A Review of the Antimicrobial Properties of Cyanobacterial Natural Products.

The development of multiple-drug-resistant pathogens has prompted medical research toward the development of new and effective antimicrobial therapies. Much research into novel antibiotics has focused on bacterial and fungal compounds, and on chemical modification of existing compounds to increase their efficacy or reactivate their antimicrobial properties. In contrast, cyanobacteria have been relatively overlooked for antibiotic discovery, and much more work is required. This may be because some cyanobacterial species produce environmental toxins, leading to concerns about the safety of cyanobacterial compounds in therapy. Despite this, several cyanobacterial-derived compounds have been identified with noteworthy inhibitory activity against bacterial, fungal and protozoal growth, as well as viral replication. Additionally, many of these compounds have relatively low toxicity and are therefore relevant targets for drug development. Of particular note, several linear and heterocyclic peptides and depsipeptides with potent activity and good safety indexes have been identified and are undergoing development as antimicrobial chemotherapies. However, substantial further studies are required to identify and screen the myriad other cyanobacterial-derived compounds to evaluate their therapeutic potential. This study reviews the known phytochemistry of cyanobacteria, and where relevant, the effects of those compounds against bacterial, fungal, protozoal and viral pathogens, with the aim of highlighting gaps in the literature and focusing future studies in this field.

The Art of War with Pseudomonas aeruginosa: Targeting Mex Efflux Pumps Directly to Strategically Enhance Antipseudomonal Drug Efficacy.

Pseudomonas aeruginosa (P. aeruginosa) poses a grave clinical challenge due to its multidrug resistance (MDR) phenotype, leading to severe and life-threatening infections. This bacterium exhibits both intrinsic resistance to various antipseudomonal agents and acquired resistance against nearly all available antibiotics, contributing to its MDR phenotype. Multiple mechanisms, including enzyme production, loss of outer membrane proteins, target mutations, and multidrug efflux systems, contribute to its antimicrobial resistance. The clinical importance of addressing MDR in P. aeruginosa is paramount, and one pivotal determinant is the resistance-nodulation-division (RND) family of drug/proton antiporters, notably the Mex efflux pumps. These pumps function as crucial defenders, reinforcing the emergence of extensively drug-resistant (XDR) and pandrug-resistant (PDR) strains, which underscores the urgency of the situation. Overcoming this challenge necessitates the exploration and development of potent efflux pump inhibitors (EPIs) to restore the efficacy of existing antipseudomonal drugs. By effectively countering or bypassing efflux activities, EPIs hold tremendous potential for restoring the antibacterial activity against P. aeruginosa and other Gram-negative pathogens. This review focuses on concurrent MDR, highlighting the clinical significance of efflux pumps, particularly the Mex efflux pumps, in driving MDR. It explores promising EPIs and delves into the structural characteristics of the MexB subunit and its substrate binding sites.

Hamamelis virginiana L. Leaf Extracts Inhibit the Growth of Antibiotic-Resistant Gram-Positive and Gram-Negative Bacteria.

Virginian witch hazel (WH; Hamamelis virginiana L.; family: Hamamelidaceae) is a North American plant that is used traditionally to treat a variety of ailments, including bacterial infections. Solvents of varying polarity (water, methanol, ethyl acetate, hexane and chloroform) were used to prepare extracts from this plant. Resuspensions of each extract in an aqueous solution were tested for growth-inhibitory activity against a panel of bacteria (including three antibiotic-resistant strains) using agar disc diffusion and broth microdilution assays. The ethyl acetate, hexane and chloroform extracts were completely ineffective. However, the water and methanolic extracts were good inhibitors of E. coli, ESBL E. coli, S. aureus, MRSA, K. pneumoniae and ESBL K. pneumoniae growth, with the methanolic extract generally displaying substantially greater potency than the other extracts. Combining the active extracts with selected conventional antibiotics potentiated the bacterial growth inhibition of some combinations, whilst other combinations remained non-interactive. No synergistic or antagonistic interactions were observed for any WH extracts/antibiotic combinations. Gas chromatography-mass spectrometry analysis of the extracts identified three molecules of interest that may contribute to the activities observed, including phthalane and two 1,3-dioxolane compounds. Putative modes of action of the active WH extracts and these molecules of interest are discussed herein.

