Serena Schippa

Gut-associated bacterial microbiota in paediatric patients with inflammatory bowel disease

Background: Clinical and experimental observations in animal models indicate that intestinal commensal bacteria are involved in the initiation and amplification of inflammatory bowel disease (IBD). No paediatric reports are available on intestinal endogenous microflora in IBD. Aims: To investigate and characterise the predominant composition of the mucosa-associated intestinal microflora in colonoscopic biopsy specimens of paediatric patients with newly diagnosed IBD. Methods: Mucosa-associated bacteria were quantified and isolated from biopsy specimens of the ileum, caecum and rectum obtained at colonoscopy in 12 patients with Crohn’s disease, 7 with ulcerative colitis, 6 with indeterminate colitis, 10 with lymphonodular hyperplasia of the distal ileum and in 7 controls. Isolation and characterisation were carried out by conventional culture techniques for aerobic and facultative-anaerobic microorganisms, and molecular analysis (16S rRNA-based amplification and real-time polymerase chain reaction assays) for the detection of anaerobic bacterial groups or species. Results: A higher number of mucosa-associated aerobic and facultative-anaerobic bacteria were found in biopsy specimens of children with IBD than in controls. An overall decrease in some bacterial species or groups belonging to the normal anaerobic intestinal flora was suggested by molecular approaches; in particular, occurrence of Bacteroides vulgatus was low in Crohn’s disease, ulcerative colitis and indeterminate colitis specimens. Conclusion: This is the first paediatric report investigating the intestinal mucosa-associated microflora in patients of the IBD spectrum. These results, although limited by the sample size, allow a better understanding of changes in mucosa-associated bacterial flora in these patients, showing either a predominance of some potentially harmful bacterial groups or a decrease in beneficial bacterial species. These data underline the central role of mucosa-adherent bacteria in IBD.

Bacterial nonspecific acid phosphohydrolases: Physiology, evolution and use as tools in microbial biotechnology

Abstract. Bacterial nonspecific acid phosphohydrolases (NSAPs) are secreted enzymes, produced as soluble periplasmic proteins or as membrane-bound lipoproteins, that are usually able to dephosphorylate a broad array of structurally unrelated substrates and exhibit optimal catalytic activity at acidic to neutral pH values. Bacterial NSAPs are monomeric or oligomeric proteins containing polypeptide components with an Mr of 25 – 30 kDa. On the basis of amino acid sequence relatedness, three different molecular families of NSAPs can be distinguished, indicated as molecular class A, B and C, respectively. Members of each class share some common biophysical and functional features, but may also exhibit functional differences. NSAPs have been detected in several microbial taxa, and enzymes of different classes can be produced by the same bacterial species. Structural and phyletic relationships exist among the various bacterial NSAPs and some other bacterial and eucaryotic phosphohydrolases. Current knowledge on bacterial NSAPs is reviewed, together with analytical tools that may be useful for their characterization. An overview is also presented concerning the use of bacterial NSAPs in biotechnology.

Violacein and biofilm production in Janthinobacterium lividum

Aims:  To analyse the environmental stimuli modulating violacein and biofilm production in Janthinobacterium lividum.

The microbiota in inflammatory bowel disease in different age groups

BACKGROUND Many efforts were made in the past decades to assess the role of gut microbiota in inflammatory bowel diseases (IBD), leading to the hypothesis that an altered microbial composition, other than the presence of a specific pathogen, could be involved in the pathogenesis of the disease. On the other hand, existing differences in gut microbial community between distinct classes of age make sense of an increasing research in microbial shifts in IBD. METHODS Cultural, molecular, metabolomic and metagenomic approaches are trying to define the human gut microbiota in different age groups. RESULTS AND CONCLUSION An increase in anaerobic bacteria (Bacteroidesvulgatus, Streptococcus faecalis) was observed in adult IBD, whereas an increase in aerobic and facultative-anaerobic (Escherichia coli) was found in pediatric IBD. Overall higher bacterial cell counts were observed in IBD, jointly with a general loss of biodiversity and a preponderance of Bacteroidetes and a parallel decrease of Firmicutes phylum: a predominance of potential harmful members of Proteobacteria (E. coli) and low abundance of beneficial species (Faecalibacterium prausnitzii) was also reported in pediatric and adult age groups, respectively. Microbial community of elderly subjects contains a wider range of different species than those of children and adults, both in healthy and IBD status.

