Broth Microdilution Technique Research Articles

Efficacy of Indolicidin, Cecropin A (1-7)-Melittin (CAMA) and Their Combination Against Biofilm-Forming Multidrug-Resistant Enteroaggregative Escherichia coli.

The present study examined the anti-biofilm efficacy of two short-chain antimicrobial peptides (AMPs), namely, indolicidin and cecropin A (1-7)-melittin (CAMA) against biofilm-forming multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC) isolates. The typical EAEC isolates re-validated by PCR and confirmed using HEp-2 cell adherence assay was subjected to antibiotic susceptibility testing to confirm its MDR status. The biofilm-forming ability of MDR-EAEC isolates was assessed by Congo red binding, microtitre plate assays and hydrophobicity index; broth microdilution technique was employed to determine minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs). The obtained MIC and MBEC values for both AMPs were evaluated alone and in combination against MDR-EAEC biofilms using crystal violet (CV) staining and confocal microscopy-based live/dead cell quantification methods. All the three MDR-EAEC strains revealed weak to strong biofilm-forming ability and were found to be electron-donating and weakly electron-accepting (hydrophobicity index). Also, highly significant (P < 0.001) time-dependent hydrodynamic growth of the three MDR-EAEC strains was observed at 48h of incubation in Dulbecco's modified Eagle's medium (DMEM) containing 0.45% D-glucose. AMPs and their combination were able to inhibit the initial biofilm formation at 24h and 48h as evidenced by CV staining and confocal quantification. Further, the application of AMPs (individually and combination) against the preformed MDR-EAEC biofilms resulted in highly significant eradication (P < 0.001) at 24h post treatment. However, significant differences were not observed between AMP treatments (individually or in combination). The AMPs seem to be an effective candidates for further investigations such as safety, stability and appropriate biofilm-forming MDR-EAEC animal models.

Antibacterial mechanism of peptide Cec4 against Acinetobacter baumannii.

BackgroundA case of Acinetobacter baumannii (A. baumannii), known as gram-negative bacteria, causes a range of nosocomial infections. Due to the continuous detection of multi-drug resistant A. baumannii in the clinic, there is an urgent need to find alternative therapies, including broad-spectrum antibacterial peptides (AMP). Recently it has been found that the peptide Cec4 has good antibacterial activity against A. baumannii, but the antibacterial mechanism remains elusive.Materials and methodsThe basic structure of Cec4 was analyzed by circular dichroism (CD) spectroscopy, and the potential antibacterial mechanism of Cec4 was detected by flow cytometry, transmission electron microscopy, fluorescence and confocal microscopy. The minimum inhibitory concentration (MIC) of antimicrobial peptides against various A. baumannii was determinated with broth microdilution techniques. The biofilm formation and the sensitivity detection of biofilms to antimicrobial peptides were detected by crystal violet staining.ResultsIn this study, the main secondary structure of the antibacterial peptide Cec4 is α-helix (99.7%) in the hydrophobic environment. Furthermore, after the treatment with Cec4, an amount of leakage of A. baumannii and the destruction of its cell membrane were detected. Moreover, it was observed that FITC-Cec4 can enter the cell, and more cells were held in the G1 phase with peptide Cec4. However, the DNA binding assay of the peptide Cec4 indicates that the peptide does not target DNA. In addition, peptide Cec4 was superior in reducing adherent biofilms of A. baumannii compared to conventional antibiotics and has no cytotoxicity.ConclusionIt is apparent that the antibacterial peptide Cec4 may achieve rapid sterilization by multi-target interaction and presents an attractive therapeutic option for the prevention and control of A. baumannii infections.

