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In this study, 177 bacterial isolates were recovered from 55 agricultural soil samples collected from various locations in Egypt. Following purification, the isolates were evaluated in solid and liquid phase assays for their capacity to decolorize several types of dyes such as Azure B (AB), methylene blue (MB), and Congo red (CR). The 16S rRNA sequence analysis was used to identify isolates with the highest decolorizing capacity. The two bacterial isolates coded 304 and 434 which exhibited potential ligninolytic activity were identified as Streptomyces griseorubens and Streptomyces intermedius , respectively. Streptomyces intermedius test isolate was selected for optimization experiments using the one-factor-at-a-time approach (OFAT) followed by a statistical method of optimization using response surface methodology (RSM). The optimization experiments resulted in a 2.6-fold increase in dye decolorization capacity after 4 h of incubation with bacterium growth compared to basal conditions and thus indicated a significant reduction in dye decolorization time, accelerating the dye decolorization process and demonstrating enhanced efficiency in ligninolytic enzyme production. In addition, the whole genome sequencing (WGS) process was performed on S. intermedius isolate to detect the relevant genes related to lignin degradation and dye decolorizing activities. After annotation and analysis of the genomic sequence, various genes encoding enzymes related to lignin degradation and dye decolorization activities were identified confirming the genetic potential of this strain for efficient ligninolytic activity. The obtained WGS data was deposited in the NCBI database under the accession code SRR25321249. Taken together, the WGS data are in alignment with phenotypic dye decolorization activity of the selected isolate. Accordingly, the test isolate S. intermedius 434 was considered a potential candidate for lignin biodegradation and textile dye effluent bioremediation.

Rhamnolipids are important biosurfactants for application in bioremediation, enhanced oil recovery, pharmaceutical, and detergent industry. In this study, rhamnolipids extracted from P. aeruginosa P6 were characterized to determine their potential fields of application. Thin-layer chromatographic analysis of the produced rhamnolipids indicated the production of two homologues: mono- and di-rhamnolipids, whose structures were verified by 1 H and 13 C nuclear magnetic resonance spectroscopy. Additionally, high performance liquid chromatography-mass spectrometry identified seven different rhamnolipid congeners, of which a significantly high proportion was di-rhamnolipids reaching 80.16%. Rha-Rha-C10-C10 was confirmed as the principal compound of the rhamnolipid mixture (24.30%). The rhamnolipids were capable of lowering surface tension of water to 36 mN/m at a critical micelle concentration of 0.2 g/L, and exhibited a great emulsifying activity (E24 = 63%). In addition, they showed excellent stability at pH ranges 4–8, NaCl concentrations up to 9% (w/v) and temperatures ranging from 20 to 100 °C and even after autoclaving. These results suggest that rhamnolipids, produced by P. aeruginosa P6 using the cheap substrate glycerol, are propitious for biotechnology use in extreme and complex environments, like oil reservoirs and hydrocarbon contaminated soil. Moreover, P. aeruginosa P6 may be considered, in its wild type form, as a promising industrial producer of di-RLs, which have superior characteristics for potential applications and offer outstanding commercial benefits.

A high cellulase-producing bacterial isolate TS4 was recovered from an Egyptian soil sample and identified using 16S rRNA gene sequencing as Streptomyces thermodiastaticus. One-factor-at-a-time (OFAT) preliminary studies were carried out to determine the key factors affecting cellulase production by S. thermodiastaticus and their optimum ranges. The initial pH of the medium, carboxymethyl cellulose (CMC), tryptone, and NaCl concentrations were further optimized using a response surface Central Composite design. Fermentation under optimized variables of initial pH 6.0, presence of CMC, tryptone, and NaCl at concentrations of 2%, 0.03%, and 0.12%, respectively, resulted in 3.24 fold increase in cellulase productivity (2023 U/L) as compared to that under basal conditions (625 U/L). Cellulase production was also improved with a 4 Kilogray (KGy) dosage of gamma radiation. In comparison to the wild-type strain under basal circumstances, S. thermodiastaticus produced 5.1 fold more cellulase after a combination of model-based optimization and gamma radiation mutation. Cellulase was partially purified using ammonium sulfate precipitation followed by dialysis. The resulting cellulase was 1.74 times purified and its specific activity was 4.21 U/mg. The molecular weight of cellulase is 63 kDa as indicated by SDS-PAGE and zymogram. Its maximum activity was achieved at 60 °C and pH 5.0. In addition, it showed outstanding thermo-tolerance as it could retain its full activity after a 12-h incubation at 90 °C.

