A metagenomic assessment of bacterial community in spices sold open-air markets in Saint-Louis, Senegal

A metagenomic assessment of bacterial community in spices sold open-air markets in Saint-Louis, Senegal | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A metagenomic assessment of bacterial community in spices sold open-air markets in Saint-Louis, Senegal Sarbanding Sané, Abou Abdallah Malick Diouara, Seynabou Coundoul, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4202002/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Natural spices are reputed to play an essential role in human nutrition and well-being due to their organoleptic and therapeutic properties. Moreover, they are increasingly being processed on various scales, exposing them to potential sources of contamination. This study aimed to describe the bacterial community in spices sold in Senegal. Thyme, Curcuma, a mixture of 7 spices and "Local Spices or Herbs" (LSH) samples were collected in selected open-air markets in August 2022 in Saint-Louis, Senegal. DNA extraction was performed using the Omega E.N.Z.A Food DNA kit. PCR assays were based on a genomic fragment encoding bacterial 16S rRNA, which was subsequently sequenced using Oxford Nanopore technology (ONT). Sequencing was carried out on two batches of samples, one containing part of the "Local Spices or Herbs" (n = 10) designated as "a mixture of food seasonings", and the other, samples of a mixture of 7 spices, Curcuma, Thyme and the other part of the "Local Spices or Herbs" (n = 39). Analysis of the sequencing data showed high bacterial diversity and the predominance of Escherichia coli , Salmonella enterica and Escherichia marmotae in the samples, with a total number of reads of 65744 and 165325 for the two batches, respectively. The sample category "Homemade mixture of food condiments (ready to use)", which includes all "Local Spices or Herbs" samples, showed remarkable bacterial diversity. These were followed by Curcuma, a blend of 7 spices and thyme, respectively. These results highlight a highly diverse genomic profile of the microbial community, including pathogenic bacteria, in spice samples. This is the first metagenomic study to assess microbial diversity and analyze microbial community structures in spices in Senegal. Spices Metagenomic Bacterial community Sequencing Figures Figure 1 Figure 2 Figure 3 1. Introduction Spices have been used for centuries as an essential component in traditional Asian medicine [ 1 – 2 ]. Some of them draw special attention due to their use in the cosmetics and pharmaceutical industries [ 3 ]. Spices are reputed to be a source of bioactive compounds [ 4 ] with sometimes rich extracts in polyphenolic compounds, including high levels of antimicrobial, antioxidant and anti-inflammatory activity [ 5 – 6 ]. Preventive and curative actions of spices against cancer, gastrointestinal disorders, psychological disorders, diabetes, obesity, metabolic syndromes and neurological disorders were also reported [ 7 – 8 ]. Aromatic plants and spices have thus become well established over time, justifying their use by around 80% of the world’s population, mainly in developing countries [ 7 ]. Spices can be divided into several groups: strong spices (pepper, chilli, ginger), mild spices (paprika, coriander), aromatic spices (cinnamon, curcuma, cloves, cumin, anise, celery), dried herbs (basil, bay leaves, dill, marjoram, tarragon, thyme) and aromatic vegetables, for instance, onions or garlic [ 9 ]. From a nutritional point of view, China and some West African countries have begun to use condiments and seasonings to remedy significant dietary deficiencies, particularly in iodine, iron and vitamin A [ 10 ]. According to several studies, various micronutrients are added to condiments depending on the deficiencies demonstrated in the target population. The bioavailability of the micronutrient remains highly important, as it must be absorbed by the body in sufficient quantities to contribute effectively to improving these deficiencies [ 10 , 11 ]. Despite the awareness messages to limit the overconsumption of processed food products while promoting natural products in the diet [ 12 – 14 ], the use of industrial ingredients remains a concern [ 15 ]. However, spices and broths made from natural products can also be at risk for consumers. Indeed, due to transformation processes such as extraction and packaging processes, these can be subject to contamination by bacteria, viruses, parasites and pathogenic fungi due to handling [ 16 – 17 ]. Furthermore, the diversity and abundance of microbial communities in these food matrices can influence their compositions, nutritional benefits and even the functioning of the gut ecosystem [ 18 – 20 ]. Traditional studies of bacterial diversity in foods have relied on culture methods, whereas only around 1% of microorganisms present in the natural environment can be grown by these methods [ 21 – 22 ]. Culture-independent approaches involving molecular biology methods such as gradient gel electrophoresis (DGGE) polymerase chain reaction (PCR) and extended later to metagenomic methods to assess microbial diversity and analyze microbial community structures in diverse environments, including food matrices [ 22 – 25 ]. This study aimed to describe the genomic footprint of the microbial community present in spices sold in open-air markets in Senegal using the sequencing method. 