In Vitro and In Vivo Survival and Colonic Adhesion of Pediococcus acidilactici MTCC5101 in Human Gut

The present study aims to investigate the probiotic nature of Pediococcus acidilactici MTCC5101 by an in vitro assay of bacterial adherence to intestinal epithelial cells of human gastrointestinal (GI) tract using Caco-2 cell line. Further to assess the in vivo survival in the GI tract, oral feeding was carried out with the help of 10 healthy volunteers. The effect on wellness was assessed by studying blood biochemical parameters of the volunteers. The survival of the bacteria was assessed using PCR-based detection of P. acidilactici MTCC5101 in fecal samples. The probiotic nature of P. acidilactici MTCC 5101 was strengthened by its adherence to the intestinal epithelial Caco-2 cell line in the in vitro SEM observations. Oral feeding study for assessing the survival of bacteria in GI tract of volunteers showed the strain to be established in the GI tract which survived for about 2 weeks after feeding.


Introduction
Human intestinal microbiota is a metabolically active microbial environment that is relatively stable in the guts of healthy individuals [1]. The commensal intestinal flora of humans and animals includes the genera Lactobacillus, Pediococcus, and Lactococcus [2,3]. Strains of these genera are frequently used on large scale as starter cultures in food industries because of their generally recognized as safe (GRAS) and probiotic status [4][5][6].
Probiotics are known to exert protective influence in the human intestine through various mechanisms and represent promising applications in prophylaxis and therapy. It has also been acknowledged by WHO that probiotic microorganisms when administered in adequate amounts confer a health benefit to the host [7]. Improvement of health status by probiotics can be explained by the fact that these bacteria reduce production of toxic substances, aid in production and absorption of vital nutrients, stimulate gastrointestinal activity as well as immunity, and reduce colonization of anaerobic pathogens by competitive exclusion [8,9].
For all the health giving effects, the probiotic bacteria should also be capable of surviving while passage through the GI tract. It is essential for them to overcome highly acidic environment of stomach, digestive enzymes, and bile salts of the small intestine, which thus become important selection criterion for probiotic strains. Apart from this, adherence of bacterial cells to intestinal epithelial cells and/or mucus is considered to be a desirable feature of probiotics, as it promotes enhanced gut residence time, pathogen exclusion, and interaction with host epithelial and immune cells [10,11].
Pediococcus acidilactici MTCC5101 is an acid and bile tolerant probiotic strain that secretes a potent antibacterial bacteriocin designated as Pediocin CP2. Pediocin CP2 exhibits a wide range of antimicrobial activity against Grampositive, Gram-negative bacteria as well as fungi [12]. Genes encoding production of Pediocin CP2 are localized to the ped operon present on the 8.9 kb plasmid pCP289 of P. acidilactici MTCC5101 [13][14][15]. These properties make this sp. of Pediococcus an attractive prophylactic and therapeutic agent against pathogenic bacteria in GI tract. However, the final confirmation of its probiotic nature can come only from human trials. It is these trials that provide evidence of the survival of the probiotic strain in vivo and provide the required basis for a credible claim.
The present study aims to investigate the probiotic nature of P. acidilactici MTCC5101 by an in vitro assay of bacterial adhesion to intestinal epithelial cells of human GI tract using 2 BioMed Research International Caco-2 cell line. Further, to assess the in vivo survival in the GI tract, oral feeding was carried out with the help of 10 healthy volunteers. The effect on wellness was assessed by studying blood biochemical parameters of the volunteers.

Bacterial Strain Procurement and
Maintenance. Pediococcus acidilactici MTCC5101, characterized in the laboratory of Department of Biotechnology, Punjabi University, Patiala [12], was used in the present study. It was revived and maintained at 37 ∘ C for 18-24 h under microaerophilic conditions in de Man, Rogosa, and Sharpe (MRS) broth [16].

