Clinical Infectious Diseases Advance Access published June 17, 2015
1
Neonatal necrotizing enterocolitis, a clostridial disease?
Butel Marie-José*, Aires Julio
rip t
DHU Risks in pregnancy, EA 4065, Faculté de Pharmacie, Université Paris Descartes
*Corresponding
Dr Julio Aires EA 4065, Faculté de Pharmacie 4 avenue de l’Observatoire
M
Paris 75006 – France e-mail [email protected] tel +33 1 53 73 99 18
Ac
ce
pt
ed
fax +33 1 53 73 99 23
© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: [email protected].
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
an us c
author : Pr Marie-José Butel, EA 4065, Faculté de Pharmacie, 4 avenue de l’Observatoire, Paris 75006 – France, e-mail [email protected], tel +33 1 53 73 99 11, fax +33 1 53 73 99 23
2
Neonatal necrotizing enterocolitis (NEC) is a devastating disease that remains an important cause of morbidity and mortality among very preterm neonates and which incidence has increased in parallel with the improve survival of this population. Despite years of research
rip t
over the past decades, its pathogenesis is still uncertain and thus prevention is difficult. Three factors are recognized to coalesce, i.e. immaturity of the preterm infant, enteral
feeding and intestinal bacterial colonization [1]. Although genetics appears to play a role,
intestinal microorganisms and the host’s inflammatory response leading to the release of mediators and hence to intestinal mucosal injury [2]. However, to date there is no consensus on specific bacteria causally associated with NEC development. Neither culture nor culture-
M
independent approaches have identified a single microbial cause of NEC.
Using culture, various microorganisms have been involved. All belonged to the commensal microbiota and are potential pathogens: Escherichia coli, Klebsiella, Enterobacter,
ed
Staphylococcus sp, and Clostridium sp [3]. Particularly, the role Clostridium perfringens, C. butyricum, and C. neonatale have been reported [4] [5]
pt
Last decade, culture-independent techniques and above all Next Generation Sequencing
ce
(NGS) tools were applied to microbiota analyses. It allowed identification of bacteria unable to grow under standard laboratory conditions and had expanded our knowledge of
Ac
microbiota ecosystem composition. Hence, NGS studies have brought new insights in microbiota associated to NEC cases showing an abnormal bacterial pattern and a reduced diversity of microbiota prior to NEC or at the onset of NEC in comparison with controls [6, 7].
However, like culture, different bacterial signatures have been proposed such as Gramnegative bacilli (Enterobacter, Citrobacter, Enterobacteriaceae, or Proteobacteria), Bacillales
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
an us c
there is mounting evidence that NEC originates from a defective interaction between
3
or staphylococci species. Finally, only few studies associated NEC and clostridia species colonization [8-10]. The article by Cassir and colleagues in the current issue of Clinical Infectious Diseases (CID)
rip t
investigated the fecal microbiota in infants suffering from NEC compared to control infants. The interest of this study was to use complementary approaches, i.e. bacterial culture and
an us c
16S rRNA gene pyrosequencing-based methods. In the first part of their study, through both
techniques, the authors found a lower diversity in NEC cases (n=15) as compared to controls
and C. butyricum with all the 15 NEC cases colonized by this species versus only 2 out of the control infants. These data were confirmed using a specific qPCR to screen a larger set of
M
samples of NEC cases (n=96) versus controls (n=270). Up to now, this is the first report analyzing fecal samples from infants suffering from NEC by culture-independent method that identified C. butyricum. Early 2015, Sim K et al published in CID a study comparing fecal
ed
samples from NEC cases (n=12) versus controls (n=44) using culture and 16S rRNA gene sequencing methods. The authors concluded that C perfringens was significantly
pt
overrepresented in NEC cases.
