Microtiter Assay Research Articles

Characterization, immobilization and evaluation of anti-Pseudomonas aeruginosa biofilm activity of alginate lyase from marine bacterium, Enterobacter tabaci RAU2C.

Alginate lyases have the potential to be used as a therapeutic agent for P. aeruginosa infections. The present work was focused on the characterization of free and immobilized alginate lyase produced by marine bacteria, Enterobacter tabaci RAU2C isolated previously in the laboratory for alginate lyase production and exploring the potential of alginate lyase as an anti-biofilm agent against the P. aeruginosa biofilm. RAU2C alginate lyase was immobilized using an epoxy-activated curdlan matrix by three different methods. Further, the free and immobilized were characterized for its optimal pH and temperature. The effect of alginate concentration on alginate lyase activity was assessed and the kinetic parameters were evaluated. The anti-biofilm activity of the crude alginate lyase was studied using biofilm inhibition and disruption assays in microtiter plates with crystal violet. The biofilm disruption by RAU2C alginate lyase was also ascertained by microscopic analysis. The immobilization matrix prepared using method 3 had a better binding capacity compared to other methods. Both soluble and immobilized alginate lyase exhibited optimal activity at 37°C and pH 7.0. Km and Vmax of soluble and immobilized alginate lyase were found to be 3.38mg/mL, 22.98mg/mL min and 3.67mg/mL and 26.59mg/mL min respectively. Both microtiter assay and microscopic analysis confirmed the prevention and dispersal of pre-existing biofilms by crude RAU2C alginate lyase, highlighting its potential as an anti-biofilm agent against P. aeruginosa. The study highlights the efficacy of RAU2C alginate lyase as an anti-biofilm agent in controlling P. aeruginosa biofilms.

Exploring Novel Benzopyran Derivatives as Antitubercular Agents

Background: Tuberculosis (TB) remains a significant global health concern, necessitating the exploration of novel therapeutic agents. Reported antitubercular activities of previously synthesized benzopyran and other oxygen-containing heterocycles motivate us to synthesize and evaluate the antitubercular potential of benzopyran derivatives. Objective: The aim of this study was to combine two scaffolds; one is coumarin (benzopyran-2-one), and another one is piperazine, as both are found in anti-tubercular derivatives. Methods: Through a four-step synthetic approach, compounds SM1-SM10 were synthesized. These derivatives were subsequently evaluated for their in vitro anti-tubercular potential using the resazurin microtiter assay (REMA), with isoniazid as the standard (MIC 0.25 to 0.5μg/mL). Results: Among the synthesized compounds, SM2 and SM8 demonstrated remarkable antitubercular activity, with MICs of 4 and 6μg/mL, respectively. Notably, these MIC values are considerable for the further development of benzopyran derivatives as potent antitubercular agents. Conclusion: Outcomes underscore the potential of benzopyran derivati ves as valuable assets in TB drug discovery, warranting further exploration and development.

Antipathogenic Activity of Betainized Polyethyleneimine Sprays Without Toxicity.

Background/Objectives: The design of alternative antipathogenic sprays has recently attracted much attention due to the limitations of existing formulations, such as toxicity and low and narrow efficacy. Polyethyleneimine (PEI) is a great antimicrobial polymer against a wide range of pathogens, but toxicity limits its use. Here, betainized PEI (B-PEI) was synthesized to decrease the toxicity of PEI and protonated with citric acid (CA), boric acid (BA), and HCl to improve antimicrobial activity. Methods: Cytotoxicity of the PEI-based solutions was determined on L929 fibroblast cells. Antibacterial/fungal activity of PEI-based antipathogenic sprays was investigated by microtiter and disc diffusion assays, in addition to bacterial viability and adhesion % of common bacteria and fungi on the PEI-treated masks. Furthermore, the antiviral effect of the PEI-based solutions was determined against SARS-CoV-2 virus. Results: The biosafe concentration of PEI was determined as 1 μg/mL with 75 ± 11% cell viability, but B-PEI and its protonated forms had great biocompatibility even at 1000 μg/mL with more than 85% viability. The antibacterial/fungal effect of non-toxic B-PEI was improved by protonation with BA and HCl with 2.5-10 mg/mL minimum bactericidal/fungicidal concentrations (MBCs/MFCs). Bacterial/fungal viability and adhesion on the mask was almost eliminated by using 50 μL with 5-10 mg/mL of B-PEI-BA. Both protonated bare and betainized PEI show potent antiviral activity against SARS-CoV-2 virus. Conclusions: The toxicity of PEI was overcome by using betainized forms of PEI (B-PEI). Furthermore, the antimicrobial and antiviral efficacy of PEI and B-PEI was improved by protonation with CA, BA, and HCl of amine groups on B-PEI. B-PEI-BA spray solution has great potential as an antipathogenic spray with broad-spectrum antimicrobial potency against harmful bacteria, fungi, and viruses without any toxicity.

