Determination of intestinal flora of newborn and breast milk using molecular methods

It is well known that the fetus can be colonized in contact with the microorganisms after but also before delivery by many ways. Several studies were done that show many factors may affect intestinal flora including of feeding type, mode of delivery, hospitalization, prematurity, administration of antibiotics and the level of development of country.A total of 60-80% of intestinal bacteria consist of uncultured bacteria for which reason the investigators prefer molecular methods to analyze the intestinal flora. The most used methods by the investigators are FISH, 16S rRNA, DGGE, TGGE analysis and sequences after cloning that have advantages compared to cultural methods.The aim of this study was to analyze the fecal flora of newborn and breast milk flora of their mothers by molecular techniques and determine the dynamics of intestinal bacterial flora during the first week of life.Ten baby-mother pairs were recruited for the present study. Fecal samples were collected at 1, 3, 7. days and breast milk samples were collected at 7. days after delivery. Thirty fecal and 10 milk samples were analyzed by 16S rRNA amplification and cloning. After analysis of sequencing results the bacterial flora was determined.Although two distinct methods were used to extract DNA from breast milk. No 16S rRNA were amplified. Culture analysis of breastmilk showed very low quantity of colonies.The bacteria defined by molecular method were classified by delivery mode, feeding type, antibiotic use, place of nursery (NICU or mother), life style, and oxygen use and analysed.The results showed that the colonization started immediately after birth. The number and diversity of microorganisms increased day by day during the study. For all days that samples were taken, the most detectable genus was Streptococcus (%26), the most detectable species was Escherichia coli (%15,4).None of the samples was positive for Bifidobacteia. Pathogen bacteria like Salmonella, Shigella and Clostridium were positive in 10 % of the feces samples of asymptomatic babies, especially at third and seventh days after birth.The most common bacteria were Bacteroides among vaginally delivered babies and Streptococcus among delivered by cesarean section. None of the fecal samples from cesarean section babies was positive for Bacteroides.The breastfed babies had an heterogeneous intestinal flora at the seventh day of life which include Clostridium, E. coli, Veillonella and Lactobacillus.Our study showed that stay at newborn intensive care unit at first week of life causes decrease in intestinal flora as well as the diversity of intestinal flora.The colonisation of intestin at first week of life affected by antibiotic use especially at first and third day samples.Our study indicated differences between intestinal flora of the babies of mothers who lives in countryside and cities; babies from countryside and low income had anaerobic heterogen flora than babies from city and high income.We conclude that intestine of newborn is colonized intensively at the first week of the life. The diversity of bacterial flora is affected by alimentation type, stay at hospital, and use of antibiotics. Our study which defined establishment of intestinal flora during first week of life will be a base for further studies on intestinal flora.Key words: 16S rRNA, intestinal microflora, newborn, breast milk