Lippia grata Essential Oil Acts Synergistically with Ampicillin Against Staphylococcus aureus and its Biofilm.

Antimicrobial resistance (AMR) presents a global challenge as microorganisms evolve to withstand the effects of antibiotics. In addition, the improper use of antibiotics significantly contributes to the AMR acceleration. Essential oils have garnered attention for their antimicrobial potential. Indeed, essential oils extracted from plants contain compounds that exhibit antibacterial activity, including against resistant microorganisms. Hence, this study aimed to evaluate the antimicrobial and antibiofilm activity of the essential oil (EO) extracted from Lippia grata and its combination with ampicillin against Staphylococcus aureus strains (ATCC 25923, ATCC 700698, and JKD6008). The plant material (leaves) was gathered in Mossoro, RN, and the EO was obtained using the hydrodistillation method with the Clevenger apparatus. The antimicrobial activity of the EO was assessed through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. Antibiofilm activity was evaluated by measuring biomass using crystal violet (CV) staining, viable cell counting, and analysis of preformed biofilms. In addition, the synergistic effects of the EO in combination with ampicillin were examined by scanning electron and confocal microscopy. The EO displayed a MIC value of 2.5 mg/mL against all tested S. aureus strains and an MBC only against S. aureus JKD6008 at 2.5 mg/mL. L. grata EO caused complete biofilm inhibition at concentrations ranging from 10 to 0.312 mg/mL against S. aureus ATCC 25923 and 10 to 1.25 mg/mL against S. aureus ATCC 700698 and S. aureus JKD6008. In the viable cell quantification assay, there was a reduction in CFU ranging from 1.0 to 8.0 logs. The combination of EO with ampicillin exhibited a synergistic effect against all strains. Moreover, the combination showed a significantly inhibiting biofilm formation and eradicating preformed biofilms. Furthermore, the EO and ampicillin (individually and in combination) altered the cellular morphology of S. aureus cells. Regarding the mechanism, the results revealed that L. grata EO increased membrane permeability and caused significant membrane damage. Concerning the synergy mechanism, the results revealed that the combination of EO and ampicillin increases membrane permeability and causes considerable membrane damage, further inhibiting bacteria synergistically. The findings obtained here suggest that L. grata EO in combination with ampicillin could be a viable treatment option against S. aureus infections, including MRSA strain.

Antimicrobial and Antibiofilm Activity of Synthetic Peptide [W7]KR12-KAEK Against Enterococcus faecalis Strains.

The emergence of infections caused by microorganisms in the oral cavity and increasing concerns regarding the use of antibiotics have resulted in the development of novel antimicrobial molecules, such as antimicrobial synthetic peptides. The purpose of this study was to evaluate the antimicrobial and antibiofilm activities of the native peptide KR-12 and its derivative, the synthetic peptide [W7]KR12-KAEK, against planktonic and biofilms Enterococcus faecalis strains. The methods used to evaluate the antimicrobial activity in planktonic cultures include minimum inhibitory concentration and minimum bactericidal concentration assays. The effects of [W7]KR12-KAEK on biofilm formation and mature biofilms were evaluated by quantifying biomass (crystal violet staining) and counting colony-forming units. Structural assessments of the biofilms and cellular morphological changes were performed using scanning electron microscopy. Peptide [W7]KR12-KAEK showed potential antimicrobial activity against planktonic cells. Interestingly, the native peptide KR-12 showed no antimicrobial activity. Moreover, it inhibited biofilm formation and disrupted the mature biofilms of E. faecalis strains. These results suggest that [W7]KR12-KAEK may be a potential molecule for the development of auxiliary antimicrobial therapies against oral infections.

Structural characterization of a galectin from the marine sponge Aplysina lactuca (ALL) with synergistic effects when associated with antibiotics against bacteria.

Lectins presents the ability to interact with glycans and trigger varied responses, including the inhibition of the development of various pathogens. Structural studies of these proteins are essential to better understand their functions. In marine sponges, so far only a few lectins have their primary structures completely determined. Thus, the objective of this work was to structurally characterize and evaluate antibacterial potential, in association with different antibiotics, of the lectin isolated from the marine sponge Aplysina lactuta (ALL). ALL is a homotetramer of 60 kDa formed by four 15 kDa-subunits. The lectin showed affinity only for the glycoproteins fetuin, asialofetuin, mucin type III, and bovine submaxillary mucin type I. The complete amino acid sequences of two isoforms of ALL, named ALL-a and ALL-b, were determined by a combination of Edman degradation and overlapped peptides sequenced by tandem mass spectrometry. ALL-a and ALL-b have 144 amino acids with molecular masses of 15,736 Da and 15,985 Da, respectively. Both structures contain conserved residues typical of the galectin family. ALL is a protein with antibacterial potential, when in association with ampicillin and oxacillin the lectin potentiates its antibiotic effect, included Methicillin-resistant Staphylococcus strains. Thus, ALL shows to be a molecule with potential for the development of new antibacterial drugs.

