Lactobacillus bulgaricus mutants decompose uremic toxins

Abstract

Background/Aims: We aim to obtain a probiotic strain from Lactobacillus bulgaricus by testing its capability to decompose uremic toxins to provide new intestinal bacteria for the treatment of chronic renal failure. Methods: Original L. bulgaricus was cultured with the serum of uremic patients and then mutated by physical (ultraviolet) and chemical (diethyl sulfate) methods repeatedly. Using creatinine decomposition rate as an observed index, we selected the best strains which decreased the most concentration of the creatinine. We then tested its ability to decompose urea, uric acid, serum phosphate, parathyroid hormone, and homocysteine and its genetic stability. Results: After inductive and mutagenic treatment, DUC3-17 was selected. Its decomposition rate of creatinine, urea nitrogen, uric acid, phosphorus, parathyroid hormone, and homocysteine were 17.23%, 36.02%, 9.84%, 15.73%, 78.26%, and 12.69%, respectively. The degrading capacity was sustained over five generations. Conclusions: After directional induction and compound mutation, L. bulgaricus has greater capacity to decompose uremic toxins, with a stable inheritance.

• Chronic renal failure
• creatinine
• Lactobacillus bulgaricus
• uremic toxins

Introduction

Uremia is a term used to describe the illness accompanying kidney failure, in particular, the nitrogenous waste products. The retention of uremic toxins in serum leads to tissue damage and functional deterioration, especially in progression of chronic kidney disease (CKD), increased risk of cardiovascular events, and accelerated death of patients with chronic renal failure.1 Hemodialysis and peritoneal dialysis are currently used to remove the uremic toxins in CKD. However, they are expensive treatments. Intestinal bacterial therapy is a new therapy for chronic renal failure. It is amenable to oral administration as a micro-capsule containing the entire organism directly yielding the enzyme or expressing exogenous enzyme through genetic engineering. Intestinal bacteria decompose of uremic toxins in the gut, and reduce the toxin levels in the blood, resulting in symptomatic relief. However, use of extra-intestinal bacteria may cause opportunistic infections. They may also carry drug resistance plasmids. The purpose of this study is to screen a new bacterial strain from probiotic L. bulgaricus (L. bulgaria), which is capable of decomposing uremic toxins. This modality may help delay progression of CKD.

Materials

Bacterial strain

L. bulgaria was provided by agricultural food and microorganism laboratory of Hunan.

We used the centrifuges (Beckman, Brea, CA); a clean bench (Aristech, Florence, KY); electro-heating standing-temperature cultivator (Shanghai Precision Experiment Equipment Co. Ltd, Shanghai, China); a 30 W UV lamp (Jizhou Jiaguang Medical Instrument Co., Ltd, Shenzhen, China); and a UV spectrophotometer (Beckman).

Preparation of culture medium

We prepared 1.0 L of ordinary MRS medium in distilled water containing: peptone 10 g, yeast extract, 5.0 g, beef extract 10 g, glucose 20 g, sodium 5.0 g, diammonium citrate 2.0 g, Twain 80 1 mL, MgSO₄ · 7H₂O 0.2 g, MnSO₄ · 4H₂O 0.05 g, K₂HPO₄ 2.0 g, agar 15 g/L in solid medium, and adjusted the pH to 6.12–6.2. It was autoclaved at 115 °C for 20 min, and stored at 4 °C. Induction medium for directional induction of primitive lactic acid bacteria comprised uremic serum with a creatinine, 960.7 μmol/L. We prepared screening medium for preliminary screening after mutagenesis by applying uremia patients’ serum on the surface of the ordinary MRS medium, and obtained standard concentrations of urine creatinine in the medium, 200 μmol/L, 400 μmol/L, 600 μmol/L, 800 μmol/L, 1000 μmol/L, and 1200 μmol/L, respectively. Preparation of main reagents: phosphate-buffered saline (PBS) (NaCl 80, KCl 2.0 g, Na₂HPO₄ · 7H₂O 11.5 g, KH₂PO₄ 2.0 g) was dissolved in deionized water and the volume adjusted to 1000 mL, with diethyl sulfate (DES), sodium thiosulfate and so on.

