Article pubs.acs.org/jpr
Antibiotic Treatment Preventing Necrotising Enterocolitis Alters Urinary and Plasma Metabolomes in Preterm Pigs Pingping Jiang,† Alessia Trimigno,⊥ Jan Stanstrup,‡ Bekzod Khakimov,§ Nanna Viereck,§ Søren Balling Engelsen,§ Per Torp Sangild,† and Lars Ove Dragsted*,‡ †
Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, DK-1870, Denmark Department of Nutrition, Sports and Exercise, and §Department of Food Science, University of Copenhagen, Frederiksberg, DK-1985, Denmark ⊥ Department of Agricultural and Food Sciences, University of Bologna, Campus di Scienze degli Alimenti, Cesena, Italy ‡
S Supporting Information *
ABSTRACT: Necrotising enterocolitis (NEC) is a serious gut inflammatory condition in premature neonates, onset and development of which depend on the gut microbiome. Attenuation of the gut microbiome by antibiotics can reduce NEC incidence and severity. However, how the antibioticssuppressed gut microbiome affects the whole-body metabolism in NEC-sensitive premature neonates is unknown. In formulafed preterm pigs, used as a model for preterm infants, plasma and urinary metabolomes were investigated by LC−MS and 1 H NMR, with and without antibiotic treatment immediately after birth. While it reduced the gut microbiome density and NEC lesions as previously reported, the antibiotic treatment employed in the current study affected the abundance of 44 metabolites in different metabolic pathways. In antibiotics-treated pigs, tryptophan metabolism favored the kynurenine pathway, relative to the serotonin pathway, as shown by specific metabolites. Metabolites associated with the gut microbiome, including 3-phenyllactic acid, 4-hydroxyphenylacetic acid, and phenylacetylglycine, all from phenylalanine, and three bile acids showed lower levels in the antibiotics-treated pigs where the gut microbiome was extensively attenuated. Findings in the current study warrant further investigation of metabolic and developmental consequences of antibiotic treatment in preterm neonates. KEYWORDS: antibiotics, metabolomics, necrotising enterocolitis, UPLC−MS, NMR, preterm pigs
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INTRODUCTION Necrotising enterocolitis (NEC) is a serious intestinal inflammatory disease with high morbidity and mortality and is mainly found in preterm neonates.1 The gut microbiome in preterm infants differs from that in term neonates, and its dysbiotic and low diversity may contribute to the onset and development of NEC.2,3 Approaches that modulate the gut microbiome, such as use of prebiotics and probiotics, may reduce NEC severity.4 Conversely, attenuation of the gut microbiome by broad-spectrum antibiotics can also prevent NEC in both humans5−7 and piglets.8 Antibiotics help to avoid overgrowth of pathogenic microbes that trigger the inflammatory processes, leading to intestinal necrosis and systemic sepsis. Our previous studies in preterm piglets demonstrated that use of antibiotics reduced the microbial density and diversity, and alleviated local inflammation, shown as decreased expression of pro-inflammatory genes,8 and systemic inflammation, documented by reduced level of acute phase proteins.9 The gut microbiome is central to acquisition of energy and nutrients in preterm neonates, which is required for their fast development of organs including the gut. A dysbiotic gut © 2017 American Chemical Society
microbiome in preterm neonates with NEC can limit availability of symbiotic metabolites, including short-chain fatty acids (SCFAs) and various neurotransmitters, and this impacts the development of the digestive, immune, and nervous systems in these neonates.10 How attenuation of the neonatal gut microbiome with antibiotics affects the whole-body metabolism of preterm neonates predisposed to NEC remains to be clarified. In this study, our clinically relevant NEC model using formula-fed preterm pigs was employed to examine the effect of antibiotic treatment on metabolism in premature neonates that were predisposed to NEC. It was hypothesized that treatment with broad-spectrum antibiotics to preterm pigs would affect their metabolism and this would be shown as changed plasma and urinary metabolomes. Both plasma and urine samples were adopted in the metabolomic analysis to increase the coverage of metabolites. Untargeted approaches based on liquid chromatography−mass spectrometry (LC−MS) and proton nuclear Received: May 1, 2017 Published: September 5, 2017 3547
DOI: 10.1021/acs.jproteome.7b00263 J. Proteome Res. 2017, 16, 3547−3557
Article
Journal of Proteome Research magnetic resonance spectroscopy (1H NMR) were both employed to assess metabolomic changes in an unbiased way. This study explores alterations in the whole-body metabolism after suppressing the gut microbiome by antibiotics in preterm neonates developing NEC. The results provide new knowledge about the metabolic effects of providing antibiotics to preterm neonates to prevent and alleviate NEC.
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followed a gradient from 0.1% formic acid to 0.1% formic acid in methanol/acetonitrile (30:70, v/v) within 7.0 min. The eluates were interfaced using electrospray ionization and analyzed by a Premier quadrupole time-of-flight MS (Q-TOF MS, Waters) in both negative and positive modes using separate runs for each mode. Ionization voltages were 2.8 kV (negative mode) and 3.2 kV (positive mode), and sampling cone voltage was 30 kV. Each sample was run in duplicates in both modes. Data were collected in centroid mode using leucine encephalin to calibrate the mass accuracy. The blank and the standard mixture were run thrice in between samples to check mass error (
