Culture, selection, isolation and characterization of lactobacilli and enterococci

Baby diapers (unidentified) containing fecal samples were collected from bins of the Bright Horizon day care center (Winston-Salem, NC). Fecal samples from 34 individual diapers (0.5 g) were resuspended in 5 mL of MRS medium (to isolate Lactobacillus strains) and LM17 supplement with 35 mM lactic acid in M17 medium (for Enterococcus strain selection), and incubated at 37 °C for 24 h. Subsequently, cell cultures were serially diluted, spread onto the corresponding selection medium agar plates, and at least 10 colonies from each sample were picked up after incubation at 37 °C for 12–24 h. Colonies were further purified by the streak plate method. We performed Gram staining52 to exclude gram-negative bacteria. We detected catalase activity using 30% of hydrogen peroxide and recording bubble formation, to eliminate gas-forming isolates.

PCR for virulence genes

Gram-positive and catalase-negative colonies were screened for the presence of virulence genes by PCR, using primers for various common virulence genes listed in Supplementary Table S5. Virulent genes including agg, gelE, cylM, cylB, cylA, esp, efaAfs, efaAfm, cpd, cob, ccf and cad were detected for Enterococcus and Streptococcus strains as described by Eaton and Gasson53. For Lactobacillus strains, besides gelE, esp, cylA and efaAfs, other virulent genes hyl, asa, ace, hdc, tdc and odc were included and detected as reported by Casarotti et al.54. PCR was carried out in a 20-µL reaction mixture with MyTaq™ Red Mix (Bioline) buffer. PCR products were detected on 1.0% agarose gel electrophoresis and using gel imaging system (Kodak Image Station 4000 R; Carestream Health Inc., Rochester, NY).

Antibiotic susceptibility assay

Antibiotic susceptibility was determined by disc diffusion tests using antimicrobial susceptibility test discs (BBL™ Sensi-Disc™, BD Life Sciences, USA) following the manufacturer’s instructions. Lactobacillus and Enterococcus strains were cultivated overnight in MRS and LM17, respectively, and 50 µL of cultures were spread onto MRS or LM17 agar plates. Antibiotic-impregnated discs containing tetracycline (30 µg), chloramphenicol (30 µg), kanamycin (30 µg), erythromycin (15 µg), rifampicin (30 µg), vancomycin (30 µg), gentamycin (10 µg), streptomycin (10 µg), amoxicillin (25 µg), or ampicillin (10 µg) were applied to agar plates inoculated with bacterial strains. After being cultivated at 37 °C for 16–18 h, the diameterd of complete inhibition zones were measured. Antibiotic susceptibility was recorded as resistant (R), intermediate (MS) and susceptible (S) according to the definition based on the zone diameter in the manufacturer’s manual.

Genetic identification of isolated strains

The selected isolates were confirmed for their genus- and species-level identity by sequencing of the 16S rRNA gene. The 16S rDNA from the selected isolates was amplified by colony PCR with the 27 F and 1492 R universal primers (27 F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492 R: 5′-GGTTACCTTGTTACGACTT-3′). The amplification mixture (20 μL) contained 10 μL PCR buffer (MyTaq® Red Mix 2×, Bioline, USA), 1 μL (10 μM) of each primer and 7μL of nuclease-free water. Single pure colony was picked up and added to 10 μL sterile ultra-pure distilled water (InVitrogen) and 1 μL of this suspension was added as a template into the PCR reaction mix. The PCR reaction conditions were: 95 °C for 4 min; 30 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 2 min; and extension at 72 °C for 10 min. Controls devoid of any template DNA were included in the amplification process to serve as a negative control. The integrity of the PCR products was verified by detection of single bands following electrophoresis on 1.0% agarose. The sequencing of amplified 16S rRNA gene from isolates was performed at GeneWiz LLC (NJ, USA); the resulting sequence data were aligned and analyzed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the identification was confirmed on the basis of the highest hit scores (Supplementary Table S1).

Probiotic attributes

Tolerance to low pH and bile salts

The resistance of short-listed bacterial isolates to low pH and different bile salt concentrations (Oxgall, Sigma-Aldrich, USA) was examined by monitoring the bacterial growth, as per our published methods55. Briefly, 900 μL of MRS or LM17 broth, which was adjusted to pH 3.0 or 7.0 (control) or supplemented with 0.3% (w/v) bile (Oxgall; Sigma-Aldrich), was inoculated with 100 μL of an overnight grown culture (≈109 cfu/ml), previously washed twice with PBS (pH 7.2). After incubation for 3 h (pH tolerance) or 4 h (bile tolerance) at 37 °C, 50 μL of serially diluted cultures was spread onto MRS or LM17 agar plates and incubated for 24 h at 37 °C, followed by colony counting (CFU 1 ). An aliquot of bacterial suspension was also collected and plated on MRS or LM17 agar plates (CFU 0 ) prior to inoculation into the broth. The percentage of viable bacteria was calculated as: Survival (%) = CFU 1 /CFU 0 × 100.

