Broth Dilution Method Research Articles

Synthesis, Characterization, and Biological Studies of Metal Complexes of Dicoumarol Derivatives of Gatifloxacin

Copper(II) complexes were prepared using gatifloxacin and dicoumarin derivatives to investigate their biological activities and to conduct X-ray studies. The complexes were synthesized through classical thermal methods. Biologically potent ligands were synthesized by the reaction of dicoumarin derivative with different aldehydes in ethanol. Cu(II) complexes were formed by mixing ethanolic solution of ligands and gatifloxacin with metal solutions in a 1:1 mole ratio. The structures of the dicoumarin derivatives and their Cu(II) complexes were analyzed by infrared, 1H NMR, 13C NMR, and mass spectrometry. The thermal behavior of the new mixed ligand Cu(II) complexes was investigated by thermogravimetric, fast atom bombardment (FAB) mass, and X-ray analysis. The iron-reducing antioxidant potential (FRAP) (antioxidant activity) of all compounds was evaluated. All the synthesized compounds were screened for antibacterial activity against Escherichia coli; Pseudomonas aeruginosa; Streptococcus pyogenes; minimum inhibitory concentration values for Candida albicans; and Aspergillus niger were determined using the Luria Broth dilution method. The ligands and their complexes were evaluated for antioxidant and antibacterial activity in vitro. The complexes exhibited better results than their respective counterparts.. KEYWORDS :Antimicrobial, Antioxidant, Fast atom bombardment mass, Thermal analysis, Thermo gravimetric analysis, X-ray.

Assessment of the Antibacterial Activity of Spilanthes acmella Against Bacteria Associated with Dental Caries and Periodontal Disease: An In-vitro Microbiological Study

Dental caries and periodontal disease are two of the most common oral diseases caused by bacterial infections. Traditional medicine in India has a long history of using plant extracts for dental care. Spilanthes acmella (S. acmella), also known as the “Toothache Plant,” is a medicinal plant that has been traditionally used for its medicinal properties but has not been extensively studied for its applicability and use in dentistry. This study aims to investigate the antimicrobial action of S. acmella ethanol extract on Streptococcus mutans (S. mutans), and Lactobacillus fermentum (L. fermentum), which causes dental caries, and Porphyromonas gingivalis (P. gingivalis), Capnocytophaga gingivalis (C. gingivalis), which causes periodontal infection. The ethanol extract of S. acmella in various dilutions of 10mg/ml, 20mg/ml, 40mg/ml, 80mg/ml, and 100mg/ml was tested for its antibacterial activity against the bacteria as mentioned above using the agar well diffusion method. Erythromycin 0.125mg/ml was used as a positive control, whereas distilled water was used as a negative control. The Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) were determined by the broth dilution method. The results of this study have shown that the ethanol extract of S. acmella demonstrated concentration-dependent inhibition of bacterial growth (13-16mm diameter), with the highest concentration of 100mg/ml showing the strongest effect. The findings of this study support the use of the S. acmella plant extract in the treatment of dental caries and periodontal infection and suggest that it may be a viable alternative to traditional antimicrobial agents.

Determining the potential targets of silybin by molecular docking and its antibacterial functions on efflux pumps and porins in uropathogenic E. coli.

One of the causes of antibiotic resistance is the reduced accumulation of antibiotics in bacterial cells through pumping out the drugs. Silybin, a key component of the Silybum marianum plant, exhibits various beneficial properties, including anti-bacterial, anti-inflammatory, antioxidant, and hepatoprotective effects. Clinical isolates of E. coli were procured from 17 Shahrivar Children's Hospital in Rasht, Guilan, located in northern Iran. Their susceptibility to six antibiotics was assessed using disc diffusion and broth dilution (MIC) methods. The antibacterial effects of silybin-loaded polymersome nanoparticles (SPNs) were investigated with broth dilution (MIC) and biofilm assays. Molecular docking was utilized to evaluate silybin's (the antibacterial component) binding affinity to efflux pumps, porins, and their regulatory elements. Additionally, qRT-PCR analysis explored the expression patterns of acrA, acrB, tolC, ompC, and ompF genes in both SPNs (sub-MIC) and ciprofloxacin (sub-MIC)-treated and untreated E. coli isolates. The combined use of SPNs and ciprofloxacin exhibited a notable reduction in bacterial growth and biofilm formation, in ciprofloxacin-resistant isolates. The study identified eight overlapping binding sites of the AcrABZ-TolC efflux pump in association with silybin, demonstrating a binding affinity ranging from -7.688 to -10.33 Kcal/mol. Furthermore, the qRT-PCR analysis showed that silybin upregulated AcrAB-TolC efflux pump genes and downregulated ompC and ompF porin genes in combination with ciprofloxacin in transcriptional level in uropathogenic E. coli. Silybin, a safe herbal compound, exhibits potential in inhibiting antibiotic resistance within bacterial isolates, potentially through the regulation of gene expression and plausible binding to target proteins.

