Phenotypic, molecular, and in silico characterization of coumarin as carbapenemase inhibitor to fight carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: Carbapenems represent the first line treatment of serious infections caused by drug-resistant Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae (CRKP) is one of the urgent threats to human health worldwide. The current study aims to evaluate the carbapenemase inhibitory potential of coumarin and to test its ability to restore meropenem activity against CRKP. Disk diffusion method was used to test the antimicrobial susceptibility of K. pneumoniae clinical isolates to various antibiotics. Carbapenemase genes (NDM-1, VIM-2, and OXA-9) were detected using PCR. The effect of sub-MIC of coumarin on CRKP isolates was performed using combined disk assay, enzyme inhibition assay, and checkerboard assay. In addition, qRT-PCR was used to estimate the coumarin effect on expression of carbapenemase genes. Molecular docking was used to confirm the interaction between coumarin and binding sites within three carbapenemases. RESULTS: K. pneumoniae clinical isolates were found to be multi-drug resistant and showed high resistance to meropenem. All bacterial isolates harbor at least one carbapenemase-encoding gene. Coumarin significantly inhibited carbapenemases in the crude periplasmic extract of CRKP. The checkerboard assay indicated that coumarin-meropenem combination was synergistic exhibiting a fractional inhibitory concentration index ≤ 0.5. In addition, qRT-PCR results revealed that coumarin significantly decreased carbapenemase-genes expression. Molecular docking revealed that the binding energies of coumarin to NDM1, VIM-2, OXA-48 and OXA-9 showed a free binding energy of -7.8757, -7.1532, -6.2064 and - 7.4331 Kcal/mol, respectively. CONCLUSION: Coumarin rendered CRKP sensitive to meropenem as evidenced by its inhibitory action on hydrolytic activity and expression of carbapenemases. The current findings suggest that coumarin could be a possible solution to overcome carbapenems resistance in CRKP.

The anti-staphylococcal fusidic acid as an efflux pump inhibitor combined with fluconazole against vaginal candidiasis in mouse model.

BACKGROUND: Candida albicans is the most common fungus that causes vaginal candidiasis in immunocompetent women and catastrophic infections in immunocompromised patients. The treatment of such infections is hindered due to the increasing emergence of resistance to azoles in C. albicans. New treatment approaches are needed to combat candidiasis especially in the dwindled supply of new effective and safe antifungals. The resistance to azoles is mainly attributed to export of azoles outside the cells by means of the efflux pump that confers cross resistance to all azoles including fluconazole (FLC). OBJECTIVES: This study aimed to investigate the possible efflux pump inhibiting activity of fusidic acid (FA) in C. albicans resistant isolates and the potential use of Fusidic acid in combination with fluconazole to potentiate the antifungal activity of fluconazole to restore its activity in the resistant C. albicans isolates. METHODS: The resistance of C. albicans isolates was assessed by determination of minimum inhibitory concentration. The effect of Fusidic acid at sub-inhibitory concentration on efflux activity was assayed by rhodamine 6G efflux assay and intracellular accumulation. Mice model studies were conducted to evaluate the anti-efflux activity of Fusidic acid and its synergistic effects in combination with fluconazole. Impact of Fusidic acid on ergosterol biosynthesis was quantified. The synergy of fluconazole when combined with Fusidic acid was investigated by determination of minimum inhibitory concentration. The cytotoxicity of Fusidic acid was tested against erythrocytes. The effect of Fusidic acid on efflux pumps was tested at the molecular level by real-time PCR and in silico study. In vivo vulvovaginitis mice model was used to confirm the activity of the combination in treating vulvovaginal candidiasis. RESULTS: Fusidic acid showed efflux inhibiting activity as it increased the accumulation of rhodamine 6G, a substrate for ABC-efflux transporter, and decreased its efflux in C. albicans cells. The antifungal activity of fluconazole was synergized when combined with Fusidic acid. Fusidic acid exerted only minimal cytotoxicity on human erythrocytes indicating its safety. The FA efflux inhibitory activity could be owed to its ability to interfere with efflux protein transporters as revealed by docking studies and downregulation of the efflux-encoding genes of both ABC transporters and MFS superfamily. Moreover, in vivo mice model showed that using fluconazole-fusidic acid combination by vaginal route enhanced fluconazole antifungal activity as shown by lowered fungal burden and a negligible histopathological change in vaginal tissue. CONCLUSION: The current findings highlight FA's potential as a potential adjuvant to FLC in the treatment of vulvovaginal candidiasis.