Cell surface hydrophobicity

The hydrophobicity of isolates to hydrocarbons was examined as per the method described earlier55. Briefly, the overnight grown culture was centrifuged at 5000 g for 10 min. The cell pellet was washed twice with PBS (pH 7.2), resuspended in PBS, and the initial absorbance was adjusted to 0.7 at 600 nm (Abs 0 ). Three mL of bacterial cell suspension was mixed with 0.6 mL of n‐hexadecane (5:1), vortexed for 2 min, and incubated at room temperature for 1 h. Following incubation and phase separation, the aqueous phase was decanted carefully and was subjected to the absorbance measurement (Abs 1 ) at 600 nm. The surface hydrophobicity was calculated as per the following formula: Hydrophobicity (%) = (1 − A 1 /A 0 ) × 100. Only those strains with considerable hydrophobicity and acid and bile tolerance were subjected to subsequent assays, including tolerance to simulated gastric and intestinal fluids and adherence to Caco-2 cells.

Tolerance to simulated gastric and intestinal fluids

Bile tolerance of selected isolates was examined as per the method described elsewhere56. Briefly, simulated gastric fluid was prepared by suspending (3 g/L) pepsin in 10 mL of sterile saline solution (0.85% NaCl, w/v). The pH was adjusted to 2.5. Cultures grown overnight (≈108 cfu/mL) were inoculated into the simulated gastric fluid and incubated for 3 h at 37 °C. Simulated intestinal fluid was also prepared by dissolving bile salts (0.3% w/v) and pancreatin (1 mg/mL) in 10 mL of sterile saline solution (0.85% NaCl, w/v). The pH was adjusted to 8.0. Cultures grown overnight (≈108 cfu/mL) were inoculated into the simulated intestinal fluid and incubated for 6 h at 37 °C. Viable counts were determined by serial-dilution and plating of the cultures on MRS or LM17 agar and incubation for 24 h at 37 °C.

Adhesion to Caco-2 cells

The assay for bacterial adhesion to Caco-2 cells was performed as per the previously described method57,58. Briefly, the cells were grown in 12-well tissue-culture plates in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Invitrogen) and penicillin–streptomycin (IU/mL or μg/mL; Sigma-Aldrich). The cells were cultured at 37 °C in an atmosphere of 5% (v/v) CO 2 and 95% air until a confluent (≈80%) monolayer was reached. Before the adhesion assay, the Caco-2 cell monolayers were washed twice with sterile phosphate-buffered saline (D-PBS, pH 7.4) and were suspended in antibiotic- and serum-free DMEM (2 mL per well) and incubated in 5% CO 2 at 37 °C for 30 min. The overnight grown probiotics bacteria were washed with PBS and suspended in 1 ml of antibiotic- and serum-free DMEM (≈108 cfu/mL). An aliquot of 100μL of this bacterial suspension was added to each well of the 12-well plate containing Caco-2 cells. The plates were incubated for 2 h at 37 °C in a 5% (v/v) CO 2 atmosphere. After the incubation, each well was washed five times with PBS (pH 7.4) and 1 mL of trypsin-EDTA (0.25%, Sigma-Aldrich, USA) was added to each well. After incubating the plate for 15 min at room temperature, the detached cells were gently aspirated several times to obtain a homogenous suspension. Finally, the cell suspension was serially diluted with PBS and a 50μL-aliquot of diluted homogenate was spread onto MRS or LM17 agar and incubated at 37 °C for 24 h. Bacterial colonies were counted (X 1 cfu/mL). Bacterial cells originally added to each well of 12-well plates were also counted prior to seeding into the plate (X 0 cfu/mL). The adhesion was calculated as: adhesion (%) = (X 1 /X 0 ) × 100. The assays were performed in three independent experiments.