2. Material and methods Collection and preparation of samples Samples of Curcuma, a mixture of 7 spices (cloves, ginger, garlic, cumin, lemon, nutmeg and salt), Thyme and “Local Spices or Herbs” (LSH, a mixture of food condiments used to season dishes) were collected from 3 markets in Saint-Louis in August 2022 in compliance with the sampling process [ 26 ]. For a mixture of 7-spices, thyme and Curcuma samples, 30 g were purchased, while 100 g was for “LSH”. Samples were placed in an individual sterile zipped bag. “Pseudo-industrial spices” are in dehydrated format and sold in their original packaging, particularly Curcuma, a mixture of 7-spices and Thyme, which were transported and stored at ambient temperature until their use. However, the individuals’ zipped bags of “LSH” samples were placed and transported in an insulated bag maintained at + 4°C to the laboratory, ensuring their preservation. The samples were then kept at -20°C until analysis. Extraction of Bacterial DNA Samples were subjected to manual extraction using the Omega E.N.Z.A Food DNA kit ( Omega Bio-Tek, GA, USA ) in accordance with the manufacturer’s instructions, with a few minor modifications. To optimize cell lysis, four beads with a diameter of 2.0 mm ( https://zymoresearch.eu/products/zr-bashing-bead-lysis-tubes ) were added to the lysis buffer before beating the sample with Analog Disruptor Genie ( https://www.scientificindustries.com/otuli-mixers-and-shakers/bead-beaters/analog-disruptor-genie.html ) during 5 minutes. The DNA concentration was determined by a Nanodrop spectrophotometer One (Thermo Scientific, USA), and the sample was stored at − 20°C until analysis. PCR Amplification and Sequencing The region coding for the 16S subunit of bacterial RNA was amplified using the universal primers Fd1 (5’ – AGA GTT TGA TCM TGG CTC AG – 3’) and Rd1 (5’ – AAG GAG GTG ATC CAG CC– 3’) [ 27 ]. The reaction volume for amplification was 25 µL and included 12.5 µL of One Taq® Quick-Load 2X Master Mix with Standard Buffer kit (New England Biolabs, M0486), 2 µL of primers, 5 µL of DNA and 5.5 µL of molecular grade water. PCR was performed under the following conditions: an initial denaturation of 94°C for 30 seconds followed by 30 cycles of 94°C for 30 seconds, 50°C for 30 seconds, 68°C for 1 minute and a final extension of 68°C for 5 minutes. Amplified fragments were visualized by electrophoresis on a 1% agarose gel using ethidium bromide staining and UV light. Amplicons were then purified using the Zymo DNA clean & concentrator TM kit-5 (Zymo D4034). Library preparation was performed using the Rapid Barcoding Kit (SQK – RBK 110–96). The sequencing was performed on Flow Cell R9.4.1 (FLO-MIN106D) on a MinIon Mk1C sequencer. A real-time workflow Epi2me platform was used for species identification. Alpha diversity, which shows the complexity of species within a sample, was obtained using four indices: Chao1, Shannon, Simpson and observed species. For this, the Vegan and Phyloseq packages of the R Studio software were used. Beta diversity, which shows the complexity of species across all samples, was measured using Jaccard and x indices. To obtain this diversity, the Vegan and Ecodist packages of the R software were used. All figures were also generated using the R software. Statistical Analysis All statistical analysis was performed on R software. For each considered spice group, the Wilcoxon test was performed for differential analysis of diversity. The significance level was set at p < 0.05. The degree of significance is proportional to the number of stars shown at the top of the figures (* corresponds to p < 0.05, ** corresponds to p < 0.01 and *** corresponds to p < 0.001). In order to assess the similarity between the different microbial communities in the considered spice groups, beta-diversity was calculated based on the Bray-Curtis dissimilarity distance and the Jaccard index. The result was then represented using a principal coordinate analysis (PCoA). 3. Results A total of 49 samples divided into two categories, including "Homemade mixture of food condiments (ready to use)" (n = 19) and "pseudo-industrial spices" sold dehydrated with primary packaging (n = 30), were collected and analyzed in two batches. The first batch consists of 10 "Local or Spices Herbs" samples. This type of spice is a blend of green onions, peppers, garlic and Sofia bell pepper (Capsicum annum). The second batch, designated as "pseudo-industrial spices, " was made up of thyme, 7-spice mix, curcuma, and the "Local or Spices Herbs" samples. For each sample type, additional details, such as the numbers and collection locations, are reported in Table 1 . Table 1 Distribution of samples according to type and collection sites Categories Types of samples Dry or dehydrated form Number of collected specimens Collection sites “Homemade mixture of food condiments (ready to use)” Local spices or herbs no 19 Sor/ Saint-Louis Pseudo-industrial spices Mixture of 7 spices yes 10 Sor, Guet Ndar and Pikine / Saint-Louis Curcuma yes 10 Sor, Guet Ndar and Pikine / Saint-Louis Thyme yes 10 Sor, Guet Ndar and Pikine / Saint-Louis Sequencing data were processed using Nanopore's Epi2me software. Quality scores were 9.87 and 9.82, with classified read counts of 65744 and 165325, respectively for the two batches analyzed. Figure 1 shows the predominant bacterial species for all the samples processed in this study. Generally speaking, Escherichia coli and Salmonella enterica are the most represented species in all samples (Fig. 1). ------------------------------------- Figure 1 Relative distribution of bacterial species in samples ------------------------------------- Analysis of bacterial diversity according to spice type showed the predominance of Escherichia coli , Salmonella enterica and Acinetobacter seifertii , with proportions differing slightly from one category to another (Supplements 1–4). All species with an abundance of less than 1% were merged into an "others" category. This group represented 21.2% of species in the "Local Spices or Herbs" and was mainly made up of Saccharothrix sp.6-C (4.7%), Shigella flexneri (4.5%) and Buchnera aphidicola (4.1%). For Curcuma, the "others" group represented 9.1% and was mainly made up of Acinetobacter baumannii (6.1%), Bacillus circulans 3.9% and Kosakonia cowanii (3%). For the 7 spices and Thyme samples, this group represented 4% and 2%, respectively. The predominant bacterial species were Acinetobacter baumannii (16.3%), Clostridium botulinium (11.3%) and Clostridium tatani (3.6%) for 7 spices, and Shigella flexneri (12.4%) and Pseudomonas fulva (6.7%) for Thyme. By considering all the types of spices, alpha-diversity analysis showed that "Local Spices or Herbs" had the most incredible diversity, with an average number of taxa per sample of 115.78. This group is followed by Curcuma, a mixture of 7 spices and Thyme, respectively. The average taxa numbers per sample were 41.1, 14.5 and 10.3, respectively (Supplement 1). About the number of species per spice group, after using the Wilcoxon test, there was a significant difference between "Local Spices or Herbs" and Thyme (p < 