Antibiofilm and staphyloxanthin inhibitory potential of terbinafine against Staphylococcus aureus: in vitro and in vivo studies.

BACKGROUND: Antimicrobial resistance is growing substantially, which necessitates the search for novel therapeutic options. Terbinafine, an allylamine antifungal agent that exhibits a broad spectrum of activity and is used in the treatment of dermatophytosis, could be a possible option to disarm S. aureus virulence. METHODS: Terbinafine inhibitory effect on staphyloxanthin was characterized by quantitative measurement of staphyloxanthin intermediates and molecular docking. The effect of terbinafine on S. aureus stress survival was characterized by viable counting. The anti-biofilm activity of terbinafine on S. aureus was assessed by the crystal violet assay and microscopy. Changes in S. aureus membrane following treatment with terbinafine were determined using Fourier transform infrared (FTIR) analysis. The synergistic action of terbinafine in combination with conventional antibiotics was characterized using the checkerboard assay. qRT-PCR was used to evaluate the impact of terbinafine on S. aureus gene expression. The influence of terbinafine on S. aureus pathogenesis was investigated in mice infection model. RESULTS: Terbinafine inhibits staphyloxanthin biosynthesis through targeting dehydrosqualene desaturase (CrtN). Docking analysis of terbinafine against the predicted active site of CrtN reveals a binding energy of - 9.579 kcal/mol exemplified by the formation of H-bonds, H-arene bonds, and hydrophobic/hydrophilic interactions with the conserved amino acids of the receptor pocket. Terbinafine treated S. aureus was more susceptible to both oxidative and acid stress as well as human blood killing as compared to untreated cells. Targeting staphyloxanthin by terbinafine rendered S. aureus more sensitive to membrane acting antibiotics. Terbinafine interfered with S. aureus biofilm formation through targeting cell autoaggregation, hydrophobicity, and exopolysaccharide production. Moreover, terbinafine demonstrated a synergistic interaction against S. aureus when combined with conventional antibiotics. Importantly, terbinafine attenuated S. aureus pathogenesis using mice infection model. qRT-PCR revealed that terbinafine repressed expression of the transcriptional regulators sigB, sarA, and msaB, as well as icaA in S. aureus. CONCLUSIONS: Present findings strongly suggest that terbinafine could be used safely and efficiently as an anti-virulent agent to combat S. aureus infections.

Quantification of 16 cephalosporins in the aquatic environment by liquid chromatography-tandem mass spectrometry.

Antimicrobial agents are essential to protect human and animal health. During the coronavirus disease 2019 pandemic, antimicrobials such as cephalosporins were widely used as prophylactics and to prevent bacterial co-infection. Undoubtedly, the prevalence of antibiotics in the aquatic environment will ultimately affect the degree of resistance against these bacteria in animals and the environmental systems. In order to monitor 16 cephalosporins in the aquatic environment, we developed a new liquid chromatography-tandem mass spectrometry method that functioned simultaneously under positive and negative electrospray ionization switching modes. The chromatographic separation has been implemented using a pentafluorophenyl propyl column kept at 40°C. The limits of detection and quantitation for the studied cephalosporins ranged from (8 × 10-4 ) to (7.11 × 10-2 ) ng/ml and from (2.61 × 10-3 ) to (2.37 × 10-1 ) ng/ml, respectively. The percent extraction efficiency (apparent recovery) and relative standard deviations for the analyzed cephalosporins ranged from 61.69% to 167.67% and 2.45% to 13.48%, respectively. The overall findings showed that the effluent from the wastewater treatment plants that receive wastewater from pharmaceutical factories had a higher detected amount of cephalosporins than that of domestic sewage. Moreover, seven cephalosporins, including cefuroxime, ceftazidime, cefradine, cefprozil, cefixime, cefalexin, and cefadroxil (0.68-105.45 ng/L) were determined in the aquatic environment.

