Sinonasal Stent Coated with Slow-Release Varnish of Chlorhexidine Has Sustained Protection against Bacterial Biofilm Growth in the Sinonasal Cavity: An In Vitro Study

The aim of the study was to develop a sustained-release varnish (SRV) containing chlorhexidine (CHX) for sinonasal stents (SNS) to reduce bacterial growth and biofilm formation in the sinonasal cavity. Segments of SNS were coated with SRV-CHX or SRV-placebo and exposed daily to bacterial cultures of Staphylococcus aureus subsp. aureus ATCC 25923 or Pseudomonas aeruginosa ATCC HER-1018 (PAO1). Anti-bacterial effects were assessed by disc diffusion assay and planktonic-based activity assay. Biofilm formation on the coated stents was visualized by confocal laser scanning microscopy (CLSM) and high-resolution scanning electron microscopy (HR-SEM). The metabolic activity of the biofilms was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method. Disc diffusion assay showed that SRV-CHX-coated SNS segments inhibited bacterial growth of S. aureus subsp. aureus ATCC 25923 for 26 days and P. aeruginosa ATCC HER-1018 for 19 days. CHX was released from coated SNS segments in a pH 6 medium up to 30 days, resulting in growth inhibition of S. aureus subsp. aureus ATCC 25923 for 22 days and P. aeruginosa ATCC HER-1018 for 24 days. The MTT assay showed a reduction of biofilm growth on the coated SNS by 69% for S. aureus subsp. aureus ATCC 25923 and 40% for P. aeruginosa ATCC HER-1018 compared to the placebo stent after repeated exposure to planktonic growing bacteria. CLSM and HR-SEM showed a significant reduction of biofilm formation on the SRV-CHX-coated SNS segments. Coating of SNS with SRV-CHX maintains a sustained delivery of CHX, providing an inhibitory effect on the bacterial growth of S. aureus subsp. aureus ATCC 25923 and P. aeruginosa ATCC HER-1018 for approximately 3 weeks.

Antimicrobial activity of endophytic fungi isolated from different plant parts of Curcuma mangga Valeton and Zijp

The endophytic fungi isolated from different plant parts including leaf, stem and rhizome of Curcuma mangga were screened for antimicrobial activity by employing agar plug diffusion assay and disc diffusion assay for primary screening and secondary screening, respectively. A total of 127 endophytic fungi that were successfully isolated from various plant parts were cultured to examine their antimicrobial activities. Qualitative screening using agar plug diffusion assay revealed that 118 isolates (92.9%) showed antimicrobial activity against at least on one test microorganisms and suggested that the rhizome part exhibited the highest percentage of antiyeast (58.3%) and antifungal (91.7%) activities compared to leaf and stem parts. Quantitative screening using disc diffusion assay indicated that ethyl acetate extract from fermentative broth (extracellular compound) demonstrated better antimicrobial activity compared to methanol extract derived from fungal biomass (intracellular compound) against all the four classes of pathogenic microorganisms tested (Gram-positive bacteria, Gram-negative bacteria, yeast and fungi). The future of endophytic fungus study is very promising as it possesses hidden potential to be developed as natural antimicrobial agent.

GC-MS ANALYSIS AND ANTIMICROBIAL ACTIVITY OF STEREOSPERMUM FIMBRIATUM AGAINST SELECTED SKIN-ASSOCIATED PATHOGENS

Stereospermum fimbriatum (local name: “Chicha”) has been used traditionally to treat postpartum illness, stomachache, earache and itchy skin in Malaysia. This study was performed to investigate the phytochemical contents of S. fimbriatum (flowers, leaves, twig and stem bark) by phytochemicals screening and GC-MS analysis as well as to determine their antimicrobial potential against eleven skin-associated pathogens. The extraction was done by soxhlet extractor using n-hexane (Hex), dichloromethane (DCM) and methanol, successively. The antimicrobial activity of all extracts was screened by disc diffusion assay and selected active extracts were evaluated for their minimum inhibitory and bactericidal concentration (MIC & MBC). Phytochemicals such as steroids, terpenoids, tannins, flavonoids and saponins were identified in different plant parts of S. fimbriatum. Hex and DCM extracts of stem bark were the most potent extracts especially against three susceptible pathogens, methicillin-resistant Staphylococcus aureus (13-15 mm), S. aureus (14-15 mm) and S. epidermidis (16 mm). The range of MIC values for Hex and DCM extracts was 0.4 to 0.8 mg/mL. GC-MS analysis of Hex and DCM extracts showed the presence of numerous bioactive compounds. The stem bark of S. fimbriatum was rich of bioactive compounds and exhibited potent antibacterial activity specifically against Staphylococci sp.

