BACKGROUND: Preterm infants are at risk of developing intestinal dysbiosis with an increased proportion of Gammaproteobacteria. In this study, we sought the clinical determinants of the relative abundance of feces-associated Gammaproteobacteria in very low birth weight (VLBW) infants. Fecal microbiome was characterized at ??2 weeks and during the 3rd and 4th weeks after birth, by 16S rRNA amplicon sequencing. Maternal and infant clinical characteristics were extracted from electronic medical records. Data were analyzed by linear mixed modeling and linear regression. RESULTS: Clinical data and fecal microbiome profiles of 45 VLBW infants (gestational age 27.9?±?2.2 weeks; birth weight 1126 ± 208 g) were studied. Three stool samples were analyzed for each infant at mean postnatal ages of 9.9?±?3, 20.7?±?4.1, and 29.4?±?4.9 days. The average relative abundance of Gammaproteobacteria was 42.5% (0-90%) at ??2 weeks, 69.7% (29.9-86.9%) in the 3rd, and 75.5% (54.5-86%) in the 4th week (p?0.001). Hierarchical and K-means clustering identified two distinct subgroups: cluster 1 started with comparatively low abundance that increased with time, whereas cluster 2 began with a greater abundance at ??2 weeks (p?0.001) that decreased over time. Both groups resembled each other by the 3rd week. Single variants of Klebsiella and Staphylococcus described variance in community structure between clusters and were shared between all infants, suggesting a common, hospital-derived source. Fecal Gammaproteobacteria was positively associated with vaginal delivery and antenatal steroids. CONCLUSIONS: We detected a dichotomy in gut microbiome assembly in preterm infants: some preterm infants started with low relative gammaproteobacterial abundance in stool that increased as a function of postnatal age, whereas others began with and maintained high abundance. Vaginal birth and antenatal steroids were identified as predictors of Gammaproteobacteria abundance in the early (??2 weeks) and later (3rd and 4th weeks) stool samples, respectively. These findings are important in understanding the development of the gut microbiome in premature infants.