ce
If 16S rRNA gene sequencing is now widely used in microbial ecology analysis allowing deep analysis of the microbiota, it has its specific limits. First, assignation to specific taxa (family,
Ac
genus, or species) depends on the length and quality of the sequence. Second, it is based on PCR amplifications using bacterial universal primers or the variable 16S rRNA gene regions that can lead to miss or underrepresent some taxa. Therefore, primers design can be challenging when considering clostridia that belongs to a complex phylogenetic heterogeneous group with some cluster having high strain genetic diversity [11]. Moreover,
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
(n=15) confirming previously reported results. Moreover, a specific association between NEC
4
clostridia can be present in a subdominant status which participates to the difficulty of a clear clostridia detection and taxa identification. For example, culture based method, showed that clostridia (including C. butyricum and C. perfringens) are part of the commensal
rip t
microbiota in preterm infants without enteropathies [12]. Cassir and colleagues chose to amplify the V6 region of the 16S rRNA gene that is not the most frequently used, but
presented as better to identify clostridia due to its hypervariability. For instance, Sim K et al
already observed concerning bifidobacteria detection in gut microbiota, it will be near to impossible to design a single PCR primer set able to generate an equally efficient
amplification yield of 16S rRNA gene sequences across all components of human gut
M
microbiota [13].
The relationship between C. butyricum and NEC is reinforced with the study of Smith and colleagues who recently reported a correlation between the presence of C. butyricum and
ed
pneumatosis intestinalis in tissue specimens from neonates with NEC [4]. Moreover, this species was widely involved in studies in the 80s which specifically isolated by culture C.
pt
butyricum in blood, peritoneal fluid, or feces from infants with NEC [14-18]. Moreover, its
ce
role in NEC pathogenesis has been strengthened in animal models. Indeed, gnotobiotic quails fed a lactose diet and monoassociated with C. butyricum display NEC-like digestive
Ac
lesions [19]. Likewise, significant role of this species has also been suggested in the preterm piglet model, where C. butyricum as well other clostridial species were largely absent in piglets without NEC and significantly overrepresented among colonic mucosa samples from piglets with NEC [20].
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
an us c
amplified the V3-V5 region of the bacterial 16S rRNA genes. This raised the fact that, as
5
Another interesting aspect of Casir and colleagues manuscript is the comparative analysis of culture and 16S rRNA gene sequencing data that shows only 11% overlap. This demonstrates that like culture, molecular techniques are far from identifying the totality of fecal
rip t
microbiota and that complementary approaches as used in this study are therefore necessary.
an us c
The fact that Cassir and colleagues used bacterial culture allowed the authors to compare the genome of the C. butyricum strains for virulence traits screening.
immature intestine compared to a mature gut leading to neutrophil chemotaxis and tissue injury. Various mechanisms have been evoked: increase in pro-inflammatory cytokine
M
release such as IL-8, defect in inhibitors of the NF-κB pathway, activation of TLR4 by lipolysaccharides from gram-negative bacteria [21]. Some intestinal diseases are associated
ed
with bacterial toxins, but this was not the case for NEC. Concerning clostridia, C. butyricum bacterial fermentation has been shown to induce NEC-like lesions in the quail NEC model [19], or in rats intrarectally perfused [22]. This is in accordance with the hypothesis of the
pt
possible key role of bacteria through overproduction of bacterial fermentation of the non-
ce
digested lactose due to intestinal immaturity of the very premature infants [23]. A cytotoxicity effect of C. butyricum strains supernatants has been observed in vitro four
Ac
decades ago by means of butyrate [24]. In the current paper, Cassir and colleagues reported a cytotoxic activity for their 16 C. butyricum isolates (including NEC cases and control strains). By sequencing strains’ whole genome, they suggest that cytotoxicity is linked to one gene coding a hemolysin analog of the etiologic agent of swine dysentery. However, their conclusion must be dampen by the facts that, first, the in silico MLST analysis performed to
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
The pathogenesis of NEC can be due to an exaggerated inflammatory response of the
6
genetically compare C. butyricum strains showed clonal relationships between several strains from the same NICU. The authors did not discuss on this aspect. Second, the cytotoxic activity has been found in strains isolated from NEC cases and controls as well.
rip t
Therefore, the potential role of a putative hemolysin analog from C. butyricum as a toxin that may participate in NEC development needs to be demonstrated.
an us c
To conclude, the manuscript by Cassir and colleagues adds new data for the involvement of Clostridium in NEC and emphasizes on a specific species, C. butyricum. It is noteworthy that
bacterial signatures (C. perfringens and Klebsiella for instance) [9].