In Vitro Adhesion Assay of Pediococcus acidilactici MTCC5101.
The enterocyte-like Caco-2 cells were obtained from National Centre for Cell Science (NCCS), Pune. Cells were routinely grown in Dulbecco modified Eagle's minimal essential medium containing 25 mM glucose, 10% fetal bovine serum, and 1% antibiotic solution containing penicillin and streptomycin (Himedia) [15].
The adherence of pediococci to Caco-2 cells was examined as described previously by Bernet et al. [17] with a few modifications. For the adhesion assay, monolayers of Caco-2 cells were prepared on glass coverslips placed in sixwell tissue culture plates seeded at a concentration of 10 6 cells/mL. Experiments were carried out at 37 ∘ C in a 5% CO 2 atmosphere. The culture medium was changed every alternate day and monolayers at late postconfluence stage (after 15 days) were used for adhesion assay. To begin with the assay, monolayers were washed twice with phosphatebuffered saline (PBS, pH 7.2). 1 mL bacterial culture containing one million CFU/mL suspended in PBS was added to the monolayers on glass coverslips placed in tissue culture plates containing DMEM without antibiotic solution. The plates were incubated for 1 h in 5% CO 2 at 37 ∘ C. After 1 h, the monolayers were washed five times with sterile PBS and fixed for SEM observations. The adhesion assay was repeated thrice.

Scanning Electron Microscopy (SEM).
For SEM analysis, Caco-2 cells cocultured with bacterial cells were fixed for 1 h with 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4) at room temperature. The cells were washed twice with PB and postfixed for 30 min with 2% osmium tetroxide in PB. After fixation, cells were again washed thrice with PB and then dehydrated in a graded series (30,50,70,80,90, and 100%) of ethanol [17]. Instead of critical point dryer, cells were treated with 100% hexamethyldisilazane for 10 min and coated with gold [18]. The specimens were examined with a JEOL JSM-6610LV scanning electron microscope at Indian Institute of Technology, Ropar, Punjab, India. A number of bacterial cells adhered onto Caco-2 cells were counted on various microscopic fields per coverslip while performing SEM. The values were represented as average number of bacterial cells adhered per 100 Caco-2 cells.

In Vivo Study
Design. The study was approved by the Institutional Clinical Ethics Committee vide clearance no. ICEC/3/2011. Experiments were strictly performed according to the guidelines of Indian Council of Medical Research for conducting research on human trials. Ten healthy volunteers were selected after taking their informed consent. It was a controlled study consisting of parallel 6 weeks trials, with 4 weeks of intervention and 2 weeks wash-out period.

Preparation of Probiotic
Buttermilk. The overnight grown P. acidilactici MTCC5101 cells were harvested by centrifugation (6000 rpm, 10 min) and resuspended slowly in PBS (pH 8.0). Cells at a concentration of 10 8 -10 10 cells/mL were added to fresh buttermilk as medium for oral feeding study (Verka Co. Ltd., Patiala, India). Cell counts were taken on a haemocytometer [19] and after thorough microbiological examination, cells at an average dosage level of 10 8 -10 10 cells/mL/day were fed to 10 healthy subjects in 1 mL buttermilk base, administered once a day for 4 weeks continuously [20]. Equivalent amount of buttermilk without P. acidilactici MTCC5101 was fed to a group of 3 control volunteers which served as negative controls. The subjects were asked to abstain from taking other fermented milk products and supplements with probiotics during the trial period. They were explained how to collect, store, and deliver the fecal samples. Participants were asked about changes in their wellbeing and/or had taken any kind of medication during study period.

Collection and Analysis of Fecal Samples.
The fecal swab samples were collected using ear buds in 2 mL of sterile normal saline (0.85% NaCl) after feeding trials of 5, 10, 20, and 30 days [38]. The fecal suspension was cultured in MRS broth for bacterial enumeration for 18 h at 37 ∘ C. Cell pellets were harvested by centrifugation and were stored at −20 ∘ C for plasmid DNA isolation.

