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Monocolonization of germ-free (GF) mice enables the study of specific bacterial species in vivo. Lactobacillus acidophilus NCFM(TM) (NCFM) is a probiotic strain; however, many of the mechanisms behind its health-promoting effect remain unknown. Here, we studied the effects of NCFM on the metabolome of jejunum, cecum, and colon of NCFM monocolonized (MC) and GF mice using liquid chromatography coupled to mass-spectrometry (LC-MS). The study adds to existing evidence that NCFM in vivo affects the bile acid signature of mice, in particular by deconjugation. Furthermore, we confirmed that carbohydrate metabolism is affected by NCFM in the mouse intestine as especially the digestion of oligosaccharides (penta- and tetrasaccharides) was increased in MC mice. Additionally, levels of α-tocopherol acetate (vitamin E acetate) were higher in the intestine of GF mice than in MC mice, suggesting that NCFM affects the vitamin E acetate metabolism. NCFM did not digest vitamin E acetate in vitro, suggesting that direct bacterial metabolism was not the cause of the altered metabolome in vivo. Taken together, our results suggest that NCFM affects intestinal carbohydrate metabolism, bile acid metabolism and vitamin E metabolism, although it remains to be investigated whether this effect is unique to NCFM.

Mono-colonization of germ-free (GF) mice enables the study of specific bacterial species in vivo. Lactobacillus acidophilus is a probiotic strain, however many of the mechanisms behind its health-promoting effect remain unsolved. Here, we studied the effects of Lactobacillus acidophilus NCFMTM (NCFM) on the intestinal metabolome (jejunum, caecum, and colon) in mice by comparing NCFM mono-colonized (MC) mice with GF mice using liquid chromatography coupled to mass-spectrometry (LC-MS). The study adds to existing evidence that NCFM in vivo affects the bile acid signature of mice by deconjugation and dehydroxylation of bile acids. Furthermore, we confirmed that carbohydrate metabolism is affected by NCFM in the mouse intestine. Especially, the digestion of larger carbohydrates (penta- and tetrasaccharides) was increased in MC mice. Interestingly, we also found vitamin E (α-tocopherol acetate) in higher levels in the intestine of GF mice compared to MC mice, suggesting that NCFM either metabolizes the compound or indirectly affects the absorption by changing the metabolome in the intestine. The use of NCFM to increase the uptake of vitamin E supplements in humans and animals is a highly relevant topic for further research.

Every single species of the gut microbiota produce low-molecular-weight compounds that are absorbed constantly from the intestinal lumen and carried to systemic circulation where they play a direct role in health and disease. However, very few studies address the host metabolome as a function of colonizing bacteria. In this study the effect of the Lactobacillus acidophilus NCFM strain was investigated by comparing the metabolome of mono-colonized and germ-free mice in several compartments. By liquid-chromatography coupled to mass spectrometry, we were able to show that the metabolome differed between the mono-colonized and germ-free mice, not only in ileum, caecum and colon, but also in plasma and liver. These observations suggest that L. acidophilus NCFM highly influence the metabolism in multiple compartments, underlying that the gut microbiota metabolism affects the host systemic metabolism.

This study was part of the large European project ISAFRUIT aiming to reveal the biological explanations for the epidemiologically well-established health effects of fruits. The objective was to identify effects of apple and apple product consumption on the composition of the cecal microbial community in rats, as well as on a number of cecal parameters, which could be influenced by a changed microbiota. Principal Component Analysis (PCA) of cecal microbiota profiles obtained by PCR-DGGE targeting bacterial 16S rRNA genes showed an effect of whole apples in a long-term feeding study (14 weeks), while no effects of apple juice, purée or pomace on microbial composition in cecum were observed. Administration of pectin derived from apples resulted in considerable changes of these DGGE profiles. A 2-fold increase in the activity of beta-glucuronidase was observed in animals fed with pectin (7% in the diet) for four weeks, as compared to control animals (P

A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMβ1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMβ1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMβ1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori . The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO , which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti .

Bacillus thuringiensis subspecies kurstaki HD73, toxic for lepidopteran larvae, contains two large self-transmissible plasmids of approximately 75 kb, pHT73 and pAW63. The conjugative plasmid pHT73 has been studied extensively and has been shown to harbour the toxin gene cry1Ac, the transposon Tn4430 and several insertion sequences. In this study it was demonstrated that the minor plasmid pAW63 is also self-transmissible and about 10-30 times more efficient in mobilizing plasmid pBC16. To facilitate direct selection for pAW63 transfer, the plasmid was tagged with the tetracycline resistance transposon Tn5401 and in intraspecies matings it was found that after 2 h, all recipients had acquired a copy of the plasmid. Mating experiments demonstrated that pAW63 could be transferred to Bacillus thuringiensis subsp. israelensis. Bacillus cereus, Bacillus licheniformis, Bacillus subtilis and Bacillus sphaericus, and that the conjugative functions were expressed in these hosts. Hybridization studies showed that the replicons of pAW63 and pHT73 were distinct from one another. Sequences homologous to transposon Tn4430 and several insertion sequences were, however, shown to reside on both plasmids.