Culturable Bacterial Community on Leaves of Assam Tea (Camellia sinensis var. assamica) in Thailand and Human Probiotic Potential of Isolated Bacillus spp.

Assam tea plants (Camellia sinensis var. assamica) or Miang are found in plantations and forests of Northern Thailand. Leaf fermentation has been performed for centuries, but little information is available about their associated microbial community. One hundred and fifty-seven bacterial isolates were isolated from 62 Assam tea leaf samples collected from 6 provinces of Northern Thailand and classified within the phyla of Firmicutes, Actinobacteria, and Proteobacteria. Phayao and Phrae provinces exhibited the highest and the lowest bacterial diversities, respectively. The bacterial community structural pattern demonstrated significant differences between the west and the east sides. Since some Bacillus spp. have been reported to be involved in fermented Miang, Bacillus spp. isolated in this study were chosen for further elucidation. Bacillus siamensis ML122-2 exhibited a growth inhibitory effect against Staphylococcus aureus ATCC 25923 and MRSA DMST 20625, and the highest survival ability in simulated gastric and intestinal fluids (32.3 and 99.7%, respectively), autoaggregation (93.2%), cell surface hydrophobicity (50.0%), and bacterial adherence with Vero cells (75.8% of the control Lactiplantibacillus plantarum FM03-1). This B. siamensis ML122-2 is a promising probiotic to be used in the food industry and seems to have potential antibacterial properties relevant for the treatment of antibiotic-resistant infections.


Isolation of Bacteria
A serial dilution (1 × 10 1 -1 × 10 2 ) of each swabbed sample was conducted using NaCl, 0.85% (w/v). The spread plate technique was subsequently performed on tryptic soy agar (TSA) (Merck™, Darmstadt, Germany) and de Man, Rogosa, and Sharpe (MRS) agar (Merck™) containing 0.004% (w/v) bromocresol purple. The culture plates were incubated at 37 °C for 24-48 h. All bacterial colonies on TSA showing different morphologies and the yellow bacterial colonies on MRS agar were further re-streaked to obtain pure cultures [5,12]. These bacterial isolates were kept at −20 °C until used.

Bacterial Genomic DNA Extraction
The chromosomal DNA of each bacterial isolate was extracted according to the method described by Pitcher et al. [19] with slight modifications. Briefly, each isolate was grown in tryptic soy broth (TSB) at 37 °C for 24-48 h. One milliliter of culture broth was centrifuged for 5 min at 6000 rpm at 4 °C. The supernatant was removed, and the pellet was resuspended in 1 mL of 1X TNE buffer (10 mM Tris, 1 mM EDTA, 0.1 M NaCl, pH 7.4). The mixture was then centrifuged for 5 min at 6000 rpm at 4 °C. Three hundred microliters of 1X TNE containing 2% (v/v) Triton X-100 were added to each pellet and mixed for 2 min using a vortex mixer. Subsequently, 300 µL of a solution containing phenol:chloroform:isoamyl alcohol (25:24:1) was added and mixed for 2 min, followed by centrifugation for 10 min at 12,000 rpm at 4 °C. The aqueous phase was transferred to a new microtube and mixed with 500 µL of ethanol at 95% (v/v). Each tube was stored at -20 °C overnight and spun at 12,000 rpm at 4 °C for 15 min. The supernatant was slowly removed and the pellet was dried at room temperature and resuspended in 30-50 µL in sterile distilled water. The DNA on each tube was kept at −20 °C.

Isolation of Bacteria
A serial dilution (1 × 10 1 -1 × 10 2 ) of each swabbed sample was conducted using NaCl, 0.85% (w/v). The spread plate technique was subsequently performed on tryptic soy agar (TSA) (Merck™, Darmstadt, Germany) and de Man, Rogosa, and Sharpe (MRS) agar (Merck™) containing 0.004% (w/v) bromocresol purple. The culture plates were incubated at 37 • C for 24-48 h. All bacterial colonies on TSA showing different morphologies and the yellow bacterial colonies on MRS agar were further re-streaked to obtain pure cultures [5,12]. These bacterial isolates were kept at −20 • C until used.