Phytochemicals properties and antimicrobial activities of Carica papaya and Balanites aegyptiaca’ seeds aqueous infusion and decoction extracts

Carica papaya and Balanites aegyptiaca seeds extract have several uses, from food to traditional medicine. This study aims to determine their phytochemicals compound and appreciate their antimicrobial properties against clinical strains. Polyphenolic content was determined with standard methods while DPPH radical scavenging activity is used to appreciate the antioxidant properties, using ascorbic acid as standard. Antimicrobial activities were performed according to the inhibition diameters, using agar diffusion method. Minimum inhibitory concentrations and Minimum fungicidal concentrations were established based on broth dilution methods. The extraction yield is ranged between 6.3 and 20.3%. The results shown polyphenolic content varying from 1256.13±21.09 to 2315.81±53.52 µg GAE/100 mL for Carica papaya and from 303.98±0.93 to 948.06±3.23 µg GAE/100 mL for Balanites aegyptiaca while the antioxidant activity is ranged between 31.76±2.35 and 58.47±4.24 µg AEAC/100 mL for Carica papaya and from 29.80±0.32 to 78.44±2.81 µg AEAC/100 mL for Balanites aegyptiaca. Infusion’s extracts always have the highest values of polyphenols content and antioxidant activity than decoction extracts, be it papaya or balanites seeds. Neitheir flavonoids content nor antibacterial activity was found. The inhibition diameter varies from 14.5±0.5 to 40.5±0.5 mm for papaya seeds extracts and from 16.5±0.5 to 21.0±0.5 mm for balanites seeds extracts without a significative effect of mixture for both extracts. MICs vary from 11.71±0.1 to 75.0±0.0 mg. mL-1 for Carica papaya seeds extracts with 11.72±0.2 to 37.30±0.2 mg. mL-1 and 18.74±0.1 to 100±0.0 mg.mL-1 for Balanites aegyptiaca. Mixture extracts presented intermediaire values. Decoction extracts have lowest MICs values and infusion reveals itself to be the sweetable aqueous extraction techniques but the appropriate infusion time need to be highlight.
 Keywords: Carica papaya, Balanites aegyptiaca, Seeds extracts, Phytochemical, antifungal

Levofloxacin susceptibility of Staphylococci from conjunctiva in patients with atopic dermatitis.

The ocular surface in patients with atopic dermatitis (AD) is known to harbor an abundance of gram-positive cocci, particularly Staphylococcus aureus (S. aureus). This study reviewed the results of microbial cultures from the conjunctiva in AD patients, with special attention to the levofloxacin susceptibility of Staphylococci. Retrospective, single-center study. This study involved 131 eyes of 112 Japanese patients with AD (87 men and 25 women, mean age: 40.4 ± 12.2 years) who underwent ocular surgery at Kyorin University Hospital. Bacterial isolates were collected from the conjunctival sacs in the preoperative period. Drug resistance to methicillin and levofloxacin was judged using the minimal inhibitory concentrations of oxacillin and levofloxacin determined by the broth dilution method. One hundred and fifty-seven strains were identified in 103 of the 131 eyes examined. S. aureus was isolated from 74 eyes (56.5%), followed by Staphylococcus epidermidis (S. epidermidis). In S. aureus, 11 strains (14.9%) were methicillin-resistant, and 18 (24.3%) were levofloxacin-resistant. In S. epidermidis, 15 strains (26.8%) were methicillin-resistant, and 17 (30.4%) were levofloxacin-resistant. No significant differences were observed in levofloxacin susceptibility with age, sex, previous ocular surgery, or duration of previous surgery. However, logistic multivariate analysis revealed that levofloxacin-resistant Staphylococci were concurrently resistant to methicillin, suggesting multidrug resistance. Distinctive bacterial distribution and drug resistance need consideration in the managing of ocular disorders among patients with AD.

