Enterobacter cloacae: a villain in CaOx stone disease?

Kidney stone is a common disease with substantial morbidity and high recurrence rate. The prevalence and recurrence rate of stone formation is on the rise worldwide [1]. In China, the incidence of kidney stones is estimated to be 5.8%, including 6.5% in men and 5.1% in women [2]. Recurrence of kidney stones can cause severe damage, even renal failure which imposes heavy financial and healthy burden on individuals and society [3]. Recently, instrument miniaturization has greatly improved the surgical treatment of kidney stones. However, medical therapies and prevention have not improved substantially because the mechanism of stone formation remains unclear [4]. About 80% of kidney stones are composed of CaOx crystals mixed with varying amounts of calcium phosphate (CaP) [5]. High urinary oxalate is considered to be a crucial risk factor [6], while the urinary calcium is often normal or transient high [7]. So far, how CaOx stones form still remains unclear. Previous studies have focused on systematic factors, such as metabolic or genetic problems [8]. However, both our clinical observation and literature reports indicate that most patients with CaOx stones have unilateral nidus [9]. And all of these can’t be explained by systematic factors. Hence, there may be some local factors in the kidney inducing the formation of CaOx stones.

Historically, it is widely acknowledged that urinary microbiota is closely related to the formation of infectious stones, bacteria can promote the formation of struvite stones via urease [10]. Recently, with the rapid development of 16S rRNA gene sequencing, detection of the microbiome has been widely improved [11]. Thus, increasing evidence shows that bacteria are more broadly associated with kidney stones, not only infectious stones, but also CaOx stones [12, 13]. Ryan et al. isolated and purified Escherichia coli and Staphylococcus epidermidis from urine and stones of patients with CaOx stones, suggesting that the formation of calcium oxalate stone may be related to urinary microbiota [14]. However, the urine samples in this study were collected from bladder of different patients, which cannot eliminate the influence of systemic factors, such as genetics and metabolism factors.

To eliminate the influence of metabolic and genetic abnormalities, we recruited patients with unilateral CaOx stones and collected their two sides (both the stone sides and the non-stone sides) pelvis urine. We applied 16S rRNA gene sequencing to detect the differences of microbiota between the stone sides and non-stone sides pelvis urine of the same patients. We also performed an in vivo experiment to preliminarily explore the relationship between microbiota and CaOx stones. This study, to our knowledge, registers the first attempt to disclose the different microbiota of renal pelvis urine between stone side and non-stone side of a same patient, trying to reveal the relationship between urinary microbiota and CaOx stones.

The components of kidney stones can be classified into following types: calcium oxalate, calcium phosphate, struvite, purine, or cystine. Infection has long been considered to be only associated with struvite, most bacteria present in these stones, such as Proteus species, Klebsiella, Pseudomonas and Corynebacterium species, promote the formation of struvite stones via urease [10]. However, struvite stones account for only 4% of all urinary stones [23]. Bacteria have shown to be more broadly associated with kidney stones, patients with kidney stones often present with concomitant urinary tract infection, regardless of stone composition [24]. The majority of urinary stones are calcium oxalate combined with apatite. Previous studies of CaOx stones used to focus on metabolic and genetic factors. However, kidney stones are often unilateral which cannot be explained by such systematic factors. There may exist some local factors contributing to stones formation. What’s more, some urease-negative organisms, for example, Escherichia coli, have been found in calcium-based stones. Therefore, bacteria may be associated with CaOx stones [14]. Though many studies have revealed the association, the role bacteria playing in CaOx stones formation remains poorly understood.

Many studies have explored the microbiome of calcium-based urinary stones. Barr-Beare E and his colleague found the dominant bacterial communities of CaOx stones including Enterobacteriaceae and the genera Pseudomonas [25]. Ryan et al. added to these bacterial communities with additional identification of Staphylococcus, Veillonella, Streptococcus, Corynebacterium, Haemophilus, Lactobacillus, and Bifidobacterium [14]. However, there are still many limitations in previous studies. Most studies collected stones or bladder urine though patients only have one-side stones, microbiota of stones or bladder urine are a mixture of two sides upper urinary tracts which cannot represent the difference between the stone sides and non-stone sides kidney. To replenish the gap, we collected pelvis urine of both stone sides and non-stone sides of patients with unilateral CaOx stones to make self-control. Thus, we can diminish the influence of systemic factors and explore the local factors which relate to CaOx stones. Moreover, patients’ antibiotic exposures prior to 4 weeks were not tracked in case antibiotics destroy the bacteria initially colonized [26]. Next-generation 16S rRNA gene sequencing was conducted to the pelvis urine collected from both sides of patients’ kidney, the results can be accurate to the species level; thus, we can have a clear cognition of the difference between stone sides and non-stone sides kidneys.