Identification and Assessment of Secondary Metabolites from Three Fungal Endophytes of Solanum mauritianum Against Public Health Pathogens

Fungal endophytes, symbiotic microorganisms residing within plants, are renowned for producing bioactive secondary metabolites with diverse beneficial properties. We investigated the antimicrobial potential of fungal endophytes isolated from Solanum mauritianum, an invasive weed, against clinically significant bacterial pathogens. Selected fungal endophytes (Penicillium chrysogenum, Fusarium sp., and Paracamarosporium leucadendri) were isolated from the plant’s leaves and fruits. Their crude extracts were tested against various referenced strains, such as Mycobacterium species (M. smegmatis ATCC 607 and M. bovis ATCC 27290), Staphylococcus aureus ATCC 6571, Bacillus subtilis ATCC 11774, Klebsiella species (K. pneumoniae ATCC 10031 and K. oxytoca ATCC 8724), Escherichia coli ATCC 10536, and Pseudomonas aeruginosa ATCC 10145, using the Kirby-Bauer disk diffusion method. Resazurin Microtiter Assay was used for the determination of the minimum inhibitory concentration. The chemical nature of the secondary metabolites in the crude extracts produced by fungal endophytes was evaluated using high-resolution liquid chromatography–mass spectrometry (LC-MS) using water and acetonitrile gradient. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS/MS) was employed for untargeted metabolomics. LC-QTOF-MS/MS identified 63 bioactive compounds across the three endophytes. P. chrysogenum had the highest activity against S. aureus and M. smegmatis (1.15 mg/mL and 0.02 mg/mL, respectively), while P. leucadendri demonstrated moderate activity against M. smegmatis (2.91 mg/mL) and E. coli (1.16 mg/mL). Fusarium sp. exhibited the broadest spectrum of antibacterial activity, with MIC values ranging from 0.03 mg/mL (B. subtilis) to 10 mg/mL (M. smegmatis). P. leucadendri produced 29 metabolites, Fusarium sp. had 23 identified metabolites, and a total of 11 metabolites were identified from P. chrysogenum. The fruits of the plant, accounting for 60%, appeared to be the most abundant in the endophyte diversity when compared to the stems and leaves. This study highlights the potential of fungal endophytes from S. mauritianum as a source of novel bioactive compounds, particularly against multidrug-resistant pathogens, contributing to the ongoing efforts to combat antimicrobial resistance.

An alternative approach in the synthesis of strontium-hydroxyapatite and strontium hydroxyapatite embedded in graphitic carbon nitride nanocomposites for potential tissue engineering applications

Sr-hydroxyapatite has drawn much attention with promising applications in biomedical engineering especially in drug delivery and tissue repair. A novel protocol for the preparation of Strontium hydroxyapatite (Sr-HAP) and Strontium hydroxyapatite encapsulated by graphitic carbon nitride (Sr-HAP@gCN) with high purity and better homogeneity was adopted for potential use in orthopaedic surgeries. The physiological pathway of Strontium and calcium inside humans follow the same trend leading to the mineral deposition of the bone. In the present study, a novel attempt in synthesizing Sr-HAP and Sr-HAP@gCN by the hydrothermal method is proposed for obtaining an ideal biomaterial with potential biocompatibility and pharmacological activity. Phase identification by X-ray diffraction (XRD), Fourier transform Infra-Red spectroscopic analysis (FTIR), Electron & atomic force microscopic studies, Thermogravimetric and Raman spectroscopic analysis was performed on the prepared Sr-HAP and Sr-HAP@gCN to characterize the incorporation efficiency of Strontium. Resazurin microtiter assay and MTT assay were used to evaluate antimicrobial susceptibility and biocompatibility. The typical crystal phase of Sr-HAP and a phase change in Sr-HAP@gCN have been demonstrated with special reference to the interfacial connection between gCN and Sr-HAP through FT-IR studies. Raman spectroscopy revealed the archetypal signature of symmetric and asymmetric PO vibrations pertaining to linearity change with the addition of Strontium. The biological effect of Sr-HAP and Sr-HAP@gCN was quite significant in inducing mineralization when kept immersed in simulated body fluid (SBF) for 21 days. Promising results pertaining to antimicrobial susceptibility and biocompatibility have been proved as an encouraging choice for drug delivery applications and bone filling applications.

