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.

Prove di ozonolisi sui fanghi biologici prodotti da impianti di depurazione delle acque reflue urbane

La tecnica di ozonolisi viene applicata ai fanghi biologici derivanti da impianti di depurazione acque reflue urbane, e consiste nell'ottenere, grazie all'ozono, una minor massa fangosa da smaltire e una miglior trattabilità del fango residue. In questo elaborato si prendono in esame le sperimentazioni effettuate a Marina di Ravenna e si estraggono le prime conclusioni gestionali, economiche e ambientali sull'applicabilità del metodo a questo tipo di fango.

Strumenti per la valutazione dello stato conservativo e per la manutenzione programmata dei ponti della Provincia di Bologna

La tesi tratta di strumenti finalizzati alla valutazione dello stato conservativo e di supporto all'attività di manutenzione dei ponti, dai più generali Bridge Management Systems ai Sistemi di Valutazione Numerica della Condizione strutturale. Viene proposto uno strumento originale con cui classificare i ponti attraverso un Indice di Valutazione Complessiva e grazie ad esso stabilire le priorità d'intervento. Si tara lo strumento sul caso pratico di alcuni ponti della Provincia di Bologna. Su un ponte in particolare viene realizzato un approfondimento specifico sulla determinazione approssimata dei periodi propri delle strutture da ponte. Si effettua un confronto dei risultati di alcune modellazioni semplificate in riferimento a modellazioni dettagliate e risultati sperimentali.

Analisi e valutazione delle performance acustiche degli aeromobili nella fase di atterraggio

Nella tesi si analizzano le principali fonti del rumore aeronautico, lo stato dell'arte dal punto di vista normativo, tecnologico e procedurale. Si analizza lo stato dell'arte anche riguardo alla classificazione degli aeromobili, proponendo un nuovo indice prestazionale in alternativa a quello indicato dalla metodologia di certificazione (AC36-ICAO) Allo scopo di diminuire l'impatto acustico degli aeromobili in fase di atterraggio, si analizzano col programma INM i benefici di procedure CDA a 3° rispetto alle procedure tradizionali e, di seguito di procedure CDA ad angoli maggiori in termini di riduzione di lunghezza e di area delle isofoniche SEL85, SEL80 e SEL75.

Tecniche di risparmio idrico a livello locale e mondiale. Approfondimento sulle buone pratiche agricole per un uso sostenibile dell'acqua

Oltre un miliardo di persone non ha oggi accesso all’acqua potabile; più di due miliardi è il numero di coloro che vivono in condizioni igienico-sanitarie realmente proibitive. Sono 80 i paesi nel mondo (con il 40% della popolazione totale) in cui si riscontra difficoltà di approvvigionamento e presenza di risorse idriche che mancano dei requisiti che dovrebbero essere assicurati per la tutela della salute: quotidianamente e sistematicamente il diritto di accesso all’acqua, che nessun individuo dovrebbe vedersi negato, viene violato. Scarsità di acqua e non omogenea distribuzione sulla superficie terrestre sono fattori che concorrono alla crisi della risorsa, cui contribuiscono processsi di natura ambientale (cambiamenti climatici, desertificazione), di natura economica (le sorti dell’industria agroalimentare, la globalizzazione degli scambi, il bisogno crescente di energia), di natura sociale (migrazioni, urbanizzazione, crescita demografica, epidemie), di natura culturale (passaggio dal rurale all’urbano, dall’agricoltura di sussistenza a quella di profitto). Nell’ottica di uno sviluppo sostenibile un aumento indiscriminato dell’offerta non può costituire soluzione al continuo incremento della domanda di acqua. Si rende pertanto necessaria la definizione di politiche e strumenti di cambiamento nei modelli di consumo e nella pianificazione che consentano una riduzione degli squilibri nella distribuzione e nella gestione della risorsa a livello domestico e civile, industriale, agricolo. L’uso efficiente, e quindi sostenibile, dell’acqua è da perseguirsi secondo le modalità: • Risparmio, inteso come minore consumo di acqua all’inizio del ciclo. • Riciclo dell’acqua in circuito chiuso, inteso come riuso dell’acqua di scarico, o uso multiplo dell’acqua. Una idonea utilizzazione dipende da una idonea progettazione, che abbia come finalità: • La destinazione in via prioritaria delle fonti e delle risorse di più elevata qualità agli usi idropotabili, con una graduale sostituzione del consumo per altri usi con risorse di minore pregio. • La regolamentazione dell’uso delle acque sotterranee, mediante la limitazione del ricorso all’impiego di pozzi solo in mancanza di forniture alternative per uso civile, industriale, agricolo. • L’incentivazione ad un uso razionale della risorsa, anche mediante l’attuazione di idonee politiche tariffarie. • L’aumento dell’efficienza delle reti di adduzione e distribuzione, sia civili che irrigue. • La promozione di uso efficiente, riciclo e recupero di acqua nell’industria. • Il miglioramento dell’efficienza ed efficacia delle tecniche di irrigazione. • La promozione del riutilizzo delle acque nei vari settori. • La diffusione nella pratica domestica di apparati e tecnologie progettati per la riduzione degli sprechi e dei consumi di acqua. In ambito agricolo la necessità di un uso parsimonioso della risorsa impone il miglioramento dell’efficienza irrigua, pari appena al 40%. La regione Emilia Romagna a livello locale, Israele a livello internazionale, forniscono ottimi esempi in termini di efficacia dei sistemi di trasporto e di distribuzione, di buona manutenzione delle strutture. Possibili soluzioni verso le quali orientare la ricerca a livello mondiale per arginare la progressiva riduzione delle riserve idriche sono: • Revisione dei costi idrici. • Recupero delle riserve idriche. • Raccolta dell’acqua piovana. • Miglioramento degli impianti di distribuzione idrica. • Scelta di metodi di coltivazione idonei alle caratteristiche locali. • Scelta di colture a basso fabbisogno idrico. • Conservazione della risorsa attraverso un sistema di irrigazione efficiente. • Opere di desalinizzazione. • Trasferimento idrico su vasta scala da un’area all’altra. Si tratta di tecniche la cui attuazione può incrementare la disponibilità media pro capite di acqua, in particolare di coloro i quali non ne posseggono in quantità sufficiente per bere o sono privi di sistemi igienico-sanitari sufficienti.