Harzianic acid exerts antimicrobial activity against Gram-positive bacteria and targets the cell membrane.

The thermophilic fungus Oidiodendron flavum is a saprobe that is commonly isolated from soil. Here, we identified a Gram-positive bacteria-selective antimicrobial secondary metabolite from this fungal species, harzianic acid (HA). Using Bacillus subtilis strain 168 combined with dynamic bacterial morphology imaging, we found that HA targeted the cell membrane. To further study the antimicrobial activity of HA, we isolated an HA-resistant strain, Bacillus subtilis strain M9015, and discovered that the mutant had more translucent colonies than the wild type strain, showed cross resistance to rifampin, and harbored five mutations in the coding region of four distinct genes. Further analysis of these genes indicated that the mutation in atpE might be responsible for the translucency of the colonies, and mutation in mdtR for resistance to both HA and rifampin. We conclude that HA is an antimicrobial agent against Gram-positive bacteria that targets the cell membrane.

Pycnoporus sanguineus Polysaccharides as Reducing Agents: Self-Assembled Composite Nanoparticles for Integrative Diabetic Wound Therapy.

Purpose: Diabetic foot ulcers (DFU) are severe complications of diabetes, posing significant health and societal challenges. Elevated levels of reactive oxygen species (ROS) at the ulcer site hinder wound healing in most patients, while individuals with diabetes are also more susceptible to bacterial infections. This study aims to synthesize a comprehensive therapeutic material using polysaccharides from Pycnoporus sanguineus to promote DFU wound healing, reduce ROS levels, and minimize bacterial infections. Methods: Polysaccharides from P.sanguineus were employed as reducing and stabilizing agents to fabricate polysaccharide-based composite particles (PCPs) utilizing silver ions as templates. PCPs were characterized via UV-Vis, TEM, FTIR, XRD, and DLS. The antioxidant, antimicrobial, and cytotoxic properties of PCPs were assessed through in vitro and cellular experiments. The effects and mechanisms of PCPs on wound healing were evaluated using a diabetic ulcer mouse model. Results: PCPs exhibited spherical particles with an average size of 57.29±22.41 nm and effectively combined polysaccharides' antioxidant capacity with silver nanoparticles' antimicrobial function, showcasing synergistic therapeutic effects. In vitro and cellular experiments demonstrated that PCPs reduced cellular ROS levels by 54% at a concentration of 31.25 µg/mL and displayed potent antibacterial activity at 8 µg/mL. In vivo experiments revealed that PCPs enhanced the activities of superoxide dismutase (SOD) and catalase (CAT), promoting wound healing in DFUs and lowering the risk of bacterial infections. Conclusion: The synthesized PCPs offer a novel strategy for the comprehensive treatment of DFU. By integrating antioxidant and antimicrobial functions, PCPs effectively promote wound healing and alleviate patient suffering. The present study demonstrates a new strategy for the integrated treatment of diabetic wounds and expands the way for developing and applying the polysaccharide properties of P. sanguineus.

Mycobacteriophages in diagnosis and alternative treatment of mycobacterial infections.

Antimicrobial resistance is an increasing threat to human populations. The emergence of multidrug-resistant "superbugs" in mycobacterial infections has further complicated the processes of curing patients, thereby resulting in high morbidity and mortality. Early diagnosis and alternative treatment are important for improving the success and cure rates associated with mycobacterial infections and the use of mycobacteriophages is a potentially good option. Since each bacteriophage has its own host range, mycobacteriophages have the capacity to detect specific mycobacterial isolates. The bacteriolysis properties of mycobacteriophages make them more attractive when it comes to treating infectious diseases. In fact, they have been clinically applied in Eastern Europe for several decades. Therefore, mycobacteriophages can also treat mycobacteria infections. This review explores the potential clinical applications of mycobacteriophages, including phage-based diagnosis and phage therapy in mycobacterial infections. Furthermore, this review summarizes the current difficulties in phage therapy, providing insights into new treatment strategies against drug-resistant mycobacteria.

Classification of antimicrobial mechanism of action using dynamic bacterial morphology imaging.

Antimicrobial resistance is a major threat to human health. Basic knowledge of antimicrobial mechanism of action (MoA) is imperative for patient care and for identification of novel antimicrobials. However, the process of antimicrobial MoA identification is relatively laborious. Here, we developed a simple, quantitative time-lapse fluorescence imaging method, Dynamic Bacterial Morphology Imaging (DBMI), to facilitate this process. It uses a membrane dye and a nucleoid dye to track the morphological changes of single Bacillus subtilis cells in response to antimicrobials for up to 60 min. DBMI of bacterial cells facilitated assignment of the MoAs of 14 distinct, known antimicrobial compounds to the five main classes. We conclude that DBMI is a simple method, which facilitates rapid classification of the MoA of antimicrobials in functionally distinct classes.

Berkchaetoazaphilone B has antimicrobial activity and affects energy metabolism.