Antibacterial Activity of Bioflavonoid from Fruit Pulps of Acacia nilotica Willd

The emergence of multi-drug resistance in bacteria has led to call for research and development of new leads as antibiotics from medicinal plants. Acacia nilotica (Linn) is a plant of multipurpose medicinal uses, three bioactive flavonoids (methyl gallate, gallic acid and catechin) were isolated from its fruit pulps through a bioassay guided fractionation technique and characterized based on High Performance Liquid Chromatography, Liquid Chromatography-Mass Spectra and Nuclear Magnetic Resonance spectra. Antibacterial activity of these compounds was determined by microplate tetrazolium dye assay of broth microdilution technique against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and clinical isolates of Salmonella typhi, Klebsiella pneumonia, Candida albicans and Bacillus subtilis. Catechin, methyl gallate and gallic acid at 19.5, 39 and 39 µg/ml respectively caused a significant bio-reduction in cells of test organisms. Time kill kinetic study of the extract shows that there was percentage of growth reduction in test organisms at 2, 4, 6, 8 and 12 hrs of contact. The extent and rate of killing of the organism by the extract at 2 x MIC followed the same trend as rate of killing was time dependent. Antibacterial effects of these compounds are within the breakpoint of control drug chloramphenicol and could serve as leads in new drug development.

Rifabutin and Furazolidone Could Be the Candidates of the Rescue Regimen for Antibiotic-Resistant H. pylori in Korea.

Background/Aim. In Korea, the rate of Helicobacter pylori (H. pylori) eradication has declined steadily as a result of increasing resistance to antibiotics, especially dual resistance to clarithromycin and metronidazole. However, microbiological culture data on drug-resistant H. pylori is lacking. This study evaluated the antimicrobial efficacy of candidate antibiotics against resistant H. pylori strains. Methods. After retrospectively reviewing the data from the Helicobacter Registry in Gil Medical Center (GMC) and Asan Medical Center (AMC), along with 4 reference strains, we selected the 31 single- or multidrug-resistant strains. The susceptibility of the H. pylori strains to seven antibiotics (clarithromycin, metronidazole, levofloxacin, amoxicillin, tetracycline, rifabutin, and furazolidone) and minimum inhibitory concentration were tested using the broth microdilution technique. Results. Among 31 antibiotic resistance strains for H. pylori, there were no strains resistant to rifabutin or furazolidone, which had MICs of <0.008 and 0.5 μg/mL, respectively. Only one tetracycline-resistant strain was found (MIC < 2 μg/mL). Amoxicillin and levofloxacin were relatively less effective against the H. pylori strains compared to rifabutin or furazolidone (resistance rates 22.6%, 1.9%, respectively). Tetracycline showed the relatively low resistance rates (3.2%) for H. pylori strains. Conclusions. Therefore, along with tetracycline which has already been used as a component for second-line eradication regimen for Helicobacter, rifabutin and furazolidone, alone or in combination, could be used to eradicate antibiotic-resistant H. pylori strains where drug-resistant Helicobacter spp. are increasing.

Antimicrobial and antioxidant activities of hydrocarbon and oxygenated monoterpenes against some foodborne pathogens through in vitro and in silico studies

The present work describes the antimicrobial action of 25 monoterpenes (six hydrocarbons, five ketones, two aldehydes, six alcohols and six acetate analogues) against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus and antifungal activity against Aspergillus flavus. The antibacterial activity was evaluated by broth microdilution technique as a minimum inhibitory concentration (MIC) and the antifungal activity was performed by mycelia radial growth technique as the effective concentration causing 50% inhibition of the mycelial growth (EC50). The results showed that thymol and α-terpineol were the most potent against E. coli (MIC = 45 and 55 mg/L, respectively) and S. aureus (MIC = 135 and 225 mg/L, respectively). The results also showed that thymol displayed the maximum antifungal action against A. flavus with EC50 20 mg/L. Furthermore, the antioxidant activity was determined using N,N-dimethyl-1,4-phenylenediamine (DMPD) and the results showed that geraniol were the most potent compound (IC50 = 19 mg/L). Molecular docking studies indicated that the compounds displayed different binding interactions with the amino acid residues at the catalytic sites of N5-carboxyaminoimidazole synthetase and oxysterol binding protein Osh4 enzymes. Non-covalent interactions including van der Waals, hydrogen bonding as well as hydrophobic were observed between the compounds and the enzymes. A significant relationship was found between the docking score and the biological activity of the tested monoterpenes compared to the ceftriaxone and carbendazim as standard bactericide and fungicide, respectively. In silico ADMET properties were also performed and displayed potential for the development of promising antimicrobial agents. For these reasons, these compounds may be considered as potential ecofriendly alternatives in food preservation to delay or prevent the microbial infection and prolong the shelf life of food products.

