Showing papers in "Journal of Applied Microbiology in 2007"

Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics

Abstract: Most studies involving prebiotic oligosaccharides have been carried out using inulin and its fructo-oligosaccharide (FOS) derivatives, together with various forms of galacto-oligosaccharides (GOS). Although many intestinal bacteria are able to grow on these carbohydrates, most investigations have demonstrated that the growth of bifidobacteria, and to a lesser degree lactobacilli, is particularly favoured. Because of their safety, stability, organoleptic properties, resistance to digestion in the upper bowel and fermentability in the colon, as well as their abilities to promote the growth of beneficial bacteria in the gut, these prebiotics are being increasingly incorporated into the Western diet. Inulin-derived oligosaccharides and GOS are mildly laxative, but can result in flatulence and osmotic diarrhoea if taken in large amounts. However, their effects on large bowel habit are relatively minor. Although the literature dealing with the health significance of prebiotics is not as extensive as that concerning probiotics, considerable evidence has accrued showing that consumption of GOS and FOS can have significant health benefits, particularly in relation to their putative anti-cancer properties, influence on mineral absorption, lipid metabolism, and anti-inflammatory and other immune effects such as atopic disease. In many instances, prebiotics seem to be more effective when used as part of a synbiotic combination.

Understanding the effects of diet on bacterial metabolism in the large intestine

Abstract: Recent analyses of ribosomal RNA sequence diversity have demonstrated the extent of bacterial diversity in the human colon, and have provided new tools for monitoring changes in the composition of the gut microbial community. There is now an excellent opportunity to correlate ecological niches and metabolic activities with particular phylogenetic groups among the microbiota of the human gut. Bacteria that associate closely with particulate material and surfaces in the gut include specialized primary degraders of insoluble substrates, including resistant starch, plant structural polysaccharides and mucin. Butyrate-producing bacteria found in human faeces belong mainly to the clostridial clusters IV and XIVa. In vitro and in vivo evidence indicates that a group related to Roseburia and Eubacterium rectale plays a major role in mediating the butyrogenic effect of fermentable dietary carbohydrates. Additional cluster XIVa species can convert lactate to butyrate, while some members of the clostridial cluster IX convert lactate to propionate. The metabolic outputs of the gut microbial community depend not only on available substrate, but also on the gut environment, with pH playing a major role. Better understanding of the colonic microbial ecosystem will help to explain and predict the effects of dietary additives, including nondigestible carbohydrates, probiotics and prebiotics.

Bacterial synthesis of biodegradable polyhydroxyalkanoates

Abstract: Various bacterial species accumulate intracellular polyhydroxyalkanoates (PHAs) granules as energy and carbon reserves inside their cells. PHAs are biodegradable, environmentally friendly and biocompatible thermoplastics. Varying in toughness and flexibility, depending on their formulation, they can be used in various ways similar to many nonbiodegradable petrochemical plastics currently in use. They can be used either in pure form or as additives to oil-derived plastics such as polyethylene. However, these bioplastics are currently far more expensive than petrochemically based plastics and are therefore used mostly in applications that conventional plastics cannot perform, such as medical applications. PHAs are immunologically inert and are only slowly degraded in human tissue, which means they can be used as devices inside the body. Recent research has focused on the use of alternative substrates, novel extraction methods, genetically enhanced species and mixed cultures with a view to make PHAs more commercially attractive.

ACC deaminase from Pseudomonas fluorescens mediated saline resistance in groundnut (Arachis hypogea) plants.

Abstract: Aim: To study the effect of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase from Pseudomonas fluorescens against saline stress under in vitro and field conditions in groundnut (Arachis hypogea) plants. Methods and Results: Four plant growth-promoting rhizobacteria (PGPR) strains were used in this study to evaluate their efficacy in groundnut plants against saline stress under in vitro. Among the four PGPR strains used, Ps. fluorescens strain TDK1 showed greater performance in improving the plant growth parameters of groundnut seedlings in vitro. PCR amplification using Pseudomonas-specific 16S-23S rRNA internal transcribed spacers (ITS) primers revealed that all the four strains belonged to the group of fluorescent pseudomonads. ITS region of Ps. fluorescens strain TDK1 was cloned and sequenced. ACC deaminase activity using biochemical and molecular (PCR) analysis revealed that among all the four strains, Ps. fluorescens strain TDK1 showed greater amount of ACC deaminase activity and positive reaction to PCR amplification. ACC deaminase gene from Ps. fluorescens strain TDK1 was isolated, cloned and sequenced. Pseudomonas bioformulations were developed and they were tested in groundnut plants under saline-affected soils. The results indicated the superior performance by Ps. fluorescens strain TDK1 possessing ACC deaminase activity in improving yield parameters in groundnut plants despite salinity. Conclusions: Pseudomonas fluorescens strain TDK1 possessing ACC deaminase activity enhanced the saline resistance in groundnut plants, which in turn resulted in increased yield when compared with the groundnuts treated with Pseudomonas strains not having ACC deaminase activity. Significance and Impact of the Study: The promising role of ACC deaminase from Ps. fluorescens strain TDK1 in alleviating saline stress has been concluded in groundnut plants. This study will be useful for exploiting the activity of ACC deaminase from microbial strains against various biotic and abiotic stresses wherever ACC accumulated as precursor for ethylene biosynthesis.

