dataesr/scientific-paragraphs-categorization huggingface dataset:
The intact prophage most common regions were identified on the strain ProNaCC2 (ΦN315, ΦSa119), ProNaCC4 (ΦN315, ΦBU01), and ProNaCC7 (ΦN315, ΦSa119, ΦNM3, ΦBU01). The phage regions included the gene for the staphylococcal enterotoxin type E in the strain ProNaCC2 and the gene for the staphylococcal enterotoxin type A in the strains ProNaCC4 and ProNaCC7 (Table  7 ). The genetic surrounding of the tst locus was further investigated for the genomic localisation, because this virulence factor can be associated with SaPIs, which includes the genes for specific staphylococcal enterotoxins. The database developed for this analysis was built for ABRicate and had sequences of 74 SaPIs included the specific sequences of SEs found in these loci (Supplementary Material Database S5). The analysis showed that all six strains contained the SaPI3 similar to the SaPI described in Staphylococcus aureus subsp.
Those genomic loci were analysed for the detection of the SEs gene sequences, but intact sequences were not found (Table  7 ). In total, 4456 S. aureus plasmid complete sequences were used to build the novel ABRicate database (Supplementary Material Database S7). The database was filtered for the presence of staphylococcal enterotoxin sequences using the database developed in this study (Supplementary Material Database S3). The plasmids positive for SEs gene sequences were 186; from this group, the plasmids with the presence of enterotoxin genes with a percentage of coverage greater than or equal to 95. 00 at the nucleotide level with the reference sequences were selected. In this selection, were found the gene sequences of the enterotoxin sed, selj, ser, ses and set, confirming the plasmid location of these genes  [23].
The sequence of the plasmid pLUH02  [75]  (ref. NZ_LJBK01000031. 1) was found almost intact (98. 11% coverage and 99. 78% identity at the nucleotide level) in the genome of the strain ProNaCC1 (Table  S8 ). The sequence of the plasmid pSK67 (ref. GQ900447. 1) was found in the genome of the strain ProNaCC7 with an identity at the nucleotide level of 99. 83% but a coverage lower than the threshold (85. 14%). Both the plasmid sequences tested for the enterotoxin database resulted positive for the sequences of sed, selj, ser (Figure  8 ). The genetic surrounding of the tst locus was further investigated for the genomic localisation, because this virulence factor can be associated with SaPIs, which includes the genes for specific staphylococcal enterotoxins. The database developed for this analysis was built for ABRicate and had sequences of 74 SaPIs included the specific sequences of SEs found in these loci (Supplementary Material Database S5).
11 at the nucleotide level and at 0. 44% in the genome of the strains ProNaCC5 and ProNaCC6. Those genomic loci were analysed for the detection of the SEs gene sequences, but intact sequences were not found (Table  7 ). In total, 4456 S. aureus plasmid complete sequences were used to build the novel ABRicate database (Supplementary Material Database S7). The database was filtered for the presence of staphylococcal enterotoxin sequences using the database developed in this study (Supplementary Material Database S3). The plasmids positive for SEs gene sequences were 186; from this group, the plasmids with the presence of enterotoxin genes with a percentage of coverage greater than or equal to 95. 00 at the nucleotide level with the reference sequences were selected. In this selection, were found the gene sequences of the enterotoxin sed, selj, ser, ses and set, confirming the plasmid location of these genes  [23].
The sequence of the plasmid pLUH02  [75]  (ref. NZ_LJBK01000031. 1) was found almost intact (98. 11% coverage and 99. 78% identity at the nucleotide level) in the genome of the strain ProNaCC1 (Table  S8 ). The sequence of the plasmid pSK67 (ref. GQ900447. 1) was found in the genome of the strain ProNaCC7 with an identity at the nucleotide level of 99. 83% but a coverage lower than the threshold (85. 14%). Both the plasmid sequences tested for the enterotoxin database resulted positive for the sequences of sed, selj, ser (Figure  8 ).
