We performed the so far largest prospective study investigating the composition of kidney stones and urine microbiome in patients with nephrolithiasis. Our analyses lead to several important findings. First, the highest amounts of confidently detectable bacterial colonization were found in Apatite and Apatite/CaOx/CaPhos stones, although these stones are classified as “non-infection stones“ in the EAU guidelines.[19] Secondly, we observed that the microbiome of stones is well reflected in the corresponding patients’ urine sample. Thus, the analysis of the microbial community composition of catheterized urine samples may help to guide therapy decisions for tailored antibiosis, in order to avoid complications after stone removal but also antimicrobial overtreatment. Moreover, our analysis revealed, that stones display an increased relative abundance of distinct microorganisms, classically associated with the gastrointestinal microbiome, but also known to be pathogenic.[20]
The systemic change of public health, with increasing cases of severe obesity, raises enormous medical expenditure and impose new challenges in patient care. Nephrolithiasis is a well-known co-morbidity of obesity, and metabolic syndrome and diabetes have been considered as risk factors for kidney stone formation for more than 20 years.[21] For a long time hyper nutrition and unilateral diet, resulting in a supersaturation of carbonates considered mainly as the underlying reasons. Moreover, obese persons are characterized by hyperinsulinemia, which is associated with increased intestinal absorption and renal excretion of calcium.[22] However, beside resulting in chemical supersaturation, nutrition has a strong effect on the gut microbiome itself, and consequently also on urogenital microbial populations.[23] Consistent with previous reports, we found significant dysbiosis in patients with SD, characterized by an enrichment of classical gastrointestinal microorganisms in urine and kidney stones.[23] While others focused on dysbiosis of the gut microbiome in SD, we were able to confirm this observation also in stone and urine of patients with nephrolithiasis.[24] We showed that the bacterial composition in stones of patients with features of metabolic syndrome is characterized by increased abundance of gastrointestinal microorganisms such as E.coli, Shigella, Klebsiella, Enterococcacea, Proteus and Sphingomonas, while SD patients without FMS display a higher relative abundance of Staphylococcaceae and Ureaplasma. This supports the study from Chen et al, where an increased abundance of Escherichia-Shigella, Klebsiella, Enterococcus and Aerococcus, but a reduction of Prevotella and Lactobacillus in patients with type II diabetes and lower urinary tract symptoms (LUTS) was described.[25] The origin of these classical fecal opportunistic pathogens and commensals is not easily explained. It is possible that poor diet and the enrichment of carbohydrates promote the colonization of certain bacteria, while overgrowing others.
We were not able to demonstrate, that the accumulation of a specific bacterial genus or combination of bacteria promotes the formation of a particular type of kidney stone. However, we showed that the incidence of pathogenic Enterobacteriaceae was high in all stone types, proving that beside struvite, also CaOx and CaPhos stones are associated with these bacteria. Barr-Baere et al, also identified E. coli and other bacteria of the family Enterobacteriaceae as the most prevalent uro-pathogen in pediatric urinary tract infections accompanied by CaOx stones.[26] Taking advantage of a murine model for CaOx nephropathy, they showed increased CaOx deposition in mice after transurethral inoculation with E. coli compared to CaOx nephropathy alone. The underlying mechanism for enhanced stone formation was both the adhesion of bacterial metabolites to crystals and increased CaOx crystal aggregation.[26]
Furthermore, we hypnotized that patients with recurrent SD display an aberrant microbiome compared to patients with primary SD. Reasons therefore might be previous interventions, transporting bacteria from skin, genitals, urethra and bladder up to the renal pelvis. Moreover, antibiosis and other therapeutics can also sustainably change the bacterial composition of the urogenital tract, resulting in dysbiosis and therefore recurrent stone formation. Surprisingly, we were not able to detect significant divergence in stone and urine microbiomes of recurrent SD patients compared to primary SD patients. However, the five patients with the highest read sequence yield per stone (> 100.000 reads/stone) had multiple stone removals within the study period, with increasing read numbers in stones but not in urine. Notably, all of them had Apatite or Apatite mix stones.
Interestingly, patients with verifiable bacteria in kidney stones displayed a prolonged LOS due to different post-operative complications. Similarly, Wagenius et al. demonstrated, that patients with positive stone cultures harbor a greater risk for postoperative urosepsis, than those with just positive urine culture.[27] So the evidence of pathogens in the kidney is a tremendous risk for infectious complications.
The major limitation of our study was the limited number of patients for which sufficient microbial biomass was retrievable, impairing statistic correlations.
Finally, we want to address the problem of a suitable control group in urological microbiome research. Catheterized urine from healthy individuals would be the most suitable control to reveal disease-related dysbiosis. Since this cohort was not available due to ethical reasons, we investigated the voided mid-stream urine of volunteers without prior stone disease. Hence, we do not consider the data obtained from voided urine as a control, but rather as a benchmark and reference, to detect bacteria unique to patients with stone disease.
As previously described, we observed a sex-specific voided urine microbiome in healthy men and women (p-value 0.002, Supplementary Fig. 3A), but not in catheterized urine of patients with SD (Supplementary Fig. 3B).[28; 29] Moreover, we observed no difference in stone microbiome between man and women. This result is certainly influenced by the approach of urine collection, since voided urine represents a combination of bladder, urethral, and perimeatal microbiota. Therefore, with the available data, we are not able to conclude if the difference of sexes between healthy urine and urine form patients with stone disease is due to dysbiosis.
Additional studies including whole metagenome sequencing are required to improve our understanding of the involvement and role of bacteria in kidney stone disease. Moreover, concordant analyses of patients` voided and catheterized urine, stones and gut microbiome are necessary to illuminate the extent of dysbiosis and interactions of the microbes to prevent stone formation, tailor antibiotic therapy and circumvent postsurgical complications.