Bacterial Diversity and Antibiotic Resistance Patterns of Community-Acquired Urinary Tract Infections in Mega Size Clinical Samples of Egyptian Patients: A Cross-Sectional Study.

BACKGROUND:  Community-acquired urinary tract infection (UTI) is one of the most common infectious diseases nowadays. Alarming increased levels of antimicrobial resistance are developing globally which limit treatment options and may lead to life-threatening problems. AIM: Our study aimed to collect surveillance data on non-hospitalized Egyptian UTI cases and to develop strategies against multidrug-resistant pathogens (MDR). According to our knowledge, this is the first study to screen this high number (15,252 urine samples) in a short period (three months), providing valuable data on resistance profiles in non-hospitalized Egyptian UTI patients. METHODS: A total of 15,252 urine samples were collected from different patients. Positive cultures were identified using a semi-quantitative method. Kirby-Bauer's disc diffusion method was used for antibiotic susceptibility testing, the double disc diffusion method was used for extended-spectrum beta-lactamases-producing strains, and the Chi-square test was used for statistical data processing. RESULTS: The results showed 61% positive cultures, females accounted for 67.5%. Infants and elderly patients showed the highest positive cultures (74.4% and 69.2%, respectively). Despite Escherichia coli being the most common uropathogen (47.19%), Klebsiella species(24.42%) were the most MDR and extended-spectrum ß-lactamase (ESBL)-producing organisms. E. coli and Klebsiella spp. displayed increased resistance to cephalosporins (75% and 81%, respectively). In contrast, both organisms displayed high sensitivity to carbapenems. Unlike Klebsiella spp., E. coli was highly sensitive (92%) to first-line treatment (nitrofurantoin) for UTI. Moreover, trimethoprim/sulfamethoxazole showed higher sensitivity rates compared to other nations. CONCLUSION:  Despite Escherichia coli being the most often identified bacteria in our isolates Klebsiella spp. displayed higher resistance to the majority of tested antibiotics. Fortunately, trimethoprim/sulfamethoxazole significantly increased sensitivity, especially against E. coli. However, both species showed high rates of cephalosporin resistance. Moreover, It is important to promote Egypt's national action plan for antimicrobial resistance in collaboration with the World Health Organization, especially in the community to minimize the chance of bacterial resistance in the Egyptian community.

Therapeutic Switching of Rafoxanide: a New Approach To Fighting Drug-Resistant Bacteria and Fungi.

Control and management of life-threatening bacterial and fungal infections are a global health challenge. Despite advances in antimicrobial therapies, treatment failures for resistant bacterial and fungal infections continue to increase. We aimed to repurpose the anthelmintic drug rafoxanide for use with existing therapeutic drugs to increase the possibility of better managing infection and decrease treatment failures. For this purpose, we evaluated the antibacterial and antifungal potential of rafoxanide. Notably, 70% (70/100) of bacterial isolates showed multidrug resistance (MDR) patterns, with higher prevalence among human isolates (73.5% [50/68]) than animal ones (62.5% [20/32]). Moreover, 22 fungal isolates (88%) were MDR and were more prevalent among animal (88.9%) than human (87.5%) sources. We observed alarming MDR patterns among bacterial isolates, i.e., Klebsiella pneumoniae (75% [30/40; 8 animal and 22 human]) and Escherichia coli (66% [40/60; 12 animal and 28 human]), and fungal isolates, i.e., Candida albicans (86.7% [13/15; 4 animal and 9 human]) and Aspergillus fumigatus (90% [9/10; 4 animal and 5 human]), that were resistant to at least one agent in three or more different antimicrobial classes. Rafoxanide had antibacterial and antifungal activities, with minimal inhibitory concentration (MICs) ranging from 2 to 128 µg/mL. Rafoxanide at sub-MICs downregulated the mRNA expression of resistance genes, including E. coli and K. pneumoniae blaCTX-M-1, blaTEM-1, blaSHV, MOX, and DHA, C. albicans ERG11, and A. fumigatus cyp51A. We noted the improvement in the activity of ß-lactam and antifungal drugs upon combination with rafoxanide. This was apparent in the reduction in the MICs of cefotaxime and fluconazole when these drugs were combined with sub-MIC levels of rafoxanide. There was obvious synergism between rafoxanide and cefotaxime against all E. coli and K. pneumoniae isolates (fractional inhibitory concentration index [FICI] values ≤ 0.5). Accordingly, there was a shift in the patterns of resistance of 16.7% of E. coli and 22.5% of K. pneumoniae isolates to cefotaxime and those of 63.2% of C. albicans and A. fumigatus isolates to fluconazole when the isolates were treated with sub-MICs of rafoxanide. These results were confirmed by in silico and mouse protection assays. Based on the in silico study, one possible explanation for how rafoxanide reduced bacterial resistance is through its inhibitory effects on bacterial and fungal histidine kinase enzymes. In short, rafoxanide exhibited promising results in overcoming bacterial and fungal drug resistance. IMPORTANCE The drug repurposing strategy is an alternative approach to reducing drug development timelines with low cost, especially during outbreaks of disease caused by drug-resistant pathogens. Rafoxanide can disrupt the abilities of bacterial and fungal cells to adapt to stress conditions. The coadministration of antibiotics with rafoxanide can prevent the failure of treatment of both resistant bacteria and fungi, as the resistant pathogens could be made sensitive upon treatment with rafoxanide. From our findings, we anticipate that pharmaceutical companies will be able to utilize new combinations against resistant pathogens.

