Repurposing Finasteride and Serotonin Reuptake Inhibitors as Novel Antimicrobials: A Dual-Action Approach to Target Bacterial and Fungal Pathogens.

In this chapter, the involvement of ATP binding cassette (ABC) and major facilitator superfamily (MFS) transporters in the export of toxic compounds and their involvement in multi-drug resistance (MDR) in different fungal plant pathogens are discussed. However, as MDR is best documented for B. cinerea, this chapter focuses mainly on this important pathogen. Recent developments to higher constitutive transporter expression levels, the combined overexpression of several efflux transporters, reduced fungicide performance under field conditions and the combination of MDR mechanisms with single fungicide resistances (target site resistance) suggest that MDR of plant pathogenic fungi could become a potential threat for fungicide performance under field conditions.

In this study, the antimicrobial activities based on the synergistic effects of traditional antibiotics (imipenem, cefepime, levofloxacin hydrochloride and vancomycin) and antimicrobial peptides (AMPs; PL-5, PL-31, PL-32, PL-18, PL-29 and PL-26), alone or in combination, against three Gram-positive bacteria (Staphylococcus aureus, Streptococcus pneumoniae and Staphylococcus epidermidis) and three Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae) were investigated. In addition, the antimicrobial activity that was based on the synergistic effects of levofloxacin hydrochloride and PL-5 against Staphylococcus aureus in vivo was explored in a mouse infection model. Traditional antibiotics and AMPs showed significant synergistic effects on the antibacterial activities against the different Gram-positive and Gram-negative bacteria in vitro. A strong synergistic effect in the PL-5 and levofloxacin hydrochloride combination against Staphylococcus aureus was observed in the mouse infection model in vivo. The mechanism of synergistic action was due to the different targets of AMPs and traditional antibiotics. The combination of AMPs and traditional antibiotics can dramatically enhance antimicrobial activity and may help prevent or delay the emergence of antibiotic resistance. Thus, this combination therapy could be a promising approach to treat bacterial infections, particularly mixed infections and multi-antibiotic-resistant infections, in the clinics.

Fungal pathogens pose an increasingly worrying threat to human health, food security and ecosystem diversity. To tackle fungal infections and improve current diagnostic and therapeutic tools it is necessary to understand virulence and antifungal drug resistance mechanisms in diverse species. Recent advances in genomics approaches have provided a suitable framework to understand these phenotypes, which ultimately depend on genetically encoded determinants. In this work, we review how the study of genome sequences has been key to ascertain the bases of virulence and drug resistance traits. We focus on the contribution of comparative genomics, population genomics and directed evolution studies. In addition, we discuss how different types of genomic mutations (small or structural variants) contribute to intraspecific differences in virulence or drug resistance. Finally, we review current challenges in the field and anticipate future directions to solve them. In summary, this work provides a short overview of how genomics can be used to understand virulence and drug resistance in fungal pathogens.

BackgroundIn this work, we represent synthesis, in silico analysis and biological activity of 1,4 diazepine linked piperidine derivatives (6a–6o). All the derivatives were screened for their anti-microbial activity against gram-positive (Staphylococcus aureus, Bacillus Subtills, Bacillus megaterium) and gram-negative (Escherichia coli, Pseudonymous, Shigella sp.) bacteria. Compounds were synthesized from reaction of tert-butyl 1,4-diazepane-1-carboxylic, butyryl chloride and varied aromatic aldehyde, further characterized by 1H NMR and LCMS spectral techniques.ResultUsing ampicillin as a positive control, the synthetic compounds 6a–6o were tested for their in-silico study and experimental anti-microbial activity against gram-positive (Staphylococcus aureus, Bacillus Subtills, Bacillus megaterium) and gram-negative (Escherichia coli, Pseudonymous, Shigella sp.) bacteria. According to in vitro assay compound 6a, compound 6c, compound 6d, compound 6m and compound 6I showed higher activity against all the tested strains. Molecule 6i, compound 6j, compound 6k, compound 6f has good to moderate antibacterial activity. DFT computations were used to optimize the molecular geometry at the B3LYP/6-31G (d, p) theoretical level. The corresponding energy values of molecular orbitals were visualized using optimized geometries. Moreover, Auto Dock Vina 1.2.0 is used to assess molecular docking against two target proteins, Bacillus subtilis (PDB ID: 6UF6) and Protease Vulgaris (PDB ID: 5HXW). The target molecule 6b displayed the best binding energies for both. Additionally, we calculated the ADME for each molecule (6a–6o).ConclusionAll fifteen synthesized compounds were screened for their in vitro and in silico analysis. In vitro analysis for anti-microbial activity was carried out against gram-positive (Staphylococcus aureus, Bacillus Subtills, Bacillus megaterium) and gram-negative (Escherichia coli, Pseudonymous, Shigella sp.) bacteria and compound 6a, compound 6c, compound 6d, compound 6m and compound 6I exhibits more potent activity towards all tested strains. Molecular docking is performed against target proteins, L-amino acid deaminase from Proteus Vulgaris and LcpA ligase from Bacillus subtilis, representing the Gram-negative bacterium and Gram-positive bacterium, respectively. Compound 6b showed the highest no. of interaction with protein according to molecular docking. With the advent of innovative techniques like ADME, we select their hit compounds early on and anticipate future pharmacokinetic and pharmacodynamic benefits and drawbacks of these promising therapeutic candidates.Graphical abstract

