Bruno Biavati

Characterization of probiotic strains: an application as feed additives in poultry against Campylobacter jejuni.

Campylobacteriosis is at present the most frequent zoonosis in humans and the main source is poultry meat contaminated by Campylobacter jejuni. An alternative and effective approach to antibiotic administration to livestock to reduce bacterial contamination is the use of probiotics, which can help to improve the natural defence of animals against pathogenic bacteria. In this study 55 lactic acid bacteria and bifidobacteria were screened for desirable properties for their application as probiotics against Campylobacter in poultry. All bacteria were examined for their antimicrobial activity against three C. jejuni strains. Strains exhibiting the highest anti-Campylobacter activity were examined for their survival in the gastro intestinal tract (low pH and presence of bile salts) and food/feed processing conditions (high temperature, high NaCl concentration and starvation) and basic safety aspects such as antibiotic susceptibility and hemolytic activity were studied. On the basis of these activities, two strains, namely Lactobacillus plantarum PCS 20 and Bifidobacterium longum PCB 133, were chosen for an in vivo trial in poultry. They were separately administered to healthy chickens in order to evaluate their capability of colonizing the GI tract of poultry and to estimate their effect on C. jejuni population. The results evidenced that L. plantarum PCS 20 was not present in poultry feces at detectable concentration, whereas B. longum PCB 133 significantly increased after two weeks of daily administration and its amount was still high after a wash-out period of 6 days. In the same period, C. jejuni concentration in poultry feces was significantly reduced in chickens administered with B. longum PCB 133. Therefore, B. longum PCB 133, possessing interesting probiotic properties and a marked anti-Campylobacter activity both in vitro and in vivo, is an excellent candidate for being employed as additives to feed for poultry for the reduction of food-borne campylobacteriosis in humans.

Characterization and antimicrobial activity of essential oils of industrial hemp varieties (Cannabis sativa L.)

The present study focused on inhibitory activity of freshly extracted essential oils from three legal (THC<0.2% w/v) hemp varieties (Carmagnola, Fibranova and Futura) on microbial growth. The effect of different sowing times on oil composition and biological activity was also evaluated. Essential oils were distilled and then characterized through the gas chromatography and gas chromatography-mass spectrometry. Thereafter, the oils were compared to standard reagents on a broad range inhibition of microbial growth via minimum inhibitory concentration (MIC) assay. Microbial strains were divided into three groups: i) Gram (+) bacteria, which regard to food-borne pathogens or gastrointestinal bacteria, ii) Gram (-) bacteria and iii) yeasts, both being involved in plant interactions. The results showed that essential oils of industrial hemp can significantly inhibit the microbial growth, to an extent depending on variety and sowing time. It can be concluded that essential oils of industrial hemp, especially those of Futura, may have interesting applications to control spoilage and food-borne pathogens and phytopathogens microorganisms.

The potential of bifidobacteria as a source of natural folate

Aims:u2002 To screen 19 strains of bifidobacteria for main folate forms composition in synthetic folate‐free and complex folate‐containing media.

A Bifidobacterium-based synbiotic product to reduce the transmission of C. jejuni along the poultry food chain

With the ban of dietary antimicrobial agents, the use of probiotics, prebiotics and synbiotics has attracted a great deal of attention in order to improve intestinal health and control food-borne pathogens, which is an important concern for the production of safe meat and meat products. Recently, Campylobacter jejuni has emerged as a leading bacterial cause of food-borne gastroenteritis in humans, and epidemiological evidences indicate poultry and poultry products as the main source of human infection. This work aimed at the development of a synbiotic mixture capable of modulating the gut microbiota of broiler chickens to obtain an increase of the beneficial bacteria (i.e. bifidobacteria, lactobacilli) and a competitive reduction of C. jejuni. The prebiotic compound used in the mixture was chosen after an in vivo trial: a fructooligosaccharide and a galactooligosaccharide were separately administered to broilers mixed with normal feed at a concentration of 0.5% and 3%, respectively. Quantitative PCR on DNA extracted from fecal samples revealed a significant (p<0.05) increase of Bifidobacterium spp. in broilers treated with the galactooligosaccharide, coupled to a decrease (p<0.05) of Campylobacter spp. The galactooligosaccharide was then combined with a probiotic Bifidobacterium strain (B. longum subsp. longum PCB133), possessing in vitro antimicrobial activity against C. jejuni. The strain was microencapsulated in a lipid matrix to ensure viability into the feed and resistance to stomach transit. Finally, the synbiotic mixture was administered to broiler chickens for 14 days mixed with normal feed in order to have an intake of 10(9)CFU of PCB133/day. Bifidobacterium spp., Lactobacillus spp., Campylobacter spp., B. longum subsp. longum and C. jejuni were quantified in fecal samples. PCB133 was recovered in feces of all animals. C. jejuni concentration in poultry feces was significantly (p<0.05) reduced in chickens administered with the synbiotic mixture. This study allowed to highlight the positive effect of the synbiotic approach for C. jejuni reduction in broiler chickens, which is of fundamental importance for the safety of poultry meat consumers.

Characterization of Bifidobacterium spp. strains for the treatment of enteric disorders in newborns

Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastrointestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to nontumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify three Bifidobacterium breve strains and a Bifidobacterium longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics. A validation clinical trial involving the selected strains is being planned.

