Safety and immunomodulatory effects of three probiotic strains isolated from the feces of breast-fed infants in healthy adults: SETOPROB study

We previously described the isolation and characterization of three probiotic strains from the feces of exclusively breast-fed newborn infants: Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036. These strains were shown to adhere to intestinal mucus in vitro, to be sensitive to antibiotics and to resist biliary salts and low pH. In the present study, a multicenter, randomized, double-blind, placebo-controlled trial with 100 healthy volunteers in three Spanish cities was carried out to evaluate the tolerance, safety, gut colonization and immunomodulatory effects of these three probiotics. Volunteers underwent a 15-day washout period, after which they were randomly divided into 5 groups that received daily a placebo, a capsule containing one of the 3 strains or a capsule containing a mixture of two strains for 30 days. The intervention was followed by another 15-day washout period. Patients did not consume fermented milk for the entire duration of the study. Gastrointestinal symptoms, defecation frequency and stool consistency were not altered by probiotic intake. No relevant changes in blood and serum, as well as no adverse events occurred during or after treatment. Probiotic administration slightly modified bacterial populations in the volunteers’ feces. Intestinal persistence occurred in volunteers who received L. rhamnosus CNCM I-4036. Administration of B. breve CNCM I-4035 resulted in a significant increase in fecal secretory IgA content. IL-4 and IL-10 increased, whereas IL-12 decreased in the serum of volunteers treated with any of the three strains. These results demonstrate that the consumption of these three bacterial strains was safe and exerted varying degrees of immunomodulatory effects.Part of the research currently in progress in the authors' laboratory is funded by the company Hero Spain, S. A. through the grant #3582 managed by the Fundacion General Empresa-Universidad de Granada

Serum biochemical parameters of Zucker-lean^+/<i>fa</i> and Zucker-Lepr<i>^fa/fa</i> rats fed either a placebo or LAB strains.

<p>Values are the means ± SEM, n = 8 per group. ^#<i>P</i><0.05 (ZL baseline vs. ZO baseline), ^†<i>P</i><0.05 (ZL + placebo vs. ZO + placebo), *<i>P</i><0.05 (ZO baseline vs. ZO + placebo). ALT, alanine aminotransferase; AST, aspartate aminotransferase; NEFA, non-esterified fatty acids. HOMA-IR, homeostasis model assessment of insulin resistance. ZL, Zucker-lean^+/<i>fa</i> rats; ZO, Zucker-Lepr<i>^fa/fa</i> rats.</p

Liver triacylglycerol content of Zucker-lean^+/<i>fa</i> and Zucker-Lepr<i>^fa/fa</i> rats that were fed either a placebo or LAB strains for 30 days.

<p>Values are the means ± SEM, n = 8 per group. ^†<i>P</i><0.05 (ZL + placebo vs. ZO + placebo), and *<i>P</i><0.05 (ZO + placebo vs. ZO + LAB strains). ZL, Zucker-lean^+/<i>fa</i> rats; ZO, Zucker-Lepr<i>^fa/fa</i> rats.</p

Concentrations of serum TNF-α (A) and IL-6 (B) of Zucker-lean^+/<i>fa</i> and Zucker-Lepr<i>^fa/fa</i> rats that were fed either a placebo or LAB strains for 30 days.

<p>Values are the means ± SEM, n = 8 per group. ^†<i>P</i><0.05 (ZL + placebo vs. ZO + placebo), and *<i>P</i><0.05 (ZO + placebo vs. ZO + probiotic strains). ZL, Zucker-lean^+/<i>fa</i> rats; ZO, Zucker-Lepr<i>^fa/fa</i> rats.</p

Representative micrographs of 7-µm-thick liver sections stained with 0.3% Oil red O in 60% isopropanol of Zucker-lean^+/<i>fa</i> and Zucker-Lepr<i>^fa/fa</i> rats that were fed either a placebo or LAB strains for 30 days (A).

<p>Percentage of the micrograph area corresponding to the lipid staining of liver sections described in panel A was calculated (B). Values are the means ± SEM, n = 2 per group. ^†<i>P</i><0.05 (ZL + placebo vs. ZO + placebo), and *<i>P</i><0.05 (ZO + placebo vs. ZO + LAB strains). ZL, Zucker-lean^+/<i>fa</i> rats; ZO, Zucker-Lepr<i>^fa/fa</i> rats.</p

Serum leptin (A) and adiponectin (B) concentrations of Zucker-lean^+/<i>fa</i> and Zucker-Lepr<i>^fa/fa</i> rats that were fed either a placebo or LAB strains for 30 days.

