Minimum Inhibitory Concentration Concentration Research Articles

Design, Synthesis and Antibacterial Studies of Novel Cationic Amphipathic Cyclic Undecapeptides and Their Linear Counterparts against Virulent Bacterial Strains

Bacteria have acquired resistance against almost all antibiotics because of the misuse of antibacterial agents and long periods of treatment. Antimicrobial peptides (AMPs) are one of the most encouraging candidates to solve this problem, as they possess high prokaryotic selectivity, and affect the bacteria by a unique mode of action. Novel cyclic undecapeptides (QNRNFYFNRNQ and QNRNFHFNRNQ) and their linear counterparts were investigated for their antibacterial activity against virulent strains. The minimal inhibitory concentration (MIC) values showed that tyrosine and histidine AMPs have promising antibacterial activity against virulent bacteria. The MIC values against the P. aeruginosa PA14, E. coli O157:H7 CR3, S. aureus 209P, and B. subtilis ATCC 6633 bacterial strains were evaluated for the cyclic peptide containing tyrosine, and their values were 6.25, 12.5, 12.5, and 12.5 µM, respectively. Meanwhile, for the linear form, they were 9.3, 12.5, 12.5, and 12.5 µM, respectively. The cyclic-peptide–containing histidines’ MIC values were 6.25, 3.1, 6.25, and 3.1 µM, respectively. Meanwhile, for the linear form, they were 3.1, 3.1, 3.1, and 6.25 µM, respectively. The antibacterial activities of the new AMPs were compared with that of gentamicin sulfate, and showed relatively higher potencies. Time-inhibition studies demonstrated the rapid antibacterial effects of the novel AMPs, which were more likely to be concentration-dependent, rather than time-dependent. At double the MIC concentration, all of the tested peptides exhibited relatively stable antibacterial effects up to 24 h, especially the peptides containing tyrosine, which showed an improved antibacterial effect.

Antibiofilm and antipersister activity of acetic acid against extensively drug resistant Pseudomonas aeruginosa PAW1

Pseudomonas aeruginosa is an ESKAPE pathogen associated with difficult-to-treat burn wound and surgical-site infections. This study aimed to characterise an extensively drug resistant (XDR) P. aeruginosa isolate (designated PAW1) and to investigate the antibiofilm and antipersister effect of acetic acid on PAW1. PAW1 was identified using biotypic (VITEK) and genotypic (16S rDNA) analysis. Minimum inhibitory concentration (MIC) and disc susceptibility testing showed high level resistance against all antibiotics from classes including beta lactams, cephems, carbapenems and fluoroquinolones. It was therefore identified as extensively drug resistant (XDR), showing resistance to all antibiotics except for, aminoglycoside (gentamicin and netilmicin) and lipopeptides (polymyxin B). Time kill assays showed antibiotic tolerant, persister cell formation in presence of 100X MICs of gentamicin and polymyxin B. Other virulence traits such as ability to produce lipase, protease, haemolysin, and siderophores and to form biofilms were additional factors which may contribute to its pathogenicity. PAW1 showed promising susceptibility against acetic acid with MIC and minimum biofilm inhibitory concentration of 0.156% (v/v). Percent viability of PAW1 was dependent on dose and treatment time of acetic acid. 0.625% acetic acid treatment of 5 minutes was effective in killing >90% planktonic cells showing lesser toxicity to L929 cells (IC50 = 0.625%). Biofilm disruption caused due to acetic acid was also dose dependent, showing 40.57% disruption after treatment with 0.625% acetic acid for 5 minutes. FESEM imaging and live dead staining of planktonic and biofilm forms of PAW1 confirmed that acetic acid treatment caused 19.04% of cell shrinkage and disruption of extracellular matrix resulting in killing of cells. Antipersister activity of acetic acid was demonstrated by showing complete killing of PAW1 at 4X MIC. Overall, this study characterised an XDR isolate P. aeruginosa showing resistance and tolerance to various antibiotics. Antipersister and antibiofilm effect of acetic acid demonstrates the importance of forgotten topical agents as an effective strategy to treat XDR pathogens.

