Diversity of Bacteroidaceae family in gut microbiota of patients with chronic kidney disease and end stage renal disease

Background: Human intestine microbiota are known to be directly and indirectly altered during some diseases such as kidney complications. Bacteroides is considered as the main and the most abundant phylum among human gut microbiota, which has been classified as enterotype 1. This study aimed to assess the abundance of Bacteroides spp. in fecal flora of end-stage renal disease (ESRD) and chronic kidney disease (CKD) patients and compare it with the Bacteroides composition among fecal flora of healthy individual. Methods: Fresh fecal samples were collected from 20 CKD/ESRD patients and 20 healthy individual without any kidney complications. The pure microbial DNA was extracted by QIAamp Stool Mini Kit from stool samples. MiSeq system was used to analyze the intestinal composition by next generation sequencing method. Results: A number of 651 bacterial strains were isolated and identified from 40 fecal samples of both patients and healthy groups. Bioinformatics analysis defined 18 different types of Bacteroides species which included 2.76% of all strains. Statistical analysis showed no significant difference between study groups (P>0.05). In both healthy and patient groups three species including B. dorei, B. uniformis, and B. ovatus have allocated the most abundance to themselves. The lowest abundance was related to B. eggerthii, A. furcosa and B. barnesiae among CKD/ESRD patients and A. furcosa, B. barnesiae, and B. coprocola had the lowest abundance among healthy people. Conclusion: This study indicates despite all previous evidence of Bacteroides role in gut microbiota, it had no different distribution between healthy persons and CKD/ESRD patients.


Introduction
Bacteroidaceae family is belonged to Bacteroidetes class and phylum.The phylum Bacteroidetes can be found in the environment for instance in sea water, soil, and sediments as well as colonized on the skin of animals and into the guts. 1 Approximately, around 20%-80% of the gut microbiota relates to Bacteroidetes phylum in healthy adults which the genera of Parabacteroides, Bacteroides, Alistipes, and Prevotella are categorized in this phylum. 2,3acteroidales order has a significant abundance in the human gut so that every gram of human feces has a high concentration, which reaches up to 10 9 -10 11 CFU. 4 The genera Bacteroides has been identified as gram-negative, obligate anaerobes, rod-shaped with round ends, nonmotile, and non-spore-forming which has known as one of the major genera of microbiota composition with more than 30 species. 5,6 large variety and myriad microorganisms have been colonized in human intestine which is called microbiota. 7,8wo major phyla: the gram-positive Firmicutes and the gram-negative high CG% Bacteroidetes have formed the microbiota population and the other phyla including Fusobacteria, Actinobacteria, and Verrucomicrobia phyla have been categorized at subdominant levels. 5ut microbiota is a vast world that has many beneficial effects on human body, such as helping to food digestion, producing of hormones and essential vitamins like K and B12, modulating and developing of immune system and powerfully forming a natural defense to limit the infections caused by intestinal pathogens. 9,10ome studies also presented that the possible changes in species/phyla levels of Bacteroidetes and Firmicutes can be considered as obesity factors in children. 11Another study has reported that Bacteroides species could be widely effective in the treatment of intestinal colitis, metabolic disorders, immune dysfunctions, and cancer prevention which Bacteroides genus is considered as a new beneficial probiotic candidates. 12In addition, the members of Bacteroidaceae family also decreased inflammation response by regulating cytokine expression and lymphocytes. 13hronic kidney disease (CKD) has known as a key determinant of noncommunicable disease which can progress toward end-stage renal disease (ESRD). 14Both developed and developing nations report high numbers of cases with CKD annually that can alter the intestinal microbiota composition and microbial metabolism quantitatively and qualitatively. 15,16Intestine microbiota has contributed to the production of important metabolic substances.On the other hand, the uremic solutes such as indoxyl sulfate and p-cresyl sulfate can be generated by gut microbiota. 17Patients who suffer from CKD have shown an altered combination of gut microbiota which have been correlated to the dietary interventions and therapeutic condition and the uremic milieu, result in high production of the uremic solutes. 18Several studies have reported a statistical association between mortality and circulating levels of the uremic toxins. 18In present study, we assessed changes in the abundance and diversity of Bacteroides spp. in intestinal flora of CKD and ESRD patients by comparing differences between healthy humans.

