Functional characterization of serine proteinase inhibitor Kazal-Type in the red claw crayfish Cherax quadricarinatus.

Serine protease inhibitors Kazal type (SPINKs) function in physiological and immunological processes across multicellular organisms. In the present study, we identified a SPINK gene, designated as CqSPINK, in the red claw crayfish Cherax quadricarinatus, which is the ortholog of human SPINK5. The deduced CqSPINK contains two Kazal domains consisting of 45 amino acid residues with a typical signature motif C-X3-C-X5-PVCG-X5-Y-X3-C-X6-C-X12-14-C. Each Kazal domain contains six conserved cysteine residues forming three pairs of disulfide bonds, segmenting the structure into three rings. Phylogenetic analysis revealed CqSPINK as a homolog of human SPINK5. CqSPINK expression was detected exclusively in hepatopancreas and epithelium, with rapid up-regulation in hepatopancreas upon Vibrio parahaemolyticus E1 challenge. Recombinant CqSPINK protein (rCqSPINK) was heterologously expressed in Escherichia coli and purified for further study. Proteinase inhibition assays demonstrated that rCqSPINK could potently inhibit proteinase K and subtilisin A, weakly inhibit α-chymotrypsin and elastase, but extremely weak inhibit trypsin. Furthermore, CqSPINK inhibited bacterial secretory proteinase activity from Bacillus subtilis, E. coli, and Staphylococcus aureus, and inhibited B. subtilis growth. These findings suggest CqSPINK's involvement in antibacterial immunity through direct inhibition of bacterial proteases, contributing to resistance against pathogen invasion.

Antibacterial Activity of Plants in Cirsium: A Comprehensive Review.

As ethnic medicine, the whole grass of plants in Cirsium was used as antimicrobial. This review focuses on the antimicrobial activity of plants in Cirsium, including antimicrobial components, against different types of microbes and bacteriostatic mechanism. The results showed that the main antimicrobial activity components in Cirsium plants were flavonoids, triterpenoids and phenolic acids, and the antimicrobial ability varied according to the species and the content of chemicals. Among them, phenolic acids showed a strong antibacterial ability against Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus faecium. The antibacterial mechanisms include: (1) damaging the cell membrane, cell walls, mitochondria and nucleus of bacteria; (2) inhibiting the synthesis of proteins and nucleic acids; (3) suppressing the synthesis of enzymes for tricarboxylic acid cycle pathways and glycolysis, and then killing the bacteria via inhibition of energy production. Totally, most research results on antimicrobial activity of Cirsium plants are reported based on in vitro assays. The evidence from clinical data and comprehensive evaluation are needed.

An insight into the effect of interaction with protein on antibacterial activity of chitosan derivatives.

Antimicrobial activity of chitosan in protein-rich media is of a particular interest for various protein-based drug delivery and other systems. For the first time, bacteriostatic activity of chitosan derivatives in the presence of caseinate sodium (CAS) was studied and discussed. Complexation of chitosan derivatives soluble in acidic (CH and RCH) or alkalescent (RCH) media with CAS was confirmed by fluorescent spectroscopy, turbodimetry, light scattering data and measurement of electrical potentials of CAS/chitosan derivative complexes. An addition of CH and RCH caused a static quenching of CAS. Binding constants Kb determined for CH/CAS and RCH/CAS complexes at pH 6.0 were equal to 29.8 × 106 M-1 and 8.9 × 106 M-1, respectively. Kb value of RCH/CAS complex at pH 7.4 was equal to 1.1 × 105'M-1. The poisoned food method was used for counting the number and the direct measurement of the size of bacterial colonies on the surfaces of turbid agar media containing CAS/chitosan derivative complexex. Complete suppression of E. coli cells growth and restriction of S. aureus cells growth were observed on the surface of acidic media. A high concentration of CAS reduced the activity. The activity of RCH in alkalescent media is low or absent. These results can be promising for preparation of microbiologically stable protein-based drug delivery systems.

Diversity, chemical constituents and biological activities of endophytic fungi from Alisma orientale (Sam.) Juzep.

