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Magdalena Strus,1 Ewa Helwich,2 Ryszard Lauterbach,3 Beata Rzepecka-Węglarz,4 Katarzyna Nowicka,2 Maria Wilińska,5 Jerzy Szczapa,6 Małgorzata Rudnicka,7 Helena Sławska,8 Marek Szczepański,9 Aneta Waśko,10 Aleksandra Mikołajczyk-Cichońska,10 Anna Tomusiak-Plebanek,1 Piotr B Heczko1 1Department of Microbiology, Jagiellonian University Medical College, Kraków, Poland; 2Department of Neonatology, Institute of Mother and Child, Warszawa, Poland; 3Clinical Department of Neonatology, University Hospital, Kraków, Poland; 4Department of Neonatal Intensive Care, “UJASTEK” Medical Centre, Kraków, Poland; 5Clinical Department of Neonatology, Independent Public Clinical Hospital CMKP, Warszawa, Poland; 6Department of Neonatology, Gynecology and Obstetrics Clinical Hospital, Poznań, Poland; 7Department of Neonatology, Regional Specialist Hospital, Wrocław, Poland; 8Department of Neonatology, Specialist Hospital No. 2, Bytom, Poland; 9Clinic Department of Neonatology and Neonatal Intensive Care, University Clinical Hospital, Białystok, Poland; 10Medical Department, IBSS BIOMED S.A., Kraków, Poland Aim: Probiotic bacteria administered directly after birth to preterm neonates may improve gastrointestinal function and may reduce the incidence of late-onset sepsis, which is a frequent complication in this group. Purpose: The main objective of this study was to evaluate whether a new probiotic bacterial mixture of Lactobacillus rhamnosus KL53A and Bifidobacterium breve PB04 given to preterm, low-birth-weight neonates would influence composition of their gut microbiota and sepsis rates. Patients and methods: This study was a multicenter, randomized, double-blind, placebo-controlled trial conducted in clinical centers of neonatal care in Poland. A probiotic or placebo preparation was given twice daily to 181 preterm low-birth-weight neonates who were eligible for enteral feeding between July 2012 and July 2013. The probiotic was given to 90 neonates, while placebo was given to 91 neonates. The gut microbiota was monitored by microbiological analysis of stool samples. Sepsis episodes were detected on the basis of clinical and laboratory findings and confirmed by blood cultures. Results: Tested probiotic administration resulted in continuous increase of the Lactobacillus and Bifidobacterium counts in the gut microbiota. The applied tested strains successfully colonized the neonates gut since they were present in over 90% of stool samples, which was confirmed by molecular analysis. Regardless of the study group (probiotic or placebo), B. breve ­colonization correlated with lower staphylococcal sepsis incidence, which was irrespective of whether ­probiotics were given. No sepsis case caused by strains included in study probiotic was recorded. Conclusion: Appropriately selected and characterized probiotic bacteria may be safely given to preterm neonates to normalize their distorted gut microbiota and may contribute to lower staphylococcal sepsis rates. Keywords: probiotics, LBW neonates, staphylococcal sepsis, gut microbiota, Lactobacillus, Bifidobacterium