Bacterial Genomic DNA Extraction
The chromosomal DNA of each bacterial isolate was extracted according to the method described by Pitcher et al. [19] with slight modifications. Briefly, each isolate was grown in tryptic soy broth (TSB) at 37 • C for 24-48 h. One milliliter of culture broth was centrifuged for 5 min at 6000 rpm at 4 • C. The supernatant was removed, and the pellet was resuspended in 1 mL of 1X TNE buffer (10 mM Tris, 1 mM EDTA, 0.1 M NaCl, pH 7.4). The mixture was then centrifuged for 5 min at 6000 rpm at 4 • C. Three hundred microliters of 1X TNE containing 2% (v/v) Triton X-100 were added to each pellet and mixed for 2 min using a vortex mixer. Subsequently, 300 µL of a solution containing phenol:chloroform:isoamyl alcohol (25:24:1) was added and mixed for 2 min, followed by centrifugation for 10 min at 12,000 rpm at 4 • C. The aqueous phase was transferred to a new microtube and mixed with 500 µL of ethanol at 95% (v/v). Each tube was stored at -20 • C overnight and spun at 12,000 rpm at 4 • C for 15 min. The supernatant was slowly removed and the pellet was dried at room temperature and resuspended in 30-50 µL in sterile distilled water. The DNA on each tube was kept at −20 • C.

16S rRNA Gene Amplification
Amplification of the bacterial 16S rRNA gene was performed by polymerase chain reaction (PCR) in a thermal cycler (Labcycler, SensoQuest, Germany). The universal bacterial primers, 27F (5 -AGA  GTT TGA TCM TGG CTC AG-3 ) and 1492R (5 -TAC GGY TAC CTT GTT ACG ACT T-3 ) were used [20]. The PCR reactions were held at 94 • C for 5 min for DNA denaturation, followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 56 • C for 30 s, extension at 72 • C for 1 min, and an elongation step at 72 • C for 5 min to ensure complete amplification. PCR samples were temporarily stored at 4 • C. Amplicons were electrophoresed on agarose gel, 0.8% (w/v), supplemented with 1X nucleic acid staining solution (RedSafe ® , iNtRON Biotechnology, Inc., Seongnam-Si, Korea) for 50 min at 95 V and 300 mA in 1X Tris-acetate-EDTA (TAE) buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) using the electrophoretic gel system (EC320, Minicell Primo, CA, USA) at room temperature. The marker used was a 1 kb DNA ladder (RBC Bioscience, New Taipei, Taiwan). The gel documentation system (SynGene, Frederick, MD, USA) was used for gel visualization under UV light. The PCR products were purified and sequenced by DNA sequencing services (First BASE Laboratories Sdn Bhd., Selangor, Malaysia). The 16S rRNA gene sequence homologies were retrieved from the National Center for Biotechnology Information (NCBI) GenBank (https://www.ncbi.nlm.nih.gov/) and EzBioCloud databases (https://www.ezbiocloud.net/). The sequence data were aligned, and the phylogenetic tree was constructed by a neighbor-joining method [21] with the MEGA 7 program [22]. The bootstrap values of 1000 replicates were performed [23] with the Tamura-Nei model [24]. Amplicon sequences were deposited in the GenBank.

Antibacterial Activity Investigation
The pathogenic bacteria consisting of Bacillus cereus TISTR 687, Escherichia coli ATCC 25922, E. coli O157:H7 DMST 12743, methicillin resistant Staphylococcus aureus DMST 20625, Salmonella Typhi DMST 22842, Shigella dysenteriae DMST 1511, Staphylococcus aureus ATCC 25923, and Vibrio cholerae DMST 2873 were used for determination of antibacterial activity. The Bacillus strains were cultured in TSB at 37 • C overnight. The supernatant was filtered through a 0.45 µm nylon syringe filter before use. Thirty-three microliters of the Bacillus culture filtrate were used to evaluate the antimicrobial properties against pathogenic bacteria using the agar well diffusion method [25].

