Impatto di rifaximina sul microbiota intestinale: selezione di bifidobatteri antibiotico resistenti

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

Changes in plant metabolism induced by dioxygenase inhibitors and their effect on the epiphytic microbial community and fire blight (Erwinia amylovora) control

Fire blight, caused by the gram negative bacterium Erwinia amylovora, is one of the most destructive bacterial diseases of Pomaceous plants. Therefore, the development of reliable methods to control this disease is desperately needed. This research investigated the possibility to interfere, by altering plant metabolism, on the interactions occurring between Erwinia amylovora, the host plant and the epiphytic microbial community in order to obtain a more effective control of fire blight. Prohexadione-calcium and trinexapac-ethyl, two dioxygenase inhibitors, were chosen as a chemical tool to influence plant metabolism. These compounds inhibit the 2-oxoglutarate-dependent dioxygenases and, therefore, they greatly influence plant metabolism. Moreover, dioxygenase inhibitors were found to enhance plant resistance to a wide range of pathogens. In particular, dioxygenase inhibitors application seems a promising method to control fire blight. From cited literature, it is assumed that these compounds increase plant defence mainly by a transient alteration of flavonoids metabolism. We tried to demonstrate, that the reduction of susceptibility to disease could be partially due to an indirect influence on the microbial community established on plant surface. The possibility to influence the interactions occurring in the epiphytic microbial community is particularly interesting, in fact, the relationships among different bacterial populations on plant surface is a key factor for a more effective biological control of plant diseases. Furthermore, we evaluated the possibility to combine the application of dioxygenase inhibitors with biological control in order to develop an integrate strategy for control of fire blight. The first step for this study was the isolation of a pathogenic strain of E. amylovora. In addition, we isolated different epiphytic bacteria, which respond to general requirements for biological control agents. Successively, the effect of dioxygenase inhibitors treatment on microbial community was investigated on different plant organs (stigmas, nectaries and leaves). An increase in epiphytic microbial population was found. Further experiments were performed with aim to explain this effect. In particular, changes in sugar content of nectar were observed. These changes, decreasing the osmotic potential of nectar, might allow a more consistent growth of epiphytic bacteria on blossoms. On leaves were found similar differences as well. As far as the interactions between E. amylovora and host plant, they were deeply investigated by advanced microscopical analysis. The influence of dioxygenase inhibitors and SAR inducers application on the infection process and migration of pathogen inside different plant tissues was studied. These microscopical techniques, combined with the use of gpf-labelled E. amylovora, allowed the development of a bioassay method for resistance inducers efficacy screening. The final part of the work demonstrated that the reduction of disease susceptibility observed in plants treated with prohexadione-calcium is mainly due to the accumulation of a novel phytoalexins: luteoforol. This 3-deoxyflavonoid was proven to have a strong antimicrobial activity.

Characterization and exploiment of Microbial Strains to be used in Meat Processing and Fermentation

The principal aim of this research project has been the evaluation of the specific role of yeasts in ripening processes of dry-cured meat products, i.e. speck and in salami produced by adding Lactobacilli starter cultures, i.e. L. sakei, L. casei, L. fermentum, L. rhamnosus, L.sakei + S.xylosus. In particular the contribution of the predominant yeasts to the hydrolytic patterns of meat proteins has been studied both in model system and in real products. In fact, although several papers have been published on the microbial, enzymatic, aromatic and chemical characterization of dry-cured meat e.g. ham over ripening, the specific role of yeasts has been often underestimated. Therefore this research work has been focused on the following aspects: 1. Characterization of the yeasts and lactic acid bacteria in samples of speck produced by different farms and analyzed during the various production and ripening phases 2. Characterization of the superficial or internal yeasts population in salami produced with or without the use of lactobacilli as starter cultures 3. Molecular characterization of different strains of yeasts and detection of the dominant biotypes able to survive despite environmental stress factors (such as smoke, salt) 4. Study of the proteolytic profiles of speck and salami during the ripening process and comparison with the proteolytic profiles produced in meat model systems by a relevant number of yeasts isolated from speck and salami 5. Study of the proteolytic profiles of Lactobacilli starter cultures in meat model systems 6. Comparative statistical analysis of the proteolytic profiles to find possible relationships between specific bands and peptides and specific microorganisms 7. Evaluation of the aromatic characteristics of speck and salami to assess relationships among the metabolites released by the starter cultures or the dominant microflora

Evaluation of Microbial Contamination in Bivalve Mollusks: Epidemiology and Diagnosis