Up to now, among clostridia species thought to be associated with NEC, none involved
M
known toxin production. By contrast, C. difficile, a toxicogenic species, is not implicated in NEC [19]. Hence, potential pathogenic characteristics shared by these clostridial species and
ed
may be by bacteria belonging to other genera deserve research. Such characteristics could be specific of the bacterial strains responsible for the disease, since all these species belong
ce
pt
to commensal microbiota, or could interact with the host displaying specific susceptibility.
Ac
The authors have no reported conflicts related to the content of this manuscript.
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
in other reports NEC was associated with other clostridial species or different separate
7 References
rip t
1. Neu J, Walker WA. Necrotizing enterocolitis. N Engl J Med 2011; 364:255-64.
2. Gephart SM, McGrath JM, Effken JA, Halpern MD. Necrotizing enterocolitis risk: state of the science. Adv Neonatal Care 2012; 12:77-87.
in the pathogenesis of necrotizing enterocolitis. Pediatrics 2010; 125:777-85.
4. Smith B, Bode S, Petersen BL, et al. Community analysis of bacteria colonizing intestinal tissue of neonates with necrotizing enterocolitis. BMC Microbiol 2011; 11:73.
5. Alfa MJ, Robson D, Davi M, Bernard K, Van Caeseele P, Harding GK. An outbreak of necrotizing
Infect Dis 2002; 35:S101-S105.
M
enterocolitis associated with a novel clostridium species in a neonatal intensive care unit. Clin
ed
6. Mai V, Young CM, Ukhanova M, et al. Fecal microbiota in premature infants prior to necrotizing
pt
enterocolitis. PLoS One 2011; 6:e20647.
7. Morrow AL, Lagomarcino AJ, Schibler KR, et al. Early microbial and metabolomic signatures
ce
predict later onset of necrotizing enterocolitis in preterm infants. Microbiome 2013; 1:13.
8. De La Cochetière MF, Piloquet H, Des Robert C, Darmaun D, Galmiche JP, Rozé JC. Early
Ac
intestinal bacterial colonization and necrotizing enterocolitis in premature infants: the putative role of Clostridium. Pediatr Res 2004; 56:1-5.
9. Sim K, Shaw AG, Randell P, et al. Dysbiosis anticipating necrotizing enterocolitis in very premature infants. Clin Infect Dis 2014.
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
an us c
3. Morowitz MJ, Poroyko V, Caplan M, Alverdy J, Liu DC. Redefining the role of intestinal microbes
8 10. Zhou Y, Shan G, Sodergren E, Weinstock G, Walker WA, Gregory KE. Longitudinal analysis of the premature infant intestinal microbiome prior to necrotizing enterocolitis: a case-control study. PLoS One 2015; 10:e0118632.
rip t
11. Kalia VC, Mukherjee T, Bhushan A, Joshi J, Shankar P, Huma N. Analysis of the unexplored features of rrs (16S rDNA) of the genus Clostridium. BMC Genomics 2011; 12:18.
an us c
12. Ferraris L, Butel MJ, Campeotto F, Vodovar M, Roze JC, Aires J. Clostridia in premature
neonates' gut: incidence, antibiotic susceptibility, and perinatal determinants influencing
13. Turroni F, Marchesi JR, Foroni E, et al. Microbiomic analysis of the bifidobacterial population in the human distal gut. ISME J 2009; 3:745-51.