Citrate synthase was purified to homogeneity from a Gram-positive bacterium (Bacillus megaterium) for the first time. The Mr of the native enzyme was determined to be 84 000 (S.E.M. +/- 5000). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in guanidinium chloride revealed a single protein species of Mr 40 300 (S.E.M. +/- 4400), indicating a dimeric enzyme. This dimeric structure was confirmed by cross-linking the native enzyme with dimethyl suberimidate and with glutaraldehyde, followed by electrophoretic analysis. The enzyme follows Michaelis-Menten kinetics with respect to both substrates, acetyl-CoA and oxaloacetate, and is sensitive to non-specific inhibition by a range of adenine nucleotides. In both molecular and catalytic properties the citrate synthase closely resembles the enzyme from eukaryotic sources and contrasts markedly with the larger, hexameric, enzyme from Gram-negative bacteria.

Background and Aim: The use of bioactive compounds of medicinal plants in prevention, control and treatment of human diseases has a long history. Most of plant bioactive compounds have highly complex chemical structures, and their chemical syntheses are often uneconomical. Moreover, their production is still dependent on plants. Plants of Papaveraceae family contain a variety of bioactive compounds that have many uses in traditional and modern medicine. In the present study, we evaluated the antimicrobial activity of alkaloid extracts of four plant species of Papaveraceae family against some human pathogens. Materials and Methods: Crude alkaloid compounds of Papaver macrostomum , Roemeria refracta , Papaver somniferum and Glaucium grandiflorum plants were extracted using cain method. The antimicrobial activity of plant alkaloid extracts against Pseudomonas aeroginosa PTCC 1310, Listeria monocytogenes PTCC 1297, Staphylococcus aureus PTCC 1189, Klebsiella pneumoniae PTCC 1290 and Candida albicans PTCC 5027 pathogens were assessed using agar diffusion method. Results: The results of this study indicated that the alkaloid extracts of tested plants were effective against fungal and bacterial pathogens. All plant alkaloid extracts exhibited more inhibitory effect against fungal pathogen than the bacterial pathogens. Comparison of MIC and MBC values for bacterial pathogens revealed that all plant alkaloid extracts showed more inhibitory effects against the gram-positive bacteria than gram-negative bacterial pathogens. The results also showed that the alkaloid extracts of R . refracta have stronger inhibitory effect against fungal and bacterial pathogens compared with other plants. Conclusion: The antimicrobial compounds present in the plant species of Papaveraceae family are suitable candidates to produce new antibiotics. The results of this study demonstrated that antimicrobial compound in R . refracta plant could be used in the treatment of infectious diseases caused by C . albicans and S . aureus pathogens.

