Antimicrobial peptides and proteins as alternative antibiotics for porcine semen preservation.

BACKGROUND: Antimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture, are promoting AMR while also encouraging the research and employment of alternative drugs. The addition of antibiotics to the cell media is strongly recommended in sperm preservation, being gentamicin the most used for boar semen. Because of its continued use, several bacterial strains present in boar semen have developed resistance to this antibiotic. Antimicrobial peptides and proteins (AMPPs) are promising candidates as alternative antibiotics because their mechanism of action is less likely to promote AMR. In the present study, we tested two AMPPs (lysozyme and nisin; 50 and 500 µg/mL) as possible substitutes of gentamicin for boar semen preservation up to 48 h of storage. RESULTS: We found that both AMPPs improved sperm plasma membrane and acrosome integrity during semen storage. The highest concentration tested for lysozyme also kept the remaining sperm parameters unaltered, at 48 h of semen storage, and reduced the bacterial load at comparable levels of the samples supplemented with gentamicin (p > 0.05). On the other hand, while nisin (500 µg/mL) reduced the total Enterobacteriaceae counts, it also decreased the rapid and progressive sperm population and the seminal oxidation-reduction potential (p < 0.05). CONCLUSIONS: The protective effect of lysozyme on sperm function together with its antimicrobial activity and inborn presence in body fluids, including semen and cervical mucus, makes this enzyme a promising antimicrobial agent for boar semen preservation.

Synthetic polyploid induction influences morphological, physiological, and photosynthetic characteristics in Melissa officinalis L.

Melissa officinalis L., a well-known herb with diverse industrial and ethnopharmacological properties. Although, there has been a significant lack in the breeding attempts of this invaluable herb. This study aimed to enhance the agronomical traits of M. officinalis through in vitro polyploidization. Nodal segments were micropropagated and subjected to oryzalin treatment at concentrations of 20, 40, and 60 mM for 24 and 48 hours. Flow cytometry, chromosome counting, and stomatal characteristics were employed to confirm the ploidy level of the surviving plants. The survival rate of the treated explants decreased exponentially with increasing oryzalin concentration and duration. The highest polyploid induction rate (8%) was achieved with 40 mM oryzalin treatment for 24 hours. The induced tetraploid plants exhibited vigorous growth, characterized by longer shoots, larger leaves, and a higher leaf count. Chlorophyll content and fluorescence parameters elucidated disparities in photosynthetic performance between diploid and tetraploid genotypes. Tetraploid plants demonstrated a 75% increase in average essential oil yield, attributed to the significantly larger size of peltate trichomes. Analysis of essential oil composition in diploid and tetraploid plants indicated the presence of three major components: geranial, neral, and citronellal. While citronellal remained consistent, geranial and neral increased by 11.06% and 9.49%, respectively, in the tetraploid population. This effective methodology, utilizing oryzalin as an anti-mitotic agent for polyploid induction in M. officinalis, resulted in a polyploid genotype with superior morpho-physiological traits. The polyploid lemon balm generated through this method has the potential to meet commercial demands and contribute significantly to the improvement of lemon balm cultivation.

Effect of Conventional Preservatives and Essential Oils on the Survival and Growth of Escherichia coli in Vegetable Sauces: A Comparative Study.

Essential oils have gained attention as natural alternatives to chemical preservatives in food preservation. However, more information is needed regarding consumer acceptance of essential oils in actual food products. This study aimed to compare the effects of conventional preservatives, heat treatment, and essential oils derived from thyme, oregano, and lemongrass on the survival and growth of pathogenic Escherichia coli in vegetable sauces. The results demonstrated a gradual decrease in pathogen numbers over time, even in untreated samples. On the fifth day of storage, heat treatment, sodium chloride, and acidification using citric acid (pH 3.2) exhibited reductions of 4.4 to 5.3 log CFU/g compared to the untreated control. Among the essential oils tested, lemongrass essential oil at a concentration of 512 mg/kg demonstrated the most remarkable effectiveness, resulting in a reduction of 1.9 log CFU/g compared to the control. Fifteen days after treatment, the control samples exhibited a contamination rate of 6.2 log CFU/g, while E. coli numbers in treated samples with heat, sodium chloride, citric acid (pH 3.2), and lemongrass essential oil (512 mg/kg) were below the detection limits. Additionally, sensory evaluation was conducted to assess the acceptability of the treated samples. The findings provide valuable insights into the potential utilisation of essential oils as natural preservatives in vegetable sauces and their impact on consumer acceptance.