Physico-Chemical Investigation and Antimicrobial Efficacy of Ozonated Oils: The Case Study of Commercial Ozonated Olive and Sunflower Seed Refined Oils.

Drug resistance represents one of the great plagues of our time worldwide. This largely limits the treatment of common infections and requires the development of new antibiotics or other alternative approaches. Noteworthy, the indiscriminate use of antibiotics is mostly responsible for the selection of mutations that confer drug resistance to microbes. In this regard, recently, ozone has been raising interest for its unique biological properties when dissolved in natural oils. Ozonated oils have been reported to act in a non-specific way on microorganisms hindering the acquisition of advantageous mutations that result in resistance. Here, we focused on the antimicrobial effect of two commercial olive (OOO) and sunflower seeds (OSO) oils. Nuclear magnetic resonance spectroscopy and thermal analysis showed the change in the chemical composition of the oils after ozonation treatment. Different ozonated oil concentrations were then used to evaluate their antimicrobial profile against Candida albicans, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli by agar diffusion and broth dilution methods. Cytotoxicity was also evaluated in keratinocytes and epithelial cells. Overall, our results revealed that both OOO and OSO showed a potent microbicidal effect, especially against C. albicans (IC50 = OOO: 0.3 mg/mL and OSO: 0.2 mg/mL) and E. faecalis (IC50 = OOO: 0.4 mg/mL and OSO: 2.8 mg/mL) albeit exerting a certain effect also against S. aureus and E. coli. Moreover, both OOO and OSO do not yield any relevant cytotoxic effect at the active concentrations in both cell lines. This indicates that the ozonated oils studied are not toxic for mammalian cells despite exerting a potent antimicrobial effect on specific microorganisms. Therefore, OOO and OSO may be considered to integrate standard therapies in the treatment of common infections, likely overcoming drug resistance issues.

Agreement of methods to assess antimicrobial susceptibility using Escherichia coli isolates as target models.

Antimicrobial susceptibility tests (AST) conducted in vitro offer a range of methods to assess the antimicrobial resistance (AMR) of microorganisms. Escherichia coli, a widely distributed bacterium, is closely linked to the issue of AMR. In this way, the present study aimed to assess the agreement among different in vitro AST methods, including disk diffusion in agar, broth dilution, and agar dilution method. A total of 100 E. coli isolates were analyzed for their resistance levels against six antibiotics: amoxicillin, ceftiofur, ciprofloxacin, chloramphenicol, tetracycline, and sulfamethoxazole+trimethoprim, using the aforementioned AST methods. Standard breakpoint values were employed to classify isolates as resistant, intermediate, or susceptible, and comparisons among the AST methods were conducted by McNemar's test (P<.05). The obtained data demonstrated equivalence among the AST methods, highlighting the reliability of these standardized classical methodologies. This standardization aids in preventing the inappropriate use of antimicrobials and the dissemination of antimicrobial-resistant microorganisms.

In vitro susceptibility of nontuberculous mycobacteria in China

ObjectivesThis study aimed to measure the prevalence of resistance to antimicrobial agents, and explore the risk factors associated with drug resistance by using nontuberculous Mycobacteria (NTM) isolates from China.MethodsA total of 335 NTM isolates were included in our analysis. Broth dilution method was used to determine in vitro drug susceptibility of NTM isolates.ResultsClarithromycin (CLA) was the most potent drug for Mycobacterium intracellulare (MI). The resistance rate of 244 MI isolates to CLA was 21%, yielding a minimum inhibitory concentrations (MIC)50 and MIC90 of 8 and 64 mg/L, respectively. 51% of 244 MI isolates exhibited resistance to amikacin (AMK). For 91 Mycobacterium abscessus complex (MABC) isolates, 6 (7%) and 49 (54%) isolates were categorized as resistant to CLA at day 3 and 14, respectively. The resistance rate to CLA for Mycobacterium abscessus subspecies abscessus (MAA) was dramatically higher than that for Mycobacterium abscessus subspecies massiliense (MAM). Additionally, the percentage of patients presenting fever in the CLA-susceptible group was significantly higher than that in the CLA-resistant group.ConclusionsOur data demonstrate that approximate one fifth of MI isolates are resistant to CLA. We have identified a higher proportion of CLA-resistant MAA isolates than MAM. The patients caused by CLA-resistant MI are at low risk for presenting with fever relative to CLA-susceptible group.

