Pyrosequencing Analysis Reveals Changes in Intestinal Microbiota of Healthy Adults Who Received a Daily Dose of Immunomodulatory Probiotic Strains

Nutrients 2015, 7, 3999-4015; doi:10.3390/nu7063999 OPEN ACCESS nutrients ISSN 2072-6643 www.mdpi.com/journal/nutrients Article Pyrosequencing Analysis Reveals Changes in Intestinal Microbiota of Healthy Adults Who Received a Daily Dose of Immunomodulatory Probiotic Strains Julio Plaza-Díaz 1,2, Jose Ángel Fernández-Caballero 3, Natalia Chueca 3, Federico García 3, Carolina Gómez-Llorente 1,2, María José Sáez-Lara 4, Luis Fontana 1,2 and Ángel Gil 1,2,* 1 2 3 4 Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, Granada 18071, Spain; E-Mails: (J.P.-D.); (C.G.-L.); (L.F.) Institute of Nutrition and Food Technology “José Mataix”, Biomedical Research Center, University of Granada, Armilla 18100, Spain Department of Microbiology, Complejo Hospitalario Universitario de Granada, Instituto de Investigación Biosanitaria (IBS), Granada 18012, Spain; E-Mails: (J.A.F.-C.); (N.C.); (F.G.) Department of Biochemistry and Molecular Biology I, School of Sciences, University of Granada, Granada 18071, Spain; E-Mail: * Author to whom correspondence should be addressed; E-Mail: ; Tel.: +34-958241000 (ext. 20307). Received: 26 January 2015 / Accepted: 8 May 2015 /Published: 26 May 2015 Abstract: The colon microbiota plays a crucial role in human gastrointestinal health. Current attempts to manipulate the colon microbiota composition are aimed at finding remedies for various diseases. We have recently described the immunomodulatory effects of three probiotic strains (Lactobacillus rhamnosus CNCM I-4036, Lactobacillus paracasei CNCM I-4034, and Bifidobacterium breve CNCM I-4035). The goal of the present study was to analyze the compositions of the fecal microbiota of healthy adults who received one of these strains using high-throughput 16S ribosomal RNA gene sequencing. Bacteroides was the most abundant genus in the groups that received L. rhamnosus CNCM I-4036 or L. paracasei CNCM I-4034. The Shannon indices were significantly increased in these two groups. Our results also revealed a significant increase in the Lactobacillus genus after the intervention with L. rhamnosus CNCM I-4036. The initially different colon microbiota became homogeneous in the subjects who received L. rhamnosus CNCM I-4036. While some orders that were initially present disappeared after the administration of L. rhamnosus CNCM I-4036, Nutrients 2015, 7 4000 other orders, such as Sphingobacteriales, Nitrospirales, Desulfobacterales, Thiotrichales, and Synergistetes, were detected after the intervention. In summary, our results show that the intake of these three bacterial strains induced changes in the colon microbiota. Keywords: gut; healthy adults; high-throughput nucleotide sequencing; microbiota; probiotics 1. Introduction The human colon microbiota is a complex ecosystem that is composed of approximately 1014 bacterial cells [1], which is ten times the number of cells in the human body, and it has been suggested to encode 100-fold more unique genes than the human genome [2]. Approximately 400–500 bacterial species comprise the colon microbiota [3]. The colon microbiota has a profound influence on human physiology and nutrition and plays a crucial role in human gastrointestinal (GI) health, affecting metabolism and the immune system and protecting against pathogens while modulating GI development [4,5]. Current attempts to manipulate the GI tract microbiota are focused on finding remedies for several health disorders, including infections and inflammatory, allergic, and immunologic conditions. Probiotics are consumed in different forms, such as yogurt, cheese and fermented foods, as a regular part of the human diet and as treatments for different GI tract dysfunctions [6]. However, the actual ability of probiotics to affect gut microorganisms is still under debate, because although it has been confirmed in several studies, [7] numerous confounding elements exist, such as diverse consumer’s susceptibilities to probiotic intake and marked differences in probiotic products (e.g., dissimilarities in microbial strains, concentrations of viable cells, and product formulations) [8]. The development of high-throughput 16S ribosomal RNA gene sequencing techniques has accelerated the knowledge of gut microbiome diversity [9]. Pyrosequencing allows for the determination of the entire phylogenetic spectrum, taxonomic characterization, and the flexibility to analyze populations at different taxonomic levels. Thus, 16S rRNA gene-based barcoded pyrosequencing has been extensively used for the characterization of gut microbial communities in healthy and diseased individuals [10–12] and has also been used to study the effects of dietary intervention on the colon microbiota [10,13,14]. Recently, we described a multicenter, randomized, double-blind, placebo-controlled trial performed to assess the effects of three novel probiotic strains (Lactobacillus rhamnosus CNCM I-4036, Lactobacillus paracasei CNCM I-4034, and Bifidobacterium breve CNCM I-4035) on healthy volunteers, including evaluations of tolerance, safety, persistence in the gut, and immunomodulatory effects [15]. We found intestinal colonization in the volunteers who received L. rhamnosus CNCM I-4036 and that the administration of B. breve CNCM I-4035 results in a significant increase in the secretory IgA level. Further, we demonstrated that IL-4 and IL-10 levels increase whereas IL-12 decreases in the sera of volunteers treated with any of the three strains. The consumption of these three bacterial strains is safe and leads to varying degrees of immunomodulatory effects [15]. The aim of the present study was to analyze the composition of the human fecal microbiota of healthy adults who received daily doses of either a placebo or one of the three aforementioned probiotic strains using high-throughput 16S ribosomal RNA gene sequencing. Additionally, we investigated the impact Nutrients 2015, 7 4001 of probiotic administration on intestinal colonization, which might contribute to the current understanding of the immunomodulatory effects of these three strains [15]. 2. Materials and Methods 2.1. Ethical Statement The probiotic strains Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 were obtained from the feces of breast-fed newborns, as previously described (SETOPROB study) [15–17]. These strains were assayed for enzymatic activity and carbohydrate utilization, and they were deposited in the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institute Pasteur [18]. This study followed the guidelines of the Declaration of Helsinki, and all procedures involving human subjects were approved by the ethics committees of the University of Granada, Murcia and Valencia. Written informed consent was obtained from the subjects after a careful explanation of the nature of the study. Additionally, this trial was registered at www.clinicaltria (...truncated)