Feeding-Induced Changes of Bacteriolytic Activity and the Pattern of Bacteriolytic Compounds in the Stomach and Small Intestine of the Haematophagous Bug Triatoma infestans (Klug, 1834) (Reduviidae, Triatominae)

Intestinal homeostasis mechanisms of the haematophagous triatomines regulate the development of mutualistic symbionts and other gut bacteria. Investigating antimicrobial compounds of these insects, we have determined spectrophotometrically that the bacteriolytic activity is between pH 3 and pH 9 using homogenates of fifth instar Triatoma infestans stomachs and small intestines from unfed bugs and up to 50 days after feeding. The activity against Gram-positive Micrococcus luteus was strongest at pH 4 and pH 7 and was higher in the stomach than in the small intestine. Symbiotic Rhodococcus triatomae were not lysed. Lysis of Gram-negative Escherichia coli showed a maximum at pH 7 in the stomach and at pH 5 in the small intestine. Bacteriolytic activity against both M. luteus and E. coli was reduced 24 h after feeding, then increased, and at 50 days after feeding was strongly reduced. In zymographs, the activity against M. luteus was mainly correlated to proteins of about 16 kDa. At different periods of time after feeding, seven bands of lysis appeared between 15 and 40 kDa and more bands using extracts of the small intestine than those of the stomach. This is the first proof for the synthesis of antibacterial proteins of 22–40 kDa in triatomines.

Polyethylene Films Coated with Antibacterial and Antiviral Layers Based on CO2 Extracts of Raspberry Seeds, of Pomegranate Seeds and of Rosemary

The main goal of the work was to create an internal coating based on super critical CO2 extracts of raspberry seeds, pomegranate seeds and rosemary that could be active against chosen bacterial strains. Additionally, the synergistic effect of these substances in the coating were then analysed. The next goal of the work was to demonstrate the antiviral activity of the coatings against phi6 bacteriophage particles (airborne viruses surrogate). The results of the study indicated that three coatings containing a mixture of extracts showed bacteriolytic activity against S. aureus cells and bacteriostatic activity against E. coli and B. subtilis strains. Two coatings showed bacteriolytic activity against a P. syringae strain. As a result of the experiments, a synergistic effect was noted in the active additives/compounds in the coatings. These coatings may be used as internal coatings for packaging films to extend the shelf life of selected food products. All seven coatings may also be used as external coatings with antiviral activity, as these coatings demonstrated significant effects on the phi6 phage, selected as a surrogate for airborne viruses, e.g., coronaviruses. It could be concluded that coatings I–VII will also show antiviral effects on SARS-CoV-2 particles.

Expression of Antimicrobial Peptide (AMP), Cecropin B, in a Fused Form to SUMO Tag With or Without Three-Glycine Linker in Escherichia coli and Evaluation of Bacteriolytic Activity of the Purified AMP

Current antibiotics have limited action mode, which makes it difficult for the antibiotics dealing with the emergence of bacteria resisting the existing antibiotics. As a need for new bacteriolytic agents alternative to the antibiotics, AMPs have long been considered substitutes for the antibiotics. Cecropin B was expressed in a fusion form to six-histidine and SUMO tags in Escherichia coli. Six-histidine tag attached to SUMO was for purification of SUMO-cecropin B fusion proteins and removal of the SUMO tag from cecropin B. Chimeric gene was constructed into pKSEC1 vector that was designed to be functional in both Escherichia coli and chloroplast. To maximize translation of the fusion protein, sequences were codon-optimized. Four different constructs were tested for the level of expression and solubility, and the construct with a linker, 6xHisSUMO3xGly-cecropin B, showed the highest expression. In addition, cleavage of the SUMO tag by SUMOase in the three fusion constructs which have no linker sequence (3xGly, three glycines) was not as efficient as the construct with the linker between SUMO and cecropin B. The cleaved cecropin B showed bacteriolytic activity against Bacillus subtilis at a concentration of 0.0625 μg/μL, while cecropin B fused to SUMO had no activity at a higher concentration, 0.125 μg/μL. As an expression system for AMPs in prokaryotic hosts, the use of tag proteins and appropriate codon-optimization strategy can be employed and further genetic modification of the fusion construct should help the complete removal of the tag proteins from the AMP in the final step of purification.