Sample collection
The present trial enrolled 20 patients with CKD and ESRD undergoing hemodialysis from the kidney transplantation ward of Imam-Reza teaching hospital, Tabriz, Iran.On the other hand, 20 healthy volunteers were joined to the study as control group.Fresh fecal samples of both case and control groups were directly collected from the anus of individuals and transferred into the sterile containers and were stored at -80 ºC until further processes.Before participating in the investigation, before participating in the research, written informed consent was signed by all patients and healthy group.Our ESRD patients in this investigation had some underlying diseases including chronic pyelonephritis, glomerulonephritis, hypertensive nephrosclerosis, polycystic kidney disease, post renal and urolithiasis, urolithiasis and systemic lupus erythematosus.Exclusion criteria include patients with some complications such as intestinal disease or colectomy, cholecystectomy, and diabetes, also patients suffering infections, inflammatory disorders, autoimmune diseases and patients who had received antibiotics within three months before enrolling in the study.

DNA extraction and PCR amplification
First of all four grams of mixed and homogenized fresh fecal samples were weighted for extraction of pure DNA.Microbial DNA was isolated from the fecal mixture using the QIAamp Stool Mini Kit (QIAGEN, Germany), according to the manufacturer's instruction. 19The Thermo NanoDrop 2000 spectrophotometer (Thermo Scientific, MA, USA) was used to find the exact amount of DNA in each fecal sample. 20Two sequence of universal bacterial 16srRNA (V3-V4 hypervariable regions) were used for amplification of template DNA and sequencing.The specific sequences in this trial were as follows 21 : The amplification of the target sequences was performed using a T100TM thermal (Bio-Rad, USA).The polymerase chain reaction (PCR) reactions were performed as follows: 95 °C for 5 minutes, followed by 35 cycles of 95 °C for 1 minute, 55 °C for 45 seconds, and 72 °C for 1 minute, with a final extension of 72 °C for 1 minute.The electrophoresis was run in 1% agarose gel in Tris-boric acid-Ethylenediaminetetraacetic acid (EDTA) buffer to assess the PCR products and the gel was stained with ethidium-bromide to be visible under UV light.MiSeq system (100k 2 x 300 bp paired-end reads) (Illumina, USA) was accomplished the sequencing of PCR products in Omega Bioservices company.Bioinformatics analyses were completed by Illumina's BaseSpace in parallel with Illumina's in-house QIIme 2 pipeline.

Statistical analysis
Statistical analysis was assessed using programs including GraphPad PRISM 8 and SPSS 20.Statistical analysis was performed to compare case and control groups using the Mann-Whitney nonparametric test and Welch's t test.P values < 0.05 were considered statistically significant.

Results
A total of 20 patients with CKD/ESRD, 14 patients were male and 6 patients were female with the mean age of 53.20 ± 12.03 years.As well as, a total of 20 healthy individuals, 10 persons were male and 10 were female with the mean age 59.3 ± 7.89 years.The results of MiSeq system demonstrated that 651 bacterial strains were found in 40 fecal samples of both patients and healthy individuals, which 18 strains (596538 reads, 257413 vs. 339125 reads) belong to family Bacteroidaceae.The strains were belonged to two genera including Bacteroides (17 species) and Anaerorhabdus (one species).The most abundance of species in patients with CKD/ESRD were B. dorei (32.66%),B. uniformis (21.03%) and B. ovatus (10.5%) and the lowest were B. eggerthii (0.01%), Anaerorhabdus furcosa (0.03%) and B. barnesiae (0.17%).As well as, the most abundance of species in healthy individuals were B. dorei (30.74%),B. uniformis (25.38%) and B. ovatus (18.3%) and the lowest were A. furcosa (0.01%), B. barnesiae (0.01%) and B. coprocola (0.07%).Using statistical analysis, the abundance of various species did not show any significant difference between the patients and control group (all P > 0.05).The abundance of various species is shown in Table 1 and Figure 1.

