Disrupted intestinal microbiota and intestinal inflammation in children with cystic fibrosis and its restoration with Lactobacillus GG: a randomised clinical trial

Eugenia Bruzzese, Maria Luisa Callegari, Valeria Raia, Sara Viscovo, Riccardo Scotto, Susanna Ferrari, Lorenzo Morelli, Vittoria Buccigrossi, Andrea Lo Vecchio, Eliana Ruberto, Alfredo Guarino, Eugenia Bruzzese, Maria Luisa Callegari, Valeria Raia, Sara Viscovo, Riccardo Scotto, Susanna Ferrari, Lorenzo Morelli, Vittoria Buccigrossi, Andrea Lo Vecchio, Eliana Ruberto, Alfredo Guarino

Abstract

Background & aims: Intestinal inflammation is a hallmark of cystic fibrosis (CF). Administration of probiotics can reduce intestinal inflammation and the incidence of pulmonary exacerbations. We investigated the composition of intestinal microbiota in children with CF and analyzed its relationship with intestinal inflammation. We also investigated the microflora structure before and after Lactobacillus GG (LGG) administration in children with CF with and without antibiotic treatment.

Methods: The intestinal microbiota were analyzed by denaturing gradient gel electrophoresis (DGGE), real-time polymerase chain reaction (RT-PCR), and fluorescence in situ hybridization (FISH). Intestinal inflammation was assessed by measuring fecal calprotectin (CLP) and rectal nitric oxide (rNO) production in children with CF as compared with healthy controls. We then carried out a small double-blind randomized clinical trial with LGG.

Results: Twenty-two children with CF children were enrolled in the study (median age, 7 years; range, 2-9 years). Fecal CLP and rNO levels were higher in children with CF than in healthy controls (184±146 µg/g vs. 52±46 µg/g; 18±15 vs. 2.6±1.2 µmol/L NO2 (-), respectively; P<0.01). Compared with healthy controls, children with CF had significantly different intestinal microbial core structures. The levels of Eubacterium rectale, Bacteroides uniformis, Bacteroides vulgatus, Bifidobacterium adolescentis, Bifidobacterium catenulatum, and Faecalibacterium prausnitzii were reduced in children with CF. A similar but more extreme pattern was observed in children with CF who were taking antibiotics. LGG administration reduced fecal CLP and partially restored intestinal microbiota. There was a significant correlation between reduced microbial richness and intestinal inflammation.

Conclusions: CF causes qualitative and quantitative changes in intestinal microbiota, which may represent a novel therapeutic target in the treatment of CF. Administration of probiotics restored gut microbiota, supporting the efficacy of probiotics in reducing intestinal inflammation and pulmonary exacerbations.

Trial registration: ClinicalTrials.gov NCT 01961661.

Trial registration: ClinicalTrials.gov NCT01961661.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Intestinal inflammation markers in CF…
Figure 1. Intestinal inflammation markers in CF and healthy children.
Fecal calprotectin (A) and rectal nitric oxide (B) concentrations in children with CF and in healthy controls (P<0.01).
Figure 2. Microbial diversity in CF and…
Figure 2. Microbial diversity in CF and healthy children.
DGGE profiles of intestinal microbiota in children with CF (indicated by numbers) and age-matched healthy controls (indicated by letters) obtained with universal Hda1-Hda2 primers.
Figure 3. A hierarchical cluster analysis of…
Figure 3. A hierarchical cluster analysis of the banding patterns in CF and healthy children.
Dendrogram constructed using Pearson’s correlation coefficients and the UPGMA algorithm of DGGE gels obtained analyzing the V2–V3 region of all fecal samples. Gels were analyzed using Fingerprinting II software (Bio-Rad). Healthy subjects are indicated with capital letters, and CF patients with numbers.
Figure 4. DGGE profiles of Bacteroide /…
Figure 4. DGGE profiles of Bacteroide/Prevotella group in CF and healthy children.
A, Nested DGGE of Bacteroide/Prevotella group profiles in children with CF (indicated by numbers) and age-matched healthy controls (indicated by letters). The bands corresponding to Bacteroides vulgatus and Bacteroides uniformis are reduced in children with CF as compared with healthy controls. B, Dendrogram of DGGE gel obtained using primers for analysis of Bacteroides/Prevotella group.
Figure 5. RT-PCR analysis of selected bacterial…
Figure 5. RT-PCR analysis of selected bacterial species in stools of CF and healthy children.
Quantitative differences in E. rectale, E. coli, Bacteroides/Prevotella group, B. uniformis, B. vulgatus, Bifidobacterium spp., B. catenulatum group, B. adolescentis, and F. prausnitzii as determined by RT-PCR in children with CF and healthy individuals. Values are means ± SD. Differences indicated with *** are extremely statistically significant (P<0.0001); differences indicated with ** are very statistically significant (P = 0.003), and differences indicated with * are statistically significant (P = 0.03).
Figure 6. Flow diagram of enrolled patients.
Figure 6. Flow diagram of enrolled patients.
Figure 7. Quantitative analysis of different bacterial…
Figure 7. Quantitative analysis of different bacterial species by FISH in children with CF, healthy controls, and children with CF after antibiotic treatment.
A significant reduction in the levels of Bacteroides spp., Eubacterium rectale, and Fecalibacterium prausnitzii spp. was observed in children with CF as compared with that in healthy controls. A further reduction in the number of Bacteroides spp. and E. rectale was observed in children with CF after antibiotic treatment as compared with that in children with CF who were not treated with antibiotics. *P<0.001; **P<0.01.
Figure 8. Modification of fecal CLP concentration…
Figure 8. Modification of fecal CLP concentration before and after LGG or placebo treatment in children with CF.
A significant reduction in fecal CLP was observed in children with CF after LGG treatment (P<0.05).

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