Bioprospecting for Antibacterial Drugs: a Multidisciplinary Perspective on Natural Product Source Material, Bioassay Selection and Avoidable Pitfalls.

Bioprospecting is the exploration, extraction and screening of biological material and sometimes indigenous knowledge to discover and develop new drugs and other products. Most antibiotics in current clinical use (eg. ß-lactams, aminoglycosides, tetracyclines, macrolides) were discovered using this approach, and there are strong arguments to reprioritize bioprospecting over other strategies in the search for new antibacterial drugs. Academic institutions should be well positioned to lead the early stages of these efforts given their many thousands of locations globally and because they are not constrained by the same commercial considerations as industry. University groups can lack the full complement of knowledge and skills needed though (eg. how to tailor screening strategy to biological source material). In this article, we review three key aspects of the bioprospecting literature (source material and in vitro antibacterial and toxicity testing) and present an integrated multidisciplinary perspective on (a) source material selection, (b) legal, taxonomic and other issues related to source material, (c) cultivation methods, (d) bioassay selection, (e) technical standards available, (f) extract/compound dissolution, (g) use of minimum inhibitory concentration and selectivity index values to identify progressible extracts and compounds, and (h) avoidable pitfalls. The review closes with recommendations for future study design and information on subsequent steps in the bioprospecting process.

Colistin causes profound morphological alteration but minimal cytoplasmic membrane perforation in populations of Escherichia coli and Pseudomonas aeruginosa.

Whilst colistin (polymyxin E) represents the last mainstream treatment option for multidrug-resistant Gram-negative pathogens, details of its mechanism of action remain to be fully resolved. In this study, the effects of sub-inhibitory, inhibitory-bactericidal, and supra-bactericidal levels of colistin on the membrane integrity and morphology of Escherichia coli and Pseudomonas aeruginosa were investigated using potassium loss, flow cytometry, and scanning electron microscopy (SEM). Supra-bactericidal colistin concentrations induced just 4-12% intracellular potassium loss from bacteria after 24 h. Flow cytometry data suggested colistin might alter cell arrangement, and SEM confirmed the antibiotic causes bacterial aggregation. Filamentation was not detected in either species at any concentration or time-point up to 24 h. These results argue against the hypotheses that colistin kills bacteria by puncturing the cytoplasmic membrane or disrupting DNA synthesis. The colistin-induced bacterial aggregation detected has implications for the interpretation of MBC, time-kill, and other test results obtained with this antibiotic.

Morphological and ultrastructural changes in bacterial cells as an indicator of antibacterial mechanism of action.

Efforts to reduce the global burden of bacterial disease and contend with escalating bacterial resistance are spurring innovation in antibacterial drug and biocide development and related technologies such as photodynamic therapy and photochemical disinfection. Elucidation of the mechanism of action of these new agents and processes can greatly facilitate their development, but it is a complex endeavour. One strategy that has been popular for many years, and which is garnering increasing interest due to recent technological advances in microscopy and a deeper understanding of the molecular events involved, is the examination of treated bacteria for changes to their morphology and ultrastructure. In this review, we take a critical look at this approach. Variables affecting antibacterial-induced alterations are discussed first. These include characteristics of the test organism (e.g. cell wall structure) and incubation conditions (e.g. growth medium osmolarity). The main body of the review then describes the different alterations that can occur. Micrographs depicting these alterations are presented, together with information on agents that induce the change, and the sequence of molecular events that lead to the change. We close by highlighting those morphological and ultrastructural changes which are consistently induced by agents sharing the same mechanism (e.g. spheroplast formation by peptidoglycan synthesis inhibitors) and explaining how changes that are induced by multiple antibacterial classes (e.g. filamentation by DNA synthesis inhibitors, FtsZ disruptors, and other types of agent) can still yield useful mechanistic information. Lastly, recommendations are made regarding future study design and execution.

Alkaloids: an overview of their antibacterial, antibiotic-enhancing and antivirulence activities.