Methods

Specimen collection

The serum was obtained from uremic patients at the Division of Nephrology, The Second Xiangya Hospital. In the morning, we drew 3 mL blood from selected patients before breakfast, and added ethylenediamine tetraacetic acid (EDTA) to the blood, then separated the plasma by centrifugation and preserved at −70 °C in a refrigerator.

Activation and growth curve of bacteria

We selected L. bulgaria saved in −70 °C refrigerator and inoculating an MRS medium with the bacteria, activated for three times. We inoculated activated bacteria on bouillon culture-medium using 4% (v/v) bacterial load, and anaerobically cultured at 37 °C. The optical density (OD) value was tested at 4 h intervals, with blank medium as control. Using OD value as ordinate and culture time as abscissa, growth curves were drawn.

Bacterial decomposition of creatinine

We resuspended the logarithmic phase suspension in saline, and adjusted their absorbance (OD₆₀₀) in each tube from 0.08 to 0.1 by McFarland turbidity. We added 200 µL bacterial suspension to 800 µL serum of patients with uremia, and cultured in the anaerobic tank at 37 °C for 24 h. A saline added to the above serum was used as a control. The serum was centrifuged for 10 min, and the upper serum was used for estimating creatinine concentration, to determine the bacterial ability to decompose creatinine.

Induction by high concentrations of urotoxin

The original bacterial suspension with bacterial load of 10% (v/v) was inoculated on uremic patients’ serum containing creatinine concentration of 960.7 µmol/L, and mixed well. Anaerobic incubation for 24 h resulted in the first generation of bacteria, oscillating and mixing until well-distributed for the second time. A 10% (v/v) of this mixture was subjected to another anaerobic incubation for 24 h later, to obtain the second generation. We used every other five generation to detect the bacterial ability to decompose creatinine until the strains with the strongest capability of degradation were obtained.

Mutation

(1) Preparation of bacterial suspension: The late logarithmic phase broth was taken, adjusting the bacterial concentration of the suspension to 108 cfu/mL by McFarland turbidity. The bacterial suspension was added into a sterile triangular bottle with glass beads, and oscillated for 20 min until sufficiently mixed for the next step. (2) Ultraviolet (UV) mutagenesis: The lights were preheated for 20 min, and 5 mL bacterial suspension was place on an empty dish (90 mm), stirred on a magnetic stirrer, and exposed under 30 W UV light at a distance of 30 cm, lasting for 5 s, 10 s, 15 s, 30 s, 45 s, 60 s, 90 s, and 120 s, respectively. (3) DES mutagenesis: We added 16 mL PBS solution to 4 mL of the prepared bacterial suspension, followed by 0.2 mL DES (specific gravity 1.18, molecular weight 154) to a final concentration of 1.0%, vibrated at 37 °C for 5 min, 10 min, 20 min, 30 min, and 40 min. We used 2.5 times of 25% sodium thiosulfate to stop mutagenesis. (4) DES-UV mutagenesis: We first treated bacteria suspension with DES for 20 min, and exposed to UV for 15 s. (5) We diluted bacterial suspension to a concentration of 10⁻⁵, 10⁻⁶, and 10⁻⁷ and added 0.1 mL of each of the dilutions on MRS-coated tablet on three plates, with matched blank and untreated broth dilution. After anaerobic culture for 36 h at 37 °C, we measured bacterial death using the equation: mortality = viable counts per mL before mutagenesis − after mutagenesis/viable counts per mL before mutagenesis. Bacteria subjected to UV and compound mutagenesis should be placed in darkroom.

First screening

The duration of mutagenesis is determined at mortality rate of 70–80%. After mutagenesis, bacteria were placed on a screening medium coated with uremic toxin and cultured for 36 h. The colonies with the largest growth diameter were inoculated in the serum of patients with uremia, serving as a positive control, while serum without bacteria was used as negative control. With each mutagen 30 of the mutant strains were selected, each strain per set. After 24 h of culture, creatinine concentration was measured in the supernatant. Creatinine composition rate was calculated in original strains and mutant strains as follows: creatinine decomposition rate (%) = serum creatinine levels at the beginning − at the end/serum creatinine levels at the beginning.

