Riccardo Valerio De Biase

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.

Lactobacillus reuteri ATCC55730 in cystic fibrosis.

Objectives: The aim of this study was to evaluate in patients with cystic fibrosis (CF) the effect of Lactobacillus reuteri (LR) on the rate of respiratory exacerbations and of the infections of both upper respiratory and gastrointestinal tracts. Methods: Prospective randomized, double-blind, placebo-controlled study enrolling 61 patients with CF with mild-to-moderate lung disease at the Regional Center for CF of the Department of Pediatrics, University of Rome “La Sapienza.” All of the patients were not hospital inpatients at the time of the enrollment. Inclusion criteria were forced expiratory volume in the first second (FEV1) >70% predicted; no inhaled or systemic steroids, no anti-inflammatory drugs, antileukotrienes, and mast cell membrane stabilizers; and no serious organ involvement. Exclusion criteria were a history of pulmonary exacerbation or upper respiratory infection in the previous 2 months; changes in medications in the last 2 months; a history of hemoptysis in the last 2 months; and colonization with Burkholderia cepacia or mycobacteria. Patients were randomly assigned to receive LR (30 patients) in 5 drops per day (1010 colony-forming units) or placebo (31 patients) for 6 months. Main outcomes were number of episodes of pulmonary exacerbations and hospital admissions for pulmonary exacerbations, number of gastrointestinal and upper respiratory tract infections. FEV1, fecal calprotectin, and cytokine profile in induced sputum and plasma were assessed at baseline and at the end of the trial. Results: Pulmonary exacerbations were significantly reduced in the LR group compared with the placebo group (P < 0.01; odds ratio 0.06 [95% confidence interval {CI} 0–0.40]; number needed to treat 3 [95% CI 2–7]). Similarly, the number of upper respiratory tract infections (in our series only otitis) was significantly reduced in the LR group compared with the placebo group (P < 0.05; odds ratio 0.14 [95% CI 0–0.96]; number needed to treat 6 [95% CI 3–102]). The 2 groups did not differ statistically in the mean number and duration of hospitalizations for pulmonary exacerbations and gastrointestinal infections. There was no significant statistical difference in the mean delta value of FEV1, fecal calprotectin concentration, and tested cytokines (tumor necrosis factor-&agr; and interleukin-8) between the 2 groups. Conclusions: LR reduces pulmonary exacerbations and upper respiratory tract infections in patients with CF with mild-to-moderate lung disease. LR administration may have a beneficial effect on the disease course of CF.

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.

Bdellovibrio bacteriovorus directly attacks Pseudomonas aeruginosa and Staphylococcus aureus Cystic fibrosis isolates

Bdellovibrio bacteriovorus is a predator bacterial species found in the environment and within the human gut, able to attack Gram-negative prey. Cystic fibrosis (CF) is a genetic disease which usually presents lung colonization by Pseudomonas aeruginosa or Staphylococcus aureus biofilms. Here, we investigated the predatory behavior of B. bacteriovorus against these two pathogenic species with: (1) broth culture; (2) “static” biofilms; (3) field emission scanning electron microscope (FESEM); (4) “flow” biofilms; (5) zymographic technique. We had the first evidence of B. bacteriovorus survival with a Gram-positive prey, revealing a direct cell-to-cell contact with S. aureus and a new “epibiotic” foraging strategy imaged with FESEM. Mean attaching time of HD100 to S. aureus cells was 185 s, while “static” and “flow” S. aureus biofilms were reduced by 74 (at 24 h) and 46% (at 20 h), respectively. Furthermore, zymograms showed a differential bacteriolytic activity exerted by the B. bacteriovorus lysates on P. aeruginosa and S. aureus. The dual foraging system against Gram-negative (periplasmic) and Gram-positive (epibiotic) prey could suggest the use of B. bacteriovorus as a “living antibiotic” in CF, even if further studies are required to simulate its in vivo predatory behavior.