Technology-assisted viva voce exams: A novel approach aimed at addressing student anxiety and assessor burden in oral assessment.

BACKGROUND AND PURPOSE: Viva voce (VIVA) exams are resource intensive, can be prone to inter-rater reliability issues, and induce anxiety in many students. Costs, reliability, validity, and student welfare have been targeted for VIVA re-design. The objective of this study is to design and assess if a less labour-intensive approach to VIVA exams is acceptable to students, reducing student anxiety, whilst maintaining authenticity of the assessment. EDUCATIONAL ACTIVITY AND SETTING: The School of Pharmacy and Medical Sciences (Griffith University) delivers undergraduate and postgraduate pharmacy degrees, which contain multiple VIVAs. We have designed and implemented a modified VIVA called the technology-assisted VIVA exam (TaVIVA) utilising remote recording, retrospective marking, and pre-recorded multimedia delivered questions to test student acceptability, impact on student anxiety, and inform potential delivery as a summative assessment. FINDINGS: Student responses were overwhelmingly positive, reporting satisfaction with the TaVIVA. There was strong agreement that the school should continue to develop the TaVIVA. Students perceived that it was fairer than traditional VIVAs and less anxiety inducing. However, students indicated that the traditional VIVA was more authentic and that they eventually need to conduct a VIVA in the presence of an assessor. SUMMARY: The TaVIVA is an innovative assessment approach with potential benefits over the traditional VIVA, including facilitation of assessment consistency and reductions in student anxiety. We postulate that the TaVIVA is a useful and valid means of scaffolding student performance in VIVA assessment.

An assessment of the growth inhibition profiles of Hamamelis virginiana L. extracts against Streptococcus and Staphylococcus spp.

Staphylococcal and streptococcal species trigger a wide variety of infections involving epithelial tissues. Virginian witch hazel (WH; Hamamelis virginiana L.; family: Hamamelidaceae) is a plant that has been used traditionally by Native Americans to treat a variety of skin conditions. Extracts from the leaves were examined for their inhibitory effects on these bacterial species. Solvents of different polarity (water, methanol, ethyl acetate, hexane and chloroform) were used to prepare extracts from WH leaves, and the aqueous resuspensions were screened for antibacterial activities using disc diffusion and liquid dilution assays. Extract phytochemical profiles and toxicities were also examined, and combinations of extracts with conventional antibiotics were tested against each bacterial strain. The methanolic and aqueous extracts inhibited the growth of S. oralis, S. pyogenes, S. epidermidis and S. aureus, but not S. mutans. The extracts were especially active against staphylococcal species, with MIC values between 200 and 500 µg/ml. Combinations of active extracts with conventional antibiotics failed to yield beneficial interactions, except for two cases where additive interactions were observed (aqueous WH extract combined with chloramphenicol against S. oralis, and methanolic WH extract combined with ciprofloxacin against S. aureus). Phytochemical assays indicated an abundance of tannins, triterpenoids and phenolics in the water and methanol extracts, with trace amounts of these components in the ethyl acetate extract. Phytochemicals were not detected in hexane and chloroform extracts. Thus, phytochemical abundance in extracts was concordant with antibacterial activities. All extracts were found to be non-toxic in Artemia nauplii assays. These findings indicate the potential for WH leaf extracts for clinical use in treating staphylococcal and streptococcal infections, while substantiating their traditional Native American uses.

Use of specific combinations of the triphala plant component extracts to potentiate the inhibition of gastrointestinal bacterial growth.