N-thiocarboxyanhydrides, amino acid-derived enzyme-activated H2S donors, enhance sperm mitochondrial activity in presence and absence of oxidative stress.

BACKGROUND: Hydrogen sulfide (H2S) donors are crucial tools not only for understanding the role of H2S in cellular function but also as promising therapeutic agents for oxidative stress-related diseases. This study aimed to explore the effect of amino acid-derived N-thiocarboxyanhydrides (NTAs), which release physiological H2S levels in the presence of carbonic anhydrase, on porcine sperm function during short-term incubation with and without induced oxidative stress. For this purpose, we employed two H2S-releasing NTAs with release half-lives (t1/2) in the range of hours that derived from the amino acids glycine (Gly-NTA) or leucine (Leu-NTA). Because carbonic anhydrase is crucial for H2S release from NTAs, we first measured the activity of this enzyme in the porcine ejaculate. Then, we tested the effect of Gly- and Leu-NTAs at 10 and 1 nM on sperm mitochondrial activity, plasma membrane integrity, acrosomal status, motility, motile subpopulations, and redox balance during short-term incubation at 38 °C with and without a reactive oxygen species (ROS)-generating system. RESULTS: Our results show that carbonic anhydrase is found both in spermatozoa and seminal plasma, with activity notably higher in the latter. Both Gly- and Leu-NTAs did not exert any noxious effects, but they enhanced sperm mitochondrial activity in the presence and absence of oxidative stress. Moreover, NTAs (except for Leu-NTA 10 nM) tended to preserve the sperm redox balance against the injuries provoked by oxidative stress, which provide further support to the antioxidant effect of H2S on sperm function. Both compounds also increased progressive motility over short-term incubation, which may translate into prolonged sperm survival. CONCLUSIONS: The presence of carbonic anhydrase activity in mammalian spermatozoa makes NTAs promising molecules to investigate the role of H2S in sperm biology. For the first time, beneficial effects of NTAs on mitochondrial activity have been found in mammalian cells in the presence and absence of oxidative stress. NTAs are interesting compounds to investigate the role of H2S in sperm mitochondria-dependent events and to develop H2S-related therapeutic protocols against oxidative stress in assisted reproductive technologies.

In Vitro Selective Combinatory Effect of Ciprofloxacin with Nitroxoline, Sanguinarine, and Zinc Pyrithione against Diarrhea-Causing and Gut Beneficial Bacteria.

