CASABURI, GIORGIO

D'ARGENIO, VALERIA

MARTINELLI, MASSIMO

MIELE, ERASMO

SACCHETTI, LUCIA

SALVATORE, FRANCESCO

STAIANO, ANNAMARIA

Vincenza Precone

PubMed

Archivio della ricerca - Università degli studi di Napoli Federico II

nature publishing group 851© 2013 by the American College of Gastroenterology The American Journal of GASTROENTEROLOGYLETTERS TO THE EDITOR To the Editor: Th e pathogenesis of Crohn ’ s disease (CD) is not known: the currently accepted hypothesis is that genetic suscep-tibility and environmental factors coop-erate to trigger the chronic infl ammation typical of the disease. Recent evidence implicating the gut microbiome in CD development has generated interest in its characterization ( 1 ). Previous studies have shown that bacterial diversity is reduced in CD patients, that bacterial populations in CD patients diff er in relation to pheno-type, and that the microbial composition diff ers signifi cantly between infl amed and non-infl amed mucosal areas ( 2 ).  Nutritional therapy is eff ective in pedi-atric CD; it induces remission of mucosal infl ammatory reactions and exerts a ben-efi cial eff ect on the child ’ s nutritional sta-tus and growth ( 3 ). It has been suggested that nutritional therapy may modify the fecal microfl ora in CD children ( 4 ). In an attempt to address this hypothesis, we char-acterized the entire gut mucosal microbi-ome in a child with CD at diagnosis and aft er nutritional therapy, and in a sex- and age-matched subject not aff ected by CD.  Th is 14-year-old boy was diagnosed with active CD (Pediatric Crohn ’ s Disease Activ-ity Index, PCDAI  =  50). Aft er colonoscopy, he underwent nutritional therapy consisting of a daily powder constituted by proteins, microbiome composition in these three ileum samples. A specifi c amplicon was designed to cover the V4 – V6 variable regions of the bacterial 16S rRNA genes and amplifi ed using hybrid-tagged primers. Ultra-deep amplicon sequencing was per-formed on the 454 Genome Sequencer FLX instrument (Roche Life Science, Branford, CT). Data were analyzed using the QIIME community analysis pipeline ( 5 ). High-quality fi ltered sequences were used to identify the operational taxonomic antioxidants, and anti-infl ammatory fats (Alicalm formula, Nutricia Advanced Medical Nutrition) for 8 weeks. Aft er this time, a clinical re-evaluation revealed dis-ease remission (PCDAI  =  0). Endoscopic ileum mucosal samples at diagnosis and aft er therapy were collected for DNA extraction. An ileum tissue sample was obtained from a 15-year-old boy with a gut polyp and without family history of CD. We used a 16S rRNA next-generation sequencing strategy to investigate the gut  An Altered Gut Microbiome Profi le in a Child Affected by Crohn ’ s Disease Normalized After Nutritional Therapy  Valeria  D ’ Argenio ,  MD 1 , 2 ,   Vincenza  Precone ,  PhD 1 , 2 ,   Giorgio  Casaburi ,  MS 1 , 2 ,  Erasmo  Miele ,  MD 3 ,  Massimo  Martinelli ,  MD 3 ,   Annamaria  Staiano ,  MD ,  PhD 3 ,   Francesco  Salvatore ,  MD, PhD 1 , 4 and   Lucia  Sacchetti ,  PhD 1 , 2   doi:10.1038/ajg.2013.46 Consensus Lineage from phylum to familyP->Bacteroidetes;C->Bacteroidia;O->Bacteroidales;F->Bacteroidaceae 24491051581661710624623675792639661694347610441P->Bacteroidetes;C->Bacteroidia;O->Bacteroidales;F->PorphyromonadaceaeP->Firmicutes;C->Clostridia;O->Clostridiales;F->LachnospiraceaeP->Proteobacteria;C->Betaproteobacteria;O->Burkholderiales;F->AlcaligenaceaeP->Proteobacteria;C->Gammaproteobacteria;O->Enterobacteriales;F->EnterobacteriaceaeP->Proteobacteria;C->Gammaproteobacteria;O->Pasteurellales;F->PasteurellaceaeP->Proteobacteria;C->Gammaproteobacteria;O->Pseudomonadales;F->PseudomonadaceaeControl Patient-BT Patient-AT0Bacteroidetes Firmicutes Fusobacteria Proteobacteria Other1020304050% Of total clones in each patient 60708090ControlPatient-BTPatient-AT Figure 1 .  Composition of the ileum microbiome characterized in the control subject and in the Crohn ’ s disease (CD) patient before therapy (patient-BT) and after therapy (patient-AT) by next-generation sequencing of the 16S rRNAs. ( a ) Phylum-level classifi cation shows the reduction of  Bacteroidetes and the signifi cant prevalence of  Proteobacteria in the patient-BT vs. the control and the patient-AT. ( b ) The heatmap table highlights in brownish red the most signifi cant alterations in the bacterial composition of gut microbiome detected in the CD patient-BT; the numerical fi gures indicate the number of bacterial families sequenced in each patient. The American Journal of GASTROENTEROLOGY VOLUME 108 | MAY 2013   www.amjgastro.com852  Letters to the Editor units (OTUs): 