Survival Evaluation in Simulated Gastric and Intestinal Fluids
The fluids resembling human gastric and intestinal fluids were prepared according to the method of Huang and Adams [27]. The Bacillus isolates were grown in TSB at 37 • C for 18-24 h before harvesting by centrifugation for 5 min at 6000 rpm at 4 • C. Then, the cell pellets were washed twice and resuspended in 1X PBS (~1 × 10 9 CFU/mL). Then, the Bacillus cell suspension was inoculated into simulated gastric fluid (TSB supplemented with pepsin, 3 mg/mL, pH 2.5) to obtain about 1 × 10 8 CFU/mL of the initial cell concentration. The mixture was incubated at 37 • C and 50 rpm in a shaker incubator. Viable cell counts were performed on TSA plates at 0, 1, 2, 3, and 4 h after incubation. In parallel, Bacillus isolates were inoculated into the simulated intestinal fluids (TSB added with pancreatin, 1 mg/mL, and bile salt, 0.3% (w/v), pH 8.0). Viable bacterial cell counts were done on TSA plates at 0, 1, 2, 4, 6, 12, and 24 h after incubation. The percentage of survival rate was calculated.

Cellular Autoaggregation Assay
Autoaggregation was assessed following the modified protocol of Valeriano et al. [28]. To harvest the Bacillus grown cells, centrifugation at 6000 rpm 4 • C for 5 min was conducted. The cells were resuspended in 1X PBS to obtain the turbidity of 0.1 at 600 nm (OD i ) and left standing undisturbed at room temperature. The absorbance of the upper suspension fluid was tested at 600 nm (OD t ) at 1, 2, 4, 6, 12, 24, 36, and 48 h. The auto-aggregation percentage was estimated in accordance with the following equation: Autoaggregation (%) = (1 − (OD t /OD i )) × 100.

Cell Surface Hydrophobicity Assay
Cellular hydrophobicity was elucidated by measuring the ability of bacterial cells to adhere to hydrocarbon [15]. The cells of Bacillus after cultivation at 37 • C for 18-24 h were harvested by centrifugation. The cell pellet was adjusted equivalent to 0.1 at 600 nm (A initial ) (approximately 1 × 10 8 CFU/mL) in 1X PBS. The resuspended cells and xylene (3 mL of each) were added into the tube and mixed well with a vortex mixer. The mixture was allowed to stand without disturbance for 30 min at room temperature to allow separation of organic and aqueous phases. Measurement of the aqueous phase final absorbance (A final ) was conducted. The control used was Lactobacillus acidophilus TISTR 2365. The surface hydrophobicity percentage was calculated as the following: surface hydrophobicity (%) = ((A intial − A final )/A intial ) × 100.

In Vitro Bacterial Adhesion Assay
Cell adhesion was carried out by using the Vero cell line, a kidney epithelial cell. The assay was performed according to the modified method of Llanco et al. [29]. The Vero cells were cultivated in 6-well plates with Dulbecco's modified Eagle medium (DMEM, Gibco ® , Loughborough, UK) containing 10% (v/v) fetal bovine serum (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and 1% (v/v) penicillin/streptomycin (Caisson Laboratories, Inc., Smithfield, UT, USA), and incubated at 37 • C in a CO 2 incubator for 48 h. Afterwards, the harvested cells were adjusted equivalent to 1 × 10 5 cell/well prior to incubation overnight at 37 • C in a CO 2 incubator, and then triple washed with 1X PBS. The cell pellets of Bacillus strain were resuspended in DMEM without antibiotics before addition into the well (1 × 10 8 CFU/well final concentration). The plate was incubated for 1 h and washed three times with 1X PBS. After that, the Vero cells were fixed in absolute methanol for 5 min prior to staining with Giemsa stain for 15 min. Control strains were Escherichia coli ATCC 25922 (known as normal gut flora) and the well-known probiotics Lactobacillus acidophilus TISTR 2365 and Lactiplantibacillus plantarum FM03-1 (GenBank accession no. MF599378). Bacterial adhesion characteristics were observed under a compound microscope, and the percentage of adhesion was elucidated

Statistical Analysis
Alpha diversity was conducted to analyze the complexity of family diversity on Assam tea leaves through four indices including Shannon's index [30], Pielou's evenness index [31], Simpson's index [32], and species richness [33]. To examine the relationship between relative abundances of bacterial families and provinces, the number of an individual family found divided by the total number of families found in the province was multiplied by 100, and the histogram was constructed using Microsoft Excel software (version 1909) [7]. Principal coordinate analysis (PCoA) was performed using PAST software (version 3.25) [34]. A one-way analysis of variance (ANOVA) was performed to investigate statistical significance of antibacterial activity, survival in simulated gastrointestinal fluids, cellular autoaggregation, cell surface hydrophobicity, and bacterial adhesion assay using SPSS 22.0 with Duncan's multiple range tests. p < 0.05 was considered to indicate significant difference.

