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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE  
Year : 2020  |  Volume : 31  |  Issue : 5  |  Page : 898-904
The Proportion of Bifidobacterium and Escherichia coli in Colon of Children with Recurrent Urinary Tract Infection


Department of Child Health, Faculty of Medicine, Universitas Indonesia, Dr. Cipto Mangunkusumo National Hospital, Jakarta, Indonesia

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Date of Web Publication21-Nov-2020
 

   Abstract 


Recurrent urinary tract infection (UTI) in children is a well-known risk factor of chronic kidney disease. Periurethral area is normally inhabited by non-pathogenic flora, such as Bifidobacterium sp., and pathogenic flora from gastrointestinal tract, such as Escherichia coli (E. coli), which can cause UTI. Dysbiosis between pathogenic and non-pathogenic bacteria leads to infections, but studies regarding dysbiosis and recurrent UTI have not yet been documented. To estimate the proportional differences between gastrointestinal E. coli and Bifidobacterium sp. in children with recurrent UTI, a cross-sectional study was conducted in children from age six months to <18 years old diagnosed with recurrent UTI in Dr. Cipto Mangunkusumo Hospital. Healthy children matched in gender and age were recruited as control group. Stool samples were obtained from all the children in the two groups. Stool DNA was extracted using real-time polymerized chain reaction method to count E. coli and Bifidobacterium sp. proportion. Children with recurrent UTI had significantly higher proportion of E. coli compared to control group (10.97 vs. 4.74; P = 0.014) and lower proportion of Bifidobacterium sp. (6.54 vs. 9.33; P = 0.594). In children with recurrent UTI group, E. coli proportion was found higher than Bifidobacterium sp. although not statistically significant (10.97 vs. 6.54; P = 0.819). In healthy controls, Bifidobacterium sp. proportion was significantly higher than E. coli (4.74 vs. 9.33; P = 0.021). The total amount of E. coli (996,004 vs. 1,099,271; P = 0.798) and Bifidobacterium sp. (835,921 vs. 1,196,991; P = 0.711) were higher in secondary UTI compared to the simple UTI. Proportion of E. coli is higher in children with recurrent UTI than in healthy children. The proportion of E. coli is higher than Bifidobacterium sp in the colon of children with recurrent UTI.

How to cite this article:
Pardede SO, Paramastri KA, Hegar B, Rafli A. The Proportion of Bifidobacterium and Escherichia coli in Colon of Children with Recurrent Urinary Tract Infection. Saudi J Kidney Dis Transpl 2020;31:898-904

How to cite this URL:
Pardede SO, Paramastri KA, Hegar B, Rafli A. The Proportion of Bifidobacterium and Escherichia coli in Colon of Children with Recurrent Urinary Tract Infection. Saudi J Kidney Dis Transpl [serial online] 2020 [cited 2020 Nov 30];31:898-904. Available from: https://www.sjkdt.org/text.asp?2020/31/5/898/301196



   Introduction Top


Recurrent urinary tract infections (UTI) refer to recurring episodes of significant bacteriuria after initial treatment and are caused by the same or different organisms identified from previous culture.[1],[2] Escherichia coli (E. coli) is the most common cause of UTI which accounts for 50%–90% of infections.[3],[4],[5] Recurrent UTI may lead to a higher risk of complications, such as sepsis and acute kidney injury, also long-term complications such as hypertension, proteinuria, and chronic kidney disease.[2],[4],[5],[6],[7] Factors influencing in children with recurrent UTI are gender, age, hygiene, status of the immune system, voiding habits, abnormal urinary tract anatomy, and urinary tract stone.[1],[2] Another contributing factor influencing recurrent UTI is the colonization of fecal pathogens on the peri-urethral region.[6],[8],[9],[10]

There is a balanced composition of bacteria in the human body between commensals and pathogenic bacteria. A greater number of commensal bacteria will account for a healthy individual while a greater number of pathogenic bacteria will, conversely, account for a sick individual.[11],[12],[13] The most common commensal bacteria studied is Bifidobacterium sp., while the pathogenic one is E. coli.[14],[15],[16],[17],[18] Large number of pathogenic bacteria may cause suppression in the population of commensal bacteria, and therefore leads to opportunistic colonization of gut bacteria such as E. coli in periurethral region causing recurrent UTI.[15],[19],[20],[21] Dysbiosis between pathogenic and non-pathogenic bacteria leads to systemic infections, but studies regarding dysbiosis and recurrent UTI have not yet been documented. The aim of this study is to evaluate the difference between gut E. coli and Bifidobacterium sp. proportions in children with recurrent UTI (simple and secondary).[22]


