Purification and molecular cloning of antimicrobial peptides from Scots pine seedlings

A novel protocol for rapid and efficient purification of antimicrobial peptides from plant seedlings has been developed. Two peptides with antimicrobial activity, designated p1 and p2, were purified nearly to homogeneity from Scots pine seedlings by a combination of sulfuric acid extraction, ammonium sulfate precipitation, heat-inactivation and ion-exchange chromatography on phosphocellulose. Purified proteins had molecular masses of 11 kDa (p1) and 5.8 kDa (p2) and were identified by mass spectrometry as defensin and lipid-transfer protein, respectively. We demonstrated their growth inhibitory effects against a group of phytopathogenic fungi. Furthermore, we report for the first time molecular cloning and characterization of defensin I cDNA from Scots pine. A cDNA expression library from 7 days Scots pine seedlings was generated and used to isolate a cDNA clone corresponding to Scots pine defensin, termed PsDef1. The full-length coding sequence of PsDef1 is 252 bp in length and has an open reading frame capable to encode a protein of 83 amino residues. The deduced sequence has the typical features of plant defensins, including an endoplasmic reticulum signal sequence of 33 aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5601.6 Da, which correlates well with the results of SDS-PAGE analysis. Finally, the antimicrobial properties of PsDef1 against a panel of fungi and bacteria define it as a member of the morphogenic group of plant defensins. (C) 2009 Elsevier Inc. All rights reserved.

Effects of drying methods and conditions on antimicrobial activity of edible chitosan films enriched with galangal extract

The aim of this work was to study the effects of drying methods and conditions (i.e., ambient drying, hot air drying at 40 degrees C, vacuum drying and low-pressure superheated steam drying within the temperature range of 70-90 degrees C at an absolute pressure of 10 kPa) as well as the concentration of galangal extract on the antimicrobial activity of edible chitosan films against Staphylococcus aureus. Galangal extract was added to the film forming solution as a natural antimicrobial agent in the concentration range of 0.3-0.9 g/100 g. Fourier transform infrared (FTIR) spectra and swelling of the films were also evaluated to investigate interaction between chitosan and the galangal extract. The antimicrobial activity of the films was evaluated by the disc diffusion and viable cell count method, while the morphology of bacteria treated with the antimicrobial films was observed via transmission electron microscopy (TEM). The antimicrobial activity, swelling and functional group interaction of the antimicrobial films were found to be affected by the drying methods and conditions as well as the concentration of the galangal extract. The electron microscopic observations revealed that cell wall and cell membrane of S. aureus treated by the antimicrobial films were significantly damaged. (C) 2009 Elsevier Ltd. All rights reserved.

Antimicrobial peptide-lipid binding interactions and binding selectivity

Surface pressure measurements, external reflection- Fourier transform infrared spectroscopy, and neutron re. flectivity have been used to investigate the lipid-binding behavior of three antimicrobial peptides: melittin, magainin II, and cecropin P1. As expected, all three cationic peptides were shown to interact more strongly with the anionic lipid, 1,2 dihexadecanoyl-sn-glycerol3-( phosphor-rac-( 1- glycerol)) ( DPPG), compared to the zwitterionic lipid, 1,2 dihexadecanoyl-sn-glycerol-3-phosphocholine ( DPPC). All three peptides have been shown to penetrate DPPC lipid layers by surface pressure, and this was confirmed for the melittin-DPPC interaction by neutron reflectivity measurements. Adsorption of peptide was, however, minimal, with a maximum of 0.4 mg m(-2) seen for melittin adsorption compared to 2.1 mg m(-2) for adsorption to DPPG ( from 0.7 mu M solution). The mode of binding to DPPG was shown to depend on the distribution of basic residues within the peptide alpha-helix, although in all cases adsorption below the lipid layer was shown to dominate over insertion within the layer. Melittin adsorption to DPPG altered the lipid layer structure observed through changes in the external reflection-Fourier transform infrared lipid spectra and neutron reflectivity. This lipid disruption was not observed for magainin or cecropin. In addition, melittin binding to both lipids was shown to be 50% greater than for either magainin or cecropin. Adsorption to the bare air-water interface was also investigated and surface activity followed the trend melittin. magainin. cecropin. External re. ection- Fourier transform infrared amide spectra revealed that melittin adopted a helical structure only in the presence of lipid, whereas magainin and cecropin adopted helical structure also at an airwater interface. This behavior has been related to the different charge distributions on the peptide amino acid sequences.