With reports of pandrug-resistant bacteria causing untreatable infections, the need for new antibacterial therapies is more pressing than ever. Alkaloids are a large and structurally diverse group of compounds that have served as scaffolds for important antibacterial drugs such as metronidazole and the quinolones. In this review, we highlight other alkaloids with development potential. Natural, semisynthetic and synthetic alkaloids of all classes are considered, looking first at those with direct antibacterial activity and those with antibiotic-enhancing activity. Potent examples include CJ-13,136, a novel actinomycete-derived quinolone alkaloid with a minimum inhibitory concentration of 0.1 ng/mL against Helicobacter pylori, and squalamine, a polyamine alkaloid from the dogfish shark that renders Gram-negative pathogens 16- to >32-fold more susceptible to ciprofloxacin. Where available, information on toxicity, structure-activity relationships, mechanisms of action and in vivo activity is presented. The effects of alkaloids on virulence gene regulatory systems such as quorum sensing and virulence factors such as sortases, adhesins and secretion systems are also described. The synthetic isoquinoline alkaloid virstatin, for example, inhibits the transcriptional regulator ToxT in Vibrio cholerae, preventing expression of cholera toxin and fimbriae and conferring in vivo protection against intestinal colonisation. The review concludes with implications and limitations of the described research and directions for future research.

Potassium loss from chlorhexidine-treated bacterial pathogens is time- and concentration-dependent and variable between species.

The membrane-active antimicrobial agent chlorhexidine is used extensively as an antiseptic during infection prophylaxis and treatment. Whilst known to induce membrane damage that results in loss of internal solutes from bacteria, the present study sought to determine the rate and extent of cytoplasmic potassium loss and whether any species-specific differences exist. Direct measurement of potassium was achieved using flame emission spectrophotometry. Exposure of selected species to minimum inhibitory (MIC) or minimum bactericidal concentration (MBC) resulted in solute loss that was both concentration and time dependent. Within 5-min treatment with MIC levels, losses of 3 % from P. aeruginosa, 9 % from E. coli, and 15 % from S. aureus were recorded, whilst at 5 % w/v chlorhexidine, elevated loss of 20, 28, and 41 % occurred, respectively. Nonlinear potassium release was evident from all species when treated with 5 % chlorhexidine over a 60-min period. After this contact time, potassium loss from E. coli and S. aureus rose to 93 or 90 %, respectively; in contrast, P. aeruginosa retained 62 % intracellular potassium. Results confirm lethal concentrations of chlorhexidine induce rapid and substantial loss of cytoplasmic potassium from common pathogens. However, bacterial responses vary between species and should be borne in mind when considering mechanism of action.

Potential applications for Annona squamosa leaf extract in the treatment and prevention of foodborne bacterial disease.

Foodborne disease is a major public health problem. The present study examined Annona squamosa leaves, which are traditionally used to treat diarrhea and other infections, for their potential to be used in modern food safety or medicine. Active constituents were partially purified by ethanol extraction and column chromatography. MICs of the extract were 62.5 to 125 microg/mL against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus, and 250 microg/mL against Campylobacter jejuni. In time-kill assays, 500 microg/mL of the extract reduced colony forming unit numbers of C. jejuni almost 10 000-fold within 12 hours. Similar decreases were seen against B. cereus, but over a longer time-frame. LC-MS analysis indicated the presence of reticuline and oxophoebine. Assessment of stability by MIC assay showed activity was heat-labile, with loss of activity greatest following high temperature treatments. Activity was relatively stable at refrigeration temperature. These results indicate A. squamosa has broad-spectrum but heat-labile activity against foodborne bacterial pathogens, and bactericidal activity against B. cereus and C. jejuni. This bactericidal activity is not sufficiently rapid for A. squamosa to be used as a food sanitizer, but the extract could potentially be developed as an additive for refrigerated foods, or a modern treatment for foodborne illness.

Production and evaluation of an antimicrobial peptide-containing wafer formulation for topical application.

A targeted approach for direct topical antimicrobial delivery involving the formulation of impregnated freeze-dried wafers prepared from a natural polymer has been assessed to consider potential for treatment of wounded skin. The synthetic cationic antimicrobial peptides (CAPs) NP101 and NP108 were found to have modest in vitro activity against bacterial species commonly associated with wound infections. Minimum inhibitory concentration/minimum bactericidal concentrations against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were found to be 0.31 mg/ml for NP101 and 0.25-0.5 mg/ml for NP108. Rapid, substantial cytoplasmic potassium loss was induced by NP108 in E. coli, but not the other species. Through scanning electron microscopy, both CAPs were observed to alter cell morphology, prevent normal septation, promote cell aggregation and trigger release or formation of extracellular filaments. Wafers harbouring these agents displayed substantial antibacterial activity when assessed by standard diffusion assay. These data confirm that topical delivery of CAPs, through their incorporation within freeze-dried wafer formulations prepared from natural polymers, represents a potential viable approach for treating skin infection.

