|Year : 2013 | Volume
| Issue : 1 | Page : 31-35
|Effect of probiotics on the fecal microflora after radiotherapy: A pilot study
Faculty of Health Care, Catholic University in Ružomberok, Institute of Clinical Microbiology of Central Military Hospital Faculty Hospital, Ružomberok, Slovakia
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|Date of Web Publication||6-Aug-2013|
| Abstract|| |
Background and Aim : The development of gastrointestinal symptoms following pelvic radiotherapy depends on morphological and functional modifications of the intestinal epithelium after radiation. The aim of this study was to evaluate and compare the effects of preventive administration of the preparation ''5'' Strain Dophilus and Hylak on the fecal microflora after radiotherapy in patients during radiotherapy. Materials and Methods : Fourteen patients were randomly selected and subdivided into two groups: The first group was administered ''5'' Strain Dophilus (L Group) and the second group was administered Hylak (H Group). Radiation was delivered by a Cobalt 60 unit by using the four field box technique. The doses were divided into 2 Gy per day over 5 to 7 weeks to give the total cumulative dose of 50 Gy (2 Gy/day). High risk patients (e.g., patients with prostate cancer), received dosage 65 67 Gy (2 Gy/day). Results : Both experimental and clinical studies have shown that probiotics can effectively modulate intestinal inflammation by altering the composition and the metabolic and functional properties of gut indigenous flora. Conclusions : Many bacteria were found to be sensitive to irradiation. It would be necessary to check the possible effects of cytostatics on bacteria in larger studies.
Keywords: Fecal microflora, probiotics, radiotherapy
|How to cite this article:|
Timko J. Effect of probiotics on the fecal microflora after radiotherapy: A pilot study. Indian J Pathol Microbiol 2013;56:31-5
| Introduction|| |
The radiation therapy plays an important role in curing or alleviating the effects of malignant disease. However, irradiation in almost all cases affecting the adjacent healthy tissue and therefore the tolerance of healthy tissue is a major limiting factor in radiation therapy.  The transient symptoms caused by damage to the intestinal mucosa occur in the majority of patients after irradiation of abdominal or pelvic tumors. There is a direct correlation between the dose of radiation used and the incidence of intestinal complications.  The usual complaint of the patients are diarrhea, nausea, and vomiting.
It is known that radiation causes radiation enteritis - changes in fecal microflora, intestinal mucosal permeability, and intestinal motility.  Interactions between the commensal bacteria and the epithelium influence the epithelial response to injury. A potential role of intestinal bacteria and their toxins in the appearance of acute radiation enteropathy has been described previously.  The findings reported by Crawford and Gordon  add a new dimension to among complex relationships in that the microbial organisms may suppress (or add) factors that mediate tissue radiosensitivity. The data presented in their article suggest that alteration of gut microbiota may be clinically useful to increase the resistance of the small bowel to radiotherapy.
They are also evidence that the initial intestinal microbial composition of each individual could be a determinant for developing postirradiation diarrhea.  Tracking changes in the number of facultative anaerobes was also previously observed.  The stability of the entire population of bacteria is an important presence of cellulolytic species. 
Mc Gough et al. reviewed the original studies in the management of gastrointestinal tract side effects in patients undergoing pelvic radiotherapy and found that probiotic supplementation may be beneficial in preventing symptoms. Some studies suggest that probiotics can modify radiation-induced injury and symptom severity. ,, Probiotics may play a key role in the enhancement of host intestinal antioxidant defense systems. Probiotic bacteria with the ability to secrete extracellularly antioxidant compounds or to promote intracellular enforcement of antioxidant systems are close enough to fulfill the criteria of the ideal intestinal radioprotector. 
The aim of this pilot study was to evaluate and compare the effects of preventive administration of the preparation ''5'' Strain Dophilus and Hylak on the fecal microflora after radiotherapy in patients during radiotherapy.
| Materials and Methods|| |
Fourteen patients diagnosed with carcinoma of the colon (7), bladder (2), uterus (2), and prostate (3) were randomly selected and subdivided into two groups: The first group was administered ''5'' Strain Dophilus (L-Group) and the second group was administered Hylak (H-Group). The treatment arms were not balanced with gender and primary tumor site.