Repurposing pantoprazole and haloperidol as efflux pump inhibitors in azole resistant clinical Candida albicans and non-albicans isolates.

Candida species have a major role in nosocomial infections leading to high morbidity and mortality. Increased resistance to various antifungals, especially azoles is a significant problem. One of the main mechanisms for azole resistance is the up-regulation of efflux pump genes including CDR1 and MDR1. In the current study, clinical Candida isolates were identified to the species level and the antifungal susceptibility (AFS) of different Candida species was determined by disk diffusion method. Furthermore, the main mechanisms of azole resistance were investigated. Finally, haloperidol and pantoprazole were tested for their potential synergistic effect against fluconazole-resistant isolates. One hundred and twenty-two Candida clinical isolates were used in this study. 70 isolates were Candida albicans (57.4%), the non-albicans Candida species include: C. krusei (20.5%), C. tropicalis (6.6%), C. parapsilosis (5.7%), C. dubliniensis (4.9%) and C. glabrata (4.9%). The AFS testing showed that resistance to fluconazole and voriconazole were 13.1% (n = 16) and 9.8% (n = 12), respectively. Among the 16 resistant isolates, eight isolates (50%) were strong biofilm producers, seven (43.8 %) formed intermediate biofilm and one had no biofilm. All resistant strains overexpressed efflux pumps. Using RT-PCR, the efflux genes CDR1, MDR1 and ABC2 were over-expressed in azole resistant isolates. Haloperidol-fluconazole and pantoprazole-fluconazole combinations reduced the MIC of fluconazole in resistant isolates. The current study showed an increase in azole resistance of Candida species. The majority of resistant isolates form biofilm, and overexpress efflux pumps. Pantoprazole and Haloperidol showed a noteworthy effect as efflux pump inhibitors which oppose the fluconazole resistance in different Candida species.

In vivo studies of the effect of PPQ-6, a quinoline-based agent against Schistosoma mansoni in mice.

Schistosomiasis is still a public health problem. Praziquantel is the only drug available for treatment of all forms of human schistosomiasis. Although praziquantel is an effective drug against all species of human schistosomes, concerns about resistance have been raised, especially in endemic areas. A hybrid compound containing several pharmacophore within a single molecule is a promising strategy. Here, we described the anti-schistosomal effect of 4-(2-Chloroquinolin-3-yl)-2-oxo-6-(p-tolyl)-1,2-dihydropyridine-3-carbonitrile (PPQ-6), a hybrid drug based on quinoline and pyridine. PPQ-6 was given as two regimens (20 or 40 mg/kg). In both regimens, PPQ-6 significantly reduced liver and spleen indices, nitric oxide production, tissue egg load, hepatic granuloma size and count, immature eggs and total worm burden especially females. Our findings suggested that PPQ-6 is a promising anti-schistosomal agent; however more research is needed to elucidate its mechanism of action and report its activity on juvenile schistosomes and other species of human schistosomes.

Biological evaluation of newly synthesized quinoline-based compound PPQ-8 in acute and chronic toxoplasmosis: An experimental study.

Toxoplasma gondii is a widely distributed protozoan parasite, which affects worm-blooded animals including human. The commonest chemotherapeutics used for treatment of symptomatic toxoplasmosis have numerous adverse effects. Thus there is an eminent need to develop new therapeutic agents. Here we described the therapeutic efficacy of 4-(2-chloroquinolin-3-yl)-6-(2,5-dimethoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (PPQ-8); a quinoline-related compound in a mouse model of acute and chronic toxoplasmosis. In acute infection, PPQ-8 decreased the parasite load in liver and spleen with amelioration of the hepatic and splenic pathology. In addition, recovered tachyzoites showed distorted shapes, reduced sizes, irregularities, surface protrusions, erosions and peeling besides apical region distortion when seen by scanning electron microscopy. In chronic toxoplasmosis, PPQ-8 produced degeneration and reduction of the brain cysts without stimulating a damaging inflammatory response within the brain. In both models acute and chronic, PPQ-8 prolonged the survival time of mice. These findings hold promise for the development of a novel anti-toxoplasmosis drug using PPQ-8, but further in vivo studies should be carried out to elucidate PPQ-8 mechanism of action and to report its efficacy in combination with other anti-toxoplasmosis agents.