   Methods Top


This was a cross-sectional study conducted in The Department of Child Health, Dr. Cipto Mangunkusumo National Hospital from April to August 2016. Subjects were children diagnosed with recurrent UTI who came to nephrology clinic and were selected consecutively. All children aged 6 months to <18 years old diagnosed with recurrent UTI were included. Exclusion criteria were children with diarrhea, treated with antibiotics or consuming probiotics (including breast milk, formula milk, or probiotics sachet) within the past two weeks, immunocompromised children and those whose parents refused to participate. Control group was taken from the community, randomly, matched on age and gender with no manifestation of fever, diarrhea, clinical symptoms of UTI, leukocyturia, and no bacterial growth on urine culture. What was excluded in the subject was also excluded in control. This study was approved by the Ethical Committee Faculty of Medicine, Universitas Indonesia- Dr. Cipto Mangunkusumo National Hospital.

Written consent was obtained from parents whose children met the criteria after clear information was given. Data recorded were identity, age, gender, probiotics consumption, antibiotics consumption, physical examination, laboratory, and radiology results to differentiate simple and secondary UTIs. During study, patients were treated according to UTI treatment protocol in the Department of Child Health, Dr. Cipto Mangunkusumo National Hospital.

Urine and stool samples were collected from every subjects. Urinalysis and urine culture were performed using catheter or mid-stream urine technique. One to five grams of stool sample was obtained during home visits using sterile fecal pot and delivered in an ice pack-filled container directly to the Biomolecular Gastrohepatology Laboratory of Department of Child Health, Dr. Cipto Mangunkusumo National Hospital, Jakarta or stored in the freezer before delivery if direct delivery was not possible. Stool samples were obtained from all the children and stored under −70°C inside a freezer in the laboratory until samples are sufficient for efficient analysis. Stool samples were analyzed using real-time polymerized chain reaction on E. coli and Bifidobacterium sp. Results were presented as proportion/200 mg feces for each bacteria. Proportion was defined as the ratio of copy number/200 mg of each bacteria to copy number/200 mg of total bacteria. The same procedures were also applied to the control group.

Fever was defined as body temperature ≥38 °C and leukocyturia was urinary leukocyte >5/hpf. UTI was suspected if leukocyturia and bacteriuria presented in urinalysis. The definition of UTI was the presence of significant bacteriuria (bacterial colony >100,000 cfu/mL) in urine culture with mid-stream or urethral catheter sampling. Recurrent UTI referred to ≥2× infections within the past three months. Secondary UTI was UTI accompanied by anatomical or functional abnormality causing static urine, while simple UTI was UTI without anatomical or functional abnormality causing static urine. E. coli was gut pathogens considered as recurrent UTI risk factor, while gut commensal bacteria considered as recurrent UTI protecting factor was Bifidobacterium sp.

Due to the fact that the presence of both identified bacteria only represented small portions of total colon bacteria, data analysis on this study was performed on colon bacteria proportion, not on the number. The proportion of bacteria was obtained by comparing copy number of each bacteria with total bacteria. The proportion of unidentified bacteria was obtained by dividing the subtraction of both genus bacteria from total bacteria (phylogenetic gap) by total colon bacteria.

Data were analyzed using computer-based IBM SPSS Statistics software version 20.0 (IBM Corp., Armonk, NY, USA) for Windows. Statistical analysis was performed using unpaired t-test, Mann-Whitney, and Wilcoxon with data normalization using the previous logarithm. Data were presented in the median due to abnormal distribution (Saphiro–Wilk test <0005). Normalization test with logarithm for both bacteria and total colon bacteria was performed due to abnormal data distribution in simple and secondary UTI analysis. Unpaired t-test was used to analyze results on normalized data. Mann–Whitney test was used to find difference on unidentified bacteria between the two groups, median proportion of E. coli was on recurrent UTI compared to the control group. Wilcoxon test was used to compare the median proportion of Bifidobacterium sp. and E. coli in healthy subjects.


   Results Top


There were 33 children with recurrent UTI who had the inclusion criteria during the study period; however, only 26 children (78.8%) had significant urine culture. One child was excluded because of having acute diarrhea during stool sample collection. Urine culture was performed on 31 subjects on the control group, but six (19.3%) children were excluded due to the presence of significant bacterial growth on two urine cultures and referred to the clinic for further management.

