UNIVERSITÀ DEGLI STUDI DI NAPOLI “FEDERICO II” DOTTORATO IN BIOLOGIA APPLICATA INDIRIZZO MICROBIOLOGIA Characterization of Lactobacilli and Bacilli of intestinal origin Tutor : Candidato: Ch.mo Prof. Fakhry S. Saad Ezio Ricca Coordinatore: Ch.mo Prof. Ezio Ricca INDICE Chapter one Introduction 1. Intestinal microflora and probiotics pag 1 2. The Lactobacillus, Bifidobacterium and Bacillus pag 3 genera 2.1 Lactobacillus pag 3 2.2. Bifidobaterium pag 6 2.3 Bacillus pag 7 2.3.1 Spores pag 7 2.3.2 Spore structure pag 9 2.3.3 Spore germination pag 11 3. Pigmented Bacilli pag 12 4. Carotenoids pag 14 5. Aim of this work pag 17 Refernces pag 19 Chapter two pag 28 Characterization of intestinal bacteria tightly pag 28 bound to the human ileal epithelium Abstract pag 29 1. Introduction pag 29 2. Material and Methods pag 32 2.1 Collection of ileal samples pag 32 2.2 Bacterial isolation and culture conditions pag 32 2.3 Physiological and biochemical analysis pag 32 2.4 Simulated gastric and intestinal fluids pag 33 2.5 PCR condition and primers pag 34 2.6 In vitro biofilm assay pag 34 I 2.7 In vitro cytotoxicity assays pag 34 2.8 Preparation of bacterial supernatant pag 35 3. Results pag 35 3.1 Isolation of Lactobacilli tightly associated to ileal pag 35 epithelial cells 3.2 Production of antimicrobial ativity pag 36 3.3 Resistance to simulated GIT conditions pag 38 3.4 Biofilm formation pag 40 3.5 Mucin degradation pag 41 3.6 In vitro cytotoxicity assay pag 43 4. Discussion pag 43 References pag 46 Chapter three pag 49 Characterization of spore forming Bacilli isolated from pag 49 The human gastrointestinal tract Abstract pag 50 1. Introduction pag 50 2. Materials and methods pag 53 2.1 Collection of ileal and faecal samples pag 53 2.2 Bacterial isolation and characterization pag 53 2.3 Physiological analysis pag 54 2.4 Analysis of enterotoxins and virulence traits pag 55 3. Results pag 55 3.1 Isolation of spore-formers from human gut pag 55 3.2 Swarming motility and biofilm formation pag 57 3.3 Production of antimicrobial activity pag 59 II 3.4 Rasistance of spores and cells to simulated GIT pag 60 conditions 3.5 Growth and sporulation in anaerobic conditions pag 62 3.6 Presence of potential virulance factors pag 63 4. Discussion pag 64 References pag 67 Chapter four pag 71 Defining the natural habitat of Bacillus spore-formers pag 71 Abstract pag 72 1. Introduction pag 72 2. Materials and methods pag 74 2.1 Collection of faecal samples pag 74 2.2 Soil samples pag 74 2.3 Determination of counts of spore-forming bacteria pag 74 2.4 Others methods pag 75 3. Results and Discussion pag 75 3.1 Spore formers are recovered from human faecal pag 79 samples 3.2 Aerobic spores present in soil pag 81 3.3 Endospores found in food products pag 82 3.4 The true habitat of Bacillus species pag 82 References pag 84 III Chapter five pag 87 Carotenoids Found in Spore-Forming Bacteria pag 87 Abstract pag 88 1. Introduction pag 88 2. Materials and methods pag 89 2.1 General methods pag 89 2.2 Isolation of pigmented spore-formers pag 92 2.3 References strains pag 92 2.4 16S rRNA analysis pag 92 2.5 Determination of UV resistance pag 93 2.6 Hydrogen peroxide assays pag 93 2.7 Pigmented extraction pag 93 2.8 Pigmented analysis pag 94 2.9 Detection of water-soluble pigments pag 95 3. Results pag 95 3.1 Biotypes of pigmented spore formers pag 95 3.2 Carotenoid profiling pag 103 3.3 Detection of water-soluble pigments pag 108 3.4 Pigment-conferred resi stance properties pag 108 4. Discussion pag 112 References pag 116 IV Appendix pag 120 V INTRODUCTION 1. Intestinal microflora and probiotics The concept that the endogenous microflora has a crucial role in host health has been indipendently suggested by Pasteur, Koch, Metchnikoff and Escherich during the very early years of modern microbiology (Hopper et al. 1998). Since then, it has been claimed that the composition of the microflora is essential for the well-being of the host and the importance of interactions between host and bacteria has been emphasized (Hopper et al. 1998). Despite these early insights, the study of bacteria- host interactions has been for many years focused exclusively on those involving pathogenic organisms. Only recently, scientists have started to study the molecular mechanisms mediating the interaction of non-pathogenic bacteria with the mucosal surfaces of their host (Hopper et al. 1998). Very recently, metagenomic data have shown that the adult human gut houses a bacterial community (microbiota) containing trillions of members comprising thousands of species-level phylogenetic types (phylotypes). Two bacterial phyla, the Firmicutes and the Bacteroidetes, commonly dominate this ecosystem (Mahowald et al. 2009), as they do in the guts of at least 60 mammalian species (Ley et al. 2008). Progress made with 16S rDNA-based enumerations has disclosed significant differences in community membership between healthy adults (Gill et al. 2006), differences that may contribute to variations in normal physiology between individuals or that may predispose to disease. For example, studies of gnotobiotic mouse