The effect of essential oils on the growth of bacteria from municipal wastewater treatment

Bacterial sensitivity to essential oils has been reported in the case of soil isolated bacteria, food isolated bacteria but there is little evidence available to support the fact that wastewater isolated bacteria show sensitivity to essential oils. Keeping in view this fact the present investigation aims to determine the wastewater isolated bacterial strains sensitivity to six commercially available plant essential oils including clove, cinnamon, oregano, tea tree, fennel, and wintergreen. The essential oils were tested against ten laboratory bacterial strains (Acinetobacter baumanii, Escherichia coli: DH5α, E.coli: AD202, Pseudomonas fluorescens, Pseudomonas poae, Pseudomonas putida, staphylococcus aureus, and Stenotrophomonas maltophilia) (2) and ten wastewater isolated bacterial strains (Acinetobacter baumanii, Acinetobacter bouretii, Aeromonas hydrophila, E.coli, Enterobacter cloaceae, Flavobacterium branchiophilum, Klebsiella pneumoniae, Pseudomonas staurtii, Serratia fonticola, and Staphylococcus muscae) using the Kirby-Bauer disc diffusion assay, and the broth tube macrodilution MIC assay. The disc-diffusion assay showed that three of the oils, clove, cinnamon and oregano, were the most effective at inhibiting the growth of all the known single isolates. The broth tube MIC assay found that the WWTP isolated bacterial strains such as E. coli, Staphylococcus muscae, Enterobacter cloaceae, Acinetobacter baumanii were most sensitive to clove oil at MIC concentration ≤ 0.52 mg/ml, cinnamon oil at MIC concentration ≤ 0.51 mg/ml, and oregano oil MIC concentration ≤ 0.47 mg/ml. Finally, wastewater microbial community samples from activated sludge, returned sludge and anaerobic digesters were reduced by 0% > 94.24%, 46% > 99%, 70% > 97% percent when tested against clove, cinnamon, and oregano oils.

HPLC Quantification of Thymoquinone Extracted from Nigella sativa L. (Ranunculaceae) Seeds and Antibacterial Activity of Its Extracts against Bacillus Species

The medicinal importance of Nigella sativa seeds for treating various ailments is portrayed by its traditional uses. Owing to its immense pharmacological importance, the thymoquinone phytoconstituent of N. sativa can prove beneficial for the South Asian countries including Pakistan, where this seed is commonly produced and healthcare facilities are limited. In this study, the antibacterial activity of various extracts of N. sativa seeds, extracted thymoquinone, and oil samples have been investigated against Bacillus subtilis and Bacillus licheniformis using well and disc diffusion assay. The inhibition zones ranged between 7 and 44 mm against both the bacterial strains by well diffusion assay, while disc diffusion assay provided inhibition zones in the range of 7–23 mm. Commercial and local Kalonji oil samples were included in the study. Oil samples dissolved in methanol showed increased inhibition of bacteria. However, the extracted thymoquinone showed highest antibacterial activity. Medicine formulated using thymoquinone will prove to be an herbal alternate against the resistant microbiota associated with bacterial infections. Antibacterial activity against some Bacillus species will help signify the effect on normal gut flora when oral therapy is followed. Trying different extraction protocols can help increase extraction efficiency. Study on extraction of thymoquinone in local produce of black seed can be fruitful for conducting the stability studies and can help to gain maximum benefits from the bioactives. The crude extracts from 10 g of these seeds were subjected to preliminary phytochemical investigation. Results showed that although methanol extract had the presence of maximum phytochemicals, hexane extract was the most potent in terms of antibacterial activity. Thymoquinone, a therapeutically important bioactive in N. sativa seed, was extracted employing both solvents. TLC assay and UV spectroscopy were used for its qualitative assessment, while HPLC-UV quantification showed that 250 mg/mL of methanol extract had 368.3 μg/mL thymoquinone, while its successive extraction yielded 32.94 μg/mL thymoquinone.

