Neveen A. Abdelaziz

Carbapenem-resistant Acinetobacter baumannii (CRAB) has become a critical concern that necessitates the development of novel antimicrobial approaches. One of the most promising innovative approaches for combating CRAB infections is using effective and lytic bacteriophages (phages), known as phage therapy. Therefore, we recovered and characterized a polyvalent lytic Salmonella_phage_VB_ST-SA173, producing lytic activity against 6 CRAB clinical isolates and 3 multidrug-resistant (MDR) Salmonella serovars. Throughout pH 2–10, and thermal stability at up to 60 °C, the phage maintained its stability and lytic activity against the tested isolates. The presence of a tailed phage with a characteristic prolate head and a contractile tail was detected by the transmission electron microscope (TEM). According to the Oxford nanopore sequencing data, the genome of Salmonella_phage_VB_ST-SA173 was 53,636 bp in size, contained 45.9% G + C, and had 53 opening reading frames (ORFs). According to the TEM, ORFs, and BLASTn analysis findings, it was proved that the Salmonella_phage_VB_ST-SA173 belongs to the Loughboroughvirus genus. The efficacy of the phage-formulated Carbopol 940 hydrogel in wound healing was assessed preclinically in an infected burn wound animal model with a CRABa clinical isolate. The survival rate was enhanced in the phage-treated group compared to the untreated control groups. Histopathological analysis showed improved wound healing in the form of apparently healthy skin with apparently normal epidermal and dermis layers. In conclusion, depending on its in vitro and physicochemical traits, the phage-loaded hydrogel is expected to be a promising candidate for clinical trials against human CRAB-related skin infections. Key points: • A polyvalent Loughboroughvirus phage showed lytic activity against CRAB and Salmonella serovars. • The phage showed stability at a wide range of pH and temperature. • The phage hydrogel enhanced healing in the burn-wound animal model infected with CRABa.

Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate (ODHP) was extracted in a previous study from the culture broth of soil isolate Alcaligenes faecalis MT332429 and showed a promising antimycotic activity. This study was aimed to formulate ODHP loaded β-cyclodextrins (CD) nanosponge (NS) hydrogel (HG) to control skin fungal ailments since nanosponges augment the retention of tested agents in the skin. Box-Behnken design was used to produce the optimized NS formulation, where entrapment efficiency percent (EE%), polydispersity index (PDI), and particle size (PS) were assigned as dependent parameters, while the independent process parameters were polyvinyl alcohol % (w/v %), polymer-linker ratio, homogenization time, and speed. The carbopol 940 hydrogel was then created by incorporating the nanosponges. The hydrogel fit Higuchi’s kinetic release model the best, according to in vitro drug release. Stability and photodegradation studies revealed that the NS-HG remained stable under tested conditions. The formulation also showed higher in vitro antifungal activity against Candida albicans compared to the control fluconazole. In vivo study showed that ODHP-NS-HG increased survival rates, wound contraction, and healing of wound gap and inhibited the inflammation process compared to the other control groups. The histopathological examinations and Masson’s trichrome staining showed improved healing and higher records of collagen deposition. Moreover, the permeability of ODHP-NS-HG was higher through rats’ skin by 1.5-folds compared to the control isoconazole 1%. Therefore, based on these results, NS-HG formulation is a potential carrier for enhanced and improved topical delivery of ODHP. Our study is a pioneering research on the development of a formulation for ODHP produced naturally from soil bacteria. Key points: • Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate was successfully formulated as a nanosponge hydrogel and statistically optimized. • The new formula exhibited in vitro good stability, drug release, and higher antifungal activity against C. albicans as compared to the fluconazole. • Ex vivo showed enhanced skin permeability, and in vivo analysis showed high antifungal activity as evidenced by measurement of various biochemical parameters and histopathological examination.