Imidazoles are a category of azole antifungals that encompass compounds such as ketoconazole, miconazole, esomeprazole, and clotrimazole. In contrast, the triazoles group, which includes fluconazole, voriconazole, and itraconazole, also plays a significant role. The rise of antibiotic resistance in fungal pathogens has evolved into a substantial global public health concern. In this study, two newly synthesized imidazo[1,2-a]pyridine derivative (Probe I and Probe II) molecules were investigated for its antimicrobial potency against of a panel of bacterial (Gram-positive and Gram-negative bacteria) and fungal pathogens. Among the different types of pathogens, we found that Probe II showed excellent antifungal activity against fungal pathogens, based on the preliminary screening the potent molecule further investigated against multidrug-resistance Candida sp. (n = 10) and compared with commercial molecules. In addition, in-silico molecular docking, its dynamics, absorption, distribution, metabolism, excretion and toxicity (ADMET) were analyzed. In this study, the small molecule (Probe II) displayed potent activity only against the Candida spp. including several multidrug-resistant Candida spp. Probe II exhibited minimum inhibitory concentration ranges from 4 to 16µg/mL and minimum fungicidal concentration in the range 4‒32µg/mL as the lowest concentration enough to eliminate the Candida spp. The selected molecules inhibit the formation of yeast to mold as well as ergosterol formation by the computational simulation against Sterol 14-alpha demethylase (CYP51) and inhibition of ergosterol biosynthesis by in-vitro model show that the Probe II completely inhibits the formation of ergosterol in yeast cells at 2× MIC. The ADMET analysis Probe II could be moderately toxic to the human being, though the in-vitro toxicity studies will help to understand the real-time toxic level. The novel compound Probe II, which was synthesized during the study, shows promise for development into a new generation of drug treatments aimed at addressing the emerging drug resistance in Candida sp.

A new crustin homologue (SpCrus6) involved in the antimicrobial and antiviral innate immunity in mud crab, Scylla paramamosain

Peptidoglycan recognition protein (PGRP) is conserved from insects to mammals. In insects, PGRP recognizes bacterial cell wall peptidoglycan (PGN) and activates prophenoloxidase cascade, a part of the insect antimicrobial defense system. Because mammals do not have the prophenoloxidase cascade, its function in mammals is unknown. However, it was suggested that an identical protein (Tag7) was a tumor necrosis factor-like cytokine. Therefore, the aim of this study was to identify the function of PGRP in mammals. Mouse PGRP bound to PGN with fast kinetics and nanomolar affinity (K(d) = 13 nm). The binding was specific for polymeric PGN or Gram-positive bacteria with unmodified PGN, and PGRP did not bind to other cell wall components or Gram-negative bacteria. PGRP mRNA and protein were expressed in neutrophils and bone marrow cells, but not in spleen cells, mononuclear cells, T or B lymphocytes, NK cells, thymocytes, monocytes, and macrophages. PGRP was not a PGN-lytic or a bacteriolytic enzyme, but it inhibited the growth of Gram-positive but not Gram-negative bacteria. PGRP inhibited phagocytosis of Gram-positive bacteria by macrophages, induction of oxidative burst by Gram-positive bacteria in neutrophils, and induction of cytokine production by PGN in macrophages. PGRP had no tumor necrosis factor-like cytotoxicity for mammalian cells, and it was not chemotactic on its own or in combination with PGN. Therefore, mammalian PGRP binds to PGN and Gram-positive bacteria with nanomolar affinity, is expressed in neutrophils, and inhibits growth of bacteria.

Mumijo is a traditional medicine used in Central Asia thousands of years. Its beneficial effect is exerted on metabolic processes and on the human immune system. It is also used as an antiseptic agent. The in vitro antibacterial activity of a mumijo preparation was investigated with agar diffusion technique against 25 Gram-positive and 6 Gram-negative bacterial isolates representing 13 different genera. Effect of different media and pH on the antibacterial action was also studied. Different concentrations of mumijo exerted bactericid and/or bacteriostatic effect against 3 Gram-negative (Acidovorax delafieldii, Serratia marcescens, Variovorax paradoxus) and 15 Gram-positive bacterial isolates (Agrococcus jenensis, Arthrobacter globiformis, Bacillus benzoevorans, Bacillus cereus var. mycoides, Bacillus megaterium,Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus) on the complete media. The maximum inhibitory potential of mumijo was observed at neutral pH (pH=7.4). Antibacterial effect of mumijo was not observed when minimal media were used for cultivation. The experimental data revealed that mumijo has remarkable growth inhibition effect against Gram-positive bacteria on complete medium at neutral pH. Key words: Mumijo, Gram-positive bacteria, Gram-negative bacteria, bacteriostatic effect, bactericid effect.