Antibacterial and cytotoxicity properties of a polyherbal mouthwash containing Achyranthes aspera and Trachyspermum ammi against selected periodontal pathogens

BackgroundChlorhexidine (CHX) is considered as a gold standard for its antibacterial efficacy and substantivity in chemical plaque control. However, some adverse effects are associated with its prolonged use. Herbal medicines like Achyranthes aspera and Trachyspermum ammi have been used in many clinical conditions, and they appear to be a valuable substitute next to CHX in the management of periodontal diseases. ObjectiveThis in vitro study was designed to assess and compare the antibacterial potential and cytotoxic effects of novel polyherbal mouthwash containing A. aspera and T. ammi with 0.2% CHX mouthwash against Porphyromonas gingivalis, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans. MethodsEthanolic extracts of A. aspera and T. ammi were prepared by the Soxhlet apparatus method and were subjected to preliminary phytochemical screening. The individual plant extracts and the plant extract mixture (PEM) of A. aspera and T. ammi in the ratio of 1:1, 2:1, 1:2 (w/v) were assessed for minimum inhibitory concentration (resazurin microtitre assay) and minimum bactericidal concentration (spread plating method) against selected periodontal pathogens in comparison to CHX. The polyherbal mouthwash was assessed for zone of inhibition (well diffusion method) and cytotoxicity (MTT assay) on adult human gingival fibroblasts. All the experiments were performed in triplicate. ResultsThe antibacterial activity was evident in the PEMs, and polyherbal mouthwash against tested periodontal pathogens and was comparable to CHX. The cytotoxicity assay findings confirmed that polyherbal mouthwash exhibited 82.1% of surviving cells which proved good biocompatibility. ConclusionA. aspera and T. ammi based mouthwash possess comparable antibacterial activity against periodontal pathogens when compared to CHX.

FTY720 Reduces the Biomass of Biofilms in Pseudomonas aeruginosa in a Dose-Dependent Manner.

Pseudomonas aeruginosa, a nosocomial pathogen, has strong biofilm capabilities, representing the main source of infection in the human body. Repurposing existing drugs has been explored as an alternative strategy to combat emerging antibiotic-resistant pathogens. Fingolimod hydrochloride (FTY720), an immunomodulatory drug for multiple sclerosis, has shown promising antimicrobial effects against some ESKAPE pathogens. Therefore, the effects of FTY720 on the biofilm capabilities of Pseudomonas aeruginosa were investigated in this study. It was determined that FTY720 inhibited the growth of P. aeruginosa PAO1 at 100 µM. The significant reduction in PAO1 cell viability was observed to be dose-dependent. Additional cytotoxicity analysis on human cell lines showed that FTY720 significantly reduced viabilities at sub-inhibitory concentrations of 25-50 µM. Microtiter assays and confocal analysis confirmed reductions in biofilm mass and thickness and the cell survivability ratio in the presence of FTY720. Similarly, virulence production and biofilm-related gene expression (rhlA, rhlB, pilA, pilI, fliC, fliD and algR) were determined. The results demonstrate that pigment production was affected and quantitative real-time PCR analysis showed a variable degree of reduced gene expression in response to FTY720 at 12.5-50 µM. These findings suggest that FTY720 could be repurposed as an alternative antibiofilm agent against Pseudomonas aeruginosa.

Enhanced Anti-Bacterial Activity of Arachidonic Acid against the Cariogenic Bacterium Streptococcus mutans in Combination with Triclosan and Fluoride.

Dental caries is a global health problem that requires better prevention measures. One of the goals is to reduce the prevalence of the cariogenic Gram-positive bacterium Streptococcus mutans. We have recently shown that naturally occurring arachidonic acid (AA) has both anti-bacterial and anti-biofilm activities against this bacterium. An important question is how these activities are affected by other anti-bacterial compounds commonly used in mouthwashes. Here, we studied the combined treatment of AA with chlorhexidine (CHX), cetylpyridinium chloride (CPC), triclosan, and fluoride. Checkerboard microtiter assays were performed to determine the effects on bacterial growth and viability. Biofilms were quantified using the MTT metabolic assay, crystal violet (CV) staining, and live/dead staining with SYTO 9/propidium iodide (PI) visualized by spinning disk confocal microscopy (SDCM). The bacterial morphology and the topography of the biofilms were visualized by high-resolution scanning electron microscopy (HR-SEM). The effect of selected drug combinations on cell viability and membrane potential was investigated by flow cytometry using SYTO 9/PI staining and the potentiometric dye DiOC2(3), respectively. We found that CHX and CPC had an antagonistic effect on AA at certain concentrations, while an additive effect was observed with triclosan and fluoride. This prompted us to investigate the triple treatment of AA, triclosan, and fluoride, which was more effective than either compound alone or the double treatment. We observed an increase in the percentage of PI-positive bacteria, indicating increased bacterial cell death. Only AA caused significant membrane hyperpolarization, which was not significantly enhanced by either triclosan or fluoride. In conclusion, our data suggest that AA can be used together with triclosan and fluoride to improve the efficacy of oral health care.