The dried tuber of Alisma orientale (Sam.) Juzep. (AOJ) is a traditional Chinese medicine with high medicinal value. The endophytic fungi of medicinal plants are a treasure house of natural compounds. However, there is a lack of research on the diversity and biological activity of endophytic fungi of AOJ. In this study, high-throughput sequencing technology was used to study the diversity of endophytic fungi in the roots and stems of AOJ, and endophytic fungi with a high output of phenols and flavonoids were screened by chromogenic reaction, and the antioxidant and antibacterial activities and chemical constituents of crude extracts of their fermentation broth were studied. A total of 3,426 amplicon sequence variants (ASVs) belonging to 9 phyla, 27 classes, 64 orders, 152 families, and 277 genera were identified from AOJ. There were significant differences in the endophytic fungal communities of AOJ roots and stems, as well as in the endophytic fungal communities of triangular AOJ and circular AOJ. In addition, 31 strains of endophytic fungi were isolated from AOJ, of which 6 strains had good antioxidant and antibacterial activities. The crude extract of YG-2 had the strongest free radical scavenging ability and bacteriostatic ability, and its IC50 DPPH, IC50 ABTS, and IC50⋅OH values were 0.009 ± 0.000 mg/mL, 0.023 ± 0.002 mg/mL, and 0.081 ± 0.006 mg/mL, respectively. The results of LC-MS showed that the main component of the crude extract of YG-2 was caffeic acid (10.12 µmol/g). Overall, the results of this study preliminarily elucidated the diversity and community composition of endophytic fungi of AOJ, indicating that AOJ endophytic fungi have abundant secondary metabolites and good antioxidant and antibacterial activities. This study provides an important reference for further research, development and utilization of AOJ endophytic fungi and a theoretical basis for the further development of the endophytic fungus YG-2 (Chaetomium globosum) as a source of antioxidants.

Nanoparticles coated by chloramphenicol in hydrogels as a useful tool to increase the antibiotic release and antibacterial activity in dermal drug delivery.

BACKGROUND: Nanocarriers for antibacterial drugs became hopeful tools against the increasing resistance of bacteria to antibiotics. This work focuses on a comprehensive study of the applicability and therapeutic suitability of dermal carbopol-based hydrogels containing chloramphenicol carried by various nanoparticles (AuNPs and SiNPs). METHODS: The different forms of carbopol-based drugs for dermal use were obtained. Five different concentrations of chloramphenicol and two types of nanoparticles (silica and gold) in carbopol-based ointments were tested. The influence of different carbopol formulations with nanocarriers on the rheological properties as well as the release profile of active substances and bacteriostatic activity on five reference strains were determined. RESULTS: The properties of the obtained hydrogels were compared to a commercial formulation, and finally it was possible to obtain a formulation that allowed improved antimicrobial activity over a commercially available detreomycin ointment while reducing the concentration of the antibiotic. CONCLUSION: The work indicates that it is possible to reduce the concentration of chloramphenicol by four times while maintaining its bacteriostatic activity, which can improve the patient's safety profile while increasing the effectiveness of the therapy.

Natural Product-Based Drugs Discovery: Semisynthesis and Biological Evaluation of Ester Derivatives from Pregn-5-ene-3ß,17α,20(S)-triol.

There is no doubt that derivation of intermediates from natural product is a very efficient way to develop new environmentally friendly pesticide. We synthesis a succession of compounds esterified with pregn-5-ene-3ß,17α,20(S)-triol to evaluate its insecticidal and bacteriostatic activity. Otherwise, their structure-activity relationships (SAR) are also discussed. As a result, compounds 7g, 7h, 7j, 7l and 7o exhibit more obvious insecticidal activity against 3rd Mythimna separata Walker (LC50 = 0.60, 0.68, 0.79, 0.85 and 0.78 mg/mL, respectively) than periplocoside F (PSF). Meanwhile, compounds 7g, 7h and 7i perform well inhibitory activity against Pseudomas syringae pv. actinidiae (Psa) in vitro (minimum inhibitory concentration (MIC) values: 0.10-0.25 mg/mL, minimum bactericidal concentration (MBC) values: 0.15-0.35 mg/mL). And SAR analysis indicates that the replacement and position of fluorine atom on benzoyl are highly vital to biological activity.

Effects of conjugates of ε-polylysine-dextran created through Maillard reaction on quality and storage stability of the chicken gel.