The aim of the present study was to evaluate the qualitative analysis of phytochemicals and antimicrobial activity of various solvent extracts of Boerhaavia diffusa L. (Family: Nyctaginaceae) leaves. The antimicrobial activity of different solvent extracts of B. diffusa L. leaves were tested against the Gram-positive and Gram-negative bacterial strains by observing the zone of inhibition. The Gram‑positive bacteria used in the test were Staphylococcus aureus, Bacillus subtilis, Streptococcus faecalis and Micrococcus luteus, and the Gram-negative bacteria were Escherichia coli, Pseudomonas aeruginosa, Salmonella typhii, Klebsiella pneumoniae, Proteus vulgaris, Serratia marcescens, Shigella flexneri and Vibrio cholerae. It was observed that ethanol, methanol, chloroform, ethylacetate and aqueous extracts showed activity against Gram-positive and Gram-negative bacteria. The ethanol extract of B. diffusa L. leaves showed more activity against Gram-positive (e.g. S. aureus, zone of diameter 11 mm) and Gram-negative bacteria (e.g. E. coli, zone of diameter 9 mm) when compared to other solvent extracts except V. cholerae. The results confirmed the presence of antibacterial activity of B. diffusa L. leaves extract against various human pathogenic bacteria. Key words: Antibacterial, Boerhaavia diffusa L., infectious diseases, inhibition zones, medicinal plants, Nyctaginaceae