Identification of novel resistance-associated mutations and discrimination within whole-genome sequences of fluoroquinolone-resistant Mycobacterium tuberculosis isolates.

This study aims to elucidate additional mutation loci associated with fluoroquinolone (FQ) resistance and evaluate the discriminatory capacity of mutation loci and allele mutation frequencies in identifying FQ-resistant Mycobacterium tuberculosis (MTB) isolates. A random selection of isolates was extracted from an ongoing collection. Drug resistance was determined using the resazurin microtiter assay (REMA) as the gold standard. Mutation loci and the burden of mutations in the quinolone resistance-determining region (QRDR) were elucidated through whole-genome sequencing (WGS). Novel amino acid mutations, namely, G520D and G520T, were identified in the gyrB and associated with FQ resistance. In the context of distinguishing FQ-resistant isolates, the AUC for the QRDR mutation frequency burden (0.969) surpassed that of the mutation locus (0.929), and this difference was statistically significant (P = 0.03). Furthermore, using the resistance mutation locus as a reference, setting the QRDR mutation frequency burden threshold at 1.31% resulted in a 3.60% increase in the accuracy of classifying FQ-resistant isolates (NRI = 3.60%, P < 0.001). The QRDR mutation frequency burden appears to offer superior diagnostic efficacy in discriminating FQ-resistant isolates compared to qualitative detection of mutant loci.IMPORTANCEFluoroquinolone (FQ) drugs are recommended as second-line drugs for the treatment of multidrug-resistant tuberculosis. With the massive use of FQ drugs in the clinical treatment of tuberculosis (TB), there is an increasing rate of drug resistance to FQ drugs. In this study, we identified and demonstrated novel amino acid mutations associated with FQ resistance in Mycobacterium tuberculosis (MTB), and we quantified the mutation sites and identified the quinolone resistance-determining region (QRDR) mutation frequency burden as a novel diagnostic method for FQ resistance. We hope that the results of this study will provide data support and a theoretical basis for the rapid diagnosis of FQ-resistant MTB.

Oleanolic acid: an antimycobacterial component of Syzygium aromaticum L. and inhibitor of efflux mediated drug resistance

Oleanolic acid (OA) was isolated from Syzygium aromaticum L. buds, and structurally characterised using different spectroscopic techniques; MS, IR,1H/13C-NMR and 2D NMR experiments. The antimycobacterial activity according to a resazurin microtiter assay (REMA) showed important inhibitory effect of OA on the virulent H37Rv strain, with the lowest minimum concentration of 50 µg/mL, compared to other fractions. Molecular docking of OA with BacA drug efflux pump resulted in good binding affinity of hydrophobic interaction type. Therefore, OA could contribute to the antimycobacterial action of clove buds, and has potential as an efflux pump inhibitor. Further studies are required on its use to combat multidrug resistant strains.

Exploring nature's antidote: unveiling the inhibitory potential of selected medicinal plants from Kisumu, Kenya against venom from some snakes of medical significance in sub-Saharan Africa.

