Core proteome mediated subtractive approach for the identification of potential therapeutic drug target against the honeybee pathogen Paenibacillus larvae.

Background & Objectives: American foulbrood (AFB), caused by the highly virulent, spore-forming bacterium Paenibacillus larvae, poses a significant threat to honey bee brood. The widespread use of antibiotics not only fails to effectively combat the disease but also raises concerns regarding honey safety. The current computational study was attempted to identify a novel therapeutic drug target against P. larvae, a causative agent of American foulbrood disease in honey bee. Methods: We investigated effective novel drug targets through a comprehensive in silico pan-proteome and hierarchal subtractive sequence analysis. In total, 14 strains of P. larvae genomes were used to identify core genes. Subsequently, the core proteome was systematically narrowed down to a single protein predicted as the potential drug target. Alphafold software was then employed to predict the 3D structure of the potential drug target. Structural docking was carried out between a library of phytochemicals derived from traditional Chinese flora (n > 36,000) and the potential receptor using Autodock tool 1.5.6. Finally, molecular dynamics (MD) simulation study was conducted using GROMACS to assess the stability of the best-docked ligand. Results: Proteome mining led to the identification of Ketoacyl-ACP synthase III as a highly promising therapeutic target, making it a prime candidate for inhibitor screening. The subsequent virtual screening and MD simulation analyses further affirmed the selection of ZINC95910054 as a potent inhibitor, with the lowest binding energy. This finding presents significant promise in the battle against P. larvae. Conclusions: Computer aided drug design provides a novel approach for managing American foulbrood in honey bee populations, potentially mitigating its detrimental effects on both bee colonies and the honey industry.

Bioaugmentation and phytoremediation wastewater treatment process as a viable alternative for pesticides removal: case of pentachlorophenol.

This study focused on the potential for pentachlorophenol removal by a biological process in secondary treated wastewater (STWW). The proposed process is a combined method of phytoremediation using a native plant, Polypogon maritimus and Lemna minor, and bioaugmentation using a fungus. The bioaugmentation process was performed by a fungal isolate capable of removing PCP, isolated from the compost. The identification of the fungus was performed by morphological, biochemical, and molecular methods. A biological treatment system by bioaugmentation and phytoremediation was set up to estimate the capacity of this process to eliminate a high concentration of PCP. physico-chemical parameters, such as pH, COD, and BOD were tested at experimentation times T0 (initial) and Tf (final). The concentration of PCP is controlled by the HPLC method. Thus, the growth of the fungus was determined by spectrophotometry and enumeration on the agar medium. The results obtained show that the isolated and selected fungus is identified by Penicillium Ilerdanum. The fungal strain used has a significant capacity for tolerance and elimination of PCP. The results of the physico-chemical parameters showed an improvement in the quality of wastewater after the treatment was carried out. The elimination of PCP came with a release of Common law- and an important decrease in the DOC value in the STWW. The results obtained show that the Polypogon treatment shows a significant elimination of PCP by a percentage of the order of 92.01% and 23.58 g. L- 1 chloride concentration. The macrophytes used showed a better ability to tolerate and eliminate PCP with an increase of chlorophyll and its longer sheets. Supplementary Information: The online version contains supplementary material available at 10.1007/s40201-023-00865-y.

Identification of Disalicyloyl Curcumin as a Potential DNA Polymerase Inhibitor for Marek's Disease Herpesvirus: A Computational Study Using Virtual Screening and Molecular Dynamics Simulations.