Antioxidant and antimicrobial activities of tea infusions

Tea polyphenols, especially the catechins, are potent antimicrobial and antioxidant agents, with positive effects on human health. White tea is one of the less studied teas but the flavour is more accepted than that of green tea in Europe. The concentrations of various catechins in 13 different kinds of infusion were determined by capillary electrophoresis. The total polyphenol content (Folin-Ciocalteu method), the trolox equivalent antioxidant capacity (TEAC value determined with the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation) and the inhibitory effects of infusions on the growth of some microorganisms were determined. Five different infusions (black, white, green and red teas and rooibos infusion) were added to a model food system, comprising a sunflower oil-in-water emulsion containing 0% or 0.2% bovine serum albumin (BSA), and the oxidative stability was studied during storage at 37 degrees C. Oxidation of the oil was monitored by determination of the peroxide value. The highest radical-scavenging activity observed was for the green and white teas. Emulsions containing these extracts from these teas were much more stable during storage when BSA was present than when it was not present, even though BSA itself did not provide an antioxidant effect (at 0.2% concentration). Rooibos infusion did not show the same synergy with BSA. Green tea and white tea showed similar inhibitions of several microorganisms and the magnitude of this was comparable to that of the commercial infusion 2 (C.I.2), "te de la belleza". This tea also had an antioxidant activity comparable to green tea. (C) 2007 Published by Elsevier Ltd.

Effect of pH on the antimicrobial activity and oxidative stability of oil-in-water emulsions containing caffeic acid

Antioxidant properties in food are dependent on various parameters. These include the pH value and interactions with food components, including proteins or metal ions. food components affect antioxidant stability and also influence the properties of microorganisms and their viability. This paper describes an investigation of the effect of pH on the antioxidant and antibacterial properties of caffeic acid in different media. The pH values studied, using an oil-in-water emulsion as model system, were 3, 5 (with and without phosphate buffer), and 9. Effects of mixtures of caffeic acid, bovine serum albumin (BSA), and Fe (III) on oxidative deterioration in the emulsion samples were studied. The results show that the antioxidant activity of caffeic acid was increased by the presence of BSA. This effect was pH dependent and was affected by the presence of iron Ions. Antibacterial properties were also pH dependent. The minimum concentration of caffeic acid required to inhibit some microorganisms in the pH range of 5 to 7 was determined. A concentration of 0.41% (w/w) caffeic acid was enough to inhibit the growth of some of the studied microorganisms in the pH range of 5 to 7. However, near-neutral pH concentrations higher than 0.4% were needed to inhibit some microorganisms, including Listeria monocytogenes, E. coli, and Staphylococcus aureus, in the medium.

Lipid binding interactions of antimicrobial plant seed defence proteins: puroindoline-a and β-purothionin

The indolines and thionins are basic, amphiphilic and cysteine-rich proteins found in cereals; puroindoline-a (Pin-a) and β-purothionin (β-Pth) are members of these families in wheat (Triticum aestivum). Pin-a and β-Pth have been suggested to play a significant role in seed defence against microbial pathogens, making the interaction of these proteins with model bacterial membranes an area of potential interest. We have examined the binding of these proteins to lipid monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) using a combination of neutron reflectometry, Brewster angle microscopy, and infrared spectroscopy. Results showed that both Pin-a and β-Pth interact strongly with condensed phase DPPG monolayers, but the degree of penetration was different. β-Pth was shown to penetrate the lipid acyl chain region of the monolayer and remove lipids from the air/liquid interface during the adsorption process, suggesting this protein may be able to both form membrane spanning ion channels and remove membrane phospholipids in its lytic activity. Conversely, Pin-a was shown to interact mainly with the head-group region of the condensed phase DPPG monolayer and form a 33 Å thick layer below the lipid film. The differences between the interfacial structures formed by these two proteins may be related to the differing composition of the Pin-a and β-Pth hydrophobic regions.

Spatial and temporal patterns in antimicrobial resistance of Salmonella Typhimurium in cattle in England and Wales

Salmonella is the second most commonly reported human foodborne pathogen in England and Wales, and antimicrobial-resistant strains of Salmonella are an increasing problem in both human and veterinary medicine. In this work we used a generalized linear spatial model to estimate the spatial and temporal patterns of antimicrobial resistance in Salmonella Typhimurium in England and Wales. Of the antimicrobials considered we found a common peak in the probability that an S. Typhimurium incident will show resistance to a given antimicrobial in late spring and in mid to late autumn; however, for one of the antimicrobials (streptomycin) there was a sharp drop, over the last 18 months of the period of investigation, in the probability of resistance. We also found a higher probability of resistance in North Wales which is consistent across the antimicrobials considered. This information contributes to our understanding of the epidemiology of antimicrobial resistance in Salmonella.

Identifying antimicrobial resistance genes of human clinical relevance within Salmonella isolated from food animals in Great Britain

