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Colonization patterns, especially by anaerobic flora, may play an important role in neonatal gut function. Probiotics could affect disease risk either directly through colonization or indirectly by promoting changes in gut microbial ecology.To study the ability of Lactobacillus GG (LGG) to colonize the neonatal gut and modify its microbial ecology, a prospective, randomized study was performed in 71 preterm infants of less than 2000 g birth weight. Infants less than 1500 g (24 treated, 15 control) received 109 LGG orally twice daily for 21 days. Those infants weighing 1500 to 1999 g (23 treated, 9 control) were treated for 8 days. Stools were collected before treatment and on day 7 to 8 (and day 14 and 21, in the infants weighing less than 1500 g) for quantitative aerobic and anaerobic cultures.Colonization with LGG occurred in 5 of 24 (21%) infants who weighed less than 1500 g versus 11 of 23 (47%) in larger infants. Colonization was limited to infants who were not on antibiotics within 7 days of treatment with LGG. There was a paucity of bacterial species at baseline, although larger infants had more bacterial species (1.59 ± 0.13 (SEM) vs 1.11 ± 0.12;P < 0.03) and higher mean log colony forming units (CFU) (8.79 ± 0.43 vs 7.22 ± 0.63;P < 0.05) compared with infants weighing less than 1500 g LGG. Treatment in infants weighing less than 1500 g resulted in a significant increase in species number by day 7, with further increases by day 21. This increase was mainly the result of increased Gram (+) and anaerobic species. No difference in species number was noted in controls. Mean log CFU of Gram (−) bacteria did not change in treated infants weighing less than 1500 g. However, Gram (+) mean log CFU showed a significant increase on day 21 (6.1 ± 0.9) compared with day 0 (3.5 ± 0.9) (P < 0.05). No significant changes in species number or quantitative counts were noted after LGG treatment in the infants weighing 1500 to 1999 g LGG was well tolerated in all infants.The neonatal response to a probiotic preparation is dependent on gestational and post-natal age and prior antibiotic exposure. Although LGG is a relatively poor colonizer in infants, especially those infants weighing less than 1500 g at birth, it does appear to affect neonatal intestinal colonization patterns.