Human Gut Microbial Taxa Metabolizing Dietary Obesogens: A BPA 1 directed-culturing and Bioinformatics Combined Approach

Background : Integrated data from culturomics and functional omics may depict holistic 22 understanding on gut microbiome eubiosis or dysbiosis, and microbial isolates can become a 23 source of differential enzymes and useful bioactive compounds. Culturing methods developed 24 during last decade swift increases the importance of gut microbial isolates, focusing on media, 25 modifications and conditions that propitiate cultured taxa that previously were considered 26 fastidious or unculturable. In this context and focusing on gut microbiota dysbiosis triggered by 27 obesogens and microbiota disrupting chemicals (MDC), we have conducted a directed- 28 culturing and bioinformatics combined approach, adding bisphenol A (BPA) and specific 29 treatments to find resistant spore-forming bacteria, to obtain isolated strains for further explore 30 their molecular BPA metabolizing or neutralizing capacities. 31 Results: Overall microbiota culturing media and conditions have been retrieved and organized 32 according to main gut taxa isolated during last decade. Furthermore, a catalogue of BPA 33 directed-cultured microorganisms has been obtained from 46 fecal samples from two 34 populations, children with obesity and normo-weight. A total of 235 BPA tolerating and 35 potentially BPA biodegrading microorganisms were mainly grouped to strictly anaerobic 36 sporuled/non-sporuled, anaerobic facultative sporuled/non-sporuled. Firmicutes, 37 Enterobacteria and Actinobacteria species showed the major representation in both groups. 38 However, differential BPA tolerant microbiota composition from the populations was detected. 39 Bioinformatics analysis disclosed and predicted the variability of harboring genes encoding 40 specific enzyme for BPA biodegradation pathways that corroborated from directed-culturing 41 microbiota consortia obtained. 42 Conclusions: Strains from Staphylococcus , Bacillus and Enterococcus genera represented the 43 majority of the successfully cultured bacteria in both population specimens. From them, the 44 bioinformatics prediction assigned to Bacillus spp. the higher potential for BPA biodegradation. 45 Therefore, extensive directed-culturomics approaches could be designed for different MDC 46 with common biodegradation pathways, such as parabens, phthalates, and benzophenones. forming, BPA-degrading Bacillus sp. GZB isolated from an electronic-waste recycling site reveals insights into BPA degradation pathways.

Therefore, extensive directed-culturomics approaches could be designed for different MDC 46 with common biodegradation pathways, such as parabens, phthalates, and benzophenones.

Background 50
Microbiota dysbiosis in obesity-related disorders triggered by exposure to ED and obesogens 51 Currently, the exposure to obesogens and ED can lead to a microbial dysbiosis [1,2]. The 52 dysbiosis are based on misbalanced taxa compositions and associated to several metabolic 53 diseases, such as type 2 diabetes, obesity, and other endocrine disorders [3,4,5]. To isolate, 54 culture and analyze the microbial taxa components that can lead towards altered functional 55 effects would allow a better understanding of the pathophysiological mechanisms and its 56 prevention through the administration of beneficial microbes, helping to regulate the 57 physiological hormonal axis [6]. Directed-culturing of microorganisms from obese and non-58 obese microbiota may lead to identify potential metabolizing and detoxifying strains, which 59 could be used as NGP [7,8].

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The importance of culturomics for the human microbiome description is advancing towards 61 more effective isolations via sophisticated culture methods of the human microbiome [9]. This  gene sequencing [9,10]. It showed its success in the isolation, description and characterization 66 of new bacterial species from the human microbiota [9,11,12]. This enabled the expansion of 67 the current human microbial database by reporting the isolation of a significant number of novel bacterial species and rendered the identification of previously considered "unclassified 69 organisms" possible in clinical settings [12].