Study participants in L-Group were administered the probiotic preparation ''5'' Strain Dophilus (Swiss Herbal Remmedies LTD Ontario, Canada) with an enteric coating and containing five probiotic cultures (55% Lactobacillus rhamnosus, 20% Bifidobacterium adolescentis, 5% L. acidophilus, 5% Bifidobacterium longum, 15% Enterococcus faecium) with a count of 6 billion active bacteria/capsule at a daily dosage of 2 × 1 capsule.
Patients in H-Group were administered the Hylak Tropfen Forte (Merckle GmbH, Germany) preparation, i.e., cell-free fermentation products of Lactobacillus helveticus and gut symbionts (100 ml containing: 24.95 g Escherichia coli metabolita, 12.5 g Streptococci faecalis metabolita, 12.5 g Lactobacilli acidophili metabolita, 49.9 g Lactobacilli helvetici metabolita) in doses of 40 drops, three times per day.
Exclusion criteria were: Previous radiation treatments, current antibiotics therapy, the using of antibiotics during the previous 2 weeks, established gastrointestinal disease (chronic diarrhea, Crohn's disease, ulcerative colitis). Any patient whose medical condition required antibiotic therapy during RT was excluded from the group.
Radiation was delivered by a Cobalt-60 unit by using the four-field box technique. The doses were divided into 2 Gy per day over 5 to 7 weeks to give the total cumulative dose of 50 Gy (2 Gy/day). High-risk patients (e.g., patients with prostate cancer), received dosage 65-67 Gy (2 Gy/day).
The design of this study was approved by the Ethics Committee at the Central Military Hospital Ruzomberok. Participants signed a written informed consent form, according to the Helsinski Declaration.
Fresh fecal samples from 14 patients were collected in sterile bags and kept for not longer than 12 hours, at 4°C, before processing. Portions (0.5 g) of each stool were suspended in 4.5 ml PBS (8 g of NaCl, 0.2 g of KCL, 1.44 g of Na 2 HPO 4 , and 0.24 g of KH 2 PO 4 each per liter) and vortexed for at least 3 min to homogenize the sample. Next serial 10-fold dilutions were prepared, after which 0.1 ml samples of the each dilution were plated on the Wilkins-Chalgren agar (bifidobacteria setting) supplemented with mupirocin (100 mg/l) and glacial acetic acid (1 ml/l) according to Rada and Petr,  Rogosa agar (lactobacilli), Slanetz-Bartley agar (enterococci), Endo agar (coliform bacteria)-all agars were prepared according to instructions from Oxoid, England. Bifidobacteria were incubated in anaerobic jars (AnaeroJar, Oxoid) at 37°C for 3 days, lactobacilli and enterococci were incubated aerobically at 37°C for 48 hours and coliform bacteria at 37°C for 24 hours.
Identification of bifidobacteria was done by using microscopy, rapid test ID 32 A (bioMérieux, France); lactobacilli using microscopy, API 50 CHL test (bioMιrieux, France), and Anaerotest 23 (PLIVA-Lachema, Brno); enterococci using EN-coccus test (PLIVA-Lachema, Brno) and E. coli using the API 10S test (bioMérieux, France).
Rest of the suspension was centrifuged at 700 × g for 1 min to remove debris. The supernatant was diluted 1:3 with 4% (wt/vol) paraformaldehyde in PBS (PFA), fixed at 4°C for 16 hours, and stored in 50% (vol/vol) ethanol-PBS at -30°C until further analysis (FISH).
Residues from the fecal samples were frozen and stored at -30°C until nucleic acid extraction was performed (DGGE).
Analyses of Fecal Samples by Molecular Method
Fecal DNA was extracted using the QIAamp® DNA stool mini kit (Qiagen, USA). Briefly, 200 mg was taken from each frozen stool sample and placed immediately into ASL buffer. Each fecal sample was homogenized by vortexing. The mixture was then heated to 70°C for 5 min to obtain bacterial lysis. Fecal DNA was further extracted and purified following the manufacturer's instructions.
Denaturing gradient gel electrophoresis (DGGE) was used for the detection and identification of different bacterial species. The method begins with PCR amplification of 16S rRNA fragments with general primers for bacteria with a clamp: FP338cl (5'- CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCG CCG CCG CAC TCC TAC GGG AGG CAG CAG - 3') and reverse primer RP534 (5'- ATT ACC GCG GCT GCT GG - 3') according to Muyzer et al. and Kopecny et al. The PCR products were analyzed on DGGE gels (gradient from 45 to 65%) using a protocol based on the method of Temmerman et al.  The electrophoresis was performed at 60°C, and at 70 V for 14 hours. The gels were stained with SYBR Green I, and bands were detected under UV.