705, 1,328, and 2,171 OTUs were obtained in the patient before and aft er therapy, and in the control subject, respectively. On the basis of the taxonomic assignment of the OTUs, we characterized the ileum microbiome of our samples at fi ve phylogenetic levels (from phylum to genus). As shown in  Figure 1 ,  Proteobacteria were more abundant, and  Bacteroidetes less abundant, in our CD patient before ther-apy than in the control. Interestingly, the composition of the ileum microbiome in the patient aft er therapy was virtually the same as in the control. Moreover, our data support the reduced bacterial diversity of the CD microbiome. In fact, the Shannon Diversity Index Score was signifi cantly lower ( P  <  0.05) in our CD patient before therapy than aft er therapy and also when compared with the control ( Figure 2 ).  Although our fi ndings were obtained in one case – control study, and therefore may be considered preliminary, they strongly suggest that nutritional therapy can improve the infl ammatory status of CD by restoring the composition of the mucosal microbiome. Th is case of CD gut microbiome dysbiosis that responded to nutritional therapy can be considered proof-of-concept to evaluate a similar approach in other pediatric clinical laboratories and may serve to prompt mul-ticenter studies and possible clinical trials.  ACKNOWLEDGMENTS  We thank Jean Ann Gilder (Scientifi c Communication srl., Naples) for edit-ing the text, and Vittorio Lucignano, CEINGE – Biotecnologie Avanzate, for technical assistance related to graphics.  CONFLICT OF INTEREST  Guarantor of the article : Francesco Salvatore, MD, PhD.  Specifi c author contributions : V.D., A.S., L.S., and F.S. designed the project; A.S., E.M., and M.M. enrolled the study subjects, performed the endoscopy, treated and monitored the patient during follow-up; V.D. and V.P. performed the next-generation sequencing experiment; V.D. and G.C. performed the bio-informatic analysis; V.D., L.S., and F.S. analyzed the data and wrote the manuscript. All the authors revised and approved the fi nal draft .  Financial support: Th is work was suppor-ted by grant L.5 / 95 (to F.S.) from Regione Campania; grant PS 35-126 / Ind and grant PON01_02589 (MICROMAP) 2012 from the Ministry of University and Research (both to F.S.); grant RF-2010-2318372 from the Ministry of Health (to F.S.).  Potential competing interests: None.  REFERENCES  1 .  Neuman  MG ,  Nanau  RM .  Infl ammatory bowel disease: role of diet, microbiota, life style .  Transl Res  2012 ; 160 : 29 – 44 .  2 .  Walker  AW ,  Sanderson  JD ,  Churcher  C  et al.  High-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and diff erences between infl amed and non-infl amed regions of the intestine in infl ammatory bowel disease .  BMC Microbiol  2011 ; 11 : 7 .  3 .  Day  AS ,  Whitten  KE ,  Sidler  M  et al.  Systematic review: nutritional therapy in paediatric Crohn’s disease .  Aliment Pharmacol Th er  2008 ; 27 :  293 – 307 .  4 .  Lionetti  P ,  Callegari  M ,  Cavicchi  M  et al.   Enteral nutrition-induced remission is associated with profound modifi cation of the intestinal microfl ora in Crohn’s disease .  J Pediatr Gastroenterol Nutr  2004 ; 39 : S106 .  5 .  Caporaso  JG ,  Kuczynski  J ,  Stombaugh  J  et al.  QIIME allows analysis of high-throughput community sequencing data .  Nature Methods  2010 ; 7 : 335 – 6 .  1 CEINGE-Biotecnologie Avanzate ,  Naples ,  Italy ;  2 Department of Biochemistry and Medical Biotechnologies, University of Naples Federico II ,  Naples ,  Italy ;  3 Department of Pediatrics, University of Naples Federico II ,  Naples ,  Italy ;  4 IRCCS-Fondazione SDN ,  Naples ,  Italy .  Correspondence:  Francesco Salvatore, MD, PhD ,  CEINGE-Biotecnologie Avanzate, Dipartimento di Biochimica e Biotecnologie Mediche – Ed. 19 ,  Via Sergio Pansini 5 ,  Naples  80131 ,  Italy . E-mail:  salvator@unina.it 8.0ControlPatient-before therapyPatient-after therapy6.0Mean shannon diversity index score7.14.02.003.96.2Control Patient-BT Patient-AT** Figure 2 .  The mean Shannon Diversity Index Score. Values were signifi cantly lower before therapy (patient-before therapy (patient-BT)) compared with values obtained after treatment (patient after therapy (patient-AT)) and with values obtained in the control subject ( * P  <  0.05). Th is work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

An Altered Gut Microbiome Profile in a Child Affected by Crohn's Disease Normalized After Nutritional Therapy

An Altered Gut Microbiome Profile in a Child Affected by Crohn's Disease Normalized After Nutritional Therapy

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