Shellfish are filter-feeding organisms that can accumulate many bacteria and viruses. Considering that depuration procedures are not effective in removal of certain microorganisms, shellfish-borne diseases are frequent in many parts of the world, and their control must rely primarily on investigation of prevalence of human pathogens in shellfish and water environment. However, the diffusion of enteric viruses and Vibrio bacteria is not known in many geographical areas, for example in Sardinia, Italy. A survey aimed at investigating the prevalence of Norovirus (NoV), hepatitis A virus (HAV), V. parahaemolyticus, V. cholerae and V. vulnificus was carried out, analyzing both local and imported purified, non-purified and retail shellfish from North Italy and Sardinia. Shellfish from both areas were found contaminated by NoVs, HAV and Vibrio, including retail and purified animals. Molecular analysis evidenced different NoV genogroups and genotypes, including bovine NoVs, as well as pathogenic Vibrio strains, underlining the risk for shellfish consumers. However, also other approaches are needed to control the diffusion of shellfish-borne diseases. It was originally thought that enteric viruses are passively accumulated by shellfish. Recently, it was proven that NoVs bind to specific carbohydrate ligands in oysters, and various NoV strains are characterized by a different bioaccumulation pattern. To deepen the knowledge on this argument, a study was carried out, analyzing bioaccumulation of up to 8 different NoV strains in four different species of shellfish. Different bioaccumulation patterns were observed for each shellfish species and NoV strain used, potentially important in setting up effective shellfish purification protocols. Finally, a novel study of evaluation of viral contamination in shellfish from the French Atlantic coast was carried out following the passage of Xynthia tempest over Western Europe which caused massive destruction. Different enteric viruses were found over a one month period, evidencing the potential of these events of contaminating shellfish.

Land use change to perennial energy crops in Northern Italy: Effects on soil organic carbon sequestration and distribution, soil enzyme activities and microbial communities.

Studies on soil organic carbon (SOC) sequestration in perennial energy crops are available for North-Central Europe, while there is insufficient information for Southern Europe. This research was conducted in the Po Valley, a Mediterranean-temperate zone characterised by low SOC levels, due to intensive management. The aim was to assess the factors influencing SOC sequestration and its distribution through depth and within soil fractions, after a 9-year old conversion from two annual systems to Miscanthus (Miscanthus × giganteus) and giant reed (Arundo donax). The 13C natural abundance was used to evaluate the amount of SOC in annual and perennial species, and determine the percentage of carbon derived from perennial crops. SOC was significantly higher under perennial species, especially in the topsoil (0-0.15 m). After 9 years, the amount of C derived from Miscanthus was 18.7 Mg ha-1, mostly stored at 0-0.15 m, whereas the amount of C derived from giant reed was 34.7 Mg ha-1, evenly distributed through layers. Physical soil fractionation was combined with 13C abundance analysis. C derived from perennial crops was mainly found in macroaggregates. Under giant reed, more newly derived-carbon was stored in microaggregates and mineral fraction than under Miscanthus. A molecular approach based on denaturing gradient gel electrophoresis (DGGE) allowed to evaluate changes on microbial community, after the introduction of perennial crops. Functional aspects were investigated by determining relevant soil enzymes (β-glucosidase, urease, alkaline phosphatase). Perennial crops positively stimulated these enzymes, especially in the topsoil. DGGE profiles revealed that community richness was higher in perennial crops; Shannon index of diversity was influenced only by depth. In conclusion, Miscanthus and giant reed represent a sustainable choice for the recovery of soils exhausted by intensive management, also in Mediterranean conditions and this is relevant mainly because this geographical area is notoriously characterised by a rapid turnover of SOC.

The role of bifidobacteria in newborn health and the intestinal microbial balance

Gut microbial acquisition during the early stage of life is an extremely important event since it affects the health status of the host. In this contest the healthy properties of the genus Bifidobacterium have a central function in newborns. The aim of this thesis was to explore the dynamics of the gut microbial colonization in newborns and to suggest possible strategies to maintain or restore a correct balance of gut bacterial population in infants. The first step of this work was to review the most recent studies on the use of probiotics and prebiotics in infants. Secondly, in order to prevent or treat intestinal disorders that may affect newborns, the capability of selected Bifidobacterium strains to reduce the amount of Enterobacteriaceae and against the infant pathogen Streptococcus agalactiae was evaluated in vitro. Furthermore, the ability of several commercial fibers to stimulate selectively the growth of bifidobacterial strains was checked. Finally, the gut microbial composition in the early stage of life in response to the intrapartum antibiotic prophylaxis (IAP) against group B Streptococcus was studied using q-PCR, DGGE and next generation sequencing. The results globally showed that Bifidobacterium breve B632 strain is the best candidate for the use in a synbiotic product coupled to a mixture of two selected prebiotic fibers (galactooligosaccharides and fructooligosaccharides) for gastrointestinal disorders in infants. Moreover, the early gut microbial composition was affected by IAP treatment with infants showing lower counts of Bifidobacterium spp. and Bacteroides spp. coupled to a decrement of biodiversity of bacteria, compared to control infants. These studies have shown that IAP could affect the early intestinal balance in infants and they have paved the way to the definition of new strategies alternative to antibiotic treatment to control GBS infection in pregnant women.