ETHNOPHARMACOLOGICAL RELEVANCE: Triphala is used in Ayurveda to treat a wide variety of diseases, including numerous bacterial infections. Interestingly, the plant components of triphala (Terminalia bellirica, Terminalia chebula and Emblica officinalis) are also good inhibitors of bacterial growth when used individually, yet plant preparations are generally used in combination in traditional medicine. Surprisingly, no previous studies have addressed the reason why the combination is preferred over the individual components to treat bacterial infections. AIM OF THE STUDY: To test and compare the antibacterial efficacy of triphala and its component parts to quantify their relative efficacies. The individual plant components will also be tested as combinations, thereby determining whether combining the individual components potentiates the antibacterial activity of the components used alone. MATERIALS AND METHODS: Triphala and the three individual plant components were extracted using solvents of varying polarity (methanol, water, ethyl acetate) and the antibacterial activity of the aqueous resuspensions was quantified by disc diffusion and broth microdilution MIC assays. Combinations of extracts produced from the individual components were also tested against each bacterial species and the ΣFICs was calculated to determine the class of interaction. Where synergy was detected, isobologram analysis was used to determine the optimal component ratios. The Artemia nauplii bioassay was used to test for toxicity and GC-MS headspace profiling analysis was used to highlight terpenoid components that may contribute to the antibacterial activity of triphala. RESULTS: The aqueous and methanolic triphala, T. bellirica, T. chebula and E. officinalis extracts displayed good inhibitory activity against all bacterial strains, with MICs often in the 250-750 µg/mL range. The methanolic extracts were generally more potent than the aqueous extracts and T. chebula was the most potent of the individual plant components. Combining the extracts of the different plant species resulted in potentiation of the growth inhibitory activity of most combinations compared to that of the individual components. Indeed, with the exception of S. flexneri, all bacterial species were potentiated by at least one combination of methanolic plant extracts, with a substantial proportion of these displaying synergistic interactions. All extracts were found to be either non-toxic, or of low to moderate toxicity in Artemia nauplii assays. CONCLUSION: Whilst the individual plant components of triphala all inhibit the growth of multiple pathogenic bacteria, the activity is potentiated for multiple combinations. Therefore, the traditional usage of the combination of the three plant materials in triphala not only extends the activity profile of the mixture over that of the individual components, but it also substantially potentiates the inhibitory activity towards multiple bacteria, partially explaining the preference of triphala compared to the individual components.

Terminalia ferdinandiana Fruit and Leaf Extracts Inhibit Methicillin-Resistant Staphylococcus aureus Growth.

The development of multiple antibiotic-resistant bacteria has vastly depleted our repertoire of effective antibiotic chemotherapies. The development of multi-ß-lactam-resistant strains are particularly concerning due to our previous reliance on this class of antibiotics because of their initial efficacy and broad-spectrum activity. With increases in extended-spectrum ß-lactam-resistance and an expanded resistance to other classes of antibiotics, there is an urgent need for the development of effective new antibiotic therapies. Terminalia ferdinandiana is an endemic Australian plant known for its high antioxidant and tannin contents. T. ferdinandiana fruit and leaf extracts have strong antibacterial activity against a wide variety of bacterial pathogens. However, T. ferdinandiana extracts have not been tested against ESBL and MRSA antibiotic-resistant pathogens. An objective of this study was to screen T. ferdinandiana fruit and leaf extracts for bacterial growth inhibitory activity by disc diffusion assay against ß-lactam-sensitive and -resistant E. coli strains and against methicillin-sensitive and -resistant S. aureus. The minimum inhibitory concentration (MIC) was quantified by liquid dilution techniques. The fruit methanolic extract, as well as the methanolic, aqueous, and ethyl acetate leaf extracts strongly inhibited the growth of the MRSA, with MICs as low as 223 µg/mL. In contrast, the extracts were ineffective inhibitors of ESBL growth. Metabolomic fingerprint analysis identified a diversity and relative abundance of tannins, flavonoids, and terpenoids, several of which have been reported to inhibit MRSA growth in isolation. All extracts were nontoxic in the Artemia nauplii and HDF toxicity assays, further indicating their potential for medicinal use.

Developing New Antimicrobial Therapies: Are Synergistic Combinations of Plant Extracts/Compounds with Conventional Antibiotics the Solution?

