Andrew J. Lamb

Antimicrobial activity of flavonoids

Abstract 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.

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.

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.

Incidence of mesophilic Aeromonas within a public drinking water supply in north-east Scotland

The motile mesophilic Aeromonas are ubiquitous to a wide variety of aquatic environments including drinking water distribution systems. Concern over the presence of mesophilic Aeromonas in public drinking water supplies has been expressed in recent years as it has been regarded as a pathogenic organism of importance in gastroenteritis. A major drinking water distribution system in north‐east Scotland was monitored over a 12 month period to determine the prevalence of mesophilic Aeromonas. These data were examined in relation to chlorine concentration, pH, temperature, rainfall and the standard bacteriological indicators of water quality. Aeromonas were isolated to varying degrees from 21 of the 31 reservoirs investigated. The maximum recovery observed during the study was 605 cfu in 300 ml. The probability of isolation generally decreased with increasing levels of chlorination, although this oxidant was found to be ineffective in many reservoirs. Certain reservoirs with poor chlorination profiles yielded very few isolates, whereas some highly chlorinated sites liberated Aeromonas frequently and in relatively high numbers. A seasonal pattern in the incidence of Aeromonas emerged with infrequent isolation during the winter period increasing to a peak during the summer, with most isolates recovered when water temperature was >12 °C. An association was demonstrated between the pattern of Aeromonas isolations and that of rainfall. No relationship was apparent between incidence of Aeromonas and total heterotrophic plate counts.

Aeromonas popoffii sp. nov., a mesophilic bacterium isolated from drinking water production plants and reservoirs.

We examined the taxonomic position of seven Aeromonas isolates, recovered from Flemish and Scottish drinking water production plants and reservoirs, which were previously recognized by numerical analysis of genomic AFLP fingerprints as members of an unknown Aeromonas taxon that most closely resembled the species Aeromonas bestiarum (DNA hybridization group [HG] 2). The new phenotypic and DNA-DNA hybridization data obtained in this study show that the A. bestiarum-like strains constitute a separate Aeromonas species, for which the name Aeromonas popoffii sp. nov. is being proposed. The new species exhibited an internal DNA relatedness ranging from 79 to 100% and was 22 to 63% related to the type or reference strains of other Aeromonas spp. The highest DNA binding values were determined with A. bestiarum (51 to 63%), followed by Aeromonas hydrophila sensu stricto (HG1; 50 to 60%) and Aeromonas salmonicida (HG3; 39 to 55%). Although fingerprints generated by ribotyping and cellular fatty acid analysis often were highly similar, minor differences between the respective fingerprints were of significance for the differentiation of A. popoffii from its closest taxonomic neighbors, HG1, HG2, and HG3. Phenotypically, all seven strains of A. popoffii were positive for acid and gas production from D-glucose and glycerol, growth in KCN broth, arginine dihydrolase, DNase, Voges-Proskauer reaction, and resistance to vibriostatic agent O/129 and ampicillin but displayed negative reactions for production of urease, tryptophan deaminase, ornithine decarboxylase, and lysine decarboxylase (LDC). None of the strains displayed strong hemolytic activity. The lack of D-sucrose fermentation and LDC production and the ability to utilize DL-lactate as the sole energy and carbon source were useful characteristics for the biochemical separation of A. popoffii from A. bestiarum. Other Aeromonas spp. could be differentiated phenotypically from the new species by at least two features. The chromosomal G+C content of A. popoffii ranges from 57.7 to 59.6 mol%. Strain LMG 17541 is proposed as the type strain.

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.

Aggregation of Staphylococcus aureus following treatment with the antibacterial flavonol galangin.

Aim:  The flavonol galangin, an antimicrobial constituent of the traditional medicines propolis and Helichrysum aureonitens, is being assessed as part of an ongoing investigation into the antibacterial activity of flavonoids. The present study sought to establish whether galangin has any aggregatory effect on bacterial cells.

False-positive coliform reaction mediated by Aeromonas in the Colilert defined substrate technology system

The Colilert defined substrate technology system allows specific, one‐step detection of both coliforms and Escherichia coli while claiming to suppress the influence of non‐coliform heterotrophs. The Colilert assay was examined in order to determine whether organisms from the genus Aeromonas could interfere and cause production of a false‐positive coliform result as aquatic Aeromonas are known to constitute a fraction of the heterotrophic population found in drinking water. Results obtained clearly demonstrate that Aeromonas sp. can elicit a positive coliform type reaction at very low densities. Cell suspensions as low as 1 × 101 cells 10 ml−1 were observed to yield a positive reaction using Colilert reagent 4 weeks short of shelf‐life expiry. Use of aged Colilert for monitoring water quality could lead to over‐estimation of coliforms as Aeromonas have been identified in many treated drinking water supplies.

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.

Improvements to source protection for private water supplies in Scotland, UK☆

Private water supplies are defined as any water supply which is not provided by a statutory water undertaker and in which the responsibility for its maintenance lies with the owner or person who uses the supply. In Scotland around 60,000 people rely on such supplies from which they will be receiving water subject to either limited or no statutory requirement for quality sampling. Further, while 60,000 is the resident population, a large number of people attending campsites, hotels, guesthouses or using food outlets will be exposed to private supplies for transitory periods of time. Of greatest concern is the threat of microbial contamination resulting in an increased threat to public health. In an effort to improve the microbiological quality of the drinking water from private supplies a microbiological risk assessment protocol was developed to enhance the source protection of such supplies. This paper details the development of this protocol.