Unlocking the therapeutic potential of Nigella sativa extract: phytochemical analysis and revealing antimicrobial and antioxidant marvels.

The growing global threat of antimicrobial resistance endangers both human and animal life, necessitating the urgent discovery of novel antimicrobial solutions. Medicinal plants hold promise as sources of potential antimicrobial compounds. In this study, we investigated the phytochemical constituents and microbicidal capabilities of the ethanolic extract from Nigella sativa (black seed). Gas chromatography analysis (GC) identified 11 compounds, among them thymoquinone, and thymol, contributing to antimicrobial and antioxidant properties. Antimicrobial assays demonstrated notable inhibition zones against broad spectra of bacteria, including Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Staphylococcus aureus, Enterobacter, and Bacillus subtilis, along with potent antifungal activity against Aspergillus niger, Penicillium, and Candida albicans. Notably, when combined with antibiotics, the extract displayed exceptional synergistic antimicrobial efficacy. The black seed extract demonstrated membrane-damaging activity and disrupted virulence factors that protect microbes from antimicrobial agents, including the formation of bacterial biofilm and protease secretion. Thymoquinone, the primary active constituent of the extract, exhibited similar antimicrobial and ant virulence properties. In silico analysis targeting key regulators of quorum sensing and biofilm formation in P. aeruginosa, such as RhlG, LasR, and PqsR, showed a remarkable affinity of thymol and thymoquinone for these targets. Moreover, the N. sativa extract exhibited dose-dependent cytotoxicity against both the promastigote and amastigote forms of Leishmania tropica parasites, hinting at potential antiparasitic activity. In addition to its antimicrobial properties, the extract displayed potential antioxidant activity at a concentration of 400 µg/mL.

Cilostazol is a promising anti-pseudomonal virulence drug by disruption of quorum sensing.

Resistance to antibiotics is a critical growing public health problem that desires urgent action to combat. To avoid the stress on bacterial growth that evokes the resistance development, anti-virulence agents can be an attractive strategy as they do not target bacterial growth. Quorum sensing (QS) systems play main roles in controlling the production of diverse virulence factors and biofilm formation in bacteria. Thus, interfering with QS systems could result in mitigation of the bacterial virulence. Cilostazol is an antiplatelet and a vasodilator FDA approved drug. This study aimed to evaluate the anti-virulence activities of cilostazol in the light of its possible interference with QS systems in Pseudomonas aeruginosa. Additionally, the study examines cilostazol's impact on the bacterium's ability to induce infection in vivo, using sub-inhibitory concentrations to minimize the risk of resistance development. In this context, the biofilm formation, the production of virulence factors and influence on the in vivo ability to induce infection were assessed in the presence of cilostazol at sub-inhibitory concentration. Furthermore, the outcome of combination with antibiotics was evaluated. Cilostazol interfered with biofilm formation in P. aeruginosa. Moreover, swarming motility, biofilm formation and production of virulence factors were significantly diminished. Histopathological investigation revealed that liver, spleen and kidney tissues damage was abolished in mice injected with cilostazol-treated bacteria. Cilostazol exhibited a synergistic outcome when used in combination with antibiotics. At the molecular level, cilostazol downregulated the QS genes and showed considerable affinity to QS receptors. In conclusion, Cilostazol could be used as adjunct therapy with antibiotics for treating Pseudomonal infections. This research highlights cilostazol's potential to combat bacterial infections by targeting virulence mechanisms, reducing the risk of antibiotic resistance, and enhancing treatment efficacy against P. aeruginosa. These findings open avenues for repurposing existing drugs, offering new, safer, and more effective infection control strategies.

Redirecting pantoprazole as a metallo-beta-lactamase inhibitor in carbapenem-resistant Klebsiella pneumoniae.