0.001), Curcuma and Thyme (p < 0.001), and Curcuma and the mixture of 7-spice (p < 0.01). However, there was no statistically significant difference between the mixture of 7-spice and Thyme, as shown in Figs. 2A and 2B. Figures 2C and 2D show that species' relative abundance and stability are greater for "Local Spices or Herbs". This group shows a significant difference with Thyme (p < 0.05). There is also a significant difference between Curcuma and Thyme (p < 0.001) and between Curcuma and the mix of 7-spice (p < 0.001). ------------------------------------- Figure 2 Alpha-diversity analysis ------------------------------------- Beta-diversity was illustrated using PcoA based on Bray-Curtis dissimilarity and Jaccard distance, which showed similarities in bacterial composition between samples from the different spice groups and showed two clusters (Fig. 3). One consisted of the species found in Thyme, Curcuma, a mixture of 7 spices samples and part of the “Local Spices or Herbs” samples. The second cluster consists solely of “Local Spices or Herbs” samples. These results show that the bacterial communities in the samples from the first cluster are closer than those in the second cluster. ------------------------------------- Figure 3 Beta-diversity analysis ------------------------------------- 4. Discussion This study aimed to describe the microbial community present in spices sold in open-air markets in Saint-Louis using the sequencing method. The high-throughput sequencing method used in this study allowed us to detect a broad-spectrum genomic footprint of micro-organisms, including those usually not detected by culture methods. Indeed, since the revolutionary improvement in DNA sequencing technologies, direct high-throughput analysis of the genomic DNA of an entire community without prior culture has become the most common approach, overcoming the constraints of conventional microbiological approaches [ 28 ]. In addition, from a technological point of view, MinION sequencing is mainly used to address metataxonomy [ 29 ]. Our results show the presence of many pathogenic bacteria in the spice samples studied, with their abundance varying according to the type considered. Studies in countries like Brazil have reported similar results [ 30 ]. In contrast to other studies in Vietnam, Iran, Indonesia, India, and the Netherlands, analyzed spices meet established microbiological standards and were of satisfactory quality [ 29 – 30 ]. These were particularly spices, herbs, and dried spices produced on a small scale. The significant bacterial communities found in the “Local Spices or Herbs” samples analyzed compared to the others could be explained by the difference in water activity. The “Local Spices or Herbs” samples, consisting of a mixture of aromatic herbs and food condiments, represent a more favourable environment for bacterial growth than the other dried spices. Nevertheless, despite their packaging giving them “stable physicochemical characteristics”, the dehydrated products, such as Curcuma, the mixture of 7-spice and thyme, have a relatively large bacterial community (Supplements 1–4). These foods generally undergo drying processes at ambient temperature at the place of production. Moreover, in the developing countries from which they originate, harvesting and production technologies do not necessarily comply with optimal sanitary conditions [ 29 – 31 ]. The non-compliance with hygienic measures at all stages of the process, particularly during agricultural production, harvesting, washing and sun-drying, may explain the results of this study. Packaging hygiene and storage conditions are not always respected either, which is a probable cause of food contamination (FAO, https://www.fao.org/3/w7429f/w7429f0r.htm ). All these factors lead to high levels of microbial contamination, which means these products may not be suitable for human consumption [ 29 , 32 , 33 ]. The presence of Escherichia coli , Salmonella spp . and Bacillus spp . in most of the analyzed samples could potentially be harmful in herbs and in various food matrices such as spices, which were the subject of this study [ 36 ]. Moreover, the bacterial spores introduced by spices can withstand various preparation processes, including heat treatment [ 37 ]. The high presence of Escherichia coli in all samples may indicate faecal contamination of various origins. In the case of fat-in-house spices, including horticultural products, faecal contamination could be irrigation water from wastewater systems used without prior biological treatment [ 37 – 42 ]. As for the other spices studied, faecal contamination could be due to a hygiene failure by operators handling the processing (reduction to powder, mincing, etc.), storage and transport stages. The analysis of distance matrices containing dissimilarity information can effectively capture significant and subtle compositional differences between the samples studied [ 44 ]. Other techniques, such as the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), an agglomerative hierarchical clustering method widely used in practice, can also be considered. Step by step, it combines the two closest groups or elements into a higher-level group, and the distance between the new group and any other group is calculated as the arithmetic mean distance between the elements in the different groups or clusters [ 45 – 46 ]. The small sample size (n = 49) in this study constitutes a limitation. Furthermore, the presence of genetic material cannot provide information on the level of bacteriological contamination. Therefore, the description of the microbial community obtained by the metagenomic approach could have been accompanied by conventional microbiological analyses to highlight the fraction of viable bacteria in the analyzed samples. 