Breast-milk samples were taken before and after the intervention. the FOS group (= 0.0007) indicating greater changes in milk microbiota compared to the control group. Linear regression models suggested that the maternal age influenced the response for FOS supplementation (= 0.02). Interestingly, the pattern of changes to genus abundance upon supplementation was not shared between mothers. We demonstrated that manipulating the human milk microbiota through prebiotics is possible, and the maternal age can affect this response. and spp. and spp. from human milk samples collected before and after 20 days of supplementation. The qPCR was performed using Taqman (ThermoFisher Scientific) or SYBR green (ThermoFisher Scientific) for spp. and spp., respectively. For spp., the amplifications were carried out in a 25 L mixture (final volume) containing 12.5 L of TaqMan? Universal PCR 2X Master Mix (ThermoFisher Scientific), 200 nM of each primer (F_Bifid 09c CGG GTG AGT AAT GCG TGA CC, R_Bifid 06 TGA TAG GAC GCG ACC CCA [29]), 250 nM of probe (P_Bifid 6FAM-CTC CTG GAA ACG GGT G [29]), and 5 L of the DNA template. Reactions were performed under the following conditions: 1 cycle at 95 oC for 10 minutes, followed by 40 cycles at 95 oC for 30 seconds, and at 60 C for 1 minute. For spp., the qPCR reactions were carried out in a 25 L mixture (final volume), containing 12.5 L of SYBR Green? PCR 2X Master Mix (ThermoFisher Scientific), 500 nM of each primer (Lac-F 5-AGCAGTAGGGAATCTTCCA-3; Lac-R 5-CACCGCTACACATGGAG-3 [30]), and 5 L of the DNA template. The PCR conditions were: 1 cycle at 95 C for 5 minutes, followed by 40 cycles at 95 C for 15 seconds, 58 C for 20 seconds, 72 C for 30 seconds and at 80 C for 30 seconds. All qPCR were performed using an ABI-PRISM 7500 sequencing detection system (Applied Biosystems, Bridge-Water, NJ, USA). For the construction of standard curves, 10-fold dilution series between 105 and 101 copies of genomic DNA from known quantities of genomic DNA extracted from a pure culture of Rabbit polyclonal to ALDH1L2 target species were applied for qPCRs. Negative besides blanks controls from the DNA extraction kit controls were included in the PCR runs. All amplification reactions were performed in triplicates. The coefficients for reaction efficiency, calculated as 10(?1/slope) ?1, ranged from 98% to 102%, and the correlation coefficients R2 obtained for the standard curve were between 0.98 and 0.99. The Ct (cycle threshold) from each sample was compared with the Ct from the standard curve in order to get the number of copies of the 16S rRNA gene in the samples. The minimum limit of detection of the qPCR technique was 1.4 log equivalent cells/mL of human milk. Below that, quantities were considered as not detected. 2.9. Statistical Analysis All statistical analyses were performed using the statistical computing language R. For differences in the human milk microbiota composition, the adonis function (PERMANOVA test) was performed using weighted and unweighted UniFrac distances to compare the groups (FOS and placebo) by day (before and after the intervention), using 999 permutations (vegan package). In addition, in order to Isavuconazole compare the abundance of individual taxa before and after the intervention trial, ANCOM [31] was applied for repeated measures.Alpha-diversity analyses were performed after applying rarefactions (10,000 sequences/sample) to standardize sequence counts (vegan package). The MannCWhitney test or Wilcoxon signed ranks test were used to compare the alpha-diversity of independent or dependent samples, respectively. The JensenCShannon distance (JSD) was used to calculate the distribution of the distance between before and after paired samples by each group (placebo or FOS group). The MannCWhitney test was used to compare the distributions. The MannCWhitney test or.Rows correspond to genera with maximum abundance higher than 0.05. trajectories covered by paired samples from the beginning to the end of the supplementation were higher for the FOS group (= 0.0007) indicating greater changes in milk microbiota compared to the control group. Linear regression models suggested that the maternal age influenced the response for FOS supplementation (= 0.02). Interestingly, the pattern of changes to genus abundance upon supplementation was Isavuconazole not shared between mothers. We demonstrated that manipulating the human milk Isavuconazole microbiota through prebiotics is possible, and the maternal age can affect this response. and spp. and spp. from human milk samples collected before and after 20 days of supplementation. The qPCR was performed using Taqman (ThermoFisher Scientific) or SYBR green (ThermoFisher Scientific) for spp. and spp., respectively. For spp., the amplifications were carried out in a 25 L mixture (final volume) containing 12.5 L of TaqMan? Universal PCR 2X Master Mix (ThermoFisher Scientific), 200 nM of each primer (F_Bifid 09c CGG GTG AGT AAT GCG TGA CC, R_Bifid 06 TGA TAG GAC GCG ACC CCA [29]), 250 nM of probe (P_Bifid 6FAM-CTC CTG GAA ACG GGT G [29]), and 5 L of the DNA template. Reactions were performed under the following conditions: 1 cycle at 95 oC for 10 minutes, followed by 40 cycles at 95 oC for 30 seconds, and at 60 C for 1 minute. For spp., the qPCR reactions were carried out in a 25 L mixture (final volume), containing 12.5 L of SYBR Green? PCR 2X Master Mix (ThermoFisher Scientific), 500 nM of each primer (Lac-F 5-AGCAGTAGGGAATCTTCCA-3; Lac-R 5-CACCGCTACACATGGAG-3 [30]), and 5 L of the DNA template. The PCR conditions were: 1 cycle at 95 C for 5 minutes, followed by 40 cycles at 95 C for 15 seconds, 58 C for 20 seconds, 72 C for 30 seconds and at 80 C for 30 seconds. All qPCR were performed using an ABI-PRISM 7500 sequencing detection system (Applied Biosystems, Bridge-Water, NJ, USA). For the construction of standard curves, 10-fold dilution series between 105 and 101 copies of genomic DNA from known quantities of genomic DNA extracted from a pure culture of target species were applied for qPCRs. Negative besides blanks controls from the DNA extraction kit controls were included in the PCR runs. All amplification reactions were performed in triplicates. The coefficients for reaction efficiency, calculated as 10(?1/slope) ?1, ranged from 98% to 102%, and the correlation coefficients R2 obtained for the standard curve were between 0.98 and 0.99. The Ct (cycle threshold) from each sample was compared with the Ct from the standard curve in order to get the number of copies of the 16S rRNA gene in the samples. The minimum limit of detection of the qPCR technique was 1.4 log equivalent cells/mL of human milk. Below that, quantities were considered as not detected. 2.9. Statistical Analysis All statistical analyses were performed using the statistical computing language R. For differences in the human milk microbiota composition, the adonis function (PERMANOVA test) was performed using weighted and unweighted UniFrac distances to compare the organizations (FOS and placebo) by day time (before and after the treatment), using 999 permutations (vegan package). In addition, in order to compare the large quantity of individual taxa before and after the treatment trial, ANCOM [31] was applied for repeated actions.Alpha-diversity analyses were performed after applying rarefactions (10,000 sequences/sample) to standardize sequence counts (vegan package). The MannCWhitney test or Wilcoxon authorized ranks test were used to compare the alpha-diversity of self-employed or dependent samples, respectively. The JensenCShannon range (JSD) was used to calculate the distribution of the distance between before and after combined samples by each group (placebo or FOS group). The MannCWhitney test was used to compare the distributions. The MannCWhitney test or t-test were used to compare spp. and spp. counts from qPCR between the interventional organizations. Wilcoxon signed ranks test for combined samples.
