microbiome in nicu -...

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Máximo Vento MD PhD University & Polytechnic Hospital La Fe

Health Research InsCtute La Fe Division of Neonatology

Research presented in this conference has been funded by the RETIC RED SAMID (INSTITUTO CARLOS III; SPANISH MINISTRY OF ECONOMY)

RD08/0072/0026 & RD12/0026/0012 & FEDER FUNDS EU

F E D E R

Gut Microbiome in the NICU

POZNAN 2016 2

Gut microbiome

•  The human microbiome is a complex system of many microbial communiCes inhabiCng a diversity of environmental niches throughout the human body.

•  The human gastrointesCnal tract is inhabited by a complex and dynamic populaCon of around 500–1000 different microbial species.

•  It houses at least 160 such species from a consorCum of 1000 to 1150 prevalent bacterial species whose collecCve genome (“microbiome”) contains at least 100 Cmes as many genes as the human genome.

POZNAN 2016 3 Collado MC et al Gut Microbes 2012

Gut microbiome

•  The human gut microbiota is an important environmental factor for human health, having evoluConarily conserved roles in the metabolism, immunity, development, and behavior of the host.

•  From an ecological point of view, colonizaCon of the infant’s gut represents the de novo assembly of a microbial community and is influenced by dietary and medical factors.

POZNAN 2016 4 Bäckhead F et al Cell Host & Microbe 2015

Gut microbiome

•  TradiConal culture-‐based techniques cannot culture the majority of bacterial cells seen microscopically in feces.

•  InnovaCve NON CULTURE techniques high throughput molecular techniques.

Murgas Torrazza R et al Clin Perinatol 2013 POZNAN 2016 5

Gut microbiome

Non Culture Techniques for Taxonomic IdenIficaIon of IntesInal Flora •  16S rRNA sequencing V3/V4 AND V4/V5 regions. •  Shotgun (whole genome) approach: –  Illumina –  454 Titanium (longer sequence reads)


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16S rRNA 454 pyrosequencing

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Collado MC et al Pediatr Res 2015

Whole genome shotgun sequencing

POZNAN 2016 8 Collado MC et al Pediatr Res 2015

Gut microbiome

Studies of funcIonal expression of the IntesInal Microbiome •  Metagenomics (profiling intesCnal microbiota, DNA): comparison to known funcConal expression of similar sequences.

•  Metabolomics: metabolomic profile (metabolites) associated with microbiota.

•  Metaproteomics: catalyCc potenCal of microbiota (Proteins)

•  Metatranscriptomics: microbiota responses to environmental changes (RNA)


Gut microbiome

POZNAN 2016 10 Murgas Torrazza R et al Clin Perinatol 2013

Gut microbiome

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PotenIal biological funcIons & metabolites

Murgas Torrazza R et al Clin Perinatol 2013

Gut microbiome

POZNAN 2016 12 Collado MC Gut Microbes 2012

Changes in gut microbiome predisposes to specific condiIons

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Gut microbiome

POZNAN 2016 13 Collado MC et al Pediatr Res 2015

Factors predisposing to changes in microbiome equilibrium

Gut Microbiome

•  Low diversity bacterial ecosystem •  Retarded Bidifdobacterium colonizaCon •  High prevalence of Staphylococcus, Enterobacteriaceae,

Enterococcaceae, Lactobacillus, Weissella. •  Generalized inflammatory response associated with the

presence of: –  Enterobacter, Enterococcus, Lactobacillus, Phtorhabdus, Tannerella

spp –  Exacerbated immune response impacCng intesCnal barrier enhancing

bacterial translocaCon and risk of sepsis, NEC and other inflammatory condiCons

POZNAN 2016 14 Deitch EA Surgeon 2012

Prematurity and Gut Microbiome CharacterisIcs

Gut Microbiome

•  Sepsis remains one of the most common causes of neonatal morbidity and mortality in the preterm populaCon.

•  EOS occurs in 1.5-‐1.9% of VLBW infant and LOS in 20% •  Mortality approaches 18% •  Most common pathogens are Coagulase-‐negaCve

Staphylococci (CONS) followed by gram-‐negaCve bacteria are the most frequently idenCfied pathogens.

•  Dysbiosis and lower microbial diversity are associated with increased risk of sepsis and NEC.

POZNAN 2016 15 Madan JC et al ADC FNE 2012

Prematurity, Gut Microbiome and Sepsis Sepsis in preterm alters gene expression and microbiota profiles

AIM •  To analyze gut microbiota and mucosal gene expression using

non-‐invasively obtained samples to provide an integraCve perspecCve of host-‐microbe interacCons in neonatal sepsis.

DESIGN •  ProspecCve observaConal cohort case control study in the

NICU of the UP Hospital La Fe (Valencia). •  Eligible paCents were twin pairs ≤1500 g •  SepCc twin was considered case and non-‐sepCc twin control.