Synthesis and Characterization of Silver Nanoparticles with Ultrasound-Assisted Reverse Micelles Method and Their Antibacterial Effects on Methicillin-Resistant Staphylococcus aureus Isolates

Background: Serious infections are associated with methicillin-resistant Staphylococcus aureus (MRSA) bacteria and this can lead to many deaths in the world. The aim of the present study was to evaluate the antibacterial effect of silver nanoparticles (AgNPs) against MRSA isolates from clinical samples. Methods: Ag nanoparticles were synthesized by ultrasound-assisted reverse micelles method. The as-prepared Ag nanoparticles were characterized by X-Ray diffraction (XRD) and scanning electron microscopy (SEM). The antibacterial effect of AgNPs was investigated using agar well diffusion assay and minimum inhibitory concentration (MIC) was determined. Results: The XRD studies showed that pure Ag nanoparticles have been produced after calcination. Synthesized AgNPs showed favorable effects on the bacteria used. MIC and minimum bactericidal concentration (MBC) values were determined to be 0.015 and 0.07 mg/mL, respectively. All MRSA isolates were susceptible to AgNPs. In contrast, they showed high resistance to multiple classes of antibiotics. Conclusions: AgNPs had high inhibitory activity against MRSA; therefore, they can be proposed as an alternative or adjuvant to antibiotics for the treatment of MRSA infections. Further investigations are required to assess the safety and efficacy of AgNPs in the body.

New Cobalt (II) Complexes with Imidazole Derivatives: Antimicrobial Efficiency against Planktonic and Adherent Microbes and In Vitro Cytotoxicity Features.

Three novel Co(II) complexes of the type [Co(C4H5O2)2L2] (where C4H5O2 is methacrylate anion; L = C3H4N2 (imidazole; HIm) (1), C4H6N2 (2-methylimidazole; 2-MeIm) (2), C5H8N2 (2-ethylimidazole; 2-EtIm) (3)) have been synthesized and characterized by elemental analysis, IR and UV-Vis spectroscopic techniques, thermal analysis and single crystal X-ray diffraction. X-ray crystallography revealed for complexes (1) and (2) distorted trigonal bipyramid stereochemistry for Co(II), meanwhile for complex (3) evidenced that the unit cell comprises three molecular units with interesting structural features. In each unit, both stereochemistry adopted by metallic ion and coordination modes of carboxylate anions are different. The screening of antimicrobial activity revealed that Candida albicans planktonic cells were the most susceptible, with minimal inhibitory concentration (MIC) values of 7.8 μg/mL for complexes (1) and (2) and 15.6 μg/mL for complex (3). Complexes (1) and (2) proved to be more active than complex (3) against the tested bacterial strains, both in planktonic and biofilm growth state, with MIC and minimal biofilm eradication concentration (MBEC) values ranging from 15.6 to 62.5 μg/mL, the best antibacterial effects being noticed against Staphylococcus aureus and Pseudomonas aeruginosa. Remarkably, the MBEC values obtained for the four tested bacterial strains were either identical or even lower than the MIC ones. The cytotoxicity assay indicated that the tested complexes affected the cellular cycle of HeLa, HCT-8, and MG63 cells, probably by inhibiting the expression of vimentin and transient receptor potential canonical 1 (TRPC1). The obtained biological results recommend these complexes as potential candidates for the development of novel anti-biofilm agents.

Biocidal Polymers: Synthesis, Characterization and Antimicrobial Activity of Bis-Quaternary Onium Salts of Poly(aspartate-co-succinimide)

Microbial multidrug resistance presents a real problem to human health. Therefore, water-soluble polymers based on poly(aspartate-co-succinimide) were synthesized via reaction of poly(aspartate-co-succinimide) with bis-quaternary ammonium or quaternary salts. The resultant copolymers were characterized by various techniques such as FTIR, TGA, 1HNMR, 13CNMR and elemental microanalysis. Antimicrobial activities of the new onium salts were investigated against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella typhi, and the fungi; Candida albicans, Aspergillus niger, Cryptococcus neoformans and Aspergillus flavus by agar diffusion method. Antimicrobial activity was studied in terms of inhibition zone diameters, in addition to the estimation of minimal inhibitory concentration (MIC) of the prepared compounds. A. niger and E. coli were the most affected microorganisms among the tested microorganisms with an inhibition zone of 19–21 (mm) in case of biocides, (V) and (VII). The obtained results showed that the quaternary onium salts have higher activity compared to the aspartate copolymer with MIC concentrations of 25 mg/mL for (VII) and (V) and 50 mg/mL for (VI) and (IV).