Antibiotic resistance in diarrhea-causing bacteria and its disruption of gut microbiota composition are health problems worldwide. The development of combinatory agents that increase the selective inhibitory effect (synergism) against diarrheagenic pathogens and, simultaneously, have a lowered impact (antagonism) or no negative action on the gut microbiota is therefore proposed as a new strategy efficient for chemotherapy against diarrheal conditions. In this study, the in vitro selective combinatory effect of ciprofloxacin with nitroxoline, sanguinarine, and zinc pyrithione, representing various classes of alkaloid-related compounds (nitroquinolines, benzylisoquinolines and metal-pyridine derivative complexes) against selected standard diarrhea-causing (Bacillus cereus, Enterococcus faecalis, Listeria monocytogenes, Shigella flexneri, and Vibrio parahaemolyticus) and gut-beneficial (Bifidobacterium adolescentis, Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, Lactobacillus casei, and Lactobacillus rhamnosus) bacteria, was evaluated according to the sum of fractional inhibitory concentration indices (FICIs) obtained by the checkerboard method. The results showed that the individual combination of ciprofloxacin with nitroxoline, sanguinarine, and zinc pyrithione produced a synergistic effect against the pathogenic bacteria, with FICI values ranging from 0.071 to 0.5, whereas their antagonistic interaction toward the Bifidobacterium strains (with FICI values ranging from 4.012 to 8.023) was observed. Ciprofloxacin-zinc pyrithione produced significant synergistic action against S. flexneri, whereas a strong antagonistic interaction was observed toward B. breve for the ciprofloxacin-nitroxoline combination. These findings suggest that certain combinations of agents tested in this study can be used for the development of antidiarrheal therapeutic agents with reduced harmful action on the gastrointestinal microbiome. However, further studies focused on their pharmacological efficacy and safety are needed before they are considered for clinical trials. IMPORTANCE Diarrheal infections, which are commonly treated by antibiotics, are still responsible for over 4 to 5 million cases of human deaths annually. Moreover, the rising incidence of antibiotic resistance and its negative effect on beneficial bacteria (e.g., Bifidobacteria) of the gut microbial community are another problem. Thus, the development of selective agents able to inhibit diarrheal bacteria and, simultaneously, that have no negative impact on the gut microbiota, is important. Our results showed that individual combinations of ciprofloxacin with nitroxoline, sanguinarine, and zinc pyrithione produced synergism against the pathogenic bacteria, whereas their antagonistic interaction toward the beneficial strains was observed. The antagonism can be considered a positive effect contributing to the safety of the therapeutic agents, whereas their synergism against diarrheal bacteria significantly potentiates total antimicrobial efficacy. The certain combinations tested in this study can be used for the development of antidiarrheal agents with reduced harmful action on the gastrointestinal microbiome.

Attraction or Repelling Effects of Commercial Plant Essential Oils on the Synanthropic Cheiracanthium mildei (Araneae: Cheiracanthiidae).

The northern yellow sac spider Cheiracanthium mildei L. Koch, is expanding its range to Central Europe, especially to synanthropic habitats. The spiders become unwanted companions because of the unreasonable fear - arachnophobia, and estetic reason - silk retreats in corners, capturing dust. The most commonly used substances against spiders are pesticides, which are, however, toxic. In our work we tested the attraction or repellence of 15 essential oils (EO) from plants representing eight families to C. mildei. Our research has shown a significant repellent effect of EO from three plants, namely Syzygium aromaticum (L.) Merr. et L. M. Perry (Myrtales: Myrtaceae), Ananas comosus (L.) Merr. (Poales: Bromeliaceae) and Musa sp. (L.) (Zingiberales: Musaceae). In contrast, some EOs appeared to have an attraction effect, particularly Carum carvi L. (Apiales: Apiaceae). Zingiber officinale Roscoe (Zingiberales: Zingiberaceae) reduced the tendency of spiders to construct the silken retreat. S. aromaticum, A. sativus, Musa sp. and Z. officinale have the potential to be used as natural repellents against spiders.

Plant-Derived Products as Antibacterial and Antifungal Agents in Human Health Care.

A number of papers reporting antimicrobial properties of extracts, essential oils, resins and various classes of compounds isolated from higher plants have been published in recent years; however, a comprehensive analysis of plant-derived antimicrobial agents currently applied in practice for the improvement of human health is still lacking. This review summarizes data on clinical efficacy, antimicrobial effects and the chemistry of commercially available antibacterial and antifungal agents of plant origin currently used in the prevention and treatment of gastrointestinal, oral, respiratory, skin, and urinary infections. As a result of an analysis of the literature, more than 40 plant-derived over-the-counter pharmaceuticals, dietary supplements, cosmetics, herbal medicines, and functional foods containing complex mixtures (e.g. Glycyrrhiza glabra extract, Melaleuca alternifolia essential oil, and Pistacia lentiscus resin), pure compounds (e.g. benzoic acid, berberine, eucalyptol, salicylic acid and thymol) as well as their derivatives and complexes (e.g. bismuth subsalicylate and zinc pyrithione) have been identified. The effectiveness of many of these products is illustrated by results of clinical trials and supported by data on there in vitro antimicrobial activity. A broad spectrum of various commercial products currently available on the market and their welldocumented clinical efficacy suggests that plants are prospective sources for the identification of new types of antimicrobial agents in future. Innovative approaches and methodologies for effective proof-of-concept research and the development of new types of plant-derived products effective against recently emerging problems related to human microbial diseases (e.g. antimicrobial resistance) are also proposed in this review.

