Neonatal antibiotic exposure impairs child growth during the first six years of life by perturbing intestinal microbial colonization
Authors: Atara Uzan-Yulzari
Oren Ziv
Aia Oz
Hadar Neuman
Himanshu Kumar
Samuli Rautava
Nicola Segata
Anna Belogolovski
Sarah Perschbacher
Regina Ensenauer
Edoardo Pasolli
Olli Turta
Erika Isolauri
Seppo Salminen
Hanna Lagström
Yoram Louzoun
Hila Ben-Amram
Helena Ollila
Omry Koren
Itai Sharon
Christina Kunz
Anne Kaljonen
Journal: Nature Communications (2021)
Discipline: Multidisciplinary
Keywords: antibiotics; neonatal period; child growth; birth cohort; weight gain
Affiliations: Azrieli Faculty of Medicine, Bar Ilan University, Safed, Israel., Department of Pediatrics, University of Turku and Turku University Hospital, Turku, Finland., Department of Mathematics, Bar-Ilan University, Ramat-Gan, Israel., Institute of Child Nutrition, Max Rubner-Institut, Federal Research Institute of Nutrition and Food, Karlsruhe, Germany., Institute for Medical Information Processing, Biometry, and Epidemiology (IBE), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany., Zefat Academic College, Safed, Israel., Department CIBIO, University of Trento, Trento, Italy., Migal - Galilee Research Institute, Qiryat Shemona, Israel., Ziv Medical center, Safed, Israel., Functional Foods Forum, University of Turku, Turku, Finland., Department of Biostatistics, Faculty of Medicine, University of Turku, Turku, Finland., Department of Public Health, University of Turku and Turku University Hospital, Turku, Finland., Tel-Hai Academic College, Qiryat Shemona, Israel., Department of General Pediatrics, Neonatology and Pediatric Cardiology, University Children's Hospital, Heinrich Heine University Düsseldorf, Düsseldorf, Germany., Department of Pediatrics, University of Turku and Turku University Hospital, Turku, Finland. ., University of Helsinki & Helsinki University Hospital, New Children's Hospital, Pediatric Research Center, Helsinki, Finland. ., Azrieli Faculty of Medicine, Bar Ilan University, Safed, Israel. .

Abstract:  Exposure to antibiotics in the first days of life is thought to affect various physiological aspects of neonatal development. Here, we investigate the long-term impact of antibiotic treatment in the neonatal period and early childhood on child growth in an unselected birth cohort of 12,422 children born at full term. We find significant attenuation of weight and height gain during the first 6 years of life after neonatal antibiotic exposure in boys, but not in girls, after adjusting for potential confounders. In contrast, antibiotic use after the neonatal period but during the first 6 years of life is associated with significantly higher body mass index throughout the study period in both boys and girls. Neonatal antibiotic exposure is associated with significant differences in the gut microbiome, particularly in decreased abundance and diversity of fecal Bifidobacteria until 2 years of age. Finally, we demonstrate that fecal microbiota transplant from antibiotic-exposed children to germ-free male, but not female, mice results in significant growth impairment. Thus, we conclude that neonatal antibiotic exposure is associated with a long-term gut microbiome perturbation and may result in reduced growth in boys during the first six years of life while antibiotic use later in childhood is associated with increased body mass index. 