Marek's disease virus (MDV) is a highly contagious and persistent virus that causes T-lymphoma in chickens, posing a significant threat to the poultry industry despite the availability of vaccines. The emergence of new virulent strains has further intensified the challenge of designing effective antiviral drugs for MDV. In this study, our main objective was to identify novel antiviral phytochemicals through in silico analysis. We employed Alphafold to construct a three-dimensional (3D) structure of the MDV DNA polymerase, a crucial enzyme involved in viral replication. To ensure the accuracy of the structural model, we validated it using tools available at the SAVES server. Subsequently, a diverse dataset containing thousands of compounds, primarily derived from plant sources, was subjected to molecular docking with the MDV DNA polymerase model, utilizing AutoDock software V 4.2. Through comprehensive analysis of the docking results, we identified Disalicyloyl curcumin as a promising drug candidate that exhibited remarkable binding affinity, with a minimum energy of -12.66 Kcal/mol, specifically targeting the DNA polymerase enzyme. To further assess its potential, we performed molecular dynamics simulations, which confirmed the stability of Disalicyloyl curcumin within the MDV system. Experimental validation of its inhibitory activity in vitro can provide substantial support for its effectiveness. The outcomes of our study hold significant implications for the poultry industry, as the discovery of efficient antiviral phytochemicals against MDV could substantially mitigate the economic losses associated with this devastating disease.

Inhibitor Assessment against the LpxC Enzyme of Antibiotic-resistant Acinetobacter baumannii Using Virtual Screening, Dynamics Simulation, and in†vitro Assays.

BACKGROUND: Bacterial resistance is currently a significant global public health problem. Acinetobacter baumannii has been ranked in the list of the World Health Organization as the most critical and priority pathogen for which new antibiotics are urgently needed. In this context, computational methods play a central role in the modern drug discovery process. The purpose of the current study was to identify new potential therapeutic molecules to neutralize MDR A. baumannii bacteria. METHODS: A total of 3686 proteins retrieved from the A. baumannii proteome were subjected to subtractive proteomic analysis to narrow down the spectrum of drug targets. The SWISS-MODEL server was used to perform a 3D homology model of the selected target protein. The SAVES server was used to evaluate the overall quality of the model. A dataset of 74500 analogues retrieved from the PubChem database was docked with LpxC using the AutoDock software. RESULTS: In this study, we predicted a putative new inhibitor for the Lpxc enzyme of A. baumannii. The LpxC enzyme was selected as the most appropriate drug target for A. baumannii. According to the virtual screening results, N-[(2S)-3-amino-1-(hydroxyamino)-1-oxopropan-2-yl]-4-(4-bromophenyl) benzamide (CS250) could be a promising drug candidate targeting the LpxC enzyme. This molecule shows polar interactions with six amino acids and non-polar interactions with eight other residues. In vitro experimental validation was performed through the inhibition assay. CONCLUSION: To the best of our knowledge, this is the first study that suggests CS250 as a promising inhibitory molecule that can be exploited to target this gram-negative pathogen.

Biological and Molecular Characterization of the Lytic Bacteriophage SoKa against Pseudomonas syringae pv. syringae, Causal Agent of Citrus Blast and Black Pit in Tunisia.

Pseudomonas syringae pv. syringae (Pss), the causal agent of citrus blast and black pit lesion of lemon fruit, continues to cause serious damage in citrus production in Tunisia. Faced with the rapid emergence of the disease and the inefficiency of conventional control methods, an alternative strategy based on the use of bacteriophages was pursued in this study. The lytic Pss bacteriophage SoKa was isolated from soil collected from Tunisian citrus orchards. Analysis of the host range showed that SoKa was able to lyse seven other Pss strains. Interestingly, Pseudomonas syringae pv. porri, pathogenic to leek, could also be infected by SoKa. The activity of SoKa was maintained at pH values between 2 and 10, at temperatures between -80 and 37 °C; the phage could resist UV radiation at an intensity of 320 nm up to 40 min. Whole genome sequencing revealed that the Pseudomonas phage SoKa is a novel phage that belongs to the Bifseptvirus genus of the Autographiviridae family. The absence of virulence proteins and lysogeny-associated proteins encoded on the phage genome, its anti-biofilm activity, and the significant reduction of tissue necrosis in different fruit bioassays make SoKa potentially suitable for use in phage biocontrol.

Diversity of rhizospheric and endophytic bacteria isolated from dried fruit of Ficus carica.

