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. 2015 Sep 15:6:952.
doi: 10.3389/fmicb.2015.00952. eCollection 2015.

The vaginal isolate Lactobacillus paracasei LPC-S01 (DSM 26760) is suitable for oral administration

Silvia Balzaretti  1 , Valentina Taverniti  1 , Greta Rondini  1 , Giorgio Marcolegio  1 , Mario Minuzzo  2 , Maria C Remagni  1 , Walter Fiore  3 , Stefania Arioli  1 , Simone Guglielmetti  1
Affiliations

The vaginal isolate Lactobacillus paracasei LPC-S01 (DSM 26760) is suitable for oral administration

Silvia Balzaretti et al. Front Microbiol. .

Abstract

Bacterial vaginosis is one of the most common urogenital diseases affecting women in reproductive age. The administration of probiotics as vaginal suppository has been proposed as a strategy to cure this condition and reduce its recurrence. Nonetheless, also oral consumption of probiotics, which is a more practical route of administration, proved to be an efficient strategy. In this perspective, we studied Lactobacillus paracasei LPC-S01 (DSM 26760), a human vaginal isolate included in commercial probiotic preparations for topical use, in order to assess if this bacterium can also perform as gastrointestinal probiotic. Comparative genomics revealed the presence of several accessory genes suggesting that LPC-S01 is a niche-generalist member of its species. According to a procedure conventionally used to predict the probiotic potential, we demonstrated that the probiotic properties of strain LPC-S01, with respect to those of the well-known probiotic references L. paracasei Shirota and DG, are equal for the bile tolerance and the reduction of NF-κB activation in Caco-2 cells, or superior for the tolerance to gastric juice and the adhesion to Caco-2 epithelial cells. We then demonstrated that LPC-S01 is susceptible to antibiotics indicated by EFSA and does not produce biogenic amines. Finally, a double-blind cross-over pilot intervention trial on healthy human volunteers showed that, after a 7-days oral consumption of capsules containing about 24 billion live cells, the fecal cell concentrations of strains LPC-S01 and DG (evaluated by qPCR) were not dissimilar. Specifically, both probiotics' cell concentrations were above the detection limit for an average of 5 days from the end of the treatment, corresponding to a mean number of evacuations of 7 ± 2. Taken together, these data demonstrate that the vaginal isolate L. paracasei LPC-S01 possesses safety and functional properties that may support its use as probiotic to be administered per os for potential intestinal as well as vaginal applications.

Keywords: Caco-2 adhesion; NF-κB; gastrointestinal persistence; in vivo trial; lactobacilli; probiotic.

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Figures

Figure 1
Figure 1
Comparative genomic analysis of Lactobacillus paracasei LPC-S01 (reference genome) with the genome sequences of other six L. paracasei strains. Gray rectangles indicate mobile genetic elements. pφ, prophage-related regions. mge, mobile genetic element; mge 1, putative integrated plasmid; mge 2, putative mobile genetic element containing a restriction-modification system. EPS, putative exopolysaccharide coding operon. Letters flanking red rectangles refers to panels of Figure 2.
Figure 2
Figure 2
In silico predicted functional organization of the chromosomal regions of Lactobacillus paracasei LPS-S01 that discriminates LPC-S01 from the others strains included in the comparative genomic analysis depicted in Figure 1. Letters in the panels of this figure refer to the letters flanking red rectangles in Figure 1. (A) Putative phospho-β-galactosidase operon. In black are indicated transposase-associated putative genes. In white are indicated putative genes that are also present in the genome of the other L. paracasei strains considered for comparative genomic analysis. (B) Putative taurine ABC-type transport and metabolization operon. (C) Multi-transport region for the uptake of sugars and other small molecules. The membrane transport mechanism is indicated above the picture, whereas the transported molecule is indicated below. ABC, ATP binding cassette transport system, PTS, phosphotransferase transport system. Gal-NAc, N-acetyl galactosamine; Glu-NAc, N-acetyl glucosamine; Co+, cobalt ions. (D) Putative ABC-type Fe3+ transport system. Gene “a,” putative acetyltransferase (COG0456) coding gene; gene “b,” putative gene coding for an uncharacterized protein (DegV family, COG1307). (E) Putative nucleotide transport and metabolism operon. MFS, major facilitator superfamily. In white are indicated putative genes that are also present in the genome of all the other L. paracasei strains considered.
Figure 3
Figure 3
BLASTN search results for the region EPS2 from Lactobacillus paracasei LPC-S01, which putatively codes for the enzymes involved in the synthesis of an exopolysaccharide (EPS). The figure has been obtained adding a picture of the putative EPS operon over the graphic representation of the BLASTN output. In white are indicated genes outside the putative EPS operon.
Figure 4
Figure 4
Tolerance of Lactobacillus paracasei strains to simulated gastric juice at pH 2 (90 min incubation) and bile (180 min incubation). Dashed lines refer to controls (i.e., bacterial cells incubated in phosphate buffer, pH 6.5). Data are reported as the number of viable bacterial cells (CFU) plotted on a semi-logarithmic diagram. Vertical bars at each point refer to standard deviation calculated on three independent experiments conducted in duplicate. Asterisk between strain LPC-S01 and Shirota at the end of the experiment indicates statistically significant difference according to two-tailed unpaired Student's t test (*P < 0.05).
Figure 5
Figure 5
Experiments on human epithelial colorectal Caco-2 cell layer. (A) Adhesion of Lactobacillus paracasei strains to the Caco-2 epithelial cell layer as observed with Giemsa staining under a light microscope. Bars, 25 μm. One Caco-2 nucleus for each layer is indicated with the letter N. LPC-S01 adhesion was specific to Caco-2 cells: no adhesion was detected on the cover glass underlying Caco-2 cells (G; top part of the panel on the left). (B) Effect of L. paracasei strains on Caco-2 cells stably transfected with an NF-κB/luciferase reporter vector, in the presence of 5 ng/ml of IL-1β. Data in the histograms are the means (± standard deviations) from two independent experiments conducted in triplicate. RLU, relative luminescence units. Asterisks indicate statistically significant differences according to two-tailed unpaired Student's t-test: ***P < 0.001; **P < 0.01; *P < 0.05.
Figure 6
Figure 6
Persistence of Lactobacillus paracasei in the gastrointestinal tract of healthy adults. (A) Bacterial concentration of L. paracasei DG and LPC-S01 in the fecal samples collected during the study per single participant. (B) Average change in the fecal concentration of L. paracasei cells after the probiotic-intake period. Vertical bars denote 0.95 confidence intervals. Current effect according to repeated measures ANOVA performed to determine the statistical significance of the treatment × time interaction: F(5, 65) = 0.794; p = 0.558.
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