Intestinal bacteria and ageing

Abstract: Advancements in science and medicine, as well as improved living standards, have led to a steady increase in life expectancy, and subsequently a rise in the elderly population. The intestinal microbiota is important for maintenance of host health, providing energy, nutrients and protection against invading organisms. Although the colonic microbiota is relatively stable throughout adult life, age-related changes in the gastrointestinal (GI) tract, as well as changes in diet and host immune system reactivity, inevitably affect population composition. Recent studies indicate shifts in the composition of the intestinal microbiota, which may lead to detrimental effects for the elderly host. Increased numbers of facultative anaerobes, in conjunction with a decrease in beneficial organisms such as the anaerobic lactobacilli and bifidobacteria, amongst other anaerobes, have been reported. These changes, along with a general reduction in species diversity in most bacterial groups, and changes to diet and digestive physiology such as intestinal transit time, may result in increased putrefaction in the colon and a greater susceptibility to disease. Therapeutic strategies to counteract these changes have been suggested in ageing people. These include dietary supplements containing prebiotics, probiotics and a combination of both of these, synbiotics. Limited feeding trials show promising results with these supplements, although further longer-term investigations are required to substantiate their use in elderly healthcare fields.

Microbial biofilms in the human gastrointestinal tract

Abstract: The human gastrointestinal tract contains rich and diverse microbiotas along its length. However, while extensive studies have been made on lumenal bacterial communities in the gut, less work has been carried out on organisms growing in biofilms, where individual groups of bacteria exist in a multiplicity of different microhabitats and metabolic niches associated with the mucosa, the mucus layer and particulate surfaces in the gut lumen. Bacteria and yeasts also occur in biofilms attached to artificial surfaces and devices implanted in the host, such as in patients being fed via enteral tubes. Although we are just beginning to investigate the composition and metabolic activities of these structures, increasing evidence suggests that they are important to the host in both health and disease. There is mounting interest in mucosal biofilms in the colon, especially with respect to their role in inflammatory bowel disease. Because bacteria growing in biofilms are more resistant to antibiotics than unattached organisms, it is often difficult to modify the structure and composition of these communities, or to eradicate them from the body. However, recent work has shown that there is considerable potential to alter the species composition of mucosal biofilms in a beneficial way using synbiotics.

Microbial diversity of intestinal contents and mucus in rainbow trout (Oncorhynchus mykiss).

Abstract: Aims: The aim of this study was to understand the microbial community of intestinal contents and mucosal layer in the intestine of rainbow trout by means of culture-dependent conventional and independent molecular techniques. Methods and Results: Forty-one culturable microbial phylotypes, and 39 sequences from 16S rRNA and two from 18S rRNA genes, were retrieved. Aeromonadaceae, Enterobacteriaceae and Pseudomonadaceae representatives were the dominant cultured bacteria. Genomic DNA isolated from intestinal contents and mucus was used to generate 104 random clones, which were grouped into 32 phylotypes at 99% minimum similarity, most of which were affiliated with Proteobacteria (>70% of the total). However, unlike library C (intestinal contents), the phyla Bacteroidetes and Fusobacteria were not found in intestinal mucus (library M), indicating that the microbiota in the gut mucus was different from that of the intestinal contents. Twelve sequences were retrieved from denaturing gradient gel electrophoresis analysis, and dominant bands were mostly related to Clostridium. Conclusions: Many novel sequences that have not been previously recognized as part of the intestinal flora of rainbow trout were retrieved. Significance and Impact of the Study: The fish gut harbours a larger bacterial diversity than previously recognized, and the diversity of gut mucus is different from that of intestinal contents.

Antimicrobial activity of flavonoids extracted from bergamot (Citrus bergamia Risso) peel, a byproduct of the essential oil industry.

Abstract: Aims: To evaluate the antimicrobial properties of flavonoid-rich fractions derived from bergamot peel, a byproduct from the Citrus fruit processing industry and the influence of enzymatic deglycosylation on their activity against different bacteria and yeast. Methods and Results: Bergamot ethanolic fractions were tested against Gram-negative bacteria (Escherichia coli, Pseudomonas putida, Salmonella enterica), Gram-positive bacteria (Listeria innocua, Bacillus subtilis, Staphylococcus aureus, Lactococcus lactis) and the yeast Saccharomyces cerevisiae. Bergamot fractions were found to be active against all the Gram-negative bacteria tested, and their antimicrobial potency increased after enzymatic deglycosylation. The minimum inhibitory concentrations of the fractions and the pure flavonoids, neohesperidin, hesperetin (aglycone), neoeriocitrin, eriodictyol (aglycone), naringin and naringenin (aglycone), were found to be in the range 200 to 800 μg ml−1. The interactions between three bergamot flavonoids were also evaluated. Conclusion: The enzyme preparation Pectinase 62L efficiently converted common glycosides into their aglycones from bergamot extracts, and this deglycosylation increased the antimicrobial potency of Citrus flavonoids. Pairwise combinations of eriodictyol, naringenin and hesperetin showed both synergistic and indifferent interactions that were dependent on the test indicator organism. Significance and Impact of the Study: Bergamot peel is a potential source of natural antimicrobials that are active against Gram-negative bacteria.