There is currently an increasing demand for the characterization of endophytic bacteria isolated from different parts of plants (rhizosphere, roots, fruit, leaf) in order to improve the organic agriculture practices. The current research was performed to identify both rhizospheric bacteria isolated from the rhizosphere of Ficus carica in three different sites in the north of Tunisia and endophytic bacteria isolated from dried figs. We then characterized them for a diversity of plant growth-promoting (PGP) activities. A collection of 120 isolates from rhizospheric soil and 9 isolates from dried figs was obtained and purified. 16SrDNA gene amplification of rhizospheric bacteria revealed significant diversity and allowed for the assigning of the isolates to 6 phyla: Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Representative strains of the collection (90 strains) were tested for numerous PGP activities and resistance to abiotic stresses. The most common PGP trait for all bacteria from the three regions was siderophore production (62%), followed by cellulase (38%), then protease activity (37%), then by lipases activity (17%) and lastly by solubilization of phosphates (9%). Twenty -three strains that showed most PGP traits were selected, 8 strains presented 12 or more, and 15 strains displayed between 7 and 11 of 17 PGP activities. The majority of the isolates manifested a possible adaptation to abiotic stress and unfavorable environments. PCR-DGGE analysis of soil rhizosphere of the three sites allowed also for the acquisition of a Cluster analysis of rhizospheric bacterial communities. Our current study identified and characterized for the first time in Tunisia rhizospheric and endophytic bacteria from dried fruit of Ficus carica.

Epiphytic Yeasts and Bacteria as Candidate Biocontrol Agents of Green and Blue Molds of Citrus Fruits.

Overall, 180 yeasts and bacteria isolated from the peel of citrus fruits were screened for their in vitro antagonistic activity against Penicillium digitatum and P. italicum, causative agents of green and blue mold of citrus fruits, respectively. Two yeast and three bacterial isolates were selected for their inhibitory activity on mycelium growth. Based on the phylogenetic analysis of 16S rDNA and ITS rDNA sequences, the yeast isolates were identified as Candida oleophila and Debaryomyces hansenii while the bacterial isolates were identified as Bacillus amyloliquefaciens, B. pumilus and B. subtilis. All five selected isolates significantly reduced the incidence of decay incited by P. digitatum and P. italicum on 'Valencia' orange and 'Eureka' lemon fruits. Moreover, they were effective in preventing natural infections of green and blue mold of fruits stored at 4 °C. Treatments with antagonistic yeasts and bacteria did not negatively affect the quality and shelf life of fruits. The antagonistic efficacy of the five isolates depended on multiple modes of action, including the ability to form biofilms and produce antifungal lipopeptides, lytic enzymes and volatile compounds. The selected isolates are promising as biocontrol agents of postharvest green and blue molds of citrus fruits.

An assessment of the air quality in apple warehouses: new records of Aspergillus europaeus, Aspergillus pulverulentus, Penicillium allii and Penicillium sumatraense as decay agents.

Airborne fungi are one of the major components of aeromycobiota known to produce several fungal diseases in fruits. Their presence in indoor environment of warehouses may limit the storage period of apples. Qualitative and quantitative analyses of airborne fungal spores were conducted using gravity settling techniques to detect fungal airspora present in the atmosphere of two apple warehouses in Tunisia. In this study, 375 fungal isolates were obtained and purified. Phylogenetic analysis of calmodulin, beta-tubulin and ITS regions coupled with phenotypic characterization helped to identify 15 fungal species. Penicillium exhibited the highest diversity with ten species detected (Penicillium allii, P. chrysogenum, P. citrinum, P. expansum, P. italicum, P. polonicum, P. solitum, P. steckii, P. sumatraense and P. viridicatum), followed by four species of Aspergillus genus (Aspergillus europaeus, A. flavus, A. niger and A. pulverulentus) and Alternaria alternata. In vivo experiments confirmed the pathogenicity of 13 species at room temperature and under cold-storage conditions. Among them, A. europaeus, A. pulverulentus, P. allii and P. sumatraense were described for the first time as pathogens on apples. The present study identified the major airborne fungi associated with postharvest rot in apple storage facilities in Tunisia and may help in efficient control of postharvest and storage fruit diseases.

Antioxidant and Antimicrobial Potentials of Seed Oil from Carthamus tinctorius L. in the Management of Skin Injuries.

