Antimicrobial Effects of Potential Probiotics of Bacillus spp. Isolated from Human Microbiota: In Vitro and In Silico Methods

The variable taxa components of human gut microbiota seem to have an enormous biotechnological potential that is not yet well explored. To investigate the usefulness and applications of its biocompounds and/or bioactive substances would have a dual impact, allowing us to better understand the ecology of these microbiota consortia and to obtain resources for extended uses. Our research team has obtained a catalogue of isolated and typified strains from microbiota showing resistance to dietary contaminants and obesogens. Special attention was paid to cultivable Bacillus species as potential next-generation probiotics (NGP) together with their antimicrobial production and ecological impacts. The objective of the present work focused on bioinformatic genome data mining and phenotypic analyses for antimicrobial production. In silico methods were applied over the phylogenetically closest type strain genomes of the microbiota Bacillus spp. isolates and standardized antimicrobial production procedures were used. The main results showed partial and complete gene identification and presence of polyketide (PK) clusters on the whole genome sequences (WGS) analysed. Moreover, specific antimicrobial effects against B. cereus, B. circulans, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Serratia marcescens, Klebsiella spp., Pseudomonas spp., and Salmonella spp. confirmed their capacity of antimicrobial production. In conclusion, Bacillus strains isolated from human gut microbiota and taxonomic group, resistant to Bisphenols as xenobiotics type endocrine disruptors, showed parallel PKS biosynthesis and a phenotypic antimicrobial effect. This could modulate the composition of human gut microbiota and therefore its functionalities, becoming a predominant group when high contaminant exposure conditions are present.


Introduction
The human gut microbiota could be considered as a new source for the identification and isolation of multiple microorganisms producing bioactive compounds and enzymes of interest such as biopolymers, antimicrobials notably demanded by the food, health, and several biotechnological industries [1,2]. Identifying the composition of cultivable gut microbiota has always been a challenge due mainly to the requested anaerobic conditions [3]. Efforts in simulating these harsh culture conditions allow isolating potential NGP [4] and even a variety of taxonomy bacterial groups which were also tolerant to xenobiotics or obesogens [5] followed by characterization through 16S rRNA gene sequencing.
Bisphenols are considered as microbiota disrupting chemicals (MDC) [5] and their presence in humans has been confirmed by detecting them in human biospecimens: feces, serum, urine, saliva, hair, tissue and blood [23,24]. Bisphenol A (BPA) is used in manufacturing polycarbonate and epoxy resins for food consumer products and packages. There is also cumulative exposure from contaminating soils, aquatic environments, drinking water, air and dust particles [25]. The estrogen activity alteration is the most widely studied effect of BPA and analogues, enhancing endocrine disruptor activities [26]. Moreover, some studies have shown obesogenic effects through microbiota dysbiosis [27], fat cell development, and lipid accumulation [28]. There are several regulations enforced concerning the hazards of Bisphenol A, as derivative of polycarbonates plastics and epoxy resins, used in food contact materials, toys, or other products. In order to protect the consumers from cumulative exposure, the tolerable daily intake (TDI) for BPA is permanently re-evaluated according to new toxicity data through specific international projects, such as U.S. National Toxicology Program (CLARITY-BPA program) [29] or European Food Safety Authority (EFSA) comprehensive re-evaluation of BPA exposure and toxicity [30].
Moreover, commensal microorganisms isolated from human microbiota could in general fulfill the criteria of safety assessment and the status of Qualified Presumption of Safety (QPS) [31,32]. Similarly, most Bacillus subtilis cluster species are considered QPS [33] and they are increasingly marketed as products [34]. Conversely, Bacillus cereus cluster species can be also present in the gut microbiota, but they are not considered as QPS [34,35].
Next-generation sequencing (NGS) platforms and WGS of microorganisms have enlarged the molecular comparison knowledge on the gene collection for encoding enzymes, and better taxonomy has supported appropriate classification. Moreover, specific WGS gene description is needed to consider the food and feed safety aspects of microbiota cultivated strains [35].
Genome mining tools and phenotypic analysis are complementary approaches to predict and demonstrate the production of active secondary metabolites such as antimicrobial products from Bacillus species [36]. Genome mining revealed the potential for known and novel PKs extensively in Bacillus ( Figure 1). Moreover, based on the prediction of the general architecture, novel clusters were identified in novel Bacillus spp. variants. In addition, more recent in silico and bioinformatics approaches seem to be successful to find and verify the microbial potential to produce valuable enzymes for biotechnological applications [36]. European Food Safety Authority (EFSA) comprehensive re-evaluation of BPA exposure and toxicity [30]. Moreover, commensal microorganisms isolated from human microbiota could in general fulfill the criteria of safety assessment and the status of Qualified Presumption of Safety (QPS) [31,32]. Similarly, most Bacillus subtilis cluster species are considered QPS [33] and they are increasingly marketed as products [34]. Conversely, Bacillus cereus cluster species can be also present in the gut microbiota, but they are not considered as QPS [34,35].
Next-generation sequencing (NGS) platforms and WGS of microorganisms have enlarged the molecular comparison knowledge on the gene collection for encoding enzymes, and better taxonomy has supported appropriate classification. Moreover, specific WGS gene description is needed to consider the food and feed safety aspects of microbiota cultivated strains [35].
Genome mining tools and phenotypic analysis are complementary approaches to predict and demonstrate the production of active secondary metabolites such as antimicrobial products from Bacillus species [36]. Genome mining revealed the potential for known and novel PKs extensively in Bacillus ( Figure 1). Moreover, based on the prediction of the general architecture, novel clusters were identified in novel Bacillus spp. variants. In addition, more recent in silico and bioinformatics approaches seem to be successful to find and verify the microbial potential to produce valuable enzymes for biotechnological applications [36]. The main objective of the present study was to determine the antimicrobial effects of catalogue of microorganisms isolated from human gut, by applying directed-culturing methods after the addition of endocrine disruptor chemicals. Taxa groups of isolated bisphenol A (BPA)-degrading Bacillus spp. will be analyzed by with in vitro assays to demonstrate the bioactive substances released against commensals and critical pathogens according to the World Health Organization (WHO). Moreover, genome mining and in silico tests will be used for disclosing the genes responsible for antimicrobial production and its enzymatic pathways.