5. Conclusion This study aimed to describe the bacterial community in spices sold in open-air markets in Saint-Louis, Senegal. It revealed that spices, whether fresh (“Local Spices or Herbs”) or dehydrated (Curcuma, Thyme, a mixture of 7 spices), can carry bacteria, which could be pathogens such as Salmonella spp , Shigella spp , Bacillus spp , Enterobacter spp , Pseudomonas spp , Klebsiella spp . In addition, it highlighted potential defective hygiene in the production chain of these spices through the presence of indicators of faecal contamination such as Escherichia coli and Salmonella enterica species. These results suggest that efforts must be made to raise awareness among the various stakeholder in the production chain. In addition, further research with a more significant number of samples of spices should be carried out to identify other potential contaminants that could harm to human health. Abbreviations DGGE : Gradient Gel Electrophoresis; DNA : Deoxyribonucleic acid; FAO : Food and Agriculture Organization of the United Nations; GRBA-BE: Applied Biotechnologies and Environmental Bioprocesses Research Group; IRESSEF : Institute for Health Research, Epidemiological Surveillance and Training; ISEP : Higher Institute for Vocational Training; ITNA : Institute of Applied Nuclear Technology; LABAAM : Biological, Agronomic, Food Sciences and Complex Systems Modelling Laboratory; LAE : Analysis and Testing Laboratory; LSH : Local Spices or Herbs; ONT : Oxford Nanopore Technology; PCoA : Principal Coordinate Analysis; PCR : Polymerase Chain Reaction; RNA : Ribonucleic Acid; UPGMA : Unweighted Pair Group Method with Arithmetic Mean; USSEIN : Sine Saloum El Hadj Ibrahima Niass University Declarations Ethics approval and consent to participate : Not applicable Consent for publication : Not applicable Data Availability : The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing Interests :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. Funding :This research was funded by the Ministry of Higher Education, Research and Innovation (MESRI) of Senegal govment through his FIRST (Fonds d'Impulsion de la Recherche Scientifique et Technique) program. Author Contributions : Conceived, obtaining funding: A.A.M.D.; coordinated the work: A.A.M.D., M.D.; drafted the manuscript: S.S., A.A.M.D.; sampling: S.S., S.C., S.D.T., A.K.; performed the work: S.S., S.C., S.D.T., A.A.M.D.; curation: S.S., A.A.M.D.; formal analysis: S.S., S.C., S.D.T, AAMD; investigation: A.A.M.D.; supervision: A.A.M.D, M.D.; validation: A.A.M.D.; reviewed the manuscript: S.S., A.A.M.D., A.K., S.F.W., A.T., M.D., M.N.M., F.T., M.D., M.M., C.M.N., A.D.S., A.S.N., C.T.K. 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Evol. , vol. 13, n o 2, pp. 309–313, févr. 10.1093/oxfordjournals.molbev.a025590 Lance GN et, Williams WT (1966) « A Generalized Sorting Strategy for Computer Classifications », Nature , vol. 212, n o 5058, Art. n o 5058, oct. 10.1038/212218a0 Additional Declarations No competing interests reported. Supplementary Files Supplement1.pptx Supplement2.pptx Supplement3.pptx Supplement4.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4202002","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":287130741,"identity":"b2863fa7-89be-4290-9d1e-91dc5dabd09a","order_by":0,"name":"Sarbanding Sané","email":"","orcid":"","institution":"Groupe de Recherche Biotechnologies Appliquées \u0026 Bioprocédés Environnementaux (GRBA-BE), École Supérieure Polytechnique (ESP) – Université Cheikh Anta 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Mbengue","email":"","orcid":"","institution":"Laboratoire de Microbiologie Appliquée et de Génie Industriel, École Supérieure Polytechnique (ESP) – Université Cheikh Anta Diop","correspondingAuthor":false,"prefix":"","firstName":"Malick","middleName":"","lastName":"Mbengue","suffix":""},{"id":287130758,"identity":"3b0bdd92-8bff-4d20-bf16-5615cbdc351c","order_by":12,"name":"Cheikh Momar Nguer","email":"","orcid":"","institution":"Groupe de Recherche Biotechnologies Appliquées \u0026 Bioprocédés Environnementaux (GRBA-BE), École Supérieure Polytechnique (ESP) – Université Cheikh Anta DIOP","correspondingAuthor":false,"prefix":"","firstName":"Cheikh","middleName":"Momar","lastName":"Nguer","suffix":""},{"id":287130759,"identity":"b57037c0-82ba-4658-a5ec-4352c59de7d7","order_by":13,"name":"Aminata Diassé Sarr","email":"","orcid":"","institution":"Institut Supérieur d’Enseignement Professionnel (ISEP) 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17:28:58","extension":"pptx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":56249,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement4.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4202002/v1/7bb4642d598a12b2496442bd.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A metagenomic assessment of bacterial community in spices sold open-air markets in Saint-Louis, Senegal","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSpices have been used for centuries as an essential component in traditional Asian medicine [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Some of them draw special attention due to their use in the cosmetics and pharmaceutical industries [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Spices are reputed to be a source of bioactive compounds [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] with sometimes rich extracts in polyphenolic compounds, including high levels of antimicrobial, antioxidant and anti-inflammatory activity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Preventive and curative actions of spices against cancer, gastrointestinal disorders, psychological disorders, diabetes, obesity, metabolic syndromes and neurological disorders were also reported [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Aromatic plants and spices have thus become well established over time, justifying their use by around 80% of the world\u0026rsquo;s population, mainly in developing countries [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Spices can be divided into several groups: strong spices (pepper, chilli, ginger), mild spices (paprika, coriander), aromatic spices (cinnamon, curcuma, cloves, cumin, anise, celery), dried herbs (basil, bay leaves, dill, marjoram, tarragon, thyme) and aromatic vegetables, for instance, onions or garlic [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom a nutritional point of view, China and some West African countries have begun to use condiments and seasonings to remedy significant dietary deficiencies, particularly in iodine, iron and vitamin A [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. According to several studies, various micronutrients are added to condiments depending on the deficiencies