M
14. Howard FM, Bradley JM, Flynn DM, Noone P, Szawatkowski M. Outbreak of necrotizing enterocolitis caused by Clostridium butyricum. Lancet 1977;1099-102.
ed
15. Sturm R, Staneck JL, Stauffer LR, Neblett III WW. Neonatal necrotizing enterocolitis associated with penicillin-resistant, toxigenic Clostridium butyricum. Pediatrics 1980; 66:928-31.
ce
3.
pt
16. Gothefors L, Blenkharn I. Clostridium butyricum and necrotizing enterocolitis. Lancet 1978;52-
17. Laverdière M, Robert A, Chicoine R, Salet D, Rosenfeld R. Clostridia in necrotising enterocolitis.
Ac
Lancet 1978; 2:377.
18. Lawrence G, Bates J, Gaul A. Pathogenesis of neonatal necrotising enterocolitis. Lancet 1982;137-9.
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
colonization. PLoS One 2012; 7:e30594.
9 19. Waligora-Dupriet AJ, Dugay A, Auzeil N, Huerre M, Butel MJ. Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model. Pediatr Res 2005; 58:629-35.
rip t
20. Azcarate-Peril MA, Foster DM, Cadenas MB, et al. Acute necrotizing enterocolitis of preterm piglets is characterized by dysbiosis of ileal mucosa-associated bacteria. Gut Microbes 2011;
an us c
2:234-43.
21. Patel RM, Denning PW. Intestinal microbiota and its relationship with necrotizing enterocolitis.
22. Lin J, Nafday SM, Chauvin SN, et al. Variable effects of short chain fatty acids and lactic acid in inducing intestinal mucosal injury in newborn rats. J Pediatr Gastroenterol Nutr 2002; 35:545-
M
50.
23. Kien CL. Colonic fermentation of carbohydrate in the premature infant : possible relevance to
ed
necrotizing enterocolitis. J Pediatr 1990; 117:S52-S58.
24. Popoff MR, Jolivet-reynaud C, Carlier JP. Cytotoxic activity of Clostridium butyricum
Ac
ce
pt
supernatants induced by butyrate. Fems Microbiol Lett 1987; 43:95-100.
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
Pediatr Res 2015.
1
Neonatal necrotizing enterocolitis, a clostridial disease?
Butel Marie-José*, Aires Julio
rip t
DHU Risks in pregnancy, EA 4065, Faculté de Pharmacie, Université Paris Descartes
*Corresponding
Dr Julio Aires EA 4065, Faculté de Pharmacie 4 avenue de l’Observatoire
M
Paris 75006 – France e-mail [email protected] tel +33 1 53 73 99 18
Ac
ce
pt
ed
fax +33 1 53 73 99 23
© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: [email protected].
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
an us c
author : Pr Marie-José Butel, EA 4065, Faculté de Pharmacie, 4 avenue de l’Observatoire, Paris 75006 – France, e-mail [email protected], tel +33 1 53 73 99 11, fax +33 1 53 73 99 23
2
Neonatal necrotizing enterocolitis (NEC) is a devastating disease that remains an important cause of morbidity and mortality among very preterm neonates and which incidence has increased in parallel with the improve survival of this population. Despite years of research
rip t
over the past decades, its pathogenesis is still uncertain and thus prevention is difficult. Three factors are recognized to coalesce, i.e. immaturity of the preterm infant, enteral
feeding and intestinal bacterial colonization [1]. Although genetics appears to play a role,
intestinal microorganisms and the host’s inflammatory response leading to the release of mediators and hence to intestinal mucosal injury [2]. However, to date there is no consensus on specific bacteria causally associated with NEC development. Neither culture nor culture-
M
independent approaches have identified a single microbial cause of NEC.
Using culture, various microorganisms have been involved. All belonged to the commensal microbiota and are potential pathogens: Escherichia coli, Klebsiella, Enterobacter,
ed
Staphylococcus sp, and Clostridium sp [3]. Particularly, the role Clostridium perfringens, C. butyricum, and C. neonatale have been reported [4] [5]
pt
Last decade, culture-independent techniques and above all Next Generation Sequencing
ce
(NGS) tools were applied to microbiota analyses. It allowed identification of bacteria unable to grow under standard laboratory conditions and had expanded our knowledge of
Ac
microbiota ecosystem composition. Hence, NGS studies have brought new insights in microbiota associated to NEC cases showing an abnormal bacterial pattern and a reduced diversity of microbiota prior to NEC or at the onset of NEC in comparison with controls [6, 7].