Structural study and antimicrobial and wound healing effects of lectin from Solieria filiformis (Kützing) P.W.Gabrielson.

The SfL-1 isoform from the marine red algae Solieria filiformis was produced in recombinant form (rSfL-1) and showed hemagglutinating activity and inhibition similar to native SfL. The analysis of circular dichroism revealed the predominance of ß-strands structures with spectra of ßI-proteins for both lectins, which had Melting Temperature (Tm) between 41 °C and 53 °C. The three-dimensional structure of the rSfL-1 was determined by X-ray crystallography, revealing that it is composed of two ß-barrel domains formed by five antiparallel ß chains linked by a short peptide between the ß-barrels. SfL and rSfL-1 were able to agglutinate strains of Escherichia coli and Staphylococcus aureus and did not show antibacterial activity. However, SfL induced a reduction in E. coli biomass at concentrations from 250 to 125 µg mL-1, whereas rSfL-1 induced reduction in all concentrations tested. Additionally, rSfL-1 at concentrations from 250 to 62.5 µg mL-1, showed a statistically significant reduction in the number of colony-forming units, which was not noticed for SfL. Wound healing assay showed that the treatments with SfL and rSfL-1 act in reducing the inflammatory response and in the activation and proliferation of fibroblasts by a larger and fast deposition of collagen.

Antimicrobial, Antibiofilm Activities and Synergic Effect of Triterpene 3ß,6ß,16ß-trihydroxyilup-20(29)-ene Isolated from Combretum leprosum Leaves Against Staphylococcus Strains.

Antimicrobial resistance is a natural phenomenon and is becoming a huge global public health problem, since some microorganisms not respond to the treatment of several classes of antibiotics. The objective of the present study was to evaluate the antibacterial, antibiofilm, and synergistic effect of triterpene 3ß,6ß,16ß-trihydroxyilup-20(29)-ene (CLF1) against Staphylococcus aureus and Staphylococcus epidermidis strains. Bacterial susceptibility to CLF1 was evaluated by minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) assay. In addition, the effect combined with antibiotics (ampicillin and tetracycline) was verified by the checkerboard method. The biofilms susceptibility was assessed by enumeration of colony-forming units (CFUs) and quantification of total biomass by crystal violet staining. The compound showed bacteriostatic and bactericidal activity against all Staphylococcal strains tested. The synergistic effect with ampicillin was observed only for S. epidermidis strains. Moreover, CLF1 significantly inhibited the biofilm formation and disrupted preformed biofilm of the all strains. Scanning electron microscopy (SEM) images showed changes in the cell morphology and structure of S. aureus ATCC 700698 biofilms (a methicillin-resistant S. aureus strain). Molecular docking simulations showed that CLF1 has a more favorable interaction energy than the antibiotic ampicillin on penicillin-binding protein (PBP) 2a of MRSA, coupled in different regions of the protein. Based on the results obtained, CLF1 proved to be a promising antimicrobial compound against Staphylococcus biofilms.

Chemical composition determination and evaluation of the antibacterial activity of essential oils from Ruellia asperula (Mart. Ex Ness) Lindau and Ruellia paniculata L. against oral streptococci.

This study investigated the chemical composition and evaluated the antibacterial and antibiofilm activities of essential oils (EOs) extracted from Ruellia asperula (EORA) and Ruellia paniculata (EORP) against oral streptococci. The EO constituents were analyzed by gas chromatography/mass spectrometry. The antimicrobial potential of EOs was evaluated using the minimum inhibitory concentration, minimum bactericidal concentration, and time-kill determination. Furthermore, the quantification of total biomass and the number of viable cells in the biofilms were evaluated. The major constituents of EORA were cariophylla-4(12)-8-(13)-dien-5ß-ol (14.1%), (ß)-caryophyllene (22.7%), and caryophyllene oxide (29.4%). For EORP, the major constituents were (ß)-caryophyllene (11.0%), spathulenol (13.1%), and δ-amorphene (14.9%). The tested EOs exhibited antibacterial activity against planktonic growth and biofilm formation. Thus, the EOs from R. asperula and R. paniculata prove to be promising alternatives for bacterial growth control and biofilm formation prevention of oral streptococci.