Nested PCR-denaturing gradient gel electrophoresis (nested DGGE) was used for the detection and identification of different bifidobacteria species. The method was performed with PCR amplification of 16S rRNA fragments with bifidobacteria-specific PCR primers.  The fragments were purified on QIAquick PCR purification Boxes Kit (Qiagen, USA). During the cleaning, 2 × preconcentration and used for PCR with primers and clamp as well as in the previous method.
Selected DNA fragments were isolated from the DDGE gels, subsequently amplified and sequenced for identification of bacterial species. DNA isolation consisted of sterile fragment cut from the gel and extraction of the cut piece of polyacrylamide gel with sterile H 2 O for 24 hours at 7°C. The V2-V3 region of the 16S rDNA was reamplified with PCR primers FP341 and RP 534.  Obtaining PCR products were cleaned using QIAquick® PCR purification kit (Qiagen, USA). Sequence of these PCR fragments was determined using BigDye® cycle sequencing kit (Applied Biosystems, UK). The sequences were automatically analyzed on ABI PRISM® 3100 Genetic Analyzer and then manually corrected. The assembled partial rDNA sequences were compared with sequences in the GenBank database using the BLAST algorithm. 
A specific fluorescence in situ hybridization (FISH) kit for Bifidobacterium spp. by the fluid method (RiboTechnologies, Groningen, The Netherlands) was used for the quantitative detection of bifidobacteria in feces.
Data were analyzed statistically. The significance of differences between the groups was evaluated by the Student's t-test (Microsoft Excel, version 2002).
| Results and Discussion|| |
The median age of the 14 patients was 64 ± 8 years. The gender ratio (male/female) was 11/3. Patients in H-Group received a higher radiation dose (62.8 Gy), compared with 51.8 Gy in L-Group. Number of patients with chemotherapy was too small for separate microbiological evaluation.
Determination of Selected Groups of Bacteria
Overview of results on changes in the numbers of different groups of bacteria can be seen in [Table 1]. The differences are non-significant due to low number of samples.
|Table 1: Total counts of the selected groups of bacteria from fecal samples|
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Relatively good compliance was achieved in the determination of bifidobacteria by culture and FISH methods. The differences were around 2.3%, a result that is at variance in both methods.
It was found that preparation Hylak increased numbers of bifidobacteria and ''5'' Strain Dophilus decreased their total number. The difference was evident in the numbers of measurements by FISH. This determination is more accurate because it is not limited by the critical numbers, where it is no longer possible to cultivate isolates. Given that the numbers of bifidobacteria should be between 10 8 to10 9 /ml, it is evident that the preparation ''5'' Strain Dophilus is not suitable for the stimulation of bifidobacteria in this type of patients.
Both drugs, Hylak and ''5'' Strain Dophilus, reduced the total numbers of lactobacilli. It is interesting that the decline in their numbers in the administration of ''5'' Strain Dophilus decline was nearly four times higher. Even from this perspective, the preparation ''5'' Strain Dophilus is less suitable.
Enterococci were in the feces of 10 5 -10 6 /ml. Both drugs increased their numbers slightly. Halyk stimulatory effect was lower in this case.
E. coli counts were increased following administration of Hylak. Conversely, ''5'' Strain Dophilus preparation of their numbers declined.
Denaturing Gradient Gel Electrophoresis
Microbiological analysis was completed by determining the spectrum of dominant bacteria using DGGE. Quantitative data on the number of individual species of bacteria cannot be obtained from the intensity of DGGE fragments. However, it is possible to obtain an overview of the number of dominant species of bacteria and those determined by sequencing.
The number of bacteria after exposure usually dropped by an average of 30%. This is reflected in the number of bands. The number of bands shows that the number of bacteria after irradiation generally decreased on average by 33.5%. Only one case was observed with an increase of 30% (L3). These changes usually correspond to the observed changes in overall numbers of bifidobacteria set using FISH or cultivation. The decrease in total numbers should be verified using cultivation.
