Francesca Gaggìa

Probiotics and prebiotics in animal feeding for safe food production

Recent outbreaks of food-borne diseases highlight the need for reducing bacterial pathogens in foods of animal origin. Animal enteric pathogens are a direct source for food contamination. The ban of antibiotics as growth promoters (AGPs) has been a challenge for animal nutrition increasing the need to find alternative methods to control and prevent pathogenic bacterial colonization. The modulation of the gut microbiota with new feed additives, such as probiotics and prebiotics, towards host-protecting functions to support animal health, is a topical issue in animal breeding and creates fascinating possibilities. Although the knowledge on the effects of such feed additives has increased, essential information concerning their impact on the host are, to date, incomplete. For the future, the most important target, within probiotic and prebiotic research, is a demonstrated health-promoting benefit supported by knowledge on the mechanistic actions. Genomic-based knowledge on the composition and functions of the gut microbiota, as well as its deviations, will advance the selection of new and specific probiotics. Potential combinations of suitable probiotics and prebiotics may prove to be the next step to reduce the risk of intestinal diseases and remove specific microbial disorders. In this review we discuss the current knowledge on the contribution of the gut microbiota to host well-being. Moreover, we review available information on probiotics and prebiotics and their application in animal feeding.

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.

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.

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.

Intracellular pH of Mycobacterium avium subsp. paratuberculosis following exposure to antimicrobial compounds monitored at the single cell level

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johnes disease; moreover, it seems to be implicated in the development of Crohns disease in humans. In the present study, fluorescence ratio imaging microscopy (FRIM) was used to assess changes in intracellular pH (pH(i)) of one strain of MAP after exposure to nisin and neutralized cell-free supernatants (NCSs) from five bacteriocin-producing lactic acid bacteria (LAB) with known probiotic properties. The evaluation of pH(i) by FRIM provides information about the physiological state of bacterial cells, bypassing the long and problematic incubations needed for methods relying upon growth of MAP such as determination of colony forming units. The FRIM results showed that both nisin and the cell-free supernatant from Lactobacillus plantarum PCA 236 affected the pH(i) of MAP within a few hours. However, monitoring the population for 24h revealed the presence of a subpopulation of cells probably resistant to the antimicrobial compounds tested. Use of nisin and bacteriocin-producing LAB strains could lead to new intervention strategies for the control of MAP based on in vivo application of probiotic cultures as feed additives at farm level.

Beneficial microorganisms for honey bees: problems and progresses

Nowadays, honey bees are stressed by a number of biotic and abiotic factors which may compromise to some extent the pollination service and the hive productivity. The EU ban of antibiotics as therapeutic agents against bee pathogens has stimulated the search for natural alternatives. The increasing knowledge on the composition and functions of the bee gut microbiota and the link between a balanced gut microbiota and health status have encouraged the research on the use of gut microorganisms to improve bee health. Somehow, we are assisting to the transfer of the “probiotic concept” into the bee science. In this review, we examine the role of the honey bee gut microbiota in bee health and critically describe the available applications of beneficial microorganisms as pest control agents and health support. Most of the strains, mainly belonging to the genera Lactobacillus, Bifidobacterium and Bacillus, are isolated from honey bee crop or gut, but some applications involve environmental strains or formulation for animal and human consumption. Overall, the obtained results show the favourable effect of applied microbial strains on bee health and productivity, in particular if strains of bee origin are used. However, it is actually not yet possible to conclude whether this strategy will ever work. In particular, many aspects regarding the overall setup of the experiments, the dose, the timing and the duration of the treatment need to be optimized, also considering the microbiological safety of the hive products (i.e. pollen and honey). In addition, a deep investigation about the effect on host immunity and physiology is envisaged. Lastly, the final users of the formulations, i.e. beekeepers, should be taken into account for the achievement of high-quality, cost-effective and easy-to-use products.