A fibrinogen-related Lectin from Echinometra lucunter represents a new FReP family in Echinodermata phylum.

Fibrinogen-related proteins (FREPs) have been identified in several animals. They are involved in the body's defense, acting as mediators of phagocytosis. Ficolins and intelectins are some of the most studied Fibrinogen-related Domain (FReD)-containing lectins. In this work, we have isolated a singular FReD-containing lectin, which cannot be classified as ficolin or intelectin. ELL (Echinometra lucunter lectin) was isolated from coelomic plasma by affinity chromatography on xanthan gum. Primary structure was determined by tandem mass spectrometry. Moreover, antimicrobial activity of ELL was evaluated against planktonic cells and biofilm of Escherichia coli, Staphylococcus aureus and S. epidermidis. ELL showed hemagglutinating activity in Ca2+ presence, which was inhibited by glycoprotein mucin and thyroglobulin. Complete amino acid sequence consisted of 229 residues, including a FReD in the N-terminal. Searches for similarity found that ELL was very close to putative proteins from Strongylocentrotus purpuratus. ELL showed moderate similarity with uncharacterized sea stars proteins and protochordate intelectins. ELL was able to inhibit the planktonic growth of the Gram-positive bacteria and significantly reduce the biofilm formation of all bacteria tested. In conclusion, we identified a new type of FReP-containing lectin with some structural and functional conservation towards intelectins.

Wound healing activity of lectin isolated from seeds of Centrolobium microchaete Mart. ex Benth. on cutaneous wounds in mice.

This study aims to evaluate the wound healing potential of lectin isolated from the seeds of Centrolobium microchaete (Mart. ex Benth) (CML) on cutaneous wounds in mice. CML did not show cytotoxicity on murine dermal fibroblasts (L929 cell line). The wounds treated with CML (200 µg/mL) showed a decrease in area within 12 days post-operative (P.O.) when compared to control. On 3rd and 7th day P.O., the CML-treated group exhibited fibroblast proliferation and neovascularization. On 12th day P.O., complete restructuring of the epithelial layer and connective tissue was observed in the CML-treated group, whereas control groups exhibited incomplete reepithelialization. CML treatment enhanced the wound closure via the wound contraction process, resulting in the restructuring of the skin layers on 12th day P.O. In conclusion, CML induced a fast and efficient wound healing, suggesting that it can be used as a promising therapeutic tool to heal acute wounds.

The biofilm inhibition activity of a NO donor nanosilica with enhanced antibiotics action.

Nitric oxide (NO) has emerged as a promising antibacterial agent, where NO donor compounds have been explored. Here, we investigated the role of a silica nanoparticle containing nitroprusside (MPSi-NP) as a NO donor agent against methicillin-sensitive (ATCC 25,923 and ATCC 12228) and methicillin-resistant (ATCC 700,698 and ATCC 35984) Staphylococcus strains. Biofilm inhibition was studied along with antibiotic activity in combination with standard antibiotics (ampicillin and tetracycline). MPSi-NP exhibited thermal release of 63% of NO within 24 h, while free nitroprusside released only 18% during a dialysis assay, indicating an assisted release of NO mediated by the nanoparticles. This nanomaterial showed only a moderate activity in blocking biofilm production, but exhibited a significant decrease in the number of viable bacterial cells (over 600-fold for Staphylococcus aureus ATCC 700,698 and Staphylococcus epidermidis ATCC 35984). Remarkably, even using MPSi-NP at concentrations below any antibacterial action, its combination with ampicillin promoted a significant decrease in MIC for resistant strains of S. aureus ATCC 700,698 (2-fold) and S. epidermidis ATCC 35,984 (4-fold). A carbopol-based gel formulation with MPSi-NP (0.5% w/w) was prepared and showed a zone of inhibition of 7.7 ± 0.6 mm for S. epidermidis ATCC 35984. Topical use of MPSi-NP in combination with antibiotics might be a manageable strategy to prevent and eventually treat complicated resistant bacterial infections.

Evaluation of Antimicrobial and Antioxidant Potential of Essential Oil from Croton piauhiensis Müll. Arg.