Microbial ecology of biotechnological processes

The investigation of phylogenetic diversity and functionality of complex microbial communities in relation to changes in the environmental conditions represents a major challenge of microbial ecology research. Nowadays, particular attention is paid to microbial communities occurring at environmental sites contaminated by recalcitrant and toxic organic compounds. Extended research has evidenced that such communities evolve some metabolic abilities leading to the partial degradation or complete mineralization of the contaminants. Determination of such biodegradation potential can be the starting point for the development of cost effective biotechnological processes for the bioremediation of contaminated matrices. This work showed how metagenomics-based microbial ecology investigations supported the choice or the development of three different bioremediation strategies. First, PCR-DGGE and PCR-cloning approaches served the molecular characterization of microbial communities enriched through sequential development stages of an aerobic cometabolic process for the treatment of groundwater contaminated by chlorinated aliphatic hydrocarbons inside an immobilized-biomass packed bed bioreactor (PBR). In this case the analyses revealed homogeneous growth and structure of immobilized communities throughout the PBR and the occurrence of dominant microbial phylotypes of the genera Rhodococcus, Comamonas and Acidovorax, which probably drive the biodegradation process. The same molecular approaches were employed to characterize sludge microbial communities selected and enriched during the treatment of municipal wastewater coupled with the production of polyhydroxyalkanoates (PHA). Known PHA-accumulating microorganisms identified were affiliated with the genera Zooglea, Acidovorax and Hydrogenophaga. Finally, the molecular investigation concerned communities of polycyclic aromatic hydrocarbon (PAH) contaminated soil subjected to rhizoremediation with willow roots or fertilization-based treatments. The metabolic ability to biodegrade naphthalene, as a representative model for PAH, was assessed by means of stable isotope probing in combination with high-throughput sequencing analysis. The phylogenetic diversity of microbial populations able to derive carbon from naphthalene was evaluated as a function of the type of treatment.

Bioremediation of PCB-contaminated marine sediments: From identification of indigenous dehalorespirers to enhancement of microbial reductive dechlorination

Marine sediments are the main accumulation reservoir of organic recalcitrant pollutants such as polychlorinated biphenyls (PCBs). In the anoxic conditions typical of these sediments, anaerobic bacteria of the phylum Chloroflexi are able to attack these compounds in a process called microbial reductive dechlorination. Such activity and members of this phylum were detected in PCB-impacted sediments of the Venice Lagoon. The aim of this work was to investigate microbial reductive dechlorination and design bioremediation approaches for marine sediments of the area. Three out of six sediment cultures from different sampling areas exhibited dechlorination activities in the same conditions of the site and two phylotypes (VLD-1 and VLD-2) were detected and correlated to this metabolism. Biostimulation was tested on enriched dechlorinating sediment cultures from the same site using five different electron donors, of which lactate was the best biostimulating agent; complementation of microbial and chemical dechlorination catalyzed by biogenic zerovalent Pd nanoparticles was not effective due to sulfide poisoning of the catalyst. A new biosurfactant-producing strain of Shewanella frigidimarina was concomitantly obtained from hydrocarbon-degrading marine cultures and selected because of the low toxicity of its product. All these findings were then exploited to develop bioremediation lab-scale tests in shaken reactors and static microcosms on real sediments and water of the Venice lagoon, testing i) a bioaugmentation approach, with a selected enriched sediment culture from the same area, ii) a biostimulation approach with lactate as electron donor, iii) a bioavailability enhancement with the supplementation of the newly-discovered biosurfactant, and iv) all possible combinations of the afore-mentioned approaches. The best bioremediation approach resulted to be a combination of bioaugmentation and bioremediation and it could be a starting point to design bioremediation process for actual marine sediments of the Venice Lagoon area.

Atmospheric plasma processes for microbial inactivation: food applications and stress response in Listeria monocytogenes

This PhD thesis is focused on cold atmospheric plasma treatments (GP) for microbial inactivation in food applications. In fact GP represents a promising emerging technology alternative to the traditional methods for the decontamination of foods. The objectives of this work were to evaluate: - the effects of GP treatments on microbial inactivation in model systems and in real foods; - the stress response in L. monocytogenes following exposure to different GP treatments. As far as the first aspect, inactivation curves were obtained for some target pathogens, i.e. Listeria monocytogenes and Escherichia coli, by exposing microbial cells to GP generated with two different DBD equipments and processing conditions (exposure time, material of the electrodes). Concerning food applications, the effects of different GP treatments on the inactivation of natural microflora and Listeria monocytogenes, Salmonella Enteritidis and Escherichia coli on the surface of Fuji apples, soya sprouts and black pepper were evaluated. In particular the efficacy of the exposure to gas plasma was assessed immediately after treatments and during storage. Moreover, also possible changes in quality parameters such as colour, pH, Aw, moisture content, oxidation, polyphenol-oxidase activity, antioxidant activity were investigated. Since the lack of knowledge of cell targets of GP may limit its application, the possible mechanism of action of GP was studied against 2 strains of Listeria monocytogenes by evaluating modifications in the fatty acids of the cytoplasmic membrane (through GC/MS analysis) and metabolites detected by SPME-GC/MS and 1H-NMR analyses. Moreover, changes induced by different treatments on the expression of selected genes related to general stress response, virulence or to the metabolism were detected with Reverse Transcription-qPCR. In collaboration with the Scripps Research Institute (La Jolla, CA, USA) also proteomic profiles following gas plasma exposure were analysed through Multidimensional Protein Identification Technology (MudPIT) to evaluate possible changes in metabolic processes.