The discovery of penicillin nearly 90 years ago revolutionized the treatment of bacterial disease. Since that time, numerous other antibiotics have been discovered from bacteria and fungi, or developed by chemical synthesis and have become effective chemotherapeutic options. However, the misuse of antibiotics has lessened the efficacy of many commonly used antibiotics. The emergence of resistant strains of bacteria has seriously limited our ability to treat bacterial illness, and new antibiotics are desperately needed. Since the discovery of penicillin, most antibiotic development has focused on the discovery of new antibiotics derived from microbial sources, or on the synthesis of new compounds using existing antibiotic scaffolds to the detriment of other lines of discovery. Both of these methods have been fruitful. However, for a number of reasons discussed in this review, these strategies are unlikely to provide the same wealth of new antibiotics in the future. Indeed, the number of newly developed antibiotics has decreased dramatically in recent years. Instead, a reexamination of traditional medicines has become more common and has already provided several new antibiotics. Traditional medicine plants are likely to provide further new antibiotics in the future. However, the use of plant extracts or pure natural compounds in combination with conventional antibiotics may hold greater promise for rapidly providing affordable treatment options. Indeed, some combinational antibiotic therapies are already clinically available. This study reviews the recent literature on combinational antibiotic therapies to highlight their potential and to guide future research in this field.

Soluble and membrane-bound Drosophila melanogaster CYP6G1 expressed in Escherichia coli: purification, activity, and binding properties toward multiple pesticides.

Cytochrome P450 CYP6G1 has been implicated in the resistance of Drosophila melanogaster to numerous pesticides. While in vivo and in vitro studies have provided insight to the diverse functions of this enzyme, direct studies on the isolated CYP6G1 enzyme have not been possible due to the need for a source of recombinant enzyme. In the current study, the Cyp6g1 gene was isolated from D. melanogaster and re-engineered for heterologous expression in Escherichia coli. Approximately 460 nmol L⁻¹ of P450 holoenzyme were obtained in 500 mL cultures. The recombinant enzyme was located predominantly within the bacterial cytosol. A two-step purification protocol using Ni-chelate affinity chromatography followed by removal of detergent on a hydroxyapatite column produced essentially homogenous enzyme from both soluble and membrane fractions. Recombinant CYP6G1 exhibited p-nitroanisole O-dealkylation activity but was not active against eleven other typical P450 marker substrates. Substrate-induced binding spectra and IC50 values for inhibition of p-nitroanisole O-dealkylation were obtained for a wide selection of pesticides, namely DDT, imidacloprid, chlorfenvinphos, malathion, endosulfan, dieldrin, dicyclanil, lufenuron and carbaryl, supporting previous in vivo and in vitro studies on Drosophila that have suggested that the enzyme is involved in multi-pesticide resistance in insects.

Intramolecular heme ligation of the cytochrome P450 2C9 R108H mutant demonstrates pronounced conformational flexibility of the B-C loop region: implications for substrate binding.

A previous study [Dickmann, L., et al. (2004) Mol. Pharmacol. 65, 842-850] revealed some unusual properties of the R108H mutant of cytochrome P450 2C9 (CYP2C9), including elevated thermostability relative to that of CYP2C9, as well as a UV-visible absorbance spectrum that was indicative of nitrogenous ligation to the heme iron. In our study, size-exclusion chromatography and UV-visible absorbance spectroscopy of CYP2C9 R108H monomers demonstrated that nitrogen ligation is indeed intramolecular. Pulsed electron paramagnetic resonance of CYP2C9 R108H monomers showed that a histidine is most likely bound to the heme as previously hypothesized. An energy-minimized model of the R108H mutant maintained a CYP fold, despite substantial movement of several loop regions of the mutant, and, therefore, represents an extreme example of a closed conformation of the enzyme. Molecular dynamics (MD) simulations of CYP2C9 were performed to study the range of energetically accessible CYP2C9 conformations. These in silico studies showed that the B-C loop region of CYP2C9 moves away from the heme to a position resembling the putative open conformation described for rabbit CYP2B4. A model involving the movement of the B-C loop region and R108 between the open and closed conformations of CYP2C9 is presented, which helps to explain the enzyme's ability to regio- and stereospecifically metabolize some ligands while allosterically activating others.

Cerivastatin in vitro metabolism by CYP2C8 variants found in patients experiencing rhabdomyolysis.