Early interactions between legumes and rhizobia: disclosing complexity in a molecular dialogue.

Abstract: The exchange of chemical signals between soil bacteria (rhizobia) and legumes has been termed a molecular dialogue. As initially conceived in the early 1990s, it involved two main groups of molecules: nod gene-inducing flavonoids from plants and the mitogenic lipochito-oligosaccharide Nod factors of rhizobia. This review considers how subsequent research revealed the existence of a more complex set of interactions, featuring expanded roles for the original participants and contributions from additional plant and bacterial metabolites. Rhizobia respond to chemoattractants and growth-enhancing compounds in root exudates, and several plant nonflavonoids possess nod gene-inducing properties. Expression of non-nod genes is induced by flavonoids; these include encoders of a type I secreted protein and the entire type III, and possibly also type IV, secretion systems. Many other genes and proteins in rhizobia are flavonoid-inducible but their functions are largely unknown. Rhizobia produce far more Nod factor variants than was previously envisaged and their structures can be influenced by the pH of the environment. Other symbiotically active compounds or systems of rhizobia, some of them universally present, are: the surface polysaccharides, quorum-sensing N-acyl homoserine lactones, plant growth-promoting lumichrome and two-component regulatory systems.

Inulin‐type fructans of longer degree of polymerization exert more pronounced in vitro prebiotic effects

Abstract: Two short chain fructans – oligofructose (DP 2–20) and inulin (DP 3–60) – were administered to the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) at 2·5 g day−1. The influence of fructan addition towards fermentation activity and microbial community composition from the different SHIME colon compartments were evaluated. Both fructans exerted prebiotic effects with significantly higher butyrate and propionate production and stimulation of lactic acid-producing bacteria. Compared with oligofructose, it was noted that it took more time before significant effects from inulin addition were observed. Yet, the higher short-chain fatty acid production and lower proteolytic activity showed that the prebiotic effects from inulin were more pronounced than oligofructose. Also, the bifidogenic effects from inulin vs oligofructose were higher in the distal colon compartments and this effect was prolonged in the distal colon once the addition was stopped.

Novel alternatives to antibiotics: bacteriophages, bacterial cell wall hydrolases, and antimicrobial peptides

Abstract: Extensive research has been conducted on the development of three groups of naturally occurring antimicrobials as novel alternatives to antibiotics: bacteriophages (phages), bacterial cell wall hydrolases (BCWH), and antimicrobial peptides (AMP). Phage therapies are highly efficient, highly specific, and relatively cost-effective. However, precautions have to be taken in the selection of phage candidates for therapeutic applications as some phages may encode toxins and others may, when integrated into host bacterial genome and converted to prophages in a lysogenic cycle, lead to bacterial immunity and altered virulence. BCWH are divided into three groups: lysozymes, autolysins, and virolysins. Among them, virolysins are the most promising candidates as they are highly specific and have the capability to rapidly lyse antibiotic-resistant bacteria on a generally species-specific basis. Finally, AMP are a family of natural proteins produced by eukaryotic and prokaryotic organisms or encoded by phages. AMP are of vast diversity in term of size, structure, mode of action, and specificity and have a high potential for clinical therapeutic applications.

Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum)

Abstract: Aim: To develop a probiotic with effectiveness against Aeromonas sp., which was pathogenic to rainbow trout. Methods and Results: When Bacillus subtilis AB1, which was obtained from fish intestine, was administered for 14 days to rainbow trout in feed at a concentration of 107 cells per gram either as viable, formalized or sonicated cells or as cell-free supernatant, the fish survived challenge with the pathogen. AB1 stimulated immune parameters, specifically stimulating respiratory burst, serum and gut lysozyme, peroxidase, phagocytic killing, total and α1-antiprotease and lymphocyte populations. Conclusions: Bacillus subtilis AB1 was effective as a probiotic at controlling infections by a fish-pathogenic Aeromonas sp. in rainbow trout. Significance and Impact of the Study: Disease control in fish is possible by means of the oral application of live and inactivated cells and their subcellular components with the mode of action reflecting stimulation of the innate immune response.