Bioengineered Silver Nanoparticle from Spirulina platensis in Attenuating Biofilm Mediated Virulence in Vibrio parahemolyticus: An in vitro and in vivo Approach

Objectives: In this study, bioengineered AgNPs (silver nanoparticles) from Spirulina platensis was used to counter the biofilm mediated virulence by Vibrio parahemolyticus. Methods: AgNPs were synthesized from crude Spirulina platensis extract and characterized using UV spectrophotometry, Transmission electron microscope (TEM). Antibacterial effect against aquatic pathogen Vibrio parahemolyticus was carried out. Further, the nanoparticle at sub MIC (minimum inhibitory concentration) level were screened for anti-virulence property under in vitro studies which include biofilm inhibition using microtiter plate assay, microscopic analysis under light microscope and SEM (Scanning electron microscope) imaging, EPS (Exopolysaccharide) inhibition, CSH(Cell surface hydrophobicity) inhibition using BATH(Bacterial adhesion to hydrocarbons) assay, swarming and swimming motility inhibition, growth pattern analysis followed by in vivo study in aquatic animal model brine shrimp and its survival percentage along with light microscopic imaging was performed. Results: AgNPs, at 320μg/ml showed bactericidal activity, minimal inhibitory concentration was found to be 160 μg/ml. All anti-virulence assay performed at below sub MIC concentration. Biofilm and EPS showed 63% and 53% inhibition respectively, CSH was found to be decreased 3-fold. Growth pattern at sub MIC compared with non-treated showed similar result. In vivo study increased survival percentage (90%) when treated with AgNPs, compared to untreated (20%). A 7-fold increased resistance to pathogen was found and light microscope also confirms the efficacy of Ag nanoparticle. Conclusion: AgNPs become a substitute to the current commercial antibiotics used in aquaculture against Vibriosis, by functioning as an efficient quorum sensing inhibitor.

In Vitro Activity of Pentamidine Alone and in Combination with Antibiotics against Multidrug-Resistant Clinical Pseudomonas aeruginosa Strains.

Multidrug-resistant (MDR) Pseudomonas aeruginosa is a public health problem causing both community and hospital-acquired infections, and thus the development of new therapies for these infections is critical. The objective of this study was to analyze in vitro the activity of pentamidine as adjuvant in combinations to antibiotics against seven clinical P. aeruginosa strains. The Minimum Inhibitory Concentration (MIC) was determined following standard protocols, and the results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints; however, the gentamicin activity was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. The bactericidal in vitro activity was studied at 1×MIC concentrations by time–kill curves, and also performed in three selected strains at 1/2×MIC of pentamidine. All studies were performed in triplicate. The pentamidine MIC range was 400–1600 μg/mL. Four of the strains were MDR, and the other three were resistant to two antibiotic families. The combinations of pentamidine at 1×MIC showed synergistic activity against all the tested strains, except for pentamidine plus colistin. Pentamidine plus imipenem and meropenem were the combinations that showed synergistic activity against the most strains. At 1/2×MIC, pentamidine plus antibiotics were synergistic with all three analyzed strains. In summary, pentamidine in combination with antibiotics showed in vitro synergy against multidrug-resistant P. aeruginosa clinical strains, which suggests its possible use as adjuvant to antibiotics for the therapy of infections from MDR P. aeruginosa.

