Denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA amplicons to monitor changes in fecal bacterial populations of weaning pigs after introduction of Lactobacillus reuteri strain MM53

The diversity and stability of the fecal bacterial microbiota in weaning pi gs was studied after introduction of an exogenous Lactobacillus reuteri str ain, MM53, using a combination of cultivation and techniques based on genes encoding 16S rRNA (16S rDNA). Piglets (n = 9) were assigned to three treat ment groups (control, daily dosed, and 4th-day dosed), and fresh fecal samp les were collected daily. Dosed animals received 2.5 x 10(10) CFU of antibi otic-resistant L. reuteri MM53 daily or every 4th day. Mean Lactobacillus c ounts for the three groups ranged from 1 x 10(9) to 4 x 10(9) CFU/g of fece s. Enumeration of strain L. reuteri MM53 on MRS agar (Difco) plates contain ing streptomycin and rifampin showed that the introduced strain fluctuated between 8 x 10(3) and 5 x 10(6) CFU/g of feces in the two dosed groups. Den aturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragm ents, with primers specific for variable regions 1 and 3 (V1 and V3), was u sed to profile complexity of fecal bacterial populations. Analysis of DGGE banding profiles indicated that each individual maintained a unique fecal b acterial population that was stable over time, suggesting a strong host inf lueuce. In addition, individual DGGE patterns could be separated into disti nct time-dependent clusters. Primers designed specifically to restrict DGGE analysis to a select group of lactobacilli allowed examination of interspe cies relationships and abundance. Based on relative band migration distance and sequence determination, L. reuteri was distinguishable within the V1 r egion 16S rDNA gene patterns, Daily fluctuations in specific bands within t hese profiles were observed, which revealed an antagonistic relationship be tween L. reuteri MM53 (band V1-3) and another indigenous Lactobacillus asse mblage (band V1-6).