demonstrated in the target population. The bioavailability of the micronutrient remains highly important, as it must be absorbed by the body in sufficient quantities to contribute effectively to improving these deficiencies [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Despite the awareness messages to limit the overconsumption of processed food products while promoting natural products in the diet [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], the use of industrial ingredients remains a concern [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, spices and broths made from natural products can also be at risk for consumers. Indeed, due to transformation processes such as extraction and packaging processes, these can be subject to contamination by bacteria, viruses, parasites and pathogenic fungi due to handling [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, the diversity and abundance of microbial communities in these food matrices can influence their compositions, nutritional benefits and even the functioning of the gut ecosystem [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTraditional studies of bacterial diversity in foods have relied on culture methods, whereas only around 1% of microorganisms present in the natural environment can be grown by these methods [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Culture-independent approaches involving molecular biology methods such as gradient gel electrophoresis (DGGE) polymerase chain reaction (PCR) and extended later to metagenomic methods to assess microbial diversity and analyze microbial community structures in diverse environments, including food matrices [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This study aimed to describe the genomic footprint of the microbial community present in spices sold in open-air markets in Senegal using the sequencing method.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cp\u003e \u003cem\u003eCollection and preparation of samples\u003c/em\u003e \u003c/p\u003e \u003cp\u003eSamples of Curcuma, a mixture of 7 spices (cloves, ginger, garlic, cumin, lemon, nutmeg and salt), Thyme and \u0026ldquo;Local Spices or Herbs\u0026rdquo; (LSH, a mixture of food condiments used to season dishes) were collected from 3 markets in Saint-Louis in August 2022 in compliance with the sampling process [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. For a mixture of 7-spices, thyme and Curcuma samples, 30 g were purchased, while 100 g was for \u0026ldquo;LSH\u0026rdquo;. Samples were placed in an individual sterile zipped bag. \u0026ldquo;Pseudo-industrial spices\u0026rdquo; are in dehydrated format and sold in their original packaging, particularly Curcuma, a mixture of 7-spices and Thyme, which were transported and stored at ambient temperature until their use. However, the individuals\u0026rsquo; zipped bags of \u0026ldquo;LSH\u0026rdquo; samples were placed and transported in an insulated bag maintained at +\u0026thinsp;4\u0026deg;C to the laboratory, ensuring their preservation. The samples were then kept at -20\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003e \u003cem\u003eExtraction of Bacterial DNA\u003c/em\u003e \u003c/p\u003e \u003cp\u003eSamples were subjected to manual extraction using the \u003cem\u003eOmega E.N.Z.A Food DNA kit (\u003c/em\u003eOmega Bio-Tek, GA, USA\u003cem\u003e)\u003c/em\u003e in accordance with the manufacturer\u0026rsquo;s instructions, with a few minor modifications. To optimize cell lysis, four beads with a diameter of 2.0 mm (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zymoresearch.eu/products/zr-bashing-bead-lysis-tubes\u003c/span\u003e\u003cspan address=\"https://zymoresearch.eu/products/zr-bashing-bead-lysis-tubes\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were added to the lysis buffer before beating the sample with Analog Disruptor Genie (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.scientificindustries.com/otuli-mixers-and-shakers/bead-beaters/analog-disruptor-genie.html\u003c/span\u003e\u003cspan address=\"https://www.scientificindustries.com/otuli-mixers-and-shakers/bead-beaters/analog-disruptor-genie.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) during 5 minutes. The DNA concentration was determined by a Nanodrop spectrophotometer One (Thermo Scientific, USA), and the sample was stored at \u0026minus;\u0026thinsp;20\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePCR Amplification and Sequencing\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe region coding for the 16S subunit of bacterial RNA was amplified using the universal primers Fd1 (5\u0026rsquo; \u0026ndash; AGA GTT TGA TCM TGG CTC AG \u0026ndash; 3\u0026rsquo;) and Rd1 (5\u0026rsquo; \u0026ndash; AAG GAG GTG ATC CAG CC\u0026ndash; 3\u0026rsquo;) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The reaction volume for amplification was 25 \u0026micro;L and included 12.5 \u0026micro;L of \u003cem\u003eOne Taq\u0026reg; Quick-Load 2X Master Mix with Standard Buffer\u003c/em\u003e kit (New England Biolabs, M0486), 2 \u0026micro;L of primers, 5 \u0026micro;L of DNA and 5.5 \u0026micro;L of molecular grade water. PCR was performed under the following conditions: an initial denaturation of 94\u0026deg;C for 30 seconds followed by 30 cycles of 94\u0026deg;C for 30 seconds, 50\u0026deg;C for 30 seconds, 68\u0026deg;C for 1 minute and a final extension of 68\u0026deg;C for 5 minutes. Amplified fragments were visualized by electrophoresis on a 1% agarose gel using ethidium bromide staining and UV light. Amplicons were then purified using the Zymo DNA clean \u0026amp; concentrator \u003csup\u003eTM\u003c/sup\u003e kit-5 (Zymo D4034). Library preparation was performed using the Rapid Barcoding Kit (SQK \u0026ndash; RBK 110\u0026ndash;96). The sequencing was performed on Flow Cell R9.4.1 (FLO-MIN106D) on a MinIon Mk1C sequencer. A real-time workflow Epi2me platform was used for species identification. Alpha diversity, which shows the complexity of species within a sample, was obtained using four indices: Chao1, Shannon, Simpson and observed species. For this, the Vegan and Phyloseq packages of the R Studio software were used. Beta diversity, which shows the complexity of species across all samples, was measured using Jaccard and x indices. To obtain this diversity, the Vegan and Ecodist packages of the R software were used. All figures were also generated using the R software.