models indicate that mutualistic relations with the gut microbiota influence maturation of the immune system, modulate responses to epithelial cell injury, affect energy balance, and support biotransformations that we are not equipped to perform on our own, including processing of xenobiotics. Several bacterial species are used in commercial preparations (probiotics) that contain live bacteria and that, when orally administered, are thought to have a positive effect on human health by restoring an active and balanced microflora after antibiotic treatments or other stressful conditions. This practice has started in a rather empirical way much before that the interactions between the microbiota and the host were studied in details. There are now many proposed mechanisms by which probiotics may positively affect the animal health. Listed below is a brief 1 description of mechanisms by which probiotics may protect the host cells against intestinal diseases. Production of inhibitory substances. Probiotic bacteria produce a variety of substances that are inhibitory to both gram-positive and gram-negative bacteria. These inhibitory substances include organic acids, hydrogen peroxide and bacteriocins. These compounds may reduce not only the number of viable cells but may also affect bacterial metabolism or toxin production. Blocking of adhesion sites. Competitive inhibition for bacterial adhesion sites on intestinal epithelial surfaces is another mechanism of action for probiotics (Conway et al. 1987; Goldin et al. 1992; Kleeman and Klaenhammer. 1982). Consequently, some probiotic strains have been chosen for their ability to adhere to epithelial cells. Competition for nutrients. Probiotics may utilize nutrients otherwise consumed by pathogenic microorganisms. However, the evidence that this occurs in vivo is lacking. Degradation of toxin receptor. The postulated mechanism by which S. boulardii protects animals against C. difficile intestinal disease is through degradation of the toxin receptor on the intestinal mucosa (Castagliuolo et al. 1996 and 1999; Pothoulakis et al. 1993). Stimulation of immunity. Recent evidence suggests that stimulation of specific and nonspecific immunity may be another mechanism by which probiotics can protect against intestinal diseases (Fukushima et al. 1998; Kaila et al. 1992; Link- Amster et al. 1994; Malin et al. 1996; Perdigon et al. 1986). For example, oral administration of Lactobacillus GG during acute rotavirus diarrhea is associated with an enhanced immune response to rotavirus (Kaila et al. 1992). This may account for the shortened course of diarrhea seen in treated patients. The underlying mechanisms of immune stimulation are not well understood, but specific cell wall components may act as adjuvants and increase humoral immune responses. Most probiotics marketed today contain live cells of various bacterial species belonging to the Lactobacillus and Bifidobacterium genera, commensal bacteria normally found in the GIT, although not as numerically predominant genera. More 2 surprising is the use in several commercial probiotic preparations of bacteria not considered as normal inhabitant of the intestine, i.e. bacteria of the Bacillus genus, aerobic spore formers normally considered as soil organisms (Serror et al. 2002). 2. The Lactobacillus, Bifidobacterium and Bacillus genera 2.1 Lactobacillus Lactobacillus is a genus belonging to the group of Lactic Acid Bacteria (LAB), a widespread group of gram-positives that are catalase-negative, anaerobic (microaerophilic) and non motile organisms. To this heterogeneous group of bacteria, that includes mesophilc as well as thermophilic species, belogs several genera of cocci (Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus) and rods (Carnobacterium, Lactobacillus). Most species of these genera are ubiquitous in the environment, occupying niches ranging from plant surfaces to the gastrointestinal tracts of many animals. They are, in general, nutritonally fastidious organisms requiring several amino acids, vitamins, cofactors and metabolic intermediates. With a few exceptions, LAB derive all of their energy from conversion of glucose to lactate or to lactate and a mixture of other products via the homofermentative or heterofermentative pathway, respectively. During homolactic fermentation, 85 to 95 % of the sugar utilized is converted to lactic acid; in contrast during heterolactic fermentation only 50 % of the sugar utilized is converted to lactic acid. The remaining products of heterolactic fermentation are carbon-dioxide and ethanol, which is sometimes further converted to acetate. These pathways generate ATP by nonoxidative substrate-level phosphorylation; in the homofermentative pathway, the net energy yield is of 2 molecules of ATP per molecule of glucose. In the heterofermentative pathway the net energy yield is 1 molecule of ATP per molecule of glucose under strictly anaerobic conditions and 2 molecules of ATP per molecule of glucose in aerobic or microaerophilic conditions, because of the presence of an alternative electron sink for cofactor regeneration.