Comparative Antimicrobial Activity of Securidaca longipedunculata (Fresen) Extracts on Selected Human Pathogens

The escalating trend of microbial resistance has become a pressing global concern, challenging the effectiveness of antimicrobial agents in treating infections. This study was aimed at evaluating the antimicrobial activity of the n-hexane, ethyl acetate, and methanol extracts of Securidaca longipedunculata against selected human pathogens. The antimicrobial screening against bacterial pathogens- (e.g., Methicillin-resistant staphylococcus aureus, Vancomycin-resistant Enterococci, Staphylococcus aureus, Staphylococcus feacalis, Escherichia coli, Salmonella typhimurium, Pseudomans fluorescens and Klebsiella pneumonieae) and fungal pathogens (which includes Candida albicans, Candida krusei, Aspergillus niger, Aspergillus fumigates, and Microsporum canis) was performed using agar well diffusion and broth dilution methods. The results demonstrated significant antimicrobial activity, with higher efficacy against S. aureus. Susceptibility test results showed that the extract inhibited the growth of all the test organisms (bacteria and fungi) with a mean zone of inhibition range of 18-29 mm, with the exception of VRE, S. typhimurium, P. fluorescens, C. krusei and M. canis in all the three extracts, while Hexane extract showed no inhibition against E. coli and A. niger. The standard drugs sparfloxacin and ciprofloxacin (500 µg/cm3) had zone of inhibition ranging from 28 - 40 mm. While the antifungal drugs fluconazole and fulcin exhibited zone of inhibition ranging from 27 mm to 34 mm. The Minimum inhibitory concentration (MIC) for the extracts ranges from 62.5 - 250µg/cm3, while the Minimum Bactericidal/Fungicidal Concentrations (MBC/MFC) range from 250 - 500 µg/cm3, respectively. The ethyl acetate extracts possessed higher antimicrobial activity among the three extracts tested against S. Aureus. The findings suggest that S. longipedunculata leaves hold promise as a natural source of antimicrobial agents, warranting further exploration for potential therapeutic applications.

Epidemiological characterization of clinical isolates of meticillin resistant Staphylococcus aureus through multilocus sequence typing and staphylococcal cassette chromosome mec typing in Northwest Iran.

Methicillin-resistant Staphylococcus aureus (MRSA), is considered a potential and aggressive nosocomial pathogen. It accounts for 50% of S. aureus isolates in tertiary hospitals in Iran, however, there is no sufficient evolutionary and epidemiological investigation about this medically important bacterium. We aimed to study the lineage and evolution of MRSA in Northwest Iran during 2021-2022 based on the obtained phenotypic and genotypic characteristics. Seventy-two non-duplicate MRSA isolates were collected from 3 referral hospitals in Tabriz, Ardebil, and Urmia cities. The antimicrobial susceptibility patterns were determined by disk diffusion test and micro broth dilution methods. Thereafter 4 virulence genes (eta, etb, pvl, tst) and 5 types of staphylococcal cassette chromosome mec (SCCmec) were detected by PCR. In the final step, representative isolates were selected to be studied by Multilocus sequence typing (MLST). The highest resistance was observed to erythromycin and clindamycin at a rate of 76.4%, followed by ciprofloxacin (61.1%), gentamicin (54.2%), rifampin (38.9%), and co-trimoxazole (27.8%). All isolates were susceptible to vancomycin. The virulence genes of etb, pvl, tst, and eta were detected in 50%, 29.2%, 21.8%, and 13.9% of isolates, respectively. SCCmec types III and I were the most prevalent types, followed by types IV, II, and V. MLST analysis revealed 6 sequence types: ST6854, ST5282, ST127, ST7804, ST1607, and ST7784. Two MLST-based clonal complexes (CC8, and CC97) were identified as well. The ST numbers were non-repetitive. CC8 as a pandemic clone and an individual lineage and clinically significant clade was reported as the most prevalent clonal complex. It is essential periodic evaluations of antibiotic susceptibility patterns and study the evolutionary characteristics of medical-challenging microorganisms in particular MRSA to effectively treat and restrict the outbreaks.