Bacterial Isolation from Assam Tea Leaves and 16S rRNA Gene Identification
Viable bacterial cells were obtained from 62 fresh Assam tea leaf surfaces collected in the 17 sampling sites ( Figure 1; Table S1). The TSA grown colonies provided 156 bacterial isolates. A maximum of seven different colony morphologies were found in samples collected from Thep Sadej subdistrict, Doi Saket district, Chiang Mai province (code ML041; 18 • (Figure 2A and Table S1). Mai = Nan > Phayao > Chiang Rai > Lampang = Phrae provinces, respectively. Meanwhile, only one bacterial isolate with acid producing capacity but catalase negative was found on MRS agar from the sample collected in Sritoi subdistrict, Mae Chai district, Phayao province (code ML151; 19°21′13.31′'N, 99°42′48.01′'E;) ( Figure 2A and Table S1).
The family Bacillaceae comprised the largest number of members (60 isolates). Interestingly, only two bacterial families, Bacillaceae and Staphylococcaceae, could be found in all provinces. Moreover, Phayao province displayed the highest number of bacterial families (10) including Bacillaceae, Enterobacteriaceae, Erwiniaceae, Leuconostocaceae, Microbacteriaceae, Micrococcaceae, Planococcaceae, Pseudomonadaceae, Sphingomonadaceae, and Staphylococcaceae, with 16S rRNA gene sequence similarity between 93.2 and 100.0%. For the MRS grown isolate, it was identified as a member of the family Leuconostocaceae, with 16S rRNA gene sequence similarity of 99.9% (Table S2).

Bacterial Community on Assam Tea Leaf Surface
The relative abundance of bacterial families on Assam tea leaves from each province is presented in Figure 3. Leaves of Assam tea collected from Phayao province demonstrated the highest diversity with 10 bacterial families and 30 different isolates, while Assam tea leaves obtained from Phrae province had the lowest bacterial diversity with 14 different isolates belonging to two families, Bacillaceae and Staphylococcaceae. The family Bacillaceae comprised the largest number of members (60 isolates). Interestingly, only two bacterial families, Bacillaceae and Staphylococcaceae, could be found in all provinces. Moreover, Phayao province displayed the highest number of bacterial families (10) including Bacillaceae, Enterobacteriaceae, Erwiniaceae, Leuconostocaceae, Microbacteriaceae, Micrococcaceae, Planococcaceae, Pseudomonadaceae, Sphingomonadaceae, and Staphylococcaceae, with 16S rRNA gene sequence similarity between 93.2 and 100.0%. For the MRS grown isolate, it was identified as a member of the family Leuconostocaceae, with 16S rRNA gene sequence similarity of 99.9% (Table S2).

Bacterial Community on Assam Tea Leaf Surface
The relative abundance of bacterial families on Assam tea leaves from each province is presented in Figure 3. Leaves of Assam tea collected from Phayao province demonstrated the highest diversity with 10 bacterial families and 30 different isolates, while Assam tea leaves obtained from Phrae province had the lowest bacterial diversity with 14 different isolates belonging to two families, Bacillaceae and Staphylococcaceae.    Table 1). The beta diversity analysis and the separation pattern of bacterial communities were shown by PCoA, suggesting that most bacterial communities from Chiang Mai province were explicitly separated from Lampang, Phayao, Phrae, and Nan provinces. Additionally, the bacterial communities of Chiang Rai and Lampang presented overlap between two main groups ( Figure 4).  Table 1). The beta diversity analysis and the separation pattern of bacterial communities were shown by PCoA, suggesting that most bacterial communities from Chiang Mai province were explicitly separated from Lampang, Phayao, Phrae, and Nan provinces. Additionally, the bacterial communities of Chiang Rai and Lampang presented overlap between two main groups ( Figure 4).

