaffold possesses a mesoporous structure (2–50 nm) and high specific surface area (232.25 m2/g) that ensure a high adsorption capacity of red blood cells in wound healing. The in vitro cytocompatibility of the scaffold is confirmed by the growth and proliferation of NIH 3T3 fibroblast cells in a 3D cell culture study. In addition, the 3D composite scaffold shows antimicrobial activity against wound-infecting pathogens, namely Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. In summary, this work integrates the uniqueness of 3D bioprinting and the inherent characteristics of TCNF/EPL-based composite into a flexible 3D scaffold to achieve antibacterial performance and cytocompatibility for biomedical applications. [ABSTRACT FROM AUTHOR]

Introduction: e-poly-L-lysine (e-PL) is a high value, widely used natural antimicrobial peptide additive for foods and cosmetic products that is mainly produced by Streptomyces albulus. In previous work, we developed the high-yield industrial strain S. albulus WG-608 through successive rounds of engineering. Methods: Here, we use integrated physiological, transcriptomic, and proteomics association analysis to resolve the complex mechanisms underlying high e-PL production by comparing WG-608 with the progenitor strain M-Z18. Results: Our results show that key genes in the glycolysis, pentose phosphate pathway, glyoxylate pathway, oxidative phosphorylation, and L-lysine biosynthesis pathways are differentially upregulated in WG-608, while genes in the biosynthetic pathways for fatty acids, various branched amino acids, and secondary metabolite by-products are downregulated. This regulatory pattern results in the introduction of more carbon atoms into L-lysine biosynthesis and e-PL production. In addition, significant changes in the regulation of DNA replication, transcription, and translation, two component systems, and quorum sensing may facilitate the adaptability to environmental pressure and the biosynthesis of e-PL. Overexpression of ppk gene and addition of polyP6 further enhanced the e-PL production. Discussion: This study enables comprehensive understanding of the biosynthetic mechanisms of e-PL in S. albulus WG-608, while providing some genetic modification and fermentation strategies to further improve the e-PL production. [ABSTRACT FROM AUTHOR]

Both Gram-negative and Gram-positive bacteria can release vesicle-like structures referred to as bacterial extracellular vesicles (BEVs), which contain various bioactive compounds. BEVs play important roles in the microbial community interactions and host-microbe interactions. Markedly, BEVs can be delivered to host cells, thus modulating the development and function of the innate immune system. To clarify the compositions and biological functions of BEVs, we need to collect these vesicles with high purity and bioactivity. Here we propose an isolation strategy based on a broad-spectrum antimicrobial epsilon-poly-L-lysine (ϵ-PL) to precipitate BEVs at a relatively low centrifugal speed (10,000 × g). Compared to the standard ultracentrifugation strategy, our method can enrich BEVs from large volumes of media inexpensively and rapidly. The precipitated BEVs can be recovered by adjusting the pH and ionic strength of the media, followed by an ultrafiltration step to remove ϵ-PL and achieve buffer exchange. The morphology, size, and protein composition of the ϵ-PL-precipitated BEVs are comparable to those purified by ultracentrifugation. Moreover, ϵ-PL-precipitated BEVs retained the biological activity as observed by confocal microscopy studies. And THP-1 cells stimulated with these BEVs undergo marked reprogramming of their transcriptome. KEGG analysis of the differentially expressed genes showed that the signal pathways of cellular inflammatory response were significantly activated. Taken together, we provide a new method to rapidly enrich BEVs with high purity and bioactivity, which has the potential to be applied to BEVsrelated immune response studies. [ABSTRACT FROM AUTHOR]

Epsilon-Poly-L-lysine (ε-PL) is a non-toxic cationic homopolypeptide with unique bioactive properties. In this study, two novel polylysine producers, Paenibacillus polymyxa HS6 and Stenotrophomonas maltophilia YS8 were isolated from different soil samples and identified by 16S rDNA sequence analysis. To obtain pure cationic polypeptide from these bacteria, chemical precipitation with sodium tetraphenylborate was used. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, High-performance liquid chromatography, and Fourier-transform infrared spectroscopy confirmed the purified cationic compound to be polylysine. The antioxidant activity of ε-PL was determined by the 2,2-diphenyl-1-picryl-hydrazyl-hydrate method. Moreover, the anticancer and cytotoxic effects were evaluated against MCF-7, HT-29, and L929 cells by thiazolyl blue tetrazolium bromide and flow cytometry. The maximum yields of 1.8 and 0.39 g/L polylysine were obtained by P. polymyxa HS6 and S. maltophilia YS8, respectively. The results showed that radical scavenging activity of polylysine at a minimum concentration (0.18 mg/mL) was at least 7% which increased to 90% at 8 mg/mL. Furthermore, the highest anticancer activity was observed against the MCF-7 cells (99.5%) at 0.5 mg/mL concentration, while almost no toxicity was observed towards L929 cells. Based on the results obtained in this study, it is suggested that the polylysine produced by P. polymyxa HS6 and S. maltophilia YS8 is a potential bioactive compound with significant anticancer and antioxidant properties. [ABSTRACT FROM AUTHOR]

Foodborne Enterobacteriaceae pathogens, especially Salmonella, still seriously threaten food safety. To establish a foundation for further developing phage- and endolysin-based methods combating these pathogens, in this study, the newly isolated Salmonella-virus-FelixO1 phage BPS15S6 for biocontrol purposes was characterised by genomic bioinformatic analysis, and then its endolysin LyS15S6 was obtained using a prokaryotic expression system, characterised in vitro and evaluated in the antibacterial efficacy. It was shown that BPS15S6 had an 87,609-bp genome with 130 open reading frames and does not appear to carry known lysogeny-associated genes and other damaging genetic determinants and is unlikely to perform generalised transduction. Furthermore, LyS15S6 was determined to possess the high enzymatic activity of 1,001,000 U mg−1 and the broad spectrum of lysing 56 tested Gram-negative strains. The assays of thermostability and optimum pH revealed that LyS15S6 was stable up to 40 °C and more active at pH 7. Notably, we demonstrate that edible ε-poly-l-lysine (EPL) can be used as an outer-membrane permeabiliser to improve the antibacterial performance of endolysins. When combined with 1 μg ml−1 EPL, 2 μM LyS15S6 could cause 3–4 log viable cell reductions of the three tested Enterobacteriaceae pathogens in vitro after 2 h of reaction at 25 °C and 2.56 and 3.14 log reductions of Salmonella ATCC13076 after 15 min of reaction at 25 °C and 2 h of reaction at 8 °C respectively. A new strategy, the combined application of endolysins and edible EPL for combating Enterobacteriaceae pathogens in food, is thus presented in this work. [ABSTRACT FROM AUTHOR]