Antimicrobial resistance has become one of the major threats to human health. Therefore, there is a strong need for novel antimicrobials with new mechanisms of action. The kingdom of fungi is an excellent source of antimicrobials for this purpose because it encompasses countless fungal species that harbor unusual metabolic pathways. Previously, we have established a library of secondary metabolites from 10,207 strains of fungi. Here, we screened for antimicrobial activity of the library against seven pathogenic bacterial strains and investigated the identity of the active compounds using ethyl acetate extraction, activity-directed purification using HPLC fractionation and chemical analyses. We initially found 280 antimicrobial strains and subsequently identified 17 structurally distinct compounds from 26 strains upon further analysis. All but one of these compounds, berkchaetoazaphilone B (BAB), were known to have antimicrobial activity. Here, we studied the antimicrobial properties of BAB, and found that BAB affected energy metabolism in both prokaryotic and eukaryotic cells. We conclude that fungi are a rich source of chemically diverse secondary metabolites with antimicrobial activity.

Functional Analysis of the Minimal Twin-Arginine Translocation System Components from Streptococcus thermophilus CGMCC 7.179 in Escherichia coli DE3.

The twin-arginine translocation (Tat) system, which is used for folded protein secretion, is rare in lactic acid bacteria (LAB). Previously, a Tat system composed of TatAS and TatCS subunits (the subscript S denotes a Streptococcus thermophilus origin) was identified in S. thermophilus CGMCC 7.179. In the present study, the tatA S and tatC S genes were cloned and functionally analyzed in Escherichia coli DE3 tat-deficient mutants. The E. coli tatABCDE-deficient mutant complemented with tatC S A S exhibited shortened cellular chains, but its ability to grow in the presence of sodium dodecyl sulfate (SDS) was not restored, suggesting that the S. thermophilus Tat system could partially replace that of E. coli. Surprisingly, the E. coli tatABE-deficient mutant complemented with tatA S and the E. coli tatC-deficient mutant complemented with tatC S displayed relatively normal cellular morphology and enhanced tolerance to SDS. These results suggest that TatAS and TatCS could serve as active protein translocases in E. coli DE3 tat-deficient mutants. Moreover, TatAS acted as a bifunctional subunit to fulfill the roles of both TatA and TatB of E. coli DE3. Thus, this minimal Tat system would be a promising candidate to translocate recombinant proteins in LAB.

Characterization of a New Cell Envelope Proteinase PrtP from Lactobacillus rhamnosus CGMCC11055.

Cell envelope proteinases (CEPs) play essential roles in lactic acid bacteria growth in milk and health-promoting properties of fermented dairy products. The genome of Lactobacillus rhamnosus CGMCC11055 possesses two putative CEP genes prtP and prtR2, and the PrtP displays the distinctive domain organization from PrtR2 reported. The PrtP was purified and biochemically characterized. The results showed that the optimal activity occurred at 44 °C, pH 6.5. p-Amidinophenylmethylsulfonyl fluoride obviously inhibited enzymatic activity, suggesting PrtP was a member of serine proteinases. Under the optimal conditions, ß-casein was a favorite substrate over αS1- and κ-casein, and 35 oligopeptides were identified in the ß-casein hydrolysate, including the phosphoserine peptide and bioactive isoleucine-proline-proline. By analysis of the amino acid sequences of those oligopeptides, proline was the preferred residue at the breakdown site. Therefore, we speculated that PrtP was a new type of CEPs from Lb. rhamnosus.

The potential of the endolysin Lysdb from Lactobacillus delbrueckii phage for combating Staphylococcus aureus during cheese manufacture from raw milk.

Phage endolysins have received increased attention in recent times as potential antibacterial agents and the biopreservatives in food production processes. Staphylococcus aureus is one of the most common pathogens in bacterial food poisoning outbreaks. In this study, the endolysin Lysdb, one of the two-component cell lysis cassette of Lactobacillus delbrueckii phage phiLdb, was shown to possess a muramidase domain and catalytic sites with homology to Chalaropsis-type lysozymes. Peptidoglycan hydrolytic bond specificity determination revealed that Lysdb was able to cleave the 6-O-acetylated peptidoglycans present in the cell walls of S. aureus. Turbidity reduction assays demonstrated that Lysdb could effectively lyse the S. aureus live cells under acidic and mesothermal conditions. To further evaluate the ability of Lysdb as a potential antibacterial agent against S. aureus in cheese manufacture, Lactobacillus casei BL23 was engineered to constitutively deliver active Lysdb to challenge S. aureus in lab-scale cheese making from raw milk. Compared with the raw milk, the viable counts of S. aureus were reduced by 10(5)-fold in the cheese inoculated with the engineered L. casei strain during the fermentation process, and the pathogenic bacterial numbers remained at a low level (10(4) CFU/g) after 6 weeks of ripening at 10 °C. Taken together, all results indicated that the Lysdb has the function as an effective tool for combating S. aureus during cheese manufacture from raw milk.