Staphylococcus aureus as a target of peripheral blood microbicidal factors from healthy donors

Objective. The aim of investigation was to evaluate the bacteriolytic activity of the peripheral blood of healthy donors against S. aureus. Currently, much attention is paid to the involvement of opportunistic microorganisms in the development of infectious and inflammatory diseases, among which one of the leading places is occupied by the processes of staphylococcal etiology. Staphylococcus aureus possesses a unique spectrum of pathogenic factors, which, together with intracellular persistence, allow staphylococci to avoid exposure to immune factors and other agents. Materials and methods. The bacteriolytic activity of the peripheral blood of 32 healthy donors, as well as the ability of the whole blood and serum to destroy biofilms, were evaluated. The phagocytic activity of peripheral blood monocytes and neutrophils and the ability to produce hydroxyl radicals were analyzed. For opsonization of S. aureus, a commercial immunoglobulin G or donor serum was used. Results. It was shown that the whole peripheral blood practically does not have a significant effect on the number of viable cells of S. aureus. However, freshly obtained blood serum significantly destroys the biofilm. It has been established that a fifth part of peripheral blood leukocytes is absorbed by S. aureus. After opsonization of microbial cells with immunoglobulin G, the indices of phagocytic activity of monocytes and neutrophils did not change significantly. When using freshly obtained serum for opsonization of objects, a stimulating effect on the production of hydroxyl radicals by leukocytes was revealed (2758.7 725.3 and 870.6 197.4 related light units, respectively; p 0.05). After heating the serum at 56 C, the stimulating effect was leveled (1091.1 234.7 related light units; p 0.05 for samples with non-opsonized objects). In general, the obtained data indicate that the complement components can be recognized as the most effective system for the elimination of S. aureus. Conclusions. Thus, S. aureus uniquely adapted to the human body that allows staphylococci to persist for a long time without clinical manifestations. It can be assumed that among the factors of the immune system, the proteins of the complement system, which destroy both S. aureus cells and the biofilm matrix, probably have the most effective bactericidal action. However, the effectiveness of this system depends on the protein-synthesizing function of the liver, the availability of microorganisms to the action of complement.

A New Family of Bacteriolytic Proteins in Dictyostelium discoideum

Phagocytic cells ingest and destroy bacteria efficiently and in doing so ensure the defense of the human body against infections. Phagocytic Dictyostelium discoideum amoebae represent a powerful model system to study the intracellular mechanisms ensuring destruction of ingested bacteria in phagosomes. Here, we discovered the presence of a bacteriolytic activity against Klebsiella pneumoniae in cellular extracts from D. discoideum. The bacteriolytic activity was detected only at a very acidic pH mimicking the conditions found in D. discoideum phagosomes. It was also strongly decreased in extracts of kil1 KO cells that were previously described to kill inefficiently internalized bacteria, suggesting that the activity observed in vitro is involved in killing of bacteria in phagosomes. We purified a fraction enriched in bacteriolytic activity where only 16 proteins were detected and focused on four proteins selectively enriched in this fraction. Three of them belong to a poorly characterized family of D. discoideum proteins exhibiting a DUF3430 domain of unknown function and were named BadA (Bacteriolytic D. discoideum A), BadB, and BadC. We overexpressed the BadA protein in cells, and the bacteriolytic activity increased concomitantly in cell extracts. Conversely, depletion of BadA from cell extracts decreased significantly their bacteriolytic activity. Finally, in cells overexpressing BadA, bacterial killing was faster than in parental cells. Together these results identify BadA as a D. discoideum protein required for cellular bactericidal activity. They also define a new strategy to identify and characterize bactericidal proteins in D. discoideum cells.