Background Methicillin-Resistant Staphylococcus aureus (MRSA) causes life-threatening infections, with narrow therapeutic options including: vancomycin and linezolid. Accordingly, this study aimed to characterize phenotypically and genotypically, the most relevant means of linezolid resistance among some MRSA clinical isolates. Methods A total of 159 methicillin-resistant clinical isolates were collected, of which 146 were indentified microscopically and biochemically as MRSA. Both biofilm formation and efflux pump activity were assessed for linezolid-resistant MRSA (LR-MRSA) using the microtiter plate and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) methods, respectively. Linezolid resistance was further characterized by polymerase chain reaction (PCR) amplification and sequencing of domain V of 23 S rRNA; rplC ; rplD ;and rplV genes. Meanwhile, some resistance genes were investigated: cfr; cfr(B); optrA; msrA;mecA; and vanA genes. To combat LR-MRSA, the effect of combining linezolid with each of 6 different antimicrobials was investigated using the checkerboard assay. Results Out of the collected MRSA isolates (n = 146), 5.48% (n = 8) were LR-MRSA and 18.49% (n = 27) were vancomycin-resistant (VRSA). It is worth noting that all LR-MRSA isolates were also vancomycin-resistant. All LR-MRSA isolates were biofilm producers (r = 0.915, p  = 0.001), while efflux pumps upregulation showed no significant contribution to development of resistance (t = 1.374, p  = 0.212). Both mecA and vanA genes were detected in 92.45% (n = 147) and 6.92% (n = 11) of methicillin-resistant isolates, respectively. In LR-MRSA isolates, some 23 S rRNA domain V mutations were observed: A2338T and C2610G (in 5 isolates); T2504C and G2528C (in 2 isolates); and G2576T (in 1 isolate). Amino acids substitutions were detected: in L3 protein ( rplC gene) of (3 isolates) and in L4 protein ( rplD gene) of (4 isolates). In addition, cfr(B) gene was detected (in 3 isolates). In 5 isolates, synergism was recorded when linezolid was combined with chloramphenicol, erythromycin, or ciprofloxacin. Reversal of linezolid resistance was observed in some LR-MRSA isolates when linezolid was combined with gentamicin or vancomycin. Conclusions LR-MRSA biofilm producers’ phenotypes evolved in the clinical settings in Egypt. Various antibiotic combinations with linezolid were evaluated in vitro and showed synergistic effects.

Background Antimicrobial resistance, particularly in clinical Enterococcus isolates, poses a serious global health threat because of difficult-to-treat nosocomial infections. The emergence of vancomycin-resistant enterococci (VRE), mediated by VanA or VanB operons, has significantly limited treatment options. This study aimed at identifying antibiotic resistance and virulence genes in enterococci and exploring potential correlations between these genetic traits. Methods A total of 100 suspected enterococci were gathered from two hospitals and identified through phenotypic methods and the VITEK 2 Compact system. The Kirby-Bauer disk diffusion and MIC by microbroth dilution methods were employed for antimicrobial susceptibility. The gelatinase production and biofilm were evaluated phenotypically, while the presence of vancomycin resistance ( van A, van B) and virulence ( esp , gel E, hyl ) genes was confirmed by PCR and sequenced for genetic characterization. Results Sixty-five Enterococcus isolates were characterized, with E. faecium (50.7%) and E. faecalis (41.5%) being the predominant species. Linezolid, teicoplanin, and chloramphenicol still retain good activity with 6.15%, 10.7%, and 29.2% resistance, respectively. About 40% of isolates were VRE, and all harbored the van A gene. Biofilm formation and gelatinase production were most prevalent in E. faecium and E. faecalis , indicating enhanced virulence. Sequencing confirmed the chromosomal location and identity of the resistance and virulence genes, supporting their accurate detection and distribution among different Enterococcus species. Statistical analysis revealed that both esp and gel E genes were significantly associated with biofilm formation and gelatinase activity; however, esp showed a positive correlation with van A and vancomycin resistance, while gel E demonstrated a negative correlation. Even though van A is typically linked to high levels of resistance to both teicoplanin and vancomycin, only seven out of the twenty-six isolates that were van A-positive showed phenotypic resistance to teicoplanin. Conclusion Enterococcus faecium and E. faecalis were identified as predominant multidrug-resistant species carrying multiple virulence determinants, with esp and gel E strongly linked to biofilm formation and gelatinase activity. Linezolid, teicoplanin, and chloramphenicol remained the most effective agents. Our findings demonstrate the coexistence of resistance and virulence traits, along with unexpected genotype–phenotype variations, underscoring the need for integrated molecular and phenotypic approaches in surveillance and clinical management.

