Data_Sheet_1_In-process real-time probiotic phenotypic strain identity tracking: The use of Fourier transform infrared spectroscopy.zip

Probiotic bacteria, capable of conferring benefits to the host, can present challenges in design, development, scale-up, manufacturing, commercialization, and life cycle management. Strain identification is one of the main quality parameters; nevertheless, this task can be challenging since established methodologies can lack resolution at the strain level for some microorganisms and\or are labor-intensive and time-consuming. Fourier transform infrared spectroscopy (FTIRS) has been largely used for the investigation of pathogenic species in the clinical field, whereas only recently has been proposed for the identification of probiotic strains. Within the probiotic industrial production, bacterial strains can be subjected to stressful conditions that may affect genomic and phenotypic characteristics; therefore, real-time monitoring of all the sequential growth steps is requested. Considering the fast, low-cost, and high-throughput features, FTIRS is an innovative and functional technology for typing probiotic strains from bench-top experiments to large-scale industrial production, allowing the monitoring of stability and identity of probiotic strains. In this study, the discriminatory power of FTIRS was assessed for four Lactiplantibacillus plantarum probiotic strains grown under different conditions, including temperatures (30 and 37°C) and medium (broth and agar), after consecutive sub-culturing steps. A comparison between the generated spectra with pulsed-field gel electrophoresis (PFGE) profiles was also performed. FTIRS was not only able to distinguish the strains of L. plantarum under different growth conditions but also to prove the phenotypic stability of L. plantarum type strain LP-CT after six growing steps. Regardless of the growth conditions, FTIRS spectra related to LP-CT constituted a unique hierarchical cluster, separated from the other L. plantarum strains. These results were confirmed by a PFGE analysis. In addition, based on FTIRS data, broth cultures demonstrated a higher reproducibility and discriminatory power with respect to agar ones. These results support the introduction of FTIRS in the probiotic industry, allowing for the step-by-step monitoring of massive microbial production while also guaranteeing the stability and purity of the probiotic strain. The proposed novel approach can constitute an impressive improvement in the probiotic manufacturing process.</p

Top association signals for IL-6, ESR, MCP-1, and hsCRP in the ImmunoChip and MetaboChip data-sets.

<p>The table summarizes top association signals for IL-6, ESR, MCP-1 and hsCRP phenotypes in the ImmunoChip and MetaboChip data-sets (Step 3). For each marker, frequency and effect estimates are given with respect to the minor allele. We also reported the r² with the SNP detected in the GWAS scan (Step 1). Novel signals are indicated in bold.</p>a<p>The effect size is measured in standard deviation units, being estimated as the β coefficient of the regression model when using the normalized trait (e.g. an effect size of 1.0 implies each additional copy of the allele being evaluated increases trait values by 1.0 standard deviations).</p>b<p>I =  ImmunoChip, M =  MetaboChip.</p>c<p>The table reports the pvalue on the primary analysis. On the conditional analysis, the pvalue for the independent SNPs were: rs12378220, 9.43×10⁻⁰⁸; rs3093077, 9.02×10⁻¹¹; rs2259816, 7.58×10⁻¹⁰.</p>d<p>Independent signals.</p

Manhattan plot and QQ plot of association findings.

<p>The figure summarizes the association results obtained on the ImmunoChip and MetaboChip markers (Step 3). The blue dotted line marks the Bonferroni threshold significance levels (1.7×10⁻⁷), and SNPs in loci exceeding this threshold are highlighted in green. The bottom panel represents the QQ plot, where the red line corresponds to all test statistics, and the blue line to results after excluding statistics at top markers (highlighted in green in the Manhattan Plot). The gray area corresponds to the 90% confidence region from a null distribution of P values (generated from 100 simulations).</p

Zoom views of the association results in the loci associated with MCP-1 and hsCRP.

<p>Each panel shows the association curve around the strongest SNP, which is highlighted with a purple dot. The SNPs are coloured according to their linkage disequilibrium (r²) with the top variant in the 1000 Genomes European data set, with symbols that reflect genomic annotation as indicated in the legend. Arrows highlight independent signals, if any, described in the manuscript; while light blue lines indicate the recombination rate, according to the right-hand Y axis. Genomic positions are as in build 37. Gene transcripts are annotated in the lower box. Plots were drawn using the standalone LocusZoom version <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002480#pgen.1002480-Pruim1" target="_blank">[65]</a>.</p

Top genome-wide association results for IL-6, ESR, MCP-1, and hsCRP.

<p>The table summarizes top genome-wide association signals for IL-6, ESR, MCP-1 and hsCRP phenotypes in the HapMap based GWAS (Step 1), as well as results in the replication independent cohort (Step 2) and in the combined data-sets. For each marker, frequency and effect estimates are given with respect to the minor allele. Imputation quality scores (RSQ) are reported for imputed SNPs. Novel signals are indicated in bold.</p>a<p>The effect size is measured in standard deviation units, being estimated as the β coefficient of the regression model when using the normalized trait (e.g. an effect size of 1.0 implies each additional copy of the allele being evaluated increases trait values by 1.0 standard deviations).</p>b<p>Independent signals.</p

Zoom views of the association results in the loci associated with IL-6 and ESR.

<p>Each panel shows the association curve around the strongest SNP, which is highlighted with a purple dot. The SNPs are coloured according to their linkage disequilibrium (r²) with the top variant in the 1000 Genomes European data set, with symbols that reflect genomic annotation as indicated in the legend. Arrows highlight independent signals, if any, described in the manuscript; while light blue lines indicate the recombination rate, according to the right-hand Y axis. Genomic positions are as in build 37. Gene transcripts are annotated in the lower box. Plots were drawn using the standalone LocusZoom version <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002480#pgen.1002480-Pruim1" target="_blank">[65]</a>.</p