Stress response of Escherichia coli to essential oil components - insights on low-molecular-weight proteins from MALDI-TOF.

The antibacterial effects of essential oils and their components (EOCs) are usually attributed to effects on membranes and metabolism. Studies of the effects of EOCs on protein expression have primarily analysed proteins larger than 10 kDa using gel electrophoresis. In the present study, we used MALDI-TOF-MS to investigate the effects of EOCs on low-molecular-weight proteins. From 297 m/z features, we identified 94 proteins with important differences in expression among untreated samples, samples treated with EOCs, and samples treated with antibiotics, peroxide, or chlorine. The targets of these treatments obviously differ, even among EOCs. In addition to ribosomal proteins, stress-, membrane- and biofilm-related proteins were affected. These findings may provide a basis for identifying new targets of essential oils and synergies with other antibiotics.

Anti-Infectivity against Herpes Simplex Virus and Selected Microbes and Anti-Inflammatory Activities of Compounds Isolated from Eucalyptus globulus Labill.

Herpes simplex virus (HSV) causes numerous mild-to-serious human diseases, including mucocutaneous herpes infections and life-threatening herpes encephalitis. Moreover, herpes viral lesions can be complicated by inflammation and secondary bacterial infections. The development of resistance to antiviral drugs along with the undesirable side effects of these drugs are relevant argue for the development of new anti-HSV drugs with diverse mechanisms of action. Eucalyptus extracts have been used for decades to combat various infectious diseases. We isolated and studied 12 pure compounds and one mixture of two constitutional isomers from the leaves and twigs of E. globulus. The structures were identified by spectroscopic methods (NMR, HR-MS, IR) and all of them were tested for antiherpetic activity against the replication of antigen types HSV-1 and HSV-2. Tereticornate A (12) (IC50: 0.96 µg/mL; selectivity index CC50/IC50: 218.8) showed the strongest activity in the anti-HSV-1 assay, even greater than acyclovir (IC50: 1.92 µg/mL; selectivity index CC50/IC50: 109.4), a standard antiviral drug. Cypellocarpin C (5) (EC50: 0.73 µg/mL; selectivity index CC50/EC50: 287.7) showed the most potent anti-HSV-2 activity, also more intensive than acyclovir (EC50: 1.75 µg/mL; selectivity index CC50/EC50: 120.0). The antimicrobial activity of the isolated compounds was also evaluated against the bacteria Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa and the yeast Candida albicans. The anti-inflammatory potential was examined using LPS-stimulated THP-1-XBlue™-MD2-CD14 and THP-1 macrophages and focusing on the influences of the NF-κB/AP-1 activity and the secretion of pro-inflammatory cytokines IL-1ß and TNF-α.

In Vitro Digestibility of Aluminum from Hibiscus sabdariffa Hot Watery Infusion and Its Concentration in Urine of Healthy Individuals.

Increased ingestion of aluminum (Al) can lead to its accumulation in the human body, especially in people with kidney problems. Al is also associated with several nervous diseases and its negative influence on embryo development during pregnancy has been proven in animal models. Hibiscus sabdariffa L. petals are widely used alone or in fruit tea formulas, which are recommended for drinking during pregnancy instead of tea. Its petals can contain similar and even higher amounts of Al as tea, which is a known Al accumulator. Our research investigated whether the regular intake of H. sabdariffa infusion leads to increased burden of Al. Sixteen days of ingestion of H. sabdariffa infusion (c Al = 0.5 mg.L-1) led to increased but unbalanced levels (15-86 µg L-1) of Al in urine compared to a period when the infusion was not ingested. The highest amounts of Al excreted were observed every third day during the ingestion. Mild health problems, such as nausea and dizziness (which could be related to plant properties) were reported by more sensitive volunteers.Our results suggest that the tea infusion from H. sabdariffa petals increases body burden of Al and, therefore, sensitive individuals as pregnant women and people with kidney problems should be cautious with excessive consumption of hibiscus infusion or fruit teas containing this plant. However, further study including more individuals is needed to fully confirm our preliminary results.