Each species of bacteria were determined on the basis of similarity of DNA sequence fragments in the NCBI database. The similarity above 97% indicates a generic consistency and the similarity above 95% indicates gender conformity [Figure 1].
|Figure 1: DGGE 16S DNA fragments of bacteria from fecal samples irradiated pati ents Fragments marked with numbers were sequenced. Before and aft er irradiati on samples were collected: Samples 1 and 2 (L1 before and aft er RT), samples 3 and 4 (L2 before and aft er RT), samples 5 and 6 (H3 before and aft er RT), samples 7 and 8 (L3 before and aft er RT),|
samples 9 and 10 (L4 before and aft er RT), samples 11 and 12 (L6 before and aft er RT), sample 13 (L5 before RT), sample 14 (H1 before RT), sample 15 (L9 before RT), sample 16 (H6 before RT), sample 17 (H2 before RT), sample 18 (L10 before RT), sample 19 (H4 aft er RT), sample 20 (H5 after RT), sample 21 - ''5'' Strain Dophilus, sample 22 - Hylak
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Bacteroides ovatus disappears after radiotherapy in patients H3 L1 and L4. Like some Eubacterium spp., this occurred prior to irradiation in patients L4, L6, L5, H1, H6, H2, H4.
Oscillatoriales cyanobacterium is a stable species and if present, it remains the H3, L3, L4, L6, L5, H1, H6, L10, H2. The incidence of Ruminococcus bromii after irradiation increased in these patients, L2, L3, L4, H2, H4, H5. The counts of Lactobacillus jensenii increased after exposure of L2, L3 L5, H1.
On the basis of sequence, similarity was determined following bacterial species:
Bifidobacterium longum species were dominant in most patients. This corresponds to the composition of the bench for the elderly. These species are commonly present before and after irradiation. The spectrum of species is narrower in comparison with the microflora of infants.
On the basis of sequence, similarity was determined following bacterial species:
PCR primers used for lactobacilli were not specific enough to confirm presence of higher numbers of Lactobacilli. This is consistent with lower overall numbers of Lactobacilli detected by culture [Table 1].
These numbers were compared with bifidobacteria, two orders of magnitude lower. Lactobacilli were detected in patients who received Hylak but not in patients who were administered ''5'' Strain Dophilus.
Electrophoresis showed the presence of other bacterial species in the studied samples. The presence of Akkermansia muciniphila before irradiation indicates normal commensal microflora.  This strain in patients L1 and L2 disappeared after irradiation. As in the analysis of total bacteria Bacteroides ovatus disappears after radiotherapy in patients H3 L1 and L4. The presence of clostridia and Faecalibacterium prausnitzii was observed before and after irradiation.
The presence of pathogenic Shigella flexneri in patient L4 is indicated by intestinal problems. It correlates well with the significantly higher numbers (2.1 × 10 9 ) of cultivable facultative anaerobes E. coli [Table 1] and watery stool type. One type of E. coli was also observed at the L4 before irradiation.
Clostridium cellobioparum was observed after irradiation of L2, L6, and H1. This is a non-pathogenic type of cellulotic bacteria, which is common in the digestive tract of various animals and landfill sites. 
Enterococcus faecium isolates are common in the digestive tract of various animals, but can cause indigestion, inflammation of the urethra and skin. It is common for them to have resistance to certain antibiotics.  Their presence has been demonstrated in patients L5, L6, L9, H1, and H2.
It needs to be noted that the DGGE, though it has several advantages, also has certain limitations such as copy number (variants) and microheterogeneity of rRNA genes could result in overestimation of diversity and also closely related or unrelated strains may be similar to electrophoretic mobilities that may result in underestimation of actual diversity. Also, 16SrRNA is just one methodology, several others are also there.
We have demonstrated changes in the intestinal microbial community. Many bacteria were sensitive to irradiation and after irradiation, their numbers have fallen significantly. The observed changes are at the genus and species levels. Further studies should focus on deeper understanding of these changes. Furthermore, it is necessary to examine the possible effect of cytostatics and its effect on bacteria in larger studies.
It needs to be noted that this is a relatively new area of research and very few papers exist on this subject in the literature.
| Conclusions|| |
Future clinical studies in this area should initially focus on clarifying how intestinal microbial compositions shift in response to radiation. Since molecular tools are rapidly evolving, the identification of changes in microbiota composition will provide important new insights into the pathogenesis as well as novel treatments of gastrointestinal symptoms following radiotherapy.
Using this knowledge, strain-specific probiotics could be administered to increase bacterial species that best confer host radioprotection. Through genetic engineering, it is now possible not only to strengthen the effects of existing strains but also to create completely new probiotics which have the ability to produce directly or promote the intestinal release of specific agents.
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Institute of Clinical Microbiology of Central Military Hospital Faculty Hospital, Ružomberok, 034 26
Source of Support: None, Conflict of Interest: None
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