Background: The present study investigated the efficacy of Conyza bonariensis, Commiphora africana, Senna obtusifolia, Warburgia ugandensis, Vernonia glabra, and Zanthoxylum usambarense against Bitis arietans venom (BAV), Naja ashei venom (NAV), and Naja subfulva venom (NSV). Methods: 40 extracts and fractions were prepared using n-hexane, dichloromethane, ethyl acetate, and methanol. In vitro efficacy against snake venom phospholipase A2 (svPLA2) was determined in 96-well microtiter and agarose-egg yolk coagulation assays. in vivo efficacy against venom-induced cytotoxicity was determined using Artemia salina. Two commercial antivenoms were used for comparison. Results: The 96-well microtiter assay revealed poor svPLA2 inhibition of BAV by antivenom (range: 20.76% ± 13.29% to 51.29% ± 3.26%) but strong inhibition (>90%) by dichloromethane and hexane fractions of C. africana, hexane and ethyl acetate extracts and fraction of W. ugandensis, dichloromethane fraction of V. glabra, and the methanol extract of S. obtusifolia. The methanol extract and fraction of C. africana, and the hexane extract of Z. usambarense strongly inhibited (>90%) svPLA2 activity in NAV. The hexane and ethyl acetate fractions of V. glabra and the dichloromethane, ethyl acetate, and methanol extracts of C. africana strongly inhibited (>90%) svPLA2 in NSV. The agarose egg yolk coagulation assay showed significant inhibition of BAV by the dichloromethane fraction of C. africana (EC50 = 3.51 ± 2.58μg/mL), significant inhibition of NAV by the methanol fraction of C. africana (EC50 = 7.35 ± 1.800μg/mL), and significant inhibition of NSV by the hexane extract of V. glabra (EC50 = 7.94 ± 1.50μg/mL). All antivenoms were non-cytotoxic in A. salina but the methanol extract of C. africana and the hexane extracts of V. glabra and Z. usambarense were cytotoxic. The dichloromethane fraction of C. africana significantly neutralized BAV-induced cytotoxicity, the methanol fraction and extract of C. africana neutralized NAV-induced cytotoxicity, while the ethyl acetate extract of V. glabra significantly neutralized NSV-induced cytotoxicity. Glycosides, flavonoids, phenolics, and tannins were identified in the non-cytotoxic extracts/fractions. Conclusion: These findings validate the local use of C. africana and V. glabra in snakebite but not C. bonariensis, S. obtusifolia, W. ugandensis, and Z. usambarense. Further work is needed to isolate pure compounds from the effective plants and identify their mechanisms of action.

Preliminary evaluation of in-Vivo and in-Vitro antifungal activity of Piper longum, Origanum majorana, Embelia ribes and Butea monosperma with Gas chromatography-mass spectrometry analysis of phytochemical property of plant extracts against Candida species causing dermatological

: Globally, fungi infections cause more than 1.5 million fatalities annually, mostly in those with weakened immune systems with Candida albicans found to be the chief pathogen. Approximately one to two billion people are affected by dermatological fungal infections involving skin and skin appendages, perhaps very difficult to estimate its exact prevalence due to under-reporting, this augments the requirements for more attention towards new molecules to combat resistance.: Evaluation of antifungal activity of Piper longum, Origanum majorana, Embelia ribes, Butea monosperma both in-vivo and in-vitro analysis against Candida species. This experimental study conducted for evaluating both in-vitro as well as in-vivo anti-fungal activity of Piper longum, Butea monosperma, Embelia ribes and Origanum majorana, and evaluation of phytoconstituent through Gas Chromatography and Mass Spectrometry analysis by GC-MS-QP-2010 plus system to identify the active bio-component. Fluconazole used as the standard control in-vivo and Itraconazole used as a standard drug for in-vitro infection and analysis. Ethical Committee approval was taken (IMS, BHU Dean/2018/CAEC/818). The antifungal activity of the extracts in-vitro was evaluated by Resazurin microtiter assay against Candida sp. Micro broth dilution methods. Rats were given an injection of 0.2 mL (intravenously/i.v) of a 10° UFC/mL inoculum made from a fresh 48-hour Candida albicans culture in sterile saline to cause infection. Induction of candidemia in rats was confirmed using qualitative Gram-stained smear. : The extracts of Butea monosperma (Alc.), Piper longum (Alc.), Origanum majorana (Aq.), and Piper longum (Aq.) showed effective antifungal activity against Candida sp. in-vitro research with a minimum inhibitory concentration (MIC) of less than 0.25 mg/mL. : This study is contributing towards the search for a natural drug candidate to combat resistance caused by the Candida genus, as it is one of the highest contributors to fungal dermatological/skin infections.

19 Schiff bases as antimycobacterial agents: synthesis, molecular docking and a plausible mechanism of action.

Aim: To discover novel anti-Mycobacterium tuberculosis (Mtb) drugs, 19 compounds were synthesized; their anti-Mtb effects were evaluated and mechanisms of action were preliminarily explored. Materials & methods: The compounds were synthesized and their anti-Mtb activity was elucidated using resazurin microtiter assays. The plausible target of the potential compound was investigated by microimaging techniques, gas chromatography-mass spectrometry analysis and molecular docking. Results: 19compounds inhibited Mtb growth with minimum inhibitory concentrationsranging from 1 to 32μg/ml. Compounds 1-17 showed inhibition of Mtb KatG enzyme. Compound 19, the most potent, might be an inhibitor of Pks13 polyketide synthase. Conclusion: This study suggests that 2-((6-fluoropyridin-3-yl)methylene) hydrazine-1-carbothioamide (19) is a potential anti-Mtb lead compound with a novel mechanism of action.