Higher Prevalence and Abundance of Bdellovibrio bacteriovorus in the Human Gut of Healthy Subjects

Introduction Members of the human intestinal microbiota are key players in maintaining human health. Alterations in the composition of gut microbial community (dysbiosis) have been linked with important human diseases. Understanding the underlying processes that control community structure, including the bacterial interactions within the microbiota itself, is essential. Bdellovibrio bacteriovorus is a gram-negative bacterium that preys other gram-negative species for survival, acting as a population-balancer. It was found in terrestrial/aquatic ecosystems, and in animal intestines, postulating its presence also in the human gut. Methods The present study was aimed to evaluate, by end-point PCR and qPCR, the presence of B. bacteriovorus in intestinal and faecal biopsy specimens from 92 paediatric healthy subjects and patients, suffering from Inflammatory Bowel Diseases (IBD), Celiac disease and Cystic fibrosis (CF). Results i) B. bacteriovorus was present and abundant only in healthy individuals, while it was heavily reduced in patients, as in the case of IBD and Celiac, while in CF patients and relative controls we observed comparable results; ii) B. bacteriovorus seemed to be mucosa-associated, because all IBD and Celiac biopsies (and related controls) were treated with mucus-removing agents, leaving only the mucosa-attached microflora; iii) B. bacteriovorus abundance was district-dependent, with a major preponderance in duodenum, and gradually decreasing up to rectum; iv) B. bacteriovorus levels significantly dropped in disease status, in duodenum and ileum. Conclusions Results obtained in this study could represent the first step for new therapeutic strategies aimed to restore a balance in the intestinal ecosystem, utilizing Bdellovibrio as a probiotic.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Allelic Variants Relate to Shifts in Faecal Microbiota of Cystic Fibrosis Patients

Introduction In this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF). CFTR mutations (F508del is the most common) lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age. Methods Thirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure. Results Patients were classified by two different criteria: 1) presence/absence of F508del mutation; 2) disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme) were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum) were reduced. Conclusions This is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a ‘systemic disease’, linking the lung and the gut in a joined axis.

Dysbiotic Events in Gut Microbiota: Impact on Human Health

The human body is colonized by a large number of microbes coexisting peacefully with their host. The most colonized site is the gastrointestinal tract (GIT). More than 70% of all the microbes in the human body are in the colon. The microorganism population is 10 times larger of the total number of our somatic and germ cells. Two bacterial phyla, accounting for more than 90% of the bacterial cells, dominate the healthy adult intestine: Firmicutes and Bacteroidetes. Considerable variability in the microbiota compositions between people is found when we look at the taxonomic level of species, and strains within species. It is possible to assert that the human microbiota could be compared to a fingerprint. The microbiota acts as a barrier from pathogens, exerts important metabolic functions, and regulates inflammatory response by stimulating the immune system. Gut microbial imbalance (dysbiosis), has been linked to important human diseases such as inflammation related disorders. The present review summarizes our knowledge on the gut microbiota in a healthy context, and examines intestinal dysbiosis in inflammatory bowel disease (IBD) patients; the most frequently reported disease proven to be associated with changes in the gut microbiota.

Dominant genotypes in mucosa-associated Escherichia coli strains from pediatric patients with inflammatory bowel disease

Background: Studies performed in adults with inflammatory bowel disease (IBD) have suggested that mucosa‐associated Escherichia coli strains may be involved in its pathogenesis. The aim of this study was to characterize E. coli strains from the intestinal mucosa of pediatric IBD patients to investigate whether a particular subset of strains could be associated with the disease. Methods: We analyzed the genomic and phenotypic traits of 60 E. coli strains isolated from biopsies of pediatric patients with Crohns disease (CD), ulcerative colitis (UC), and from age‐matched controls. Results: No noteworthy differences were found in the distribution of phylogroups. The percentage of adhesive E. coli strains was similar in biopsies from patients and controls. However, the adhesion ability of E. coli strains differed between ileal and colonic or rectal areas, only in the strains from CD and UC patients. The percentage of E. coli possessing more than 1 of the adhesive/virulence determinants was significantly higher in strains from UC than from CD and controls. Interestingly, the genetic profile examination revealed 2 large clusters of genetically linked E. coli strains from IBD patients. Ninety‐two percent of the strains isolated from CD patients were in the first cluster (A) and were distributed between 2 genetic subclusters (A1 and A2), while a second cluster (B) contained most of the strains isolated from UC (78%; subcluster B1), and control strains (77%; subcluster B2). Conclusions: Genomic analysis of mucosa‐associated E. coli strains found a close genetic association between strains isolated from CD and UC patients.