The inhibitory activity of magnolol on Candida tropicalis

Background: Candida tropicalis (C. tropicalis) is isolated third in non- albicans Candida species. Antifungal agents used for the treatment of Candida species are mainly Fluconazole (FLCZ) and amphotericin-B (AMPH-B) and so on. However, according to the SENTRY Antifungal Surveillance Program, it has been reported that the C. tropicalis FLCZ resistance rate is increasing. It is also known that AMPH-B has nephrotoxicity. Magnolol first extracted from traditional Chinese medical plant magnolia officinalis. Previous studies demonstrated that magnolol inhibit the growth of candida species. Nevertheless, there few studies of C. tropicalis on magnolol. In this study, we investigated the activities of magnolol against C. tropicalis. Methods and materials: We used 20 strains of C. tropicalis clinical isolates (C. t 1–20) and C. tropicalis ATCC 750. We investigated MIC (minimum inhibitory concentration), MBIC (minimum biofilm inhibitory concentration), Biofilm formation (BF) ability and Time-kill (TK) assay (6h, 12h, 24h, 48h). MBIC used 3 strains (C. t 1, 4, 9) with different BF ability and was assayed 4 time point (6h, 12h, 24, 48h). Time-kill assay (6h, 12h, 24h, 48h) used C. tropicalis ATCC 750, the magnolol concentration was determined as MIC and sub-MIC concentration. Results: The MIC90 of FLCZ, AMPH-B and magnolol with respect to C. tropicalis were 0.5 μg/mL, 1 μg/mL and 64 μg/mL, respectively. BF ability was produced in all strains on microtiter plate. All antifungal drugs and magnolol against MBIC were >512 ug/mL at all 4 time point. TK assay was inhibited growth for 48 hours at MIC concentrations, but not inhibited growth for 24 hours at concentration sub-MIC. Conclusion: It was suggested in MIC that magnolol exhibits antifungal activity against C. tropicalis. However, magnolol showed no antifungal activity in MBIC like other antifungal agents. These indicate that magnolol may not show antifungal activity after C. tropicalis BF.

Allicin modulates expression of cell wall-related genes in Aspergillus fumigatus

Background: Aspergillus fumigatus causes life-threatening iinvasive aspergillosis, especially in immunocompromised hosts, and resistance of this filamentous fungus to commonly used antifungal agents has been on the rise. Allicin (diallyl-dithiosulfinate), an allyl-sulfur compound from garlic has been proven to exhibit antimicrobial properties. However, there have been few studies on the antifungal mechanism of allicin against A. fumigatus. Based on our previous studies on the antifungal mechanism of allicin against Candida albicans, an opportunistic yeast pathogen, we hypothesize that allicin would similarly target cell-wall biosynthesis and integrity in A. fumigatus. This study evaluated the expression of cell-wall related genes in A. fumigatus ATCC 36607 upon treatment with allicin. Methods and materials: The minimum inhibitory concentration (MIC) of allicin against A. fumigatus ATCC 36607was determined according to the CLSI M38-A2 guidelines. A. fumigatus was incubated in the presence of allicin at subinhibitory concentrations of 1/4X MIC, 1/2X MIC, and 1X MIC for 48 hours. The cells were harvested post-incubation with allicin and RNA was extracted and reverse-transcribed into cDNA for evaluation of expression of selected genes via real time PCR. Results: The minimum MIC of allicin against A. fumigatus was found to be 3.2 μg/ml. Scanning electron micrographs exhibited significant morphological alteration of A. fumigatus hyphae and conidia after 48 h incubation at 1× MIC, 2× MIC and sub-MIC concentrations of allicin 1/4× MIC and 1/2× MIC in comparison with untreated (growth) control. Upon treatment at 1/4× MIC (0.8 μg/ml), hyphae were seen as wrinkled with patchy and rough surface implying decrease in structural integrity. Further, at 1/2× MIC (1.6 μg/ml), aberrant shape of hyphae with irregular constriction was observed along the length of hypha. At 1X MIC, complete suppression of hyphae growth was observed and only conidia were visible. Real-time PCR findings revealed that significant down-regulation of chsE, chsG, fksA, and gel2 genes were observed at various MICs of allicin tested. On the contrary, rhoD showed an upregulated expression. Conclusion: This study suggests the ability of allicin to affect the expression of cell wall-related genes of A. fumigatus.

Antifungal Activity of Rosmarinus Officinalis Essential Oil and Nystatin on Store Isolate of Candida species from HIV/AIDS Patients with Oral Candidiasis

Background: Oral candidiasis is caused by the mycotic activity of Candida albicans present in the oral cavity, and it is one of the most common opportunistic infections found in patients with Human Immunodeficiency Virus (HIV)/acquired immune deficiency syndrome (AIDS). The growing resistance and side effects to common antifungal drugs have promoted herbal essential oils as antifungal agents in recent years. In this study, essential oils (EO) of Rosmarinus officinalis (Lamiaceae) were examined for in vitro antifungal activ­ity against Candida species. Purpose: To evaluate the antifungal activity of essential oils of Rosmarinus officinalis (Lamiaceae) and nystatin using the microdilution technique by determining the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of Candida spesies. Methods: This was an experimental laboratory study with a post-test-only design conducted in Dr. Soetomo General Academic Teaching Hospital, Surabaya. Forty isolates consisted of twenty isolates of Candida albicans and twenty isolates of Candida non-albicans were collected. The isolates were tested for antifungal activity using the microdilution on 96-well plates. Result: There was a significant difference from the results of the MIC concentration of rosemary essential 100% to 6.25% microdilution method between nystatin and rosemary essential oil (p < 0.05). Conclusion: The antifungal activity of rosemary essential oil was better than nystatin that the lowest MIC value, which was 6.25%, has been obtained the microdilution method. The minimum fungicidal concentration of rosemary essential oil was 25%, while the minimum fungicidal concentration nystatin was higher than 100%.