There were 25 subjects and 25 controls which consisted of 14 (56%) boys and 11 (44%) girls. Mean age of subject and control group were six years six months old and six years five months old, respectively. Subjects with simple UTI were eight (32%) and secondary UTI were 17 (68%) children. Hydronephrosis and hydroureter on neurogenic bladder with vesicoureteral reflux (VUR) were the most common abnormality found in 16 secondary UTI subjects (64.7%). The most common bacteria found on recurrent UTI group, either simple or secondary UTIs, were E. coli on 13 children (52%), six children had Pseudomonas (24%) and the remaining were Acinetobacter iwofii, Proteus mirabilis, Klebsiela pneumonia, Morganela morgagni, Streptococcus alpahemolyticus, Citrobacter freudi.

Gut bacteria composition on recurrent UTI and healthy subjects

The median comparison of phylogenetic gap colon bacteria proportion of recurrent UTI and control patients were 78.7073 (21.83–98.91) and 88.0324 (15.59–99.63). To facilitate analysis, proportion of phylogenetic gap was converted to proportion of UTI and control, 78.7% and 88%, respectively. There were no significant difference on unidentified bacteria between two groups (P = 0.443). This result showed that phylogenetic gap did not affect the composition of colon bacteria.

[Table 1] shows that the median proportion of E. coli was significantly higher on recurrent UTI compared to the control group (P = 0.014). Although not significant, the median pro-portion of Bifidobacterium sp. was higher on control than recurrent UTI group. Proportion of Escherichia coli was higher than Bifidobacterium sp. in children with recurrent UTI. In contrast, the median proportion of Bifidobacterium sp. was significantly higher than E. coli in healthy subjects (P = 0.021).
Table 1: The proportion of colon bacteria in recurrent UTI and control.

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To determine the effect of colon bacteria proportion on simple and complicated UTI incidence, the analysis was performed on the proportion of bacteria without control. Median proportion evaluation of colon bacteria proportion in simple and secondary UTI were presented on [Table 2] and [Table 3]. Analysis result on normalized data showed no significant difference as presented on [Table 3].
Table 2: Proportion of colon bacteria in simple and secondary urinary tract infection.

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Table 3: Mean logarithm of colon bacteria proportion in simple and secondary urinary tract infection.

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They were found higher in secondary than simple UTI. Proportion of both bacterias in simple and secondary UTI was presented on [Table 2]. The proportion of E. coli in simple UTI was higher than in secondary UTI, while the proportion of Bifidobacterium sp. was higher in secondary than simple UTI without statistical significant difference.


   Discussion Top


The number and median proportion of E. coli were significantly higher in recurrent UTI compared to the control group as analyzed with Mann–Whitney test (P = 0.014). Study by Monira in Bangladesh reported dysbiosis with higher number of pathogenic and commensal bacteria than beneficial bacteria in severely malnourished than in healthy children. Higher number of E. coli was associated with ninefold increased risk of developing malnutrition.[22] This result was also consistent with E. coli urine culture results found in majority of the study subjects. E. coli is known as the most common cause of UTI. Colonizing periurethral gut bacteria have a great opportunity to enter the urethra via turbulent urinary flow which facilitates bacterial translocation. Pathogens ability to strongly adhere to mucosal wall renders the “wash-out” mechanism ineffective. Pathogens presence on periurethral region leads to recurrent UTI.[15] Despite a higher proportion of E. coli than Bifidobacterium sp. in children with recurrent UTI, there was no significant difference in proportion.

Comparison of both bacteria in children with recurrent UTI and healthy children revealed that Bifidobacterium sp. proportion was significantly higher in healthy children (P = 0.021). This result describes the possibility that Bifidobacterium sp. may take a role in UTI prevention caused by pathogens because of the competitive nature of Bifidobacterium as beneficial bacteria which inhibit entero-pathogens growth. Beneficial bacteria produce lactic and acetic acid, which decreases gut pH and create unfavorable milieu for pathogens such as E. coli, Salmonella, and Staphylococcus aureus to grow. Bifidobacterium sp. may prevent increase of intestinal mucosal permeability, which will cause bacterial translocation because it stimulates intestinal epithelial cell response so that intestinal mucosal glycoprotein is not damage and protects microvilli integrity. The decrease of Bifidobacterium sp. proportion as compared with other intestinal flora plays a key role in bacterial translocation.[22],[23]

Disorder of bladder voiding is a risk factor of recurrent UTI. UTI is initiated by ascending colonization of peri-urethral gut bacteria into the bladder and kidney.[7],[14],[15] This study showed that the number of both genuses and the total number of gut bacteria were higher in secondary than in simple UTI. The median proportion of E. coli was higher in simple than secondary UTI although it was not statistically significant. The insignificant difference could be caused by abnormal data distribution. Despite data normalization using logarithm, there was no statistical significant difference between both bacterias. Thus, E. coli and Bifidobacterium sp. had no effect on the incidence of recurrent simple nor secondary UTI.