Green Synthesis of Silver Nanoparticles and Evaluation of Their Antibacterial Activity against Multidrug-Resistant Bacteria and Wound Healing Efficacy Using a Murine Model

Green nanotechnology has significant applications in various biomedical science fields. In this study, green-synthesized silver nanoparticles, prepared by using Catharanthus roseus and Azadirachta indica extracts, were characterized using UV–Vis spectroscopy, dynamic light scattering, X-ray diffraction, scanning electron microscopy, and transmission electron microscopy. Silver nanoparticles (Ag NPs) synthesized from leaf extracts of C. roseus and A. indica effectively inhibited the growth of multidrug-resistant (MDR) bacteria isolated from patients with septic wound infections. The maximum bacteriolytic activity of the green-synthesized Ag NPs of C. roseus and A. indica against the MDR bacterium K. Pneumoniae was shown by a zone of inhibition of 19 and 16 mm, respectively. C. roseus Ag NPs exhibited more bacteriolytic activity than A. indica Ag NPs in terms of the zone of inhibition. Moreover, these particles were effective in healing wounds in BALB/c mice. Ag NPs of C. roseus and A. indica enhanced wound healing by 94% ± 1% and 87% ± 1%, respectively. Our data suggest that Ag NPs from C. roseus and A. indicia ameliorate excision wounds, and wound healing could be due to their effective antimicrobial activity against MDR bacteria. Hence, these Ag NPs could be potential therapeutic agents for the treatment of wounds.

Two Novel Bacteriophages Improve Survival in Galleria mellonella Infection and Mouse Acute Pneumonia Models Infected with Extensively Drug-Resistant Pseudomonas aeruginosa

ABSTRACT Extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) is a life-threatening pathogen that causes serious global problems. Here, we investigated two novel P. aeruginosa bacteriophages (phages), Bϕ-R656 and Bϕ-R1836, in vitro, in silico, and in vivo to evaluate the potential of phage therapy to control XDR-PA clinical strains. Bϕ-R656 and Bϕ-R1836 belong to the Siphoviridae family and exhibited broad host ranges which could lyse 18 (64%) and 14 (50%) of the 28 XDR-PA strains. In addition, the two phages showed strong bacteriolytic activity against XDR-PA host strains from pneumonia patients. The whole genomes of Bϕ-R656 and Bϕ-R1836 have linear double-stranded DNA of 60,919 and 37,714 bp, respectively. The complete sequence of Bϕ-R656 had very low similarity to the previously discovered P. aeruginosa phages in GenBank, but phage Bϕ-R1836 exhibited 98% and 91% nucleotide similarity to Pseudomonas phages YMC12/01/R24 and PA1/KOR/2010, respectively. In the two in vivo infection models, treatment with Bϕ-R656 and Bϕ-R1836 enhanced the survival of Galleria mellonella larvae (50% and 60%, respectively) at 72 h postinfection and pneumonia-model mice (66% and 83%, respectively) at 12 days postinfection compared with untreated controls. Treatment with Bϕ-R656 or Bϕ-R1836 also significantly decreased the bacterial load in the lungs of the mouse pneumonia model (>6 log10 CFU and >4 log10 CFU, respectively) on day 5. IMPORTANCE In this study, two novel P. aeruginosa phages, Bϕ-R656 and Bϕ-R1836, were evaluated in vitro, in silico, and in vivo for therapeutic efficacy and safety as an alternative antibacterial agent to control XDR-PA strains collected from pneumonia patients. Both phages exhibited potent bacteriolytic activity and greatly improved survival in G. mellonella larva infection and a mouse acute pneumonia model. Based on these results, we strongly predict that these two new phages could be used as fast-acting and safe alternative biological weapons against XDR-PA infections.