Microbiota Sampling Bank and Directed Culturing Approach
Ten isolates from fecal human microbiota collections of 0-1 year old infants (Isolates B-Project INFABIO) appropriately maintained at −80 °C underwent a directed culturing approach using 0.5 g of the fecal specimen in 1.5 mL of Brain Heart Infusion or Man The main objective of the present study was to determine the antimicrobial effects of catalogue of microorganisms isolated from human gut, by applying directed-culturing methods after the addition of endocrine disruptor chemicals. Taxa groups of isolated bisphenol A (BPA)-degrading Bacillus spp. will be analyzed by with in vitro assays to demonstrate the bioactive substances released against commensals and critical pathogens according to the World Health Organization (WHO). Moreover, genome mining and in silico tests will be used for disclosing the genes responsible for antimicrobial production and its enzymatic pathways.

Microbiota Sampling Bank and Directed Culturing Approach
Ten isolates from fecal human microbiota collections of 0-1 year old infants (Isolates B-Project INFABIO) appropriately maintained at −80 • C underwent a directed culturing approach using 0.5 g of the fecal specimen in 1.5 mL of Brain Heart Infusion or Man Rogosa and Sharpe (BHI/MRS) broths, adding different concentrations of BPA (0.5, 10, 20, and 50 ppm), in order to search tolerant and/or potentially BPA biodegrading microorganisms, incubation for 72 h. Further serial dilutions and spreading onto BHI/MRS solid media plus incubation under aerobic and anaerobic conditions (anaerobic jars anaerocult ® ) at 37 • C over 72 h were applied. BPA-tolerant colonies with distinguishing features were isolated as pure culture for subsequent morphological, phenotypic, and genotypic identifications: bacterial cell counts, gram staining, spore staining, capsule staining, catalase activity, oxidase, and motility tests.

BPA Microbiota Tolerance Testing
BPA biodegradation microbiota capacity was tested directly adding BPA to the human fecal samples. The specimens were exposed to 25 ppm concentration of BPA at 30 • C during 72 h. BPA was measured in the extracts and supernatants through Liquid chromatographymass spectrometry (LC-MS/MS) system for BPA quantification. Chemicals, reagents, instrumentation, and software for bisphenols determination were provided by CIC services under validated procedures previously described by García-Córcoles et al. [38].