Discussion
In both CKD patients and healthy individuals, Bacteroidetes (~40%), Firmicutes (~40%) and Proteobacteria (~10%) counted as the predominant phyla in gut microbiota composition. 22According to Faith et al 23 report, during lifetime and human generations, Bacteroidetes phyla was more stable in comparison with the phyla Firmicutes.Other study has specified Bacteroidetes as the most plenteous phylum were accounted around 41% in both healthy clients and patients. 24In present study, among both CKD/ESRD patients and healthy volunteers, 18 strains of a total 651 bacterial strains were related to Bacteroidaceae family.On the other hand, the novelty of this study was to compare the abundance and diversity of Bacteroides species between fecal samples of CKD/ESRD patients and control group.Consequently, Statistical analysis calculations proved that there was no significant difference in variety of Bacteroides species between patients with CKD/ESRD and healthy individuals.
Numerous studies have examined Bacteroides at different levels of phylum, family, genus and species.Gut microbiota balance changed qualitatively and quantitatively through CKD patients who this imbalance is accompanied with decrease in Bacteroidaceae, some Prevotellaceae, and particular Bifidobacterium and Lactobacillus, and increase in the count of Enterobacteriaceae, Lachnospiraceae, and certain Ruminococcaceae. 22,25Jiang et al demonstrated that a significant reduction of total bacteria quantity was shown in ESRD patients.Bacteroides was prevalent in ESRD patients whereas Prevotella was enriched in healthy individuals.Also, among ESRD patients, the count of the butyrate producing bacteria such as Faecalibacterium, Roseburia, Coprococcus, Prevotella, and Clostridium were declined. 22Crespo-Salgado et al 26 found that the gut microbiota composition in pediatric patients with hemodialysis was different compared with healthy controls.Unlike healthy individual, a decrease was shown in Proteobacteria members while Bacteroidetes was considerably increased in hemodialysis (HD) patients. 26n addition to phylum Bacteroidetes, this raises in HD patients, phylum Firmicutes decreases in ESRD patients undergoing peritoneal dialysis (PD). 25n other study about kidney stone disease (KSD), scientists found that gut microbiome can have an essential role in kidney stone formation. 27A unique gut microbiota was shown in patients who suffer from nephrolithiasis compared with healthy clients. 27Among kidney stone formers, Bacteroides spp. was more prevalent while healthy controls significantly had higher Prevotella spp. in microbiota composition. 27In a comparison of Bacteroides count among KSD and healthy group, Bacteroidetes was 3.4 times more plenty in KSD patients. 27Li et al assessed patients with both CKD and high systolic blood pressure and observed altered bacterial composition and a reduction in bacterial abundance.Their achievement demonstrated which in hypertension models, the richness of the intestinal Bacteroidetes and Firmicutes was associated with increased blood pressure. 28otal quantity of fecal microbiota was decreased in ESRD patients unlike healthy controls.Human intestinal microbiota has 3 main enterotype including Bacteroides categorized as enterotype 1, Prevotella as enterotype 2, and Ruminococcus as enterotype 3. 29 A research study showed that from healthy individual through patients with ESRD, the mentioned enterotype shift from enterotype 2 (Prevotella) to enterotype 1 (Bacteroides), especially Bacteroidaceae which have the ability to produce p-cresol increase in ESRD patients. 27On the other hand, those bacteria that tend to produce short chain fatty acids like butyrate were declined among patients with ESRD. 27everal studies demonstrated that Bacteroides increase greatly in a variety of diseases, for instance Bacteroides in genus level has increased significantly in diabetes mellitus group. 30In addition, the segmented filamentous bacteria specially colonization by Bacteroidetes can induce the intestinal infiltration of pro-inflammatory TH17 cells, which is necessary to balance TH1 and TH2 responses. 31n present research, Bacteroides in genus and species level were assessed among both CKD/ESRD patients and healthy people which B. dorei, B. uniformis, and B. ovatus had the highest abundant among both group without any difference.Boente et al 32

Conclusion
In summary, several previous knowledge have demonstrated the correlation between increased abundance of Bacteroides members and some disease particularly Kidney problems.Therefore, our findings around comparison of Bacteroides species abundance with CKD/ESRD patients and healthy individuals can extend the previous findings, which there was no significant difference in distribution of Bacteroidaceae members among both assessed patients and healthy groups.
The limitation of our research is that due to financial limitations, it was not possible to conduct research on a larger number of individuals.
have evaluated the members of Bacteroidaceae among other disease like hypertension that some species of Bacteroides including B. eggerthii, B. cellulosilyticus, and 3 unclassified Bacteroides had important function in patient with hypertension and other Bacteroides spp.such as B. dorei, B. nordii, and B. uniformis have enriched in control group.These findings refer to that the composition of Bacteroidaceae members alter not only among CKD/ESRD patients, but also in some other disorders.

Table 1 .
The abundance of different species of Bacteroidaceae family identified in fecal samples of both patients with CKD/ESRD and healthy individuals STDEV, standard deviation; Min, minimum; Max, maximum.