Gram-positive bacteria are part of the normal flora of fish from different aquatic environments. They are mesophilic bacteria and demonstrate optimum growth at ambient temperature. In the sub-tropics, marine fish are caught from seas at temperatures of 16 to 34C, they are usually not iced and are handled at ambient temperature. It was hypothesized that under these conditions Gram-positive bacteria will be abundant in sub-tropical marine fish and will have roles in the spoilage of fish. A review of literature showed that there is a gap in understanding the Gram-positive bacterial populations in sub-tropical marine fish. This is partly due to the fact that the selective media used for isolating Gram-positive bacteria have limitations. Ecological and speciation studies have revealed that the ecology and speciation of many Gram-positive bacteria have not been clearly elucidated. The effect of ambient storage on the individual genera and species of Gram-positive bacteria in fish has been rarely studied. The spoilage potential of Gram-positive bacteria of marine fish origin has not been clearly determined. Therefore, the main aims of this study were to isolate Gram-positive bacteria from fresh and ambient-temperature-stored sub-tropical marine fish, speciate the isolates and study the spoilage potential of the isolates. The practical components of this study were conducted in four parts. The first part dealt with validation of tryptone soya agar with 0.25% phenylethyl alcohol (PEA-TSA) to enumerate Gram-positive bacteria. The second part enumerated Gram-positive bacteria from the muscles, gills and gut of Pseudocaranx dentex (Silver Trevally), Pagrus auratus (Snapper) and Mugil cephalus (Sea Mullet) stored at 25C for 15 hours using PEA-TSA. The third part dealt with the speciation of the isolates using appropriate methods such as polymerase chain reaction, 16S rRNA gene sequence, the VITEK JR system and conventional biochemical methods. In the fourth part, the isolates were assayed qualitatively for their ability to produce volatile sulphur compounds (VSC), reduce trimethylamine oxide and decarboxylate histidine, lysine and ornithine at mesophilic temperature, 32C. Initial studies indicated that PEA-TSA significantly (P 0.05). Gram-positive aerobic bacterial counts (GABC) significantly (P 0.05) differences were found in GABC between muscles and gills. Moreover, there were no significant differences (P> 0.05) in GABC between fish species during storage. In total, 390 bacteria were isolated from the fresh and stored fish; 339 isolates (87%) were found to be Gram-positive. Two hundred and sixty-six isolates (78%) of Gram-positive bacteria were identified to fall into 13 genera, namely Staphylococcus, Micrococcus, Bacillus, Virgibacillus, Brevibacillus, Corynebacterium, Streptococcus, Enterococcus, Aerococcus, Exiguobacterium, Carnobacterium, Vagococcus and Sporosarcina and 30 species. In fresh fish, Staphylococcus epidermidis and Micrococcus luteus were the most frequent isolates. The effect of storage at 25C for 15 hours resulted in a change of Gram-positive bacterial populations; while S. epidermidis, S. xylosus and Bacillus megaterium were no longer present, S. warneri, B. sphaericus, Brevibacillus borstelensis, Enterococcus faecium and Streptococcus uberis increased. Three species, E. faecium, Str. uberis and B. sphaericus, were the most prevalent at the end of storage. Micrococcus luteus and S. warneri were the most prevalent isolates from Pseudocaranx dentex, but E. faecium and Str. uberis were the most frequently isolated from Pagrus auratus and Mugil cephalus. With respect to different parts of the fish body, E. faecium, Str. uberis and B. sphaericus were the most frequent isolates from the muscles, E. faecium, Str. uberis from the gills and M. luteus from the gut. Among the 228 isolates examined, Br. borstelensis 73, Br. borstelensis 291, Str. uberis 339, Vagococcus fluvialis 31 and Vag. fluvialis 132 produced VSC from sodium thiosulphate, cysteine and methionine. However, strains varied in sulphur source utilization. Exiguobacterium acetylicum 5, Exiguobacterium spp. 191, Carnobacterium spp. 338, Br. borstelensis 73, Br. borstelensis 291, Str. uberis 30, Str. uberis 339, Vag. fluvialis 31 and Vag. fluvialis 132 reduced TMAO. No histidine decarboxylase activity was found in the Gram-positive bacterial species tested. Lysine and ornithine were decarboxylated mainly by different strains of S. warneri, S. epidermidis and M. luteus. During ambient storage of fish, the frequency of lysine-decarboxylating bacteria increased and became more diverse after 5 hours of storage. Among fish species examined, the frequencies of lysine- and ornithine-decarboxylating bacteria were higher and more diverse in Pseudocaranx dentex than in Pagrus auratus and Mugil cephalus. This study found that Gram-positive bacteria were abundant and diverse in sub-tropical marine fish; however, their frequencies were affected by fish habitat and fish body part. Ambient temperature storage determined which Gram-positive bacterial species were dominant. With the exception of one isolate of S. aureus, Gram-positive bacteria isolated from sub-tropical marine fish caught from unpolluted water were not potential pathogens. The study also showed that Gram-positive bacteria had greater ability to decarboxylate lysine and ornithine than to produce VSC or reduce TMAO, and the spoilage potential of a bacterial species was a strain-dependent behaviour. This is a significant study as it is the first study on this aspect sub-tropical marine fish. It validated a selective medium that can be used to enumerate most Gram-positive bacteria from a marine environment. Most of the Gram-positive bacterial species from sub-tropical marine fish identified in this study were documented for the first time. The effects of ambient storage and the spoilage potential of Gram-positive bacteria from sub-tropical marine were clearly elucidated.

Aim: In vitro antibacterial activity of 6-substituted-3(2H)-pyridazinone-2-acetyl-2-(substituted/nonsubstitutedbenzal/ acetophenone) hydrazone derivatives were tested in common species causing hospital-acquired infections. Material and Method: Antimicrobial activities of the compounds were performed by determining minimum inhibitory concentration (MIC) value against four Gram-positive, five Gram-negative and four Candida species fungi. Modified serial microdilution method was carried out. Reference strains of American Type Culture Collection (ATCC) were used. Results: In general, eleven compounds exhibited considerable activity. Comparatively, compound 3 exhibited strong activity against Enterobacter hormaechei and 5, 11 were the most active against Acinetobacter baumannii at 31.25 μg/mL. Compounds 1,2,3,4,8 and 10 were found to be as active as positive control ampicillin trihidrate against Stenotrophomonas maltophilia. On the other hand, compounds 1,2,3,4,7,8,9,10 and 11 showed strong antifungal activitiy as much as fluconazole against Candida tropicalis. Compound 1 was mostly active against Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis. It was also revealed that the antifungal activity of compounds 1, 6, 7, 8 and 9 were higher than the others. Compound 1 and 8 exhibited the best activity against Candida glabrata and Candida parapsilosis respectively. Conclusions: All tested compounds showed better activity against Gram-negative bacteria and yeast than Gram-positive bacteria. These compounds may be considered as alternative antimicrobial agents in the treatment of multiple drug resistant Gram-negative, Gram-positive bacteria and fungal pathogens. Especially, we suggested that Compound 1 and 8 might be a promising candidate of new antifungal agents