Comparative Analysis of In vitro Antimicrobial and Antioxidant Potential of Cinnamomum tamala Extract and their Essential Oils of Two Different Chemotypes

Background: The essential oils of aromatic plants have wide range of biological applications. Natural food preservatives have been always a demanding for food industries in both developed and developing countries to prevent bacterial growth in food stuffs. Therefore, focused on Cinnamon leaves essential oils components against food pathogens have been investigated to confirm its potential use in food products. Methods: The antimicrobial activity of two Cinnamon leaves oils and extracts (T-2 and T-19) were examined by disc diffusion assay and the minimum inhibitory concentration by two-fold serial dilution method against foodborn pathogenic microorganisms i.e. E.coli (MTCC 723), B. Cereus (MTCC 430), S. aureus (MTCC 3381), S. typhi (MTCC 734) and C. perfringens (MTCC 1349). The antioxidant activity of both essential oils and extract was determined by DPPH assay. The chemical profiling of Cinnamon essential oils were determined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Result: The antimicrobial activity of both Cinnamon leaves oils and extract were evaluated by disc diffusion assay and it showed that in essential oils and extracts exhibited the highest zone of inhibition (ZOI) against S. aureus and E.coli. Minimum inhibitory concentration (MIC) of both oils and extracts ranged from 0.156 mg/ml to 5mg/ml and the antioxidant properties of oils and extract of cinnamaldehyde type Cinnamon possessed the highest antioxidant activity than linalool type. The chemical constituent of Cinnamon oil was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) revealed that T-2 contains cinnamaldehyde (75%) and T-19 contains Linalool (63.77%) were found as major constituents. Therefore the results shows that essential oil of cinnamaldehyde type Cinnamon could be a potential rich source of natural antioxidants and also more effective against food borne pathogens than linalool type and could be used as natural antibacterial agents in food preservation.

Synthesis, Characterization, Antibacterial and Wound Healing Efficacy of Silver Nanoparticles From Azadirachta indica

Bacteria are the causative agents of numerous diseases. Ever increasing number of bacterial infections has generated the need to find new antibiotic materials and new ways to combat bacterial infections. Our study investigated Azadirachta indica (AI) as an alternate source of antibiotic compounds. Phytochemical and GC-MS analysis revealed presence of flavonoids, phenolic compounds, terpenoids and terpenes. Aqueous extracts of leaves were used to synthesize silver nanoparticles (AI-AgNPs), as established by colorimetric confirmation with maximum absorbance peak at 400 nm. Optimized reaction parameters produced high yield of stable AI-AgNPs, which were characterized by UV-Vis spectroscopy, energy-dispersive X-ray spectroscopy, scanning electron microscopy, and transmission electron microscopy. Results confirmed particle diameter of 33 nm and spherical shape of AI-AgNPs. Fourier transform infrared spectroscopy inferred the presence of functional groups in bioactive constituents involved in conversion of silver ions into elemental silver by acting as capping and reducing agents during formation of AI-AgNPs. X-ray diffraction revealed their crystalline nature. Toxicity studies on Drosophila validated normal egg laying capacity and eclosion of F1 generation on AI-AgNPs (100 μg/mL). DPPH (65.17%) and ABTS (66.20%) assays affirmed strong radical scavenging effect of AI-AgNPs (500 μg/mL). The antibacterial activity of AI-AgNPs (1,000 μg/mL) was confirmed by disc diffusion assay with zone of inhibition against Bacillus cereus (17.7 mm), Escherichia coli (18.7 mm), Pseudomonas aeruginosa (10.3 mm), and Staphylococcus aureus (17.7 mm). Minimum inhibitory concentration and minimum bactericidal concentration values for AI-AgNPs ranged between 390 and 780 μg/mL. Higher bacterial suppression by AI-AgNPs in comparison with AI-extract was further divulged by prominent damage to the bacterial cell walls, disintegration of cell membranes and outflow of intercellular content as evident in SEM images. AI-AgNPs were loaded on PF127 (biocompatible-biodegradable polymer) to form a viscous, spreadable, hydrogel that demonstrated enhanced antibacterial properties in disc diffusion assay (13–18.7 mm). When topically applied on mice, AI-AgNPs-PF127 hydrogel did not show symptoms of skin irritation. Application of AI-AgNPs-PF127 hydrogel on wound sites in mice, significantly increased the wound contraction rate. Our studies present a simple green route to synthesize AI-AgNPs with enhanced antibacterial and free-radical scavenging efficacy; and AI-AgNPs-PF127 hydrogel as a low-toxic, eco-friendly delivery vehicle with potential in wound healing.

