Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis

Abstract

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.

FIG. 1

Primers for the specific amplification of 16S rDNA sequences of species of the Lactobacillus, Pediococcus, Leuconostoc, and Weissella group and alignment of the primer binding regions within the 16S rDNA sequences of related and nonrelated intestinal bacteria.

FIG. 2

PCR-DGGE analysis of 16S rDNA fragments generated by PCR with the specific primers Lac1 and Lac2GC and DNA extracted from Lactobacillus species or mixed populations. Lanes: 1, L. sharpeae DSM 20505^T; 2, L. acidophilus DSM 20079^T; 3, L. salivarius subsp. salicinius DSM 20554^T; 4, L. paracasei subsp. paracasei DSM 5622^T; 5, L. reuteri DSM 20016^T; M1 and M2, mixture of the Lactobacillus species containing 5 × 10⁷ and 5 × 10⁸ cells of each species per ml, respectively.

FIG. 3

DGGE analysis of PCR-amplified 16S rDNA fragments obtained with primer pair Lac1 and Lac2GC and DNA isolated from human feces. The fragments were excised and sequenced. Based on sequence comparisons, the fragments were allotted to the following species: 1A to 1C, Lactobacillus sakei; 2A to 2C, L. curvatus; 3, L. acidophilus; 4, L. crispatus; 5, W. confusa; 6, P. pentosaceus; 7A and 7B, Leuconostoc mesenteroides; 8, L. fructivorans; 9A and 9B, L. casei group. Bands not resulting in sequences upon purification and sequencing are indicated by arrows.

FIG. 4

DGGE of PCR products obtained with primer pair Lac1 and Lac2 GC of fecal samples from subjects C and D taken each month over a period of 6 months. Sequence characterization of the excised fragments indicated the presence of 1A to 1D, L. sakei; 2A to 2D, L. curvatus; 3A, L. delbrueckii; and 4A, L. plantarum.

FIG. 5

Comparison of PCR-DGGE and bacteriological culture to monitor the species composition of the Lactobacillus population in human feces. DGGE analyses were performed with PCR-amplified 16S rDNA fragments which were obtained with the primer pair Lac1 and Lac2GC and DNA extracted from fecal samples (lane 1) or the corresponding Lactobacillus isolates (subject C, lanes 2 to 5; subject D, lanes 2 to 4) cultured on Rogosa medium. The DNA fragments were allotted by sequence analysis to the following species. Isolates from subject C were as follows: lane 2, L. plantarum; lane 3, L. acidophilus; lane 4, L. crispatus; lane 5, L. casei group. DGGE profile of subject C revealed the following: fragment 1, L. sakei; fragment 2, L. curvatus; fragment 3, L. acidophilus; fragment 4, L. crispatus; fragment 5, Leuconostoc mesenteroides. Isolates from subject D were identified as follows: lane 2, W. confusa; lane 3, Pediococcus acidilactici; lane 4, L. casei group. DGGE profile of subject D identified the following: fragment 1, L. curvatus; fragment 2, W. confusa; fragment 3, Lactobacillus fructivorans; fragment 4, L. casei group. Fragments that did not result in sequences upon purification and sequencing are indicated by arrows.

FIG. 6

DGGE analysis of 16S rDNA fragments obtained from the fecal samples of subjects 2 and 4 before, during, and after consumption of a probiotic product. Subject 2: R1, L. ruminis DSM 20403^T; R2, L. rhamnosus DSM 20021^T; 1 to 8, fecal samples taken during the control ( to 3), test ( and 5), and posttest period ( to 8). Subject 4: R2, L. rhamnosus DSM 20021^T; R3, L. acidophilus DSM 20079^T; 4 to 8, fecal samples taken during the test ( and 5) and posttest period ( and 8). Samples from the control period ( to 3) and sample 7 of the posttest period of subject 4 did not give PCR products. DNA fragments which did not match fragments of the reference strains were allotted upon sequencing to the following species: 1, L. sakei; 2, L. delbrueckii; 3, L. curvatus; and 4, Leuconostoc mesenteroides.