Composition and Antimicrobial Activity of Euphrasia rostkoviana Hayne Essential Oil.

Eyebright, Euphrasia rostkoviana Hayne (Scrophulariaceae), is a medicinal plant traditionally used in Europe for the treatment of various health disorders, especially as eyewash to treat eye ailments such as conjunctivitis and blepharitis that can be associated with bacterial infections. Some Euphrasia species have been previously reported to contain essential oil. However, the composition and bioactivity of E. rostkoviana oil are unknown. Therefore, in this study, we investigated the chemical composition and antimicrobial activity of the eyebright essential oil against some organisms associated with eye infections: Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, and Candida albicans. GC-MS analysis revealed more than 70 constituents, with n-hexadecanoic acid (18.47%) as the main constituent followed by thymol (7.97%), myristic acid (4.71%), linalool (4.65%), and anethole (4.09%). The essential oil showed antimicrobial effect against all organisms tested with the exception of P. aeruginosa. The best activity was observed against all Gram-positive bacteria tested with the minimum inhibitory concentrations of 512 µg/mL. This is the first report on the chemical composition of E. rostkoviana essential oil and its antimicrobial activity.

In vitro combinatory antimicrobial effect of plumbagin with oxacillin and tetracycline against Staphylococcus aureus.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a bicyclic naphthoquinone naturally distributed among Plumbago species, has been reported to have antimicrobial activity against a wide range of microorganisms. In this study, plumbagin was examined for its combinatory antimicrobial effect with tetracycline or oxacillin against nine strains of Staphylococcus aureus, including its methicillin- and multidrug-resistant strains. Minimum inhibitory concentrations were determined through the broth microdilution method, whereas the combinatory effect was evaluated according to the sum of fractional inhibitory concentration (ΣFIC) indices. Additive interactions were obtained for both combinations against most of the strains tested. Synergy was obtained for combination with oxacillin against two out of seven strains (ΣFIC range 0.273-0.281), both were methicillin resistant. Our results proved plumbagin as a compound suitable for anti-Staphylococcal combinatory testing. Moreover, to the best of our knowledge, this is the first report of plumbagin synergy with oxacillin against S. aureus strains, including its resistant forms.

Thymoquinone vapor significantly affects the results of Staphylococcus aureus sensitivity tests using the standard broth microdilution method.

The broth microdilution (BMD) method is widely used for the determination of minimum inhibitory concentrations of antimicrobial agents, including volatile oils and their components. In this series of various experiments, we have demonstrated the influence of thymoquinone (TQ) vapor on the results of the BMD test performed with Staphylococcus aureus as a model organism. The spread of vapor from the TQ containing wells (32-512 µg/mL) caused the complete inhibition of staphylococcal growth in adjoining wells initially containing bacterium-inoculated pure Mueller-Hinton broth only and thus produced false positive results of the test. The ability of TQ to pass into the adjoined wells was subsequently confirmed by gas chromatography-mass spectrometry, whereas TQ at concentrations up to 84 µg/mL was detected in these wells after five hours. Based on these results, we suppose that vapors of TQ as well as of other naturally occurring volatile compounds and their mixtures (for example essential oils and plant extracts) can significantly influence results of the standard BMD assay. These observations, therefore, call for development of new appropriate BMD method suitable for assessment of antimicrobial activity of volatile substances.

Synergistic interactions of epigallocatechin gallate and oxytetracycline against various drug resistant Staphylococcus aureus strains in vitro.