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EDs are considered as MDC [13]. Concretely, BPA is used in polycarbonate and epoxy resins 72 and packages. Its cumulative contamination reaches all kinds of environments, such as soils, 73 sediments, and aquatic environments, water, air and dust particles [14]. Several routes of 74 human exposure to BPA have been described, including the digestive system (ingestion) 75 through exposure to food packaging, drinking containers, dental monomers [15,16]; the vertical 76 transmission (maternofetal) [17]; the respiratory system (inhalation) [18]; and the integumentary 77 system (skin and eye contact) though the thermal paper of the receipts, eyeglass lenses and 78 feminine hygiene products [19,20]. The presence of this obesogen or MDC in humans has been 79 confirmed by detecting it in human serum, urine, saliva, hair, tissue and blood [21,22]. Thus,

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BPA removal from the natural environment is an increasing worldwide concern and several 81 studies identified biological effective via to remove BPA from the environment through 82 organisms such as bacteria, fungi, algae and plants [23,24]. However, there are still no clear 83 clinical studies aimed at eliminating or reducing the amount of exposure to BPA in the human 84 body. The demonstrated evidence of the effects of BPA as an ED and its transfer to foods has 85 led the industry to use analogous compounds such as bisphenol S (BPS). However, recently 86 studies have shown that some of these analogues may be even more harmful than BPA [25]. In 87 this case, BPS has also been shown to act as an ED but investigation in this field has remained 88 limited [26]. Moreover, the use of NGP is increasing due to the specific knowledge of the The BPA-degradation capabilities from some microorganisms, like Bacillus spp., have been 93 studied as an environmental and bioremediation resource [27,28]. Furthermore, species from 94 this genus have been isolated from infant fecal samples with the four complete molecular 95 pathways of BPA degradation [29]. However, while the use of BPA-degrading microorganisms 96 is widely extended in bioremediation, based on a previous review [30]

Material and Methods 105
Culturomics review data for increasing the microbiota taxa isolates 106 Literature search and review of studies were developed in collaboration with Granada 107 librarian support using medical subject headings (MeSH) and the key words (see below) under 108 a stepwise procedure search and adapted to each database's tutorials. The following electronic and microbiota and "metabolic syndrome" "endocrine disrupt*"; Culturomics * and microbiota 117 and "metabolic syndrome" and xenobiotic*; Culturomics * and microbiota and "metabolic 118 syndrome" and hormon*; Culturomics * and microbiota and "metabolic syndrome" and "drug 119 metabol*"; Culturomics * and microbiota and diabetes and "endocrine disrupt*"; Culturomics * 120 and microbiota and diabetes and xenobiotic*; Culturomics * and microbiota and diabetes and 121 hormon*; Culturomics * and microbiota and diabetes and "drug metabol*"; Culturomics * and 122 microbiota and fertility.

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Experimental Culturomics approach to isolate gut microbes metabolizing obesogenic ED 124 BPA Directed-Culturing approach for the isolation of microbiota strain catalogue

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A common approach to isolate microbial strains from microbiota has been pursued in our 126 research team [29]. For this study, 235 microbial isolates from fecal human microbiota

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In parallel, a specific treatment was carried out to favor the isolation of spore-forming bacteria.

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For this, after the exposure to BPA and before the spread on the media, the samples were

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In order to discover the presence of BPA biodegradation gene potential of cultured microbiota, 162 several bioinformatics tools were used to perform genome mining. A data retrieving program has been specifically computed using Pascal programming language to obtain the BPA 164 pathways enzymes ID and the corresponding Loci from the microbial genomes.

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Type strains genomes from the closest species isolated were retrieved from NCBI Genome Data 166 Bank in GenBank file format in order to list the proteins that they were able to potentially 167 encode the enzymes.

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A more detailed prediction of the clusters was performed by checking the downstream and 169 upstream genes of those involved in BPA biodegradation using NCBI genome map viewer.

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The identification of BPA genes encoding enzymes involved on the four biodegradation 171 pathways was carried out by the analysis of the WGS T of type strains, following the same 172 approach explained above.