Antibacterial activity of three medicinal Thai plants against Campylobacter jejuni and other foodborne pathogens.

Leaves of Adenanthera pavonina, Moringa oleifera and Annona squamosa are used in traditional Thai medicine to treat dysentery and other diseases. This study investigated the antibacterial activity of these plants against six species of foodborne pathogen. Methods and solvents employed to extract active constituents were optimised using the disc diffusion assay. Phytochemical analysis of the optimised extracts was performed by thin layer chromatography (TLC). Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined by broth microdilution. A. pavonina contained flavonoids, terpines and tannins, and was the most active extract against Campylobacter jejuni, inhibiting growth at 62.5-125 µg mL(-1). The A. squamosa extract contained flavonoids, terpines, tannins and alkaloids, and had the broadest spectrum of antibacterial activity, inhibiting Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus and C. jejuni between 62.5 and 500 µg mL(-1). MBCs were 2- to 4-fold higher than MICs against C. jejuni and B. cereus, suggesting the extracts are bactericidal against these species. Negligible activity was detected from M. oleifera. The data presented here show that A. pavonina and A. squamosa could potentially be used in modern applications aimed at the treatment or prevention of foodborne diseases.

Recent advances in understanding the antibacterial properties of flavonoids.

Antibiotic resistance is a major global problem and there is a pressing need to develop new therapeutic agents. Flavonoids are a family of plant-derived compounds with potentially exploitable activities, including direct antibacterial activity, synergism with antibiotics, and suppression of bacterial virulence. In this review, recent advances towards understanding these properties are described. Information is presented on the ten most potently antibacterial flavonoids as well as the five most synergistic flavonoid-antibiotic combinations tested in the last 6 years (identified from PubMed and ScienceDirect). Top of these respective lists are panduratin A, with minimum inhibitory concentrations (MICs) of 0.06-2.0 µg/mL against Staphylococcus aureus, and epicatechin gallate, which reduces oxacillin MICs as much as 512-fold. Research seeking to improve such activity and understand structure-activity relationships is discussed. Proposed mechanisms of action are also discussed. In addition to direct and synergistic activities, flavonoids inhibit a number of bacterial virulence factors, including quorum-sensing signal receptors, enzymes and toxins. Evidence of these molecular effects at the cellular level include in vitro inhibition of biofilm formation, inhibition of bacterial attachment to host ligands, and neutralisation of toxicity towards cultured human cells. In vivo evidence of disruption of bacterial pathogenesis includes demonstrated efficacy against Helicobacter pylori infection and S. aureus α-toxin intoxication.

Antimicrobial activity of Blumea balsamifera (Lin.) DC. extracts and essential oil.

Leaves of Blumea balsamifera (Lin.) DC. are used in traditional Thai and Chinese medicine for the treatment of septic wounds and other infections. In this study, the essential oil, hexane, dichloromethane and methanol extracts of these leaves were evaluated for antibacterial and antifungal activities using the disc diffusion assay and agar microdilution method. The essential oil was the most potent, with a minimum inhibitory concentration (MIC) of 150 µg mL⁻¹ against Bacillus cereus and an MIC of 1.2 mg mL⁻¹ against Staphylococcus aureus and Candida albicans. Activity was also detected from the hexane extract against Enterobacter cloacae and S. aureus. Minimum bactericidal and fungicidal concentrations were typically equal to or two-fold higher than the MICs for both extracts, indicating microbicidal activity. The present data show that B. balsamifera extracts have activity against various infectious and toxin-producing microorganisms. This plant's active constituents could potentially be developed for use in the treatment and/or prevention of microbial disease.

Variables to be considered when assessing the photocatalytic destruction of bacterial pathogens.

The current study sought to assess the importance of three common variables on the outcome of TiO(2) photocatalysis experiments with bacteria. Factors considered were (a) ability of test species to withstand osmotic pressure, (b) incubation period of agar plates used for colony counts following photocatalysis and (c) chemical nature of suspension medium used for bacteria and TiO(2). Staphylococcus aureus, Escherichia coli, Salmonella ser. Typhimurium and Pseudomonas aeruginosa were found to vary greatly in their ability to withstand osmotic pressure, raising the possibility that osmotic lysis may be contributing to loss of viability in some photocatalytic disinfection studies. Agar plate incubation time was also found to influence results, as bacteria treated with UV light only grew more slowly than those treated with a combination of UV and TiO(2.) The chemical nature of the suspension medium used was found to have a particularly pronounced effect upon results. Greatest antibacterial activity was detected when aqueous sodium chloride solution was utilised, with approximately 1 x 10(6) CFU mL(-1)S. aureus being completely killed after 60 min. Moderate activity was observed when distilled water was employed with bacteria being killed after 2h and 30 min, and no antibacterial activity at all was detected when aqueous tryptone solution was used. Interestingly, the antibacterial activity of UV light on its own appeared to be very much reduced in experiments where aqueous sodium chloride was employed instead of distilled water.

