Quantification of Changes in Bifidobacteria and Lactobacilli Colony-forming Units During Rifaximin Treatment and Synbiotic Gel in Healthy Volunteers

Summary

This clinical trial evaluated counts of bifidobacteria and lactobacilli after 1 week of rifaximin treatment and a Synbiotic Gel composed of Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi07, with agave inulin, during the rifaximin treatment and 4 weeks after, compared with a placebo gel, showing that the Synbiotic Gel may help maintain lactobacilli and bifidobacteria.

Abstract

Although the gut microbiota is generally stable, it can be influenced by antibiotic treatment. Rifaximin, which is used to treat gastrointestinal disorders, is frequently administered with probiotics to reduce the risk of dysbiosis. Lactobacillus acidophilus interacts with intestinal cells and uses prebiotic compounds. Bifidobacteria, particularly Bifidobacterium infantis ssp. lactis Bi07, is a probiotic that has been associated with beneficial effects in the gut. In vitro studies have demonstrated the superiority of agave fructans over chicory inulin. No studies have documented changes in fecal lactobacilli and bifidobacteria counts following the concomitant administration of rifaximin and synbiotic. Our objective was to evaluate changes in the CFU counts of gut bifidobacteria and lactobacilli in healthy volunteers after consumption of Synbiotic Gel, which contains Lactobacillus acidophilus NCFM, Bifidobacterium lactis Bi07, and agave inulin as a prebiotic, during 1 week of rifaximin treatment and for 4 weeks after. In a randomized, placebo-controlled, double-blind clinical trial, healthy participants without antimicrobial treatment in the previous month were randomized 1:1 in two groups (10 participants per group). The Synbiotic Gel group received 10.32 g of agave inulin as a prebiotic and 4 × 10⁹ CFU/g of Lactobacillus acidophilus NCFM and 4 × 10⁹ CFU/g of Bifidobacterium lactis Bi07 per day, for 5 weeks, and 200 mg rifaximin twice a day for the first week. The Placebo Gel group received placebo gel for 5 weeks and 200 mg rifaximin twice a day for the first week. Bacterial changes were estimated by quantifying CFU by microbiological assessment of feces. Five weeks later, the lactobacilli and bifidobacteria populations did not change. The detection of Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi07 in some stool samples suggests transient and partial colonization, highlighting the need for dose-response studies with this synbiotic product.

Introduction

The human gut microbiota is a densely populated community that includes permanently present native species and other transient microorganisms. Although its composition can vary between individuals, it is generally considered stable. However, it can be influenced by age, infection, and antibiotic treatment^1,2,3. Rifaximin, a broad-spectrum antibiotic with low intestinal absorption, is used to treat intestinal disorders and can alter microbiota. Probiotic bacteria from the genera Lactobacillus and Bifidobacterium are used to mitigate these effects².

Strains of Bifidobacterium spp. have been reported to be rifaximin-resistant², which has an interesting effect of promoting a gut biota containing populations of Bifidobacterium spp. in sufficient numbers for beneficial probiotic effects.

Moreover, Lactobacillus acidophilus has been widely recognized for its ability to inte​ract with the gastrointestinal epithelium⁴ and its capacity to utilize prebiotics, such as polysaccharides, oligosaccharides, and fructooligosaccharides. However, only a few probiotic strains, such as Lactobacillus acidophilus North Carolina Food Microbiology (NCFM) strain, have been identified. Therefore, studying their in vivo effects is important, as is the presence of Bifidobacterium lactis Bi07 in the gastrointestinal tract, as has been associated with various health-promoting benefits^2,5. These probiotics require a prebiotic to grow optimally, which is why a synbiotic containing both a probiotic and a prebiotic works synergistically; that is, its favorable effect is higher than that of the probiotic alone. Together with prebiotics, which serve as substrates for probiotics, this symbiosis is expected to enhance the survival of probiotic microorganisms in the gastrointestinal tract, leading to greater restoration of the microbiota⁶.