<p>Values are the means ± SEM, n = 8 per group. ^#<i>P</i><0.05 (ZL baseline vs. ZO baseline), and ^†<i>P</i><0.05 (ZL + placebo vs. ZO + placebo). ZL, Zucker-lean^+/<i>fa</i> rats; ZO, Zucker-Lepr<i>^fa/fa</i> rats.</p

Haematoxylin-eosin stained, 5-µm-thick sections of ileal (top panels, A–D) and colonic (bottom panels, E–H) mucosa of Zucker-lean^+/<i>fa</i> and Zucker-Lepr<i>^fa/fa</i> rats that were fed either a placebo or LAB strains for 30 days.

<p>Two rats per group were used for this staining. Three pieces of tissue from each animal were fixed and embedded in the same paraffin block. Four to 8 sections per block were cut, stained and analyzed. Representative micrographs from various groups are shown. A and E: Zucker-lean^+/<i>fa</i> rats at baseline; B and F: Zucker-Lepr<i>^fa/fa</i> rats + placebo; C and G: Zucker-Lepr<i>^fa/fa</i> rats +<i>L. rhamnosus</i>; and D and H: Zucker-Lepr<i>^fa/fa</i> rats + LAB mixture.</p

Beyond CC398: Characterisation of Other Tetracycline and Methicillin-Resistant Staphylococcus aureus Genetic Lineages Circulating in Spanish Hospitals

Tetracycline resistance (Tet(R)) has been evidenced as a good phenotypic marker for detection of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) isolates of the clonal complex CC398. The aim of this study was to characterise a collection of 95 Tet(R)-MRSA isolates, not belonging to the lineage CC398, that were obtained in a previous multicentre study, to detect other MRSA clonal complexes that could be associated with this phenotypic Tet(R) marker. The Tet(R)-MRSA isolates were recovered from 20 Spanish hospitals during 2016 and they were characterised to determine their antimicrobial resistance and virulence phenotypes/genotypes as well as the presence of the immune evasion cluster (IEC). A high proportion of isolates belonging to the CC1 lineage (46%) were observed, as well as to the CC5, CC8 and CC45 lineages (11% each one). Thirty-two different spa-types were identified, being predominantly CC1-t127 (40%) and CC45-t1081 (11%). The IEC system (with the gene scn as marker) was present in 73% of isolates and 16% produced the Panton Valentine leucocidin (PVL). A high proportion of MRSA-CC1 isolates were scn-negative (38.6%) and 52.9% of them were blaZ-negative. A multidrug resistance (MDR) phenotype was identified in 86% of MRSA isolates. The knowledge of other Tet(R)-MRSA genetic lineages, in addition to CC398, is highly relevant, since most of them were MDR and some of them presented important virulence factors. Strains potentially associated with livestock (as the subpopulation CC1-t127-scn-negative) or with humans (as the CC45 lineage or the subpopulation CC1-scn-positive) have been found in this study. The use of tetracycline-resistance for detection, not only of CC398 but also of other LA-MRSA lineages should be tracked in the future

Effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 on Hepatic Steatosis in Zucker Rats

We have previously described the safety and immunomodulatory effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 in healthy volunteers. The scope of this work was to evaluate the effects of these probiotic strains on the hepatic steatosis of obese rats. We used the Zucker rat as a genetic model of obesity. Zucker-Leprfa/fa rats received one of three probiotic strains, a mixture of L. paracasei CNCM I-4034 and B. breve CNCM I-4035, or a placebo for 30 days. An additional group of Zucker-lean+/fa rats received a placebo for 30 days. No alterations in intestinal histology, in the epithelial, lamina propria, muscular layers of the ileal or colonic mucosa, or the submucosae, were observed in any of the experimental groups. Triacylglycerol content decreased in the liver of Zucker-Leprfa/fa rats that were fed L. rhamnosus, B. breve, or the mixture of B. breve and L. paracasei. Likewise, the area corresponding to neutral lipids was significantly smaller in the liver of all four groups of Zucker-Leprfa/fa rats that received probiotics than in rats fed the placebo. Zucker-Leprfa/fa rats exhibited significantly greater serum LPS levels than Zucker-lean+/fa rats upon administration of placebo for 30 days. In contrast, all four groups of obese Zucker-Leprfa/fa rats that received LAB strains exhibited serum LPS concentrations similar to those of Zucker-lean+/fa rats. Serum TNF-α levels decreased in the Zucker-Leprfa/fa rats that received B. breve, L. rhamnosus, or the mixture, whereas L. paracasei feeding decreased IL-6 levels in the serum of Zucker-Leprfa/fa rats. In conclusion, the probiotic strains reduced hepatic steatosis in part by lowering serum LPS, and had an anti-inflammatory effect in obese Zucker rats.Part of the research currently in progress in the authors' laboratory is funded by the company Hero Spain, S. A. through the grant #3545 managed by the Fundacion General Empresa-Universidad de Granada

CONSORT flow diagram of the subjects in the SETOPROB study (NCT01479543).

<p>CONSORT flow diagram of the subjects in the SETOPROB study (NCT01479543).</p