Diana Di Gioia

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.

Vanillin production using metabolically engineered Escherichia coli under non-growing conditions.

BackgroundVanillin is one of the most important aromatic flavour compounds used in the food and cosmetic industries. Natural vanillin is extracted from vanilla beans and is relatively expensive. Moreover, the consumer demand for natural vanillin highly exceeds the amount of vanillin extracted by plant sources. This has led to the investigation of other routes to obtain this flavour such as the biotechnological production from ferulic acid. Studies concerning the use of engineered recombinant Escherichia coli cells as biocatalysts for vanillin production are described in the literature, but yield optimization and biotransformation conditions have not been investigated in details.ResultsEffect of plasmid copy number in metabolic engineering of E. coli for the synthesis of vanillin has been evaluated by the use of genes encoding feruloyl-CoA synthetase and feruloyl hydratase/aldolase from Pseudomonas fluorescens BF13. The higher vanillin production yield was obtained using resting cells of E. coli strain JM109 harbouring a low-copy number vector and a promoter exhibiting a low activity to drive the expression of the catabolic genes. Optimization of the bioconversion of ferulic acid to vanillin was accomplished by a response surface methodology. The experimental conditions that allowed us to obtain high values for response functions were 3.3 mM ferulic acid and 4.5 g/L of biomass, with a yield of 70.6% and specific productivity of 5.9 μmoles/g × min after 3 hours of incubation. The final concentration of vanillin in the medium was increased up to 3.5 mM after a 6-hour incubation by sequential spiking of 1.1 mM ferulic acid. The resting cells could be reused up to four times maintaining the production yield levels over 50%, thus increasing three times the vanillin obtained per gram of biomass.ConclusionFerulic acid can be efficiently converted to vanillin, without accumulation of undesirable vanillin reduction/oxidation products, using E. coli JM109 cells expressing genes from the ferulic acid-degrader Pseudomonas fluorescens BF13. Optimization of culture conditions and bioconversion parameters, together with the reuse of the biomass, leaded to a final production of 2.52 g of vanillin per liter of culture, which is the highest found in the literature for recombinant strains and the highest achieved so far applying such strains under resting cells conditions.

Bifidobacteria: their impact on gut microbiota composition and their applications as probiotics in infants

This review is aimed at describing the most recent advances in the gut microbiota composition of newborns and infants with a particular emphasis on bifidobacteria. The newborn gut microbiota is quite unstable, whereas after weaning, it becomes more stable and gets closer to the typical adult microbiota. The newborn and infant gut microbiota composition is impaired in several enteric and non-enteric pathologies. The core of this review is the description of the most recent documented applications of bifidobacteria to newborns and infants for their prevention and treatment. Acute diarrhea is the most studied disease for which bifidobacteria are applied with great success, Bifidobacterium longum and Bifidobacterium breve being the most applied species. Moreover, the most recent updates in the use of bifidobacteria for the prevention and treatment of pathologies typical of newborns, such as necrotizing enterocolitis, colics, and streptococcal infections, are presented. In addition, a number of not strictly enteric pathologies have in recent years evidenced a strict correlation with an aberrant gut microbiota in infants, in particular showing a reduced level of bifidobacteria. These diseases represent new potential opportunities for probiotic applications. Among them, allergic diseases, celiac disease, obesity, and neurologic diseases are described in this review. The preliminary use of bifidobacteria in in vitro systems and animal models is summarized as well as preliminary in vivo studies. Only after validation of the results via human clinical trials will the potentiality of bifidobacteria in the prevention and cure of these pathologies be definitely assessed.

