Table 2.

Microbiological (compositional and functional) and inflammatory biomarkers affected by probiotics.

Reference	Microbiota (Faeces)/Immunological Markers *	Method
Aloisio et al., 2018 [45]	Whole cohort: ↑ probiotic B. breve counts. Bottle fed newborns: ↓ potentially pathogenic B. fragilis group members. ↑ Bifidobacterium spp. and ↓ Clostridium spp over time with no treatment effect, no differences in E. coli abundance over time.	qPCR
Baldassare et al., 2018 [46]	No differences in total bacteria counts and Bifodobacterium spp. over time and treatment. A statistical insignificant tendency toward elevated Lactobacillus spp. with probiotic supplementation. ↑ faecal propylene glicol in probiotic group and ↑ alanine, 2-hydroxyisovalerate and 2-oxoisocaproate in placebo group.	qPCR/H-NMR
Fatheree et al., 2017 [52]	No differences in faecal alpha-diversity (Shannon, Chao1, or Simpson diversity indices) over time and by treatment ↓ of dominant faecal gram (-) bacteria, i.e., Klebsiella, Proteus, and Veillonella ↓ faecal calprotectin over time but not by the Tx blood: ↓ IL-2; ↓ population of Tregs: CD4+Foxp3+CD25+ within CD4+Foxp3+, and CD4+Foxp3+HELIOS+ within CD4+Foxp3+ (thymus-derived) with colic resolution in probiotic treated group (no statistical analysis was done)	NGS/flow cytometry/ELISA
Mentula et al., 2009 [48]	↑ of total counts of anaerobic bacteria, bifidobacteria, L. rhamnosus GG, enterococci. Faecal fermentation parameters (SCFA, CFA) were measured but no statistical analysis was performed.	culture-dependent technique/GC
*** Nation et al., 2017 [56]	Crying time reduction regardless L. reuteri colonisation No differences in E. coli colonization rates or densities and microbial diversity regarding L. reuteri colonization status. E. coli density negatively correlated with microbial diversity L. reuteri concentration positively correlated with crying time No difference in faecal calprotectin levels regarding probiotic colonization status	qPCR/T-RFLP/ELISA
Nocerino et al., 2020 [54]	No difference in microbiota composition and alpha-diversity index by Tx ↑ Bifidobacterium spp. only in the responder infants treated with BB-12 Bifidobacterium abundance was correlated with the reduction of crying time ↑ Proteobacteria in the placebo group ↑ butyrate levels in responders Blood: ↑ HBD-2, LL-37, sIgA levels and ↓n faecal calprotectin level in responders	high-throughput sequencing of 16S rRNA, ELISA, indirect enzyme immunoassays
Savino et al., 2010 [57]	↑ in Lactobacillus spp. (including probiotic L. reuteri) by Tx ↓ in faecal E. coli and ammonia	culture-dependent techniques, enzymatic colorimetric test
Savino et al., 2018 [19]	↑ of Lactobacillus spp. by T ↓ feacal calprotectin Blood: ↑ of FOXP3 concentration thus decreased RoRg/FOXP3 mRNA ratio	qPCR, ELISA
Savino et al., 2018a [50]	- Blood: ↑ mRNA expression of TREGs, FOXP3	real time PCR/Qpcr
Savino et al., 2019 [33]	-↑ expression of CC-chemokine receptor 7	qPCR
Sung et al., 2014 [58]	No differences between in faecal microbial diversity, and E. coli load by Tx ↓ feacal calprotectin in responders from probiotic and placebo group	16SrDNA amplification (T-RFLP) ELISA, qPCR

IL-2 = interleukin 2; CD4+Foxp3+CD25+ =; CD4+Foxp3+HELIOS+ within CD4+Foxp3+ =; Helios-positive (thymus-derived) Tregs =; SCFA = short chain fatty acids; CFA = cellular fatty acids; HBD-2 = human β-defensin 2; LL-37 = cathelecidin; sIgA = secretory IgA; FOXP3 = forkhead box P3; RORγ = retinoid-related orphan receptor-γ; mRNA = messenger RNA; Th17 = T helper cell 17; Treg = regulatory T cell; qPCR = quantitative polymerase chain reaction; Tx – treatment; T-RFLP = terminal restriction fragment length polymorphism; NGS = next generation sequencing. rDNA = ribosomal DNA; ELISA = enzyme-linked immunosorbent assay; *—when not specified faecal biomarkers are listed; ***—partly same cohort as Sung et al., 2014