In addition, the genes arlR and arlS, which are involved in the cascade reaction for the norA activation, were found in all the strains except the strain ProNaCC5. The penicillin resistance gene blaZ was identified in the strains ProNaCC1 and ProNaCC7. The gene was present in both the strains with a sequence identity at the nucleotide level of 96. 45 relative to the CP000732 reference sequence. The fusidic acid resistance gene fusC was found only in the strain ProNaCC6 with a sequence identity of 100% with the NC_002953 reference sequence. The lanthionine-containing peptide antibiotic (lantibiotic) gene (bsaA2)  [77] , was the only bacteriocin found among the six enterotoxigenic S. aureus genomes that had a sequence identity at the nucleotide level of 100% with the reference sequence of the bagel3 database.
aureus genomes that had a sequence identity at the nucleotide level of 100% with the reference sequence of the bagel3 database. The sequence was found in the genome of the strains ProNaCC1, ProNaCC6, and ProNaCC7. The second highest percentage identity bacteriocin gene was the delta-hemolysin (hld), which had 80. 00% of identity with the reference sequence, and it was found in the genome of ProNaCC1, ProNaCC4, ProNaCC5, and ProNaCC7. In total, 38 other bacteriocins (Table  S9 ) with a sequence identity at the nucleotide level were found to range between 3. 33% and 54. 55%.
Core Genomes A pan-genome of 5651 genes was generated from the six enterotoxigenic strains and 29 enterotoxigenic strains reported in the literature (Table  S2 ). The total number of core genes was 1364, which represents the number of genes that are shared in a percentage higher than 99% among the six strains. Based on the output of Roary, insight into their pan-genomic property was also gained. In Figure  10 , presents the tree constructed by clustering strains based on accessory genes and the matrix plot, which denotes the presence and absence of every gene over all strains by blue and white, respectively. Figure  10  presents the reported the geographic locations (Australia, Denmark, France, Italy, Japan, Russian Federation, Switzerland, United Kingdom), isolation sources (Staphylococcal Food Poisoning event, milk, clinical and food samples), and the enterotoxins identified with the ABRicate database for each strain.
Food Poisoning event, milk, clinical and food samples), and the enterotoxins identified with the ABRicate database for each strain. Figure  10. Pan-genomic analysis of the 35 enterotoxigenic strains. On the left side is the phylogenetic tree of the strains included in this study. On the central heatmap is the the geographical location of isolation and sources (colour legend in the picture). The detection of the gene coding for enterotoxins is reported as presence and absence with the colours purple and orange respectively. At least two genes coding for staphylococcal enterotoxins were detected in all the genomes included in the analysis. The genes selx and selw were the most prevalent among the 39 strains; nevertheless, no correlation was found between the country of isolation and origin of the samples.
The functional annotation of the six-enterotoxigenic S. aureus pan-genome was 66. 2% of the COGs database with known functions of the total pan-genome (Figure  11 ). The pangenome of the six enterotoxigenic S. aureus was assembled to highlight the genetic and pathogenic diversity of the strains, and the locations of the SE genes compared to the core genome were validated. The gene of the staphylococcal enterotoxin-like x gene (selx) was detected in the core genome, as already described, along with the gene selw, in almost all the 35 strains included in the pangenome study. However, the genes coding for enterotoxins were detected mostly in specific accessory genomic regions, demonstrating the importance and the role of these genomic structures for the evolution of S. aureus as foodborne pathogens.
aureus enterotoxigenic strains isolated from foods and from SFP outbreaks by PCR  [80, 81]  with the use of microarray-based methods  [82]  and analysing the whole genome sequences  [83, 84]. However, these studies focused on the presence of few VFs or specific staphylococci related to host  [85]. In this study, 149 genes were detected in the genome of the enterotoxigenic strains and the genes related to staphylococcal enterotoxins were further characterised with the use of dedicated databases developed in this study. The VFs were grouped into six functional categories and their distribution was determined in the six enterotoxigenic strains. In addition, the strains with other 29 enterotoxigenic strains reported in the literature were used for constructing the pan-genome of enterotoxigenic S.
aureus, highlighting the genomic location of the enterotoxins and their role in the evolution of its genome. The virulence of S. aureus is facilitated by the attachment to the host cell and is mostly mediated by adhesins  [86]. In the genome of the six enterotoxigenic S. aureus strains 31 genes coding for proteins that mediate the adherence to damaged tissues, extra-cellular matrix, and the surface of cells were annotated. The intercellular adhesion (ica) locus consists of different genes (icaA, icaB, icaC, icaD) and the promotor icaR, which are involved in the biofilm formation initiated by atl  [87, 88]. In other studies, the strains ica-positive were isolated in both the food and clinical environment, but their presence was not always associated with the production of biofilm  [89]. The database included other genes involved in the formation of biofilms, i.
The toxins produced by S. aureus are different molecules that can be grouped in hemolysins, leukocidins/leukotoxins, toxic shock syndrome toxin, exfoliative toxins, exotoxins and enterotoxins. In addition, the genes involved in the secretion system were included in this group of genes and all the six strains contained at least one of them (esaA/esxA/yueB, esaB/esxB/essB, essC/esxC, essD/esxD) previously described as a versatile secretion system in S. aureus that might be involved in the transport of protein and or DNA  [71]. The strains resulted positive for the staphylococcal enterotoxins genes commonly reported as a causative agent of food poisoning. With the use of the novel ABRicate database for enterotoxins and their variants, further enterotoxin genes were identified, which completed the comprehensive portraits of the strains presented in this study.