Molecular Identification of P. acidilactici MTCC5101
in Fecal Samples. A metagenomic approach was followed for the isolation of plasmid DNA from fecal samples [39]. Isolated DNA was stored at −20 ∘ C until further use and its quality, quantity, and purity were determined using agarose gel electrophoresis and absorbance measurements on spectrophotometer (UV mini-1240, UV-Vis spectrophotometer, Shimadzu Corporation). PCR was performed in PROGENE (Techne) using forward primer 5 -CTGCGTTGATAGGCCAGGT-3 and reverse primer 5 -ACCTTGATGTCCACCAGTAGC-3 which are specific for 323 bp fragment of pedA gene located on the cryptic plasmid pCP289 of P. acidilactici MTCC5101 [13]. The amplification program consisted of 1 cycle of 94 ∘ C for 3 min and 30 cycles of amplification (94 ∘ C for 1 min, 63 ∘ C for 1 min, and 72 ∘ C for 1 min).

Bacteriocin Activity Assay.
Well diffusion assay was performed as per standard methodology of Sarkar and Banerjee [40]. MRS hard agar (1% w/v) was overlayed with MRS soft agar (0.75% w/v) preseeded with approximately one million cells of indicator Enterococcus faecalis. Wells were filled with heat inactivated supernatant of MRS grown fecal   count data that indicates detectable levels of P. acidilactici MTCC5101 in all the volunteers after a 4-week intervention. Figures 2(a) and 2(b) depict agarose gel analysis of 323 bp fragment of partial pedA gene amplified from isolated whole LAB plasmids of 5th and 10th day's fecal samples. Intensity of the PCR amplicon is lesser in the 5th day's samples which show that the establishment of the probiotic strain requires a little longer time to persist and colonize in the human gut. The fragment of the expected size was obtained in nearly all 10th day's samples and the bands in agarose gels were more prominent as compared to 5th day which confirms that the pediococci have started gut colonization. A similar pattern is observed in the 20th and 30th day's fecal samples on 2% agarose (Figures 2(c) and 2(d)). The band intensity was more prominent as compared to 5th and 10th day samples which indicate that the strain has colonized in the GI tract of volunteers, hence proving that colonization progresses in a time-dependent manner.

Bacteriocin Activity Assay.
To check for pediocin CP2 profile in the mixed fecal cell cultures of 30th day, standard well diffusion assay was carried out using E. faecalis as the indicator strain. After 24 h, definite zone of inhibition was seen in the plates that also confirms our previous findings on successful establishment of P. acidilactici MTCC5101 in GI tract of volunteers (Figure 3).

Wellness Parameters Performed.
Routine blood tests were performed to estimate the effect of probiotics on wellness parameters of volunteers. The tests include WBC and RBC counts, levels of Hb in blood, bleeding time, and clotting time (Table 3). Haematological survey was carried out before as well as after feeding trial to estimate the effect of probiotics on some of the wellness parameters of test subjects. Results indicated a small yet insignificant increase in the values of RBC counts and Hb levels of subjects. The findings confirm the safe oral consumption and health improvement capacity of probiotic strain.

Discussion
Bacterial adhesion to epithelial cells in gut is initially based on nonspecific physical interactions between the two surfaces [41,42]. After primary attachment to epithelial surface, secondary interactions between bacterial adhesins and complementary epithelial receptors play a key role in adhesion of bacterial cells to intestinal mucin and enterocytes [22,[43][44][45][46][47]. Since there is difficulty in studying bacterial adhesion in vivo, intestinal cell lines are widely used as in vitro models for assessment [48]. We have used a well-characterized cultured colon carcinoma Caco-2 cell line displaying typical features of enterocytic differentiation in the form of villi to study adhesion of P. acidilactici MTCC5101. A strong adherence of probiotic strains to intestinal epithelial cells has been reported previously in a number of studies (Table 4). In a recent study by Jensen et al. [49], it has been reported that the adhesion capacity of probiotics varies from species to species as a variation from 1% to 25% has been observed in case of 18 known probiotic lactobacilli and pediococci. B. longum SBT2928 Healthy volunteers 10 11 40-day trial; survival in the gut [28] Lactobacillus GG Children with acute infectious diarrhea 10 9 Prophylactic; reduction in duration of diarrhea [29] L. casei subsp. rhamnosus Lcr35 Healthy volunteers 10 8 -10 12 1-week study; successful colonization of gut [30] L. reuteri ATCC 55730 Healthy volunteers 10 8 28-day trial; gut colonization; immune modulation [31] B. animalis subsp. lactis BB-12