Infection of skin injuries by pathogenic microbial strains is generally associated if not treated with a lasting wound bed oxidative stress status, a delay in healing process, and even wound chronicity with several human health complications. The aim of the current study was to explore the antioxidant and antimicrobial potentialities of safflower (Carthamus tinctorius L.) extracted oil from seeds by cold pressing which would be beneficial in the management of skin wounds. Antioxidant capacity of the oil was evaluated (scavenging ability against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and ferric reducing antioxidant power (FRAP)). Total phenolic, total flavonoid, total carotenoid, and total chlorophyll contents were determined. Antimicrobial activities of safflower oil were tested against 10 skin pathogenic microorganisms: 4 bacterial strains (Escherichia coli, Enterobacter cloacae, Staphylococcus aureus, and Streptococcus agalactiae), 3 yeast species strains (Candida albicans, Candida parapsilosis, and Candida sake), and 3 fungi species (Aspergillus niger, Penicillium digitatum, and Fusarium oxysporum). A notable antioxidant capacity was demonstrated for the tested oil that exhibited moreover high antibacterial effects by both bacteriostatic and bactericidal pathways including lysozyme activity. An antifungal effect was further observed on the spore's germination. Safflower oil could be considered as a good natural alternative remedy in the management of skin wounds and their possible microbial infections.

Diversity of pathogenic Pseudomonas isolated from citrus in Tunisia.

The damages observed in Tunisian citrus orchards have prompted studies on the Pseudomonas spp. responsible for blast and black pit. Prospective orchards between 2015 and 2017 showed that the diseases rapidly spread geographically and to new cultivars. A screening of Pseudomonas spp. isolated from symptomatic trees revealed their wide diversity according to phylogenetic analysis of their housekeeping rpoD and cts genes. The majority of strains were affiliated to Pseudomonas syringae pv. syringae (Phylogroup PG02b), previously described in Tunisia. However, they exhibited various BOX-PCR fingerprints and were not clonal. This work demonstrated, for the first time in Tunisia, the involvement of Pseudomonas cerasi (PG02a) and Pseudomonas congelans (PG02c). The latter did not show significant pathogenicity on citrus, but was pathogenic on cantaloupe and active for ice nucleation that could play a role in the disease. A comparative phylogenetic study of citrus pathogens from Iran, Montenegro and Tunisia revealed that P. syringae (PG02b) strains are closely related but again not clonal. Interestingly P. cerasi (PG02a) was isolated in two countries and seems to outspread. However, its role in the diseases is not fully understood and it should be monitored in future studies. The diversity of pathogenic Pseudomonas spp. and the extension of the diseases highlight that they have become complex and synergistic. It opens questions about which factors favor diseases and how to fight against them efficiently and with sustainable means.

Identification of a Potential Inhibitor Targeting MurC Ligase of the Drug Resistant Pseudomonas aeruginosa Strain through Structure-Based Virtual Screening Approach and In Vitro Assay.

BACKGROUND & OBJECTIVE: Pseudomonas aeruginosa shows resistance to a large number of antibiotics, including carbapenems and third generation cephalosporin. According to the World Health Organization global report published in February 2017, Pseudomonas aeruginosa is on the priority list among resistant bacteria, for which new antibiotics are urgently needed. Peptidoglycan serves as a good target for the discovery of novel antimicrobial drugs. METHODS: Biosynthesis of peptidoglycan is a multi-step process involving four mur enzymes. Among these enzymes, UDP-N-acetylmuramate-L-alanine ligase (MurC) is considered to be an excellent target for the design of new classes of antimicrobial inhibitors in gram-negative bacteria. RESULTS: In this study, a homology model of Pseudomonas aeruginosa MurC ligase was generated and used for virtual screening of chemical compounds from the ZINC Database. The best screened inhibitor i.e. N, N-dimethyl-2-oxo-2,3-dihydro-1H-1,3-benzodiazole-5-sulfonamide was then validated experimentally through inhibition assay. CONCLUSION: The presented results based on combined computational and in vitro analysis open up new horizons for the development of novel antimicrobials against this pathogen.

The Antimicrobial and Wound Healing Potential of Opuntia ficus indica L. inermis Extracted Oil from Tunisia.