An important source of prebiotics is agave fructans, which have been reported to have superior in vitro effects on the microbiota than those of chicory root (Cichorium intybus L.)⁷. Studies have reported the effects of probiotic consumption; for instance, Engelbrektson et al. evaluated whether probiotic consumption during and after antibiotic administration could reduce the alterations in the microbiota caused by antibiotics⁸. They found that the group that received the probiotic blend showed a faster recovery of normal microbiota than those without it⁸. Furthermore, Koning et al. studied dysbiosis in the intestinal microbiota of 40 healthy volunteers treated with 500 mg amoxicillin twice daily for 7 days and the possible recovery of the microbiota following the consumption of a probiotic blend. The results showed that amoxicillin treatment affected the composition of the native microbiota. When a probiotic blend was administered, the microbiota significantly recovered after two months of consumption⁹.

However, the literature reports that no studies have investigated the effect of synbiotic consumption (with agave fructans as a prebiotic) on intestinal microbiota during and after antibiotic administration, such as rifaximin; we found only studies with probiotics. Therefore, this study evaluated changes in the CFU counts of gut bifidobacteria and lactobacilli in healthy volunteers after consumption of Synbiotic Gel, which contains Lactobacillus acidophilus NCFM, Bifidobacterium lactis Bi07, and agave inulin as a prebiotic, during 1 week of rifaximin treatment and for 4 weeks after.

Protocol

The protocol was approved by the Institutional Committee for Biomedical Research in Humans (CIIBH) of the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubiran (INCMNSZ) (REFERENCE 1734). All participants provided informed consent before being included in this study.

Study design and population

This was a randomized, placebo-controlled, double-blind clinical trial with a 5-week follow-up period. Workers of the INCMNSZ were invited to participate in the study, and healthy volunteers aged 20–50 years without antimicrobial treatment in the previous month who accepted and signed the informed consent form were included between 2011 and 2012. Study subjects were randomized 1:1 to the Synbiotic Gel group with 10.32 g of agave inulin as a prebiotic and 4 × 10⁹ CFU/g of Lactobacillus acidophilus NCFM and 4 × 10⁹ CFU/g of Bifidobacterium lactis Bi07 per day, contained in 4 sachets for 5 weeks, and 200 mg rifaximin twice a day for the first week. The Placebo Gel group received 4 packs of 15 g of placebo gel, with the same presentation and sensory characteristics as the Synbiotic Gel, for 5 weeks, and 200 mg rifaximin twice a day for the first week (Figure 1).

Clinical evaluation

In both study groups, symptoms and stool appearance were assessed using the Bristol Scale. Weight, height, and body mass index (BMI) were also measured. A diary was used to evaluate the consumption of synbiotic gel, placebo gel, and rifaximin and to record the presence or absence of side effects related to gel ingestion.

Fiber consumption

Fiber intake was assessed using a food diary and a 24 h recall each week in both groups.

Treatment Adherence

Adherence to rifaximin and gel consumption was evaluated by counting the number of rifaximin doses and the number of sachets of synbiotic and placebo gels consumed per participant. The presence of Lactobacillus acidophilus and Bifidobacterium lactis was detected using Polymerase Chain Reaction (PCR).

Microbiological Assessment of Feces

At visit 0 and weekly for 5 visits (6 visits) in each group, the numbers of Lactobacilli, Bifidobacteria, and Enterobacteria were evaluated in 120 fecal samples by the plate count method. This method measures colony-forming units (CFU) by placing diluted samples onto specialized solid media¹⁰. Stool samples were collected at the start of each visit.

To identify microorganisms, fecal samples were collected and stored in BBL tubes. The PBG vial was used for anaerobic bacteria, and fecal samples were transported in a Styrofoam cooler containing frozen gel packs to maintain a low temperature before processing in the laboratory. The cap of the PBG vial for coproculture was removed, and the stool sample was collected using a swab, ensuring that the swab was impregnated with the sample. The applicator was then placed into a tube containing a solid culture medium.