POZNAN 2016 16 Cernada M et al Sci Reports 2016

Flow diagram


Eligible twin

Clinical suspicion of sepsis/non sepIc control

2 blood cultures

RNA extracIon and hybridizaIon (28000 annotated genes) Three dimensional unsupervised PCA (PRINCIPAL COMPONENT ANALYSIS)

Unsupervised hierarchical clustering FuncIonal annotaIons, upstream regulators & signaling pathways

Microbial diversity and composiIon AssociaIon between microbes and neonatal intesInal gene expression

Feces sampling IsolaIon of EIC

Sepsis in preterm alters gene expression and microbiota profiles


CharacterisIcs of the populaIon

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ORGAN or SYSTEM affected SEPTIC SYMPTOMS CARDIOVASCULAR Hypotension (BP<5th cenIle)

Tachycardia (HR>180 bpm) Bradycardia (HR<100 bpm) Poor perfusion

TEMPERATURE INSTABILITY >38ºC or >36ºC RESPIRATORY Distress; Apnoea, Cyanosis NEUROLOGICAL Seizures; hypotonia; letargy GASTROINTESTINAL Abdominal distension; vomiIng;

poor feeding; feeding intolerance


Suspicion of Clinical Sepsis


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PCA of the gene expression of EIC SepCc: red; Non sepCc: green

Unsupervised hierarchical cluster analysis for significantly regulated genes (p<0.05) Upregulated: red; downregulated: blue

Cernada M et al Sci Reports 2016



Pathway Studio Analysis Protein-‐Protein interacIon network Genes related to oxidaIve stress



Pathway Studio Analysis Protein-‐Protein interacIon network Genes related to NFkB and NrF2



a)  Genes related to cell death & survival, inflammatory response b)  Upstream regulaIon: evidence of acIvaIon of IL1B pathways

a b



RelaIve abundance of bacterial distribuIon at class level in the Sepsis and Control groups.

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Conclusions •  Gene expression of EIC in sepCc preterm showed an

inducCon of inflammatory and oxidaCve stress pathways in the gut and pro-‐oxidant profile.

•  As a consequence dysbiosis in the gut microbiota with predominance of Enterobacteria and reducCon of Bacteroides and Bifidobacterium spp was reported.

•  This lead to a global reducCon of beneficial anaerobic bacteria.

•  Sepsis in preterm infants induced low-‐grade inflammaCon and oxidaCve stress in the gut mucosa, and also changes in the gut microbiota.

•  This study highlights the role of inflammaCon and oxidaCve stress in neonatal sepsis on gut microbial profiles.


Gut microbiome & NEC

•  PRENATAL FACTORS –  PCR-‐ based studies reveal microbial invasion of the amnioCc cavity in fetal life especially CulCvaCon-‐resistant anaerobes Fusobacteriaceae are prevalent.

– An associaCon between PCR posiCve microbes and preterm birth has been assessed.

– Microbial DNA present in meconium (swallowed AF) may trigger intesCnal inflammaCon in utero and exposure to TLR agonists may tolerize for further inflammatory sCmuli.

POZNAN 2016 27 Di Julio DB et al J Perinatol 2010


•  POSTNATAL FACTORS: C-‐secIon vs. vaginal – Vaginal à Lactobacillus – C-‐secCon à Staphylococcus; Acinetobacter –  Increased incidence of: •  Type 1 diabetes •  Allergic diseases (asthma, riniCs, atopic dermaCCs) •  Crohn’s disease •  MulCple sclerosis

– Not associated with NEC!

POZNAN 2016 28 Dominguez Bello MG et al Gastroenterology 2011

Gut microbiome and NEC

•  POSTNATAL FACTORS: HM vs. formula – Beneficial factors in HM reduce the incidence of NEC.

– HM fed have predominance of Firmicutes (Lactobacillus, Bacteroides, AcCnobacteria)

– FORMULA fed have predominance of Proteobacteria (E Coli, Clostridia, Staphylococcus)

– Human milk significantly reduces clinical NEC (50%) and surgical NEC (90%) (NNT=10)

POZNAN 2016 29 Le Huerou-‐Luron I et al Nutr Res Rev 2010


•  POSTNATAL FACTORS: AnIbioIcs – Reduces de diversity of intesCnal microbiota – Delays colonizaCon of beneficial bacteria – PotenCally predisposes to NEC and/or death

POZNAN 2016 30 Le Huerou-‐Luron I et al Nutr Res Rev 2010

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•  IntesCnal microbiota exists in a commensal or symbioCc relaConship with the host; however in preterm this relaCon needs to develop.

•  AlteraCons of the microbiota in preterm favors NEC. •  ComposiCon of microbiota depends on perinatal factors, type of delivery, feeding and anCbioCcs.

•  New molecular technologies are capable of idenCfying specific organisms not detectable by classical microbiological techniques.

•  This novel approach has enhanced our understanding of the microbial environment that may predispose to NEC.


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