Anti-pathogenic activity

LB (lysogeny broth) medium was used to grow pathogenic strains E. coli CFT073 and K. pneumoniae KPPR1. However, these strains were also tested for growth on MRS and LM17 and inhibitory effect of probiotic strains was tested through well diffusion and spot-on-lawn assays as described by Aspri et al.59 with minor modifications. Cultures of five Lactobacillus and five Enterococcus strains after 24 h cultivation were centrifuged at 12,000 g for 10 min. Supernatants were passed through 0.22 µm cellulose acetate membrane filters (2×) to obtain cell free samples and 150 μL of each supernatant was added into the holes dug on the LB agar plate spread with 50 μL of the pathogenic strain. The plates were incubated at 37 °C overnight, and inhibition zone diameters were recorded. In the spot-on-lawn assay, 10 μL of cell culture from each strain was used to spot on MRS or LM17 agar plates spread with indicator strains, and plates were cultivated overnight to check inhibition zones. Hydrogen peroxide strip analyses were performed using peroxide test strips (Quantofix® Peroxides-100; Sigma-Aldrich) according to the manufacturer’s instructions. Furthermore, heat sensitivity of the active substance was tested by comparing the antimicrobial activity before and after being treated at 70 °C for 1 h. Cell free supernatant was incubated with proteinase K (0.1 mg/mL) and testing the residual activity after 2 h through a well diffusion assay against E. coli CFT073 to determine involvement of proteinacious components in antimicrobial activity.

Mice studies

Preparation of probiotics for animal treatment

Pure probiotic strains were grown in corresponding media for 6–8 h to achieve the peak of log phase, and washed twice with PBS to remove traces of culture media. Equal counts (1 × 1011 CFU) of freshly prepared bacterial cells were pooled in three groups; (1) Lactobacilli: containing five selected strains of lactobacilli only; (2) Enterococci: containing five selected strains of enterococci only; and (3) Probiotic cocktail: containing all 10 strains i.e., five lactobacilli and five enterococci. The final dose (1 × 1011 CFU/mice/dose) was chosen on the basis of published literature60,61,62 and was optimized in 300 μL of PBS for oral gavage.

Single-dose study

C57BL/6 J mice were randomly divided into four groups (n = 6 per group): (1) control, (2) lactobacilli, (3) enterococci and (4) probiotic cocktail. Group 2, 3 and 4 were given a single dose of 1 × 1011 CFU/mice of lactobacilli, enterococci, and probiotic cocktail mixtures, respectively, by gavage. Control group was given equal volume of sterile PBS. Fecal samples were collected before treatment (0 h) and after 8 h, 1 day (24 h), 3 days and 10 days to analyze gut microbiome and SCFAs content.

Five-dose study

Similar to single dose study, C57BL/6J mice were randomized in four groups as described above and treated once-daily with 1 × 1011 CFU/mice of lactobacilli, enterococci and probiotics cocktail mixtures for 5 consecutive days by gavage; the control group was given equal volumes of sterile PBS once-daily by gavage for 5 days. Fecal samples were collected before treatment (0 h), after 5 days, 2, 3 and 5 weeks (i.e., 1, 2 and 4 weeks post-treatment).

All mice in both studies were allowed to eat normal chow and drinking water ad-libitum throughout the study. Body weight, food and water intakes were measured before treatments and at each time-point when fecal samples were collected in both studies. However, no significant differences were observed in any measures throughout the study period (Supplementary Fig. S9a,b). All the animal studies were conducted following procedures approved by the Wake Forest School of Medicine, Animal Research Program’s Institutional Animal Care and Use Committee (IACUC).

Fecal slurry fermentation assay

The culture media used for in-vitro fecal fermentation was prepared as described by Boler et al.63. Fecal samples were collected from 2 healthy donors, and snap-frozen in liquid nitrogen followed by storage at −80 °C until further use. Fecal collection is approved by Wake Forest School of Medicine’s Institutional Review Board. In anaerobic chamber, fecal samples were thawed, diluted (1:10 w/v) in anaerobic dilution solution (Nacl 5; glucose 2; Cystein-Hcl 0.3; g/L) and vortexed for 15 minutes for complete homogenization. The mixture was filtered through four layers of cheesecloth and was immediately used for inoculation of tubes containing media inoculated with probiotics. The final fecal suspension was divided into four groups: (a) control: fermentation media without probiotics, (b) lactobacilli-alone, (c) enterococci-alone, and (d) probiotic cocktail. Each probiotic combination (1 × 106 CFU/mL) was added to 26 mL fermentation media in sterile 50-mL falcon tubes. The tubes were kept inside the anaerobic chamber for 24 h to allow hydration of samples before starting the fermentation. Four mL of the freshly prepared fecal inoculum was added to each tube, and transferred to the built-in incubator in an anaerobic chamber at 37 °C with periodic mixing for 24 h. Samples were taken at 0, 9 and 24 h during fermentation, and pH of samples was measured using laboratory pH meter. The samples were centrifuged at 14000 g for 10 minutes at 4 °C; the supernatant was immediately frozen for SCFAs analysis while the pellets were stored at −80 °C for microbiome analyses. All experiments were repeated twice, each time in triplicate.