S. aureus acquires VFs encoded in the mobile genetic elements such as prophages but also plasmids, pathogenicity, and genomic islands, providing adaptation but also a genetic rearrangement resulting in hypervirulence of some strains but also the loss of portion of genes  [44]. Interestingly, the sequences entA_1-3 were found in the genome of all the strains (Figure  6 ). These sequences are classified as enterotoxin family protein elements but they are not complete or have a low percentage of coverage and identity at the nucleotide level to the reference sequence of the enterotoxin type A. For this reason, the sequences of SEA genes were not confirmed with the database ABRicate for enterotoxins, confirming the importance of a validated database for staphylococcal enterotoxins that considers the manual validation of sequences.
The SaPIs database was used for evaluating the presence of enterotoxins in these genetic structures and at least one SaPI was found in each strain showing that SaPIs are widespread among the six enterotoxigenic strains. The sequences did not carry out SE genes but identical sequences among the strains were detected, also in the form of small fragments, explaining their phylogenetic relationship due probably to horizontal transfer. The genes for the plasmid encoded enterotoxins SED, SElJ and SER were found in the genome of the strains ProNaCC1 and ProNaCC7. The plasmid sequences found in the genome of the strain ProNaCC1 had a 98. 11% coverage and 99. 78% identity at the nucleotide level with the plasmid pLUH02 (accession number NZ_LJBK01000031. 1) (Table  S8 ), which was described in a strain responsible for two outbreaks in a maternity hospital in Belgorod, Russia  [75].
The sequences of the S. aureus plasmid pSK67 (accession number GQ900447. 1) were identified in the genome of the strain ProNaCC7 and had an 85. 14% of coverage and 99. 83% identity with the reference sequence (GQ900447. 1) (Table  S8 ). The gene coding enterotoxins (sed, selj, ser) were found within the sequences of the plasmids confirming their origin from mobile genetic elements. The sequences of the plasmids detected in this study had similarity at nucleotide identity between 98. 87% and 99. 96% with the plasmids pIB485 (accession number AF053140. 1 and M94872. 1) and pF5 (accession number AB330135. 1) reported in strains responsible for food poisoning  [23, 103, 104]. However, the coverage corresponds only for the sequences of sed and selj, which are carried out in pIB485 and the gene selj and ser carried out in pF5, while both the strains (ProNaCC1 and ProNaCC7) carried out the sequences of the genes sed, selj, and ser in a unique putative plasmid region.
06 g/L) (Table  S11 ), and the strains ProNaCC6 resulted resistant (≥0. 5 g/L). The gene of the S. aureus fluoroquinolone efflux transporter protein NorA was detected in all the sequenced strains. The overexpression of this gene causes the resistance mechanism, but unfortunately transcriptional data were not included in this study. Nevertheless, all the strains resulted resistant to enrofloxacin at a concentration of ≤0. 5 g/L. The pangenome of the six enterotoxigenic S. aureus was assembled and applied to highlight the genetic, metabolic, and pathogenic diversity of the species in comparison with other 29 enterotoxigenic strains. Consistent with prior studies, the numbers of new genes per genome indicated substantial intraspecific variation and overall pangenome "openness" associated with a broad niche range  [110, 111]  but it is also a characteristic of populations that undergo frequent horizontal gene transfer  [112].
A particular emphasis was placed on the analysis and distribution of enterotoxin genes among the six enterotoxigenic strains (Figure  11 ) and, the gene coding for the novel SAg staphylococcal enterotoxin-like toxin X (gene selx) was found in the core genome of the six enterotoxigenic strains; this result is comparable with another study where the gene was found in the core genome of 95% of a diverse S. aureus population, including isolates from human and animal infections  [102]. The other genes coding for staphylococcal enterotoxins were detected onto mobile genetic elements (Table  7 ) and is one of the reasons why the pangenome resulted closed. In fact, the addition of other enterotoxigenic strains in the dataset does not provide new genes to the species pangenome. This feature is typical for species that live in isolated niches with limited access to the global microbial gene pool; indeed, the genomic and phenotypical diversity is due to the transfer of genetic material, including resistance genes and enterotoxins.