Synthesis and antimicrobial evaluation of a pyrazoline-pyridine silver(I) complex: DNA-interaction and anti-biofilm activity.

The emergence of resistant bacterial strains mainly due to misuse of antibiotics has seriously affected our ability to treat bacterial illness, and the development of new classes of potent antimicrobial agents is desperately needed. In this study, we report the efficient synthesis of a new pyrazoline-pyridine containing ligand L1 which acts as an NN-donor for the formation of a novel silver (I) complex 2. The free ligand did not show antibacterial activity. High potency was exhibited by the complex against three Gram-negative bacteria, namely Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumanii with the minimum inhibitory concentration (MIC) ranging between 4 and 16μg/mL (4.2-16.7μM), and excellent activity against the fungi Candida albicans and Cryptococcus neoformans (MIC ≤ 0.25μg/mL = 0.26μM). Moreover, no hemolytic activity within the tested concentration range was observed. In addition to the planktonic growth inhibition, the biofilm formation of both Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa was significantly reduced by the complex at MIC concentrations in a dose-dependent manner for Pseudomonas aeruginosa, whereas a biphasic response was obtained for MRSA showing that the sub-MIC doses enhanced biofilm formation before its reduction at higher concentration. Finally, complex 2 exhibited strong DNA binding with a large drop in DNA viscosity indicating the absence of classical intercalation and suggesting the participation of the silver ion in DNA binding which may be related to its antibacterial activity. Taken together, the current results reveal that the pyrazoline-pyridine silver complexes are of high interest as novel antibacterial agents, justifying further in vitro and in vivo investigation.

The Anti-Biofilm Activity of Oregano Essential Oil Against Dental Plaque-Forming Streptococcus mutans In Vitro and In Vivo

Background: The oral and dental infections that are mainly caused by bacterial biofilms are among the most prevalent human infections worldwide. Objectives: The present study aimed to investigate the in vitro and in vivo inhibitory and anti-biofilm effects of oregano essential oil on the Streptococcus mutans isolates obtained from elementary school students. Methods: This experimental study was conducted on 150 samples collected from the buccal and lingual surfaces of the posterior teeth of elementary school students. S. mutans strains were identified using conventional microbiological and biochemical tests, and biofilm formation was assessed using the microtiter plate assay. The minimum inhibitory concentration (MIC) of the oregano essential oil against the isolates was determined using the broth microdilution method. In addition, the effective constituents of the essential oil were measured via gas chromatography-mass spectroscopy. The in vitro and in vivo anti-biofilm activities of the oregano essential oil were also evaluated using the modified microtiter plate assay and on the tooth surfaces of male NMRI mice, respectively. Results: The frequency of S. mutans was 15.3%, 87% of which were capable of biofilm formation. The MIC of the oregano essential oil was 50 µl/ml against the S. mutans isolates, and 82% of the isolates did not grow at the concentrations of ≥ 512 µl/ml. However, none of the isolates were capable of biofilm formation at the MIC and sub-MIC concentrations of the essential oil. Limonene and myrcene were the most effective constituents of the essential oil. Furthermore, a significant correlation was observed between treatment with the oregano essential oil and biofilm formation by the streptococci isolates (P = 0.05). Conclusions: According to the results, the presence of biofilm and incidence of dental caries were significantly correlated. Moreover, the essential oil of oregano and its main constituents had potent anti-biofilm and antibacterial properties and could be utilized for the production of new plant-based mouthwashes.