Nephthea sp. inhibits biofilm, DNA gyrase, HSP90, and DHFR: in vitro, in silico, and pharmacokinetics studies.

This study attempts to identify and assess a novel marine-derived antibiofilm agent. The antibacterial activity of n-hexane, dichloromethane, ethyl acetate, and butanol fractions from the crude extract of soft coral Nephthea sp. was evaluated against six microorganisms.Ethyl acetate fraction considered the most effective one against Bacillus subtilis, Escherichia coli, and Candida, investigated potential biofilm inhibition against the tested strains. Seventeen secondary metabolites were identified using (UPLC-Q/TOF-MS) responsible for these biological activities of the active fraction. Additionally, a molecular docking study showed free binding energy of -7.5 kcal/mol; Azamial A had the highest binding affinity for the DNA gyrase enzyme, while Sinularectin had -8.3 and -7.6 kcal/mol for the DHFR and HSP90 enzymes, respectively. Moreover, pharmacokinetics and (ADME) studies for Azamial A and Sinularectin were performed. Finally, results were confirmed by the in vitro enzymatic inhibitory effect of ethyl acetate fraction suggested in the in-silico study.

Optimization of Supercritical Carbon Dioxide Extraction of Saussurea costus Oil and Its Antimicrobial, Antioxidant, and Anticancer Activities.

Saussurea costus is a medicinal plant with different bioactive compounds that have an essential role in biomedicine applications, especially in Arab nations. However, traditional extraction methods for oils can lead to the loss of some volatile and non-volatile oils. Therefore, this study aimed to optimize the supercritical fluid extraction (SFE) of oils from S. costus at pressures (10, 20, and 48 MPa). The results were investigated by GC/MS analysis. MTT, DPPH, and agar diffusion methods assessed the extracted oils' anticancer, antioxidant, and antimicrobial action. GC/MS results showed that elevated pressure from 10 to 20 and 48 MPa led to the loss of some valuable compounds. In addition, the best IC50 values were recorded at 10 MPa on HCT, MCF-7, and HepG-2 cells at about 0.44, 0.46, and 0.74 µg/mL, respectively. In contrast, at 20 MPa, the IC50 values were about 2.33, 6.59, and 19.0 µg/mL, respectively, on HCT, MCF-7, and HepG-2 cells, followed by 48 MPa, about 36.02, 59.5, and 96.9 µg/mL. The oil extract at a pressure of 10 MPa contained much more of á-elemene, dihydro-à-ionone, patchoulene, á-maaliene, à-selinene, (-)-spathulenol, cedran-diol, 8S,13, elemol, eremanthin, á-guaiene, eudesmol, ç-gurjunenepoxide-(2), iso-velleral, and propanedioic acid and had a higher antioxidant activity (IC50 14.4 µg/mL) more than the oil extract at 20 and 48 MPa. In addition, the inhibitory activity of all extracts was higher than gentamicin against all tested bacteria. One of the more significant findings from this study is low pressure in SFE enhancement, the extraction of oils from S. costus, for the first time. As a result, the SFE is regarded as a good extraction technique since it is both quick and ecologically friendly. Furthermore, SFE at 10 MPa increased the production and quality of oils, with high antioxidant activity and a positive effect on cancer cells and pathogens.