Assessment of Minimum Inhibitory Concentration and Anti-biofilm Activity of Plectranthus Amboinicus Solvent Extract against Pure Strains of Putative Periodontal Pathogens: An In-vitro Study

Introduction: Antiseptic agents used in periodontics as anti-plaque and anti-gingivitis agents are primarily chemical substances such as Bis-biguanide derivatives (chlorhexidine) or essential oils. Herbal derivatives have gained prominence in the recent past due to their activity against putative periodontal pathogens; however, only a few have achieved commercialisation. This study focusses on determining the efficacy of an extract from a widely available herb, Indian mintPlectranthus Amboinicus Methanolic extract (PAM), which has known anti-microbial and anti-inflammatory properties against periodontal pathogens in-vitro. Aim: To assess the Minimum Inhibitory Concentration (MIC) and anti-biofilm property of PAM solvent extract against pure strains of putative periodontal pathogens, namely Porphyromonas gingivalis (American Type Culture Collection-ATCC 33277), Fusobacterium nucleatum (ATCC 25586), and Aggregatibacter actinomycetemcomitans (ATCC 43718). Materials and Methods: The extract of PA was prepared using methanol and a Soxhlet extractor. An in-vitro analysis of the MIC and anti-biofilm efficacy of the extract was performed against standard strains of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum using the broth dilution method and microtitre-crystal violet assay, respectively. The MIC activity of the PAM extracts was compared with Chlorhexidine as a standard. Results: The MIC value of P. amboinicus extract was nearly similar to Chlorhexidine as assessed by the broth dilution method. The MIC of P. amboinicus extract for A.a and P.g was 0.4 ug/mL, F.n was 0.8 ug/mL, and the Chlorhexidine values against all three periodontal pathogens were 0.2 ug/mL. The anti-biofilm activity of The extract of PAMwas evaluated using the microtitrecrystal violet assay, and the Optical Density (OD) values were reduced after exposure to the extract, with a significant reduction (p&lt;0.001) of the biofilm-forming bacteria observed. Conclusion: The methanol extract of PAM demonstrated a noteworthy MIC, exhibiting effectiveness at a low concentration of 0.4 μg/mL against Aggregatibacter actinomycetemcomitans in three repeated trials. Moreover, this extract displayed significant inhibitory effects on the biofilm formation of periodontal pathogens Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum (Pg, Aa, Fn), suggesting its potential as an alternative to conventional chemical anti-microbials.

The potency of Actinomycetes InaCC A758 against dual-species biofilms: Candida albicans and Staphylococcus aureus

Candida albicans and Staphylococcus aureus can coexist to form a biofilm, leading to infections associated with biofilms. Actinomycetes produce secondary metabolites known as antibiotics, antifungals, antibiofilm, anticancer, and antimalarials. This study was aimed to explore the antibiofilm activity of secondary metabolites of Actinomycetes InaCC A758 extracts (InaCC A758) against dual-species biofilms, i.e., C. albicans and S. aureus. Ethyl acetate and chloroform were used as solvents in a maceration extraction technique to isolate the compound designated InaCC A758. The fractionation of the InaCC A758 extracts was carried out using semi-preparative HPLC. Identification of the compounds from the InaCC A758 extracts was performed using gas chromatography-mass spectrometry analysis. Antimicrobial and antibiofilm testing of the InaCC A758 extracts was done utilizing the micro broth dilution method. The morphology of the biofilms following treatment with the InaCC A758 extracts was visualized using scanning electron microscopy (SEM). The minimum inhibitory concentration of InaCC A758 against dual-species biofilms was 400 μg/ml. The concentrations of InaCC A758 required to inhibit 50% and 80% of dual-species biofilm formation (BIC50 and BIC80) ranged from 3.57–6.66 and 29.25–30.50 μg/ml, respectively. The concentrations needed to reduce formed dual-species biofilms by 50% and 80% (BRC50 and BRC80) ranged from 100.62–131.85 and 596.4–849.6 μg/ml, respectively. SEM observation showed a reduced number of cells and disruption of the cell membranes when exposed to the InaCC A758 extracts. The InaCC A758 contains seven compounds, i.e., dodecanoic acid 3-hydroxy-; 1,3,5-pentanetriol,3-methyl-; 1,3-dioxolane-4-methanol, 2-ethyl-; 2-cyclopropylcarbonyloxytridecane; 2-hepten-1- ol,(E); methyl 6-methyl heptanoate; and 11-octadecenoic acid, methyl ester. The InaCC A758 extract altered cell morphology and exhibited potential as an anti-biofilm agent, particularly against dual-species biofilms.