INTRODUCTION: Opuntia ficus indica L. inermis (OFI) is used in traditional medicine pharmacopeia for its richness in natural bioactive compounds. It has been proven to be effective in the improvement of the healing of laser-induced skin burns. The aim of the present study was to investigate the wound healing effect of OFI extracted oil on full-thickness skin wound. MATERIALS AND METHODS: The OFI seeds were firstly isolated from mature prickly pears, washed, dried, and then cold-pressed. The antimicrobial activities of OFI seed oil were estimated in vitro against bacteria, yeast, and fungi. Minimum Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) were calculated. Skin wound healing was investigated using an excisional wound healing model in rats. The skin wounds of three experimental groups of rats were topically treated once/day with saline solution (control group), 0.15 mg/mm2 of a reference drug Esth'Elle Pharma Cicaplaie cream (reference group), and 0.6 µl/mm2 of OFI seed oil (OFI oil group). The healing process was monitored daily and the percentage of wound contraction was calculated. A histological study was carried on skin biopsies. RESULTS: The extracted oil has shown an interesting antimicrobial effect on Enterobacter cloacae, antiyeast effect against Candida parapsilosis and Candida sake, and antifungal activity against three opportunistic cutaneous molds (Penicillium, Aspergillus, and Fusarium). Moreover, OFI oil has shown a good wound healing effect. It prevents cutaneous infections and reduces the reepithelialization phase. CONCLUSION: OFI extracted oil has in vitro antimicrobial/fungal properties and in vivo wound healing activity. It seems to be efficient in the treatment of cutaneous infections and the promoting of the scarring process.

New species of pathogenic Pseudomonas isolated from citrus in Tunisia: Proposal of Pseudomonas kairouanensis sp. nov. and Pseudomonas nabeulensis sp. nov.

A collection of Pseudomonas strains was isolated in different regions of Tunisia in the period 2016-2017 from the fruits and leaves of Citrus sinensis cv. 'Valencia Late' and Citrus limon cv. 'Eureka' plants with symptoms of blast and black pit disease. A phylogenetic analysis of the housekeeping gene rpoD was used for strain identification at the species level. The results demonstrated the affiliation of these strains with the genus Pseudomonas and revealed the presence of 11 strains representing two putative new species in two monophyletic branches. These strains were analyzed morphologically and genotypically by multilocus sequence analyses of the rpoD, gyrB and 16S rRNA (rrs) gene sequences, and their phenotypic characteristics by API 20NE and Biolog GEN III. Plant pathogenic properties were confirmed on fruits and detached leaves of C. limon cv. 'Eureka'. Fatty acids and WC MALDI-TOF MS major protein profiles were determined. The genomes of both representatives were sequenced. The average nucleotide index and genome-to-genome distance from KC12T and E10BT are below the cut-off established for a described species. These results support the conclusion that the strains KC12T, KC17, KC20, KC22, KC24A, KC25 and KC26 represent a novel species of Pseudomonas, for which the name of Pseudomonas kairouanensis is proposed. The type strain is KC12T (=CECT9766 and CFBP 8662). The strains E10BT, E10AB, E10CB1 and Iy3BA represent another novel species of Pseudomonas for which the name of Pseudomonas nabeulensis is proposed; the type strain is E10BT (=CECT9765 and CFBP 8661).

In vitro biofilms and antifungal susceptibility of dermatophyte and non-dermatophyte moulds involved in foot mycosis.

Tinea pedis and onychomycosis are among the commonest fungal diseases in the world. Dermatophytes and, less frequently, non-dermatophyte moulds are aetiological agents of foot mycosis and are capable of forming biofilms. Fungal biofilm has demonstrated increasing drug resistance. This work aims to evaluate, in vitro, the ability to form biofilm and the susceptibility to antifungal drugs of sessile dermatophytes and non-dermatophyte moulds involved in foot mycosis. Thirty-six dermatophytes and non-dermatophyte moulds isolated from Tunisian patients with foot mycoses, and identified with MALDI-TOF have been tested. MICs of fluconazole, econazole, itraconazole, terbinafine and griseofulvin were carried out using CLSI broth microdilution method. The ability to form biofilm and antifungal activities of drugs against fungal biofilm formation has been quantified by Crystal Violet and Safranin Red staining. Biomass quantification revealed that all species studied were able to form biofilms in vitro after 72 hours. Fluconazole, econazole, itraconazole and terbinafine inhibited fungal growth with MIC values ranging from 0.031 to >64 µg mL-1 . The best antifungal activity has been obtained with terbinafine against Fusarium solani. Econazole showed the highest activity against fungal biofilm formation. These findings can help clinicians to develop the appropriate therapy of foot mycosis.