OBJECTIVES: Cerivastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor withdrawn from the market because of serious adverse effects, is metabolized primarily by CYP2C8. The occurrence of associated myotoxicity and rhabdomyolysis were attributed to altered cerivastatin pharmacokinetics on account of gemfibrozil-inhibition or genetic variations in CYP2C8 and drug transporters involved in cerivastatin clearance. However, the effect of CYP2C8 genetic variation on cerivastatin metabolism has not been fully elucidated. METHODS: In this study, patients (n=126) with confirmed cases of rhabdomyolysis after cerivastatin administration had their CYP2C8 gene resequenced and the metabolism of cerivastatin by the discovered CYP2C8 variants was assessed in proteins expressed in Escherichia coli. RESULTS: In this unique patient population, 12 novel single nucleotide polymorphisms were discovered of which six were exclusively found in patients not using gemfibrozil. Three rare exonic variants resulted in amino acid substitutions and a frame shift deletion (V472fsL494 generating a defective mostly heme-free CYP2C8 protein). A particular promoter located deletion (-635_-634delTA) was tightly linked to CYP2C8*3. Heterologously expressed CYP2C8.3 and CYP2C8.4 displayed an increase in cerivastatin metabolic clearance of up to six-fold compared with the wild-type enzyme. Similarly, an independent sample of microsomes from human livers carrying the CYP2C8*3 and CYP2C8*4 alleles exhibited a 2-fold to 14-fold increase in normalized cerivastatin intrinsic clearance, compared with microsomes from livers carrying only the wild type allele. CONCLUSION: Gain or loss of catalytic function found in the CYP2C8 gene could certainly alter cerivastatin pharmacokinetics and may influence, at least in part, susceptibility to the development of myotoxicity.

Heterologous expression of the methyl carbamate-degrading hydrolase MCD.

The methyl carbamate-degrading hydrolase (MCD) of Achromobacter WM111 has considerable potential as a pesticide bioremediation agent. However this potential has been unrealisable until now because of an inability to express MCD in heterologous hosts such as Escherichia coli. Herein, we describe the first successful attempt to express appreciable quantities of MCD in active form in E. coli, and the subsequent characterisation of the heterologously expressed material. We find that the properties of this material closely match the previously reported properties of MCD produced from Achromobacter WM111. This includes the presence of two distinct forms of the enzyme that we show are most likely due to the presence of two functional translational start sites. The purified enzyme catalyses the hydrolysis of a carbamate (carbaryl), a carboxyl ester (alpha-naphthyl acetate) and a phophotriester (dimethyl umbelliferyl phosphate) and it is relatively resistant to thermal and solvent-mediated denaturation. The robust nature and catalytic promiscuity of MCD suggest that it could be exploited for various biotechnological applications.

The enzymatic basis for pesticide bioremediation.

Enzymes are central to the biology of many pesticides, influencing their modes of action, environmental fates and mechanisms of target species resistance. Since the introduction of synthetic xenobiotic pesticides, enzymes responsible for pesticide turnover have evolved rapidly, in both the target organisms and incidentally exposed biota. Such enzymes are a source of significant biotechnological potential and form the basis of several bioremediation strategies intended to reduce the environmental impacts of pesticide residues. This review describes examples of enzymes possessing the major activities employed in the bioremediation of pesticide residues, and some of the strategies by which they are employed. In addition, several examples of specific achievements in enzyme engineering are considered, highlighting the growing trend in tailoring enzymatic activity to a specific biotechnologically relevant function.

Selective induction of intestinal CYP3A23 by 1alpha,25-dihydroxyvitamin D3 in rats.

Enhancement of CYP3A transcription in both the small intestine and liver of the mouse by activation of a VDR signaling pathway was shown recently by Makishima et al. (Science, 2002). However, in humans and rats, hepatic VDR content is much lower than that found in small intestine, suggesting the possibility of tissue-selective responses to 1,25(OH)(2)D(3). The purpose of this study was to determine the effect of 1,25(OH)(2)D(3) on intestinal and hepatic CYP3A expression in the rat. We found that an acute intraperitoneal treatment (every 48 h) in adult male rats with 1,25(OH)(2)D(3) induced CYP3A transcription selectively in small intestine, but not in liver. At a dose of 100 ng, there was a 6.6-fold increase in intestinal CYP3A23 mRNA after the third treatment (p < 0.05). There were concordant effects of 1,25(OH)(2)D(3) treatment on intestinal CYP3A23 protein levels; 2.2-fold (p < 0.05), 3.5-fold (p < 0.05) and 4.8-fold (p < 0.01) increase following 1-3 doses of 100 ng 1,25(OH)(2)D(3), respectively. In contrast, there was no significant change of CYP3A23 protein content in liver at the 1,25(OH)(2)D(3) doses tested. In support of these findings, there was a 366-fold and 77-fold higher level of VDR mRNA expression in the respective rat and human jejunal mucosa, compared to the liver. These data suggest that the human liver will be less sensitive than the intestine to the transcriptional effects of 1,25(OH)(2)D(3) and that this regulatory pathway may contribute to inter-individual variability in constitutive intestinal CYP3A4 expression.