Rescreening

The mutation rate in decomposing bacteria increased more than 5% compared with the bacteria prior to mutational rescreening. The selected bacterial suspension was inoculated into the serum of uremic patients for 24 h, and then creatinine decomposition rate was calculated.

Mutant stability and creatinine decomposition

The selected mutant strains were cultured in serum of uremic patients for 24 h each. After five consecutive cycles, creatinine decomposition rate was calculated to estimate the bacterial stability of creatinine decomposition.

Mutants comprehensively decompose urinary toxins

The target strains obtained as above possess the strongest ability to decompose creatinine. They were cultured in anaerobic incubator at 37 °C to logarithmic phase. The OD₆₀₀ of bacteria suspension of each tube was adjusted from 0.08 to 0.1 by McFarland turbidity, mixed 200 µL bacteria suspension into 800 µL serum from uremic patients in an anaerobic tank at 37 °C. A normal saline compound with 200 µL uremic serum served as negative control. A 200 µL original strain induced strain composite mutagenesis alone. The decomposition rate of the strain showing highest creatinine bacterial suspension served as a positive control. The upper layer was used to test the level of creatinine by Jaffe’s assay; urea nitrogen was measured by UV-GLDH assay; uric acid by enzyme colorimetric detection; serum phosphorus by UV; parathyroid hormone by chemiluminescence detection; and circulating homocysteine by enzymatic cycling assay to estimate the bacterial ability to decompose such toxins.

Statistical analysis

Data are expressed as means ± standard error (SE). Measurement data were evaluated by one-way analysis of variance (ANOVA), two samples were compared by t-test, respectively. Values of p < 0.05 were regarded as statistical significance, p < 0.01 as significant. The above analyses were conducted with SPSS 17.0 statistical software.

Results

Growth curve of the original strain

As shown in Figure 1, L. bulgaria enters the late logarithmic phase in 18 h, with active chromosome replication and easy induction of mutagenesis.

Figure 1. Growth curve of original strain.

Differential ability of bacterial decomposition of creatinine: original L. bulgaria versus strain induced by high concentration urine toxin

Incubating the original L. bulgaria with the serum of uremic patients, resulted in for 24 h, no significant decline in creatinine concentration compared to the serum control group (p > 0.05) (Figure 2). However, compared to the serum control group and original strain, induced bacteria significantly decreased serum creatinine concentration (p < 0.05).

Figure 2. Lactobacillus bulgaricus degradation of creatinine (n = 10). ^ap < 0.05 versus the serum control group, ^bp < 0.05 versus original strain.

Creatinine degradation rate: L. bulgaria compound mutant versus L. bulgaria single mutant

After three rounds of consecutive mutagenesis, the strain with the highest rates of creatinine decomposition was obtained. Strain from physical mutagenesis was labeled UV₃-11, and from chemical mutagenesis labeled DES₃-29, from compound mutagenesis labeled DU₃-12 (Table 1). Compared with the serum group and original strain UV₃-11 group, creatinine concentration decreased significantly in DES₃-29 group and DU₃-12 group (p < 0.05). The difference was not statistically significant between the UV₃-11 and DES₃-29 groups (p > 0.05). Compared with UV₃-11 group and DES₃-2 group, creatinine concentration decreased significantly in DU₃-12 group (p < 0.05). Compound mutations increased the bacterial creatinine decomposition rate higher than the single factor mutation.

Table 1. Creatinine degradation rate among Lactobacillus bulgaricus mutants.

Strain label	Creatinine (µmol/L)	Creatinine decomposition rate (1%)
Physical mutagenesis UV3-11	570.9 ± 11.6^a,b	8.71
Chemical mutagenesis DES3-29	571.6 ± 10.5^a,b	8.59
Compound mutagenesis DU3-12	557.2 ± 11.6^a,b,c	10.90
Original strain group	618.9 ± 7.6	1.04
The serum control group	625.4 ± 6.5

Notes: Compared with the serum, ^ap < 0.05; compared with original strain, ^bp < 0.05; compared with UV₃-11, DES₃-29, ^cp < 0.05.