Potential efficacy of garlic lock therapy in combating biofilm and catheter-associated infections; experimental studies on an animal model with focus on toxicological aspects

BackgroundLife-threatening central venous catheter-related infections are primarily initiated by biofilm formation on the catheter surface. Antibiotic lock therapy is recommended for eradicating intraluminal biofilm. In the era of antibiotic resistance, antibiotics of natural origins provide an effective and cheap option for combating resistant strains. Garlic especially stole the spotlight because of its impressive antimicrobial effectiveness against such superbugs. AimIs to estimate the potential use of fresh garlic extract (FGE) as a lock agent against multi-drug resistant (MDR) bacteria. MethodsThe agar well diffusion and broth microdilution techniques were employed to test the antimicrobial activities of FGE against five MDR strains; E. coli, Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), Serratia marscens (S. marscens) and Methicillin-resistant Staphylococcus aureus (MRSA). Then the protective and therapeutic efficiencies of FGE against bacterial biofilms were in-vitro evaluated; at concentrations of 100, 75, 50 and 25%; in tissue culture plate (TCP) and on the polyurethane (PU) sheets using the crystal violet (CV) assay and colony-forming unit (CFU), respectively. Scanning electron microscopy (SEM) was also used to confirm eradication of biofilms on PU sheets. Finally, systemic and deep tissue infections by P. aeruginosa and MRSA were induced in mice that were then treated by FGE at either 100 or 200 mg/kg for seven days. Where the antibacterial activity was assessed by tissue and blood culturing at the end of the treatment period. Biochemical, hematological and histological parameters were also investigated. ResultsFGE exhibited potent in-vitro and in-vivo antibacterial and antibiofilm activities against MDR strains. It not only didn’t exhibit toxicological effects at the hematological and the histological levels but also provided protective effects as demonstrated by the significant drop in the biochemical parameters. ConclusionFGE has the potential to be used as a prophylactic and/or therapeutic lock agent against biofilm-associated infections caused by MDR bacteria.

In vitro activities of antimicrobial peptides and ceragenins against Legionella pneumophila.

Legionella pneumophila is a waterborne intracellular pathogenic bacterium, the most frequent cause of human legionellosis and a relatively common cause of community-acquired and nosocomial pneumonia. Some legionellosis outbreaks are related to the presence of biofilms, which provide a reservoir for L. pneumophila strains. We investigated the in vitro activities of antibiotics; erythromycin and doxycycline, antimicrobial peptides AMPs; melittin, LL-37 and CAMA (cecropin A (1-7)-Melittin A (2-9) and ceragenins; CSA-8, CSA-13, CSA-44, CSA-131 and CSA-138 against L. pneumophila. Isolation of Legionella strains was conducted according to ISO 1998. Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and minimum biofilm eradication concentrations (MBECs) were determined using microbroth dilution techniques. MIC ranges for melittin, LL-37, and CAMA were 0.25-1, 1-4, and 2-8 µg ml-1, respectively. MIC ranges for CSA-8, 13, 44, 131, and 138 were 0.5-2, 0.5-1, 1-4, 0.5-2, and 1-2 µg ml-1, respectively, and MBEC values for the ceragenins were 10-160 µg ml-1. These results demonstrate that AMPs and ceragenins display broad-spectrum, in vitro activity against L. pneumophila. In particular, CSA-8, CSA-13 and melittin gave the lowest MICs and MBCs. We also observed that ceragenins are active against established L. pneumophila biofilms.

