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In last decades, bacterial resistance to first choice antibiotics has been drastically increasing, therefore, the research of new antimicrobial substances is of great importance. This rising problem with bacterial resistance to existing antibiotics affects not only the health care institutes but also food plants. S. aureus and S. enteritidis pose a number of challenges to the food industry and cause foodborne illness in humans. In addition, due to their favourable elemental composition, oyster mushrooms (Pleurotus sp.) are a reservoir of bioactive compounds that give them remarkable antibacterial potential. P. ostreatus is of great economic importance and is the second most cultivated edible mushroom, therefore information about its possible targets on bacterial cells is of great importance for use as a dietary supplement or medicinal purposes. The results of the antibacterial assay showed that tested bacterial strains were susceptible to the methanol extract of P. ostreatus (PoME), while microbicidal activity was only detected against Gram-positive bacteria. Scanning electron microscopy (SEM) micrographs suggested that extract acted on cytoplasmic membrane of S. aureus, while the cell envelope of S. Enteritidis was the most likely target. Natural extracts may outperform individual bioactive compounds due to the synergistic interaction between the metabolites, which can enhance the effects of the individual components. Extracts rich in antibacterials are emerging as alternatives to synthetic antibiotics in the food and health sectors. Among these, crude mushroom extracts are particularly sought after for their diverse bioactive ingredients, as they can combat resistant strains of bacteria due to their different targets and modes of action. PoME can be used as an effective antimicrobial agent, suitable for applications aiming to eradicate foodborne pathogens, thus enhancing food safety.

Pseudomonas aeruginosa has been recognized as a priority pathogen by World Health Organization, due to the emergence of multidrug-resistant (MDR) strains. Thus, new treatment options such as antivirulence strategy is urgently needed. This strategy is based on the disruption of quorum sensing (QS) activity of this pathogen. The focus of this research was to explore the anti-QS activity of four selected medicinal mushrooms (Lentinula edodes, Cantharellus cibarius, Trametes versicolor and Pleurotus ostreatus) extracts on MDR clinical isolate P. aeruginosa MMA83. Another aim was to check their cytotoxicity on Caenorhabditis elegans AU37 (glp-4(bn2) I; sec-1(km4). Among three types of mushroom extracts - hot water polysaccharide extracts (WPE), hot alkali polysaccharide extracts (APE) and methanol extracts (Met), APE extracts downregulated all tested QS and virulence factors genes of P. aeruginosa MMA83. The most prominent effect was observed for C. cibarius APE extract, lowering expression from 2-fold (for lasI gene) to 20-fold for lasB gene. Extracts didn’t show cytotoxic effect on C. elegans. The efficacy of APE extracts in lowering the expression of QS and virulence factors genes of P. aeruginosa MMA83 indicate that these extracts can reduce pathogenicity of P. aeruginosa. Also, they possess one of the desirable biotechnology features – the absence of cytotoxicity. Anti-QS and antivirulence effect of APE extracts on P. aeruginosa envisages these extracts as the promising therapeutic candidates for the development of next-generation antivirulence agents.

Introduction: Brevibacillus laterosporus is a promising microbiological agent that can be used to prevent and control destructive diseases affecting honey bee colonies. In the presentstudy, the short-term effect of the B. laterosporus BGSP11 bee diet on microbiota and mycobiota was investigated. Methods: The honey bee diet was supplemented with spores of B. laterosporus BGSP11 at a concentration of 1×108 CFU/mL in sucrose solution. Metabarcoding analysis of the bee microbial community profile was performed based on 16S RNA (bacteriobiota) and Internally Transcribes Spacer (ITS) region (mycobiota) obtained using MiSeq Illumina sequencing. The QIIME2 v2021.4 pipeline was used to analyze the obtained amplicon data library. Results: The results show that the BGSP11 bee diet slightly altered the bee microbiota and did not lead to potentially harmful changes in the bacterial microbiota. Moreover, it can potentially induce positive changes, mainly reflected in the reduction of opportunistic bacteria. On the other hand, the treatment had a greater effect on mycobiota. However, the changesin the bee mycobiome caused by the treatment cannot be considered a priori as beneficial or harmful,since the interaction between the bee and its mycobiome is not sufficiently studied. The observed positive changes in the bee mycobiome are mainly reflected in the reduction of phytopathogenic fungi that may affect the organoleptic and techno-functional properties of honey. Conclusion: This pilot study suggests that the introduction of BGSP11 in beekeeping practice as a biological agent could be considered due to no harmful effects observed on the microbiota of bees.