However, like culture, different bacterial signatures have been proposed such as Gramnegative bacilli (Enterobacter, Citrobacter, Enterobacteriaceae, or Proteobacteria), Bacillales
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
an us c
there is mounting evidence that NEC originates from a defective interaction between
3
or staphylococci species. Finally, only few studies associated NEC and clostridia species colonization [8-10]. The article by Cassir and colleagues in the current issue of Clinical Infectious Diseases (CID)
rip t
investigated the fecal microbiota in infants suffering from NEC compared to control infants. The interest of this study was to use complementary approaches, i.e. bacterial culture and
an us c
16S rRNA gene pyrosequencing-based methods. In the first part of their study, through both
techniques, the authors found a lower diversity in NEC cases (n=15) as compared to controls
and C. butyricum with all the 15 NEC cases colonized by this species versus only 2 out of the control infants. These data were confirmed using a specific qPCR to screen a larger set of
M
samples of NEC cases (n=96) versus controls (n=270). Up to now, this is the first report analyzing fecal samples from infants suffering from NEC by culture-independent method that identified C. butyricum. Early 2015, Sim K et al published in CID a study comparing fecal
ed
samples from NEC cases (n=12) versus controls (n=44) using culture and 16S rRNA gene sequencing methods. The authors concluded that C perfringens was significantly
pt
overrepresented in NEC cases.
ce
If 16S rRNA gene sequencing is now widely used in microbial ecology analysis allowing deep analysis of the microbiota, it has its specific limits. First, assignation to specific taxa (family,
Ac
genus, or species) depends on the length and quality of the sequence. Second, it is based on PCR amplifications using bacterial universal primers or the variable 16S rRNA gene regions that can lead to miss or underrepresent some taxa. Therefore, primers design can be challenging when considering clostridia that belongs to a complex phylogenetic heterogeneous group with some cluster having high strain genetic diversity [11]. Moreover,
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
(n=15) confirming previously reported results. Moreover, a specific association between NEC
4
clostridia can be present in a subdominant status which participates to the difficulty of a clear clostridia detection and taxa identification. For example, culture based method, showed that clostridia (including C. butyricum and C. perfringens) are part of the commensal
rip t
microbiota in preterm infants without enteropathies [12]. Cassir and colleagues chose to amplify the V6 region of the 16S rRNA gene that is not the most frequently used, but
presented as better to identify clostridia due to its hypervariability. For instance, Sim K et al
already observed concerning bifidobacteria detection in gut microbiota, it will be near to impossible to design a single PCR primer set able to generate an equally efficient
amplification yield of 16S rRNA gene sequences across all components of human gut
M
microbiota [13].
The relationship between C. butyricum and NEC is reinforced with the study of Smith and colleagues who recently reported a correlation between the presence of C. butyricum and
ed
pneumatosis intestinalis in tissue specimens from neonates with NEC [4]. Moreover, this species was widely involved in studies in the 80s which specifically isolated by culture C.
pt
butyricum in blood, peritoneal fluid, or feces from infants with NEC [14-18]. Moreover, its
ce
role in NEC pathogenesis has been strengthened in animal models. Indeed, gnotobiotic quails fed a lactose diet and monoassociated with C. butyricum display NEC-like digestive
Ac
lesions [19]. Likewise, significant role of this species has also been suggested in the preterm piglet model, where C. butyricum as well other clostridial species were largely absent in piglets without NEC and significantly overrepresented among colonic mucosa samples from piglets with NEC [20].
