Comparison of the Antibacterial Activity of Selected Deep Eutectic Solvents (DESs) and Deep Eutectic Solvents Comprising Organic Acids (OA-DESs) Toward Gram-Positive and Gram-Negative Species.

Deep eutectic solvents (DESs) have been intensively investigated in recent years for their antibacterial properties, with DESs that comprise organic acids (OA-DESs) showing promising antibacterial action. However a majority of the reports focused only on a limited number strains and techniques, which is not enough to determine the antibacterial potential of a substance. To bridge this gap, the antibacterial activity of classical DESs and OA-DESs is assessed on twelve Gram-negative and Gram-positive bacteria strains, with some of them exhibiting specific resistance toward antibiotics. The investigated formulations of OA-DESs comprise glycolic, malic, malonic, and oxalic acids as representatives of this group. Using a range of microbiological assays as well as physicochemical characterization methods, a major difference of the effectiveness between the two groups is demonstrated, with OA-DESs exhibiting, as expected, greater antibacterial effectiveness than classical DESs. Most interestingly, slight differences in the minimum inhibitory and bactericidal concentration values as well as time-kill kinetics profiles are observed between Gram-positive and Gram-negative strains. Transmission electron microscopy analysis reveals the effect of the treatment of the bacteria with the representatives of both groups of DESs, which allows us to better understand the possible mechanism-of-action of these novel materials.

Phages and engineered lysins as an effective tool to combat Gram-negative foodborne pathogens.

One of the biggest challenges faced by food producers is ensuring microbiological safety. Despite strict criteria for food products, foodborne diseases are a global problem and pose a real risk to consumers. Therefore, it is necessary to identify new and more effective methods for eliminating pathogens from food and the food processing environment. According to the European Food Safety Authority (EFSA), the most common foodborne diseases are caused by Campylobacter, Salmonella, Yersinia, Escherichia coli, and Listeria. Out of the five listed, four are Gram-negative bacteria. Our review focuses on the use of bacteriophages, which are ubiquitous bacterial viruses, and bacteriophage endolysins to eliminate Gram-negative pathogens. Endolysins cleave specific bonds within the peptidoglycan (PG) of the bacterial cell, causing the cell to burst. Single phages or phage cocktails, which are, in some instances, commercially available products, eliminate pathogenic bacteria in livestock and various food matrices. Endolysins have matured as the most advanced class of antibacterial agents in the clinical sector, but their use in food protection is highly unexplored. Advanced molecular engineering techniques, different formulations, protein encapsulation, and the addition of outer membrane (OM) permeabilization agents enhance the activity of lysins against Gram-negative pathogens. This creates space for groundbreaking research on the use of lysins in the food sector.

Molecular characterization of the PhiKo endolysin from Thermus thermophilus HB27 bacteriophage phiKo and its cryptic lytic peptide RAP-29.

Introduction: In the era of increasing bacterial resistance to antibiotics, new bactericidal substances are sought, and lysins derived from extremophilic organisms have the undoubted advantage of being stable under harsh environmental conditions. The PhiKo endolysin is derived from the phiKo bacteriophage infecting Gram-negative extremophilic bacterium Thermus thermophilus HB27. This enzyme shows similarity to two previously investigated thermostable type-2 amidases, the Ts2631 and Ph2119 from Thermus scotoductus bacteriophages, that revealed high lytic activity not only against thermophiles but also against Gram-negative mesophilic bacteria. Therefore, antibacterial potential of the PhiKo endolysin was investigated in the study presented here. Methods: Enzyme activity was assessed using turbidity reduction assays (TRAs) and antibacterial tests. Differential scanning calorimetry was applied to evaluate protein stability. The Collection of Anti-Microbial Peptides (CAMP) and Antimicrobial Peptide Calculator and Predictor (APD3) were used to predict regions with antimicrobial potential in the PhiKo primary sequence. The minimum inhibitory concentration (MIC) of the RAP-29 synthetic peptide was determined against Gram-positive and Gram-negative selected strains, and mechanism of action was investigated with use of membrane potential sensitive fluorescent dye 3,3'-Dipropylthiacarbocyanine iodide (DiSC3(5)). Results and discussion: The PhiKo endolysin is highly thermostable with melting temperature of 91.70°C. However, despite its lytic effect against such extremophiles as: T. thermophilus, Thermus flavus, Thermus parvatiensis, Thermus scotoductus, and Deinococcus radiodurans, PhiKo showed moderate antibacterial activity against mesophiles. Consequently, its protein sequence was searched for regions with potential antibacterial activity. A highly positively charged region was identified and synthetized (PhiKo105-133). The novel RAP-29 peptide lysed mesophilic strains of staphylococci and Gram-negative bacteria, reducing the number of cells by 3.7-7.1 log units and reaching the minimum inhibitory concentration values in the range of 2-31 µM. This peptide is unstructured in an aqueous solution but forms an α-helix in the presence of detergents. Moreover, it binds lipoteichoic acid and lipopolysaccharide, and causes depolarization of bacterial membranes. The RAP-29 peptide is a promising candidate for combating bacterial pathogens. The existence of this cryptic peptide testifies to a much wider panel of antimicrobial peptides than thought previously.