Outbreak of Achromobacter xylosoxidans in an Italian Cystic fibrosis center: genome variability, biofilm production, antibiotic resistance, and motility in isolated strains.

Cystic fibrosis (CF) patients have chronic airway infection and frequent exposure to antibiotics, which often leads to the emergence of resistant organisms. Achromobacter xylosoxidans is a new emergent pathogen in CF spectrum. From 2005 to 2010 we had an outbreak in A. xylosoxidans prevalence in our CF center, thus, the present study was aimed at deeply investigating virulence traits of A. xylosoxidans strains isolated from infected CF patients. To this purpose, we assessed A. xylosoxidans genome variability by randomly amplified polymorphic DNA (RAPD), biofilm production, antibiotic resistances, and motility. All A. xylosoxidans strains resulted to be biofilm producers, and were resistant to antibiotics usually employed in CF treatment. Hodge Test showed the ability to produce carbapenemase in some strains. Strains who were resistant to β-lactamics antibiotics, showed the specific band related to metal β-lactamase (blaIMP-1), and some of them showed to possess the integron1. Around 81% of A. xylosoxidans strains were motile. Multivariate analysis showed that RAPD profiles were able to predict Forced Expiratory Volume (FEV1%) and biofilm classes. A significant prevalence of strong biofilm producers strains was found in CF patients with severely impaired lung functions (FEV1% class 1). The outbreak we had in our center (prevalence from 8.9 to 16%) could be explained by an enhanced adaptation of A. xylosoxidans in the nosocomial environment, despite of aggressive antibiotic regimens that CF patients usually undergo.

Adherent-invasive Escherichia coli (AIEC) in pediatric Crohn’s disease patients: phenotypic and genetic pathogenic features

BackgroundAdherent-invasive Escherichia coli (AIEC) have been implicated in the ethiopathogenesis of Crohn’s disease (CD). In this study, we analyzed a collection of intestinal mucosa-associated E. coli isolates, presenting AIEC phenotypes, isolated from biopsies of CD pediatric patients and non-inflammatory bowel diseases (IBD) controls, in order to investigate their genetic and phenotypic pathogenic features.ResultsA total of 616 E. coli isolates from biopsies of four pediatric CD patients and of four non-IBD controls were collected and individually analyzed. For AIEC identification, adherent isolates were assayed for invasiveness, and the capacity of the adhesive-invasive isolates to survive and replicate intracellularly was determined over macrophages J774. In this way we identified 36 AIEC-like isolates. Interestingly, their relative abundance was significantly higher in CD patients (10%; 31/308) than in non-IBD controls (1%; 5/308) (χ 2 = 38.96 p < 0.001). Furthermore pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) techniques were applied to analyze the clonality of the 36 AIEC-like isolates. The results obtained allowed us to identify 27 distinct genotypes (22 from CD patients and 5 from non-IBD controls). As for the AIEC prototype strain LF82, all 27 AIEC genotypes presented an aggregative pattern of adherence (AA) that was inhibited by D-mannose, indicating that adhesiveness of AIEC is likely mediated by type 1 pili. PCR analisys was used to investigate presence of virulence genes. The results indicated that among the 27 AIEC isolates, the incidence of genes encoding virulence factors K1 (χ 2 = 6.167 P = 0.013), kps MT II (χ 2 = 6.167 P = 0.013), fyuA (χ 2 = 6.167 P = 0.013), and ibeA (χ 2 = 8.867 P = 0.003) was significantly higher among AIEC strains isolated from CD patients than non-IBD controls.ConclusionsThe identification of AIEC strains in both CD and non-IBD controls, confirmed the “pathobiont” nature of AIEC strains. The finding that AIEC-like isolates were more abundant in CD patients, indicates that a close association of these strains with CD may also exists in pediatric patients.