Retracted: No

PubPeer comments:
 *This exchange was prepared as a Matters Arising article for Nature Communications, but was not accepted for publication. Other readers might have questions about or be interested in the issue of confounding by indication. Given the complexity involved in testing this hypothesis, this discussion may be useful to those working in this area.* **Confounding by indication: Untangling the effect of neonatal antibiotics on the microbiome from the indication for antibiotics in the infant** Response to: Uzan-Yulzari A et al. Neonatal antibiotic exposure impairs child growth during the first six years of life by perturbing intestinal microbial colonization Amee R. Manges, MPH, PhD; Alissa J. Wright, MSc, MD School of Population and Public Health, University of British Columbia, Vancouver, BC, CANADA; British Columbia Centre for Disease Control, Vancouver, BC CANADA; and Division of Infectious Diseases, Faculty of Medicine, University of British Columbia, Vancouver, BC CANADA The report published by Uzan-Yulzari et al is an important and well-designed study addressing whether neonatal antibiotic exposure has lasting effects on the intestinal microbiome, with consequences for child growth1. In the report, investigators examine whether exposure to antibiotics in the neonatal period lead to growth deficits during the first six years of life. They identify weight and height deficits in male infants following neonatal antibiotic exposure in a large infant cohort, and confirm these observations in a second cohort. The authors then experimentally test and confirm the hypothesis using fecal microbiota transfer to germ-free mice, showing that the neonatal antibiotic-exposed infant microbiota leads to deficits in weight in male germ-free mice, but not female mice. However, there is one major challenge in examining a question like this, especially in human observational (non-randomized) studies. The challenge is termed u2018confounding by indicationu2019. The authors are concerned about this issue and state, u201cWhile the detailed data from the SFBC enabled us to differentiate between neonates briefly exposed to experimental antibiotic treatment and those who were diagnosed with infection by the clinician, it is possible that our results have been confounded by the underlying causes leading to antibiotic exposure also in the subjects in whom infection was ruled out.u201d Confounding by indication refers to the inherent differences in subjects who receive a treatment versus those who do not, because those who are treated are being treated for one or more underlying reasons or clinical indications2. The infants receiving the antibiotics are receiving antibiotics for a reason (probably for many different clinical reasons) and these reasons may be responsible for the observed impairment in growth, including non-infectious indications. The potentially confounded relationship between neonatal antibiotic exposure and growth seen in male infants and not female infants may be because more male infants exhibited clinical indications for neonatal antibiotic treatment and therefore were more frequent recipients of antibiotics in the neonatal period (10.7% vs. 7.8%, males and females, respectively, Supplemental Table 1)3. There is evidence that male infants exhibit higher morbidity and mortality than female infants4. The investigators confirm the observation of poor growth in male infants in a second cohort; however, this independent validation may just recapitulate the same u2018confounding by indicationu2019 in a second population. The fecal transfer experiments in mice, which show a lower percent change in weight in mice receiving feces from antibiotic-exposed infants, should represent a non-biased assessment of the effect of antibiotic exposure on growth. However, the same underlying health issue(s) that led to the neonatal antibiotic exposure could be the cause of the changes to the infant microbiome that lead to poorer growth. The altered microbiome, transferred to germ-free mice, could then affect growth irrespective of antibiotic exposure. Confounding by indication is a big methodological challenge, which epidemiologists wrestle with frequently. One possible option available to the investigators would be to add one or more non-antibiotic comparator groups, in addition to the brief antibiotic exposure group, and test whether infants requiring other non-antibiotic interventions, such as blood pressure support, hydrocortisone or other medications, would also show impaired growth. This could help untangle the effect of neonatal antibiotics on the microbiome, from the indication for antibiotics in the infant. 1.tUzan-Yulzari, A. et al. Neonatal antibiotic exposure impairs child growth during the first six years of life by perturbing intestinal microbial colonization. Nat. Commun. 12, 443 (2021). 2.tKyriacou, D. N. Lewis, R. J. Confounding by Indication in Clinical Research. JAMA 316, 1818 (2016). 3.tBindl, L. et al. Gender-based differences in children with sepsis and ARDS: The ESPNIC ARDS Database Group. Intensive Care Med. 29, 1770u20131773 (2003). 4.tZhao, D., Zou, L., Lei, X. Zhang, Y. Gender Differences in Infant Mortality and Neonatal Morbidity in Mixed-Gender Twins. Sci. Rep. 7, 3u20138 (2017). *Response from Drs. Rautava and Koren* Dear Dr. Manges Thank you for your interest in our work. Your point concerning potential confounding by indication is well taken. As you mention, we attempted to dissect the impact of neonatal antibiotic exposure and infection by separately examining neonates who had been empirically treated with antibiotics and in whom infection was ruled out. Still, as you and we both point out, confounding by indication is possible. Most full-term infants who receive empirical antibiotic therapy but are later deemed not to be infected are likely to suffer from respiratory adaptation issues (transient tachypnea of the newborn or u2018wet lungu2019). The condition is known to be more common in boys but the impact it might have on the gut microbiota or growth is not known. It is also important to note that a stay in the neonatal intensive care unit (NICU) per se may drastically alter gut colonization as a result of exposure to the microbial environment in the NICU and potentially reduced skin-to-skin contact and breastfeeding. We were able to assess breastfeeding rates in the PEACHES cohort, but the other factors remain uninvestigated. The suggestion to examine neonates admitted in the NICU and not subjected to antibiotic therapy is excellent. However, the NICU guideline for commencement of antibiotics at the Turku University Hospital at the time of the study recommended empirical antibiotic therapy for all symptomatic neonates (altogether 9% of the entire cohort of full-term neonates received antibiotics) and, consequently, the number of infants admitted in the NICU and not exposed to antibiotics is low. We presume that most of these infants were moderately to late preterm and or treated for hypoglycemia and may not be comparable to the full-term antibiotic-exposed neonates. Future research will be needed to address this question. Our conclusion that the causal reason for the gut microbiota disturbances and the sex-specific growth failure in boys is most likely antibiotic exposure is to some extent corroborated by data from experimental studies. In a landmark report by Cox et al. (Cell 158, 705-721, doi:10.1016 j.cell.2014.05.052 (2014)), mice exposed to antibiotics in the perinatal period and until weaning exhibited increased weight gain and fat accumulation which was causally related to antibiotic-induced changes in the gut microbiota. The phenomenon was markedly more pronounced in male as compared to female mice. While the mouse phenotype was different than that observed in our study (which may be explained by differences in the type, dose and duration of the antibiotic exposure), the experimental design is free of confounding by indication and the results corroborate the sex-specificity of the antibiotic effect. The matter is currently being investigated further in our laboratory models as we try to tease out the direct effects of antibiotics without concomitant infection. We are conducting experiments in neonatal mice and hope to have answers in the coming months. Yours sincerely, Samuli Rautava and Omry Koren ,