The development of resistance to carbapenems in Klebsiella pneumoniae due to the production of metallo-ß-lactamases (MBLs) is a critical public health problem because carbapenems are the last-resort drugs used for treating severe infections of extended-spectrum ß-lactamases (ESBLs) producing K. pneumoniae. Restoring the activity of carbapenems by the inhibition of metallo-ß-lactamases is a valuable approach to combat carbapenem resistance. In this study, two well-characterized clinical multidrug and carbapenem-resistant K. pneumoniae isolates were used. The sub-inhibitory concentrations of pantoprazole and the well-reported metallo-ß-lactamase inhibitor captopril inhibited the hydrolytic activities of metallo-ß-lactamases, with pantoprazole having more inhibiting activities. Both drugs, when used in combination with meropenem, exhibited synergistic activities. Pantoprazole could also downregulate the expression of the metallo-ß-lactamase genes bla NDM and bla VIM. A docking study revealed that pantoprazole could bind to and chelate zinc ions of New Delhi and Verona integron-encoded MBL (VIM) enzymes with higher affinity than the control drug captopril and with comparable affinity to the natural ligand meropenem, indicating the significant inhibitory activity of pantoprazole against metallo-ß-lactamases. In conclusion, pantoprazole can be used in combination with meropenem as a new strategy for treating serious infections caused by metallo-ß-lactamases producing K. pneumoniae.

Effect of a novel benzimidazole derivative in experimental Schistosoma mansoni infection.

Currently, praziquantel is the only drug of choice for treatment of schistosomiasis. Reports of praziquantel resistance raise concerns about future control of the disease. Therefore, the search for new schistosomicidal drugs is eminent. In this study, the effect of a novel benzimidazole-derived compound (compound BTP-Iso) was assessed in mice harboring adult Schistosoma mansoni (Egyptian strain). Mice were treated 42 days p.i. with compound BTP-Iso using two treatment regimens (200 or 300 mg/kg). In both regimens, there were significant reductions in the number of recovered S. mansoni worms especially females and in immature ova, in addition to a significant reduction in the number and size of hepatic granulomata. A dose of 300 mg/kg resulted in a significant decrease in intestinal and hepatic tissue egg loads. Effect on schistosomes was confirmed by scanning electron microscopy, where adult worms recovered from mice treated with 200 mg/kg of compound BTP-Iso revealed tegumental alternations, characterised by swelling of tegumental ridges, bleb formation, and mild erosion in male worms; however in females, there were extensive erosion and destruction of the tegumental surface. These promising results may encourage future use of compound BTP-Iso in the treatment of schistosomiasis. However, more research is needed to detect the effect of compound BTP-Iso on early developmental stages of S. mansoni and on other species of human schistosomes.

Cyanopyridinone- and Cyanopyridine-Based Cancer Cell Pim-1 Inhibitors: Design, Synthesis, Radiolabeling, Biodistribution, and Molecular Modeling Simulation.

In this study, two new series of 3-cyanopyridinones (3a-e) and 3-cyanopyridines (4a-e) were synthesized and evaluated for their cytotoxicity and Pim-1 kinase inhibitory activity adopting 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay and in vitro Pim-1 kinase inhibition assay, respectively. Most of the tested compounds revealed promising cytotoxicity against HepG-2, HCT-116, MCF-7, and PC-3 cell lines. Among them, compounds 4c and 4d showed more potent cytotoxicity against the HePG2 cell line with IC50 = 8.02 ± 0.38 and 6.95 ± 0.34 µM, respectively, than that of the reference 5-FU (IC50 = 9.42 ± 0.46 µM). Moreover, compound 4c was more potent against HCT-116 (IC50 = 7.15 ± 0.35 µM) than 5-FU (IC50 = 8.01 ± 0.39 µM), while compound 4d with IC50 = 8.35 ± 0.42 µM displayed comparable activity to that of the reference drug. Furthermore, high cytotoxic activity was manifested by compounds 4c and 4d against MCF-7 and PC3 cell lines. Our results have also indicated that compounds 4b, 4c, and 4d elicited remarkable inhibition of Pim-1 kinase; 4b and 4c showed equipotent inhibitory activity to that of the reference quercetagetin. Meanwhile, 4d displayed IC50 = 0.46 ± 0.02 µM, showed the best inhibitory activity among the tested compounds, and was more potent than quercetagetin (IC50 = 0.56 ± 0.03 µM). For optimization of the results, docking study of the most potent compounds 4c and 4d in the Pim-1 kinase active site was carried out and compared with both quercetagetin and the reported Pim-1 inhibitor A (VRV), and the results were consistent with those of the biological study. Consequently, compounds 4c and 4d are worthy of further investigations toward the discovery of Pim-1 kinase inhibitors as drug candidates for cancer therapy. Compound 4b was successfully radiolabeled with radioiodine-131, and its biodistribution in Ehrlich ascites carcinoma (EAC)-bearing mice showed more observable uptake in tumor sites, and hence, it can be introduced as a new radiolabeled agent for tumor imaging and therapy.