Phytochemical profiling and evaluation of antimicrobial activities of common culinary spices: Syzygium aromaticum (clove) and Piper nigrum (black pepper).

The increasing resistance of microbial pathogens to conventional antibiotics necessitates the exploration of alternative antimicrobial agents. This study aims to evaluate the antimicrobial potential and phytochemical properties of Syzygium aromaticum (clove) and Piper nigrum (black pepper) extracts, both of which are known for their historical use in traditional medicine and culinary applications. Hydroalcoholic and aqueous extracts of clove and black pepper were prepared. The antimicrobial activity of these extracts was assessed using the disk diffusion method against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger. Minimum inhibitory concentration (MIC) was determined using the broth dilution method. Qualitative phytochemical screening identified the presence of key bioactive compounds, while quantitative analysis measured total phenolic and flavonoid contents. LC-HRMS/MS analysis of ethanolic extracts was performed. Both spices extracts exhibited significant antimicrobial activity, with inhibition zones ranging from 14 to 18 mm. clove showed superior antimicrobial efficacy compared to black paper, particularly against fungi. MIC values ranged between 3 mg/mL and 6 mg/mL for both spices. Phytochemical analysis revealed higher total phenolic and flavonoid contents in clove, with hydroalcoholic extracts showing greater concentrations than aqueous extracts. HPLC quantified higher eugenol content in clove extracts and higher piperine content in black pepper extracts. The differences in bioactive compound content were statistically significant (p < 0.05). The study confirms that both spices possess significant antimicrobial properties, attributable to their rich phytochemical composition, particularly phenolics and flavonoids. Clove exhibited slightly superior antimicrobial activity compared to black paper. These findings support the potential use of these spices as complementary antimicrobial agents. Further research should investigate their synergistic effects with conventional antibiotics and explore their applications in food preservation and alternative medicine.

Antimicrobial Activity and Molecular Docking of Benzoyl-N,N’-dialkylurea against Target Proteins in Microbial Cells

This research aims to evaluate the antimicrobial activity of benzoyl dialkylurea derivatives to meet the global need for new antibiotic lead compounds. Four compounds were tested in vitro against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans by broth dilution method using ciprofloxacin and nystatin as standards. Docking simulations were performed to target essential proteins including DNA gyrase, FabH, RNA polymerase in bacterial cells and DHFR in C. albicans. BDMU, BEU1, BEU2, BEU3 exhibited antibacterial activity while BEU1 and BEU2 showed weak antifungal activity against C. albicans. The BDMU, BEU2, BEU3 considered promising growth inhibition against P. aeruginosa. In silico molecular docking on DNA gyrase from P. aeruginosa (PDB. 6M1S) was proposed as a model for mechanism of action in bacterial cells. The monobenzoyl of dialkylurea containing urea functionality with chloro-substituent at position-2 and 4 on aromatic rings were potential as lead compound to generate new antibacterial agent.

Antimicrobial evaluation of crude extracts of bark and stem of virginia creeper (Parthenocissus quinquefolia (L.) Planch)