Antibacterial Activity
The culture filtrates of all isolates belonging to a family Bacillaceae (60 isolates) were tested for their antibacterial activity against test pathogenic bacteria. B. clausii ML062-2 was the only isolate promoting growth inhibition of E. coli O157:H7 (7.

Antibacterial Activity
The culture filtrates of all isolates belonging to a family Bacillaceae (60 isolates) were tested for their antibacterial activity against test pathogenic bacteria. B. clausii ML062-2 was the only isolate promoting growth inhibition of E. coli O157:H7 (7.  (Table 2). Based on the data mentioned above, B. licheniformis ML075-1 and B. siamensis ML122-2 were selected for further probiotic potential evaluation. Table 2. Antibacterial activity of Bacillus culture filtrates. Gentamicin, 0.1 and 50 (for MRSA) mg/mL were used as positive controls. Data were expressed as mean ± standard deviation of three independent experiments. The difference letters were considered statistically significant at p < 0.05.  (Table 3 and Figure 6).

Discussion
Assam tea plants or Camellia sinensis var. assamica are widely found in the highlands of Northern Thailand at altitudes over 200 m above sea level. The microbial composition of Assam tea leaves in Thailand first described by Rungsirivanich et al. [11] revealed the presence of culturable bacteria from the genera Bacillus, Floricoccus, Kocuria, Lysinibacillus, Micrococcus, and Staphylococcus. To gain further insight into the bacterial community of Assam tea leaves and understand their role in fermentation, as well as to identify beneficial bacteria for healthy food development and