Carbapenem-Resistant Klebsiella pneumoniae: Diversity, Virulence, and Antimicrobial Resistance.

Background: Klebsiella pneumoniae (K. pneumoniae) is one of the most important pathogens in nosocomial infections. It has resistance to most antibiotics, even carbapenem, resulting in restricted therapeutic options. Purpose: We tried to assess the antimicrobial resistance and virulence fitness of carbapenem-resistant K. pneumoniae (CRKP) in addition to their phenotypic and genotypic diversity. Materials and Methods: The conventional methods, automated Vitek-32 system, and antimicrobial susceptibility pattern were used to detect CRKP isolates. Virulence and resistance genes profiles were created by using PCR technique. The correlation analysis was done by using R-program. Results: The antimicrobial resistance profile for all our K. pneumoniae isolates was shocking as the MDR and CRKP were the most prominent phenotypes. Unfortunately, high degrees of heterogeneity among our CRKP isolates were recorded, as 97.5% of them were differentiated into different clusters. We found a negative correlation between the existence of virulence and antimicrobial resistance genes. In contrast to sputum and urine CRPK isolates, the blood isolates showed high antimicrobial resistance and low virulence fitness. Finally, K. pneumoniae creates several outbreaks and crises in Egypt owing to the highly heterogeneity and the wide spread of multidrug-resistant (MDR) and multi-virulent CRKP phenotypes. Conclusion: Our results are significant and alarming to health organizations throughout the world for the severity and heterogeneity of K. pneumoniae infections. Therefore, the traditional method for treatment of CRKP infections must be renewed. Additionally, the treatment protocols must be well correlated with the site of infections, phenotypes, and genotypes of CRKP strains.

What Is behind the Correlation Analysis of Diarrheagenic E. coli Pathotypes?

The treatment failure recorded among patients and animals infected with diarrheagenic Escherichia coli (DEC) was increased due to the presence of specific virulence markers among these strains. These markers were used to classify DEC into several pathotypes. We analyzed the correlations between DEC pathotypes and antimicrobial resistances, the existence of virulence genes, serotypes, and hosts. The ETEC pathotype was detected with a high prevalence rate (25%). Moreover, the ETEC and EPEC pathotypes were highly associated with human infections in contrast to the EIEC and EAEC phenotypes, which were commonly recognized among animal isolates. Interestingly, the antimicrobial resistance was affected by E. coli pathotypes. With the exception of EIEC and STEC, imipenem represented the most effective antibiotic against the other pathotypes. There were fixed correlations between the DEC pathotypes and the presence of virulence markers and hosts; meanwhile, their correlation with serotypes was variable. Additionally, the vast majority of our isolates were highly diverse, based on both phenotypic and ERIC molecular typing techniques. Our promising results gave a clear indication for the heterogeneity and weak clonality of DEC pathotypes in Egypt, which can be utilized in the evaluation of the current therapeutic protocols and infection control guidelines.

Cholyl 1,3,4-oxadiazole hybrid compounds: design, synthesis and antimicrobial assessment.

A new chemical library based on the hybridization of cholic acid with the heterocyclic moiety 1,3,4-oxadizole was synthesized, and tested for antimicrobial activity against Gram-positive, Gram-negative bacteria, and fungi. Among the synthesized compounds, the most potent derivatives against S. aureus were 4t, 4i, 4p, and 4c with MIC values between 31 and 70 µg/mL, while compound 4p was the most active one against Bacillus subtilis with a MIC value of 70 µg/mL. Interestingly, compounds 4a and 4u exerted selective activity against Gram-positive bacteria. The synthesized compounds showed good activity against A. fumigatus and C. albicans and compound 4v exhibited selective activity against fungi only.

Development of isatin-thiazolo[3,2-a]benzimidazole hybrids as novel CDK2 inhibitors with potent in vitro apoptotic anti-proliferative activity: Synthesis, biological and molecular dynamics investigations.