Role of triclosan microemulsion against triclosan resistant clones of bacterial pathogens

Triclosan although banned for the use in many cosmetic products is still used in endodontic therapy for the elimination of microorganisms. At minimum inhibitory concentrations (MIC), triclosan is bacteriostatic and selects resistant clones with prolonged exposure. This study aims to demonstrate the use of eugenol microemulsions as a vehicle to improve the activity of triclosan and prevent the selection of resistant clones. The solubility of triclosan in various oils (cinnamon oil, clove oil, eugenol and peppermint oil) and the release kinetics of triclosan from the microemulsions was determined spectrophotometrically at 281 nm. Microemulsions were formulated by the water titration method. The MIC and minimum bactericidal concentration (MBC) were determined by the broth microdilution method. Triclosan resistant clones were selected by serial passage method. Triclosan has 4 fold difference between its MIC and MBC against S. aureus, E. faecalis and S. mutans isolates. The triclosan loaded microemulsion (5%:25%:70% ratio of oil, Tween 20 & water) (EuTT20-5), containing a final concentration of 3 mg ml−1 of triclosan, eliminated this difference. On repeated passages with increasing concentrations of triclosan, mutant strains of E. faecalis with 8-fold increase in MBC (250 μg ml−1 to 2 mg ml−1) and mutant strains of S. aureus and S. mutans with 8-fold increase in MBC from 125 μg ml−1 to 1 mg ml−1 were observed. Such mutants were not generated in the EuTT20-5 treated cultures. The resistant mutants demonstrated only a 2-fold increase in MBC of EuTT20-5 which is insignificant. Thus, EuTT20-5 can not only improve the efficacy of triclosan, but it can act with equal potency against triclosan resistant isolates. Eugenol loaded triclosan microemulsions can be used instead of conventional biocidal solutions in endodontic therapy where complete elimination of microorganisms from the treatment site is required.

In vitro effect of antimicrobial photodynamic therapy with phycocyanin on Aggregatibacter actinomycetemcomitans biofilm on SLA titanium discs.

The aim of this in vitro study was to evaluate antimicrobial photodynamic therapy (aPDT) with phycocyanin on Aggregatibacter actinomycetemcomitans biofilm formed on sandblasted, large-grit, and acid-etched (SLA) titanium discs. In this in vitro experimental study, the minimum inhibitory concentration (MIC), sublethal dose of diode laser irradiation time, and sublethal dose of aPDT were first determined. Next, 30 SLA titanium discs with 8 mm diameter and 2 mm thickness were incubated with A. actinomycetemcomitans in order for the bacterial biofilm to form, and were randomly divided into 5 groups (n = 6): (I) negative control with no treatment, (II) positive control, immersed in 0.2 % chlorhexidine (CHX) for 5 min, (III) phycocyanin alone with ×2 MIC concentration for 5 min, (IV) diode laser alone (635 nm wavelength, 220 mW power), and (V) PDT with diode laser and phycocyanin. The samples were then sonicated, and the number of colony-forming units (CFUs) on each disc was calculated. Data were analyzed using one-way ANOVA, t-test, and a post-hoc test. aPDT with 125 μg/mL phycocyanin and 4 min irradiation of 635 nm diode laser decreasedA. actinomycetemcomitans biofilm by 40.07 %. The lowest mean colony count (CFUs/mL) was noted in 0.2 % CHX group (0.0 × 105 CFU/mL) while the highest mean was observed in the negative control group (4.55 ± 0.08 × 105 CFUs/mL). Using phycocyanin alone significantly decreased the A. actinomycetemcomitans count by 27.54 % (3.29 ± 0.06 × 105 CFUs/mL) compared with the negative control group (P < 0.0001). Significant differences were noted between the negative control and other groups (P < 0.0001). aPDT with phycocyanin and diode laser can decrease the A. actinomycetemcomitans biofilm on SLA titanium implant surfaces and can be used as a safe and non-invasive decontamination method for reduction of A. actinomycetemcomitans biofilm on implant surfaces.