Antibacterial activity of Morinda citrifolia Linneo seeds against Methicillin-Resistant Staphylococcus spp

In traditional medicine, Morinda citrifolia (Noni) is used to treat various ailments, including skin and respiratory-tract infections. In this work, a bio-directed study (seed extracts) with five bacteria was carried out against four clinical isolates of Methicillin-Resistant Staphylococcus (MRS) and Staphylococcus aureus ATCC 29213 strain to find molecules capable of inhibiting them. Three organic extracts were obtained by maceration of the noni seeds with ascending polarity solvents (n-hexane, dichloromethane and methanol) that were evaluated as antibacterial in the model of bioautography and broth microdilution techniques. The results showed that the methanolic extract was the most active against all bacteria (MIC = 16 mg/mL). The chromatographic fractionation performed on this extract allowed obtaining six fractions (EMF1-EMF6), of which F1, F2 and F5 exhibited activity against some of the bacteria. EMF1 fraction reached an MIC of 25 μg/mL against S. haemolyticus twice as much as the positive control, in which the chemical content is mainly composed of a mixture of γ-butyrolactones (1–2) and esterified fatty acids (3–9); chemical characterization of the nine compounds was carried out based on gas chromatography coupled to masses. EMF2 fraction, presented an MIC of 200 μg/mL against S. aureus 0198 and S. haemolyticus 562B, where a coumarin known as scopoletin (10) was isolated and active against S. aureus 0198 (MIC = 100 μg/mL). EMF5 fraction demonstrated an MIC of 200 μg/mL against S. aureus 0198, S. haemolyticus 562B and S. epidermidis 1042, in which a neolignan known as americanin A (11) was identified, showing activity against S. haemolyticus 562B and S. epidermidis 1042 (MIC = 100 μg/mL). The chemical characterization of isolated compounds 10 and 11 was performed by the analysis of 1H and 13C NMR. Therefore, the methanolic extract, identified and isolated compounds showed important antibacterial activity against the MRS, validating its use in traditional medicine.

1,4,7-Triazacyclononane Restores the Activity of β-Lactam Antibiotics against Metallo-β-Lactamase-Producing Enterobacteriaceae: Exploration of Potential Metallo-β-Lactamase Inhibitors.

Metallo-β-lactamase (MBL)-producing Enterobacteriaceae are of grave clinical concern, particularly as there are no metallo-β-lactamase inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available β-lactams are thus imperative. We investigated a zinc-chelating moiety, 1,4,7-triazacyclononane (TACN), for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae MICs, minimum bactericidal concentrations (MBCs), the serum effect, fractional inhibitory concentration indexes, and time-kill kinetics were determined using broth microdilution techniques according to Clinical and Laboratory Standards Institute (CSLI) guidelines. Enzyme kinetic parameters and the cytotoxic effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem regained its activity against carbapenemase-producing Enterobacteriaceae, with the MIC decreasing from between 8 and 64 mg/liter to 0.03 mg/liter in the presence of TACN. The TACN-meropenem combination showed bactericidal effects with an MBC/MIC ratio of ≤4, and synergistic activity was observed. Human serum effects on the MICs were insignificant, and TACN was found to be noncytotoxic at concentrations above the MIC values. Computational studies predicted that TACN inhibits MBLs by targeting their catalytic active-site pockets. This was supported by its inhibition constant (Ki ), which was 0.044 μM, and its inactivation constant (Kinact), which was 0.0406 min-1, demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor.IMPORTANCE Carbapenem-resistant Enterobacteriaceae (CRE)-mediated infections remain a significant public health concern and have been reported to be critical in the World Health Organization's priority pathogens list for the research and development of new antibiotics. CRE produce enzymes, such as metallo-β-lactamases (MBLs), which inactivate β-lactam antibiotics. Combination therapies involving a β-lactam antibiotic and a β-lactamase inhibitor remain a major treatment option for infections caused by β-lactamase-producing organisms. Currently, no MBL inhibitor-β-lactam combination therapy is clinically available for MBL-positive bacterial infections. Hence, developing efficient molecules capable of inhibiting these enzymes could be a promising way to overcome this phenomenon. TACN played a significant role in the inhibitory activity of the tested molecules against CREs by potentiating the activity of carbapenem. This study demonstrates that TACN inhibits MBLs efficiently and holds promises as a potential MBL inhibitor to help curb the global health threat posed by MBL-producing CREs.

In Silico and In Vitro Investigation of the Antifungal Activity of Isoeugenol against Penicillium citrinum.