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
an us c
amplified the V3-V5 region of the bacterial 16S rRNA genes. This raised the fact that, as
5
Another interesting aspect of Casir and colleagues manuscript is the comparative analysis of culture and 16S rRNA gene sequencing data that shows only 11% overlap. This demonstrates that like culture, molecular techniques are far from identifying the totality of fecal
rip t
microbiota and that complementary approaches as used in this study are therefore necessary.
an us c
The fact that Cassir and colleagues used bacterial culture allowed the authors to compare the genome of the C. butyricum strains for virulence traits screening.
immature intestine compared to a mature gut leading to neutrophil chemotaxis and tissue injury. Various mechanisms have been evoked: increase in pro-inflammatory cytokine
M
release such as IL-8, defect in inhibitors of the NF-κB pathway, activation of TLR4 by lipolysaccharides from gram-negative bacteria [21]. Some intestinal diseases are associated
ed
with bacterial toxins, but this was not the case for NEC. Concerning clostridia, C. butyricum bacterial fermentation has been shown to induce NEC-like lesions in the quail NEC model [19], or in rats intrarectally perfused [22]. This is in accordance with the hypothesis of the
pt
possible key role of bacteria through overproduction of bacterial fermentation of the non-
ce
digested lactose due to intestinal immaturity of the very premature infants [23]. A cytotoxicity effect of C. butyricum strains supernatants has been observed in vitro four
Ac
decades ago by means of butyrate [24]. In the current paper, Cassir and colleagues reported a cytotoxic activity for their 16 C. butyricum isolates (including NEC cases and control strains). By sequencing strains’ whole genome, they suggest that cytotoxicity is linked to one gene coding a hemolysin analog of the etiologic agent of swine dysentery. However, their conclusion must be dampen by the facts that, first, the in silico MLST analysis performed to
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
The pathogenesis of NEC can be due to an exaggerated inflammatory response of the
6
genetically compare C. butyricum strains showed clonal relationships between several strains from the same NICU. The authors did not discuss on this aspect. Second, the cytotoxic activity has been found in strains isolated from NEC cases and controls as well.
rip t
Therefore, the potential role of a putative hemolysin analog from C. butyricum as a toxin that may participate in NEC development needs to be demonstrated.
an us c
To conclude, the manuscript by Cassir and colleagues adds new data for the involvement of Clostridium in NEC and emphasizes on a specific species, C. butyricum. It is noteworthy that
bacterial signatures (C. perfringens and Klebsiella for instance) [9].
Up to now, among clostridia species thought to be associated with NEC, none involved
M
known toxin production. By contrast, C. difficile, a toxicogenic species, is not implicated in NEC [19]. Hence, potential pathogenic characteristics shared by these clostridial species and
ed
may be by bacteria belonging to other genera deserve research. Such characteristics could be specific of the bacterial strains responsible for the disease, since all these species belong
ce
pt
to commensal microbiota, or could interact with the host displaying specific susceptibility.
Ac
The authors have no reported conflicts related to the content of this manuscript.
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
in other reports NEC was associated with other clostridial species or different separate
7 References
rip t
1. Neu J, Walker WA. Necrotizing enterocolitis. N Engl J Med 2011; 364:255-64.
2. Gephart SM, McGrath JM, Effken JA, Halpern MD. Necrotizing enterocolitis risk: state of the science. Adv Neonatal Care 2012; 12:77-87.
in the pathogenesis of necrotizing enterocolitis. Pediatrics 2010; 125:777-85.
4. Smith B, Bode S, Petersen BL, et al. Community analysis of bacteria colonizing intestinal tissue of neonates with necrotizing enterocolitis. BMC Microbiol 2011; 11:73.
5. Alfa MJ, Robson D, Davi M, Bernard K, Van Caeseele P, Harding GK. An outbreak of necrotizing
Infect Dis 2002; 35:S101-S105.
M
enterocolitis associated with a novel clostridium species in a neonatal intensive care unit. Clin
ed
6. Mai V, Young CM, Ukhanova M, et al. Fecal microbiota in premature infants prior to necrotizing
pt
enterocolitis. PLoS One 2011; 6:e20647.