A Novel Cryptic Clostridial Peptide That Kills Bacteria by a Cell Membrane Permeabilization Mechanism.

This work reports detailed characteristics of the antimicrobial peptide Intestinalin (P30), which is derived from the LysC enzyme of Clostridium intestinale strain URNW. The peptide shows a broader antibacterial spectrum than the parental enzyme, showing potent antimicrobial activity against clinical strains of Gram-positive staphylococci and Gram-negative pathogens and causing between 3.04 ± 0.12 log kill for Pseudomonas aeruginosa PAO1 and 7.10 ± 0.05 log kill for multidrug-resistant Acinetobacter baumannii KPD 581 at a 5 µM concentration. Moreover, Intestinalin (P30) prevents biofilm formation and destroys 24-h and 72-h biofilms formed by Acinetobacter baumannii CRAB KPD 205 (reduction levels of 4.28 and 2.62 log CFU/mL, respectively). The activity of Intestinalin is combined with both no cytotoxicity and little hemolytic effect against mammalian cells. The nuclear magnetic resonance and molecular dynamics (MD) data show a high tendency of Intestinalin to interact with the bacterial phospholipid cell membrane. Although positively charged, Intestinalin resides in the membrane and aggregates into small oligomers. Negatively charged phospholipids stabilize peptide oligomers to form water- and ion-permeable pores, disrupting the integrity of bacterial cell membranes. Experimental data showed that Intestinalin interacts with negatively charged lipoteichoic acid (logK based on isothermal titration calorimetry, 7.45 ± 0.44), causes membrane depolarization, and affects membrane integrity by forming large pores, all of which result in loss of bacterial viability. IMPORTANCE Antibiotic resistance is rising rapidly among pathogenic bacteria, becoming a global public health problem that threatens the effectiveness of therapies for many infectious diseases. In this respect, antimicrobial peptides appear to be an interesting alternative to combat bacterial pathogens. Here, we report the characteristics of an antimicrobial peptide (of 30 amino acids) derived from the clostridial LysC enzyme. The peptide showed killing activity against clinical strains of Gram-positive and Gram-negative pathogens. Experimental data and computational modeling showed that this peptide forms transmembrane pores, directly engaging the negatively charged phospholipids of the bacterial cell membrane. Consequently, dissipation of the electrochemical gradient across cell membranes affects many vital processes, such as ATP synthesis, motility, and transport of nutrients. This kind of dysfunction leads to the loss of bacterial viability. Our firm conviction is that the presented study will be a helpful resource in searching for novel antimicrobial peptides that could have the potential to replace conventional antibiotics.

Nutritional strategies for autophagy activation and health consequences of autophagy impairment.

