Clostridium butyricum Strains and Dysbiosis Linked to Necrotizing Enterocolitis in Preterm Neonates Nadim Cassir, Samia Benamar, Jacques Bou Khalil, Olivier Croce, Marie Saint-Faust, Aurélien Jacquot, Matthieu Million, Said Azza, Nicholas Armstrong, Mireille Henry, et al. To cite this version: Nadim Cassir, Samia Benamar, Jacques Bou Khalil, Olivier Croce, Marie Saint-Faust, et al.. Clostrid- ium butyricum Strains and Dysbiosis Linked to Necrotizing Enterocolitis in Preterm Neonates. Clinical Infectious Diseases, Oxford University Press (OUP), 2015, 61 (7), pp.1107–1115. hal-01985792 HAL Id: hal-01985792 https://hal.archives-ouvertes.fr/hal-01985792 Submitted on 18 Jan 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Clostridium butyricum Strains and Dysbiosis Linked to Necrotizing Enterocolitis in Preterm Neonates Nadim Cassir,1 Samia Benamar,1 Jacques Bou Khalil,1 Olivier Croce,1 Marie Saint-Faust,2 Aurélien Jacquot,3 Matthieu Million, 1 Said Azza,1 Nicholas Armstrong,1 Mireille Henry,1 Priscilla Jardot,1 Catherine Robert,1 Catherine Gire,4 Jean-Christophe Lagier,1 Eric Chabrière,1 Eric Ghigo,1 Hélène Marchandin,5 Catherine Sartor,6 Patrick Boutte,2 Gilles Cambonie,3 Umberto Simeoni,7 Didier Raoult,1 and Bernard La Scola1 1Facultés de Médecine et de Pharmacie, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UM63, CNRS 7278, IRD 198, INSERM U1095, Aix-Marseille Université, 2Service de néonatalogie, Centre Hospitalier Universitaire de Nice GCS-CHU Lenval, 3Service de néonatalogie, Centre Hospitalier Universitaire de Montpellier, Hôpital Arnaud de Villeneuve, 4Service de néonatalogie, Hôpital Nord, Assistance Publique des Hôpitaux de Marseille, Aix-Marseille Université, 5Laboratoire de bactériologie, Centre Hospitalier Universitaire de Montpellier, Hôpital Arnaud de Villeneuve, 6Equipe opérationnelle d’hygiène hospitalière, Hôpital la Conception, and 7Service de néonatalogie, Hôpital la Conception, Assistance Publique des Hôpitaux de Marseille, Aix-Marseille Université, France Background. Necrotizing enterocolitis (NEC) is the most common and serious gastrointestinal disorder among preterm neonates.We aimed to assess a specific gut microbiota profile associated with NEC. Methods. Stool samples and clinical data were collected from 4 geographically independent neonatal intensive care units, over a 48-month period. Thirty stool samples from preterm neonates with NEC (n = 15) and controls (n = 15) were analyzed by 16S ribosomal RNA pyrosequencing and culture-based methods. The results led us to develop a specific quantitative polymerase chain reaction (qPCR) assay for Clostridium butyricumand, we tested stool samples from preterm neonates with NEC (n = 93) and controls (n = 270). We sequenced the whole genome of 16 C. butyricum strains,analyzed their phylogenetic relatedness,tested their culture supernatants for cytotoxic activity, and searched for secreted toxins. Results. Clostridium butyricum was specifically associated with NEC using molecular and culture-based meth- ods (15/15 vs 2/15; P < .00011) or qPCR (odds ratio, 45.4 [95% confidence interval, 26.2–78.6]; P < .00011). Culture supernatants of C. butyricum strains from preterm neonates with NEC (n = 14) exhibited significant cytotoxic activity (P = .008), and we identified in all a homologue of the β-hemolysin toxin gene shared by Brachyspira hyo- dysenteriae,the etiologic agent of swine dysentery.The corresponding protein was secreted by a NEC-associated C. butyricum strain. Conclusions. NEC was associated with C. butyricum strains and dysbiosis with an oxidized,acid, and poorly diversified gut microbiota.Our findings highlight the plausible toxigenic mechanism involved in the pathogenesis of NEC. Keywords. necrotizing enterocolitis;Clostridium butyricum;;;;gut microbiota;dysbiosis;toxins. Necrotizing enterocolitis (NEC) is the most common and serious acquired gastrointestinal disorder among Correspondence:Bernard La Scola, MD, PhD,Unité de Recherche sur les Mala- dies Infectieuses et Tropicales Emergentes, URMITE,UM63, CNRS 7278, IRD 198, preterm neonates.Its incidence rises to 12% in infants INSERM 1095, Faculté de médecine, Aix-Marseille Université, 27 Boulevard Jean with very low birth weight (ie, 500–1000 g) [1]. The mor- Moulin, 133385 Marseille cedex 05, France ([email protected]). Clinical Infectious Diseases ® 2015;61(7):1107–15 tality associated with NEC ranges from 20% to 50% [2]. A high morbidity rate has also been reported; in partic- ular, neurodevelopmentalimpairment is observed [1]. Low birth weight and prematurity have been identified as critical risk factors for NEC [3]. Although intestinal immatu- were collected the day of the symptom’s onset.We matched 15 rity and highly immunoreactive intestinal mucosa appear to be control patients by sex,gestational age,birth weight,days of life, important elements of the disease, it could not be reproduced in type of feeding, mode of delivery, and duration of previous anti- germ-free animals, suggesting a critical role of microbial coloni- biotic therapy (Supplementary Table 1). All controls were present zation [4]. Interestingly, NEC does not occur until at least 8–10 in the same NICU at the same time as the cases of NEC. None of days postpartum in preterm neonates, the period when gut mi- the selected patients had received probiotics or been included in crobiota diversification and anaerobic bacteria colonization be- therapeutic protocols before the stool sampling. All of them were Bacteria and viruses gins [3, 5]. To date, multiple causative organisms have been negative for routine microbial investigation. proposed, including viruses, Staphylococcus species, various as well as Clostridium difficile toxins were screened on stool sam- gram-negative bacilli, and Clostridium species [6]. However, ples using standard methods as previously described [13]. no etiologic organism has been definitively established. In the second part of the study, we tested for the presence of C. The gut microbiota from preterm neonates both prior to and butyricum 363 stool samples from preterm neonates with NEC during NEC has recently been studied using molecular methods (n = 93), preterm control neonates (n = 70), infants with gastroen- [6–8]. These studies suggested an association between the patteern teritis (n = 100), and control infants with no symptoms of gastroen- of gut microbiota and the occurrence of NEC. An interindividual teritis (n = 100). Patients with NEC included over a period of 48 variation and overall reduction in the diversity of microbiota, es- months and sampled at the onset of the disease (Figure 1) were also pecially when there has been a prolonged previous antibiotic matched with the same criteria as for the first part of the study to therapy, was retrieved. However, using genomic tools induces control preterm neonates (Supplementary Table 2). Stool samples several biases. Microbial diversities measured from the same sam- from infants with and without gastroenteritis were collected inde- ple as well as relative taxa abundances may diffeer depending on pendently at the pediatric ward, corresponding to external controls. the choice of 16S ribosomal RNA (rRNA) gene variable region(s) studied [9]. There is also a depth bias in that clinically relevant Study Design and Procedures bacteria such as Salmonella Typhi can be missed by sequence- Bacterial Composition of Stool Samples based methods in concentrations <10 5/mL [10]. We compared the bacterial composition of stool samples from As microbial culturing is the only method to characterize the 30 preterm neonates with and without NEC. Samples were test- physiological properties of bacteria and evaluate their virulence ed by pyrosequencing of a 16S rRNA amplicon targeting the V6 expression, there has been a renewed interest in culture methods region. This region was used because of its considerable se- allowing the identification of species hitherto considered noncul- quence variability that distinguishes among most bacterial spe- turable [11]. A previous comprehensive approach has allowed the cies [9]. The mothur program and the recommended procedure culture of 341 diffeerent bacterial species from 3 diffeerent stool for trimming the 16S rRNA gene amplicon data were used [14]. samples with only 15% of the species overlapping, with the This program was also used to determine Shannon diversity in- bacteria detected by concomitant pyrosequencing [10]. This dices at the 0.02 level, based on operational taxonomic unit work has facilitated the selection of optimal conditions and (OTU) sequences. The Ribosomal Database Project classifier media for bacterial cultivation from stool samples. was used for taxonomic assignments from phylum to genus In the present study, we compared the gut microbiota between [15]. Assignment at the species level from 16S rRNA pyrose- preterm neonates with NEC and controls using complementary quences was atteempted using basic local alignment search tool 16S rRNA pyrosequencing and culture-based methods. We then (BLAST) similarity