The present study mainly focused on the effects of the conjugates of PL-dextran produced through the Maillard reaction on the quality and storage stability of chicken gel for 5 days at 4 ℃. According to the results of the texture profile, water retention capacity (WRC), low-field nuclear magnetic resonance (LF NMR), aerobic plate count (APC), and total volatile basic nitrogen (TVBN), ε-polylysine (PL) could improve chicken gel storage stability while decreasing the quality of protein gels (p < 0.05). Additionally, adding dextran with high or low molecular weight could significantly increase the quality of gel during storage (p < 0.05), whereas decreased storage stability could be obtained (p < 0.05). In general, conjugates formed by PL and dextran with high molecular weight were beneficial for quality maintenance. In comparison, the polymers produced from the low molecular weight of dextran could modify the storage stability of gels. Adding conjugates of dextran and PL benefited the structure formation of protein gel, while PL would retain part of antibacterial activity when crosslinked with dextran. Therefore, it could be concluded that the quality improvement effect of PL-dextran addition on gel quality was greater than its antibacterial effect, which would impact the formulation design of novel emulsion-type meat products.

A new method for the rapid detection of the antibacterial and bacteriostatic activity of disinfectants based on Propidium Monoazide combined with real-time PCR.

Rapid detection of antibacterial and bacteriostatic properties is an important part of the quality and safety supervision of disinfectants. In this study, propidium monoazide (PMA) was used in combination with real-time PCR (PMA-qPCR) to detect the antibacterial and bacteriostatic activity of disinfectants against three commonly used indicator bacteria, Escherichia coli, Staphylococcus aureus, and Candida albicans, utilizing specifically designed primers. The method for preparing membrane-damaged bacteria was optimized to improve the ability of the PMA dye to distinguish between live and dead indicator bacteria. Finally, this method could simultaneously detect viable numbers of the indicator bacteria after the disinfectants were used. The R 2 values of the PMA-qPCR standard curves were 0.9986, 0.9980, and 0.9962 for E. coli, S. aureus, and C. albicans, respectively, and the detection range was 103 ~ 106 CFU/ml, showing no significant difference in accuracy compared to that of the plate counting method (p > 0.05). The method established here is the first application of PMA-qPCR to detect the antibacterial and bacteriostatic activity of disinfectants. This technique markedly simplifies the detection steps of antibacterial and bacteriostatic activity, reduces the detection time (3 h compared to 48 ~ 72 h for the plate counting method), improves the quality supervision efficiency of disinfectants, and guarantees healthy and safe lives.

Antibiofilm effect and mechanism of protocatechuic aldehyde against Vibrio parahaemolyticus.

This study investigated the effect of protocatechuic aldehyde (PCA) on Vibrio parahaemolyticus biofilm formation and its effects on gene expression. Crystal violet assay, metabolic activity assay, and fluorescence experiments were used to evaluate the antibiofilm activities of PCA and to reveal its possible antibiofilm mechanisms using transcriptomic analysis. The results indicated that the minimum antibacterial concentration of PCA against V. parahaemolyticus was 300 µg/mL. PCA (9.375 µg/mL) inhibited biofilm generation and adhesion of the mature biofilm. PCA (75 µg/mL) significantly reduced the metabolic viability of V. parahaemolyticus, reduced polysaccharide production, and inhibited cell surface flagella-mediated swimming and aggregation phenotypes. Meanwhile, transcriptome analysis showed that the key genes of V. parahaemolyticus expressed under PCA (75 µg/mL) inhibition were mainly related to biofilm formation (pfkA, galE, narL, and oppA), polysaccharide production and adhesion (IF, fbpA, and yxeM), and motility (cheY, flrC, and fliA). By regulating these key genes, PCA reduced biofilm formation, suppressed polysaccharide production and transport, and prevented the adhesion of V. parahaemolyticus, thereby reducing the virulence of V. parahaemolyticus. This study demonstrated that protocatechuic aldehyde can be used to control V. parahaemolyticus biofilm to ensure food safety.

Production of Helvolic Acid in Metarhizium Contributes to Fungal Infection of Insects by Bacteriostatic Inhibition of the Host Cuticular Microbiomes.