OBJECTIVES: Currently, the plague of chronic diseases, such as overweight, obesity, diabetes, and cardiovascular diseases, is associated with chronic inflammation as an effect of homeostasis disbalance. One of the processes involved in homeostasis maintenance is autophagy, which is also referred to as self-eating or cellular recycling. Due to the correlation between the epidemic scale of chronic diseases and autophagy impairment, strategies for autophagy activation are urgently needed. METHODS: In this review, we comprehensively summarized the current data on autophagy types, dysfunction, associated diseases, and ways of activation available in scientific databases and published up until 2022. RESULTS: Our review indicates that impaired autophagy is associated with inflammatory bowel diseases, cancer, overweight, obesity, type I diabetes, diseases of the cardiovascular system, neurodegenerative diseases, depression, and anxiety. We highlight nutritional behavior as one of the factors behind autophagy induction and homeostasis restoration. CONCLUSIONS: Autophagy is involved in different dysfunctions and diseases; thus, activation strategies are urgently needed. A high potential in the prevention and therapies of chronic diseases by means of autophagy induction can be expected from nutritional behaviors. To date, most studies were carried out in vitro or in a murine model. Thus, further, well-designed, clinical trials are needed to provide the missing understanding of the nutritional potential to regulate specific signaling pathways that keep autophagy running smoothly.

Overview of the Importance of Biotics in Gut Barrier Integrity.

Increased gut permeability is suggested to be involved in the pathogenesis of a growing number of disorders. The altered intestinal barrier and the subsequent translocation of bacteria or bacterial products into the internal milieu of the human body induce the inflammatory state. Gut microbiota maintains intestinal epithelium integrity. Since dysbiosis contributes to increased gut permeability, the interventions that change the gut microbiota and correct dysbiosis are suggested to also restore intestinal barrier function. In this review, the current knowledge on the role of biotics (probiotics, prebiotics, synbiotics and postbiotics) in maintaining the intestinal barrier function is summarized. The potential outcome of the results from in vitro and animal studies is presented, and the need for further well-designed randomized clinical trials is highlighted. Moreover, we indicate the need to understand the mechanisms by which biotics regulate the function of the intestinal barrier. This review is concluded with the future direction and requirement of studies involving biotics and gut barrier.

High-Pressure-Induced Sublethal Injuries of Food Pathogens-Microscopic Assessment.

High Hydrostatic Pressure (HHP) technology is considered an alternative method of food preservation. Nevertheless, the current dogma is that HHP might be insufficient to preserve food lastingly against some pathogens. Incompletely damaged cells can resuscitate under favorable conditions, and they may proliferate in food during storage. This study was undertaken to characterize the extent of sublethal injuries induced by HHP (300-500 MPa) on Escherichia coli and Listeria inncua strains. The morphological changes were evaluated using microscopy methods such as Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Epifluorescence Microscopy (EFM). The overall assessment of the physiological state of tested bacteria through TEM and SEM showed that the action of pressure on the structure of the bacterial membrane was almost minor or unnoticeable, beyond the L. innocua wild-type strain. However, alterations were observed in subcellular structures such as the cytoplasm and nucleoid for both L. innocua and E. coli strains. More significant changes after the HHP of internal structures were reported in the case of wild-type strains isolated from raw juice. Extreme condensation of the cytoplasm was observed, while the outline of cells was intact. The percentage ratio between alive and injured cells in the population was assessed by fluorescent microscopy. The results of HHP-treated samples showed a heterogeneous population, and red cell aggregates were observed. The percentage ratio of live and dead cells (L/D) in the L. innocua collection strain population was higher than in the case of the wild-type strain (69%/31% and 55%/45%, respectively). In turn, E. coli populations were characterized with a similar L/D ratio. Half of the cells in the populations were distinguished as visibly fluorescing red. The results obtained in this study confirmed sublethal HHP reaction on pathogens cells.

Operational culture conditions determinate benzalkonium chloride resistance in L. monocytogenes-E. coli dual species biofilms.