The assessment of antibiofilm activity of chitosan-zinc oxide-gentamicin nanocomposite on Pseudomonas aeruginosa and Staphylococcus aureus

In the present study, chitosan-zinc oxide (CS-ZnO) nanocomposite with/without gentamicin was synthesized and characterized which used as an antibiofilm agent to inhibit the biofilm formation of Pseudomonas aeruginosa (P. aeruginosa) PAO1 and Staphylococcus aureus (S. aureus). Synthesized CS-ZnO nanocomposite was characterized with the DLS (Dynamic Light Scattering), FTIR (Fourier Transform Infrared), XRD (X-ray Diffraction) and SEM (Scanning Electron Microscope). The minimum inhibitory concentrations (MICs) against P. aeruginosa PAO1 and S. aureus determined using broth microdilution methods. The influence of sub-MIC (1/4 MIC) and MIC concentration of CS-ZnO nanocomposite and gentamicin alone and in combination on biofilm formation was also determined. A four-fold MIC reduction in S. aureus and P. aeruginosa PAO1 treated by the gentamicin loaded CS-ZnO nanocomposite, and 84% reduction of biofilm formation for P. aeruginosa PAO1 and 77% reduction of biofilm formation for S. aureus, was observed compared to the gentamicin alone (P < 0.05).This study showed the important role of nanocomposite in designing novel antibacterial and antibiofilm agents to combat the P. aeruginosa and S. aureus biofilm-related infections.

Antimicrobial screening and hemolytic activity of products obtained from Piper montealegreanum Yuncker and effect in vitro on growth of Staphylococcus aureus

This study aimed to determine spectrum of antimicrobial activity of crude ethanolic extract (CEE), chloroform (CHCl3) and ethyl acetate (EtOAc) fractions, to determine influence of CHCl3 fraction on Staphylococcus aureus growth and hemolytic activity. Antimicrobial screening of CEE, CHCl3 and EtOAc fractions was carried out using broth microdilution technique against standard strains: Staphyloccocus aureus ATCC25923, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853 and Candida albicans ATCC76645 to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Time kill curves were performed to determine influence of CHCl3 fraction on MIC and ½ MIC concentrations on S. aureus growth. Hemolytic activity of CHCl3 was performed in human erythrocytes to report the cytotoxic effect. CHCl3 fraction showed activity against S. aureus with MIC=1024 µg mL-1 and MBC=2048 µg mL-1. This fraction did not show activity against other tested strains, as well as the CEE and EtOAc fraction. Time kill curve of S. aureus treated with CHCl3 fraction showed that at MIC this fraction has bacteriostatic activity, which was not observed with the ½ MIC. Hemolytic activity demonstrated that when CHCl3 fraction in MIC does not present membrane damage of erythrocytes, since it did not cause hemolysis. Studies should be conducted to evaluate the activity of CHCl3 fraction against S. aureus with different antimicrobial resistance profiles, in addition to evaluating the potential for resistance reversion by in vitro combination studies. The study demonstrated that the species P. montealegreanum can be an important source of compounds of importance for combating microorganisms of clinical importance.

Seagrass Halodule pinifolia active constituent 4-methoxybenzioic acid (4-MBA) inhibits quorum sensing mediated virulence production of Pseudomonas aeruginosa

Biofilm associated, multidrug resistant Pseudomonas aeruginosa infection remain a challenging problem in the clinical field since the conventional antibiotic therapy are largely inefficient and new approaches are needed. Inactivating the QS virulence mechanism with anti-infective agent is an attractive approach to prevent bacterial infections without resistance development. Seagrass Halodule pinifolia (Miki) Hartog has been shown to exhibit potential antimicrobial activities against harmful pathogens. Our study investigated the effects of seagrass H. pinifolia leaf extract and its bioactive constituents on QS-mediated virulence factors and biofilm formation in P. aerugonasa PAO1. Preliminary screening on antibiofilm activity showed that the methanolic extract of H. pinifolia exhibited potential inhibition of biofilm formation (96%) as compared to the control respectively. Further, the potential extract was column fractionated and the active fraction was characterized by GC-MS. In total eight active compounds (protocatacheuic acid (69.3%), rosmarinic acid (63.5%), caffeic acid (59.18%), p-coumaric acid (59.08%), 4-methoxybenzoic acid (53.19%), naringenin (52.9%), vanillic acid (49.19%), 4-hydroxybenzoic acid (41.73%)) were profiled from fraction 2 and were purified by HPLC, structurally confirmed by NMR. Among the eight compounds studied, 4-methoxybenzoic acid (4-MBA) showed an effective inhibition of bacterial growth and was considered as a lead molecule with minimum inhibitory concentration (MIC) of 62.5 μg/mL. Further the effect of 4-MBA on QS mediated virulence factors demonstrated that the compound at MIC concentration reduced the virulence factor production such as elastase (87.5%), protease (79.38%), pyocyanin (91.46%), rhamnolipid (86%), alginate (86%), chitinase (55%), exopolysaccharide production (83.72%) and CSH (78.39%) over the control respectively. Moreover, 4-MBA down regulated the QS-mediated virulence transcript levels upon treatment with 4-MBA. The present findings suggests that seagrasses may act as a newer source for the marine based drug discovery and the lead compound 4-MBA derived from H. pinifolia may act as anti-infective agent against P. aeruginosa as it controls the QS-mediated virulence production.

