Gut bacteria dysbiosis and necrotising enterocolitis in very low birthweight infants: a prospective case-control study

Summary

Background

Gut bacteria might predispose to or protect from necrotising enterocolitis, a severe illness linked to prematurity. In this observational prospective study we aimed to assess whether one or more bacterial taxa in the gut differ between infants who subsequently develop necrotising enterocolitis (cases) and those who do not (controls).

Methods

We enrolled very low birthweight (1500 g and lower) infants in the primary cohort (St Louis Children's Hospital) between July 7, 2009, and Sept 16, 2013, and in the secondary cohorts (Kosair Children's Hospital and Children's Hospital at Oklahoma University) between Sept 12, 2011 and May 25, 2013. We prospectively collected and then froze stool samples for all infants. Cases were defined as infants whose clinical courses were consistent with necrotising enterocolitis and whose radiographs fulfilled criteria for Bell's stage 2 or 3 necrotising enterocolitis. Control infants (one to four per case; not fixed ratios) with similar gestational ages, birthweight, and birth dates were selected from the population after cases were identified. Using primers specific for bacterial 16S rRNA genes, we amplified and then pyrosequenced faecal DNA from stool samples. With use of Dirichlet multinomial analysis and mixed models to account for repeated measures, we identified host factors, including development of necrotising enterocolitis, associated with gut bacterial populations.

Findings

We studied 2492 stool samples from 122 infants in the primary cohort, of whom 28 developed necrotising enterocolitis; 94 infants were used as controls. The microbial community structure in case stools differed significantly from those in control stools. These differences emerged only after the first month of age. In mixed models, the time-by-necrotising-enterocolitis interaction was positively associated with Gammaproteobacteria (p=0·0010) and negatively associated with strictly anaerobic bacteria, especially Negativicutes (p=0·0019). We studied 1094 stool samples from 44 infants in the secondary cohorts. 18 infants developed necrotising enterocolitis (cases) and 26 were controls. After combining data from all cohorts (166 infants, 3586 stools, 46 cases of necrotising enterocolitis), there were increased proportions of Gammaproteobacteria (p=0·0011) and lower proportions of both Negativicutes (p=0·0013) and the combined Clostridia–Negativicutes class (p=0·0051) in infants who went on to develop necrotising enterocolitis compared with controls. These associations were strongest in both the primary cohort and the overall cohort for infants born at less than 27 weeks' gestation.

Interpretation

A relative abundance of Gammaproteobacteria (ie, Gram-negative facultative bacilli) and relative paucity of strict anaerobic bacteria (especially Negativicutes) precede necrotising enterocolitis in very low birthweight infants. These data offer candidate targets for interventions to prevent necrotising enterocolitis, at least among infants born at less than 27 weeks' gestation.

Funding

National Institutes of Health (NIH), Foundation for the NIH, the Children's Discovery Institute.

Introduction

Necrotising enterocolitis is a catastrophic necroinflammatory injury to the intestines in very low birthweight infants. Its incidence (5–10% of very low birthweight infants), mortality (about 25%), and treatment (bowel resection for severe cases) have changed little in more than three decades.¹ Necrotising enterocolitis does not occur in utero, and is positively associated with exposure to antibiotics2, 3, 4 and negatively associated with exposure to human milk⁵ and probiotics.⁶ These observations underlie the long-standing belief that necrotising enterocolitis and gut bacteria are causally associated.

Stool cultures cannot quantify individual fastidious bacteria and so culture-independent analyses have been used to study gut microbes in children at risk for necrotising enterocolitis. Improved technologies and databases have enabled direct sequencing and interpretation of bacterial DNA from stools, but researchers using these methods have reported either no correlation between bacteria and necrotising enterocolitis,7, 8, 9 incriminated disparate organisms such as Gram-negative bacilli,10, 11, 12, 13 Actinobacteria,¹³ Clostridia11, 12 and other Firmicutes,¹⁰ or suggested age-dependent differences between cases and controls.¹¹ Additionally, certain taxa (Propionibacteria,¹⁰ Bifidobacteria, and Bacteroidetes¹³) have been proposed as preventers of development of necrotising enterocolitis. However, these studies analysed sequences from a median of only three stools (IQR two to seven) of infants before they developed necrotising enterocolitis.

Research in context

Evidence before this study

We did PubMed and Medline searches before undertaking this study in 2009. We used the following keywords to identify papers of interest: “Gammaproteobacteria”, “microbiome”, “necrotising enterocolitis”, and “preterm birth”, with no date or language limitations. We identified only efforts to assess gut bacteria using gradient gel electrophoresis of amplified bacterial DNA and, occasionally, sequencing of selected bands excised from the gels. Our searches did not identify any studies that applied modern sequencing technology directly to stools to establish the role of bacterial populations in the development of necrotising enterocolitis.

During the assembly of our cohort, several groups reported data for gut microbial content before necrotising enterocolitis by sequencing bacterial DNA directly from stool. These studies produced conflicting data, probably because of the dynamic bacterial population content in this age group and the relatively few patients and samples in any single study. This problem is compounded by an inherently unstable gut bacterial population in this age group.

Added value of this study

We identified dysbiosis preceding necrotising enterocolitis through analysis of 10·7 gigabases of DNA from 22 945 218 reads of 3586 stool samples from 166 infants in three US hospitals. This dysbiosis consists of over-representation of Gammaproteobacteria in stools of cases before necrotising enterocolitis develops, and under-representation of anaerobic bacteria, in particular Negativicutes.

Implications of all the available evidence

Our data might form the basis for informed discussion about management strategies of gut microbials in very low birthweight infants to avoid this catastrophic complication of preterm birth.

There are many sampling challenges encountered by investigators attempting to define causative or protective bacterial communities in necrotising enterocolitis. Necrotising enterocolitis occurs unpredictably throughout the first 2 months of age,¹⁴ a period during which gut bacteria assemble into consensus communities in children who do not develop necrotising enterocolitis (rapidly rising then slowly declining proportions of Gammaproteobacteria and increasing proportions of anaerobic bacteria) at rates that vary according to duration of gestation at birth. Also, despite this non-random choreographed progression, sudden population shifts are common.¹⁵ Moreover, the variable frequency of stool production within and between infants of this age results in non-uniformly timed sample sets before the onset of necrotising enterocolitis. Hence, dynamic gut bacterial communities and the widely varying frequency with which stool can be obtained necessitate studying many samples from many very low birthweight infants to determine if dysbiosis precedes, and therefore might cause, necrotising enterocolitis. Finally, the resulting data must undergo statistical testing that accounts for both the broad span of gestational ages at birth among at-risk infants, the extended risk of necrotising enterocolitis over the first several months of age, diverse clinical data such as antibiotic use and feeds, and repeated measures from the same individuals. It is likely that these challenges contributed to differing conclusions in previous studies, which were drawn on relatively few pre-event samples.

In this three-site prospective case-control study, we describe an intensive culture-independent analysis of a large specimen set using a mixed model approach to control for confounding clinical variables from infants at risk for necrotising enterocolitis. We aimed to establish whether development of necrotising enterocolitis is associated with altered microbial content (dysbiosis).