To investigate the occurrence of antimicrobial resistance genes of human clinical relevance in Salmonella isolated from livestock in Great Britain. Two hundred and twenty-five Salmonella enterica isolates were characterized using an antimicrobial resistance gene chip and disc diffusion assays. Plasmid profiling, conjugation experiments and identification of Salmonella genomic island 1 (SGI1) were performed for selected isolates. Approximately 43% of Salmonella harboured single or multiple antimicrobial resistance genes with pig isolates showing the highest numbers where 96% of Salmonella Typhimurium harboured one or more resistance genes. Isolates harbouring multiple resistances divided into three groups. Group 1 isolates harboured ampicillin/streptomycin/sulphonamide/tetracycline resistance and similar phenotypes. This group contained isolates from pigs, cattle and poultry that were from several serovars including Typhimurium, 4,[5],12:i:-, Derby, Ohio and Indiana. All Group 2 isolates were from pigs and were Salmonella Typhimurium. They contained a non-sul-type class 1 integron and up to 13 transferrable resistances. All Group 3 isolates harboured a class 1 integron and were isolated from all animal species included in the study. Most isolates were Salmonella Typhimurium and harboured SGI1. Salmonella isolated from livestock was shown to harbour antimicrobial resistance genes although no or little resistance to third-generation cephalosporins or ciprofloxacin, respectively, was detected. The preponderance in pigs of multidrug-resistant Salmonella Typhimurium makes it important to introduce control measures such as improved biosecurity to ensure that they do not pass through the food chain and limit human therapeutic options.

Virulotyping and antimicrobial resistance typing of salmonella enterica serovars relevant to human health in Europe

The combination of virulence gene and antimicrobial resistance gene typing using DNA arrays is a recently developed genomics-based approach to bacterial molecular epidemiology. We have now applied this technology to 523 Salmonella enterica subsp. enterica strains collected from various host sources and public health and veterinary institutes across nine European countries. The strain set included the five predominant Salmonella serovars isolated in Europe (Enteritidis, Typhimurium, Infantis, Virchow, and Hadar). Initially, these strains were screened for 10 potential virulence factors (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by polymerase chain reaction. The results indicated that only 14 profiles comprising these genes (virulotypes) were observed throughout Europe. Moreover, most of these virulotypes were restricted to only one (n = 9) or two (n = 4) serovars. The data also indicated that the virulotype did not vary significantly with host source or geographical location. Subsequently, a representative subset of 77 strains was investigated using a microarray designed to detect 102 virulence and 49 resistance determinants. The results confirmed and extended the previous observations using the virulo-polymerase chain reaction screen. Strains belonging to the same serovar grouped together, indicating that the broader virulence-associated gene complement corresponded with the serovar. There were, however, some differences in the virulence gene profiles between strains belonging to an individual serovar. This variation occurred primarily within those virulence genes that were prophage encoded, in fimbrial clusters or in the virulence plasmid. It seems likely that such changes enable Salmonella to adapt to different environmental conditions, which might be reflected in serovar-specific ecology. In this strain subset a number of resistance genes were detected and were serovar restricted to a varying degree. Once again the profiles of those genes encoding resistance were similar or the same for each serovar in all hosts and countries investigated.

High prevalence of multidrug-tolerant bacteria and associated antimicrobial resistance genes isolated from ornamental fish and their carriage water

Background: Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely added to the water these fish are shipped in to suppress the growth of potential pathogens during transport. Methodology/Principal Findings: To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp. isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representative isolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray and conventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriage water were tolerant to >= 15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone and fluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR). Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR, blaTEM21, tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferase coding genes were also detected in carriage water samples and bacterial isolates. Conclusions: These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistant bacteria and resistance genes.

A high prevalence of antimicrobial resistant Escherichia coli isolated from pigs and a low prevalence of antimicrobial resistant E-coli from cattle and sheep in Great Britain at slaughter

The incidence of antimicrobial resistance and expressed and unexpressed resistance genes among commensal Escherichia coli isolated from healthy farm animals at slaughter in Great Britain was investigated. The prevalence of antimicrobial resistance among the isolates varied according to the animal species; of 836 isolates from cattle tested only 5.7% were resistant to one or more antimicrobials, while only 3.0% of 836 isolates from sheep were resistant to one or more agents. However, 92.1% of 2480 isolates from pigs were resistant to at least one antimicrobial. Among isolates from pigs, resistance to some antimicrobials such as tetracycline (78.7%), sulphonamide (66.9%) and streptomycin (37.5%) was found to be common, but relatively rare to other agents such as amikacin (0.1%), ceftazidime ( 0.1%) and coamoxiclav (0.2%). The isolates had a diverse range of resistance gene profiles, with tet(B), sul2 and strAB identified most frequently. Seven out of 615 isolates investigated carried unexpressed resistance genes. One trimethoprim-susceptible isolate carried a complete dfrA17 gene but lacked a promoter for it. However, in the remaining six streptomycin-susceptible isolates, one of which carried strAB while the others carried aadA, no mutations or deletions in gene or promoter sequences were identified to account for susceptibility. The data indicate that antimicrobial resistance in E. coli of animal origin is due to a broad range of acquired genes.

Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum beta-lactamases. Validation of the array with control strains demonstrated a 99% correlation between polymerase chain reaction and array results. There was also good correlation between phenotypic and genotypic results for a large panel of Escherichia coli and Salmonella isolates. Some differences were also seen in the number and type of resistance genes harboured by E. coli and Salmonella strains. The array provides an effective, fast and simple method for detection of resistance genes in clinical isolates suitable for use in diagnostic laboratories, which in future will help to understand the epidemiology of isolates and to detect gene linkage in bacterial populations. (C) 2008 Published by Elsevier B.V. and the International Society of Chemotherapy.

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.