Development of antioxidant activity in milk whey during fermentation with lactic acid bacteria

Abstract: Aims: To investigate the production of antioxidant activity during fermentation with commonly used dairy starter cultures. Moreover, to study the development of antioxidant activity during fermentation, and the connection to proteolysis and bacterial growth. Methods and Results: Antioxidant activity was measured by analysing the radical scavenging activity using a spectrophotometric decolorization assay and lipid peroxidation inhibition was assayed using liposomal model system with a fluorescence method. Milk was fermented with 25 lactic acid bacterial (LAB) strains, and from these six strains, exhibiting the highest radical scavenging activity was selected for further investigation. Leuconostoc mesenteroides ssp. cremoris strains, Lactobacillus jensenii (ATCC 25258) and Lactobacillus acidophilus (ATCC 4356) showed the highest activity with both the methods used. However, the radical scavenging activity was stronger than lipid peroxidation inhibition activity. The development of radical scavenging activity was connected to proteolysis with four strains. Molecular distribution profiles showed that fermentates with high scavenging activity also possessed a higher proportion of peptides in the molecular mass range of 4–20 kDa, while others had mostly large polypeptides and compounds below 4 kDa. In addition, the amount of hydrophobic amino acids was higher in these fermentates. Conclusions: The development of antioxidant activity was strain-specific characteristic. The development of radical scavengers was more connected to the simultaneous development of proteolysis whereas, lipid peroxidation inhibitory activity was related to bacterial growth. However, high radical scavenging activity was not directly connected to the high degree of proteolysis Significance and Impact of the Study: To the best of our knowledge, this seems to be the first report, which screens possible antioxidant activity among most common dairy LAB strains. Use of such strains improve nutritional value of fermented dairy products.

Isolation and characterization of antagonistic Bacillus subtilis strains from the avocado rhizoplane displaying biocontrol activity.

Abstract: Aim: This study was undertaken to isolate Bacillus subtilis strains with biological activity against soil-borne phytopathogenic fungi from the avocado rhizoplane. Methods and Results: A collection of 905 bacterial isolates obtained from the rhizoplane of healthy avocado trees, contains 277 gram-positive isolates. From these gram-positive isolates, four strains, PCL1605, PCL1608, PCL1610 and PCL1612, identified as B. subtilis, were selected on the basis of their antifungal activity against diverse soil-borne phytopathogenic fungi. Analysis of the antifungal compounds involved in their antagonistic activity showed that these strains produced hydrolytic enzymes such as glucanases or proteases and the antibiotic lipopeptides surfactin, fengycin, and/or iturin A. In biocontrol trials using the pathosystems tomato/Fusarium oxysporum f.sp. radicis-lycopersici and avocado/Rosellinia necatrix, two B. subtilis strains, PCL1608 and PCL1612, both producing iturin A, exhibited the highest biocontrol and colonization capabilities. Conclusions: Diverse antagonistic B. subtilis strains isolated from healthy avocado rhizoplanes have shown promising biocontrol abilities, which are closely linked with the production of antifungal lipopeptides and good colonization aptitudes. Significance and Impact of the Study: This is one of the few reports dealing with isolation and characterization of B. subtilis strains with biocontrol activity against the common soil-borne phytopathogenic fungi F. oxysporum f.sp. radicis-lycopersici and R. necatrix.

Hydrolysis and microbial community analyses in two-stage anaerobic digestion of energy crops

Abstract: Aims: The roles of the diverse populations of micro-organisms responsible for biodegradation of organic matter to form methane and carbon dioxide are rudimentarily understood. To expand the knowledge on links between microbial communities and the rate limiting, hydrolytic stage of two-stage biogas production from energy crops, this study was performed. Methods and Results: The process performance. and microbial communities (as determined by fluorescence in situ hybridization) in two separate two-stage batch digestions of sugar beets and grass/clover were studied. The microbial populations developed in the hydrolytic stage of anaerobic digestion of beets and grass/clover showed very few similarities, despite that the hydrolysis dynamics were similar. In both substrates, the solubilization of organic material was rapid for the first 10 days and accompanied by a build-up of volatile fatty acids (VFAs) and lactate. Between days 10 and 15, VFA and lactate concentrations decreased, as did the solubilization rates. For both substrates, Archaea started to appear in the hydrolytic stage between days 10 and 15, and the fraction of Bacteria decreased. The major bacterial group detected in the leachate fraction for beets was Alphaproteobacteria, whereas for grass/clover it was Firmicutes. The number of cells that bound to probes specifically targeting bacteria with cellulolytic activity was higher in the digestion of grass than in the digestion of beet. Conclusions: This study allowed the identification of the general bacterial groups involved, and the identification of a clear shift in the microbial population when hydrolysis rate became limiting for each of the substrates investigated. Significance and Impact of the Study: The findings from this study could be considered as a first step towards the development of strategies to stimulate hydrolysis further and ultimately increasing the methane production rates and yields from reactor-based digestion of these substrates.

