The effect of copper(II), iron(II) sulphate and vitamin C combinations on the weak antimicrobial activity of (+)-catechin against Staphylococcus aureus and other microbes

Few attempts have been made to improve the activity of plant compounds with low antimicrobial efficacy. (+)-Catechin, a weak antimicrobial tea flavanol, was combined with putative adjuncts and tested against different species of bacteria. Copper(II) sulphate enhanced (+)-catechin activity against Pseudomonas aeruginosa but not Staphylococcus aureus, Proteus mirabilis or Escherichia coli. Attempts to raise the activity of (+)-catechin against two unresponsive species, S. aureus and E. coli, with iron(II) sulphate, iron(III) chloride, and vitamin C, showed that iron(II) enhanced (+)-catechin against S. aureus, but not E. coli; neither iron(III) nor combined iron(II) and copper(II), enhanced (+)-catechin activity against either species. Vitamin C enhanced copper(II) containing combinations against both species in the absence of iron(II). Catalase or EDTA added to active samples removed viability effects suggesting that active mixtures had produced H2O2via the action of added metal(II) ions. H2O2 generation by (+)-catechin plus copper(II) mixtures and copper(II) alone could account for the principal effect of bacterial growth inhibition following 30 minute exposures as well as the antimicrobial effect of (+)-catechin–iron(II) against S. aureus. These novel findings about a weak antimicrobial flavanol contrast with previous knowledge of more active flavanols with transition metal combinations. Weak antimicrobial compounds like (+)-catechin within enhancement mixtures may therefore be used as efficacious agents. (+)-Catechin may provide a means of lowering copper(II) or iron(II) contents in certain crop protection and other products.

Modulation of anti-pathogenic activity in canine-derived Lactobacillus species by carbohydrate growth substrate

Aims: To investigate the effect of various carbon sources on the production of extracellular antagonistic compounds against two Escherichia coli strains and Salmonella enterica serotype Typhimurium by three canine-derived lactobacilli strains. Methods and Materials: Cell-free preparations, pH neutralized, were used in antibiotic disc experiments as an initial screening. The bacteria/carbohydrate combinations that showed inhibition of the growth of those pathogens, were further investigated in batch co-culture experiments. The cell-free supernatants of the cultures, that decreased the population number of the pathogens in the co-culture experiments to log CFU ml(-1) less than or equal to 4, were tested for inhibition of the pathogens in pure cultures at neutral and acidic pH. Conclusions: The results showed that the substrate seems to affect the production of antimicrobial compounds and this effect could not just be ascribed to the ability of the bacteria to grow in the various carbon sources. L. mucosae, L. acidophilus and L. reuteri, when grown in sugar mixtures consisting of alpha-glucosides (Degree of Polymerization (DP) 1-4) could produce antimicrobial compounds active against all three pathogens in vitro. This effect could not be attributed to a single ingredient of those sugar mixtures and was synergistic. This inhibition had a dose-response characteristic and was more active at acidic pH. Significance and Impact of the Study: Knowledge of the effect that the carbon source has on the production of antimicrobial compounds by gut-associated lactobacilli allows the rational design of prebiotic/probiotic combinations to combat gastrointestinal pathogens.

Relationship between sublethal injury and microbial inactivation by the combination of high hydrostatic pressure and citral or tert-butyl hydroquinone

The aim was to investigate (i) the occurrence of sublethal injury in Listeria monocytogenes, Escherichia coli, and Saccharomyces cerevisiae after high hydrostatic pressure (HHP) treatment as a function of the treatment medium pH and composition and (ii) the relationship between the occurrence of sublethal injury and the inactivating effect of a combination of HHP and two antimicrobial compounds, tert-butyl hydroquinone (TBHQ) and citral. The three microorganisms showed a high proportion of sublethally injured cells (up to 99.99% of the surviving population) after HHP. In E. coli and L. monocytogenes, the extent of inactivation and sublethal injury depended on the pH and the composition of the treatment medium, whereas in S. cerevisiae, inactivation and sublethal injury were independent of medium pH or composition under the conditions tested. TBHQ alone was not lethal to E. coli or L. monocytogenes but acted synergistically with HHP and 24-h refrigeration, resulting in a viability decrease of >5 log(10) cycles of both organisms. The antimicrobial effect of citral depended on the microorganism and the treatment medium pH. Acting alone for 24 h under refrigeration, 1,000 ppm of citral caused a reduction of 5 log(10) cycles of E. coli at pH 7.0 and almost 3 log(10) cycles of L. monocytogenes at pH 4.0. The combination of citral and HHP also showed a synergistic effect. Our results have confirmed that the detection of sublethal injury after HHP may contribute to the identification of those treatment conditions under which HHP may act synergistically with other preserving processes.