New Pyrazole-Clubbed Pyrimidine or Pyrazoline Hybrids as Anti-Methicillin-Resistant Staphylococcus aureus Agents: Design, Synthesis, In Vitro and In Vivo Evaluation, and Molecular Modeling Simulation.

Two hybrid series of pyrazole-clubbed pyrimidines 5a-c and pyrazole-clubbed pyrazoline compounds 6a,b and 7 were designed as attractive scaffolds to be investigated in vitro and in vivo for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. From the results of the in vitro antibacterial screening, compound 5c showed excellent activity (minimal inhibitory concentration, MIC = 521 µM) when compared with that of the reference antibiotic levofloxacin (MIC = 346 µM). The inhibition of the target dihydrofolate reductase (DHFR) enzyme by compounds 4 and 5a-c (IC50 = 5.00 ± 0.23, 4.20 ± 0.20, 4.10 ± 0.19, and 4.00 ± 0.18 µM, respectively) was found to be better than the reference drug trimethoprim (IC50 = 5.54 ± 0.28 µM). Molecular modeling simulation results have justified the order of activity of all the newly synthesized compounds as DHFR enzyme inhibitors, and compound 5c exhibited the best binding profile (-13.6169386 kcal/mol). Hence, the most potent inhibitor of the DHFR enzyme, 5c, was chosen to be evaluated in vivo for its activity in treating MRSA-induced keratitis in rats and that, in turn, significantly (P < 0.0001) reduced infection in rats when compared to MRSA-treated group results.

In vitro cytotoxicity and docking study of novel symmetric and asymmetric dihydropyridines and pyridines as EGFR tyrosine kinase inhibitors.

Quinolines have a weighty effect as anticancer agents and 1,4-DHPs have demonstrated efficacy as anticancer agents in several studies, as well. New hybrid models of symmetric and asymmetric 1,4-DHPs and pyridines linked at C3 of 2-chloroquinoline as a new anticancer scaffold, were designed and synthesized. Hantszch 1,4-DHPs method was adopted for chemical synthesis. MTT assay was performed for the evaluation of cytotoxicity, and EGFR tyrosine kinase assay was performed to investigate binding to our selected compounds, measured by ELISA. The IC50 expressed in µM values revealed that compounds 4a,b, and 5i,k showed the best results against the tested four cell lines than the reference drug 5-Flurouuracil. Compound 5k displayed the most potent cytotoxic activity with IC50  values in the low µM range (12.03 ± 1.51: 20.09 ± 2.16 µM), compared with 5-Fu IC50 range (40.74 ± 2.46: 63.81 ± 2.69 µM). The incorporation of 2-chloroquinoline at C3 to C4 of 1,4-DHP could be proposed as an anticancer scaffold rather than its analogous pyridines. Ester fragments connected to 1,4-DHPs ring as a lipophilic part are essential for anticancer activity. The chirality at C4 improved the anticancer activity. The hydrogen and halogen bond facilitated protein-ligand binding mode and affinity.

In vitro activity of celastrol in combination with thymol against carbapenem-resistant Klebsiella pneumoniae isolates.