Culturing-Isolation of Bacillus Catalogue
A common approach to isolate Bacillus strains from microbiota has been pursued in our research team [39]. For this study, ten isolates from fecal human microbiota collections of 0 to 1 year old infants (Isolates B-Project INFABIO) and 6-8 year-old children (Isolates C-Project OBEMIRISK) were obtained by a serial dilution method, with exposure to different BPA concentrations (0.5, 10, 20, and 50 ppm) over 72 h and further spreading in BHI/MRS media incubated under aerobic and anaerobic conditions (anaerobic jars anaerocult ® ) at 37 • C. The BPA-tolerant bacterial colonies with distinguishing features were isolated as pure culture for subsequent morphological, phenotypic, and genotypic identifications: bacterial cell counts, gram staining, spore staining, capsule staining, catalase activity, oxidase, and motility tests.

Genomic DNA Extraction, Taxonomy Identification and Phylogenetic Analysis
Genomic DNA was extracted using DNeasy columns (Qiagen ® , Hilden, Germany) following the manufacturing instructions. The isolated DNA was quantified using Nanodrop (Thermo Scientific ® Waltham, MA, USA) and biophotometer (Eppendorf ® D30). The quality of DNA was monitored through gel electrophoreses. Complete 16S RNA gene sequencing of selected bacterial strains was done by Sanger method (Institute of Parasitology and Biomedicine "López-Neyra" IPBLN Service). Forward and reverse sequences were provided separately. Reverse sequence was converted to complementary sequence with Chromas Pro 2.0 software (Technelysium Pty Ltd., Tewantin, Australia). Sequences were examined for maximum homology against GenBank using National Center for Biotechnology Information NCBI's BLASTn program. The collection and comparison of complete 16S rRNA gene sequences were performed using the Ezbiocloud platform [40].

Enzymes Tests
Relevant enzymatic production assays were carried out to verify the potential of gut microbiota strains to synthetize relevant enzymes in the biotechnological and industrial context. Starch, carboxymethylcellulose, inulin, tween 20 and 80, and DNase supplemented media were used to determine the degradation of different substrates according to complementary methodologies [41][42][43][44][45][46].

Antimicrobial In Vitro Tests
Antimicrobial activity was tested by agar well diffusion method. Under Joint FAO/ WHO Expert Committee on Food (JECFA) procedures [47] and the study carried out by Powthong & Suntornthiticharoen [48], nine different bacteria were used as indicators to verify the antimicrobial capacity of the Bacillus spp. isolated from the gut microbiota.
To determine the synthesis of antimicrobial compounds, several isolated strains were selected according to preliminary antimicrobial tests and the main taxonomy groups: strains close/represented by rB1 (Bacillus sp. AM1), strains close/represented by rB3 (Bacillus siamensis (KCTC 13613)), strains close/represented by rB7 (Bacillus cereus (AFS039342)). Plates with 20 mL of Müller-Hinton agar were prepared and test microorganisms used as indicators: Bacillus cereus, Bacillus circulans, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Serratia marcescens, Klebsiella spp., Pseudomonas spp., and Salmonella spp., were adjusted to a cell density of 0.5 on the McFarland scale in sterile 0.85% NaCl solution. The data were expressed as mean of the three replicates. Tests were done spreading the indicator microbial strains over the surface of the Müller-Hinton agar using sterile cotton swab. Inside six mm diameter oxford wells generated in agar, 20 µL of antibiotic producing bacteria extract was added. Standards appropriate positive controls (ampicillin, gentamycin, and streptomycin at 10 µg) and negative/blank (sterile media/ethanol) were used. The plates were incubated at 37 • C for 24 h and the inhibition zones were measured.

Genome Mining Tools for PKs Gene Searching
In order to discover the presence of secondary metabolites, several bioinformatics tools were used to perform genome mining. A data retrieving software has been specifically computed using Pascal programming language to obtain the PKs enzymes ID and the corresponding Loci from the genomes.
Type strain genomes from the closest species isolated were retrieved from NCBI Genome Data Bank in GenBank file format in order to list the proteins that they were able to potentially produce.
A more detailed prediction of the clusters was performed by checking the downstream and upstream genes of those involved in PKs synthesis using NCBI genome map viewer [49].