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical Analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAll statistical analysis was performed on R software. For each considered spice group, the Wilcoxon test was performed for differential analysis of diversity. The significance level was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The degree of significance is proportional to the number of stars shown at the top of the figures (* corresponds to p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** corresponds to p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and *** corresponds to p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In order to assess the similarity between the different microbial communities in the considered spice groups, beta-diversity was calculated based on the Bray-Curtis dissimilarity distance and the Jaccard index. The result was then represented using a principal coordinate analysis (PCoA).\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA total of 49 samples divided into two categories, including \"Homemade mixture of food condiments (ready to use)\" (n\u0026thinsp;=\u0026thinsp;19) and \"pseudo-industrial spices\" sold dehydrated with primary packaging (n\u0026thinsp;=\u0026thinsp;30), were collected and analyzed in two batches. The first batch consists of 10 \"Local or Spices Herbs\" samples. This type of spice is a blend of green onions, peppers, garlic and Sofia bell pepper (Capsicum annum). The second batch, designated as \"pseudo-industrial spices, \" was made up of thyme, 7-spice mix, curcuma, and the \"Local or Spices Herbs\" samples. For each sample type, additional details, such as the numbers and collection locations, are reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of samples according to type and collection sites\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCategories\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTypes of samples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDry or dehydrated form\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNumber of collected specimens\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCollection sites\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ldquo;Homemade mixture of food condiments (ready to use)\u0026rdquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocal spices or herbs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSor/ Saint-Louis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePseudo-industrial spices\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMixture of 7 spices\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSor, Guet Ndar and Pikine / Saint-Louis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCurcuma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSor, Guet Ndar and Pikine / Saint-Louis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThyme\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSor, Guet Ndar and Pikine / Saint-Louis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSequencing data were processed using Nanopore's Epi2me software. Quality scores were 9.87 and 9.82, with classified read counts of 65744 and 165325, respectively for the two batches analyzed. Figure\u0026nbsp;1 shows the predominant bacterial species for all the samples processed in this study. Generally speaking, \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eSalmonella enterica\u003c/em\u003e are the most represented species in all samples (Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e-------------------------------------\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFigure 1\u003c/strong\u003e \u003cp\u003eRelative distribution of bacterial species in samples\u003c/p\u003e \u003c/p\u003e \u003cp\u003e-------------------------------------\u003c/p\u003e \u003cp\u003eAnalysis of bacterial diversity according to spice type showed the predominance of \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eSalmonella enterica\u003c/em\u003e and \u003cem\u003eAcinetobacter seifertii\u003c/em\u003e, with proportions differing slightly from one category to another (Supplements 1\u0026ndash;4). All species with an abundance of less than 1% were merged into an \"others\" category. This group represented 21.2% of species in the \"Local Spices or Herbs\" and was mainly made up \u003cem\u003eof Saccharothrix sp.6-C\u003c/em\u003e (4.7%), \u003cem\u003eShigella flexneri\u003c/em\u003e (4.5%) and \u003cem\u003eBuchnera aphidicola\u003c/em\u003e (4.1%). For Curcuma, the \"others\" group represented 9.1% and was mainly made up of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e (6.1%), \u003cem\u003eBacillus circulans\u003c/em\u003e 3.9% and \u003cem\u003eKosakonia cowanii\u003c/em\u003e (3%). For the 7 spices and Thyme samples, this group represented 4% and 2%, respectively. The predominant bacterial species were \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e (16.3%), \u003cem\u003eClostridium botulinium\u003c/em\u003e (11.3%) and \u003cem\u003eClostridium tatani\u003c/em\u003e (3.6%) for 7 spices, \u003cem\u003eand Shigella flexneri\u003c/em\u003e (12.4%) and \u003cem\u003ePseudomonas fulva\u003c/em\u003e (6.7%) for Thyme.\u003c/p\u003e \u003cp\u003eBy considering all the types of spices, alpha-diversity analysis showed that \"Local Spices or Herbs\" had the most incredible diversity, with an average number of taxa per sample of 115.78. This group is followed by Curcuma, a mixture of 7 spices and Thyme, respectively. The average taxa numbers per sample were 41.1, 14.5 and 10.3, respectively (Supplement 1). About the number of species per spice group, after using the Wilcoxon test, there was a significant difference between \"Local Spices or Herbs\" and Thyme (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), Curcuma and Thyme (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and Curcuma and the mixture of 7-spice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, there was no statistically significant difference between the mixture of 7-spice and Thyme, as shown in Figs.