Diversity of the Lactobacillus group in breast milk and vagina of healthy women and potential role in the colonization of the infant gut

Abstract: Aims: To evaluate the diversity of the Lactobacillus group in breast milk and the vagina of healthy women and understand their potential role in the infant gut colonization using the 16S rRNA gene approaches. Methods and Results: Samples of breast milk, vaginal swabs and infant faeces were aseptically collected from five mothers whose neonates were born by vaginal delivery and another five that had their babies by caesarean section. After polymerase chain reaction (PCR) amplification using Lactobacillus group-specific primers, amplicons were analysed by denaturing gradient gel electrophoresis (DGGE). Clone libraries were constructed to describe the Lactobacillus group diversity. DGGE fingerprints were not related to the delivery method. None of the species detected in vaginal samples were found in breast milk-derived libraries and only few were detected in infant faeces. Conclusions: The bacterial composition of breast milk and infant faeces is not related to the delivery method. Significance and Impact of the Study: It has been suggested that neonates acquire lactobacilli by oral contamination with vaginal strains during delivery; subsequently, newborns would transmit such bacteria to the breast during breastfeeding. However, our findings confirm, at the molecular level that in contrast to the maternal vagina, breast milk seems to constitute a good source of lactobacilli to the infant gut.

Difference between the spore sizes of Bacillus anthracis and other Bacillus species

Abstract: Aims: To determine the size distribution of the spores of Bacillus anthracis, and compare its size with other Bacillus species grown and sporulated under similar conditions. Methods and Results: Spores from several Bacillus species, including seven strains of B. anthracis and six close neighbours, were prepared and studied using identical media, protocols and instruments. Here, we report the spore length and diameter distributions, as determined by transmission electron microscopy (TEM). We calculated the aspect ratio and volume of each spore. All the studied strains of B. anthracis had similar diameter (mean range between 0·81 ± 0·08 μm and 0·86 ± 0·08 μm). The mean lengths of the spores from different B. anthracis strains fell into two significantly different groups: one with mean spore lengths 1·26 ± 0·13 μm or shorter, and another group of strains with mean spore lengths between 1·49 and 1·67 μm. The strains of B. anthracis that were significantly shorter also sporulated with higher yield at relatively lower temperature. The grouping of B. anthracis strains by size and sporulation temperature did not correlate with their respective virulence. Conclusions: The spores of Bacillus subtilis and Bacillus atrophaeus (previously named Bacillus globigii), two commonly used simulants of B. anthracis, were considerably smaller in length, diameter and volume than all the B. anthracis spores studied. Although rarely used as simulants, the spores of Bacillus cereus and Bacillus thuringiensis had dimensions similar to those of B. anthracis. Significance and Impact of the Study: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefence against B. anthracis. The data presented here should help in the selection of simulants that better resemble the properties of B. anthracis, and thus, more accurately represent the performance of collectors, detectors and other countermeasures against this threat agent.

Microbe-aliphatic hydrocarbon interactions in soil: implications for biodegradation and bioremediation.

Abstract: Aliphatic hydrocarbons make up a substantial portion of organic contamination in the terrestrial environment. However, most studies have focussed on the fate and behaviour of aromatic contaminants in soil. Despite structural differences between aromatic and aliphatic hydrocarbons, both classes of contaminants are subject to physicochemical processes, which can affect the degree of loss, sequestration and interaction with soil microflora. Given the nature of hydrocarbon contamination of soils and the importance of bioremediation strategies, understanding the fate and behaviour of aliphatic hydrocarbons is imperative, particularly microbe-contaminant interactions. Biodegradation by microbes is the key removal process of hydrocarbons in soils, which is controlled by hydrocarbon physicochemistry, environmental conditions, bioavailability and the presence of catabolically active microbes. Therefore, the aims of this review are (i) to consider the physicochemical properties of aliphatic hydrocarbons and highlight mechanisms controlling their fate and behaviour in soil; (ii) to discuss the bioavailability and bioaccessibility of aliphatic hydrocarbons in soil, with particular attention being paid to biodegradation, and (iii) to briefly consider bioremediation techniques that may be applied to remove aliphatic hydrocarbons from soil.

Boza, a natural source of probiotic lactic acid bacteria.

Abstract: Aims: To evaluate the probiotic properties of strains isolated from boza, a traditional beverage produced from cereals. Methods and Results: The strains survived low pH conditions (pH 3·0), grew well at pH 9·0 and were not inhibited by the presence of 0·3% (w/v) oxbile. Cytotoxicity levels of the bacteriocins, expressed as CC50, ranged from 38 to 3776 μg ml−1. Bacteriocin bacST284BZ revealed high activity (EC50 = 735 μg ml−1) against herpes simplex virus type 1. Growth of Mycobacterium tuberculosis was 69% repressed after 5 days in the presence of bacST194BZ. Various levels of auto-cell aggregation and co-aggregation with Listeria innocua LMG 13568 were observed. Adhesion of the probiotic strains to HT-29 cells ranged from 18 to 22%. Conclusions: Boza is a rich source of probiotic lactic acid bacteria. All strains survived conditions simulating the gastrointestinal tract and produced bacteriocins active against a number of pathogens. Adherence to HT-29 and Caco-2 cells was within the range reported for Lactobacillus rhamnosus GG, a well-known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics. Significance and Impact of the Study: Boza contains a number of different probiotic lactic acid bacteria and could be marketed as a functional food product.