Prediction of Polyketides in WGS of Bacillus sp. AM1 Isolated from Microbiota
The identification of PKs gene cluster was carried out by the analysis of the WGS of Bacillus sp. AM1, GenBank CP047644.1, following the same approach explained above. The microbiota composition of each fecal sample was specific and contributed differentially to the biodegradation of BPA exposure levels ( Figure 2). Each fecal sample (340, 349, and 437) showed a differential ability to eliminate BPA due to its taxa compositional and functional characteristics, showing sample 340 a maximum percentage of BPA degradation of 89.3% while sample 349 degraded 76% and 437 was able to eliminate 21% of the BPA concentration. Previous studies have shown the same effects in the environment [50], where they observed that different microbial communities presented a specific elimination rate dependent on their composition.

Results and Discussion
Cumulative exposure to a wide range of xenobiotics, such as BPA and its analogues, affects the microbiota diversity possessed by each individual, causing a selection of bacteria strains to populate the gut, and consequently modify its equilibrium through MDC [5]. This dysbiosis has been proven to be responsible for well-known diseases, such as obesity, diabetes, and even some hormonal-related cancers. Therefore, identification of the triggered main taxa variations and their functions remains a challenge. Moreover, the appropriate use of probiotics [50][51][52] or search for NGP to mitigate or reverse these dysbiosis are crucial [53,54]. A directed culturing approach allow us to select tolerant bacteria and mimic an ecological environment to understand better the impact of the specific enriched communities and their capacities to impact the taxa microbiota colonization. Cumulative exposure to a wide range of xenobiotics, such as BPA and its analogues, affects the microbiota diversity possessed by each individual, causing a selection of bacteria strains to populate the gut, and consequently modify its equilibrium through MDC [5]. This dysbiosis has been proven to be responsible for well-known diseases, such as obesity, diabetes, and even some hormonal-related cancers. Therefore, identification of the triggered main taxa variations and their functions remains a challenge. Moreover, the appropriate use of probiotics [50][51][52] or search for NGP to mitigate or reverse these dysbiosis are crucial [53,54]. A directed culturing approach allow us to select tolerant bacteria and mimic an ecological environment to understand better the impact of the specific enriched communities and their capacities to impact the taxa microbiota colonization.  (Table 2).   Data obtained by parallel experimental work showed a BPA directed human fecal culturing catalogue that contained different BPA tolerant species from the following genera and percentages: Enterococcus 28%, Bacillus 27%, Staphylococcus 10%, Escherichia 8%, Clostridium 5%, and Lactobacillus 4% (data not shown). Representing Bacilli taxa (Bacillus and Lactobacillus) was a major taxa with approximately a 30% of BPA tolerant isolated strains from microbiota samples, which corroborates the predominant presence of these genera being able to overcome the impact of xenobiotics, such as BPA, as previous assays showed [39].
In line with these results, interesting properties and uses are specifically described for Bacillus spp. Recently, several Bacilli strains have been extensively proposed for use as human and animal probiotics [55,56]. Most of the species used belong to Bacillus subtilis and Bacillus amyloliquefaciens groups and special attention should be paid to the food and clinical studies with strains that showed special enzyme capacities [57] or those able to modulate and mitigate pathophysiological disorders [58].

Taxonomical and Phylogenetic Clustering
The phylogenetic tree based on complete 16S rRNA gene of Bacillus strains isolated from microbiota treated with BPA grouped the clusters to B. subtilis, B. amyloliquefaciens, B. velezensis, B. siamensis, B.cereus, and B. pacificus (Figure 3). The two main clustering of closely related Bacillus strains belong to B. subtilis and B.amyloliquefaciens taxonomic group (green) and B. cereus group (yellow). Three representative strains (rB1, rB3, and rB7) were further processed by bioactive compounds production tests. They were organized as follows: rB1 represented B1, B4, B5, B6, B7, B8, B9, and B9.2; rB3 represented B2 and B3; rB7 represented B7 and B12. 8%, Clostridium 5%, and Lactobacillus 4% (data not shown). Representing Bacilli taxa (Bacillus and Lactobacillus) was a major taxa with approximately a 30% of BPA tolerant isolated strains from microbiota samples, which corroborates the predominant presence of these genera being able to overcome the impact of xenobiotics, such as BPA, as previous assays showed [39].
In line with these results, interesting properties and uses are specifically described for Bacillus spp. Recently, several Bacilli strains have been extensively proposed for use as human and animal probiotics [55,56]. Most of the species used belong to Bacillus subtilis and Bacillus amyloliquefaciens groups and special attention should be paid to the food and clinical studies with strains that showed special enzyme capacities [57] or those able to modulate and mitigate pathophysiological disorders [58].