Inoculation with microorganisms of Lolium perenne L.: evaluation of plant growth parameters and endophytic colonization of roots

Turfgrasses are not only designed for recreation activities, but they also provide beneficial environmental effects and positively influence the human wellness. Their major problems are predisposition to tearing out and microbial diseases. The aim of this study was to investigate whether the inoculation of microorganisms can be effective to improve plant growth and root development of perennial ryegrass, to evaluate new sustainable practice for green preservation. A microorganism-based commercial product was used to amend hydroponically grown Lolium perenne L. and results compared with the use of the same filtered product, a phytohormone solution and an untreated control. Plants were grown for five weeks, shoots cut and measured at one-week interval and, at the end, roots were measured for length and weight. Shoot resistance to tearing out was also tested. Moreover, the main microbial groups present in the product were characterized and the microbial profile of sand and root samples was investigated by PCR-DGGE. The plants treated with the product showed an increased resistance to tearing out with respect to other treatments and roots were longer with respect to the control. Microbial analyses of the product evidenced bacterial and yeast species with plant growth promoting activity, such as Stenothrophomonas maltophilia, Candida utilis and several Lactobacillus species. Some Lactobacillus strains were also found to be able to colonize plant roots. In conclusion, the treatment with microorganisms has a great potential for the maintenance and increased performance of turfgrass surfaces.

Identification of species belonging to the Bifidobacterium genus by PCR-RFLP analysis of a hsp60 gene fragment

BackgroundBifidobacterium represents one of the largest genus within the Actinobacteria, and includes at present 32 species. These species share a high sequence homology of 16S rDNA and several molecular techniques already applied to discriminate among them give ambiguous results.The slightly higher variability of the hsp60 gene sequences with respect to the 16S rRNA sequences offers better opportunities to design or develop molecular assays, allowing identification and differentiation of closely related species. hsp60 can be considered an excellent additional marker for inferring the taxonomy of the members of Bifidobacterium genus.ResultsThis work illustrates a simple and cheap molecular tool for the identification of Bifidobacterium species. The hsp60 universal primers were used in a simple PCR procedure for the direct amplification of 590xa0bp of the hsp60 sequence. The in silico restriction analysis of bifidobacterial hsp60 partial sequences allowed the identification of a single endonuclease (HaeIII) able to provide different PCR-restriction fragment length polymorphism (RFLP) patterns in the Bifidobacterium spp. type strains evaluated. The electrophoretic analyses allowed to confirm the different RFLP patterns.ConclusionsThe developed PCR-RFLP technique resulted in efficient discrimination of the tested species and subspecies and allowed the construction of a dichotomous key in order to differentiate the most widely distributed Bifidobacterium species as well as the subspecies belonging to B. pseudolongum and B. animalis.

Role of intestinal microbiota in colon cancer prevention

Environmental and hereditary factors, together with lifestyle, are important factors in colon cancer development. Considering the increasing incidence of this disease, especially in the developed western world, the last decade has seen much attention directed towards understanding possible prevention strategies. Efforts to study the intestinal microbiota and its interaction with the host have underlined that disbiosis in colonic bacterial composition is a risk factor for colon cancer. Modulation of the composition of intestinal microbiota through the use of probiotic, prebiotic and synbiotic products could therefore represent a strategy for prevention of cancer development. The mechanisms underlying the probiotic-prebiotic anticarcinogenic effect involve a combination of events: e.g. binding of mutagens, suppression of bacteria that convert pro-carcinogens into carcinogens, immune system stimulation, and a reduction in the level of certain intestinal bacterial enzymes that promote carcinogen formation.

Understanding and monitoring pathogen behaviour in the food chain

Abstract: Understanding and monitoring of microbial behaviour of pathogens in the entire food chain is seen as one of the most important prerequisites for a lower prevalence of foodborne pathogens. For this purpose, novel methods have been developed to analyse the interactions of pathogens and food and feed matrices at cellular and molecular levels as well as contact surfaces in the food chain, including the intestinal tract of farm animals. The methods are mainly based upon advanced techniques of microscopy allowing analysis of single cells in time lapse studies and the rapidly growing information available in the field of functional genomics. Further emphasis has been given to development of functional mammalian cell models to study host pathogen interactions and to select novel probiotic cultures. Molecular culture-independent methods and microarrays are useful in monitoring microbial behaviour in the food chain and are often superior to the classical methods for detection of pathogens. Effective interventions to break the transmission of pathogens along the food chain are addressed, with special attention given to the present gap between food technology, hygienic design and food safety. Finally, the fact that effective food safety management is necessary for safe food is emphasised.

Carbohydrate stress-related response in Bifidobacterium pseudolongum subsp. globosum

Bifidobacteria are indigenous components of human and animal gastrointestinal microbiota, and their health-promoting benefits have long been recognized. Of the 36 currently described species of the Bifidobacterium genus, 8 contain plasmids, most of which are cryptic. It is possible that plasmid presence is related very closely to environmental change, so in conditions of stress this presence could be specifically controlled. For plasmid-positive Bifidobacterium pseudolongum subsp. globosum RU809/1, the influence of the type and concentration of the carbohydrate source is evident in the dramatic pVS809 curing effect when growth is conducted in the presence of 0.15% (w/v) glucose, lactose, maltose, melibiose, raffinose or starch. The effect is linked to carbohydrate starvation, not to carbohydrate abundance, and is independent of biomass growth. Plasmid curing was achieved after one or two consecutive transfers, also in cells grown on medium containing 0.15% arabinose, fructose, galactose and sucrose, but not mannose, ribose or xylose. Knowing plasmid behavior in stressful conditions, like carbon source availability, has allowed an early insight into carbohydrate starvation as a curing agent for bifidobacteria. Furthermore, knowledge of plasmid behavior in stressful conditions could be important not only in genetics and ecology but also in food-grade and pharmaceutical applications for the development of cloning and expression vector systems for bifidobacteria.