Klebsiella pneumoniae is an opportunistic pathogen causing nosocomial and community-acquired infections. Klebsiella has developed resistance against antimicrobials including the last resort class; carbapenem. Currently, treatment options for carbapenem-resistant-Klebsiella (CRK) are very limited. This study aims to restore carbapenem effectiveness against CRK using celastrol and thymol. Clinical Klebsiella isolates were identified using biochemical and molecular methods. Antimicrobial susceptibility was determined using disk-diffusion method. Carbapenemase-production was tested phenotypically and genotypically. Celastrol and thymol-MICs were determined and the carbapenemase-inhibitory effect of sub-MICs was investigated. Among 85 clinical Klebsiella isolates, 72 were multi-drug-resistant and 43 were meropenem-resistant. Phenotypically, 39 isolates were carbapenemase-producer. Genotypically, blaNDM1 was detected in 35 isolates, blaVIM in 17 isolates, blaOXA in 18 isolates, and blaKPC was detected only in 6 isolates. Celastrol showed significant inhibitory effect against carbapenemase-hydrolytic activity. Meropenem-MIC did not decrease in presence of celastrol, only 2-fold decrease was observed with thymol, while 4-64 fold decrease was observed when meropenem was combined with both celastrol and thymol. Furthermore, thymol increased CRK cell wall-permeability. Molecular docking revealed that celastrol is superior to thymol for binding to KPC and VIM-carbapenemase. Our study showed that celastrol is a promising inhibitor of multiple carbapenemases. While meropenem-MIC were not affected by celastrol alone and decreased by only 2-folds with thymol, it decreased by 4-64 folds in presence of both celastrol and thymol. Thymol increases the permeability of CRK-envelope to celastrol. The triple combination (meropenem/celastrol/thymol) could be useful for developing more safe and effective analogues to restore the activity of meropenem and other ß-lactams.

Synthesis and Molecular Docking Study of New Thiazole Derivatives as Potential Tubulin Polymerization Inhibitors.

A new series of 2,4-disubstituted thiazole derivatives containing 4-(3,4,5-trimethoxyphenyl) moiety was synthesized and evaluated for their potential anticancer activity as tubulin polymerization inhibitors. All designed compounds were screened for cytotoxic activity against four human cancer cell lines, namely, HepG2, MCF-7, HCT116, and HeLa, using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, with combretastatin A-4 as a reference drug. Compounds 5c, 6d, 7c, 8, and 9a,b showed superior activity against the tested cell lines, with IC50 values ranging from 3.35 ± 0.2 to 18.69 ± 0.9 µM. Further investigation for the most active cytotoxic agents as tubulin polymerization inhibitors was also performed in order to explore the mechanism of their antiproliferative activity. The obtained results suggested that compounds 5c, 7c, and 9a remarkably inhibit tubulin polymerization, with IC50 values of 2.95 ± 0.18, 2.00 ± 0.12, and 2.38 ± 0.14 µM, respectively, which exceeded that of the reference drug combretastatin A-4 (IC50 2.96 ± 0.18 µM). Molecular docking studies were also conducted to investigate the possible binding interactions between the targeted compounds and the tubulin active site. The interpretation of the results showed clearly that compounds 7c and 9a were identified as the most potent tubulin polymerization inhibitors with promising cytotoxic activity and excellent binding mode in the docking study.

The Link between Occurrence of Class I Integron and Acquired Aminoglycoside Resistance in Clinical MRSA Isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infections because of its high resistance. Here, we study the antibiotic resistance in MRSA clinical isolates and their relation to integron I occurrence. A total of 88 clinical Staphylococcusaureus isolates were collected. MRSA were identified by the disk diffusion method (DDM) and confirmed by PCR, and antibiogram was determined by DDM. Integron I, II and the aacA4 gene were investigated by PCR. Integrase-positive strains were analyzed for the presence of resistance gene cassettes by sequencing. All isolates were identified as MRSA by DDM and confirmed by PCR. All isolates were resistant to ampicillin and cefoxitin. Concerning aminoglycosides, the frequency of resistance was reported for streptomycin (60.7%), tobramycin (37.1%) gentamicin (36%), and for amikacin (15.9%). Integron I was detected in 41 isolates (46.6%), while integron II was detected in three isolates (3.4%). Sequencing of the integron I-cassette indicated the exclusive prevalence of addA gene variants mediating aminoglycoside resistance. The aacA4 gene was found in DNA of 31 isolates (35.22%). This study revealed the high existence of MRSA. Furthermore, the AacA4 gene and class I integron harboring aadA gene were predominant in MRSA isolates.