In the current medical era, human health is experiencing numerous challenges, particularly the human malignancies. Therefore, the therapeutic arsenal for these malignancies is to be inexorably enhanced with new treatments that target tumor cells in a selective manner. In this regard, the present work aims at developing a new set of small molecules featuring the privileged isatin scaffold conjugated with a thiazolo[3,2-a]benzimidazole (TBI) motif through a cleavable hydrazide linker (7a-e and 10a-i) as potential anticancer CDK2 inhibitors. The large tricyclic TBI motif is anticipated to achieve a plethora of hydrophobic interactions within the CDK2 binding site. The growth of the two examined cell lines was significantly inhibited by most the prepared hybrids with IC50 ranges; (2.60 ± 1.47-20.90 ± 1.17 µM, against MDA-MB-231) and (1.27 ± 0.06-16.83 ± 0.95 µM, against MCF-7). In particular, hybrids 7a, 7d and 10a displayed potent dual activity against the examined cell lines, and thus selected for further investigations. They exerted a significance alteration in the cell cycle progression, in addition to an apoptosis induction within both MDA-MB-231 and MCF-7 cells. Furthermore, 7a, 7d and 10a displayed potent CDK2 inhibitory action (IC50 = 96.46 ± 5.3, 26.24 ± 1.4 and 42.95 ± 2.3 nM, respectively). The docking simulations unveiled, as expected, the ability of the TBI ring to well-accommodate and establish several hydrophobic interactions within a hydrophobic pocket in the CDK2 binding site. Also, the docking simulations highlighted the significance of incorporation of the hydrazide linker and isatin unsubstituted (NH) functionality in the H-bonding interactions. Interestingly, the most potent CDK2 inhibitor 7d achieved the best binding score (-11.2 Kcal/mole) and formed the most stable complex with CDK2 enzyme (RMSD = 1.24 Å) in a 100 ns MD simulation. In addition, the MM-PBSA calculations ascribed the lowest binding free energy to the 7d-CDK2 complex (-323.69 ± 15.17 kJ/mol). This could be attributed to an incorporation of the 5-OCH3 group that was engaged in an extra hydrogen bonding with key THR14 amino acid residue. Finally, these results suggested hybrid 7d as a good candidate for further optimization as promising breast cancer antitumor agent and CDK2 inhibitor.

Antifungal action and induction of resistance by ß-aminobutyric acid against Penicillium digitatum to control green mold in orange fruit.

Green mold, caused by Penicillium digitatum, is the most economically important postharvest disease of orange fruit worldwide. The aim of this study was to evaluate the effect of ß-aminobutyric acid (BABA) treatment on the inhibition of P. digitatum both in orange fruit and in vitro as well as the possible mechanisms of action. BABA at 125 mM significantly inhibited mycelial growth, spore germination, and germ tube elongation of P. digitatum by 93.3, 90.3, and 90.5%, respectively. The relative electrical conductivity of mycelium was increased for a period of 0-36 h after treated with BABA at 125 mM. Furthermore, BABA caused a high level of malondialdehyde (MDA) in P. digitatum mycelia during four days of incubation. The ergosterol content in the plasma membrane of P. digitatum was significantly lower in BABA-treated mycelia. Also, protein and sugar leakage were increased with BABA treatment compared with that in the control. Besides, BABA caused a considerable reduction in the total lipid content of P. digitatum mycelia at 125 mM. Scanning electron microscopy (SEM) of P. digitatum treated with BABA at 125 mM showed shrunken, distorted, and collapsed mycelia. The application of BABA at 125 mM in orange fruit inoculated with P. digitatum suppressed disease incidence and disease severity by 74.6 and 77.3%, respectively, compared to untreated fruit. Moreover, the activity of defense-related enzymes, including peroxidase (POD), polyphenoloxidase (PPO), and phenylalanine ammonia-lyase (PAL) were significantly enhanced in the orange fruit treated with BABA at 125 mM.

New thiobarbituric acid scaffold-based small molecules: Synthesis, cytotoxicity, 2D-QSAR, pharmacophore modelling and in-silico ADME screening.