Cyclodextrin effects on the ex-situ bioremediation of a chronically polychlorobiphenyl-contaminated soil

The possibility of enhancing the intrinsic ex-situ bioremediation of a chronically polychlorinated biphenyl-contaminated soil by using cyclodextrins was studied in this work. The soil, contaminated with a large array of polychlorinated biphenyls and deriving from a dump site where it has been stored for about 10 years, was found to contain indigenous cultivable aerobic bacteria capable of utilising biphenyl and chlorobenzoic acids. The soil was amended with inorganic nutrients and biphenyl, saturated with water, and treated in aerobic batch slurry- and fixed-phase reactors. Hydroxypropyl-beta-cyclodextrin and gamma-cyclodextrin, added to both reactor systems at the concentration of 10 g/L at the 39th and 100th days of treatment, were found to generally enhance the depletion rate and extent of the soil polychlorobiphenyls. Despite some abiotic losses could have affected the depletion data, experimental evidence, such as the production of metabolites tentatively characterized as chlorobenzoic acids and chloride ion accumulation in the reactors, indicated that cyclodextrins significantly enhanced the biological degradation of the soil polychlorobiphenyls. This result has been ascribed to the capability of cyclodextrins of enhancing the availability of polychlorobiphenyls in the hydrophilic soil environment populated by immobilised and suspended indigenous soil microorganisms. Both cyclodextrins were metabolised by the indigenous soil microorganisms at the concentration at which they were used. Therefore, cyclodextrins, both for their capability of enhancing the biodegradation of soil polychlorobiphenyls and for their biodegradability, can have the potential of being successfully used in the bioremediation of chronically polychlorinated biphenyl-contaminated soils. Copyright 1998 John Wiley & Sons, Inc.

Metabolic engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid.

Vanillin is one of the most important flavors in the food industry and there is great interest in its production through biotechnological processes starting from natural substrates such as ferulic acid. Among bacteria, recombinant Escherichia coli strains are the most efficient vanillin producers, whereas Pseudomonas spp. strains, although possessing a broader metabolic versatility, rapidly metabolize various phenolic compounds including vanillin. In order to develop a robust Pseudomonas strain that can produce vanillin in high yields and at high productivity, the vanillin dehydrogenase (vdh)-encoding gene of Pseudomonas fluorescens BF13 strain was inactivated via targeted mutagenesis. The results demonstrated that engineered derivatives of strain BF13 accumulate vanillin if inactivation of vdh is associated with concurrent expression of structural genes for feruloyl-CoA synthetase (fcs) and hydratase/aldolase (ech) from a low-copy plasmid. The conversion of ferulic acid to vanillin was enhanced by optimization of growth conditions, growth phase and parameters of the bioconversion process. The developed strain produced up to 8.41 mM vanillin, which is the highest final titer of vanillin produced by a Pseudomonas strain to date and opens new perspectives in the use of bacterial biocatalysts for biotechnological production of vanillin from agro-industrial wastes which contain ferulic acid.

Antibacterial effectiveness of dentin bonding systems

This study examined the antibacterial activities of several commercially available glass ionomer cements, dentin bonding systems and luting agents by employing both agar plate diffusion (APD) and growth inhibition (GI) methods. Amalgam and resin composites were also tested as control materials. In both methods (APD and GI), cylindrical specimens were used. Four bacteria strains were tested: Streptococcus mutans, S. salivarius, S. mitis and S. sanguis. These studies were performed using standardized innoculums with selective media, and the assayed materials were directly applied on the assay cultures and plates. The results of agar plate assay were in accordance with the results of growth inhibition method. The glass ionomer cements showed marked antibacterial activity. On the contrary, amalgam, composites, luting agents and dentinal bonding systems did not affect bacterial growth. The sensitivity of the growth method showed that all the strains were inhibited in the same way by each inhibitory material. The data suggest that the use of glass ionomer cements as cavity liners/bases may reduce the consequences of microleakage due to its antibacterial properties.

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.