Biofilms pose a serious challenge to the food industry. Higher resistance of biofilms to any external stimuli is a major hindrance for their eradication. In this study, we compared the growth dynamics and benzalkonium chloride (BAC) resistance of dual species Listeria monocytogenes-Escherichia coli 48 h biofilms formed on stainless steel (SS) coupons surfaces under batch and fed-batch cultures. Differences between both operational culture conditions were evaluated in terms of total viable adhered cells (TVAC) in the coupons during 48 h of the mixed-culture and of reduction of viable adhered cells (RVAC) obtained after BAC-treatment of a 48 h biofilm of L. monocytogenes-E. coli formed under both culture conditions. Additionally, epifluorescence microscopy (EFM) and confocal scanning microscopy (CLSM) permitted to visualize the 2D and 3D biofilms structure, respectively. Observed results showed an increase in the TVAC of both strains during biofilm development, being the number of E. coli adhered cells higher than L. monocytogenes in both experimental systems (p < 0.05). Additionally, the number of both strains were higher approximately 2.0 log CFU/coupon in batch conditions compared to fed-batch system (p < 0.05). On the contrary, significantly higher resistance to BAC was observed in biofilms formed under fed-batch conditions. Furthermore, in batch system both strains had a similar reduction level of approximately 2.0 log CFU/coupon, while significantly higher resistance of E. coli compared to L. monocytogenes (reduction level of 0.69 and 1.72 log CFU/coupon, respectively) (p < 0.05) was observed in fed-batch system. Microscopic image visualization corroborated these results and showed higher complexity of 2D and 3D structures in dual species biofilms formed in batch cultures. Overall, we can conclude that the complexity of the biofilm structure does not always imply higher resistance to external stimuli, and highlights the need to mimic industrial operational conditions in the experimental systems in order to better assess the risk associated to the presence of pathogenic bacterial biofilms.

Utilization of physiological and taxonomic fluorescent probes to study Lactobacilli cells and response to pH challenge.

pH stress is recognized as an important feature for Lactobacillus in relation to lifestyle and commercial utility. Hence, this study aims to investigate the cell function of Lactobacilli cells subjected to pHs between 7.0 and 2.0. For this purpose, the Lactobacilli isolates of vegetable origin were first hybridized with fluorescent oligonucleotide rRNA probes for detecting Lactobacillus species. Then, cells were exposed to pH stress and labelled with fluorescent probes, carboxyfluorescein diacetate (CFDA) and propidium iodine (PI), which provided the insight into esterase activity and membrane integrity of cells. Among isolates, fluorescence in situ hybridization (FISH) enabled us to specifically detect L. plantarum and L. brevis. Interestingly, FCM analysis revealed that at pHs between 7.0 and 4.0 the cell membrane was intact, while after the exposure at pH 3.0, and 2.0 became perturbed or impaired. Finally, L. brevis and L. plantarum differed from each other in fluorescence labeling behaviour and culturability. However, the results showed that the same standard protocol for labeling enables discrimination of subpopulations of tested species. Depending on the species, the substantial culturability loss was observed at pH 3.0 and 2.0. These results suggest that the taxonomic and physiological fluorescent probes could be suitable for in situ detection of specific bacteria and rapid assessment of the physiological status of cells.

Physiological functions at single-cell level of Lactobacillus spp. isolated from traditionally fermented cabbage in response to different pH conditions.

Changes in pH are significant environmental stresses that may be encountered by lactobacilli during fermentation processes or passage through the gastrointestinal tract. Here, we report the cell response of Lactobacillus spp. isolated from traditionally fermented cabbage subjected to acid/alkaline treatments at pH 2.5, 7.4 and 8.1, which represented pH conditions of the gastrointestinal tract. Among six isolates, four species of Lactobacillus plantarum and two of Lactobacillus brevis were identified by fluorescence in situ hybridization (FISH). The fluorescence-based strategy of combining carboxyfluorescein diacetate (CFDA) and propidium iodine (PI) into a dual-staining assay was used together with epifluorescence microscopy (EFM) and flow cytometry (FCM) for viability assessment. The results showed that the cells maintained esterase activity and membrane integrity at pH 8.1 and 7.4. There was also no loss of culturability as shown by plate counts. In contrast, the majority of 2.5 pH-treated cells had a low extent of esterase activity, and experienced membrane perturbation. For these samples, an extensive loss of culturability was demonstrated. Comparison of the results of an in situ assessment with that of the conventional culturing method has revealed that although part of the stressed population was unable to grow on the growth media, it was deemed viable using a CFDA/PI assay. However, there was no significant change in the cell morphology among pH-treated lactobacilli populations. These analyses are expected to be useful in understanding the cell response of Lactobacillus strains to pH stress and may facilitate future investigation into functional and industrial aspects of this response.