The nortriterpenoid helvolic acid (HA) has potent antibiotic activities and can be produced by different fungi, yet HA function remains elusive. Here, we report the chemical biology of HA production in the insect pathogen Metarhizium robertsii. After deletion of the core oxidosqualene cyclase gene in Metarhizium, insect survival rates were significantly increased compared to those of insects treated with the wild type and the gene-rescued strain during topical infections but not during injection assays to bypass insect cuticles. Further gnotobiotic infection of axenic Drosophila adults confirmed the HA contribution to fungal infection by inhibiting bacterial competitors in an inoculum-dependent manner. Loss of HA production substantially impaired fungal spore germination and membrane penetration abilities relative to the WT and gene-complemented strains during challenge with different Gram-positive bacteria. Quantitative microbiome analysis revealed that HA production could assist the fungus to suppress the Drosophila cuticular microbiomes by exerting a bacteriostatic rather than bactericidal effect. Our data unveil the chemical ecology of HA and highlight the fact that fungal pathogens have to cope with the host cuticular microbiomes prior to successful infection of hosts. IMPORTANCE Emerging evidence has shown that the plant and animal surface microbiomes can defend hosts against fungal parasite infections. The strategies employed by fungal pathogens to combat the antagonistic inhibition of insect surface bacteria are still elusive. In this study, we found that the potent antibiotic helvolic acid (HA) produced by the insect pathogen Metarhizium robertsii contributes to natural fungal infection of insect hosts. Antibiotic and gnotobiotic infection assays confirmed that HA could facilitate fungal infection of insects by suppression of the host cuticular microbiomes through its bacteriostatic instead of bactericidal activities. The data from this study provide insights into the novel chemical biology of fungal secondary metabolisms.

Antibacterial effect of the combination of terpenoids.

Terpenoids are natural compounds originating from five-carbon isoprene units. Over 60,000 terpenoid structures have been identified, and they contribute to the flavor, color, growth, and development of plants. There are several reports on various physiological activities of terpenoids, such as antioxidative and anticancer activities. This study revealed that combinations of terpenoids have activities against a spectrum of bacteria. The combination of carvacrol and thymol has bacteriostatic and bactericidal activities. Four terpenoids (carvacrol, thymol, eugenol, and nootkatone) exhibited bacteriostatic and bactericidal activities when used at low concentrations for 5‒10 min. The most effective bactericidal activity was observed for gram-negative bacteria. A very weak bactericidal activity was observed against Staphylococcus aureus and Enterococcus faecalis. This study revealed the antibacterial potential of different combinations of terpenoids against several bacteria that were tested. Thus, new candidates for the development of antibacterial medicines are reported here for the effective treatment of infectious bacterial diseases.

Nickel oxide nanoparticles synthesis using plant extract and evaluation of their antibacterial effects on Streptococcus mutans.

Dental decay is known in the world as the most common human infectious disease. Ascending process of dental caries index in the world shows the failure of oral disease prevention. Streptococcus mutans bacteria cause acid damage and tooth decay by producing acid over time. Nanomaterials with suitable functionality, high permeability, extremely large surface area, significant reactivity, unique mechanical features, and non-bacterial resistance can be considered as promising agents for antimicrobial and antiviral applications. In this study, nickel oxide (NiO) nanoparticles with size range from 2 to 16 nm containing Stevia natural sweetener were eco-friendly synthesized via a simple method. Additionally, their various concentrations were evaluated on S. mutans bacteria by applying the broth dilution method. The results demonstrated that these spherical NiO nanoparticles had efficient bacteriostatic activity on this gram-positive coccus.

Immune defence mechanisms presented in liver homogenates and bile of gilthead seabream (Sparus aurata).

