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Stuivenberg et al. Microbiome Res Rep 2025;4:11 https://dx.doi.org/10.20517/mrr.2024.22 Page 9 of 18

Figure 2. Plasma levels of GDUT by quartile of eGFR. Boxplots show plasma levels of GDUT in mmol/L by quartile of eGFR (calculated
2
using the CKD-EPI equations). eGFR quartiles: Q1, ,66 mL/min per 1.73 m ; Q2, 66-78; Q3, 79-90; Q4, $ 91. (A) TMAO; (B) P-cresyl
sulfate; (C) hippuric acid; (D) indoxyl sulfate; (E) P-cresyl glucuronide; (F) phenylacetyl glutamine; (G) phenyl sulfate. Plasma levels of
all the metabolites were significantly higher in lower quartiles of eGFR; P values were computed for differences in median values by the
Kruskal-Wallis test because the distribution of plasma levels was not normally distributed. Reproduced by permission of Elsevier
from [20] . GDUT: Gut-derived uremic toxin; eGFR: estimated glomerular filtration rate; CKD: chronic kidney disease; TMAO:
trimethylamine N-oxide.

rhamnosus may extend beyond the gut. While present in both stable and unstable (or actively growing)
atherosclerotic plaques, L. rhamnosus DNA had greater enrichment in stabilized plaques . Indeed, the
[6]
details of how nucleic acid components of probiotics are enriched in plaques and how they may slow
atherosclerosis progression are not clear. Certainly, the DNA of other bacterial species is present in these
plaques, and thus, stability may rely on community dynamics as opposed to a single species or strain.
Enterobacter aerogenes ZDY01 was also inquired for its probiotic potential and was shown to attenuate
choline-induced atherosclerosis in mice by decreasing cecal TMA and promoting reverse cholesterol
transport . While these results highlight the real possibility that probiotics could reduce TMAO burden,
[30]
most data come from animal studies or small-cohort clinical trials. Further testing of these probiotics in
larger populations of human atherosclerosis patients is required to establish efficacy and the potential for
any associated risks.

Indoxyl sulfate and p-cresyl sulfate
As shown in Table 1, p-cresyl sulfate was a stronger predictor of TPA than TMAO. It would be desirable,
therefore, to develop therapies to reduce not only the plasma levels of TMAO, but also p-cresyl sulfate and
other toxic GDUTs, such as indoxyl sulfate, which have been shown to promote atherosclerosis. Indoxyl
sulfate and p-cresyl sulfate are produced by the gut microbiota through the fermentation of tryptophan and
tyrosine, respectively. These toxins are commonly associated with chronic kidney disease, where their
accumulation occurs due to impaired renal function [31-33] . However, indoxyl sulfate and p-cresyl sulfate are
also closely linked to cardiovascular diseases and mortality [34-36] . In atherosclerosis patients, these toxins are
elevated in the plasma, but this observation cannot be explained by kidney function or the intake of dietary
precursors . This suggests the gut microbiota of atherosclerosis patients has a heightened ability to produce
[1]
GDUTs, leaving them at risk of the deleterious effects of these toxins. Furthermore, there are very few
[37]
strategies available for their elimination, and most of these strategies impart an incomplete effect .
To date, bifidobacteria have demonstrated the greatest success in preventing the build-up of p-cresyl sulfate
and its microbial precursor, p-cresol. Probiotic formulations containing at least one strain of
Bifidobacterium longum have been shown to reduce plasma p-cresol in two independent studies after 3
months of supplementation [38,39] . Another study that used a microbial mixture of Bifidobacterium infantis,
Lactobacillus acidophilus, and Enterococcus faecalis showed oral supplementation of these microbes reduced
fecal and serum p-cresol. A synbiotic therapy (probiotic + prebiotic) containing bifidobacteria was also
shown to significantly reduce p-cresyl sulfate in a placebo-controlled trial . These findings indicate the
[40]
strong potential to use probiotics as a treatment to reduce GDUT. However, due to the existence of a
handful of studies reporting probiotic mixtures did not reduce levels of p-cresol or p-cresyl sulfate, the
efficacy of probiotics for this purpose remains unclear [41-44] . It is of interest to note, however, that the trials
where no decrease in GDUTs was observed utilized multi-strain probiotics and not an individual
bacterium [40-45] . Delivering bacteria in different combinations alters their activity and potentially blocks their
beneficial functions. Highlighting this point, the oral delivery of five different combinations of beneficial
bacteria to nephrectomized rats showed that only two could reduce blood urea nitrogen and serum
creatinine despite the similar consortia . While the use of multi-strain probiotics certainly has merit in
[46]