For anaerobic bacteria, the vials were weighed, and 5–10 g of stool was collected using a spoon. The stool was then mixed with the contents of the vial and tightly sealed. For sample processing, the vials containing the samples and sterile Stomacher bags were weighed. The sample was emptied into a bag, centrifuged for 60 s, and then 4.5 mL of 0.1% peptone water was added. Subsequently, 500 µL of the sample was transferred to a vial and vortexed to homogenize the mixture. After 100 µL of the sample was inoculated in duplicate on each plate of the specific selective medium, Man Rogosa Sharpe (MRS) medium for lactobacilli and DP Beerens medium with cysteine + cycloheximide (to eliminate unwanted flora) for bifidobacteria, up to dilution 8 (dilution factor 0.1 mL). For enterobacteria, MacConkey medium was used at dilutions 5–8 (dilution factor 0.1 mL), where bacterial colonies were separated and quantified¹¹. Each medium powder was dissolved in purified distilled water, the solution was heated to boiling to ensure complete dissolution, and it was sterilized in an autoclave at 121 °C for 15 min. The mixture was cooled to 46.5 °C to perform the pour plate technique, pouring 1 mL per plate in duplicate into sterile Petri dishes containing 15–20 mL of culture medium, and allowing the plates to solidify. Samples inoculated were incubated at 37 °C in a CO₂-enriched atmosphere in anaerobic GasPak systems to maintain anaerobic conditions for 24–48 h. The colony-forming units (CFU) were marked with a pen and counted¹². The following formula was applied:

CFU/mL = (number of colonies × total dilution factor)/volume of culture plated (mL)

Identification of morphological characteristics of lactobacilli and bifidobacteria

Gram Stain was used to identify the specimen's morphological characteristics. A sample of bacterial colonies from each strain was placed on a microscope slide, stained with crystal violet for 1 min, and then rinsed with distilled water. An iodine solution was applied for 1 min and then rinsed with distilled water. An alcohol-acetone solution was used for 10 s as a decolorizer and rinsed with distilled water. Finally, safranin was applied as a counterstain for 1 min and rinsed with distilled water. The microscope slides were observed under an optical microscope using immersion oil with a 100x objective.

Total quantification was performed using conventional polymerase chain reaction (PCR) on randomly selected fecal samples from five subjects per group, obtaining a total of 60 samples over 5 weeks. Isolates from culture media were processed for genetic material extraction. The PCR was performed using universal bacterial primers to amplify a fragment of the 16S rRNA gene¹³. PCR products were purified and sequenced. The sequences were verified and submitted to the GenBank database to confirm the presence of lactobacillus and bifidobacterial strains isolated from the fecal samples.

Statistical analysis

Data are presented as absolute and relative frequencies or median (p25th-P75th). The cumulative severity of symptoms was determined using the Likert score (mild, 1; moderate, 2; severe, 3) at each follow-up. Intergroup comparisons were performed using the Wilcoxon rank-sum test and chi-square or Fisher’s exact test. Intra-group comparisons between weeks 0 and 5 were performed using the Friedman test and multiple comparisons with Durbin-Conover. Statistical significance was set at P < 0.05.

Results

Twenty healthy volunteers were randomly divided into the Synbiotic Gel group (n = 10) and Placebo Gel group (n = 10) (Figure 2 and Table 1). In the Synbiotic Gel group, seven were men, and three were women, with a median age of 31 years (range, 27–35 years); in the Placebo Gel group, three were men, and seven were women, with a median age of 33 years (range, 28–38 years). The groups were similar in terms of age, sex, weight, and BMI. Treatment adherence was ...

Discussion

In the present study, we found a significant reduction in enterobacterial counts in the fecal samples of subjects in the placebo group from the first week of intervention and in subjects in the Synbiotic Gel group in the first and second weeks. This change can be explained using rifaximin, which has been described as mitigating pathogenic bacterial overgrowth¹⁴. In our study, the Synbiotic Gel group following rifaximin showed a positive effect: the median population of Lactobacillus strain did not...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BBL tube	NA	NA	Any brand can be used
DP medium	NA	NA	Any brand can be used
Frozen gel packs
MacConkey medium	NA	NA
MRS medium	NA	NA	Any brand can be used
PBG vial			Phosphate buffer, gelatine, salts 0.05% cysteine as reducing agent for02
Petri dishes
Placebo Blend	KuragoBiotek-BabyCat.	NA	Same physical and sensory properties as the sym iotic gel, but without the active ingredients
Styrofoam continer
Symbiotic blend	KuragoBiotek-BabyCat.	NA	Non-dairy product composed of Lactobacillus acidophilus NCMF and Bifidobacterium infantis Bi-07 (2 x 109 CFU/g), and 17.2 g of agave inulin as a prebiotic in a pack of 15 g