This increase in the prevalence of drug-resistant pathogens occurs at a time when the discovery and development of new antimicrobial agents occur slowly. In this context, the objective of this study was to investigate the antifungal activity of isoeugenol, a phenylpropanoid, by in vitro and in silico assays against Penicillium citrinum strains. For in silico analysis, the software PASS online, Molinspiration and Osíris were used. For the determination of Minimum Inhibitory Concentration (MIC) and Minimal Fungicide Concentration (MFC) of isoeugenol and voriconazole were carried out using the broth microdilution technique. PASS online has shown that isoeugenol has the opportunity to present antiseptic, antifungal, antibacterial, antimycobacterial activities. Molinspiration showed that the phytoconstituent has good potential for oral bioavailability. In the analysis with the Osiris program, it was demonstrated that isoeugenol has low irritant and tumorigenic risk. The MIC of isoeugenol varied between 256 and 32 µg/mL, MIC50 of 64 µg/mL and MIC90 was 128 µg/mL. The MFC50, MFC90 and MFC of the isoeugenol for P. citrinum species were 64, 256 and 518 μg/mL, respectively. After analysis, it was verified that the isoeugenol have bactericidal effect against the strains of P. citrinum. After these results, it is important to discover the mechanism of action involved in the antifungal action of the compound, as well as in vitro and in vivo toxicity tests.

Hop Extract Acts as an Antioxidant with Antimicrobial Effects against Propionibacterium Acnes and Staphylococcus Aureus

Acne is associated with hyperkeratosis, elevated levels of skin sebum and growth of Propionibacterium acnes (P. acnes) and Staphylococcus aureus (S. aureus). Furthermore, P. acnes promotes inflammation by inducing IL-6 production and oxidative stress. The aim of this study was to assess the antioxidant, anti-inflammatory and antibacterial potential of a hop-CO2-extract with 50% humulone and lupulone. The susceptibility of P. acnes and S. aureus to the hop extract was tested by using the broth microdilution technique. The minimal inhibitory concentrations (MIC) for P. acnes and S. aureus were 3.1 and 9.4 µg/mL, respectively. In addition, the hop extract showed an antioxidative effect with a half maximal inhibitory concentration (IC50) of 29.43 µg/mL as well as additional anti-inflammatory effects by reducing the IL-6 expression (IC50: 0.8 µg/mL). In addition, a gel formulation with 0.3% hop extract (w/w) had antibacterial activity against P. acnes and S. aureus (inhibition zone value: 5.5 mm and 3 mm, respectively) which was significantly superior to the placebo gel. The positive control (a gel with the antibiotic clindamycin) showed an inhibition zone of 9 mm. Due to its antioxidant, anti-inflammatory and antibacterial effects hop extract might be a treatment option for acne-prone skin.

Antimicrobial and antibiofilm activity of the essential oil from dried leaves of Eucalyptus staigeriana

ABSTRACT: In recent years, compounds with biological properties produced by plants have received attention as an alternative to control microorganisms. Essential oils extracted from green leaves of Eucalyptus sp. have been demonstrated to have antimicrobial activities, but so far there are no reports of antimicrobial activity of essential oils extracted from dried leaves of Eucalyptus staigeriana. So, the objectives of this study were to determine the chemical composition of the essential oils obtained from dried leaves of E. staigeriana (EOdlES) and to evaluate in vitro antimicrobial and antibiofilm activities of EOdlES against gram-positive and gram-negative, resistance and multiresistant Enterococcus faecalis isolated from food and clinical samples. The characterization of EOdlES was performed by gas chromatography-mass spectrometry (GC/MS). For this study, 26 bacterial strains were used, which included 11 reference strains and 15 antibiotic resistant and multiresistant E. faecalis strains. Antimicrobial activities of EOdlES against gram-positive and gram-negative were determined using the disc diffusion method. The minimum inhibitory concentration (MIC) value was evaluated by a microbroth dilution technique. The antibiofilm effects were assessed by microtiter plate method. As a result, 21 compounds were identified, being oxygenated monoterpenes (69.58%) the major chemical family. EOdlES showed only antimicrobial activity against gram-positive strains. E. faecalis resistant and multiresistant strains show the lowest MIC (3.12 to 6.25%), when compared with reference E. faecalis strain. EOdlES has the ability to inhibit the biofilm formation, but little or none ability to inhibit the preformed biofilm. This study demonstrates that EOdlES is a promising alternative to control important foodborne and clinic gram-positive resistant bacteria.