Survival of Lactobacillus plantarum in model solutions and fruit juices

The aim of the work was to study the survival of Lactobacillus plantarum NCIMB 8826 in model solutions and develop a mathematical model describing its dependence on pH, citric acid and ascorbic acid. A Central Composite Design (CCD) was developed studying each of the three factors at five levels within the following ranges, i.e., pH (3.0-4.2), citric acid (6-40 g/L), and ascorbic acid (100-1000 mg/L). In total, 17 experimental runs were carried out. The initial cell concentration in the model solutions was approximately 1 × 10(8)CFU/mL; the solutions were stored at 4°C for 6 weeks. Analysis of variance (ANOVA) of the stepwise regression demonstrated that a second order polynomial model fits well the data. The results demonstrated that high pH and citric acid concentration enhanced cell survival; one the other hand, ascorbic acid did not have an effect. Cell survival during storage was also investigated in various types of juices, including orange, grapefruit, blackcurrant, pineapple, pomegranate, cranberry and lemon juice. The model predicted well the cell survival in orange, blackcurrant and pineapple, however it failed to predict cell survival in grapefruit and pomegranate, indicating the influence of additional factors, besides pH and citric acid, on cell survival. Very good cell survival (less than 0.4 log decrease) was observed after 6 weeks of storage in orange, blackcurrant and pineapple juice, all of which had a pH of about 3.8. Cell survival in cranberry and pomegranate decreased very quickly, whereas in the case of lemon juice, the cell concentration decreased approximately 1.1 logs after 6 weeks of storage, albeit the fact that lemon juice had the lowest pH (pH~2.5) among all the juices tested. Taking into account the results from the compositional analysis of the juices and the model, it was deduced that in certain juices, other compounds seemed to protect the cells during storage; these were likely to be proteins and dietary fibre In contrast, in certain juices, such as pomegranate, cell survival was much lower than expected; this could be due to the presence of antimicrobial compounds, such as phenolic compounds.

Volatile profile of Spanish Cistus plants as sources of antimicrobials for industrial applications

Cistus is a plant genus traditionally used in folk medicine as remedy for several microbial disorders and infections. The abundance of Cistus spp. in the Iberian Peninsula together with their ability to renew after wildfire contribute to their profitability as suppliers of functional ingredients. The aim of this study was to provide a comprehensive characterization of the volatile profile of different Cistus plants grown in Spain:Cistus ladanifer L., Cistus albidus L., Cistus salviifolius L., and Cistus clusii Dunal (the latter has not been studied before). A system combining headspace solid-phase microextraction and gas chromatography coupled to mass spectrometry (HS-SPME-GC–MS) was implemented; thereby, the volatile compounds were extracted and analyzed in a fast, reliable and environment-friendly way. A total of 111 volatile compounds were identified, 28 of which were reported in Cistus for the first time. The most abundant components of the samples (mono and sesquiterpenes) have been previously reported as potent antimicrobial agents. Therefore, this work reveals the potential use of the Cistus spp. studied as natural sources of antimicrobial compounds for industrial production of cosmeceuticals, among other applications.

In vitro evaluation of the antimicrobial activity of a range of probiotics against pathogens: Evidence for the effects of organic acids

The aim of this study was to investigate the antimicrobial properties of fifteen selected strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera against Gram-positive and Gram-negative pathogenic bacteria. In vitro antibacterial activity was initially investigated by an agar spot method. Results from the agar spot test showed that most of the selected strains were able to produce active compounds on solid media with antagonistic properties against Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Clostridium difficile. These results were also confirmed when cell-free culture supernatants (CFCS) from the putative probiotics were used in an agar well diffusion assay. Neutralization of the culture supernatants with alkali reduced the antagonistic effects. These experiments are able to confirm the capacity of potential probiotics to inhibit selected pathogens. One of the main inhibitory mechanisms may result from the production of organic acids from glucose fermentation and consequent lowering of culture pH. This observation was confirmed when the profile of organic acids was analysed demonstrating that lactic and acetic acid were the principal end products of probiotic metabolism. Furthermore, the assessment of the haemolytic activity and the susceptibility of the strains to the most commonly used antimicrobials, considered as basic safety aspects, were also studied. The observed antimicrobial activity was mainly genus-specific, additionally significant differences could be observed among species.