Fungal infections currently pose a real threat to human lives. In the current study, soil bacterial isolates were screened for the production of antifungal compounds to combat human fungal pathogens. Notably, the bacterial F1 isolate exhibited antimycotic action towards the Candida albicans ATCC 10231 and Aspergillus niger clinical isolates. By employing phenotypic and molecular techniques, we identified the F1 isolate as the Bacillus toyonensis isolate OQ071612. The purified extract showed stability within a pH range of 6–7 and at temperatures of up to 50 °C. It demonstrated potential antifungal activity in the presence of various surfactants, detergents, and enzymes. The purified extract was identified as 6-methoxy-1H-Indole-2-carboxylic acid using advanced spectroscopic techniques. To optimize the antifungal metabolite production, we utilized response surface methodology (RSM) with a face-centered central composite design, considering nutritional and environmental variables. The optimal conditions were as follows: starch (5 g/L), peptone (5 g/L), agitation rate of 150 rpm, pH 6, and 40 °C temperature. A confirmatory experiment validated the accuracy of the optimization process, resulting in an approximately 3.49-fold increase in production. This is the first documented report on the production and characterization of 6-methoxy-1H-Indole-2-carboxylic acid (MICA) antifungal metabolite from Bacillus toyonensis.

Despite the mounting global burden of antimicrobial resistance (AMR), the generation of new classes of effective antimicrobials still lags far behind. The interplay between multidrug resistance and biofilm formation in Acinetobacter baumannii has drastically narrowed the available therapeutic choices. The use of natural compounds holds promise as an alternate option for restoring the activity of existing antibiotics and attenuating virulence traits through reduced biofilm formation. This study aimed to evaluate the modulatory effect of combining cinnamic and gallic acids at ½MIC with various antibiotics against multidrug-resistant (MDR) A. baumannii clinical isolates as well as study the effect on the expression of the biofilm-associated genes (bap, csuE, ompA) via quantitative, real-time PCR. Combining cinnamic or gallic acid with imipenem, amikacin or doxycycline resulted in significant reduction of resistance (p < 0.05). On the contrary, no effect was recorded when both acids were combined with levofloxacin, and only cinnamic acid had a synergistic effect with colistin. The transcriptomic changes of biofilm-related genes in the presence of gallic acid at ½MIC were compared with untreated control samples. The fold expression values proved that gallic acid substantially down-regulated the respective genes in all five strong biofilm formers. Molecular docking studies of gallic and cinnamic acids on target genes revealed good binding affinities and verified the proposed mechanism of action. To the best of our knowledge, this is the first report on the effect of gallic acid on the expression of bap, csuE and ompA genes in A. baumannii, which may permit its use as an adjunct anti-virulence therapeutic strategy.

Background: Carbapenem-resistant Enterobacterales (CRE), currently listed by the World Health Organization (WHO) as top priority critical pathogens, are a major global menace to human health. In low- and middle-income countries (LMICs) the threat is mounting fueled by selective pressures caused by antibiotic abuse and inadequate diagnostic resources. Methods: This study phenotypically and genotypically characterized carbapenem resistance among 115 Enterobacterales isolates including 76 Klebsiella (K.) pneumoniae, 19 Escherichia (E.) coli, 14 Shigella (S.) sonnei, 5 Enterobacter (E.) cloacae, and 1 Proteus (P.) mirabilis. Results: Ninety-three isolates (80.9%) were carbapenem-resistant with an alarming 57.5% carbapenem non-susceptibility in isolates collected from the outpatient department. Molecular characterization of the carbapenemases (CPases) encoding genes showed that bla_NDM (80.5%) was the most prevalent; it was detected in 62 isolates (54 K. pneumoniae, 6 E. coli and 2 S. sonnei), followed by bla_VIM (36.4%) which was observed in 28 isolates (24 K. pneumoniae, 3 E. coli and 1 E. cloacae). Other CPases included bla_KPC (28.6%; in 20 K. pneumoniae, 1 E. coli and 1 S. sonnei), bla_OXA-48 (26%; in 17 K. pneumoniae, 1 E. coli,1 E. cloacae and 1 P. mirabilis), bla_IMP (6.5%; in 5 K. pneumoniae) and bla_SPM (1.3%; in K. pneumoniae). Notably more than half of the Enterobacterales isolates (54.5%) co-harboured more than one CPase-encoding gene. Co-existence of bla_NDM and bla_VIM genes was the most dominant (31.2%), followed by association of bla_NDM and bla_KPC (24.7%), then bla_VIM and bla_KPC (13%). Moreover, the effects of different genotypes on meropenem MIC values were assessed, and a statistically significant difference between the genotype (Ambler classes A and B) and the genotype (Ambler classes B and D) was recorded. Conclusion: The current findings may serve for a better understanding of the context of CRE in Egypt, associated drivers and CPases.