Influence of intrapartum antibiotic prophylaxis against group B Streptococcus on the early newborn gut composition and evaluation of the anti-Streptococcus activity of Bifidobacterium strains

Several factors are known to influence the early colonization of the gut in newborns. Among them, the use of antibiotics on the mother during labor, referred to as intrapartum antibiotic prophylaxis (IAP), has scarcely been investigated, although this practice is routinely used in group B Streptococcus (GBS)-positive women. This work is therefore aimed at verifying whether IAP can influence the main microbial groups of the newborn gut microbiota at an early stage of microbial establishment. Fifty-two newborns were recruited: 26 born by mothers negative to GBS (control group) and 26 by mothers positive to GBS and subjected to IAP with ampicillin (IAP group). Selected microbial groups (Lactobacillus spp., Bidobacterium spp., Bacteroides fragilis, Clostridium difficile, and Escherichia coli) were quantified with real-time PCR on DNA extracted from newborn feces. Further analysis was performed within the Bidobacterium genus by using DGGE after amplification with genus-specific primers. Results obtained showed a significant decrease of the bifidobacteria counts after antibiotic treatment of the mother. Bifidobacteria were found to be affected by IAP not only quantitatively but also qualitatively. In fact, IAP determined a decrement in the frequency of Bidobacterium breve, Bidobacterium bifidum, and Bidobacterium dentium with respect to the control group. Moreover, this study has preliminarily evaluated that some bifidobacterial strains, previously selected for use in infants, have antibacterial properties against GBS and are therefore potential candidates for being applied as probiotics for the prevention of GBS infections.

Effects of Triton X-100 and Quillaya Saponin on the ex situ bioremediation of a chronically polychlorobiphenyl-contaminated soil

Abstract The possibility of enhancing the ex situ bioremediation of a chronically polychlorinated biphenyl (PCB)-contaminated soil by using Triton X-100 or Quillaya Saponin, a synthetic and a biogenic surfactant, respectively, was studied. The soil, which contained about 350 mg/kg of PCBs and indigenous aerobic bacteria capable of growing on biphenyl or on monochlorobenzoic acids, was amended with inorganic nutrients and biphenyl, saturated with water and treated in aerobic batch slurry- and fixed-phase reactors. Triton X-100 and Quillaya Saponin were added to the reactors at a final concentration of 10 g/l at the 42nd day of treatment, and at the 43rd and 100th day, respectively. Triton X-100 was not metabolised by the soil microflora and it exerted inhibitory effects on the indigenous bacteria. Quillaya Saponin, on the contrary, was readily metabolised by the soil microflora. Under slurry-phase conditions, Triton X-100 negatively influenced the soil bioremediation process by affecting the availability of the chlorobenzoic acid degrading indigenous bacteria, whereas Quillaya Saponin slightly enhanced the biological degradation and dechlorination of the soil PCBs. In the fixed-phase reactors, where both the surfactant availability and the mixing of the soil were lower, Triton X-100 did not exert inhibitory effects on the soil biomass and enhanced significantly the soil PCB depletion, whereas Quillaya Saponin did not influence the bioremediation process.

Biodegradation of synthetic and naturally occurring mixtures of mono-cyclic aromatic compounds present in olive mill wastewaters by two aerobic bacteria

Abstract. Two bacterial strains, Ralstonia sp. LD35 and Pseudomonas putida DSM 1868, were assayed for their ability to degrade the monocyclic aromatic compounds commonly found in olive mill wastewaters (OMWs). The goal was to study the possibility of employing the two strains in the removal of these recalcitrant and toxic compounds from the effluents of anaerobic treatment plants fed with OMWs. At first, the two strains were separately assayed for their ability to degrade a synthetic mixture of nine aromatic acids present in OMWs, both in growing- and resting-cell conditions. Then, due to the complementary activity exhibited by the two strains, a co-culture of the two bacteria was tested under growing-cell conditions for degradation of the same synthetic mixture. Finally, the degradation activity of the co-culture on two fractions was studied. Both fractions one deriving from natural OMWs through reverse osmosis treatment and containing low-molecular weight organic molecules, and the other obtained from an anaerobic lab-scale treatment plant fed with OMWs, were rich in monocyclic aromatic compounds. The co-culture of the two strains was able to biodegrade seven of the nine components of the tested synthetic mix (2, 6-dihydroxybenzoic acid and 3, 4, 5-trimethoxybenzoic acid were the two undegraded compounds). In addition, an efficient biodegrading activity towards several aromatic molecules present in the two natural fractions was demonstrated.