Because the role of the liver of fishes in providing possible immunity remains largely unknown, the aim of this work was to identify and characterize different humoral defence mechanisms in the liver homogenates and bile of gilthead seabream (Sparus aurata) for the first time. Total protein levels and several immune parameters (complement activity, lysozyme and immunoglobulin M level) were studied. Furthermore, the activity of some lytic (proteases, antiproteases, esterase, alkaline phosphatase) and antioxidant (superoxide dismutase, catalase and peroxidase) enzymes was determined. Finally, bacteriostatic activity on three opportunist fish pathogens (Vibrio harveyi, Vibrio angillarum and Photobacterium damselae) was measured. Lysozyme and antiprotease activity were undetected in liver and bile, while natural haemolytic complement activity was only detected in bile, and immunoglobulin M was detected in both samples. The levels of proteases, esterase and antioxidant enzymes were greater in bile than in liver homogenates, while the level of alkaline phosphatase was very low in both samples. In addition, while no bacteriostatic activity was detected on liver homogenates, the bile revealed a very potent bacteriostatic activity against all the tested pathogenic bacteria. These results corroborate that fish liver - especially fish bile - contains many factors involved in innate immunity that could be useful for better understanding the role of the liver as an organ involved in fish immune functions as well as the possible contribution of bile to gut mucosal immunity.

Inhibition of Haemophilus parasuis by berberine and proteomic studies of its mechanism of action.

Haemophilus parasuis is the main agent of Glässer's disease, which causes substantial losses in pig production. However, the pathogenic mechanism and virulence factors of H. parasuis have not been fully determined. In this study, berberine is shown to have a good therapeutic effect in vivo against H. parasuis; the minimal inhibitory concentration (MIC) in vitro was 2 µg/mL. Berberine inhibited H. parasuis adhesion to and invasion of PK-15 pig kidney cells. Proteomics studies of H. parasuis after berberine treatment identified a total of 97 differentially-expressed proteins; 35 upregulated and 62 downregulated. Bioinformatics analysis showed that berberine may inhibit the growth of H. parasuis by affecting outer membrane proteins, transferrins, and energy metabolism. This study provides a basis for the development of new antibacterial agents.

Antibacterial activity and mechanism of a type-I ubiquitin from the clam Ruditapes philippinarum.

In the present study, a ubiquitin (designated as RpUbi) was identified and characterized from clam Ruditapes philippinarum. Phylogenetic analysis strongly suggested that RpUbi was a member of the ubiquitin family. In non-stimulated clams, RpUbi transcripts were constitutively expressed in all examined tissues, especially in the gills and hemocytes. After Vibrio anguillarum challenge, expression of RpUbi mRNA in hemocytes was significantly up-regulated. Recombinant RpUbi (rRpUbi) showed high antibacterial activity against Gram-positive and Gram-negative bacteria. Notably, membrane integrity and electrochemical assay indicated that rRpUbi could invade the inner layer. Moreover, DNA migration could be inhibited by rRpUbi in a concentration-dependent manner. In general, our results suggested that RpUbi played an important role in host defense against invading bacteria, perhaps through a DNA-binding process.

Antimicrobial and bacteriostatic activity of surfactants against B. subtilis for microbial cleaner formulation.

Cleaning products containing live bacteria that form spores of Bacillus spp. as active substances are becoming increasingly common in probiotic cleaner formulation. The quality of cleaning performance for the production of probiotic cleaners does not only depend on the potential of the bacterial strains used, but also on the chemical components of the formulations. In this study, the surfactants and other additives were investigated as biocidal or bacteriostatic against B. subtilis, and the viability of B. subtilis was examined at different pH ranges for microbial cleaner formulation. As a result, it was discovered that the B. subtilis, which can be used in the microbial cleaner formulation, shows higher growth and viability at the neutral pH, and it passes into the death phase at pH 3. According to antagonistic activity results, the Gram-positive S. aureus and K. pneumoniae were the most sensitive bacteria while B. cereus was the most resistant bacteria. The anionic surfactants such as linear alkylbenzene sulfonic acid and sodium lauryl ether sulfate act as bacteriostatic on Bacillus spp. and do not cause cell death. In the view of these results, the usage of appropriate bacterial cultures and the correct stabilization of the formulations are also critical elements in the development of microbial cleaner formulations.

Effect of milk serum proteins on aggregation, bacteriostatic activity and digestion of lactoferrin after heat treatment.