Epigallocatechin gallate (EGCG), the major catechin contained in tea leaves, is known to possess the synergistic anti-staphylococcal activity in combination with various ß-lactam antibiotics and tetracycline. In the present study, we explored the in vitro combinatory effect of EGCG in combination with oxytetracycline against eight standard strains and clinical isolates of Staphylococcus aureus, including erythromycin, methicillin and tetracycline resistant strains. The minimum inhibitory concentrations were determined by the broth microdilution assay and the data were evaluated according to the sum of fractional inhibitory concentrations (∑FIC). Our results showed synergistic and additive interactions against all S. aureus strains tested (∑FIC 0.288-0.631), two of which were multidrug resistant. According to our best knowledge, it is the first report on the EGCG synergy with oxytetracycline. Considering its significant synergistic antimicrobial effect and low toxicity, we suggest EGCG as a promising compound for the development of new anti-staphylococcal formulations.

Distribution of isoflavones and coumestrol in neglected tropical and subtropical legumes.

BACKGROUND: Isoflavones and coumestrol from dietary legumes are plant constituents showing multiple beneficial effects on humans. Owing to their ability to bind with mammalian estrogenic receptors and thereby intervention in several kinds of hormone-related cancers, they have received much attention. Soybean (Glycine max) is currently the major source of isoflavonoids in human diet. However, dozens of tropical and subtropical leguminous species remain unexplored for their isoflavonoids content. RESULTS: We have analyzed 55 extracts from 41 tropical and subtropical legume species used either in human or animal diet by high-performance liquid chromatography for the content of soy isoflavones, biochanin A, daidzein, daidzin, formononetin, genistein, genistin, sissotrin, ononin and the coumestan coumestrol. Genistein and biochanin A were the most abundant compounds. The highest content of genistein was found in aerial parts of Andira macrothyrsa, seeds of Pachyrhizus tuberosus and aerial parts of Calopogonium mucunoides (598, 250 and 184 µg g(-1), respectively) and biochanin A in aerial parts of Cratylia argentea, C. mucunoides and flowers of A. macrothyrsa (76, 53 and 40 µg g(-1), respectively). CONCLUSION: None of the samples tested was richer overall source of soy isoflavones and coumestrol than soybean; nevertheless several species (C. mucunoides or A. macrothyrsa) may serve as a promising source of individual compounds.

Potentiation of the in vitro antistaphylococcal effect of oxacillin and tetracycline by the anti-inflammatory drug diacetyl rhein.

BACKGROUND: The anti-inflammatory drug diacetyl rhein has been found to possess promising antistaphylococcal effects against various drug-resistant strains in our previous study. In the present work, we explored the in vitro combinatory interactions of diacetyl rhein with oxacillin and tetracycline against 13 standard strains and clinical isolates of Staphylococcus aureus, including those resistant to erythromycin, methicillin and tetracycline. METHODS: Minimum inhibitory concentrations were determined by broth microdilution assay, and the effects of combinations were evaluated according to the sum of fractional inhibitory concentrations (ΣFICs). RESULTS: Synergistic or additive effects were observed against all S. aureus strains (ΣFIC 0.258-1), whereas diacetyl rhein-oxacillin appeared to be the most effective combination, synergistically inhibiting the growth of 4 strains tested. CONCLUSION: To our best knowledge, this is the first report on a synergistic antibacterial effect of diacetyl rhein. Our results suggest this promising compound for further evaluation of its synergistic anti-infective potential as an agent with a combined anti-inflammatory and synergistic antibacterial action.

In vitro synergistic effects of baicalin with oxytetracycline and tetracycline against Staphylococcus aureus.

OBJECTIVES: In this study we examined the in vitro synergistic effect of baicalin, the flavone constituent of Scutellaria spp., in combinations with oxytetracycline and tetracycline on the growth of Staphylococcus aureus. METHODS: The MICs were determined by the broth microdilution method and the effect of combinations was evaluated according to the sum of fractional inhibitory concentration indices (FICIs). RESULTS: Synergistic activity (FICI  ≤  0.5) was observed for combinations of baicalin with oxytetracycline or tetracycline against 4 of 10 strains tested, whereas the baicalin/oxytetracycline combination possessed the strongest synergistic effect (FICI = 0.418). CONCLUSIONS: Baicalin acts synergistically with oxytetracycline and tetracycline, enhancing its antimicrobial activity against S. aureus, including methicillin- and tetracycline-resistant strains.