Fecal microbiome analysis

We conducted 16S rRNA gene amplification and sequencing as per our previously described methods64. In brief, nearly 200 mg of mice feces or fecal slurry pellets were used to extract genomic DNA using the Qiagen DNA Stool Mini Kit (Qiagen, CA, USA) per the manufacturer’s instructions. We modified the lysis temperature to 95 °C instead of 75 °C (as recommended by the manufacturer) for more efficient lysis and DNA yield of gram-positive bacteria. The V4 region of bacterial 16S rRNA gene was amplified using the primers 515 F (barcoded) and 806 R in accordance with the Earth Microbiome Project protocol65, with a minor modification as described in our previous reports64. The resulting amplicons were purified with AMPure® magnetic purification beads (Agencourt) and the purified products quantified using the Qubit-3 fluorimeter (InVitrogen). Equal amounts of purified PCR products were pooled; the resulting pool was quantified, normalized to 4 nm, denatured and diluted to 8 pM, and sequenced on an Illumina MiSeq sequencer (using Miseq reagent kit v3). The sequences were de-multiplexed, quality filtered, clustered, and analyzed using the Quantitative Insights into Microbial Ecology (QIIME, version 1.9.1) software. To avoid bias due to different sequencing depth, the OTU tables were rarefied to the lowest number of sequences per sample (single-dose study: 6650 sequences per sample; 5-dose study: 3800 sequences per sample; human fecal slurry study: 20,000 sequences per sample) for computing alpha-diversity metrics within QIIME. Linear discriminant analysis (LDA) analysis and cladograms were developed on genus level data using LDA effect size (LefSe)66 on Galaxy platform (https://huttenhower.sph.harvard.edu/galaxy/). Each dot in the PCA analysis of the gut microbiome signature represents an independently pooled equal amount of DNA of 2 mice from same group and three independent experiments of human fecal slurry.

SCFA analysis

To determine the organic acid/SCFA production of the screened isolates, overnight cultures of Lactobacillus and Enterococcus strains were inoculated into MRS/LM17 medium (inoculum of 1% and 2%, respectively). Through 24 h cultivation at 37 °C, supernatants were collected after centrifugation (12,000 g for 10 min), followed by passing through membrane filter. SCFAs from fecal slurry supernatant were obtained through centrifugation (12,000 g for 10 min) twice, and the supernatant was passed through a 0.45 µm membrane filter. SCFAs were extracted from fecal samples by taking 50 mg feces in a 1.5 mL Eppendorf tube, grinding the slurry with a pellet pestle motor, and suspending it in 1 mL PBS buffer (0.1 M, pH 7.4), followed by vortexing for 1 min at 20-min intervals for about 4 h. Thereafter, samples were immediately centrifuged and passed through a 0.45 µm membrane filter. Cell-free samples were used for determining concentrations of SCFAs (lactate, acetate, propionate and butyrate) using a high-performance liquid chromatography (Waters-2695 Alliance HPLC system, Waters Corporation, Milford, MA, USA) with DAD detector at 210 nm, equipped with an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA). Samples (10 μL) were injected each time and H 2 SO 4 (0.005 N) was used to elute the column with a flow rate of 0.6 mL/min at 35 °C.

Statistical analyses

OTUs with abundances higher than 1% were included in the subsequent analyses. Taxonomy assignment and diversity analyses were computed within QIIME to compare richness of bacterial species among experimental groups. Alpha-diversity (rarefaction curve for observed OTUs, Chao1, PD Whole Tree and Shannon) indices were computed with core_diversity_analysis.py script. Beta-diversity was generated within QIIME by using weighted and unweighted Unifrac distance matrices. Principal components analysis was performed (using EMPeror version 0.9.3-dev) to determine the influence of treatments on the overall microbiome composition. Bacterial diversity and abundance between groups within the same study were compared using non-parametric tests in R statistical software package (version 3.4.3; https://www.r-project.org/). Statistically significant differences between different groups were calculated by Kruskal-Wallis test followed by Dunn’s post-hoc analysis. SCFA contents were compared among groups by unpaired t-test. Unless otherwise stated, all assays were performed in either triplicate or three independently repeated experiments, and all the values presented here are means ± SEM. P < 0.05 was considered statistically significant.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

All protocols and procedures related to the sampling, care, and management of animals were reviewed and approved by the Institutional Animal Care and Use Committee at the Wake Forest School of Medicine. All experiments and samplings were carried out in accordance with ethical and biosafety protocols approved by Institutional guidelines.