Through the 16S rRNA gene sequencing of urine samples, we found there were significant differences in 26 species of bacteria in bilateral pelvis urine, these bacteria were enriched in PWY-6478, PWY-6572, PWY-7374, PWY-6944 (MetaCyc database), the bacteria enriched in stone sides pelvis urine were mostly enriched in PWY-6944 pathway (androstenedione degradation). Androstenedione is an androgen, urinary androgens are significantly associated with urinary calcium and citrate excretion, which will finally lead to stones formation [7]. What’s more, among the 26 differentially expressed bacteria in bilateral pelvis urine, Enterobacter cloacae and Chryseobacterium sp. were most enriched on the stone sides’ pelvis urine. Chryseobacterium sp. is a phosphate-solubilizing bacteria (PBS), it was often isolated from soil [27]. At present, the studies on it mostly focus on agriculture. What’s more, Ryan A. Dornbier and his colleagues have found that Enterobacter cloacae was isolated from CaOx stones with concordant enrichment on 16S rRNA gene sequencing. Thus, we did not pay much attention to chryseobacterium. Meanwhile, the exploration of Enterobacter cloacae has increased yearly. Enterobacter cloacae is important symbiotic bacteria with intestine, belonging to Enterobacter genus, Enterobacter family, Enterobacter order. It usually exists in the intestinal tract of 40–80% human body, and can also exist in the respiratory and urinary tract, causing urinary and respiratory tract infections [28]. Enterobacter cloacae has strong adhesion and weak invasion, and can play a role by rearranging cytoskeleton proteins into host cells [28, 29], which are mainly involved in the regulation of pathological processes, such as inflammatory response, oxidative stress and lipid metabolism of host cells [30]. In addition, studies have found that Enterobacter cloacae can activate inflammatory and autophagy pathways, triggering metabolic disorders in the body, resulting in heterotopic calcification, namely the formation of atherosclerotic plaques [31]. These suggest that the formation of Randall’s calcium plaques as ectopic calcification of the kidney may also be regulated by Enterobacter cloacae. To the best of our understanding, we are the first to discover Enterobacter cloacae existing in pelvis urine of patients with CaOx stones. The mechanism of Enterobacter cloacae inducing CaOx stones still remains unclear.

To further investigate the role Enterobacter cloacae plays in CaOx stones formation, we conducted Whole Genome Sequencing of Enterobacter cloacae. Bacteria act on the receptor cells through their virulence factors, or their metabolites, extracellular vesicles. Virulence factors are components of bacterial virulence, and they act in two forms: invasiveness and toxin. By comparing the gene coding protein sequence of the Enterobacter cloacae with the amino acid sequence in the VFDB (Virulence Factors of Pathogenic Bacteria) database, we found that flagellum, lipopolysaccharide, aeromycin and urease had high homology, among which flagellum-related protein abundance was the highest. Flagellin is an important scaffold of bacterial movement, dynamic element and virulence factor. Flagellin is the only specific ligand of Toll-like receptor 5, the host membrane protein receptor, which has strong immunogenicity. As a unique structural protein of bacteria, flagellin is also one of the most abundant proteins in bacteria. As a virulence factor, flagellin can promote the invasion of pathogenic bacteria. Therefore, flagellin is a key target for host immune monitoring. When pathogenic bacteria invade the host, the natural immune receptor TLR5 can easily recognize flagellin, thus inducing MAPK- and NF⁃κB-mediated inflammatory response [32]. Some investigators have reported that Flagellin could accelerate CaOx deposits by promoting its crystallization, growth and aggregation [12]. What’s more, flagellin could also facilitate Ca²⁺ influx via calcium cellular membrane channel [33]. However, the association of flagellin with CaOx deposits has been only superficially studied, more studies should be applied to explore the mechanism of flagellin inducing CaOx deposits.

KEGG and GO analysis revealed Enterobacter cloacae may play important roles in ion binding and signaling transduction. Cherng et al. found that bacterial infection contributed to the formation of CaOx deposits via calcium-related ion channels after injected bacteria [34]. Cell signal transduction is a process in which cells experience the stimulation of information molecules through cellular membrane or intracellular receptors, and then transform by intracellular signal transduction system, thus affecting the biological functions of cells. It broadly existed in many biological processes. There are many signaling transduction pathways involved in CaOx stones formation. The P-38/JNK MAPK transduction pathway is turned on in the process of CaOx stone formation [35].

To validate our previous claims, we have conducted in vivo experiments. We assessed the apoptosis condition and crystal depositions of kidneys between rats with different treatments. Pre-injected with Enterobacter cloacae could apparently accelerate the apoptosis and crystal depositions of metabolic disorders rats induced by Glyoxylic Acid. While injected Glyoxylic or Enterobacter cloacae alone did not make any difference in apoptosis condition and crystal depositions comparing to control groups. What’s more, the expression of IL-6, MCP-1, BMP2 and OPN was higher in rats injected with Glyoxylic Acid or Enterobacter cloacae than control. Treated with both Enterobacter cloacae and Glyoxylic Acid could enhance the increases of these proteins. It has been previously demonstrated that IL-6 and MCP-1 indicated a close relationship between inflammation and kidney stones, while our previous research has confirmed that osteogenic transformation-related proteins BMP2 and OPN played important roles in stone formation [36, 37]. Thus, we could presume that pre-existence of Enterobacter cloacae will enhance the effects of metabolic disorders in CaOx stones formation through inflammation process and osteogenic transformation. However, the process of CaOx stones formation is complex, bacteria themselves might cause infection that induce inflammatory response and macrophages recruitment [38]. We cannot be sure that Enterobacter cloacae is the special regulator of CaOx stones formation via inflammation and osteogenic transformation, or if this is a general phenomenon with kidney infection. But our study is just a preliminary exploration based on a finding that there exist different microbiota in stone sides and non-stone sides pelvis urine of patients with unilateral CaOx stones and our in vivo experiments demonstrates that Enterobacter cloacae has associations with CaOx stones formation, while the specific mechanism still needs to be further explored. In addition, we look forward to future studies which can explore the relationship of other bacteria with stone formation.

In summary, our results revealed that the occurrence of bacteria is considerably correlated with CaOx stones formation. Enterobacter cloacae and its flagellin are supposed to have effects on CaOx stone formation through ion binding and signaling transduction. In vivo experiments we conducted have identified that Enterobacter cloacae could accelerate the stone formation of rats with metabolic disorders. However, we just hypothesized the possible process of CaOx stone formation; further studies are required to explore the detailed mechanism of Enterobacter cloacae and its Flagellin inducing CaOx stone formation. What’s more, more patients should be recruited in our studies to increase the trustworthiness of our outcomes.