In Vitro Antimicrobial and Antioxidant Activities of Monoterpenes against some Food-Borne Pathogens

The present work describes the antimicrobial activity of twenty-five monoterpenes against Gram-negative Escherichiacoli and Gram-positive Staphylococcusaureus and antifungal activity against Aspergillus flavus. The antibacterial activity was evaluated by broth microdilution technique as a minimum inhibitory concentration (MIC) and the antifungal activity was estimated by mycelia radial growth technique as (EC50). The results showed that thymol and α-terpineol were the most potent against E. coli with MIC of 45 and 55 mg/L, 135 and 225 mg/L against S.aureus, respectively. The results also showed that thymol exhibited the highest antifungal activity against A. flavus with EC50 20 mg/L. Furthermore, the antioxidant properties were explored using N,N-dimethyl-1,4-phenylenediamine (DMPD) and the results showed that geraniol were the most potent compound (IC50 = 19 mg/L).

In vitro Antibacterial Effect of Hydroalcoholic Extract of Lawsonia inermis, Malva sylvestris, and Boswellia serrata on Aggregatibacter actinomycetemcomitans.

Background:Considering the increased rate of microbial resistance to antibiotics and chemical side effects of antibiotics and antiseptics used for the treatment of periodontal disease, there is a need for an alternative antimicrobial agent with fewer complications. Medicinal herbs have recently become popular as novel antimicrobial agents. This study aimed to assess the antibacterial effects of hydroalcoholic extracts of Lawsonia inermis, Malva sylvestris, and Boswellia serrata on Aggregatibacter actinomycetemcomitans.Materials and Methods:Hydroalcoholic extracts of the three medicinal plants were obtained by the maceration technique and A. actinomycetemcomitans was cultured. Antimicrobial efficacy of the three medicinal plants was compared with that of 0.2% chlorhexidine (CHX) according to the Clinical and Laboratory Standards Institute protocol using agar disc diffusion and broth microdilution techniques. All tests were repeated three times.Results:Hydroalcoholic extracts of all three plants had antimicrobial activity against A. actinomycetemcomitans. The minimum inhibitory concentration (MIC) of L. inermis, M. sylvestris, and B. serrata was 78.1, 156.2, and 1666 μg/mL with no significant difference between them. The MIC of CHX was 3.33 μg/mL, which was significantly higher than that of B. serrata extract.Conclusion:Given that further in vivo studies confirm other properties of these extracts and their safety in terms of cytotoxicity and mutagenicity, hydroalcoholic extracts of L. inermis and M. sylvestris may be used in mouthwashes or local delivery systems to affect periodontal biofilm.

Anti-Helicobacter pylori and Anti-inflammatory Properties of Eugenia uniflora L.

Abstract Helicobacter pylori is a bacterium that reaches half of the world population and it’s recognized as the main cause of chronic gastritis and peptic ulcer. In this study, we evaluated the anti-H. pylori, antioxidant and immunomodulatory activities of the methanolic (MeOH) extract of Eugenia uniflora leaves and chemical profile. Anti-H. pylori activity was evaluated by spectrophotometric broth microdilution technique by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), in addition to the evaluation of the effect on the urease enzyme. The antioxidant activity was evaluated by capturing O2 •-, HOCl e NO• radicals. The immunomodulatory effect was evaluated on the cytokines TNF-α, IL-6 and on nitric oxide through inhibition in LPS-stimulated macrophages. The chemical profile was performed by total phenolic, tannin and flavonoid contents and mass spectrometry analysis by ESI-FT-ICR MS. In the anti-H. pylori assay the extract showed MIC of 128 μg/mL, however it did not obtain MBC. The extract also showed ability to inhibit the urease enzyme about 20%. The antioxidant activity of the MeOH extract showed EC50 values of 29.77 µg/mL, 15.71 µg/mL and 442.10 µg/mL to O2 •-, HOCl and NO•, respectively. The extract also showed influence on the release of TNF-α, IL-6 and NO in LPS-stimulated macrophages, ranging from 39% to 97% inhibition. Flavonoids, phenylpropanoids, tannins, triterpenoids and carbohydrates were the major classes of compounds present in the MeOH extract as identified by (-)-ESI-FT-ICR MS. The results indicate important anti-H. pylori, antioxidant and immunomodulatory activities from Eugenia uniflora highlighting its importance in the prevention and treatment of diseases caused by H. pylori infection.