Female vulvovaginitis was one of the most common gynecological diseases. It had a great negative impact on their work and quality of life. This retrospective study evaluated the clinical and laboratory data of patients with vulvovaginitis in Hangzhou, China. To analyze the clinical situation, species distribution and antibiotic resistance of pathogenic fungi and bacteria in 626 cases of vulvovaginitis in Hangzhou. Microorganism culture, identification, and antibiotic susceptibility testing were conducted. The study aimed to provide a theoretical value for an effective treatment of vulvovaginitis. In total, 626 outpatients and inpatients diagnosed with vulvovaginitis were selected from January 2018 to January 2023. Data of all the patients were collected from the hospital's electronic medical records. Vaginal secretion was collected for testing and SPSS 25.0 software was used to perform statistical analysis. A total of 626 strains of fungi, Gram-positive, and -negative bacteria were detected. Clinical situations of patients infected with the top five pathogenic fungi and bacteria were analyzed. Pathogenic fungi and bacteria were slightly different in each age group and in each onset time group. The results of antibiotic susceptibility testing showed that the resistance rates of itraconazole and fluconazole were high and Gram- negative and -positive bacteria were multidrug resistant. Gram-negative bacteria were more sensitive to carbenicillins and compound antibiotics, while Gram-positive bacteria were sensitive to rifampicin and daptomycin. MRSA and non vancomycin-resistant strains were detected. Fungi and bacteria were usually detected as pathogenes in patients with vulvovaginitis in Hangzhou. Some factors, such as age and onset time, often affected the incidence. Pathogenic fungi and bacteria were resistant to some common antibiotics, and clinical treatments should be carried out in a timely and reasonable manner according to the results of antibiotic susceptibility testing.

BackgroundTraditional antibiotics are increasingly suffering from the emergence of multidrug resistance amongst pathogenic bacteria leading to a range of novel approaches to control microbial infections being investigated as potential alternative treatments. One plausible antimicrobial alternative could be the combination of conventional antimicrobial agents/antibiotics with small molecules which block multidrug efflux systems known as efflux pump inhibitors. Bioassay-driven purification and structural determination of compounds from plant sources have yielded a number of pump inhibitors which acted against gram positive bacteria.Methodology/Principal FindingsIn this study we report the identification and characterization of 4′,5′-O-dicaffeoylquinic acid (4′,5′-ODCQA) from Artemisia absinthium as a pump inhibitor with a potential of targeting efflux systems in a wide panel of Gram-positive human pathogenic bacteria. Separation and identification of phenolic compounds (chlorogenic acid, 3′,5′-ODCQA, 4′,5′-ODCQA) was based on hyphenated chromatographic techniques such as liquid chromatography with post column solid-phase extraction coupled with nuclear magnetic resonance spectroscopy and mass spectroscopy. Microbial susceptibility testing and potentiation of well know pump substrates revealed at least two active compounds; chlorogenic acid with weak antimicrobial activity and 4′,5′-ODCQA with pump inhibitory activity whereas 3′,5′-ODCQA was ineffective. These intitial findings were further validated with checkerboard, berberine accumulation efflux assays using efflux-related phenotypes and clinical isolates as well as molecular modeling methodology.Conclusions/SignificanceThese techniques facilitated the direct analysis of the active components from plant extracts, as well as dramatically reduced the time needed to analyze the compounds, without the need for prior isolation. The calculated energetics of the docking poses supported the biological information for the inhibitory capabilities of 4′,5′-ODCQA and furthermore contributed evidence that CQAs show a preferential binding to Major Facilitator Super family efflux systems, a key multidrug resistance determinant in gram-positive bacteria.