Present study
The current study provides clear evidence that P. acidilactici MTCC5101 adheres strongly to villi of Caco-2 cells. These results further strengthened the claim of this strain for selection as a probiotics for human use. Oral consumption of probiotics has been advocated with prophylactic and curative properties that have been observed in case of intestinal disorders such as antibioticinduced diarrheal disease, viral and bacterial diarrhea, lactose intolerance, and inflammatory bowel diseases [27,29,34,[50][51][52][53][54][55]. Recently, there has been accumulation of evidence from rigorous clinical studies on well-characterized probiotics having real health-promoting properties [56,57].
Although minimum effective dose is not known exactly, usually an oral dose of 10 6 CFU/day or more than this has been followed in most studies (Table 5). Previous clinical and colonization studies prove successful intestinal colonization of probiotic bacteria and prevention of diseased condition. A comparative analysis of such in vivo clinical studies demonstrates that the persistence time of probiotic bacteria in gut varies from strain to strain (Table 5). The relative strain-specific persistence in vivo correlates accurately and significantly with in vitro outcomes as evident from a recent study on Lactobacillus plantarum [58]. Studies on probiotic Lactobacillus casei strain DN-114 001 and L. casei strain Shirota have proven the capacity of these strains to survive and colonize human gut [59,60]. Persistence of probiotic strains in GI tract is also demonstrated by their bile and acid resistance properties, as shown in earlier study carried out on the present strain [12,[61][62][63][64].
Survival in the GI tract depends on both the strain and the food matrix involved [65]. Fecal recovery of several probiotic strains has been demonstrated in different food matrices, including fermented milk and yoghurt [66,67], fruit drinks [68], supplements [36,69], and infant formula [65]. The survival of probiotics in human GI tract should lead to shedding of live cells in fecal samples which can be detected using quantitative methods like PCR [37,70]. In the present parallel, controlled human intervention study, in vivo persistence and colonization of P. acidilactici MTCC5101 in GI tract provides a clear evidence for intimate interactions between the selected probiotic bacteria and intestinal mucosal surface. These interactions allow probiotic strain to persist in gut for a considerable time period, regardless of the dietary and physiological differences among individuals selected in the study. Furthermore, results indicate that buttermilk is a suitable carrier medium for P. acidilactici MTCC5101 strengthening the use of buttermilk as a probiotic product.
Abnormal blood biochemical parameters are an indicator of a number of clinical disorders. Oral consumption of probiotics has not been linked to any adverse subclinical effects on blood biochemistry so far [71, this study]. Probiotics have been reported to enhance absorption of essential vitamins and minerals from the diet into the body [72,73]. The enhanced absorption of vitamins and minerals has led to improved haematological environment and gut health. A slight increase in RBC counts and Hb levels of volunteer subjects after oral feeding with probiotics strain was observed. Both in vitro models and in vivo studies have suggested the successful establishment of P. acidilactici MTCC5101 in human gut that is being proposed herein with the possibility of providing beneficial health effects to the host.

Conclusions
In conclusion, P. acidilactici MTCC5101 can survive passage through the human GI tract when administered orally in a buttermilk food base. Overall, results indicate that P. acidilactici MTCC5101 is a safe and potent probiotic strain with strong adhesive and health-improving characteristics. The findings suggest an opportunity for successful use of P. acidilactici MTCC5101 in functional food applications in future.