Commercial Essential Oil Combinations against Topical Fungal Pathogens

Essential oils are amongst the most popular natural products recommended for the treatment of topical fungal infections. This study investigated the antifungal activity of 128 essential oil combinations against fungal pathogen reference strains using the broth microdilution technique to determine the minimum inhibitory concentration (MIC). The essential oils were investigated at a volume of 100 μL and the combinations comprised of 50:50 μL. Each combination was tested in triplicate and the mean recorded. The fractional inhibitory concentration index was calculated for combinations, and synergistic interactions were investigated further at different ratios and plotted on isobolograms. The fungal pathogens were found to be highly susceptible to the essential oil combinations, with Trichophyton mentagrophytes being inhibited by all but one combination. The essential oil combinations containing Cinnamomum verum or Santalum austrocaledonicum were found to display the strongest inhibition with MIC values as low as 0.06 mg/mL. Potential combinations against fungal pathogens have been presented that could be studied in clinical settings with the goal of decreasing the need for systemic or prolonged antifungal treatments that may result in treatment failure or resistance.

ANTIMICROBIAL ACTIVITY OF ETHANOLIC EXTRACTS FROM Commiphora leptophloeos (MART.) J. B. GILLETT AGAINST Staphylococcus SPP. ISOLATED FROM CASES OF MASTITIS IN RUMINANTS

Abstract Considering the therapeutic potential of medicinal plants as alternatives to antibiotic therapy, the research aims to evaluate the antimicrobial activity of Commiphora leptophloeos against isolates of Staphylococcus spp. from the milk of ruminants with subclinical mastitis. For this, the crude ethanolic extract from the bark and leaves of Commiphora leptophloeos was prepared, with these being chemically characterized by HPLC-DAD-MS and by MALDI-TOF. The extracts were then evaluated as to their antimicrobial effects against 60 isolates of Staphylococcus spp. through the broth microdilution technique to determine the minimum bactericidal concentration. In addition, the extracts were evaluated as to their ability to interfere with biofilm formation and with the already established biofilm. Although all tested extracts showed antimicrobial action, lower MBC values were recorded for the bark extract in the concentration 781.2 µg/mL (25/60). The extracts of the bark and leaves were able to interfere with the initial stages of biofilm formation, but there was no interference of the extract on the established biofilm. There was a high sensitivity of Staphylococcus spp. isolates from subclinical mastitis cases in ruminants when subjected to the extracts from bark and leaves of Commiphora leptophloeos, as well as regarding the ability of extracts to interfere in biofilm formation, indicating their potential in the use for ruminant mastitis therapy.

ANTIBACTERIAL ACTIVITY AGAINST BACILLUS SUBTILIS AND ANTIOXIDANT PROPERTIES OF METHANOL EXTRACTS FROM GARCINIA LATISSIMA MIQ. LEAVES