Effect of chitosan on hyphal growth and spore germination of plant pathogenic and biocontrol fungi

Abstract: Aims: To investigate the toxic effect of chitosan on important root pathogenic and biocontrol fungi (nematophagous, entomopathogenic and mycoparasitic). Methods and Results: We have used standard bioassays to investigate the effect of chitosan on colony growth and developed bioassays to test spore germination. The results showed that the root pathogenic and mycoparasitic fungi tested were more sensitive to chitosan than nematophagous and entomopathogenic fungi. Chitosanases (and perhaps related enzymes) are involved in the resistance to chitosan. Two fungi, one sensitive to chitosan, Fusarium oxysporum f. sp. radicis-lycopersici, and one less sensitive, Pochonia chlamydosporia, were selected for ultrastructural investigations. Transmission electron microscopy revealed differences in the ultrastructural alterations caused by chitosan in the spores of the plant pathogenic fungus and in those of the nematophagous fungus. Confocal laser microscopy showed that Rhodamine-labelled chitosan enters rapidly into conidia of both fungi, in an energy-dependent process. Conclusions: Nematophagous and entomopathogenic fungi are rather resistant to the toxic effect of chitosan. Resistance of nematophagous and entomopathogenic fungi to chitosan could be associated with their high extracellular chitosanolytic activity. Furthermore, ultrastructural damage is much more severe in the chitosan sensitive fungus. Significance and impact of the study: The results of this paper suggest that biocontrol fungi tested could be combined with chitosan for biological control of plant pathogens and pests.

Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils.

Abstract: Aim: To establish a rapid, improved soil environmental DNA extraction and purification protocol. Methods and Results: Three different soil DNA isolation and four purification strategies were compared on different soil samples with variable rates of success. Bead beating extraction gave significantly higher DNA yields than microwave-based and liquid nitrogen grinding DNA extraction methods. The inclusion of soil washing prior to cell lysis decreased the amount of purification steps required. Although these soil types differed, polyvinylpolypyrrolidone (PVPP)-sepharose 2B column elution was sufficient for all three samples, yielding DNA pure enough for successful application in molecular studies. One soil sample retained 80% of the initial DNA after successful purification. Conclusions: Optimization of a purification protocol confirmed that only a combination of previously described methods proved sufficient in yielding pure environmental DNA from humic-rich soils. Total processing time for DNA extraction and subsequent purification from multiple samples was considerably more rapid than the previously described methods. Significance and Impact of the Study: This study developed a new optimized soil DNA extraction and purification protocol that is suitable for different environmental sources that are rich in humic acid content.

High prevalence of antimicrobial-resistant genes and integrons in Escherichia coli isolates from Black-headed Gulls in the Czech Republic.

Abstract: Aims: To carry out an assessment of the occurrence of resistance to antimicrobials in Escherichia coli that has been isolated from young Black-headed Gulls in three nesting colonies. Methods and Results: A total of 257 isolates were tested for sensitivity to eight antibacterial substances by disk diffusion method. The polymerase chain reaction was used for detecting specific genes of antibacterial resistance and class 1 integrons in resistant E. coli isolates. A total 75 (29·9%) of 257 isolates were resistant to one or more antimicrobial agents. The dominant type of resistance was to tetracycline, detected in 49 (19·1%) isolates. Resistance to ampicillin was detected in 30 (11·7%), cephalothin in 11 (4·3%), streptomycin in 24 (9·3%), sulphonamides in 20 (7·8%) and chloramphenicol in 5 (1·9%) isolates. Nine isolates carrying integrons were detected. Conclusions: The study suggests that young Black-headed Gulls are an important host reservoir of resistant E. coli strains, probably reflecting the presence of such strains in their sources of food and/or water. Significance and Impact of the Study: Although Black-headed Gulls do not naturally come into contact with antibiotics, these birds can be infected with resistant E. coli and potentially serve as their reservoirs, vectors and bioindicators in the environment.

Antiobesity effect of trans-10,cis-12-conjugated linoleic acid-producing Lactobacillus plantarum PL62 on diet-induced obese mice

Abstract: Aims: To observe the antiobesity activity of trans-10,cis-12-conjugated linoleic acid (CLA)-producing lactobacillus in mice. Methods and Results: Lactobacillus plantarum PL62, which can grow in the presence of linoleic acid, was selected and studied. The culture supernatant of Lact. plantarum PL62 contained trans-10,cis-12-conjugated linoleic acid (6·4 μg ml−1), and the crude enzyme prepared from washed cells produced trans-10,cis-12 CLA (1395 μg mg−1 protein). Lact. plantarum PL62 reduced the weights of epididymal, inguinal, mesenteric, and perirenal white adipose tissues and significantly reduced the blood levels of total glucose and body weights of mice (P < 0·01). Conclusions: trans-10,cis-12-CLA-producing Lact. plantarum PL62 can exert the same antiobesity activity as trans-10,cis-12-CLA in mice. Significance and impact of the study: trans-10,cis-12-CLA-producing Lactobacillus can be a replacement for CLA for obesity treatment via the continuous production of trans-10,cis-12-CLA. The results provide a novel opportunity to develop foods with antiobesity activity.