Resistance to antifungal agents represents a major clinical challenge, leading to high morbidity and mortality rates, especially in immunocompromised patients. In this study, we screened soil bacterial isolates for the capability of producing metabolites with antifungal activities via the cross-streak and agar cup-plate methods. One isolate, coded S6, showed observable antifungal activity against Candida (C.) albicans ATCC 10231 and Aspergillus (A.) niger clinical isolate. This strain was identified using a combined approach of phenotypic and molecular techniques as Lysinibacillus sp. MK212927. The purified metabolite displayed fungicidal activity, reserved its activity in a relatively wide range of temperatures (up to 60 °C) and pH values (6–7.8) and was stable in the presence of various enzymes and detergents. As compared to fluconazole, miconazole and Lamisil, the minimum inhibitory concentration of the metabolite that showed 90% inhibition of the growth (MIC₉₀) was equivalent to that of Lamisil, half of miconazole and one fourth of fluconazole. Using different spectroscopic techniques such as FTIR, UV spectroscopy, 1D NMR and 2D NMR techniques, the purified metabolite was identified as terbinafine, an allylamine antifungal agent. It is deemed necessary to note that this is the first report of terbinafine production by Lysinibacillus sp. MK212927, a fast-growing microbial source, with relatively high yield and that is subject to potential optimization for industrial production capabilities.

In the current study, a soil bacterial isolate F2 expressed a significant antagonistic activity against Candida albicans ATCC 10231 and Aspergillus niger clinical isolate confirmed through cross streak, dual culture, and agar well diffusion methods. The isolate F2 was identified using phenotypic and molecular approaches as Alcaligenes (A.) faecalis MT332429. The identification and structural characterization of the antifungal compound was performed using advanced spectroscopic techniques including UV absorbance, ¹H and ¹³C NMR and 2D NMR (COSY, HSQC, and HMBC) and was identified as octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate. Response surface methodology (RSM) using a central composite design was employed to optimize the nutritional and cultural variables affecting the antifungal metabolite yield. The optimum conditions were found to be temperature 30 °C, agitation 150 rpm, glucose 1 g/l, peptone 2 g/l, and pH 8. A confirmatory experiment was performed to assess the accuracy of the optimization procedure, where an increase in the antifungal metabolite production by about 2.48-fold was obtained. To the best of our knowledge, this is the first report of octadecyl 3-(3, 5-di-tert-butyl-4-hydroxyphenyl) propanoate recovered from the culture broth of A. faecalis MT332429 with a promising antifungal activity along with its optimized production through RSM. Key points • A novel soil bacterial isolate, F2, identified as Alcaligenes faecalis MT332429, showed significant antagonistic activity against Candida albicans ATCC 10231 and Aspergillus niger clinical isolate. • This stable fungicidal extracellular metabolite was identified as octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate. • Optimization using central composite design resulted in 2.48-fold increase in production reaching 213.82 μg/ml.

Purpose: We aimed to optimize the factors affecting the production of the allylamine antifungal, terbinafine, by Lysinibacillus isolate MK212927, a natural producer of this broad-spectrum fungicidal compound. Methods: We employed a central composite design to optimize the five most important variables influencing the production of terbinafine which were carbon source, nitrogen source, temperature, pH and agitation. Results: The optimum conditions were found to be starch 5 g/L, ammonium chloride 5 g/L, temperature 32°C, agitation 150 rpm and pH 7. The actual response (inhibition zone diameter) was highly comparable to the value predicted by the model, indicating a valid model. Using the standard calibration curve of terbinafine, the optimized conditions resulted in an increase in the antifungal metabolite production (terbinafine) by about 1.6-fold (1814.662 μg/mL compared to 1165.550 μg/mL under standardized conditions). Conclusion: This is the first report, to the best of our knowledge, on optimized production of terbinafine by Lysinibacillus species. Hence, these findings may be useful as baseline data for scaling up the production of terbinafine from a natural microbial source.