Homology modelling, molecular dynamics simulation and docking evaluation of ß-tubulin of Schistosoma mansoni.

Schistosomiasis is one of the neglected diseases causing considerable morbidity and mortality throughout the world. Microtubules with its main component, tubulin play a vital role in helminthes including schistosomes. Benzimidazoles represent potential drug candidates by binding ß-tubulin. The study aimed to generate a homology model for the ß-tubulin of S. mansoni using the crystal structure of O visaries (Sheep) ß-tubulin (PDB ID: 3N2G D) as a template, then different ß-tubulin models were generated and two previously reported benzimidazole derivatives (NBTP-F and NBTP-OH) were docked to the generated models, the binding results indicated that both S. mansoni, S. haematobium were susceptible to the two NBTP derivatives. Additionally, three mutated versions of S. mansoni ß-tubulin wild-type were generated and the mutation (F185Y) seems to slightly enhance the ligand binding. Dynamics simulation experiments showed S. haematobium ß-tubulin is highly susceptible to the tested compounds; similar to S. mansoni, moreover, mutated models of S. mansoni ß-tubulin altered its NBTPs susceptibility. Moreover, additional seven new benzimidazole derivatives were synthesized and tested by molecular docking on the generated model binding site of S. mansoni ß-tubulin and were found to have good interaction inside the pocket.

Back to Nature: Combating Candida albicans Biofilm, Phospholipase and Hemolysin Using Plant Essential Oils.

Candida albicans is the causative agent of fatal systemic candidiasis. Due to limitations of antifungals, new drugs are needed. The anti-virulence effect of plant essential oils (EOs) was evaluated against clinical C. albicans isolates including cinnamon, clove, jasmine and rosemary oils. Biofilm, phospholipase and hemolysin were assessed phenotypically. EOs were evaluated for their anti-virulence activity using phenotypic methods as well as scanning electron microscopy (SEM) and atomic force microscopy (AFM). Among the C. albicans isolates, biofilm, phospholipase and hemolysins were detected in 40.4, 86.5 and 78.8% of isolates, respectively. Jasmine oil showed the highest anti-biofilm activity followed by cinnamon, clove and rosemary oils. SEM and AFM analysis showed reduced adherence and roughness in the presence of EOs. For phospholipase, rosemary oil was the most inhibitory, followed by jasmine, cinnamon and clove oils, and for hemolysins, cinnamon had the highest inhibition followed by jasmine, rosemary and clove oils. A molecular docking study revealed major EO constituents as promising inhibitors of the Als3 adhesive protein, with the highest binding for eugenol, followed by 1,8-cineole, 2-phenylthiolane and cinnamaldehyde. In conclusion, EOs have a promising inhibitory impact on Candida biofilm, phospholipase and hemolysin production, hence EOs could be used as potential antifungals that impact virulence factors.

Soil-Associated Bacillus Species: A Reservoir of Bioactive Compounds with Potential Therapeutic Activity against Human Pathogens.

Soil hosts myriads of living organisms with the extensive potential to produce bioactive compounds. Bacteria are the major soil inhabitants that represent a rich reservoir for antibiotic production along with their role in recycling nutrients and maintenance of the soil ecosystem. Here, from 55 tested soil samples, we isolated and identified a novel antibiotic-producing bacterial strain with a phylogenetically closest match to Bacillus subtilis sp. based on BLASTN search of GenBank for the 16S rRNA gene sequence. We characterized this novel strain through microscopic, biochemical, and molecular techniques, combined with testing its potential antimicrobial activity. Chemical studies revealed that the antibiotic produced by this strain is a glycopeptide. It exhibited profound activity against both methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans. The antibiotic is optimally produced at 37 °C after 28 h of growth. The biocompatibility of the extracted antibiotic was tested over a wide range of factors including temperature, pH, surfactants, and metal salts. To confirm its therapeutic potential, a sterile solution of the antibiotic was tested in vivo against bacteria-induced keratitis in rats where significant healing activity was recorded. Hence, this soil Bacillus strain may lead to the development of novel antibiotics for the treatment of human pathogens.