In the current study, honey bees’ diet was supplemented with spores of Brevibacillus laterosporus BGSP11 at concentration of 108 CFU/ml in sucrose solution and its short-term effects on their micro- and mycobiota have been analyzed using Illumina MiSeq sequencing. Obtained results indicate that this treatment does not lead to potentially harmful changes in the bacterial microbiome of worker bees, slightly affecting the composition of core microbiota. Moreover, several potentially beneficial changes have been observed. The treatment has led to a significant increase in the abundance of Snodgrassella alvi, and species from Lactobacillus and Bifidobacterium genera which play important roles in protection against several honey bee pathogens. Simultaneously, B. laterosporus enriched diet have led to almost complete eradication of Enterobacteriaceae family, the taxon that contains several putative pathogen species. On the other hand, the treatment affected mycobiota more profoundly, which was expected considering the greater instability compared to microbiota. Although the observed changes in honey bee mycobiome cannot be considered a priori beneficial or harmful, since the interaction between the bee and its mycobiome has not been sufficiently studied, certain beneficial consequences of the treatment have been observed. They are primarily reflected in the reduction of phytopathogenic fungi that can affect the organoleptic and techno-functional characteristics of honey. In addition, before introducing B. laterosporus in beekeeping practice as a biological agent for pathogen control it is necessary to perform more thorough studies of the impact on the honey bee microbiome, immune system, physiology and economic characteristics of honey bee colonies.

Lactic acid bacterium Lactococcus lactis BGBU1-4 produces 43 amino acids (aa) long bacteriocin, lactolisterin BU (LBU), a 5.161 kDa peptide with potent antibacterial activity against many Gram-positive pathogens. In addition, BGBU1-4 produces an additional unknown product of 3.642 kDa with antibacterial activity. Here, we determined that the significant amount of naturally produced LBU breaks down to create a 3.642 kDa truncated form of LBU bacteriocin consisting of 31 N-terminal aa (LBU1-31) that exhibits 12.5% the antibacterial activity of the full-length LBU. We showed that chemically synthesized LBU is stable and 50% less active than native LBU, and so we used the synthetic peptides of LBU and its variants to further study their activities and antibacterial potential. Deletion analysis of LBU revealed that the 24 N-terminal aa of LBU (LBU1-24) are responsible for antibacterial activity, while downstream aa (25–43) determine the species-specific effectiveness of LBU. Although LBU1-31 contains aa 1–24, the truncation at position 31 is predicted to change the structure within aa 15–31 and might impact on antibacterial activity. Intriguingly, whole genome sequencing and genome mining established that BGBU1-4 is abundant in genes that encode potential antibacterials, but produces LBU and its breakdown product LBU1-31 exclusively.

Signal transduction systems are the key players of bacterial adaptation and survival. The orthodox two-component signal transduction systems perceive diverse environmental stimuli and their regulatory response leads to cellular changes. Although rarely described, the unorthodox three-component systems are also implemented in the regulation of major bacterial behavior such as the virulence of clinically relevant pathogen P. aeruginosa. Previously, we described a novel three-component system in P. capeferrum WCS358 (RclSAR) where the sensor kinase RclS stimulates the intI1 transcription in stationary growth phase. In this study, using rclS knock-out mutant, we identified RclSAR regulon in P. capeferrum WCS358. The RNA sequencing revealed that activity of RclSAR signal transduction system is growth phase dependent with more pronounced regulatory potential in early stages of growth. Transcriptional analysis emphasized the role of RclSAR in global regulation and indicated the involvement of this system in regulation of diverse cellular activities such as RNA binding and metabolic and biocontrol processes. Importantly, phenotypic comparison of WCS358 wild type and Delta rclS mutant showed that RclS sensor kinase contributes to modulation of antibiotic resistance, production of AHLs and siderophore as well as host cell adherence and cytotoxicity. Finally, we proposed the improved model of interplay between RclSAR, RpoS and LasIR regulatory systems in P. capeferrum WCS358.

Aims The aim of this study was to construct the improved pMAL expression vector to increase the efficacy of purification of small native peptides and their clear-cut separation from MBP tag. The modifications we introduced can be applied to many expression vectors. Methods and Results To improve the pMAL expression vector, we introduced the His(6) tag and the enterokinase cleavage site (Ek) downstream from the MBP tag and Xa cleavage site on the original vector. For cloning of a desired peptide DNA, the enterokinase site contains a unique BsaBI restriction site adjacent to the original multi-cloning site. This redesigned pMAL vector was optimized for the purification of cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides. The purification of native and active peptide (P) was obtained following two-step affinity chromatography. In the first step, the entire MBP-His(6)-Ek-P fusion protein is purified using the Ni-NTA agarose column. This fusion protein was cleaved with active His(6) tagged enterokinase. In the second step, the further purification was performed by column containing the mixture of amylose and Ni-NTA agarose resins. This removes both the MBP-His(6) and His(6)-enterokinase leaving pure native protein in solution. These new vectors and the two-step purification protocol were successfully applied in purification of active native small antimicrobial peptides (AMPs), lactococcin A and human beta-defensin. Conclusions We constructed the improved pMAL expression vectors and established the pipeline and optimal conditions for their use in efficient purification of large amounts of active native small peptides. Significance and Impact of the Study Choice of expression vector impacts on the efficiency of expression and purification of desired proteins. The idea of redesigning pMAL vector was driven by the need for rapid purification of larger amounts of active native AMPs. This newly improved pMAL vector, the cloning strategy, expression conditions a