Development of antimicrobial synbiotics using potentially-probiotic faecal isolates of Lactobacillus fermentum and Bifidobacterium longum

The aims of the present study were to investigate in vitro the antimicrobial activity of Lactobacillus fermentum and Bifidobacterium longum, isolated from faeces of healthy elderly individuals, against enterohaemorrhagic Escherichia coli (E. coli O157:H7) and enteropathogenic E. coli (E. coli O86), to determine the capability of the selected strains to tolerate acid and bile in vitro, to select suitable carbohydrates in order to enhance the growth and maximise antimicrobial activity of the putative probiotic organisms and examine the adhesion properties of the synbiotics. Antimicrobial activity of the putative probiotics and synbiotics was investigated by a microtitre method using cell-free culture supernatants (CFCS). Results of the antimicrobial assay showed that both putative probiotic strains produced compounds at pH 5 that lead to higher lag phases of both E. coli O157:H7 and E. coli O86. When half the quantity of cell-free culture supernatants of both probiotic strains was used at pH 5, B. longum maintained the same antimicrobial effect against both strains of E. coli, whereas L. fermentum lead to a higher lag phase of E. coli O86 only. Neutralization of the culture supernatants with alkali reduced the antimicrobial effect with only cell-free supernatant of L. fermentum causing lower maximum growth rates of E. coli O157:H7 and E. coli O86. L. fermentum appeared to be acid tolerant whereas B. longum was more susceptible to acid and both isolates were bile tolerant. A short chain fructooligosaccharide (scFOS) and an isomalto-oligosaccharide (IMO) proved to be the most effective substrates, enhancing antimicrobial activity for L. fermentum and B. longum respectively. The adhesion of the synbiotic combinations showed that L. fermentum, exhibited higher percentage of adhesion when grown on glucose and as a synbiotic combination with scFOS whereas B. longum exhibited lowest percentage of adhesion when grown on both glucose and IMO.

Paralog re-emergence: a novel, historically contingent mechanism in the evolution of antimicrobial resistance

Evolution of resistance to drugs and pesticides poses a serious threat to human health and agricultural production. CYP51 encodes the target site of azole fungicides, widely used clinically and in agriculture. Azole resistance can evolve due to point mutations or overexpression of CYP51, and previous studies have shown that fungicide-resistant alleles have arisen by de novo mutation. Paralogs CYP51A and CYP51B are found in filamentous ascomycetes, but CYP51A has been lost from multiple lineages. Here, we show that in the barley pathogen Rhynchosporium commune, re-emergence of CYP51A constitutes a novel mechanism for the evolution of resistance to azoles. Pyrosequencing analysis of historical barley leaf samples from a unique long-term experiment from 1892 to 2008 indicates that the majority of the R. commune population lacked CYP51A until 1985, after which the frequency of CYP51A rapidly increased. Functional analysis demonstrates that CYP51A retains the same substrate as CYP51B, but with different transcriptional regulation. Phylogenetic analyses show that the origin of CYP51A far predates azole use, and newly sequenced Rhynchosporium genomes show CYP51A persisting in the R. commune lineage rather than being regained by horizontal gene transfer; therefore, CYP51A re-emergence provides an example of adaptation to novel compounds by selection from standing genetic variation.

Inactivation of the antibacterial and cytotoxic properties of silver ions by biologically relevant compounds

There has been a recent surge in the use of silver as an antimicrobial agent in a wide range of domestic and clinical products, intended to prevent or treat bacterial infections and reduce bacterial colonization of surfaces. It has been reported that the antibacterial and cytotoxic properties of silver are affected by the assay conditions, particularly the type of growth media used in vitro. The toxicity of Ag+ to bacterial cells is comparable to that of human cells. We demonstrate that biologically relevant compounds such as glutathione, cysteine and human blood components significantly reduce the toxicity of silver ions to clinically relevant pathogenic bacteria and primary human dermal fibroblasts (skin cells). Bacteria are able to grow normally in the presence of silver nitrate at >20-fold the minimum inhibitory concentration (MIC) if Ag+ and thiols are added in a 1:1 ratio because the reaction of Ag+ with extracellular thiols prevents silver ions from interacting with cells. Extracellular thiols and human serum also significantly reduce the antimicrobial activity of silver wound dressings Aquacel-Ag (Convatec) and Acticoat (Smith & Nephew) to Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli in vitro. These results have important implications for the deployment of silver as an antimicrobial agent in environments exposed to biological tissue or secretions. Significant amounts of money and effort have been directed at the development of silver-coated medical devices (e.g. dressings, catheters, implants). We believe our findings are essential for the effective design and testing of antimicrobial silver coatings.