In the present study, we utilized 16S rRNA gene sequencing to explore the urinary microbiota in male calcium-based kidney stone formers and age-matched healthy individuals. The improvement noted in our study was the strict inclusion criteria, aiming to control various confounding factors. Our results showed significantly reduced species diversity and altered microbial profile in the urine of kidney stone patients compared to controls. Several differentially represented taxa and functional pathways were found in HB and KB groups. In addition, we found the overall bacterial composition and predicted functional pathways of bladder urine was similar to that of renal pelvis urine in kidney stone patients.
Urolithiasis is a common urological disorder with diverse pathologies and the factors contributing to the increasing prevalence are currently unknown. In the previous literature, the mechanisms that urea-producing bacteria promoting struvite stone formation have been well documented. However, the potential mechanism that bacteria contribute to calcium-based stone, the vast majority of kidney stones, remains obscure. One hypothesis is that bacteria adhere to crystal and promote its growth and aggregation. This is supported by findings that bacteria such as Enterobacteriaceae selectively aggregated to oxalate calcium crystal and increased the number of aggregations [17]. Similar crystal aggregation ability was observed in Staphylococcus and Streptococcus species in vitro [18]. Another possibility is that bacteria may alter urine supersaturation via production of citrate lyase, which decreases the urine citrate levels and lead to crystal formation [19]. Lastly, bacteria may induce an inflammatory response and the release of proinflammatory proteins, which form the stone matrix inner core and progress from crystal to stone [20].
The main finding of this study was that we demonstrated distinct urinary microbiota in kidney stone patients compared to healthy subjects. Our results showed that nephrolithiasis patients had significant lower species diversity in urine. According to previous literature, decreased microbiome diversity was related to inflammation and implicated in diseases such as obesity and type II diabetes [21]. Moreover, we found several bacterial taxa associated with inflammation were overrepresented or underrepresented in the urine of kidney stone patients. The most differentially represented taxa at genus level were Acinetobacter in kidney stone patients, and Prevotella in healthy controls. As opportunistic pathogens, Acinetobacter are associated with urinary tract infection in individuals with underlying medical risk factors, such as diabetes mellitus and immunosuppression [22]. Interestingly, the abundance of Acinetobacter was showed to be higher in the faeces of nephrolithiasis patients and the urine of bladder cancer patients compared to controls, indicating its close association with urological diseases [23, 24]. Prevotella are classically considered as commensal bacteria and known to colonize the gastrointestinal tract, vaginal tract and urinary tract. It could synthetize short-chain fatty acids, which were able to protect against inflammation in acute kidney injury [25]. The decreased level of Prevotella favours inflammatory processes and has been implicated in several pathological conditions, including type 2 diabetes, diabetic nephropathy and chronic prostatitis [26, 27].
In summary, our results revealed significantly decreased species diversity, enrichments of proinflammatory bacteria and underrepresentation of anti-inflammatory taxa in the urinary microbiota of kidney stone patients. Similar trends were showed by Zampini and colleagues that a long-term shift in urinary tract microbiome may increase the risk for urinary stones, although not excluding subjects using antibiotics [15]. We also predicted several functional pathways which were significantly enriched in the urine of kidney stone patients compared to healthy controls. Our findings indicated an association between urinary microbiota dysbiosis and kidney stones, and we speculated that bacteria might influence the formation of calcium-based stones via modulation of inflammatory processes.
Another important finding in this study was the similarity of overall bacterial composition between bladder urine and renal pelvis urine in kidney stone patients. Traditionally, bacteria are considered to access the upper urinary tract under certain conditions, such as urinary reflux or bacteria translocation in severe systemic disease. However, a preliminary study showed bacteria could be detected in the upper tract urine of kidney stone patients without urinary tract infections [17]. Due to the small sample size, the author did not compare the microbiota between bladder urine and upper tract urine. In a recent study, Dornbier and colleagues found that there was no significant difference in the microbial composition of bladder urine and upper tract urine in urinary stone patients [16]. It is worth noting that ureteral stents were placed in the majority of patients (50/52, 96.1%) in that study, which may potentially influence the urinary microbiota. In the present study, we found that the species diversity and overall composition of microbiota was similar between KB and KP groups, after excluding confounding factors such as antibiotic use and ureteral stent placement. In addition, our PICRUSt results showed no significant difference with regard to the predicted functional pathways between KB and KP groups. Meanwhile, we also noted that there were a few taxa (e.g., Anoxybacillus) differentially represented in KP group, remaining an area for future research.
Some limitations should be noted when interpreting our results. First, all participants were Chinese and the sample size is relatively small, limiting generalizability and comparison of stone subtypes. Further largescale studies are necessary to investigate the urinary tract microbiota across ethnicity and stone type. Second, this study did not include female subjects, mainly due to their lower morbidity of kidney stones and higher positive rate of urine routine tests. In the future, we will conduct a more comprehensive research after recruiting adequate females in line with our inclusion criteria. Additionally, the association of risk factors for lithogenesis in urine and urine microbiota was not evaluated, because the vast majority of kidney stone patients were first onset and 24 hours urine analyses were not performed. Finally, like most metagenomic studies, we cannot comment as to whether altered urinary microbiota in kidney stone patients was a contributor or the result of kidney stone formation. All these questions will certainly be the focus of future research.