Sites of covalent attachment of CYP4 enzymes to heme: evidence for microheterogeneity of P450 heme orientation.

Typical cytochrome P450s secure the heme prosthetic group with a cysteine thiolate ligand bound to the iron, electrostatic interactions with the heme propionate carboxylates, and hydrophobic interactions with the heme periphery. In addition to these interactions, CYP4B1 covalently binds heme through a monoester link furnished, in part, by a conserved I-helix acid, Glu310. Chromatography, mass spectrometry, and NMR have now been utilized to identify the site of attachment on the heme. Native CYP4B1 covalently binds heme solely at the C-5 methyl position. Unexpectedly, recombinant CYP4B1 from insect cells and Escherichia coli also bound their heme covalently at the C-8 methyl position. Structural heterogeneity may be common among recombinant CYP4 proteins because CYP4A3 exhibited this duality. Attempts to evaluate functional heterogeneity were complicated by the complexity of the system. The phenomenon of covalent heme binding to P450 provides a novel method for assessing microheterogeneity in heme orientation and raises questions about the fidelity of heme incorporation in recombinant systems.

Critical role of histidine residues in cyclohexanone monooxygenase expression, cofactor binding and catalysis.

Cyclohexanone monooxygenase (CMO) is a member of the flavin monooxygenase superfamily of enzymes that catalyze both nucleophilic and electrophilic reactions involving a common C4a hydroperoxide intermediate. To begin to probe structure-function relationships for these enzymes, we investigated the roles of histidine residues in CMO derived from Acinetobacter NCIB 9871, with particular emphasis on the wholly conserved residue, His163 (H163). CMO activity was readily inactivated by diethyl pyrocarbonate (DEPC), a selective chemical modifier of histidine residues. Each of the seven histidines in CMO was then individually mutated to glutamine and the mutants expressed and purified from Escherichia coli. Only the H59Q mutant failed to express at significant levels. The H96Q enzyme was found to have a greatly reduced flavin adenine dinucleotide (FAD) content, indicative of compromised cofactor retention. The only significant effect on kcat occurred with the H163Q mutant, which exhibited an approximately 10-fold lower turnover of the prototypical substrate, cyclohexanone. This was accompanied by a doubling in the Km [NADPH] compared to the wild-type enzyme, suggesting that the functional decrement in H163Q is probably not solely a reflection of impaired NADPH binding. These data establish a critical role for H163 in CMO catalysis and prompt the hypothesis that this conserved residue plays a similarly important functional role across the flavin monooxygenase family of enzymes.

Rabbit CYP4B1 engineered for high-level expression in Escherichia coli: ligand stabilization and processing of the N-terminus and heme prosthetic group.

Modifications at the N-terminus of the rabbit CYP4B1 gene resulted in expression levels in Escherichia coli of up to 660 nmol/L. Solubilization of the enzyme from bacterial membranes led to substantial conversion to cytochrome P420 unless alpha-naphthoflavone was added as a stabilizing ligand. Mass spectrometry analysis and Edman sequencing of purified enzyme preparations revealed differential N-terminal post-translational processing of the various constructs expressed. Notably, bacterial expression of CYP4B1 produced a holoenzyme with >98.5% of its heme prosthetic group covalently linked to the protein backbone. The near fully covalently linked hemoproteins exhibited similar rates and regioselectivities of lauric acid hydroxylation to that observed previously for the partially heme processed enzyme expressed in insect cells. These studies shed new light on the consequences of covalent heme processing in CYP4B1 and provide a facile system for future mechanistic and structural studies with the enzyme.