The In vitro antimicrobial potential of bark and stem of virginia creeper (Parthenocissus quinquefolia (L.) Planch) was measured in order to authenticate its ethnopharmacological significance. Bark and stem parts were extracted by static maceration technique in non-polar and polar solvents, such as n-hexane, chloroform, ethanol and double distilled water. Agar well diffusion technique was used to verify the antimicrobial activity of these extracts against two of each Gram-negative, Gram-positive and fungal strains. Minimum inhibitory concentration (MIC) was measured through modified broth dilution method. However, ethanolic and aqueous extracts of stem resulted in highest inhibition zone (52.5±1.1 mm) against Pseudomonas aeruginosa, and augmented antifungal activity (46±0.48 mm) against Fusarium solani, respectively. Whereas, n-hexane extract of bark exhibited least antibacterial activity in the form of inhibition zone of 15±0.22mm against S. aureus. Similarly, minimum activity (16.5±0.21 mm) was observed in ethanolic extract of bark, against F. solani. The most resistant MIC value was shown by ethanolic bark extract, i.e. 0.5 mg/mL against P. aeruginosa. Thus, virginia creeper can be recommended as a remedy to combat efficaciously with the resilient contagious strains.

An efficient synthesis, characterization, antimicrobial and anticancer activities of azo dyes derived from eugenol

A new series of azo dyes of the naturally occurring phenol i.e., eugenol (4-allyl-2-methoxyphenol) was produced by the conventional method. For the synthesis of azo compounds, diazonium salts were coupled with eugenol. Six substituted aryl-azo-eugenol derivatives (EU (a) to EU (f)) were synthesized and confirmed their structures with 1H NMR,13C NMR, FT-IR and Mass spectroscopy. All newly synthesized azo dyes of eugenol were evaluated for their in vitro antibacterial activity against S. aureus, S. pyogenus, E. coli, P. aeruginosa strains and in vitro antifungal activity against C. albicans, A. niger and A. clavatus strains by using micro broth dilution method. The results indicate that the synthesized azo dyes of eugenol display variable inhibitory effects on the growth of bacterial and fungal strains. Further, all compounds were evaluated for their anticancer activity against human breast cancer cell lines MDA-MB-241, the compound EU(d) displays excellent anticancer activity.

In silico and in vitro evaluation of newly synthesized pyrazolo-pyridine fused tetrazolo-pyrimidines derivatives as potential anticancer and antimicrobial agents

Diversely functionalized pyrazolo-pyridine fused tetrazolo-pyrimidines 10aa-am and 10ba-bn were successfully synthesized via a catalyst-free synthetic protocol with moderate to very good yields. The compounds were evaluated for cytotoxicity against MCF-7 and HEK-293 cells using MTT assay. Among the tested compounds, 10ab (IC50- 23.83 µM) and 10ah (IC50- 23.30 µM) demonstrated the highest potency against MCF-7 cells, while 10bc (IC50- 14.46 µM) and 10bh (IC50- 2.53 µM) exhibited excellent cytotoxicity against HEK-293 cells. Additionally, antibacterial screening was performed against three Gram-negative bacteria (E. coli, P. aeruginosa, and S. enterica) and three Gram-positive bacteria (S. aureus, B. megaterium, and B. subtilis) using broth dilution method, while antifungal activity was assessed against three fungal strains (A. niger, Penicillium, and S. cerevisiae) using agar well diffusion method. In antimicrobial screening, the majority of the compounds demonstrated significant antibacterial efficacy compared to antifungal activity. We also conducted comprehensive computational studies, including DFT calculations, molecular docking and dynamics, and drug-likeness assessments. In the DFT study, compounds 10ac and 10bc displayed stable conformations, indicating their potential for higher therapeutic activity. Molecular docking analyses revealed compelling interactions, with compound 10ah demonstrating docking score −7.42 kcal/mol against catalytical domain PARP1 (PDB ID: 7KK4) and 10bh exhibiting a best docking score −10.77 kcal/mol against human corticotropin-releasing factor receptor 1 (PDB ID: 4Z9G). A 100 ns molecular dynamics (MD) simulation study of compounds 10ah and 10bh revealed the stable conformation and binding energy in a stimulating environment. In drug-likeness assessments, both the compounds 10ah and 10bh adhere all the established guidelines. Communicated by Ramaswamy H. Sarma

High-Throughput Monitoring of Pathogenic Fungal Growth Using Whole Slide Imaging for Rapid Antifungal Susceptibility Assessment

