16SrRNA Sequencing Analysis Of Urinary Microbiome And Stone Microbiome Of Kidney Stone Patients

Background and objective:Kidney stone is one of the most frequent urological disease with high recurrence rates.According to a recent study in China,the prevalence of kidney stones in Chinese adults is 5.8%,the prevalence of males is 6.5%and of females is 5.1%.Kidney stone formation is a multifactorial disease in which the defense mechanisms and risk factors are imbalanced in favors of stone formation.At present,the cause of kidney stone is considered to include:dietary change,obesity,global climate change,gender,and many other factors.The microbiome is increasingly considered as an essential factor in human health and disease.Researchesindicated that there might be relationship between microbiome and kidney stone.People with kidney stones were inclined to develop UTIs than the general population.The relationship between struvite stone formation and urease-producing organisms,such as Proteus species,has been well described.Recent researches prove that nanobacteria and oxalate-degrading bacteria also plays an important role in stone formation.The recurrence rates of calcium oxalate stones in China are 10%,35%and 50%in 1,5,10 years.Due to the high recurrence rate and the rapid development of infectious stones,there is a crucial need to dissect mechanisms that contribute to stone formation with an aim to identify potential therapeutic targets for intervention.16SrRNA is a conserved piece of sequence in the bacteria genome,with specificity region V1-V9.So,we can obtain complete information of specimen through 16SrRNA sequencing and explore the relationship between urinary microbiome and kidney stone.Methods:1.Explore the microbiome of kidney stone patients through 16SrRNA sequencing method and mid-stage urine culture.36 kidney stone patients underwent PCNL were included.We collected their urinary samples for urinary culture and kidney stone samples for 16SrRNA sequencing.The total DNA in the stone was extracted from the kidney stone specimen.The V3-V4 region of the bacterial 16SrRNA gene was sequenced by Illumina to analyze the microbiome in the stone.The sequencing results were compared with the culture results.2.Analysis of abundance and microbiome composition in kidney stone of different components.We used a stone infrared spectrometer to analyze the stone composition of kidney stones in 36 samples.The microbiome composition,Alpha diversity,Beta diversity and the species difference analysis through different component were compared.3.The 16SrRNA sequencing results of urine and kidney stone were compared.Preoperative mid-stage urine specimens,intraoperative renal pelvis urine specimens,and kidney stone specimens were collected from 13 patients undergoing percutaneous nephrolithotomy in our hospital.Mid-stage urine culture,renal pelvis culture,stone culture,and whole-DNA of preoperative mid-stage urine samples were extracted.The V3-V4 region of the bacterial 16SrRNA gene was sequenced by Illumina to analyze the microbiome communities of urine.The results between culture and 16SrRNA sequencing results were compared.Results:1.The positive rate of stone sequencing in stone patinets are much higher than that in middle-stage urine culture.Proteobacteria are the most abundant bacteria in the microbiome of stones.2.The microbial composition of kidney stones in varycomponent are different.The abundance of microorganisms in struvite stones is significantly less than that of uric acid stones and calcium oxalate stones,and the species diversity is less than calcium oxalate stones and uric acid stones.The dominant phylum and genus of bacteria in kidney stones of different component are considered to be associated with urinary tract inflammation and stone formation.3.The positive rate of urine sequencing and stone sequencing in patients with kidney stones is 100%,which is higher than that of middle urine culture,renal pelvis culture and stone culture.There is a difference in composition between the urine microbiota and the stone microbiota.The dominant species in the urine are related to the components of stones.Conclusion:This study confirmed that the 16SrRNA sequencing has a higher positive rate than the traditional urine culture technique for the analysis of urinary tract microbiome in patients with kidney stones.The urinary tract of kidney stone patients has its own microbiota.Further detailed data analysis explored the differences in the composition and structure of microbiome communities between different components of kidney stones,confirming the relationship between urinary microbiome and kidney stones.The dominant species in stone patient are associated with urinary tract inflammation and stone formation.We also screen out the species that may interact with stones of different compositions.In addition,wefound that there are some differences in microecology between urine and stones.