This study was limited to the genomic and in-silico prediction of the presence of VFs and SEs. Future developments and validation of methods for the detection of not-classical SEs and proteomic approaches will allow for a better understanding of in vivo production of the enterotoxins. The strains were whole-genome sequenced and were detected virulence factors, antimicrobial resistance genes, and mobile genetic elements with a specific focus on the staphylococcal enterotoxin and enterotoxin-like genes. Using novel databases offers a comprehensive portrait of enterotoxigenic strains of S. aureus used for the production of naturally-contaminated cheeses, and in the future, the identification of SEs and the development of innovative computational approaches will allow more accurate identification of additional genetic factors and genes necessary for the production of enterotoxins in food.
Supplementary Materials: The following are available online at http://www. mdpi. com/2073-4425/11/1/33/s1, Database S1: Multifasta file containing the sequences of the virulence factors genes included in this study; Table  S2 : List of the strains used for building the pan-genome of enterotoxigenic S. aureus. Database S3: Multifasta file containing the 99 sequences of staphylococcal enterotoxins genes and their variants included in the ABRicate database; Table  S4 : Prophage most common regions within the six enterotoxigenic genomes detected in this study; Database S5: Multifasta file containing the 74 sequences of SaPIs included in the ABRicate database; Table  S6 : Details of the SaPIs detected in the samples with an identity percentage higher than 95. 0; Database S7: Multifasta.
Acknowledgments: Research in the Decastelli's laboratory is funded by the  Italian Ministry of Health  in the form of a centre grant for the "National Reference Laboratory for Coagulase Positive Staphylococci, including S. aureus" The authors thanks  Noémie Vingadassalon  for the analysis of the EURL-CPS strains collection and selection;  Isabelle Mutel  and  Florence Guillier  for the toxin detection;  Pascal Bouchez  for the viability and curation of the collection and  Jacques-Antoine Hennekinne  for the scientific support.
56 UniProtKB-G0Z025 SEG 9 13,778 14,550 99. 87 86. 16 UniProtKB-P0A0L8 ProNaCC5 SEN SElU 9 9 14,836 15,630 15,609 16,397 100 98. 08 85. 92 84. 42 UniProtKB-A0A0H3JS72 UniProtKB-Q6XXM3 SEI 9 16,560 17,279 99. 17 87. 5 UniProtKB-O85383 SEM 9 17,320 18,033 99. 58 85. 01 UniProtKB-A0A0H3K005 SEO 9 18,317 19,096 100 87. 82 UniProtKB-A0A0H3JS76 SElW 1 1,068,149 1,068,933 99. 87 97. 84 GB-KX655711. 1 ProNaCC6 SElX 2 96,391 96,999 100 82. 92 UniProtKB-G0Z026 SEH 5 98,979 99,700 99. 86 86. 29 UniProtKB-P0A0M0 SED 11 6058 6830 99. 87 84. 73 UniProtKB-R9SA89 SElJ 11 7725 8509 97. 64 83. 95 UniProtKB-O85217 ProNaCC7 SER SEA 11 2 8626 88,812 9400 89,579 99. 74 99. 61 -84. 9 85. 81 UniProtKB-Q76LS8 UniProtKB-P0A0L2 SElW 5 93,897 94,682 100 100 GB-KX655710. 1 SElX 7 241,512 242,120 100 84. 24 UniProtKB-G0Z026 1Sequence Start: start coordinate in the sequence; 2 Sequence End: end coordinate in the sequence; 3 % Coverage: proportion of gene covered; 4 % Identity: proportion of exact nucleotide matches; 5 Reference: the genomic source of the sequence (UniProtKB: UniProt Knowledgebase; GB: GenBank nucleotide reference number.
BrdU and Ki-67 detection with Flow BrdU or Ki-67 detection kit (BD Pharmingen) were performed according to the manufacturer's protocol. Flow cytometry acquisitions were performed with a FACS Gallios TM system (Beckman Coulter) and data was analyzed with Flowjo software (TreeStar, Ashland, OR). Epidermal sheet preparation and labeling. Tail skin epidermis from C57Bl/6, Tabby or control mice were isolated from dermis by dispase II treatment as described above. Epidermal sheets were fixed with cold acetone on ice for 20min. Sheets were then washed in TRIS-buffered saline (TBS) and non-specifics sites were blocked in TBS supplemented with 5% donkey serum for 1 11/6/2019 hour at room temperature. Tissues were then incubated with 1µg/mL anti-Langerin-AF488 or 1µg/mL uncoupled anti-Ia antibody (2G9, BD) for 3 hours at room temperature.