Synthesis, Antioxidant and Antituberculosis Evaluation of Eugenol Derivatives from Mannich Condensation

AbstractMannich bases are interesting class of molecules thanks to their various pharmacological properties. This paper presents the synthesis and characterization of eugenol derivatives following Mannich condensation. The in vitro antituberculosis activity of the synthesized derivatives was evaluated by the Resazurin microtiter assay (REMA). The 2,2′‐diphenyl‐1‐picrylhydrazyl (DPPH) free radical scavenging and ferrous cation (FeCl2) chelating tests were used to estimate the antioxidant capacity. All eugenol Mannich bases inhibited the Mycobacterium tuberculosis (Mtb) H37Rv growing. Those of 2‐dimethylaminomethyl‐6‐methoxy‐4‐propenylphenol (1) and 2‐methoxy‐4‐propenyl‐6‐pyrrolidinylmethylphenol (4) were the most active at 10 μg/mL. Additionally, significant antioxidant profile was exhibited by the synthesized derivatives, especially for 2‐dicyclohexylaminomethyl‐6‐methoxy‐4‐propenylphenol (5) that showed an antiradical activity of EC50=20 μg/mL, twice as strong as that of eugenol precursor, and for 2‐methoxy‐6‐methylpiperazinylmethyl‐4‐propenylphenol (3) and compound 4, which were promising iron chelators with EC50 of 0.016 and 0.018 mg/mL, respectively, better than EDTA standard. The molecular docking experiments on two different antituberculosis receptors showed excellent binding affinity of compounds 4 and 5 into the active site of pantothenate kinase (PanK, type I), suggesting PanK as potential molecular target. These encouraging findings showed the importance of Mannich reaction for the development of antitubercular candidates and the improvement of natural products bioactivity.

Insights into the in-vitro Susceptibility and Drug-Drug Interaction Profiles Against Drug-Resistant and Susceptible Mycobacterium tuberculosis Clinical Isolates in Amhara, Ethiopia.

In Ethiopia, tuberculosis (TB) is a major public health problem. The aim of the study was to determine the in vitro susceptibility level of drugs and drug interaction profiles against drug-resistant and susceptible M. tuberculosis clinical isolates. A laboratory-based cross-sectional study was conducted between January 2023 and August 2023. GenoType MTBDRplus v.2.0 was facilitated in genetic mutation detection. Minimum inhibitory concentration (MIC) was determined using resazurin microtitre assay (REMA), while fractional inhibitory concentration index (FICI) using resazurin drug combination microtitre assay (REDCA) for in vitro quantitative susceptibility and drug interaction prediction. Among 32 clinical isolates, a total of 14 (43.8%) RIF, 20 (62.5%) INH, 2 (6.3%) EMB-related resistant and 14 (43.8%) MDR isolates were identified. Five of RIF-resistant isolates (55.6%) carrying rpoB common mutations at codon S450L were associated with high levels of RIF-resistance with MICs of ≥ 2μg/mL, whereas 100% of isolates harboring rpoB substitutions at codons D435V and H445Y were linked with moderate or low-level RIF-resistance in the MIC ranges from 0.5 to 1μg/mL. A proportion of 81.8% of isolates harboring katG S315T mutations were associated with high-level INH resistance (MIC ≥ 1μg/mL), while the 18.2% of isolates with S315T katG mutations and 100% of isolates with inhA C-15T mutations were linked to the low-level of INH resistance with MIC variability from 0.25 to 0.5μg/mL. Our results indicated that most FICIs of the dual drugs INH+RIF and INH+LEV combination for 9 (28.1%) and 4 (12.5%) INH-resistant isolates, respectively, were ≤0.5, whereas triple drugs INH+RIF+EMB, INH+RIF+LEV and INH+EMB+LEV combination for 6 (18.8%), 11 (34.4%) and 8 (25%) INH-resistant isolates were from 0.62 to 0.75, all showed synergistic effect. The study highlights that isolates with rpoB S450L and katG S315T substitutions were associated with high level of RIF and INH resistance. It is concluded that REDCA can quantitatively determine anti-mycobacterial synergy and that LEV being of potential use against INH-resistant isolates including MDR-TB when combined with RIF+INH and INH+EMB.