Bioavailability and biological effect of engineered silver nanoparticles in a forest soil

The extensive use of silver nanoparticles (SNPs) as antimicrobial in food, clothing and medicine, leads inevitably to a loss of such nanomaterial in soil and water. Little is known about the effects of soil contamination, in particular, on microbial cells, which play a fundamental ecological role. In this work, the impact of SNPs on forest soil has been studied, investigating eco-physiological indicators of microbial biomass and microbial diversity with culture-dependent and independent techniques. Moreover, SNPs bioavailability and uptake were assessed. Soil samples were spiked with SNPs at two different concentrations (10 and 100 μg g(-1)dw) and incubated with the relative controls for 30, 60 and 90 days. The overall parameters showed a significant influence of the SNPs on the soil microbial community, revealing a marked shift after 60 days of incubation.

Effect of dietary supplementation of Bifidobacterium and Lactobacillus strains in Apis mellifera L. against Nosema ceranae.

Nosema ceranae is a widespread microsporidium of European honeybee Apis mellifera L. affecting bee health. The ban of Fumagillin-B (dicyclohexylammonium salt) in the European Union has driven the search for sustainable strategies to prevent and control the infection. The gut microbial symbionts, associated to the intestinal system of vertebrates and invertebrates and its impact on host health, are receiving increasing attention. In particular, bifidobacteria and lactobacilli, which are normal inhabitants of the digestive system of bees, are known to protect their hosts via antimicrobial metabolites, immunomodulation and competition. In this work, the dietary supplementation of gut bacteria was evaluated under laboratory conditions in bees artificially infected with the parasite and bees not artificially infected but evidencing a low natural infection. Supplemented bacteria were selected among bifidobacteria, previously isolated, and lactobacilli, isolated in this work from healthy honeybee gut. Four treatments were compared: bees fed with sugar syrup (CTR); bees fed with sugar syrup containing bifidobacteria and lactobacilli (PRO); bees infected with N. ceranae spores and fed with sugar syrup (NOS); bees infected with N. ceranae and fed with sugar syrup containing bifidobacteria and lactobacilli (NP). The sugar syrup, with or without microorganisms, was administered to bees from the first day of life for 13 days. N. ceranae infection was carried out individually on anesthetised 5-day-old bees. Eight days after infection, a significant (P<0.05) lower level of N. ceranae was detected by real-time PCR in both NP and PRO group, showing a positive effect of supplemented microorganisms in controlling the infection. These results represent a first attempt of application of bifidobacteria and lactobacilli against N. ceranae in honeybees.

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.

Molecular characterisation of an endophyte showing a strong antagonistic activity against Pseudomonas syringae pv. actinidiae

Background and aimsEndophytic bacteria have been often studied as biological control agents of plant pathogens and many of their secondary metabolites involved in antagonism are Non Ribosomal Peptides (NRPs). In this study, the molecular basis of the biocontrol properties of the endophyte Pseudomonas synxantha, isolated from Actinidia chinensis, against Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial kiwi canker, has been investigated.MethodsAntagonism-deficient mutants of P. synxantha strain DLS65 were generated by insertion of minitransposon mTn5-GNm in its genome. Southern blot analysis allowed the selection of single transposon insertion-mutants. Amplification of the transposon flanking regions by means of arbitrary and single primer PCR in selected mutants was perfomed to obtain amplicons for sequencing purposes.ResultsSequencing results of the amplicons obtained from three antagonism-deficient mutants led to the localization of the transposonin three genes, which implies their involvement in the antagonism of P. synxantha: an acyl-homoserine lactone acylase gene (pvdQ), a glucose-6-phosphate dehydrogenase gene (zwf) and an mbtH-like gene were identified. It is known from the literature that these three genes are involved directly or indirectly in NRPs synthesis.ConclusionsWe suggest that a molecule with antibiotic properties, produced by NRP synthetases, contributes to the antagonistic activity of P. synxantha.