Background Response surface methodology (RSM) employing Box-Behnken design was used to optimize the environmental factors for the production of paromomycin, a 2 deoxystreptamine aminocyclitol aminoglycoside antibiotic, (2DOS-ACAGA) from Streptomyces (S.) rimosus NRRL 2455. Emergence of bacterial resistance caught our attention to consider the combination of antimicrobial agents. The effect of paromomycin combination with other antimicrobial agents was tested on some multiple drug resistant isolates. To the best of our knowledge, this is the first report on optimization of paromomycin production from S. rimosus NRRL 2455. A Quadratic model and response surface method were used by choosing three model factors; pH, incubation time and inoculum size. A total of 17 experiments were done and the response of each experiment was recorded. Concerning the effect of combining paromomycin with different antimicrobial agents, it was tested using the checkerboard assay against six multidrug resistant (MDR) pathogens including; Pseudomonas (P.) aeruginosa (2 isolates), Klebsiella (K.) pneumoniae , Escherichia (E.) coli , methicillin sensitive Staphylococcus aureus (MSSA) and methicillin resistant Staphylococcus aureus (MRSA). Paromomycin was tested in combination with ceftriaxone, ciprofloxacin, ampicillin/sulbactam, azithromycin, clindamycin and doxycycline. Results The optimum conditions for paromomycin production were a pH of 6, an incubation time of 8.5 days and an inoculum size of 5.5% v /v using the optimized media (soybean meal 30 g/L, NH 4 CL 4 g/L, CaCO 3 5 g/L and glycerol 40 ml/L), 28 °C incubation temperature, and 200 rpm agitation rate that resulted in 14 fold increase in paromomycin production as compared to preliminary fermentation level using the basal medium. The tested antibiotic combinations showed either synergistic effect on paromomycin activity on most of the tested MDR pathogens (45.83%), additive effect in 41.67% or indifferent effect in 12.5%. Conclusion RSM using multifactorial design was a helpful and a reliable method for paromomycin production. Paromomycin combination with ceftriaxone, ciprofloxacin, ampicillin/sulbactam, azithromycin, clindamycin or doxycycline showed mostly synergistic effect on certain selected clinically important MDR pathogens.

Accumulating evidence has denoted the danger of resistance in tenacious organisms like methicillin-resistant Staphylococcus aureus (MRSA). MRSA, a supple bacterium that adopts a variety of antibiotic resistance mechanisms, is the cause of multiple life-threatening conditions. Approaching a post-antibiotic era, bacteria-specific natural predators, bacteriophages, are now given the chance to prove eligible for joining the antibacterial weaponry. Considering the foregoing, this study aimed at isolating bacteriophages with promising anti-MRSA lytic activity, followed by characterization and optimization of the production of the bacteriophage with the broadest host range. Five phages were isolated from different environmental sources including the rinse of raw chicken egg, raw milk, and, remarkably, the raw meat rinses of chicken and fish. Examined for lytic activity against a set of 23 MRSA isolates collected from various clinical specimens, all five phages showed relatively broad host ranges with the bacteriophage originally isolated from raw fish rinse showing lytic activity against all the isolates tested. This phage is suggested to be a member of Siphoviridae family, order Caudovirales, as revealed by electron microscopy. It also exhibited good thermal stability and viability at different pH grades. Moreover, it showed reasonable stability against UV light and all viricidal organic solvents tested. Optimization using D-optimal design by response surface methodology was carried out to enhance the phage yield. The optimum conditions suggested by the generated model were a pH value of 7, a carbon source of 0.5% w/v sucrose, and a nitrogen source of 0.1% w/v peptone, at a temperature of 28°C and a bacterial inoculum size of 10 7 CFU/ml, resulting in a 2 log-fold increase in the produced bacteriophage titer. Overall, the above findings indicate the lytic ability inflicted by this virus on MRSA. Apparently, its stability under some of the extreme conditions tested implies its potential to be a candidate for pharmaceutical formulation as an anti-MRSA therapeutic tool. We hope that bacteriophages could tip the balance in favor of the human front in their battle against multidrug-resistant pathogens.