\u0026nbsp;2A and 2B. Figures\u0026nbsp;2C and 2D show that species' relative abundance and stability are greater for \"Local Spices or Herbs\". This group shows a significant difference with Thyme (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There is also a significant difference between Curcuma and Thyme (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and between Curcuma and the mix of 7-spice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e-------------------------------------\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFigure 2\u003c/strong\u003e \u003cp\u003eAlpha-diversity analysis\u003c/p\u003e \u003c/p\u003e \u003cp\u003e-------------------------------------\u003c/p\u003e \u003cp\u003eBeta-diversity was illustrated using PcoA based on Bray-Curtis dissimilarity and Jaccard distance, which showed similarities in bacterial composition between samples from the different spice groups and showed two clusters (Fig.\u0026nbsp;3). One consisted of the species found in Thyme, Curcuma, a mixture of 7 spices samples and part of the \u0026ldquo;Local Spices or Herbs\u0026rdquo; samples. The second cluster consists solely of \u0026ldquo;Local Spices or Herbs\u0026rdquo; samples. These results show that the bacterial communities in the samples from the first cluster are closer than those in the second cluster.\u003c/p\u003e \u003cp\u003e-------------------------------------\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFigure 3\u003c/strong\u003e \u003cp\u003eBeta-diversity analysis\u003c/p\u003e \u003c/p\u003e \u003cp\u003e-------------------------------------\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study aimed to describe the microbial community present in spices sold in open-air markets in Saint-Louis using the sequencing method. The high-throughput sequencing method used in this study allowed us to detect a broad-spectrum genomic footprint of micro-organisms, including those usually not detected by culture methods. Indeed, since the revolutionary improvement in DNA sequencing technologies, direct high-throughput analysis of the genomic DNA of an entire community without prior culture has become the most common approach, overcoming the constraints of conventional microbiological approaches [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In addition, from a technological point of view, MinION sequencing is mainly used to address metataxonomy [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur results show the presence of many pathogenic bacteria in the spice samples studied, with their abundance varying according to the type considered. Studies in countries like Brazil have reported similar results [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In contrast to other studies in Vietnam, Iran, Indonesia, India, and the Netherlands, analyzed spices meet established microbiological standards and were of satisfactory quality [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These were particularly spices, herbs, and dried spices produced on a small scale.\u003c/p\u003e \u003cp\u003eThe significant bacterial communities found in the \u0026ldquo;Local Spices or Herbs\u0026rdquo; samples analyzed compared to the others could be explained by the difference in water activity. The \u0026ldquo;Local Spices or Herbs\u0026rdquo; samples, consisting of a mixture of aromatic herbs and food condiments, represent a more favourable environment for bacterial growth than the other dried spices. Nevertheless, despite their packaging giving them \u0026ldquo;stable physicochemical characteristics\u0026rdquo;, the dehydrated products, such as Curcuma, the mixture of 7-spice and thyme, have a relatively large bacterial community (Supplements 1\u0026ndash;4). These foods generally undergo drying processes at ambient temperature at the place of production. Moreover, in the developing countries from which they originate, harvesting and production technologies do not necessarily comply with optimal sanitary conditions [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The non-compliance with hygienic measures at all stages of the process, particularly during agricultural production, harvesting, washing and sun-drying, may explain the results of this study. Packaging hygiene and storage conditions are not always respected either, which is a probable cause of food contamination (FAO, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.fao.org/3/w7429f/w7429f0r.htm\u003c/span\u003e\u003cspan address=\"https://www.fao.org/3/w7429f/w7429f0r.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e All these factors lead to high levels of microbial contamination, which means these products may not be suitable for human consumption [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe presence of \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eSalmonella spp\u003c/em\u003e. and \u003cem\u003eBacillus spp\u003c/em\u003e. in most of the analyzed samples could potentially be harmful in herbs and in various food matrices such as spices, which were the subject of this study [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Moreover, the bacterial spores introduced by spices can withstand various preparation processes, including heat treatment [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The high presence of \u003cem\u003eEscherichia coli\u003c/em\u003e in all samples may indicate faecal contamination of various origins. In the case of fat-in-house spices, including horticultural products, faecal contamination could be irrigation water from wastewater systems used without prior biological treatment [\u003cspan additionalcitationids=\"CR38 CR39 CR40 CR41\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. As for the other spices studied, faecal contamination could be due to a hygiene failure by operators handling the processing (reduction to powder, mincing, etc.), storage and transport stages.