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Theoretical searching on culturomics data, which were thoroughly analyzed, allowed retrieving 176 main culturing media and conditions used for isolation of relevant gut microbiota taxa 177 components are summarized in Table 1. This data extraction analysis displays at once a batery 178 of media for susscessful isolating of specific species belonging to genera from phyla Firmicutes,

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Similarly, useful information on favoured cultured isolates from gut microbiota acting as  [8]. Therefore, culturomics efforts contributed to enlarge the repertoire of isolated bacterial 195 species from humans by 28% and provided biological material to the scientific community that 196 can be further studied for its role and interaction with other bacterial species and host [33].

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Conversely, the efficient molecular methods, such as metagenomics, which aims to describe the 198 human microbiota with no culture efforts, needed complementary developing fields. However, 199 some drawbacks are encountered that require the use and development of comparing culture 200 approaches [34], such as sequencing depth bias [34,35], incomplete genomic databases [12, 33, 201 36] or the ability to distinguish between live and dead bacterial DNA in the studied samples 202 [36]. In a recent study that examined the gut microbiota composition of 8 healthy individuals, it 203 was shown that culturomics enabled 20% higher bacterial richness in comparison to 204 metagenomics [37]. Interestingly, isolated species' genome sequences enlarged by 22% the data 205 obtained by metagenomics analyses and showed that the number of species recovered by 206 culture is higher than the number of species detected by metagenomics [37]. 207 208  Table 2 for normo-weight children specimens analyzed and Table 3 for specimens from children with obesity. A phylum grouping data analysis showed differences in relative abundance of cultured Firmicutes, Proteobacteria and Actinobacteria between both populations.
Firmicutes were the most abundant phylum with BPA tolerance found, representing 72% in normoweight children and 73% in children with obesity. Proteobacteria was differentially represented in both groups by 17% and 20%, respectively. However, dataset showed differences in Actinobacteria and uncultured bacteria groups, Actinobacteria group represented 6% of the bacteria isolated in normo-weight children and 5% in children with obesity, in comparison to uncultured bacteria that represented 5% of the total bacteria isolated in normo-weight group, and 3% in population with obesity. Similarly, xenobiotics and specifically BPA tolerance by specific gut microorganisms was previously described for the traditional probiotics Bifidobacterium breve strain Yakult (BbY) and Lactobacillus casei strain Shirota (LcS) that showed protective effects against BPA dietary exposure in rats by reducing the intestinal absorption of BPA and facilitating its excretion [54]. Similarly, Lactococcus lactis strains adsorbed BPA but not degrade it [55]. Bioaccessible BPA decreased after digestion and this exposure changed microbial community, up-regulating the abundance of BPAdegrading bacteria, such as Microbacterium and Alcaligenes [56].  both populations (Fig. 1) representing near 75% of the taxa found. However, we can see differences between both groups in the minority BPA tolerant genera, some of these genera are exclusive of each population, conforming differential microbiota composition according to normo-weight children or children with obesity. The minority BPA tolerant genera found exclusively in normo-weight children were Rothia sp., Paraclostridium sp. and Bifidobacterium sp. However, Kocuria sp., Micrococcus sp., Burholderia sp., Raoultella sp., Shigella sp., and Latilactobacillus sp. were found exclusively in overweight and obese children.
(a) Normo-weight microbiota specimens. (b) Overweight and Obesity microbiota specimens. Microbial community from the culturomics approach and the statistical analyses showed that the obese group had more diversity, richness (Chao1, Observe) and evenness (Shannon, InvSimpson) than the normo-weight group. As for other culturomics studies, these results lead to complement already adapted approaches by highlighting the bacteria that were considered "un-cultivable" as they might be playing an important role in the health balance and disease development [36].
Importantly, culturomics for isolating new bacterial species included toxicogenomics approach to describe novel organisms able to metabolize toxicants [6]. New bacterial species are subjected to a series of phenotypic, biochemical and genomic characterization (habitat, sporulation, shape, antibiotics profile, metabolism, fatty acids contents, genome sequencing/ assembly and annotation).