Taxonomical and Phylogenetic Clustering
The phylogenetic tree based on complete 16S rRNA gene of Bacillus strains isolated from microbiota treated with BPA grouped the clusters to B. subtilis, B. amyloliquefaciens,  B. velezensis, B. siamensis, B.cereus, and B. pacificus (Figure 3). The two main clustering of closely related Bacillus strains belong to B. subtilis and B.amyloliquefaciens taxonomic group (green) and B. cereus group (yellow). Three representative strains (rB1, rB3, and rB7) were further processed by bioactive compounds production tests. They were organized as follows: rB1 represented B1, B4, B5, B6, B7, B8, B9, and B9.2; rB3 represented B2 and B3; rB7 represented B7 and B12 The strains isolated in the present work were clustered in the two main groups: B. subtilis-like (non-pathogenic) [59] and B. cereus-like (pathogenic) [60], as shown in Figure   Figure 3. Phylogenetic tree based on gene sequences of isolated gut microbiota strains. The tree was obtained by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and Kimura 2-parameter model. The species and strain names are shown. Bootstrap values shown after 1000 resamplings. Main clusters are highlighted: in green close to B.subtilis group and yellow close to B.cereus group.
The strains isolated in the present work were clustered in the two main groups: B. subtilis-like (non-pathogenic) [59] and B. cereus-like (pathogenic) [60], as shown in Figure 3, however the pathogenicity features are strain-specific dependent. The work approach is based on potential uses and predictive data analysis, but for further commercial uses, a safety assessment should be performed for each strain, to demonstrate that they do not pose any safety and/or pathogenicity concerns. The battery of tests usually requested is: antibiotic resistance test no greater than existing regulatory cutoffs against clinically important antibiotics, incapacity to induce hemolysis or produce surfactant factors, and the absence of virulence or toxigenic activity in vitro.

Enzymatic Activity Tests
B. subtilis, B. amyloliquefaciens, and B. licheniformis have been used as bacterial resources in the industrial context for the production of a wide range of enzymes and bioactive compounds for decades. Bacillus sp. AM1 and other strains belonging to Bacillus genus have shown remarkable hydrolytic enzyme capacity (Table 3), being related to the performance of key roles in several biotechnological and many manufacturing processes [61][62][63].

Antimicrobial Activity Tests
The results obtained from antimicrobial experimental tests carried out with the representative isolated microorganisms from different taxonomic clusters confirmed the ability of the strains B1 and B3 to inhibit Gram-negative and Gram-positive bacteria ( Table 4).
Preliminary results grouped the strains according to their capacity of antibiotic production with very similar inhibiting zone value, which were also in agreement with the main taxonomic clusters. rB1 represented B1, B4, B5, B6, B7, B8, B9, and B9.2; rB3 represented B2 and B3; rB7 represented B7 and B12. rB1 and rB3 strains were found to be antagonistic against Gram-positive Bacillus cereus, Bacillus circulans, Staphylococcus aureus, Streptococcus pyogenes (diameter of zone of growth inhibition 10-17 mm) and also against Gram-negative food-borne pathogenic bacteria Serratia marcescens, Escherichia coli, Salmonella, and Klebsiella pneumoniae (diameter of zone of growth inhibition 10-20 mm). Conversely, the strains rB7 did not show any production of antimicrobial effects.
Minimum inhibitory concentration (MIC) values were similar to those resultant of other polyketides antimicrobial effects previously described, being significant differential and higher the effects found against Klebsiella [64]. Therefore, the search for a putative biosynthetic pathway of the pks gene product proceeded after the validated molecular antimicrobial attributions.

WGS Mining in Type Strains
The bioinformatics analysis carried out on the type strains of closest species identified as cultivable Bacillus species from microbiota showed specific enzymes involved in PKs biosynthesis ( Table 5). The genome mining identified the clusters with the genomes from closest homologue type strains available in the database. Bioinformatic tools and Pascal ad hoc software allowed the exhaustive analysis of genomes making it a powerful prediction tool.
According to the results, Bacillus amyloliquefaciens, B. siamenensis, B. velezensis, B. subtilis and B. atrophaeus harbor almost complete pks genetic macroclusters for the production of polyketides. While B. licheniformis, B. cereus, B. pacificus, and the probiotics B. clausii, B. coagulans did not contained the PKs loci. The antimicrobial effects of polyketides are site colonization specific and the strains are scarcely used for health biotechnological interests [65]. Moreover, the ecological impact of these antimicrobial substances on the gut microbiota composition may have a huge impact, beyond the modification and control of the colonization of commensals and pathogenic bacteria, e.g., to cause weight gain effects in humans as well as in animals [66].