Anti-fungal activity of Carica papaya leaf extract against candida albicans and its synergy with fluconazole: an in-vitro study

Background: In this study objectives were to evaluate the antifungal activity in increasing concentrations of ethanolic extract and aqueous extract of Carica papaya against Candida albicans and to assess the synergistic activity of ethanolic extract of Caricia papaya with flucanazole as a potential antifungal.Methods: The aim of the study was to evaluate the antifungal activity in increasing concentrations, 500 µl/ml, 750 µl/ml and 1000 µl/ml of ethanolic extract and aqueous extract of caricia papaya against Candida albicans and the synergistic activity with Fluconazole was assessed by observing the zone of inhibition in agar disc diffusion assay and by observing the turbidity in minimum inhibitory concentration (MIC) assay.Results: It was observed that ethanolic extract of Carica papaya leaf showed significant antifungal activity in higher concentration of 1000µg/ml with zone diameter of 11.97±0.15 mm in disc diffusion assay and MIC of 350 µg/ml. The ethanolic extract of Carica papaya leaf with fluconazole showed synergistic activity with zone diameter of 13.6±0.45 mm in disc diffusion assay and MIC was 125µg/ml, whereas the standard drug Fluconazole’s zone of inhibiton was 12.83±0.9 in disc diffusion and MIC was 500 µg/ml.Conclusions: From this study, we can safely conclude that the Carica papaya leaf extract has a significant antifungal property and exhibit synergistic effect when used with fluconazole.

Antibacterial activity of silver nanoparticles-Clinacanthus nutans (AgNP-CN) against Streptococcus mutans

Clinacanthus nutans was found to possess anti-venom, anti-inflammatory, analgesic, anti- diabetic, anti-rheumatism, antiviral and antioxidant properties. Silver nanoparticles are nanoparticles between 1nm to 100nm in size and play significant role in medicinal fields. Silver nanoparticles exhibit unique properties, such as excellent conductivity, chemical stability, catalytic and antimicrobial activity. Streptococcus mutans is usually discovered in human oral cavity and the main aetiological of tooth decay. There is no study on antibacterial effect of silver nanoparticles Clinacanthus nutans (AgNP-CN) against Streptococcus mutans reported to date. Therefore, objective of this study is to investigate antibacterial properties of silver nanoparticles Clinacanthus nutans against Streptococcus mutans. Streptococcus mutans was subcultured in brain heart infusion (BHI) broth and agar. AgNP-CN with different concentrations was tested against Streptococcus mutans via disc diffusion assay, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Amoxicillin was used as positive control while DMSO and blank silver nanoparticles were used as negative control. Disc diffusion assay shows AgNP-CN inhibit Streptococcus mutans growth. AgNP-CN shows the greatest inhibition properties (15.3±1.53 mm) in comparison to Clinacanthus nutans leaves extract (6.0±0.01 mm) and blank silver nanoparticles (6.0±0.01 mm). MIC value for AgNP-CN is 2.5±0.01 mg/mL while amoxicillin is 0.007±0.01 mg/mL. Whereas MBC values for AgNP-CN is 2.5 mg/mL. Results are concentration dependent, with higher concentration shows better inhibition property. It can be concluded AgNP-CN possesses bactericidal properties against Streptococcus mutans.

Comprehensive Statistical Evaluation of Etest®, UMIC®, MicroScan and Disc Diffusion versus Standard Broth Microdilution: Workflow for an Accurate Detection of Colistin-Resistant and Mcr-Positive E. coli

Four colistin susceptibility testing methods were compared with the standard broth microdilution (BMD) in a collection of 75 colistin-susceptible and 75 mcr-positive E. coli, including ST131 isolates. Taking BMD as reference, all methods showed similar categorical agreement rates (CA) of circa 90%, and a low number of very major errors (VME) (0% for the MicroScan system and Etest®, 0.7% for UMIC®), except for the disc diffusion assay (breakpoint ≤ 11 mm), which yielded false-susceptible results for 8% of isolates. Of note is the number of mcr-positive isolates (17.3%) categorized as susceptible (≤2 mg/L) by the BMD method, but as resistant by the MicroScan system. ST131 mcr-positive E. coli were identified as colistin-resistant by all MIC-based methods. Our results show that applying the current clinical cut-off (>2 mg/L), many mcr-positive E. coli remain undetected, while applying a threshold of >1 mg/L the sensitivity of detection increases significantly without loss of specificity. We propose two possible workflows, both starting with the MicroScan system, since it is automated and, importantly, it categorized all mcr-positive isolates as colistin-resistant. MicroScan should be followed by either BMD or MIC-based commercial methods for colistin resistance detection; or, alternatively, MicroScan, followed by PCR for the mcr screening.