To establish the effect of the presence of milk serum proteins on heat-induced changes to lactoferrin, lactoferrin alone, and lactoferrin mixed with either milk serum or ß-lactoglobulin was heated at 65 °C, 70 °C and 75 °C for 30 min. After heating, the effect of milk serum proteins on aggregation of lactoferrin was characterized, after which the effect of such aggregation on digestion and bacteriostatic capacity of lactoferrin were determined. The presence of milk serum proteins accelerated the aggregation of lactoferrin during heating through thiol/disulphide interchange. Lactoferrin also formed disulphide-linked aggregates when it was heated with ß-lactoglobulin. Protein aggregates formed at 75 °C were much more resistant to infant digestion, causing decreased peptide release from lactoferrin. Heating lactoferrin and milk serum proteins together accelerated the loss of bacteriostatic activity upon heating. In conclusion, heat-induced aggregation of lactoferrin with milk serum proteins affected both its digestion and its bacteriostatic activity.

[Isolation, structural identification of secondary metabolites from Pseudomonas aeruginosa HBD-12 with antibacterial and antitumor activities].

To screen strains with antibacterial and antitumor activity, pregnenolone was used as the sole carbon source for screening bacteria from soil. Based on bacteriostatic activity assay, Pseudomonas aeruginosa HBD-12 was found to be effectively inhibiting the growth of Escherichia coli, Bacillus thuringiensis, Penicillium digitatum and Penicillium italicum, and its fermentation broth was separated and purified using column chromatography. Then, structure of the obtained monomeric compounds was analyzed by spectrum analysis, and their antitumor activity was measured using HTRF kinase detection kit. The isolated monomeric compounds 1-hydroxy-9,10-phenanthroline and 3-hydroxy-9,10-dihydrophenanthroline had significant antitumor activity. At 20 µg/mL, 1-hydroxy-9,10-phenanthroline and 3-hydroxy-9,10-dihydrophenanthroline inhibited 78.39±2.29% and 60.34±8.35% Aurora kinase A, respectively. Therefore, the secondary metabolites of Pseudomonas aeruginosa HBD-12 have the potential to develop antibacterial and antitumor drugs.

Antimicrobial Efficacy of Five Probiotic Strains Against Helicobacter pylori.

Treatment of Helicobacter pylori (H. pylori) infection is a challenge for clinicians. The large increase in drug-resistant strains makes the formulation of new therapeutic strategies fundamental. The frequent onset of side effects during antibiotic treatment (mainly due to intestinal dysbiosis) should not be underestimated as it may cause the interruption of treatment, failure of H. pylori eradication and clonal selection of resistant bacteria. Probiotic integration during antibiotic treatment can exert a dual function: a direct antagonistic effect on H. pylori and a balancing effect on dysbiosis. Therefore, it fulfills the definition of a new therapeutic strategy to successfully treat H. pylori infection. Data reported in literature give promising but discrepant results. AIM: To assess in vitro bacteriostatic and bactericidal activity of probiotic strains against H. pylori. MATERIALS AND METHODS: L. casei, L. paracasei, L. acidophilus, B. lactis and S. thermophilus strains were used. Agar well diffusion and time-kill curves were carried out to detect bacteriostatic and bactericidal activity, respectively. RESULTS: All probiotic strains showed both bacteriostatic and bactericidal activity vs. H. pylori. CONCLUSIONS: Such findings prompted us to plan a protocol of treatment in which probiotics are given to infected patients in association with antibiotic therapy.

Comparative Evaluation of Piper nigrum, Rosmarinus officinalis, Cymbopogon citratus and Juniperus communis L. Essential Oils of Different Origin as Functional Antimicrobials in Foods.

Essential oils can be used as preservatives in foods because of their ability to inhibit bacteria growth in low concentration, which does not influence on foods' organoleptic properties and does not generate the resistance mechanisms in cells. The aim of that work was to compare the effectiveness of commercial oils from black pepper (Piper nigrum), rosemary (Rosmarinus officinalis), lemongrass (Cymbopogon citratus) and juniper (Juniperus communis L.) with oils obtained in our laboratory. The typical cultivation method was supported by the flow cytometry to detect the cells of very low physiologic and metabolic activity. Our investigation demonstrated that both types of oils can effectively inhibit the growth of saprophytic bacteria P. orientalis. The oils distilled in our laboratory had a bacteriostatic effect at a lower concentration, which is important for application in the food industry. Flow cytometry analyzes and confirmed the thesis that essential oils do not have a germicidal effect on bacteria cells.