Assessment of invitrosynergy of daptomycin or vancomycin plus ceftaroline for daptomycin non-susceptible Staphylococcus aureus

The combination of vancomycin or daptomycin plus ceftaroline has showed synergistic results in vitro. This study aimed to investigate in vitro synergy of vancomycin or daptomycin plus ceftaroline for seven patients with daptomycin non-susceptible Staphylococcus aureus (SA) bacteremiaThirteen isolates from seven patients were evaluated: two methicillin-susceptible and five methicillin-resistant SA infections. All patients were treated with daptomycin and became non-susceptible (minimum inhibitory concentration (MIC) >1 μg/mL) with therapy or had resistant strains initially. Time kill experiments were completed with 0.25 × MIC, 0.5 × MIC, and 0.75 × MIC concentrations. No synergy was seen at 0.25 × MIC. Synergy was observed for 4 isolates with vancomycin plus ceftaroline and with daptomycin plus ceftaroline for 2 isolates at 0.5 × MIC.These results are in accordance with literature that supports synergistic combinations of daptomycin or vancomycin with ceftaroline for SA bacteremia. Daptomycin non-susceptible SA bacteremia presents a treatment challenge.

Chemical Composition and Antimicrobial Activity of Essential Oil Extracted from Eucalyptus citriodora Leaf

Eucalyptus oil is a rich source of bioactive compounds with a variety of biological activities and is widely used in traditional medicine. Eucalyptus citriodora is cultivated for the production of essential oils. However, the mode of antibacterial action of essential oils from E. citriodora is not well-known. This study aimed to determine the chemical components, microbial inhibitory effect, and mechanism of action of the essential oil from E. citriodora. The oil was extracted from E. citriodora leaves by hydro-distillation and the chemical components were analyzed using gas chromatography-mass spectrometry. The antibacterial activities of eucalyptus oil against gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, and Staphylococcus intermedius) and gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) were screened by disc diffusion method and quantitative analysis was conducted by the microdilution method. The mechanism of action of the extracted essential oil was observed using SEM and analyzed by SDS-PAGE. The major components of E. citriodora oil were citronellal (60.55 ± 0.07%), followed by dl-isopulegol (10.57 ± 0.02%) and citronellol (9.04 ± 0.03%). The antibacterial screening indicated that E. citriodora oil exhibited prominent activity against all tested strains. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against B. subtilis were 0.5% and 1.0%, respectively. The MIC and MBC concentrations against S. aureus, S. intermedius, E. coli, and P. aeruginosa were 1% and 2%, respectively. As observed by SEM, the antibacterial mechanism of E. citriodora oil involved cell wall damage; SDS-PAGE revealed decrease in protein bands compared to untreated bacteria. Thus, E. citriodora oil showed significant antimicrobial properties and caused cellular damage.

The Effect of Growth Medium Strength on Minimum Inhibitory Concentrations of Tannins and Tannin Extracts against E. coli.

In this study the effect of growth medium strength on the minimum inhibitory concentration (MIC) of different tannins and tannin extracts against Escherichia coli was systematically investigated for the first time. Three pure compounds (vescalagin, castalagin and gallic acid) and five extracts (chestnut, quebracho, mimosa, Colistizer and tannic acid) were studied. Broth microdilution was assayed and bacteria were grown using different growth medium strengths varying from half to double the concentration recommended by the producer. MICs were determined using the iodonitrotetrazolium chloride (INT) dye or turbidity measurements. It was observed that MIC values depend on the growth medium strength. With an increase in the growth medium concentration MIC values rose roughly linearly for all samples, while their relative order remained unchanged, indicating that a direct interaction of tannins with growth medium nutrients represents the likely source of their antimicrobial activity. Understanding the effect of growth medium strength can finally yield a plausible explanation for the observed variation in MIC values reported in the scientific literature as well as provide help in planning proper applications of tannins in the livestock production.