Effect of administration of bifidobacteria on intestinal microbiota in low-birth-weight infants and transition of administered bifidobacteria: a comparison between one-species and three-species administration.

The effects of administration of bifidobacteria on the intestinal microbiota in low-birth-weight infants, and the transition of each strain of administered bifidobacteria were investigated. A single strain of Bifidobacterium breve M-16V (5 × 10(8); one-species group) or a mixture of three species composed of B. breve M-16V, Bifidobacterium longum subsp. infantis M-63 and B. longum subsp. longum BB536 (5 × 10(8) of each strain; three-species group) were administered daily for 6 weeks. Bifidobacterial administration significantly increased the detection rates and cell numbers of bifidobacteria in the feces (weeks 1-6). The proportion of bifidobacteria was significantly higher in the one-species group at weeks 1-4, and in the three-species group at weeks 1-6 compared with the control group. Furthermore, the proportion of bifidobacteria in the three-species group was significantly higher than that in the one-species group at weeks 1 and 6. The proportion of infants with bifidobacteria-predominant microbiota was significantly higher in the three-species group than in the control group during the test period. The detection rates of Clostridium were lower in the bifidobacteria-administered groups. The proportions of Enterobacteriaceae were significantly lower in the three-species group compared to the other groups (weeks 4 and 6). Among the three strains administered, B. breve M-16V and Bifidobacterium infantis M-63 were detected in 85% or more of the infants during the administration period, while B. longum BB536 was detected in 40% or less. Compared with administration of one species, administration of three species of bifidobacteria resulted in earlier formation of a bifidobacteria-predominant fecal microbiota and maintenance of this microbiota.

Detection and quantification of four species of the genus Clostridium in infant feces.

To determine the composition of Clostridium in the feces of infants approximately 30 days old, we have developed a detection and quantification method of Clostridium paraputrificum, Clostridium perfringens, Clostridium tertium, and Clostridium difficile by species-specific primers. C. perfringens and C. difficile were detected in four fecal samples from 22 infants (18.2%), whereas C. paraputrificum was detected in three samples (16.7%). C. tertium was detected in two samples (9.1%). Moreover, the occurrences of the four species in bottle-and mix-fed infants were relatively higher than in breast-fed infants (P< 0.05). Subsequently, positive samples detected by nested PCR (polymerase chain reaction) were subjected to realtime PCR. The results showed that the numbers of C. paraputrificum, C. perfringens, C. tertium, and C. difficile ranged from about 1x10(5) to 3x10(7) cells/g wet feces.

Culture-independent analysis of fecal microbiota in infants, with special reference to Bifidobacterium species.

Fecal microbiota of 31 breast-fed, 26 mix-fed, and 11 bottle-fed infants were analyzed by using terminal restriction fragment length polymorphism (T-RFLP), and culture method. We first determined the total and cultivated bacterial counts in infant fecal microbiota. Only approximately 30% of bacteria present in fecal microbiota were cultivable while the remainder was yet-to-be cultured bacteria. Sixty-eight fecal samples were divided into two clusters (I and II) by T-RFLP analysis, and then subdivided into five subclusters (Ia, Ib, IIa, IIb and IIc). There was no clear relationship between clusters and feeding method. A proportion of bifidobacteria was detected in the fecal material by PCR method using species-specific primers. The predominant Bifidobacterium spp. was Bifidobacterium longum longum type (43 samples (63.2%)), followed by B. longum infantis type (23 samples (33.8%)) and B. breve (16 samples (23.5%)). The distribution of Bifidobacterium spp. was similar in the three feeding groups. In contrast, the high incidence of B. breve in cluster I, especially subcluster Ia and B. longum longum type in cluster II, especially subcluster IIa and IIc were characterized by T-RFLP method. Our results showed that the colonization of Bifidobacterium spp. in infant feces correlated with the T-RFLP clusters.