The median proportion of Bifidobacterium sp. was higher in secondary than simple UTI but the number of Bifidobacterium sp. did not decrease recurrent UTI incidence in secondary UTI. This might be caused by other factors of recurrent secondary UTI such as type of anatomical abnormality (VUR and bowel and bladder dysfunction), antibiotic compliance, long-term prophylactic antibiotics, and genetic host immune system variation.[15],[22],[23] This study revealed that the proportion of E. coli was higher in children with recurrent UTI than in healthy children, probably related to recurrent UTI incidence. The number and proportion of Bifidobacterium sp. was higher than E. coli in healthy children, probably related to UTI prevention. The number and proportion of both gut bacteriae were not different statistically in affecting simple or secondary UTI incidence. However, further studies are necessary to prove it.

Awareness of UTI in children should be increased due to unspecific clinical manifestations, which may delay diagnosis and increases the risk of progressive kidney damage up to 25%.[1],[2] Asymptomatic UTI were found in 6/31 (19.3%) healthy subjects. It showed high percentage of children with asymptomatic UTI in the population although this condition do not need treatment. One of recurrent UTI risk factors is secondary UTI because functional and/or anatomical disorders disrupt the bladder voiding process and cause significant residual volume. Disorder of bladder voiding process in secondary UTI is caused by voiding dysfunction, neurogenic bladder and VUR.[7],[14],[15] In this study, 17 (68%) subjects had secondary UTI and eight (32%) subjects had simple UTI. The incidence of secondary UTI was higher due to cases found in Dr. Cipto Mangunkusumo Hospital as a national referral hospital were mostly referral cases requiring multidisciplinary management. Neurogenic bladder causing VUR was the most common cause of complicated UTI, account for 11 (64.7%) of all children with secondary UTI or 44% of all recurrent UTI. This incidence was consistent with a cohort prospective multicenter study reporting that the incidence of VUR in children with UTI was 56%. Higher recurrent UTI risk within two years on VUR compared with non-VUR children (25.4% vs. 17.3%).[16]

Colonic bacteria can be classified as beneficial bacteria (genus Lactobacillus and Bifidobacterium), harmful or pathobionts (genus Streptococcus, Enterococcus, Bacteroides) and pathogenic potential or opportunistic (genus Clostridium, Staphylococcus, Escherichia, Proteus).[12],[17] Bifidobacterium sp. is the most common bacteria found in the stool of healthy neonates (29.49%).[6] Healthy individuals have natural balance among the three kinds of bacteria; however, imbalance (dysbiosis) may lead to immune system disruption and trigger inflammation response, causing diseases.[12],[17],[18],[19]

The most dominant bacterial composition in gut is Bacteroides, Bifidobacterium and Clostridium sp.[12],[18] Gut bacterial composition is affected by diet, antibiotics, host genetic properties, lifestyle, and systemic diseases.[12],[17],[18] Among Indian American and Malawian people, one of the most dominant fecal bacteria is Bifidobacterium sp.[20] There are some studies of gut bacterial composition conducted in Indonesia.

During the study, there was difficulty in recruiting subjects because children with recurrent UTI who visited the nephrology clinic Department of Child Health, Dr. Cipto Mangunkusumo Hospital mostly had already consumed antibiotics and not all children with suspected UTI showed significant bacterial growth on urine culture. Therefore, a lot of recurrent UTI children cannot be enrolled. Consumption of food and beverages, which might affect colon bacteria composition were also difficult to record. Another challenge was in collecting stool samples. Most children felt uncomfortable to defecate into the sterile fecal pot or sterile plastic, causing the time of fecal collection was not always simultaneously with the urine sample.


   Conclusions Top


The proportion of E.coli was higher significantly in recurrent UTI than healthy children. In children with recurrent UTI, the proportion of E. coli was higher than Bifidobacterium sp, but not statistically significant. The total number of colon bacteria in secondary UTI was higher than in simple UTI. No difference was found in the proportion of Bifidobacterium sp. and E. coli in both groups.

Conflict of interest: None declared.



 
   References Top

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Jones KV, Asscher AW. Urinary tract infection and vesicoureteral reflux. In: Edelmann CM, Bernstein J, Meadow SR, Spitzer A, Travis LB, editors. Pediatric Kidney Disease. 2nd ed. London: Little, Brown and Co.; 1992. p. 1943-82.  Back to cited text no. 14
    
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Correspondence Address:
Sudung Oloan Pardede
Department of Child Health, Faculty of Medicine, Universitas Indonesia, Dr. Cipto Mangunkusumo National Hospital, Jakarta
Indonesia
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DOI: 10.4103/1319-2442.301196

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