A large number of infections are caused by Gram-positive and Gram-negative multi-resistant bacteria worldwide, adding up to a figure of around 700,000 deaths per year. The indiscriminate uses of antibiotics, as well as their misuse, resulted in the selection of bacteria resistant to known antibiotics, for which it has little or no treatment. In this way, the strategies to combat the resistance of microorganisms are extremely important and, essential oils of Croton species have been extensively studied for this purpose. The aim of this study was to carry the evaluation of antibacterial, antibiofilm, antioxidant activities, and spectroscopic investigation of essential oil from Croton piauhiensis (EOCp). The EOCp exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria with required MICs ranging from 0.15 to 5% (v/v). In addition, the MBC of the EOCp for Staphylococcus aureus ATCC 25923 and ATCC 700698, were 0.15 and 1.25%, respectively. Moreover, the EOCp significantly reduced significantly the biofilm production and the number of viable cells from the biofilm of all bacterial strains tested. The antioxidant potential of the EOCp showed EC50 values ranging from 171.21 to 4623.83 µg/mL. The EOCp caused hemolysis (>45%) at the higher concentrations tested (1.25 to 5%), and minor hemolysis (17.6%) at a concentration of 0.07%. In addition, docking studies indicated D-limonene as a phytochemical with potential for antimicrobial activity. This study indicated that the EOCp may be a potential agent against infections caused by bacterial biofilms, and act as a protective agent against ROS and oxidative stress.

Antimicrobial activity and antibiotic synergy of a biphosphinic ruthenium complex against clinically relevant bacteria.

The aim of this study was to investigate the antibacterial activity, antibiotic-associated synergy, and anti-biofilm activity of the ruthenium complex, cis-[RuCl2 (dppb) (bqdi)]2+ (RuNN). RuNN exhibited antimicrobial activity against Gram-positive bacteria with minimum inhibitory concentration (MIC) values ranging from 15.6 to 62.5 µg ml-1 and minimum bactericidal concentration (MBC) values ranging from 62.5 to 125 µg ml-1. A synergistic effect against Staphylococcus spp. was observed when RuNN was combined with ampicillin, and the range of associated fractional inhibitory concentration index (FICI) values was 0.187 to 0.312. A time-kill curve indicated the bactericidal activity of RuNN in the first 1-5 h. In general, RuNN inhibited biofilm formation and disrupted mature biofilms. Furthermore, RuNN altered the cellular morphology of S. aureus biofilms. Further, RuNN did not cause hemolysis of erythrocytes. The results of this study provide evidence that RuNN is a novel therapeutic candidate to treat bacterial infections caused by Staphylococcus biofilms.

A new mucin-binding lectin from the marine sponge Aplysina fulva (AFL) exhibits antibiofilm effects.

A new mucin-binding lectin (AFL) was isolated from the marine sponge Aplysina fulva. AFL was purified by affinity chromatography on Sepharose™ matrix. Its hemagglutinating activity was independent of divalent ions, and it was weakly inhibited by simple sugars. However, porcine stomach mucin was a powerful inhibitor. In SDS PAGE, piridylethylated AFL showed one band of approximately 16 kDa, whereas in the non-reducing conditions, AFL showed at least two bands of 30 and 70 kDa. Mass spectrometry MALDI-ToF analysis showed one major ion of 31,652 ±â€¯5 Da, which corresponded to a dimer formed by subunits linked by disulfide bonds. The first fifteen amino acids of AFL were determined, and no sequence similarity was observed with any known protein. Internal sequences were obtained by mass spectrometry analysis of tryptic digestion of AFL spots. These peptides showed similarity with a lectin from marine sponge Aplysina lactuca. Secondary structure of AFL was predominantly formed by ß-conformations, which were stable at variations of pH and temperature. AFL did not inhibit planktonic growth of Gram-positive and Gram-negative bacteria tested. However, the lectin did significantly reduce the biomass biofilm of the bacteria Staphylococcus aureus, S. epidermidis, and Escherichia coli.

Antibacterial activity of a new lectin isolated from the marine sponge Chondrilla caribensis.

A new lectin from the marine sponge Chondrilla caribensis (CCL) was isolated by affinity chromatography in Sepharose 6B media. CCL is a homotetrameric protein formed by subunits of 15,445 ±2Da. The lectin showed affinity for disaccharides containing galactose and mucin. Mass spectrometric analysis revealed about 50% of amino acid sequence of CCL, which showed similarity with a lectin isolated from Aplysina lactuca. Secondary structure consisted of 10% α-helix, 74% ß-sheet/ß-turn and 16% coil, and this profile was unaltered in a broad range of pH and temperatures. CCL agglutinated Staphylococcus aureus, S epidermidis and Escherichia coli, and it was able to reduce biofilm biomass, but showed no inhibition of planktonic growth of these bacteria. CCL activity was inhibited by α-lactose, indicating that Carbohydrate Recognition Domain (CRD) of the lectin was involved in antibiofilm activity.