WGS Representative Bacillus sp. AM1 from Microbiota: Genome Mining Data
From the analysis of the specific Bacillus sp. AM1 WGS, the cluster genes and enzymes related to PKs biosynthesis were identified (bae, mln, and dfn) and they were related to the production of bacillaene, and two other polyketides macrolactin and difficidin.
This complex microbial ecosystem seems to be enriched in new bacterial strains belonging to Bacillus genus that produce PKs with a wide range of applications in the current biotechnological context. Among these applications, PKs stand out for their antimicrobial capacity against certain bacterial species. Therefore, further identification through bioinformatics tools and experimental data will confirm the functionality of these bioactive substances.
Advances in NGS and in silico tools allow to perform an appropriate screening of genes of concern or interest in microbiota, such as antimicrobial resistance genes and the capacity of antimicrobial production of cultivable isolates WGS. A better understanding of the microbiota ecology, driven by the bioactive compounds released by its components, will lead to better clinical interventions. Antimicrobials naturally synthetized by gut microorganisms are mainly described as bacteriocins [12]. However, it is important to consider other molecules acting as antimicrobial as polyketides. Isolation and elucidation of PKs structures by nuclear magnetic resonance (NMR) methods are limited by the concentration needed for analysis [67]. Thus, it is possible to predict the types of PKs and their variants, as showed for Bacillales [37]. Genome mining performed in the present study allowed BLAST driven search for predicted PKs clusters. Pascal ad hoc software analysed the type strain genomes making it a powerful prediction tool. Similarly, another useful prediction tool could be used as nonribosomal peptide-synthetase NRPS/PKs substrate predictor [68]. Importantly, Bacillus and specific WGS genes description is needed to verify the safety assessment of different strains if they are proposed to be used in food or feed chain [70]. Moreover, the safety of a beneficial microbe or probiotic strain must be sufficiently characterized by high-throughput technologies, safe for the intended use, and assessed through pathogenicity, immunotoxicity, and colonization, in addition to its antibiotic resistance profile [71]. However currently, there is no consensus or standardization for the interventional use of probiotics [72]. In addition to general guidelines for the qualification of the QPS, European Food Safety Authority (EFSA) made a supplementary requirement for Bacillus species other than the Bacillus cereus group, where a cytotoxicity test should be performed to determine whether the strain produces high levels of non-ribosomal synthesised peptides. One of the criteria for strains to fulfill and meet the requirements for QPS and generally recognized as safe (GRAS) standards is antimicrobial activity and the absence of antimicrobial resistance genes as a possible safety concern against critically important antimicrobials (CIAs) or highly important antimicrobials (HIAs), which might eventually be transferred via horizontal gene transfer to pathogenic bacteria during food manufacture or after consumption [33,73]. According to the general guidelines for the qualifications of the QPS, unless the strain qualifies for the QPS approach or belongs to a taxonomic unit, known not to produce antimicrobials relevant to use in humans and animals, assessment should be made to determine the inhibitory activity of culture supernatants against reference strains, known to be susceptible to a range of antibiotics and the inhibitory substance [47]. A slight adjustment has been made for the production strains, which have to demonstrate the absence of carry-over into the final product together with the exact phase of the industrial scale manufacturing process, and whether any CIAs or HIAs are used during the manufacturing of the product, to determine compatibility with other additives showing antimicrobial activity and, furthermore, possible co-/cross-resistance [35].

Conclusions
Bacillus strains isolated from human gut microbiota, and taxonomically closest to the safely qualified B. subtilis and B. amyloliquefaciens groups, became cultivable predominant taxa when high bisphenol exposure conditions were tested. In parallel, these strains harbored PKS molecular gene biosynthetic loci and showed phenotypic antimicrobial effects. Therefore, they might be proposed as beneficial microorganisms with molecular features that would contribute to modulate the ecological taxa composition and functionality of human gut microbiota. Intervention studies will be further needed to demonstrate the ability to recover from microbiota dysbiosis, triggered by high MDC exposure diets and lifestyles, towards eubiosis and healthier status.  Institutional Review Board Statement: Ethical review and approval were waived for this specific study, due to the specimens collection belonged to our previous approved project INFABIO.