Antimicrobial activity of flavonoids.

Flavonoids are ubiquitous in photosynthesising cells and are commonly found in fruit, vegetables, nuts, seeds, stems, flowers, tea, wine, propolis and honey. For centuries, preparations containing these compounds as the principal physiologically active constituents have been used to treat human diseases. Increasingly, this class of natural products is becoming the subject of anti-infective research, and many groups have isolated and identified the structures of flavonoids possessing antifungal, antiviral and antibacterial activity. Moreover, several groups have demonstrated synergy between active flavonoids as well as between flavonoids and existing chemotherapeutics. Reports of activity in the field of antibacterial flavonoid research are widely conflicting, probably owing to inter- and intra-assay variation in susceptibility testing. However, several high-quality investigations have examined the relationship between flavonoid structure and antibacterial activity and these are in close agreement. In addition, numerous research groups have sought to elucidate the antibacterial mechanisms of action of selected flavonoids. The activity of quercetin, for example, has been at least partially attributed to inhibition of DNA gyrase. It has also been proposed that sophoraflavone G and (-)-epigallocatechin gallate inhibit cytoplasmic membrane function, and that licochalcones A and C inhibit energy metabolism. Other flavonoids whose mechanisms of action have been investigated include robinetin, myricetin, apigenin, rutin, galangin, 2,4,2'-trihydroxy-5'-methylchalcone and lonchocarpol A. These compounds represent novel leads, and future studies may allow the development of a pharmacologically acceptable antimicrobial agent or class of agents.

Detection of galangin-induced cytoplasmic membrane damage in Staphylococcus aureus by measuring potassium loss.

Galangin is one of the active antimicrobial principles of propolis or 'bee glue' and Helichrysum aureonitens, a perennial herb used by South African indigenes to treat infection. The effect of this compound and antibacterial agents with known mechanisms of action upon the cytoplasmic membrane integrity of Staphylococcus aureus was investigated by comparing potassium loss profiles from bacterial cell suspensions. Using an agar dilution assay, the minimum inhibitory concentrations (MICs) of the flavonol galangin, the bacteriostatic antibiotic novobiocin and the bactericidal antibiotic penicillin G against Staphylococcus aureus NCTC 6571 were determined as being 50 microg/mL, 62.5 ng/mL and 31.3 ng/mL, respectively. When 5x10(7)cfu/mL Staphylococcus aureus were suspended in 'potassium-free' media containing 50 microg/mL galangin, a 60-fold decrease in viability was observed after 12 h. Populations of 1x10(9) cfu/mL Staphylococcus aureus incubated for 12 h in 50 microg/mL galangin lost 21% more potassium than untreated control populations. Novobiocin had no effect on potassium loss, but populations incubated in 31.3 ng/mL penicillin G exhibited a 6% increase in potassium loss. This data clearly demonstrates that galangin causes a significant increase in potassium loss from Staphylococcus aureus cells, which may be attributed to either direct damage to the cytoplasmic membrane or indirect damage effected through autolysis/weakening of the cell wall and consequent osmotic lysis.

Assessment of the antibacterial activity of selected flavonoids and consideration of discrepancies between previous reports.

Activity of the flavonoids apigenin, baicalin and galangin against sensitive and antibiotic resistant strains of Staphylococculs aureus, Enterococcus faecalis, E. faecium, Escherichia coli and Pseudomonas aeruginosa was investigated. Using an agar dilution assay, galangin was shown to have a minimum inhibitory concentration (MIC) of 25 to 50 microg/mL against all six strains of S. aureus but negligible activity against the othe species. Apigenin displayed only marginal activity against S. aureus and no activity was detected from baicalin. In inhibition curve studies, galangin caused a 100,000-fold decrease in the viability of a growing population of S. aureus NCTC 6571 within the first two hours of treatment. Decreases in viability of S. aureus NCTC 11561 and NCIMB 9968 populations were also observed.