Objective: The objective of this study was to identify fractions with the highest antibacterial activity against Bacillus subtilis and to determineantioxidant activities and establish the chromatographic fractions as candidate antibacterial and antioxidant agents.Methods: Extracts were fractionated using column chromatography, and antibacterial activities were assayed by the analyses of inhibition zones andbioautography, as well as by broth microdilution techniques. Antioxidant activities were evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH)assay.Results: The strongest antibacterial activity against B. subtilis (ATCC 6633) was observed with fractions B and C obtained in this research, witha minimum inhibitory concentration value of 312.5 μg/mL. The effective percentage (EP) value of crude extract at 10 μg/mL was 29.47±2.01%.Fractions C and D had greater EP values than the crude extract, whereas fraction D had the highest scavenging activity against DPPH free radicals(37.73±1.44%) when used at 10 μg/mL. The half effective concentration of the extract was 23.40 μg/mL, whereas that of the most active fraction Dwas 19.38 μg/mL and quercetin as positive control was 3.72 μg/mL.Conclusion: The present data confirm that fractions of methanol extract from Garcinia latissima Miq. leaves possess antibacterial and antioxidantactivities. These observations may facilitate the development of antimicrobial phytomedicines with a wide spectrum of activities and standardizedantioxidant properties.

Non-Antimicrobial Drugs: Etodolac as a Possible Antimicrobial or Adjuvant Agent Against ESKAPE Pathogens.

Introduction Multiple-drug resistant bacteria are emerging exponentially in healthcare units, threatening public health and requiring novel therapeutic approaches. In 2017, World Health Organization published a list that frames antimicrobial resistant bacteria into priority levels for research of novel drugs to fight them. Methods & Materials Antimicrobial resistant ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter sp.) and Enterococcus faecalis and Escherichia coli pathogens are present in this list. Representative isolates of each species were used to test the Antibacterial and anti-biofilm formation activities of Etodolac (a Non-Steroidal Anti-Inflammatory Drug, NSAID) at 10 and 1 mM using a broth microdilution technique. Results & Discussion Statistically significant (p< 0,05) results were observed against all tested gram-positives, particularly anti-biofilm activity against E. faecium. Etodolac had an almost null influence on tested gram-negatives, with the exception of one A. baumannii clinical isolate regarding biofilm formation inhibition. Observed differences deserve further analysis and prospection of the involved mechanisms, to unravel possible novel bacterial targets for drug development. Similar work with other NSAID's may also be worth exploring to ascertain novel therapeutic applications for these drugs, particularly regarding biofilm formation inhibition, per si or as adjuvants of current antibiotherapy, mainly against gram-positives, as suggested by present work. Conclusion Already approved drugs in terms of pharmacokinetics and safety may deploy faster solutions for antimicrobial therapy against priority pathogens. Current work intends to bring attention to that possibility, particularly regarding NSAIDs, anti-biofilm formation and top priority pathogens.

In vitro evaluation of local antibiotic delivery via fibrin hydrogel

Background/purposeFibrin hydrogel is commonly used as hemostatic agent and scaffold but it is questionable for carrying antibiotics. Thus, this study aimed to investigate whether the fibrin hydrogel can be used to deliver the optimal concentration of ciprofloxacin against oral pathogen. Materials and methodsThe optimal concentration of ciprofloxacin was investigated from broth microdilution technique against three common oral bacteria. Ten times the bactericidal concentration of ciprofloxacin loaded to 0.4% fibrin hydrogel was observed by using a confocal laser scanning microscope and then was left in tris-buffer saline solution (TBS) for 0, 1, 12, 24, 72 and 168 h in parallel with the control group of ciprofloxacin loaded to 0.5% alginate hydrogel and ciprofloxacin solution. Spectrophotometer was used to analyze the accumulated drug release from the collected TBS, of which the measurement method was calibrated. The efficacy of the released ciprofloxacin was tested using an agar well diffusion assay. The inhibition zone of the released ciprofloxacin from fibrin hydrogel was statistically compared with 150 and 1500 μg/ml ciprofloxacin solution, while non-loaded fibrin hydrogel served as the control. ResultsThe results revealed that minimum inhibitory concentration was 1–2 μg/ml and minimum bactericidal concentration was 4–15 μg/ml. The fibrin hydrogel gradually released ciprofloxacin until 168 h while the alginate hydrogel immediately liberated all the loaded ciprofloxacin within an hour. The agar well diffusion significantly showed greater clear zone in fibrin hydrogel loaded ciprofloxacin compared to non-loaded fibrin hydrogel but not with ciprofloxacin in TBS. ConclusionThe results suggested that fibrin hydrogel can be used for local ciprofloxacin delivery without interfering the efficacy of ciprofloxacin.