Prevalence, Etiology, and Risk Factors of Tinea Pedis and Tinea Unguium in Tunisia.

BACKGROUND: Foot mycoses are a frequent disease that represents a public health problem worldwide. OBJECTIVES: This study aims to evaluate the epidemiology of foot mycoses among Tunisian patients, in order to determine the fungal etiological agents and to identify possible risk factors. PATIENTS AND METHODS: A prospective study of three hundred and ninety-two patients was undertaken during one year (2013-2014). All subjects were asked to collect demographic data related to the risk factors of foot mycoses. A complete mycological diagnosis was carried out on all patients. RESULTS: A total of 485 samples were collected; tinea pedis and tinea unguium were confirmed in 88.2% of cases. Dermatophytes were isolated in 70.5% and the most frequent pathogen was Trichophyton rubrum (98.1%), followed by yeasts (17.7%) commonly Candida parapsilosis. Non-dermatophyte molds (NDMs) were observed in 8.02% cases and Fusarium sp. was the frequent genus (29.1%). The main predisposing factors of fungal foot infections were practicing ritual washing (56.6%) and frequentation of communal showers (50.5%). CONCLUSION: This is a recent survey of foot mycoses in Tunisia. Epidemiological studies can be useful to eradicate these infections and to provide further measures of hygiene and education.

Phenotypical and molecular characterization of Microsporum canis strains in North-Tunisia.

In this study, 40 Microsporum canis isolates were obtained from different patients from the Mycology Unit of the Hospital La Rabta (Tunis) during a 3 month period. The phenotypic identification was done by morphological characterization and biochemical tests. Molecular analysis was performed by amplification of the ITS region of rDNA, the amplified region was subjected to enzymatic digestion and sequenced to evaluate phylogenetic relationships. The morphological analysis showed a considerable diversity of colonies as well as different morphologies of conidia and we have noted variability in the assimilation of the nitrogen and carbon sources. The PCR-RFLP results showed only one restriction pattern for each enzyme. The phylogenetic tree proves that all the strains from Tunisian patients are clonal and related with other strains from different origins. The classical methods used in the mycological laboratories are time-consuming, the PCR-RFLP analysis of the ITS is a reliable tool for the identification of M. canis strains. M. canis from infected Tunisian patients are clonal, although the isolates had different phenotypic characteristics.

Synthesis, crystal structure and potential antimicrobial activities of di (4-sulfamoyl-phenyl-ammonium) sulphate.

A new organic-inorganic hybrid SO4[C6H9N2O2S]2, has been synthesized and characterized by X-ray diffraction. This compound crystallizes in the orthorhombic system, spaces group Pbcn. In the title compound, the packing is stabilized by intermolecular N-H⋯O hydrogen bonds and π-π stacking interactions between the phenyl rings, linking the molecules into three-dimensional network. The in vitro antimicrobial activity of di (4-sulfamoyl-phenyl-ammonium) sulphate was determined by the broth dilution method against several strains selected to define their spectrum and potency. Here we show that the synthetic sulfanilamide exhibits promising antibacterial potency. High inhibition was also detected against Candida albicans. In this paper we firstly showed the antifungal activity of the sulfanilamide against two serious phytopathogenic fungi. Interestingly, the new compound was able to suppress mycelial growth as well as the spores germination of tested fungi, values of spore germination vary from 97.6% to 37.5%, respectively for Botrytis cinerea and Fusarium species. The minimal inhibitory concentrations (MIC) ranging from 8 to 100 µg ml(-1) and IC50 values varying from 5.81 to less than 100 µg ml(-1)), showed that the sulfanilamide sulphate had high activity against bacteria, yeast and fungi, compared to others published antifungal compounds.