Congruent Strain Specific Intestinal Persistence of <em>Lactobacillus plantarum</em> in an Intestine-Mimicking <em>In Vitro</em> System and in Human Volunteers

<div><h3>Background</h3><p>An important trait of probiotics is their capability to reach their intestinal target sites alive to optimally exert their beneficial effects. Assessment of this trait in intestine-mimicking <em>in vitro</em> model systems has revealed differential survival of individual strains of a species. However, data on the <em>in situ</em> persistence characteristics of individual or mixtures of strains of the same species in the gastrointestinal tract of healthy human volunteers have not been reported to date.</p> <h3>Methodology/Principal Findings</h3><p>The GI-tract survival of individual <em>L. plantarum</em> strains was determined using an intestine mimicking model system, revealing substantial inter-strain differences. The obtained data were correlated to genomic diversity of the strains using comparative genome hybridization (CGH) datasets, but this approach failed to discover specific genetic loci that explain the observed differences between the strains. Moreover, we developed a next-generation sequencing-based method that targets a variable intergenic region, and employed this method to assess the <em>in vivo</em> GI-tract persistence of different <em>L. plantarum</em> strains when administered in mixtures to healthy human volunteers. Remarkable consistency of the strain-specific persistence curves were observed between individual volunteers, which also correlated significantly with the GI-tract survival predicted on basis of the <em>in vitro</em> assay.</p> <h3>Conclusion</h3><p>The survival of individual <em>L. plantarum</em> strains in the GI-tract could not be correlated to the absence or presence of specific genes compared to the reference strain <em>L. plantarum</em> WCFS1. Nevertheless, <em>in vivo</em> persistence analysis in the human GI-tract confirmed the strain-specific persistence, which appeared to be remarkably similar in different healthy volunteers. Moreover, the relative strain-specific persistence <em>in vivo</em> appeared to be accurately and significantly predicted by their relative survival in the intestine-mimicking <em>in vitro</em> assay, supporting the use of this assay for screening of strain-specific GI persistence.</p> </div

Relative <i>L. plantarum</i> strain abundance of 4 independent replicates^a.

a<p>Four mixtures were designed that each contained 10 <i>L. plantarum</i> strains with 10 distinctive 339-IR-340 sequences. The variable amount of the tenth strain (reference WCFS1) was a dilution series and is subtracted from the other strains.</p>b<p>Nr indicates sample number.</p>c<p>St dev indicates standard deviation of the 4 replicates.</p

<i>L. plantarum</i> strain WCFS1, NCTH19-2, and NC8 relative abundance after human consumption as assessed by pyrosequencing.

<p>Relative strain abundances from subjects 1 to 10 are depicted in red, green, blue, purple, yellow, pink, brown, orange, white and grey diamonds, respectively. The graphs represent the number of strain specific sequences in the fecal amplicons, divided by the number of strain-specific sequences identified in the input mixture amplicon. The total number of sequences per sample was set at 1 for normalization purposes. Axis-scaling in all the graphs is the same as depicted for strain WCFS1.</p

Strain-specific<i>L. plantarum</i> relative abundance after human consumption as detected by pyrosequencing.

<p>Relative strain abundances of the bacterial preparations consumed by the volunteers are depicted in black diamonds and those determined in time-specified post-consumption fecal material from the subjects 1 to 5 in red, green, blue, purple, and yellow diamonds, respectively. The graphs represent the number of strain specific sequences in the amplicons generated from DNA derived from fecal samples, divided by the number of strain-specific sequences identified in the input mixture amplicon. The total number of sequences per sample was set at 1 for normalization purposes. Axis-scaling in all the graphs is the same as depicted for strain WCFS1.</p

Schematic representation of the 339-IR-340 region of <i>L. plantarum</i> strains.

<p>Panel A: Schematic representation of the variable region (grey area) between the <i>lp_0339</i> and <i>lp_0340</i> genes (white open arrows) of <i>L. plantarum</i> WCFS1 with the single nucleotide polymorphism positions (yellow areas) detected in the other strains. Primers used to generate amplicons for sequencing are displayed. Panel B: Sequence comparison of the 10 sequence variations in the 339-IR-340 intergenic region. Yellow circles indicate the nucleotide(s) that distinguish the 339-IR-340 sequence types.</p

Co-clustering of 34 <i>L. plantarum</i> strains based on the presence/absence gene profiles and the 339-IR-340 region distribution.

<p>The previously published comparative genome hybridization datasets <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044588#pone.0044588-Siezen1" target="_blank">[19]</a> were used to construct the genomic relatedness tree presented, which was complemented with the distribution of the 10 distinct 339-IR-340 sequence types, indicated by the colored bars.</p

Combinations of 10 strains consumed as mixtures by the 10 volunteers (subjects).

a<p>Strains indicated in bold are consumed by all volunteers.</p

Relative survival of <i>L. plantarum</i> strains subjected to an <i>in vitro</i> GI-tract assay.

<p><b> </b> Relative viability loss of <i>L. plantarum</i> strains harvested from logarithmic phase (panel A) or stationary phase (panel B) of growth after 60 min (dark grey) gastric juice incubation and subsequent 60 min (light grey) pancreatic juice incubation. The starting population size is set at 0 Log₁₀ CFU ml⁻¹, the data presented are averages of technical triplicates (+ standard deviation). Strains depicted in bold in panel B were present in the bacterial preparation consumed by subjects 1 to 5.</p

CRISPR-Cas loci identified in the strains of the VSL#3 product.

<p>The CRISPR-Cas loci were identified using CRISPRFinder [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192452#pone.0192452.ref056" target="_blank">56</a>] and their gene order and predicted annotations are depicted along with their juxtaposing array of spacers. Legend: *, split gene.</p

General predicted genomic features of bacterial strains from VSL#3 product.

<p>General predicted genomic features of bacterial strains from VSL#3 product.</p