&lt;i&gt;Nocardiopsis synnemataformans&lt;/i&gt; NBRM9, an extremophilic actinomycete producing extremozyme cellulase, using lignocellulosic agro-wastes and its biotechnological applications

&lt;abstract&gt; &lt;p&gt;Actinomycetes are an attractive source of lignocellulose-degrading enzymes. The search for actinomycetes producing extremozyme cellulase using cheap lignocellulosic waste remains a priority goal of enzyme research. In this context, the extremophilic actinomycete NBRM9 showed promising cellulolytic activity in solid and liquid assays. This actinomycete was identified as &lt;italic&gt;Nocardiopsis synnemataformans&lt;/italic&gt; based on its phenotypic characteristics alongside phylogenetic analyses of 16S rRNA gene sequencing (OQ380604.1). Using bean straw as the best agro-waste, the production of cellulase from this strain was statistically optimized using a response surface methodology, with the maximum activity (13.20 U/mL) achieved at an incubation temperature of 40 °C, a pH of 9, an incubation time of 7 days, and a 2% substrate concentration. The partially purified cellulase (PPC) showed promising activity and stability over a wide range of temperatures (20–90 °C), pH values (3–11), and NaCl concentrations (1–19%), with optimal activity at 50 °C, pH 9.0, and 10% salinity. Under these conditions, the enzyme retained &amp;gt;95% of its activity, thus indicating its extremozyme nature. The kinetics of cellulase showed that it has a V&lt;sub&gt;max&lt;/sub&gt; of 20.19 ± 1.88 U/mL and a Km of 0.25 ± 0.07 mM. The immobilized PPC had a relative activity of 69.58 ± 0.13%. In the in vitro microtiter assay, the PPC was found to have a concentration-dependent anti-biofilm activity (up to 85.15 ± 1.60%). Additionally, the fermentative conversion of the hydrolyzed bean straw by &lt;italic&gt;Saccharomyces cerevisiae&lt;/italic&gt; (KM504287.1) amounted to 65.80 ± 0.52% of the theoretical ethanol yield. Overall, for the first time, the present work reports the production of extremozymatic (thermo, alkali-, and halo-stable) cellulase from &lt;italic&gt;N. synnemataformans&lt;/italic&gt; NBRM9. Therefore, this strain is recommended for use as a biotool in many lignocellulosic-based applications operating under harsh conditions.&lt;/p&gt; &lt;/abstract&gt;

Antitubercular evaluation of dihydropyridine-triazole conjugates: design, synthesis, in vitro screening, SAR and in silico ADME predictions.

This study investigates the potential of click chemistry for the development of novel anti-tuberculosis agents. A targeted library of 1,4-dihydropyridine-1,2,3-triazole conjugates was synthesized and evaluated for their in vitro activity against Mycobacterium tuberculosis H37Ra using the resazurin microtiter assay (REMA). Among the synthesized derivatives, compounds J10, J11, J14, J22 and J23 demonstrated significant antimycobacterial activity. These compounds exhibited low MIC values ranging from 6.24 to 6.64 μg mL-1, highlighting their promising potential as lead compounds for further developing novel tuberculosis therapeutics. In addition to the promising in vitro activity, structure-activity relationship (SAR) analysis revealed that electron-withdrawing groups on the aryl-substituted ring of the dihydropyridines (J10-J24), a triazole with an unsubstituted aryl ring or with electron-donating groups (methyl or methoxy), and a geminal dimethyl group are essential structural features for the observed antitubercular activity. Furthermore, in silico ADME (absorption, distribution, metabolism, and excretion) parameters and pharmacokinetic studies supported the potential of these conjugates for oral bioavailability. These findings collectively highlight the 1,4-dihydropyridine-1,2,3-triazole scaffold as a promising platform for developing novel orally active anti-tuberculosis drugs.

Investigating genomic diversity of Staphylococcus aureus associated with pediatric atopic dermatitis in South Africa.

Staphylococcus aureus frequently colonizes the skin and nose of patients with atopic dermatitis (AD), a disease associated with skin barrier dysfunction and chronic cutaneous inflammation. Published genomic studies on AD-associated S. aureus in pediatric populations in sub-Saharan Africa are limited. To investigate the phenotypic and genomic diversity of S. aureus in children with and without AD during early childhood. A cross-sectional study of 220 children (aged 9-38 months) with AD (cases) and without AD (controls) from Cape Town and Umtata, South Africa. S. aureus phenotypic and genomic diversity were investigated using whole-genome sequencing, antibiotic susceptibility testing and biofilm microtiter assay. Of the 124 S. aureus isolates recovered from 220 children, 96 isolates (79 cases and 17 controls) with high-quality sequences were analyzed. Isolates from cases showed greater phenotypic resistance to gentamicin (10%), rifampicin (4%), chloramphenicol (4%), and exhibited multidrug resistance (9%) than in controls. Furthermore, the isolates from cases formed stronger biofilms than those from controls (76% vs. 35%, p = 0.001), but showed no dominance of any virulence factor gene or mobile genetic elements. There was no significant difference in the distribution of immune evasion cluster types between cases and controls. However, IEC type G was identified only among cases. AD-associated S. aureus has phenotypic and genetic features that are important for successful pathogenic colonization and survival. Further studies are needed to assess the pathological implications of colonization of various S. aureus lineages in vivo to elucidate their pathological contribution to AD pathogenesis and pathophysiology.