Antifungal effects of essential oils from oregano and fennel on Sclerotinia sclerotiorum.

Abstract: Aims: The antifungal effects of essential oils of oregano (Origanum syriacum var. bevanii) and fennel (Foeniculum vulgare) were evaluated against Sclerotinia sclerotiorum. Effects of the essential oils on morphological structures of hyphae and sclerotia were studied under light and scanning electron microscopes (SEM). Methods and Results: Inhibitory effects of volatile and contact phases of the essential oils used were determined on hyphae and sclerotia. Both essential oils have a marked antifungal effect against S. sclerotiorum. Soil amendment with essential oils has significant effect on reducing sclerotial viability. Both essential oils significantly inhibited the fungal growth in soil, thereby increasing the number of surviving tomato seedling by 69·8% and 53·3%, respectively. Light and SEM observations on pathogen hyphae and sclerotia revealed considerable morphological alterations in hyphae and sclerotia. Conclusions: The significant reduction in the mycelial growth and germination of sclerotia would greatly reduce the pathogen inoculum source. This may influence the rate of disease development in soil. Significance and Impact of the Study: Considering the reduction in the number of diseased plants in infested soil amended with essential oils, we concluded that oregano and fennel essential oils could be used as possible bio fungicides alternative to synthetic fungicides against phytopathogenic fungi.

Effects of yogurt and bifidobacteria supplementation on the colonic microbiota in lactose-intolerant subjects.

Abstract: Aims: Colonic metabolism of lactose may play a role in lactose intolerance. We investigated whether a 2-week supplementation of Bifidobacterium longum (in capsules) and a yogurt enriched with Bifidobacterium animalis could modify the composition and metabolic activities of the colonic microbiota in 11 Chinese lactose-intolerant subjects. Methods and Results: The numbers of total cells, total bacteria and the Eubacterium rectale/Clostridium coccoides group in faeces as measured with fluorescent in situ hybridization and the faecal beta-galactosidase activity increased significantly during supplementation. The number of Bifidobacterium showed a tendency to increase during and after supplementation. With PCR-denaturing gradient gel electrophoresis, in subjects in which B. animalis and B. longum were not detected before supplementation, both strains were present in faeces during supplementation, but disappeared after supplementation. The degree of lactose digestion in the small intestine and the oro-caecal transit time were not different before and after supplementation, whereas symptom scores after lactose challenge decreased after supplementation. Conclusions: The results suggest that supplementation modifies the amount and metabolic activities of the colonic microbiota and alleviates symptoms in lactose-intolerant subjects. The changes in the colonic microbiota might be among the factors modified by the supplementation which lead to the alleviation of lactose intolerance. Significance and Impact of the Study: This study provides evidence for the possibility of managing lactose intolerance with dietary lactose ( yogurt) and probiotics via modulating the colonic microbiota.

Quantification by real-time PCR of cellulolytic bacteria in the rumen of sheep after supplementation of a forage diet with readily fermentable carbohydrates: effect of a yeast additive.

Abstract: Aim: To examine the effect of concentrate and yeast additive on the number of cellulolytic bacteria in the rumen of sheep. Methods and Results: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens were quantified using real-time PCR (targeting 16S rDNA) in parallel to cellulolytic flora enumeration with cultural techniques. Whatever the conditions tested, R. flavefaciens was slightly more abundant than F. succinogenes, with both species outnumbering R. albus. Before feeding, the shift from hay to hay plus concentrate diet had no effect on rumen pH and on the number of the three specie; while after feeding, the concentrate-supplemented diet induced a decrease (−1 log) of the number of the three species concomitant with the rumen acidification. Overall, the presence of the live yeast resulted in a significant increase (two- to fourfold) of the Ruminococci. Conclusion: The use of real-time PCR allowed us to show changes in the number of cellulolytic bacterial species in vivo in response to diet shift and additives that could not be as easily evidenced by classical microbial methods. Significance and Impact of the Study: This study contributes to the understanding of the negative impact of readily fermentable carbohydrates on rumen cellulolysis and the beneficial effect of yeast on rumen fermentation.

Antifungal activity of essential oils against filamentous fungi determined by broth microdilution and vapour contact methods.