BPA biodegradation metabolic maps through WGS T data mining
The bioinformatics analysis carried out on the WGS of Type strains of closest species identified as cultivable species from microbiota showed a differential potential of BPA biodegradation and specific enzymes arsenal involved (  Genome mining data based on WGS T representative BPA biodegradation analyses was achieved through the advances in next generation sequencing (NGS) and in silico tools allows performing an appropriate screening of genes of concern or interest in microbiota, such as biodegradation capacities or toxicomicrobiomics potential through bioinformatics, metagenomics or in silico analysis of cultivable isolates WGS [58,59]. A better understanding of the microbiota ecology driven by the bioactive compounds, which are released by gut microbial components may drive towards better clinical interventions [60]. Genome mining done in the present study allowed BLAST driven searching for predicted BPA pathways. Pascal ad hoc programme analysed the type strain genomes making it a powerful prediction tool. Similarly, another useful prediction tool could be used as well as for BPA biodegradation pathways [61].
Interestingly, the species found exclusively in normo-weight children microbiota (Paraclostridium sp. and Bifidobacterium sp.) had a low BPA biodegradation potential, being clustered as BPA tolerant or resistant.
However, the species from obese children (Kouria sp., Micrococcus sp., Burkolderia sp., Raoultella sp. and Shigella sp.) showed higher BPA degradation potential, being grouped as BPA biodegrader. Thus, a first trend of this analysis showed that microorganisms from obese children seemed to present more BPA biodegradation potential than normo-weight children.
Moreover, comparative data from wide metagenomics analysis regarding the variability of taxa composition in individuals with obesity and normo-weight, Firmicutes/Bacteroidetes (F/B) ratio constitutes a recognized biomarker for comparisons, as well as Actinobacteria and Proteobacteria relative abundances. F/B ratio showed higher values in obese than normo-weight individuals [62] as Actinobacteria appeared usually also higher in obese population. Conversely, Bacteroides and Proteobacteria were slightly higher in normo-weight populations [62]. In this sense, it is important to consider the ecological role of those enzymes and their impact on the gut microbiota composition may have a huge influence on metabolizing and neutralizing BPA, by releasing metabolites that contribute to the modification of individual taxa microbial components on long-term basis [63].
Interestingly, specific transitory gut taxa identified with high potential of BPA biodegradation could be also used for environmental bioremediation purposes or plant probiotics. Several authors investigated the BPA removal capacity using bacterial strains from dessert soil that belong to Pseudomonas putida, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella sp. and Pantoea sp. [64]. Degradation of BPA by Pseudomonas putida YC-AE1 was considered as a low cost effective and eco-friendly method compared to physical and chemical methods [65]. Similarly, a consortium isolated from river sediment (Terrimonas pekingensis and Pseudomonas sp.) was able to use BPS as the sole carbon source and was highly efficient to degrade 99% with an initial concentration of 50 mg/L in 10 days [66]. Gut bacteria harbouring laccases could be used for detoxification of several hazardous dietary contaminants and emerging ED through bioreactor with novel biocatalytic system based on active membranes and immobilized laccase technology [67].

Conclusions
We are exposed to obesogenic MDC, such as bisphenols and concretely to BPA. The pathophysiological impact of these obesogens seem to depend on inter-individual and diverse microbial gut composition, and we are just starting to understand how these microbiota consortia interact with host and how their enzymatic arsenals would shape those communities to build a functional human microbiome. Our results indicate that specific and differential gut enriched microbial isolates or consortia that resist, tolerate or biodegrade BPA were present in human-associated microbial communities and they harboured the specific gene encoding enzymes involved in biodegrading BPA and other obesogens, and that such enhancing enzymatic properties of the gut communities could perpetuate their modulation ecological actions, even after the exposure to obesogens or BPA should be present, impacting in health and disease host status.

Ethics approval and consent to participate
Fecal sample library was obtained after corresponding approval of CEIC 20/12/2019.

Consent for publication
Not Applicable.