7. Morrow AL, Lagomarcino AJ, Schibler KR, et al. Early microbial and metabolomic signatures
ce
predict later onset of necrotizing enterocolitis in preterm infants. Microbiome 2013; 1:13.
8. De La Cochetière MF, Piloquet H, Des Robert C, Darmaun D, Galmiche JP, Rozé JC. Early
Ac
intestinal bacterial colonization and necrotizing enterocolitis in premature infants: the putative role of Clostridium. Pediatr Res 2004; 56:1-5.
9. Sim K, Shaw AG, Randell P, et al. Dysbiosis anticipating necrotizing enterocolitis in very premature infants. Clin Infect Dis 2014.
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
an us c
3. Morowitz MJ, Poroyko V, Caplan M, Alverdy J, Liu DC. Redefining the role of intestinal microbes
8 10. Zhou Y, Shan G, Sodergren E, Weinstock G, Walker WA, Gregory KE. Longitudinal analysis of the premature infant intestinal microbiome prior to necrotizing enterocolitis: a case-control study. PLoS One 2015; 10:e0118632.
rip t
11. Kalia VC, Mukherjee T, Bhushan A, Joshi J, Shankar P, Huma N. Analysis of the unexplored features of rrs (16S rDNA) of the genus Clostridium. BMC Genomics 2011; 12:18.
an us c
12. Ferraris L, Butel MJ, Campeotto F, Vodovar M, Roze JC, Aires J. Clostridia in premature
neonates' gut: incidence, antibiotic susceptibility, and perinatal determinants influencing
13. Turroni F, Marchesi JR, Foroni E, et al. Microbiomic analysis of the bifidobacterial population in the human distal gut. ISME J 2009; 3:745-51.
M
14. Howard FM, Bradley JM, Flynn DM, Noone P, Szawatkowski M. Outbreak of necrotizing enterocolitis caused by Clostridium butyricum. Lancet 1977;1099-102.
ed
15. Sturm R, Staneck JL, Stauffer LR, Neblett III WW. Neonatal necrotizing enterocolitis associated with penicillin-resistant, toxigenic Clostridium butyricum. Pediatrics 1980; 66:928-31.
ce
3.
pt
16. Gothefors L, Blenkharn I. Clostridium butyricum and necrotizing enterocolitis. Lancet 1978;52-
17. Laverdière M, Robert A, Chicoine R, Salet D, Rosenfeld R. Clostridia in necrotising enterocolitis.
Ac
Lancet 1978; 2:377.
18. Lawrence G, Bates J, Gaul A. Pathogenesis of neonatal necrotising enterocolitis. Lancet 1982;137-9.
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
colonization. PLoS One 2012; 7:e30594.
9 19. Waligora-Dupriet AJ, Dugay A, Auzeil N, Huerre M, Butel MJ. Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model. Pediatr Res 2005; 58:629-35.
rip t
20. Azcarate-Peril MA, Foster DM, Cadenas MB, et al. Acute necrotizing enterocolitis of preterm piglets is characterized by dysbiosis of ileal mucosa-associated bacteria. Gut Microbes 2011;
an us c
2:234-43.
21. Patel RM, Denning PW. Intestinal microbiota and its relationship with necrotizing enterocolitis.
22. Lin J, Nafday SM, Chauvin SN, et al. Variable effects of short chain fatty acids and lactic acid in inducing intestinal mucosal injury in newborn rats. J Pediatr Gastroenterol Nutr 2002; 35:545-
M
50.
23. Kien CL. Colonic fermentation of carbohydrate in the premature infant : possible relevance to
ed
necrotizing enterocolitis. J Pediatr 1990; 117:S52-S58.
24. Popoff MR, Jolivet-reynaud C, Carlier JP. Cytotoxic activity of Clostridium butyricum
Ac
ce
pt
supernatants induced by butyrate. Fems Microbiol Lett 1987; 43:95-100.
Downloaded from http://cid.oxfordjournals.org/ at Freie Universitaet Berlin on July 2, 2015
Pediatr Res 2015.