Selection of the mutant with the best creatinine decomposition rate

The above-induced strains were first treated by compound mutation. The strain with the highest creatinine degradation rate (10.41%) was the target strain, which was marked as DUC1-4 and later as DUC2-4 (11.90%) after three rounds of compound mutation, ultimately the strain with the highest creatinine degradation rate (17.23%) was the target strain, which was marked as DUC₃-17 (Table 2).

Table 2. Lactobacillus bulgaricus’ creatinine degradation capability with compound mutation (n = 10).

Strain label	Creatinine (µmol/L)	Creatinine decomposition rate (%)
The first round of mutagenesis DUC1-4	736.7 ± 8.4^a,c	10.41
The second round of mutagenesis DUC2-4	688.5 ± 8.6^a,b,c	16.27
The third round of mutagenesis DUC3-17	680.6 ± 6.23^a,b,c	17.23
The strain-induced group	778.5 ± 4.7^a	5.33
The serum control group	822.3 ± 8.7

Notes: Compared with the serum group, ^ap < 0.01; compared with the strain-induced group, ^bp < 0.01; compared with DUC₁-4 group, ^cp < 0.01.

Testing DUC₃-17 strain for genetic stability in creatinine degradation

DUC₃-17 was cultured in uremic serum for five generations successively. The serum creatinine concentration decreased significantly compared with the control group, and the decline level was stable, indicating that the DUC₃-17's ability to degrade creatinine is relatively stable (Figure 3).

Figure 3. DUC₃-17 strain: genetic stability of creatinine degradation (n = 5).

Decomposition capability of DUC₃-17 strains

DUC₃-17 strain reduced the level of serum creatinine, blood urea nitrogen, phosphorus, parathyroid hormone, and homocysteine concentrations. Induced strain significantly reduced the level of serum creatinine, blood urea nitrogen, uric acid concentrations (p < 0.05), while the level of serum phosphorus, parathyroid hormone, and homocysteine showed no significant change (p > 0.05). The original strain showed no decomposition capability. The results are shown in Table 3.

Table 3. Resolving uremic toxin with DUC₃-17 (n = 10).

Strain label	The serum control group	Original strain	Strain-induced group	DUC3-17	Creatinine decomposition rate (%)
Creatinine (µmol/L)	460.2 ± 12.6	451.8 ± 5.1	435.4 ± 7.9^a,d	381.6 ± 3.6^a,c,f	17.23
Nitrogen (mmol/L)	21.4 ± 1.6	21.1 ± 0.9	15.5 ± 0.4^a,d	13.66 ± 0.19^a,c,f	36.02
Uric acid (mmol/L)	475.5 ± 16.4	474.3 ± 5.4	455.4 ± 9.2^b,d	428.5 ± 9.9^b,d,f	9.84
Phosphorus (mmol/L)	3.75 ± 0.22	3.75 ± 0.18	3.71 ± 0.20	3.16 ± 0.16^a,c,e	15.73
Parathyroid hormone (mmol/L)	10.5 ± 0.8	10.1 ± 0.6	9.9 ± 0.4	2.3 ± 0.5^a,c,e	78.26
Homocysteine (mmol/L)	27.7 ± 0.8	27.4 ± 0.5	27.1 ± 1.4	24.2 ± 1.6^a,d,f	12.69

Notes: Compared with the serum control group, ^ap < 0.01, ^bp < 0.05; compared with original strain group, ^cp < 0.01, ^dp < 0.05; compared with the strain-induced group, ^ep < 0.01, ^fp < 0.05.