Invasive fungal infections are a major health threat with high morbidity and mortality, highlighting the urgent need for rapid diagnostic tools to detect antifungal resistance. Traditional culture-based antifungal susceptibility testing (AFST) methods often fall short due to their lengthy process. In our previous research, we developed a whole-slide imaging (WSI) technique for the high-throughput assessment of bacterial antibiotic resistance. Building on this foundation, this study expands the application of WSI by adapting it for rapid AFST through high-throughput monitoring of the growth of hundreds of individual fungi. Due to the distinct “budding” growth patterns of fungi, we developed a unique approach that utilizes specific cell number change to determine fungi replication, instead of cell area change used for bacteria in our previous study, to accurately determine the growth rates of individual fungal cells. This method not only accelerates the determination of antifungal resistance by directly observing individual fungal cell growth, but also yields accurate results. Employing Candida albicans as a representative model organism, reliable minimum inhibitory concentration (MIC) of fluconazole inhibiting 100% cells of Candida albicans (denoted as MIC100) was obtained within 3h using the developed method, while the modified broth dilution method required 72h for the similar reliable result. In addition, our approach was effectively utilized to test blood culture samples directly, eliminating the need to separate the fungi from whole blood samples spiked with Candida albicans. These features indicate the developed method holds great potential serving as a general tool in rapid antifungal susceptibility testing and MIC determination.

RNA-Seq-Based Transcriptomics and GC-MS Quantitative Analysis Reveal Antifungal Mechanisms of Essential Oil of Clausena lansium (Lour.) Skeels Seeds against Candida albicans.

Infections caused by Candida albicans (C. albicans) and increasing resistance to commonly used drugs lead to a variety of mucosal diseases and systemic infectious diseases. We previously confirmed that the essential oil of Clausena lansium (Lour.) Skeels seeds (CSEO) had antifungal activity against C. albicans, but the detailed mechanism between the chemical components and antifungal activity is unclear. In this study, a quantitative analysis of five volatile components of CSEO, including sabinene, α-phellandrene, β-phellandrene, 4-terpineol, and β-caryophyllene, was carried out using the gas chromatography-mass spectrometry (GC-MS) method. Both the broth dilution and kinetic growth methods proved that the antifungal activity of CSEO against fluconazole-resistant C. albicans was better than that of its main components (sabinene and 4-terpineol). To further investigate the inhibitory mechanism, the transcriptional responses of C. albicans to CSEO, sabinene, and 4-terpineol treatment were determined based on RNA-seq. The Venn diagram and clustering analysis pattern of differential expression genes showed the mechanism of CSEO and 4-terpineol's anti-C. albicans activity might be similar from the perspective of the genes. Functional enrichment analysis suggested that CSEO regulated adherence-, hyphae-, and biofilm-formation-related genes, which may be CSEO's active mechanism of inhibiting the growth of fluconazole-resistant C. albicans. Overall, we preliminarily revealed the molecular mechanism between the chemical components and the antifungal activity of CSEO against C. albicans. This study provides new insights to overcome the azole resistance of C. albicans and promote the development and application of C. lansium (Lour.) Skeels seeds.

Antibacterial Potency of Various Concentrations of Pineapple Peel Extract (Ananas comosus) against Enterococcus faecalis

Introduction: Enterococcus faecalis is a Gram-positive bacterium commonly found in cases of failed root canal treatment. Irrigation material with antibacterial effects are important in supporting the success of root canal treatment. Pineapple peel as a natural ingredient is reported to contain compounds such as flavonoids, saponins, tannins, and bromelain enzymes that have been proven antibacterial effects. Purpose: To determine antibacterial potency of pineapple peel extract (Ananas comosus) with concentrations of 1.56%, 3.125%, 6.25% and 12.5% against E. faecalis. Methods: In vitro quasi-laboratory experimental study with a post-test only control group design consisting of pineapple peel extract with concentrations of 1.56%, 3.125%, 6.25%, 12.5%, and aquadest as negative control with repetition 6 times. The inhibition test was determined using the disc diffusion method on Mueller-Hinton Agar media. The MIC value can be determined using the broth dilution method and the MBC value can be determined using the solid dilution method. The inhibition zone diameter data were analyzed using One Way ANOVA test and Post Hoc Tukey test. Results: Pineapple peel extract with concentrations 12,5% had the largest mean inhibition zone, which is was 3.13 mm. The MIC and MBC values of pineapple peel extract were determined at a concentration of 6.25%. Conclusion: Pineapple peel extract (Ananas comosus) has antibacterial potency against Enterococcus faecalis.