Discussion
Assam tea plants or Camellia sinensis var. assamica are widely found in the highlands of Northern Thailand at altitudes over 200 m above sea level. The microbial composition of Assam tea leaves in Thailand first described by Rungsirivanich et al. [11] revealed the presence of culturable bacteria from the genera Bacillus, Floricoccus, Kocuria, Lysinibacillus, Micrococcus, and Staphylococcus. To gain further insight into the bacterial community of Assam tea leaves and understand their role in fermentation, as well as to identify beneficial bacteria for healthy food development and biological control, a larger area of sampling sites with various environmental conditions (altitude, soil, and climate) was explored in this study, covering six provinces (Chiang Mai, Chiang Rai, Lampang, Phayao, Phrae, and Nan) in the upper northern region of Thailand. Through molecular taxonomic markers, the isolated bacteria were classified within 15 families and 20 genera, some of which were adapted to extreme environments. For example, the genera Anoxybacillus and Lysinibacillus are reported as thermophilic [35,36], which is related to the formation of endospores. Most strains of the genus Cellulomonas have been described as cellulolytic bacteria, which can be found in cellulose-rich habitats [37]. On the other hand, Pantoea spp. Curtobacterium spp., Microbacterium spp., Sphingomonas spp. Lysinibacillus spp., Solibacillus spp., Kocuria spp., and Pseudomonas spp. have been grouped as endophytic bacteria implicated in plant growth promotion with some species producing antimicrobial substances [38][39][40][41][42][43][44], which may be involved in growth enhancement and biological control in Assam tea orchards [7]. The genera Acinetobacter and Brevibacillus have been reported to be found not only in the environment (involved in bioremediation [45] and biogeochemical cycles [46]) but also in animals and humans [47,48]. The Weissella and Floricoccus genera are grouped as lactic acid bacteria (LAB) [49,50]. Several reports reveal that some LAB strains can produce bacteriocin or bacteriocin-like inhibitory substances that play a role in inhibition of pathogenic microbes and in biological control. Moreover, LAB are also important in fermentation processes, and some species present probiotic properties [51]. Bacillus spp. have been widely found in various environments. Some species of Bacillus are pathogenic (e.g., B. cereus), probiotics (e.g., B. clausii, B. subtilis, B. licheniformis), endophytic (with the ability to produce the plant hormone indole-3-acetic acid or IAA; e.g., B. altitudinis, B. paramycoides, B. tequilensis), and antimicrobial (e.g., B. niacin, B. mobilis) [52][53][54][55][56]. Staphylococcus spp., Macrococcus spp., and Micrococcus spp. have been reported as normal flora on human and animal skins, and opportunistic pathogens such as S. epidermidis, S. xylosus, Macrococcus canis, and Micrococcus luteus [57][58][59] may lead to bloodstream infections. Besides that, Corynebacterium aurimucosum is also reported to be the cause of urinary tract infection in humans [60], while Enterobacter hormaechei is described as the causal agent of opportunistic infections in urinary and respiratory tracts [61]. The genus Pantoea has been generally found in plant surfaces as well as human feces. Some species have also been reported as human pathogens leading to bacteremia [62]. The existence of mammal microbial flora on Assam tea leaves indicates the close relationship between human, animals, and Assam tea plants. Assam tea orchards in the study area exist both in the forests and in the area around tea farmers' houses. Possibly, the human and animal flora spread to the plants via their activities, but how these bacteria affect the tea plants is not known.
The microbial community structure has been suggested to be associated with temperature, mineralization, hydrocarbon content, soil organic matter, and moisture [63,64]. In this study, the family Microbacteriaceae and Staphylococcaceae were explicitly dominant in Chiang Mai and Phrae provinces with 72.8 and 84.9% relative abundance, respectively. Moreover, the elevation above sea level is also considered to affect microbial biodiversity [65]. Although, the number of bacterial isolates identified within the family Bacillaceae was higher than that of Staphylococcaceae (38 and 28%, respectively), the Staphylococcaceae family presented higher relative abundance in Chiang Rai, Lampang, Phayao, and Phrae provinces than did the family Bacillaceae. By interview and observation, the collecting sites in these four provinces were quite inactive in terms of tea leaf plucking for fermented Miang production during the sample collecting period. Hence, the tea plants were undisturbed, which allowed certain bacteria to establish, thrive, and succeed in this microecosystem. The active Assam tea orchards displayed higher numbers of bacterial species, possibly due to the more turns of tea leaves which were often taken off. Moreover, altitude and temperature seemed to be unrelated to the occurrence.
The microbes found on Assam tea leaf surfaces revealed an abundance of Actinobacteria, Firmicutes, and Proteobacteria phyla, which have been commonly found in the environment including soils, leaves, stems, and roots, as well as skin of human and animals [66]. In this study, all diversity indices indicated that Phayao province had the highest bacterial biodiversity, while Phrae province had the lowest. Interestingly, the family Micrococcaceae was not found only in Chiang Mai and Phrae provinces, which clearly displayed low diversity. The absence of the family Micrococcaceae in Chiang Mai and Phrae may be due to the geographical location ( Figure 1). The microbial biodiversity clearly indicated the difference of bacteria existing on Assam tea leaves between two areas (referred to as the west and the east sides of Northern Thailand). The west (Mae Hong Son, Chiang Mai and Lamphun provinces) and the east (Chiang Rai, Lampang Phayao, Phrae, and Nan provinces) sides of Northern Thailand can be separated by western Phi Pan Nam range or known as Khun Tan range. Moreover, the difference of bacterial communities may be related to flow through rivers, especially the Wang River, which runs from Chiang Rai, upper Northern Thailand, passes through Lampang to Tak provinces, and lower Northern Thailand. These data support the various processes of Assam tea leaf fermentation and microbes in fermented Miang found in both areas, which have been reported by Kanpiengjai et al. [2], Khanongnuch et al. [3], and Chaikaew et al. [5].
In general, slow shifts from the original states of any microbial communities are demonstrated when continuous cultivation has been performed for a long time [7]. In this study, bacterial communities on Assam tea leaves at Nan province in March 2016 and at Chiang Mai province in 2015 were completely replaced by the Bacillaceae and Staphylococcaceae families within 2 years. This occurrence may be due to the involvement of human activity, wildfire, and deforestation in the study area and surroundings. "Fermented Miang" or fermented Assam tea leaves made by the local wisdom of people in the areas of Northern Thailand, the Lao People's Democratic Republic, and the Republic of the Union of Myanmar for centuries has been unpopular with the new generation for many reasons, such as its physical appearance and old-fashioned packaging. However, the hidden market share has been noticed recently. The use of probiotics in fermented Assam tea leaves has attracted people with healthy lifestyles. Hence, information on bacterial communities would benefit further product development and elucidation of properties in the field of the gut microbiome or related subjects.
A previous study described the probiotic properties and utilization of Bacillus [67]. In fermented Miang, B. tequilensis, B. siamensis, B. megaterium, B. toyonensis, and B. aryabhattai presented great probiotic potential [68]. Some Bacillus strains mentioned above could be also found in this study, suggesting a persistence of these bacteria from fresh to fermented leaves.
B. licheniformis ML075-1 and B. siamensis ML122-2 demonstrated antagonistic activity against pathogenic bacteria, especially against B. cereus TISTR 687 and MRSA DMST 20625, respectively. Previous studies reported that Bacillus spp. can produce antimicrobial substances such as peptides, bacteriocins, or BLIS [16]. Interestingly, B. siamensis ML122-2 could inhibit growth of MRSA DMST 20625, a cause of resistance to many antibiotics. Culture broth pH seemed not to be responsible for the antagonistic activity of these isolates (data not shown). In the future, its antimicrobial active principle should be further elucidated.
In this study, B. licheniformis ML075-1 and B. siamensis ML122-2 were susceptible to all tested antibiotics. This susceptibility results may ensure that when these Bacillus isolates are used as probiotics, the chance that an antibiotic resistance gene will be transferred to any recipient bacteria in the intestine is scarce [15].
Acid and base tolerance are among the most important requirements for probiotics potential due to their need to survive in the stomach and intestines [69]. B. siamensis ML122-2 demonstrated higher survival rate in the gastric fluid than B. licheniformis ML075-1 and L. acidophilus TISTR 2365, respectively. The previous study by Kristoffersen et al. [70] indicated that B. cereus ATCC 14579 spores were germinated in the distal parts of the small intestine, which contains lower amounts of bile salt. In this study, the alkaline condition in simulated intestinal fluid was tested against Bacillus and was consistent with the previous study in that at the start of the experiment where high concentration of bile salt was present, Bacillus strain count was reduced, with an increasing rate after incubation for four (B. licheniformis ML075-1) and six (B. siamensis ML122-2) hours. This might be because the bacteria adapted to the test fluid or produced some metabolites to neutralize the fluid.
The adhesion ability of probiotics has been considered to be a critical prerequisite for bacterial colonization on the host gut [69]. In this study, B. siamensis ML122-2 demonstrated autoaggregation (higher than 90%), high hydrophobicity, and high adherence ability to Vero cells, therefore presenting the highest probiotics potential among the identified bacteria. In the future, it will be worth further investigating the potential of these species for the treatment of antibiotic-resistant infections. Furthermore, B. siamensis ML122-2 may provide a functional ingredient useful in synbiotic production, which will be investigated in the near future.