Biofilms: A developmental niche for vancomycin-intermediate Staphylococcus aureus

Staphylococcus aureus are gram-positive bacteria responsible for a wide array of diseases, ranging from skin and soft tissue infections to more chronic illnesses such as toxic shock syndrome, osteomyelitis, and endocarditis. Vancomycin is currently one of the most effective antibiotics available in treating patients infected with methicillin-resistant S. aureus (MRSA), however the emergence of vancomycin-resistant S. aureus (VRSA), and more commonly vancomycin-intermediate S. aureus (VISA), threaten the future efficacy of vancomycin. Intermediate resistance to vancomycin occurs due to mutations within the loci of Staphylococcal genes involved in cell wall formation such as rpoB, graS, and yycG. We hypothesized the VISA phenotype may also arise as a result of the natural stress occurring within S. aureus biofilms, and that this phenomenon is mediated by the RecA/SOS response. Wildtype and recA null mutant/lexAG94E strains of S. aureus biofilms were established in biofilm microtiter assays or planktonic cultures with or without the addition of sub-inhibitory concentrations of vancomycin (0.063 mg/l - 0.25 mg/L ciprofloxacin, 0.5 mg/l vancomycin). Efficiency of plating techniques were used to quantify the subpopulation of biofilm-derived S. aureus cells that developed vancomycin-intermediate resistance. The results indicated that a greater subpopulation of cells from wildtype biofilms (4.16 × 102 CFUs) emerged from intermediate-resistant concentrations of vancomycin (4 μg/ml) compared with the planktonic counterpart (1.53 × 101 CFUs). Wildtype biofilms (4.16 × 102 CFUs) also exhibited greater resistance to intermediate-resistant concentrations of vancomycin compared with strains deficient in the recA null mutant (8.15 × 101 CFUs) and lexA genes (8.00 × 101 CFUs). While the VISA phenotype would be an unintended consequence of genetic diversity and potentially gene transfer in the biofilm setting, it demonstrates that mutations occurring within biofilms allow for S. aureus to adapt to new environments, including the presence of widely used antibiotics.

Antibiotic Resistance Genes, Virulence Factors, and Biofilm Formation in Coagulase-Negative Staphylococcus spp. Isolates from European Hakes (Merluccius merluccius, L.) Caught in the Northeast Atlantic Ocean.

The indiscriminate use of antibiotics has contributed to the dissemination of multiresistant bacteria, which represents a public health concern. The aim of this work was to characterize 27 coagulase-negative staphylococci (CoNS) isolated from eight wild Northeast Atlantic hakes (Merluccius merluccius, L.) and taxonomically identified as Staphylococcus epidermidis (n = 16), Staphylococcus saprophyticus (n = 4), Staphylococcus hominis (n = 3), Staphylococcus pasteuri (n = 2), Staphylococcus edaphicus (n = 1), and Staphylococcus capitis (n = 1). Biofilm formation was evaluated with a microtiter assay, antibiotic susceptibility testing was performed using the disk diffusion method, and antibiotic resistance and virulence determinants were detected by PCR. Our results showed that all staphylococci produced biofilms and that 92.6% of the isolates were resistant to at least one antibiotic, mainly penicillin (88.8%), fusidic acid (40.7%), and erythromycin (37%). The penicillin resistance gene (blaZ) was detected in 66.6% (18) of the isolates, of which 10 also carried resistance genes to macrolides and lincosamides (mphC, msr(A/B), lnuA, or vgaA), 4 to fusidic acid (fusB), and 3 to trimethoprim-sulfamethoxazole (dfrA). At least one virulence gene (scn, hla, SCCmecIII, and/or SCCmecV) was detected in 48% of the isolates. This study suggests that wild European hake destined for human consumption could act as a vector of CoNS carrying antibiotic resistance genes and/or virulence factors.