Abstract: Aims: The in vitro activity of some essential oils (EO) (thyme red, fennel, clove, pine, sage, lemon balm and lavender) against clinical and environmental fungal strains was determined. Methods and Results: The minimal inhibitory concentrations were determined by a microdilution method in RPMI 1640 and by a vapour contact assay. The composition of oils was analysed by gas chromatography (GC) and GC/mass spectrometry. The results indicated that the oils antifungal activity depended on the experimental assay used. The inhibiting effects of EO in vapour phase were generally higher than those in liquid state. According to both methods thyme red and clove were found to be the oils with the widest spectrum of activity against all fungi tested. Conclusions: Despite the differences between the two methods, our results demonstrate that some EO are very active on dermatophytes and dematiaceous fungi. However, more data will be necessary to confirm this good in vitro efficacy. Significance and Impact of the Study: This study could identify candidates of EO for developing alternative methods to control environmental and clinically undesirable filamentous fungi.

Two Lactobacillus strains, isolated from breast milk, differently modulate the immune response

Abstract: Aims: The ability of two different Lactobacillus strains (Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716), isolated from human breast milk, to modulate the immune response was examined. Methods and Results: In rodent bone-marrow-derived macrophages (BMDM), the presence of Lact. fermentum CECT5716 induced pro-inflammatory cytokines, in contrast to the activation of IL-10 induced by Lact. salivarius CECT5713. Although both strains reduced the lipopolysaccharide (LPS)-induced inflammatory response in BMDM, the effect of Lact. salivarius CECT5713 was more efficient, probably because of the production of higher amounts of IL-10 cytokine. In vivo assays in mice showed similar results; the consumption of Lact. fermentum CECT5716 enhanced the production of Th1 cytokines by spleen cells and increased the IgA concentration in faeces. However, the consumption of Lact. salivarius CECT5713 induced IL-10 production by spleen cells. Conclusion: Therefore, in general, the effect of Lact. fermentum CECT5716 is immunostimulatory in contrast to the anti-inflammatory effect of Lact. salivarius CECT5713. Significance and Impact of the Study: The results of this study show that two Lactobacillus strains isolated from breast milk can exert different and even opposing effects on immune response demonstrating the specificity of each strain.

Production of cellulases and hemicellulases by Penicillium echinulatum grown on pretreated sugar cane bagasse and wheat bran in solid-state fermentation.

Abstract: Aim: To evaluate the solid-state fermentation (SSF) production of cellulase and hemicellulases (xylanases), by Penicillium echinulatum 9A02S1, in experiments carried out with different concentrations of the pretreated sugar cane bagasse (PSCB) and wheat bran (WB). Methods and Results: This study reports the production of xylanolytic and cellulolytic enzymes by P. echinulatum 9A02S1 using a cheap medium containing PSCB and WB under SSF. The highest amounts of filter paper activity (FPA) could be measured on mixtures of PSCB and WB (32·89 ± 1·90 U gdm−1). The highest β-glucosidase activity was 58·95 ± 2·58 U gdm−1 on the fourth day. The highest activity for endoglucanases was 282·36 ± 1·23 U gdm−1 on the fourth day, and for xylanases the activity was around 10 U gdm−1 from the second to the fourth day. Conclusions: The present work has established the potential of P. echinulatum for FPA, endoglucanase, β-glucosidase and xylanase productions in SSF, indicating that WB may be partially substituted by PSCB. Significance and Impact of the Study: The incorporation of cheap sources, such as sugar cane bagasse, into media for the production of lignocellulose enzymes should help decrease the production costs of enzymatic complexes that can hydrolyse lignocellulose residues for the formation of fermented syrups, thus contributing to the economic production of bioethanol.

New water disinfection system using UVA light-emitting diodes

Abstract: Aim: To evaluate the ability of high-energy ultraviolet A (UVA) light-emitting diode (LED) to inactivate bacteria in water and investigate the inactivating mechanism of UVA irradiation. Methods and Results: We developed a new disinfection device equipped with high-energy UVA-LED. Inactivation of bacteria was determined by colony-forming assay. Vibrio parahaemolyticus, enteropathogenic Escherichia coli, Staphylococcus aureus and Escherichia coli DH5α were reduced by greater than 5-log10 stages within 75 min at 315 J cm−2 of UVA. Salmonella enteritidis was reduced greater than 4-log10 stages within 160 min at 672 J cm−2 of UVA. The formation of 8-hydroxy-2′-deoxyguanosine in UVA-LED irradiated bacteria was 2·6-fold higher than that of UVC-irradiated bacteria at the same inactivation level. Addition of mannitol, a scavenger of hydroxyl radicals (OH˙), or catalase, an enzyme scavenging hydrogen peroxide (H2O2) to bacterial suspensions significantly suppressed disinfection effect of UVA-LED. Conclusion: This disinfection system has enough ability to inactivate bacteria and OH˙ and H2O2 participates in the disinfection mechanism of UVA irradiation. Significance and Impact of the Study: We newly developed UVA irradiation system and found that UVA alone was able to disinfect the water efficiently. This will become a useful disinfection system.