Conclusions
Assam tea leaves are rich in microbial biodiversity. These microbial communities may play a major role in biological control and Assam tea leaf fermentation. The microbiodiversity on Assam tea leaves presented a stabilized community representing good sources of probiotics. Particularly, B. siamensis ML122-2 isolated from Assam tea leaf surfaces demonstrated high probiotic potential. It represents antibacterial activity, especially MRSA; hence, its antimicrobial substance should be elucidated in the future.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/8/10/1585/ s1: Figure S1, Phylogenetic relationships of some bacterial isolates (bold) isolated from Assam tea leaves in Northern Thailand with their closest species and related taxa based on 16S rRNA gene sequence analysis. The branching pattern was generated by the neighbor-joining method. Bootstrap values (expressed as percentages of 1000 replications). Bar, 0.05 substitutions per nucleotide position. Saccharolobus caldissimus JCM 32116 T (GenBank accession no. LC275065) is presented as outgroup sequence. Table S1, Assam tea leaf collecting site from different regions in Northern Thailand. The data presented number of Assam tea plants, locations, altitude, bacterial cell count, number of isolates per sample, and number of species per sample. Table S2, Classification of bacteria isolated from Assam tea leaf surfaces compared with the type strain and the similarity (%) of 16S rRNA gene sequence.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.