A series of twenty five new thiobarbituric acid derivatives, viz. 3a-h, 4-7, 8a-c, 9, 10a-c, 11 and 12a-d, were designed and synthesized as potential cytotoxic agents. In-vitro screening of the new compounds against the three human cancer cell lines Caco-2, HepG-2 and MCF-7 was performed to assess their intrinsic activity. Compound 12d exhibited potent sub-micromolar activity against HepG-2 and MCF-7 (IC50 = 0.07 and 0.08 µM, respectively). In-silico pharmacophore modelling of this chemotype compounds disclosed a five features' pharmacophore model representing essential steric and electronic fingerprints essential for activity. Finally, a 2D-QSAR model was devised to quantitatively correlate the 2D molecular feature descriptors of this series of thiobarbiturates with their cytotoxic activity against MCF-7. Finally, in silico evaluation of the physicochemical and ADME properties of these derivatives was performed.

Quercetin 3-O-glucoside recovered from the wild Egyptian Sahara plant, Euphorbia paralias L., inhibits glutamine synthetase and has antimycobacterial activity.

Tuberculosis remains a major health problem accentuated by the rise of resistance to all available drugs. Therefore, this study was launched to discover a novel antituberculosis agent from wild Egyptian Sahara plants. Twelve such plants were screened, in vitro, for their activity against various Mycobacterium species. The most active plant, Euphorbia paralias, was further fractionated with different organic solvents, and the activity of the obtained fractions was determined by the agar diffusion and broth microdilution methods. The methanol fraction was the most active against Mycobacterium spp., and was non-toxic in doses up to 10 g/kg of animal weight. Its main component was separated by column chromatography, and then identified by ultraviolet spectroscopy and nuclear magnetic resonance analysis as quercetin-3-O-ß-D-glucoside. Docking analysis suggested that quercetin-3-O-ß-D-glucoside inhibits the glutamine synthetase enzyme, a promising target for the development of antituberculosis drugs. This prediction was confirmed by an in vitro glutamine synthetase biosynthetic assay. To the best of our knowledge, and based on bioinformatics mining of the BioPhytMol database, this is the first report on the antimycobacterial activity of Euphorbia paralias plant. It is also the first report on the inhibition of mycobacterial glutamine synthetase by the flavonoid quercetin.

Novel [(3-indolylmethylene)hydrazono]indolin-2-ones as apoptotic anti-proliferative agents: design, synthesis and in vitro biological evaluation.

On account of their significance as apoptosis inducing agents, merging indole and 3-hydrazinoindolin-2-one scaffolds is a logic tactic for designing pro-apoptotic agents. Consequently, 27 hybrids (6a-r, 9a-f and 11a-c) were synthesised and evaluated for their cytotoxicity against MCF-7, HepG-2 and HCT-116 cancer cell lines. SAR studies unravelled that N-propylindole derivatives were the most active compounds such as 6n (MCF-7; IC50=1.04 µM), which displayed a significant decrease of cell population in the G2/M phase and significant increase in the early and late apoptosis by 19-folds in Annexin-V-FTIC assay. Also, 6n increased the expression of caspase-3, caspase-9, cytochrome C and Bax and decreased the expression of Bcl-2. Moreover, compounds 6i, 6j, 6n and 6q generated ROS by significant increase in the level of SOD and depletion of the levels of CAT and GSH-Px in MCF-7.

Biological evaluation of some new N-(2,6-dimethoxypyrimidinyl) thioureido benzenesulfonamide derivatives as potential antimicrobial and anticancer agents.

A series of novel heterocyclic thioureas 3a-u containing sulfonamide moiety have been synthesized by the condensation of isothiocyanatobenzenesulfonamide 2 with a variety of heterocyclic amines. The newly synthesized heterocyclic thioureas were investigated for their antimicrobial and anticancer activity. The in vitro antibacterial and antifungal activity were done using well diffusion method. Interestingly, compounds 3j and 3m, showed similar or better activity compared with the reference drug against the tested microorganisms. Although, 3j was less active among its analogues to inhibit the breast carcinoma cells, it exhibit strong broad spectrum antimicrobial activities. However, The results of the cytotoxic activity revealed that compound 3p was the most active against the breast carcinoma cell line (MCF-7) giving promising IC50 value of 1.72 µg/mL, compared with reference drug (5-flourouracil) with IC50 value of 4.8 µg/mL. The most potent compounds in cytotoxic activity 3b and 3p were further docked inside the active site of CAIX and were found to exhibit a proper binding with the active site amino acids according to their bond lengths, angles and conformational energy.