Background: This study aimed to produce, purify, structurally elucidate, and explore the biological activities of metabolites produced by Streptomyces (S.) griseus isolate KJ623766, a recovered soil bacterium previously screened in our lab that showed promising cytotoxic activities against various cancer cell lines. Methods: Production of cytotoxic metabolites from S. griseus isolate KJ623766 was carried out in a 14L laboratory fermenter under specified optimum conditions. Using a 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium-bromide assay, the cytotoxic activity of the ethyl acetate extract against Caco2 and Hela cancer cell lines was determined. Bioassay-guided fractionation of the ethyl acetate extract using different chromatographic techniques was used for cytotoxic metabolite purification. Chemical structures of the purified metabolites were identified using mass, 1D, and 2D NMR spectroscopic analysis. Results: Bioassay-guided fractionation of the ethyl acetate extract led to the purification of two cytotoxic metabolites, R1 and R2, of reproducible amounts of 5 and 1.5 mg/L, respectively. The structures of R1 and R2 metabolites were identified as β- and γ-rhodomycinone with CD50 of 6.3, 9.45, 64.8 and 9.11, 9.35, 67.3 µg/mL against Caco2, Hela and Vero cell lines, respectively. Values were comparable to those of the positive control doxorubicin. Conclusions: This is the first report about the production of β- and γ-rhodomycinone, two important scaffolds for synthesis of anticancer drugs, from S. griseus.

Vitamin D 3 is a fat-soluble prohormone that is activated inside the liver to produce 25-hydroxyvitamin D 3 (calcidiol), and in the kidney to produce the fully active 1α, 25-dihydroxy vitamin D 3 (calcitriol). A previous work piloted in our laboratory, resulted in a successful recovery of a local soil-promising Actinomyces hyovaginalis isolate CCASU-A11-2 capable of converting vitamin D 3 into calcitriol. Despite the rising amount of research on vitamin D 3 bioconversion into calcitriol, further deliberate studies on this topic can significantly contribute to the improvement of such a bioconversion process. Therefore, this work aimed to improve the bioconversion process, using the study isolate, in a 14 L laboratory fermenter (4 L fermentation medium composed of fructose (15 g/L), defatted soybean (15 g/L), NaCl (5 g/L), CaCO 3 2 g/L); K 2 HPO 4 , (1 g/L) NaF (0.5 g/L) and initial of pH 7.8) where different experiments were undertaken to investigate the effect of different culture conditions on the bioconversion process. Using the 14 L laboratory fermenter, the calcitriol production was increased by about 2.5-fold (32.8 µg/100 mL) to that obtained in the shake flask (12.4 µg/100 mL). The optimal bioconversion conditions were inoculum size of 2% v/v, agitation rate of 200 rpm, aeration rate of 1 vvm, initial pH of 7.8 (uncontrolled); addition of vitamin D 3 (substrate) 48 h after the start of the main culture. In conclusion, the bioconversion of vitamin D 3 into calcitriol in a laboratory fermenter showed a 2.5-fold increase as compared to the shake flask level where, the important factors influencing the bioconversion process were the aeration rate, inoculum size, the timing of substrate addition, and the fixed pH of the fermentation medium. So, those factors should be critically considered for the scaling-up of the biotransformation process. Key points Bioconversion of vitamin D into calcitriol has been performed in a 14 L laboratory fermenter using a local soil-promising Actinomyces hyovaginalis isolate CCASU-A11-2. The calcitriol production was increased by about 2.5-fold (32.8 µL/100 mL) to that obtained in the shake flask (12.4 µL/100 mL). Aeration rate, inoculum size, the timing of substrate addition, and the fixed pH were important factors influencing the bioconversion process.

Solid-state fermentation has a special advantage of preventing the foaming problem that obstructs submerged fermentation processes for rhamnolipid production. In the present work, a 50:50 mixture of sugarcane bagasse and sunflower seed meal was selected as the optimum substrate for rhamnolipid production using a Pseudomonas aeruginosa mutant 15GR and an impregnating solution including 5% v/v glycerol. Using Box–Behnken design, the optimum fermentation conditions were found to be an inoculum size 1% v/v, temperature 30 °C and unlike other studies, pH 8. These optimized conditions yielded a 67% enhancement of rhamnolipid levels reaching 46.85 g rhamnolipids per liter of impregnating solution, after 10 days, which was about 5.5 folds higher than that obtained by submerged liquid fermentation. Although maximum rhamnolipids concentration was obtained after 10 days of incubation, rhamnolipids concentration already reached high levels (41.87 g/l) after only 6 days. This rhamnolipid level was obtained in a shorter time and using lower carbon source concentrations than most studies reported so far. The findings obtained indicate an enormous potential for employing solid-state fermentation for rhamnolipid production by the studied isolate.