Discussion

Intestinal bacterial therapy in the treatment of chronic renal failure is a new research hotspot.2,3 Ranganathan et al.4 had isolated a Sporosarcina pasteurii (sp) in the gut, which decomposed many kinds of uremic toxins. Its capacity to decompose urine toxin is close to hemodialysis, except for electrolyte imbalance, however, a large number of SP bacteria are associated with dysbacteriosis and severe infections caused by bacteria carrying antibiotic resistant plasmids, and hence contraindicated for clinical application. Lactobacillus was the first natural bacteria for treating chronic renal failure,5 albeit with weak toxin clearance. In recent years, engineered bacteria have been used to clear urine toxins and used as biological agents. Currently, engineered bacteria only produced a single enzyme, such as urease6 and uricase.7 However, specific enzymes only decompose specific substrates. Bacteria that break down a variety of urinary toxins are greatly desirable to treat patients with chronic renal failure, harboring a variety of toxins.

Gene mutation is an important biological phenomenon8 and is the source of natural mutation and artificial mutagenesis. However, artificial mutation is associated with low mutation frequency, and uncertain direction and variation. Increasing the efficiency of mutagenesis and exploring directional mutagenesis is the focus of current research. Directional mutagenesis using high-tech industrial radiation and chemical induced mutation can control the direction of mutation. Based on the bacterial mutant’s advantages and disadvantages, the mutagen dose and mutagenesis methods are determined. Directional mutagens react with nuclear genes, which change the genetic structure or DNA sequence. Base deletion, insertion, and replacement are widely used to cultivate favorable genetic traits in plant breeding, agriculture and the food industry,9 and recently in the pharmaceutical industry. Deng Xiaoli et al.10 used UV + NTG mutagenesis combined with fosfomycin separation in the screening of a stable high yielding strain which produce fosfomycin. Using fosfomycin as directional inducer, and UV and nitrosoguanidine as mutagen fosfomycin production was increased from 1.15 mg/mL to 2.30 mg/mL, transforming production by 100% and the conversion rate by 10.16%. We used the serum of uremic patients as directional inducer. Original L. bulgaria was induced by the serum of uremic patients and mutated repeatedly by physical (ultraviolet) and chemical (DES) methods. We used creatinine decomposition rate as an observed index, and selected the strains which most decreased the concentration of the creatinine as engineered bacteria. Uremic serum contains almost all toxins related to uremic symptoms, organ damage, and biochemical abnormalities. Therefore, such serum-induced bacterial mutants may have the ability to produce the same kind of urinary toxin enzymes. Existing mutagenesis studies focused on induction of a high yielding strain, which only produced certain enzyme. In our study, we aimed at inducing a variety of enzymes by mutation. Our results show that untreated lactobacilli do not possess the ability to decompose creatinine, urea, uric acid, phosphate, parathyroid hormone, and homocysteine, but after induction by uremic serum containing high concentrations of creatinine for 20 generations, lactic acid bacteria exhibited the ability to degrade creatinine, urea nitrogen, and uric acid, but not phosphorus, parathyroid hormone, and homocysteine. The results suggest that enzymatic degradation of lactic acid creatinine, urea nitrogen, and uric acid can be induced by its substrate, but not the other three kinds of toxins. It also suggests that changing mutagenesis technique is the key to improving enzyme decomposition rate. The induced strains received three cycles each of UV mutagenesis, DES mutagenesis, and DES-UV compound mutation. The results showed that the L. bulgaria that received compound mutagenesis manifested a stronger decomposition rate of creatinine than the strains receiving single factor mutagenesis. The L. bulgaria which received many cycles mutagenesis was stronger than the strains that received single mutation. Eventually we obtained a mutant DUC₃-17 whose creatinine decomposition rate significantly increased compared with the original strain: Creatinine decomposition rate was 17.23% with an increase of about 6.33% compared to DU3-12, a primitive L. bulgaria receiving compound mutation. It increased by almost 11.90% compared to DU3-12 that was induced by uremic patients’ serum. It increased by about 16.19% compared to original L. bulgaria without treatment. In five generations, its decomposition rate was still stable. The bacterial strain degraded creatinine, urea nitrogen, uric acid, phosphorus, parathyroid hormone, and homocysteine. Our result indicated the benefits of exploring intestinal bacterial therapy in the treatment of chronic renal failure. Further experiments are required to confirm the bacterial decomposition rate of various urinary toxins. We will undertake in vivo studies to confirm its use to treat kidney failure.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This study was supported by a specialized research fund from the National Natural Science Foundation (Grant 81270853).