ACKNOWLEDGEMENTS The authors would like to thank  Astrid Hoste  for assistance with immunohistochemistry, Monique Duval and  Delphine Lamon  for mouse care,  Dr. Jean-Daniel Fauny  for his help with confocal microscopy analyses and  Claudine Ebel  ( Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France ) for cell sorting. We also thank  Dr. Hélène Haegel  ( Transgene, Strasbourg, France ) who provided anti-CSF-1R blocking antibody. We are indebted to  Pr. Nikolaus Romani  for critical reading of the manuscript as well as  Pr. Ralf Paus  for discussion. This work is supported by the  Centre National pour la Recherche Scientifique  and the  Agence Nationale pour la Recherche  ( Program "Investissements d'Avenir ",  ANR-10-LABX-0034   MEDALIS  ;  ANR-11-EQPX-022 ). B.
Voisin is funded by the  French Ministry of Research and Higher Education  and by the  Fondation pour la Recherche Médicale  ( FDT20130928345 ). V. Flacher  and  C. G. Mueller  are supported by the  CNRS and European Union  grants ( Marie-Curie Career Integration Grant  " Dermacro PCIG12-GA-2012-334011 " to  V. Flacher  and  Internal Training Network  " STROMA ITN-289720 " to  C. G. Mueller). CONTRIBUTIONS B. Voisin ,  D. Chartoire ,  C. Devouassoux  and C. Kowalczyk-Quintas performed experiments. F. Clauss and  P. Schneider  provided Tabby mice. F. Lezot provided IL-34tm1a mice. B. Voisin ,  V. Flacher  and  C. G. Mueller  wrote the manuscript. The authors have no conflicting financial interests.
Data analysis. The antibacterial effect of aztreonam in combination with avibactam was investigated by modeling the interaction between both compounds, using WinNonlin software (version 6. 2, Certara, Princeton, NJ, USA). Aztreonam MICs versus avibactam concentrations were fitted according an inhibitory E max model with a baseline effect parameter (Equation  2 ). A Gauss-Newton minimization method with the Levenberg and Hartley modification was used for data fitting. Data were not logtransformed before analysis and we used the weighted least squares method for minimization based on observations (Yobs i ) i. e. the weight applied to each observation (w i ) was: w i = 1/(Yobs i )². MIC CAVI =MIC 0 - (MIC 0 -MIC ∞ )×C AVI (C AVI +IC 50 ) (Equation 2) Where, MIC 0 is the MIC of aztreonam in the absence of avibactam and MIC ∞ the asymptotic value of MIC when avibactam concentration (C AVI ) tends toward infinity.
Funding This research project was funded by the  Innovative Medicine Initiative Joint undertaking under COMBACTE-CARE  grant agreement  115620  resources of which are composed of financial contribution from the  European Union Seventh Framework Programme  ( FP7/2007-2013 ) and  European Federation of Pharmaceutical Industries and Associations (EFPIA)  companies in kind contribution. Avibactam dry powder was provided by  AstraZeneca.
4 ). After 7 days, we observed a reduction of 2 logs in the total number of spores in BHIS with DOC when compared to cells grown in BHIS or BHIS with CHO (Fig. 4a ). We also found that toxin-encoding genes were downregulated in the DOC-induced biofilm (Supplementary Data 1), a finding confirmed by qRT-PCR. However, the RNA samples used for the transcriptome were obtained from biofilms produced in BHIS containing glucose, which is also known to repress toxin synthesis. 36 To determine the impact of DOC on toxin synthesis, we prepared RNA from cells grown for 48 h in BHIS and BHIS with DOC or CHO. The expression of tcdA and tcdB was down-regulated by 6-or 3-fold in BHIS with DOC when compared to BHIS or BHIS with CHO, respectively (Fig. 4b ). We confirmed by ELISA that the addition of DOC to BHIS strongly reduced production and release of toxin A, while the addition of CHO had little or no effect (Fig.
2). Three stacks of images were acquired randomly on three independent samples. Fluorophores were excited and emissions were captured as prescribed by the manufacturer. Simulated 3D fluorescence projections were generated using IMARIS 7. 0 software (Bitplane, Zürich, Switzerland). RNA isolation and quantitative reverse-transcriptase PCR One full 24-wells plate was used to produce one replicate for one condition. After 48 h, the supernatant was removed by inverting the plate. The unwashed biofilms from BHISG were recovered in 20 mL of PBS. In BHISG with 240 μM DOC, the biofilm formed was washed twice and resuspended in 20 mL of PBS. The recovered washed (BHISHG + DOC) and unwashed (BHISG) biofilms were then centrifuged and total RNA was extracted from cell pellets as previously described. 57 cDNA synthesis and qRT-PCR were carried as described before  57  using primers listed in Supplementary Table  2.