\u003c/p\u003e \u003cp\u003eThe analysis of distance matrices containing dissimilarity information can effectively capture significant and subtle compositional differences between the samples studied [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Other techniques, such as the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), an agglomerative hierarchical clustering method widely used in practice, can also be considered. Step by step, it combines the two closest groups or elements into a higher-level group, and the distance between the new group and any other group is calculated as the arithmetic mean distance between the elements in the different groups or clusters [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe small sample size (n\u0026thinsp;=\u0026thinsp;49) in this study constitutes a limitation. Furthermore, the presence of genetic material cannot provide information on the level of bacteriological contamination. Therefore, the description of the microbial community obtained by the metagenomic approach could have been accompanied by conventional microbiological analyses to highlight the fraction of viable bacteria in the analyzed samples.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study aimed to describe the bacterial community in spices sold in open-air markets in Saint-Louis, Senegal. It revealed that spices, whether fresh (\u0026ldquo;Local Spices or Herbs\u0026rdquo;) or dehydrated (Curcuma, Thyme, a mixture of 7 spices), can carry bacteria, which could be pathogens such as \u003cem\u003eSalmonella spp\u003c/em\u003e, \u003cem\u003eShigella spp\u003c/em\u003e, \u003cem\u003eBacillus spp\u003c/em\u003e, \u003cem\u003eEnterobacter spp\u003c/em\u003e, \u003cem\u003ePseudomonas spp\u003c/em\u003e, \u003cem\u003eKlebsiella spp\u003c/em\u003e. In addition, it highlighted potential defective hygiene in the production chain of these spices through the presence of indicators of faecal contamination such as \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eSalmonella enterica\u003c/em\u003e species. These results suggest that efforts must be made to raise awareness among the various stakeholder in the production chain. In addition, further research with a more significant number of samples of spices should be carried out to identify other potential contaminants that could harm to human health.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eDGGE\u003c/strong\u003e: Gradient Gel Electrophoresis;\u0026nbsp;\u003cstrong\u003eDNA\u003c/strong\u003e: Deoxyribonucleic acid;\u0026nbsp;\u003cstrong\u003eFAO\u003c/strong\u003e: Food and Agriculture Organization of the United Nations;\u0026nbsp;\u003cstrong\u003eGRBA-BE:\u003c/strong\u003e Applied Biotechnologies and Environmental Bioprocesses Research Group;\u0026nbsp;\u003cstrong\u003eIRESSEF\u003c/strong\u003e : Institute for Health Research, Epidemiological Surveillance and Training; \u003cstrong\u003eISEP\u003c/strong\u003e : Higher Institute for Vocational Training; \u003cstrong\u003eITNA\u003c/strong\u003e : Institute of Applied Nuclear Technology;\u0026nbsp;\u003cstrong\u003eLABAAM\u003c/strong\u003e : Biological, Agronomic, Food Sciences and Complex Systems Modelling Laboratory;\u0026nbsp;\u003cstrong\u003eLAE\u003c/strong\u003e : Analysis and Testing Laboratory;\u0026nbsp;\u003cstrong\u003eLSH\u003c/strong\u003e: Local Spices or Herbs; \u003cstrong\u003eONT\u003c/strong\u003e: Oxford Nanopore Technology;\u0026nbsp;\u003cstrong\u003ePCoA\u003c/strong\u003e: Principal Coordinate Analysis;\u0026nbsp;\u003cstrong\u003ePCR\u003c/strong\u003e:\u0026nbsp;Polymerase Chain Reaction;\u0026nbsp;\u003cstrong\u003eRNA\u003c/strong\u003e: Ribonucleic Acid; \u003cstrong\u003eUPGMA\u003c/strong\u003e: Unweighted Pair Group Method with Arithmetic Mean;\u0026nbsp;\u003cstrong\u003eUSSEIN\u003c/strong\u003e : Sine Saloum El Hadj Ibrahima Niass University\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e: Not applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e:\u0026nbsp;Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e: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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e:This research was funded by the Ministry of Higher Education, Research and Innovation (MESRI) of Senegal govment through his FIRST (Fonds d\u0026apos;Impulsion de la Recherche Scientifique et Technique) program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: Conceived, obtaining funding: A.A.M.D.; coordinated the work: A.A.M.D., M.D.; drafted the manuscript: S.S., A.A.M.D.; sampling: S.S., S.C., S.D.T., A.K.; \u0026nbsp;performed the work: S.S., S.C., S.D.T., A.A.M.D.; curation: S.S., A.A.M.D.; formal analysis: S.S., S.C., S.D.T, AAMD; investigation: A.A.M.D.; supervision: A.A.M.D, M.D.; validation: A.A.M.D.; reviewed the manuscript: S.S., A.A.M.D., A.K., S.F.W., A.T., M.D., M.N.M., F.T., M.D., M.M., C.M.N., A.D.S., A.S.N., C.T.K. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eWe are grateful to the top management of the Ecole Sup\u0026eacute;rieure Polytechnique (ESP) for their support to the GRBA-BE.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eScazzocchio B, Minghetti L, et, D\u0026rsquo;Archivio M (2020) \u0026laquo; Interaction between Gut Microbiota and Curcumin: A New Key of Understanding for the Health Effects of Curcumin \u0026raquo;, \u003cem\u003eNutrients\u003c/em\u003e, vol. 12, n\u003csup\u003eo\u003c/sup\u003e 9, p. 2499, ao\u0026ucirc;t \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/nu12092499\u003c/span\u003e\u003cspan address=\"10.3390/nu12092499\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWillenbacher E et al (2019) \u0026laquo; Curcumin: New Insights into an Ancient Ingredient against Cancer \u0026raquo;, \u003cem\u003eInt. 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Moreover, they are increasingly being processed on various scales, exposing them to potential sources of contamination. This study aimed to describe the bacterial community in spices sold in Senegal. Thyme, Curcuma, a mixture of 7 spices and \"Local Spices or Herbs\" (LSH) samples were collected in selected open-air markets in August 2022 in Saint-Louis, Senegal. DNA extraction was performed using the Omega E.N.Z.A Food DNA kit. PCR assays were based on a genomic fragment encoding bacterial 16S rRNA, which was subsequently sequenced using Oxford Nanopore technology (ONT). Sequencing was carried out on two batches of samples, one containing part of the \"Local Spices or Herbs\" (n\u0026thinsp;=\u0026thinsp;10) designated as \"a mixture of food seasonings\", and the other, samples of a mixture of 7 spices, Curcuma, Thyme and the other part of the \"Local Spices or Herbs\" (n\u0026thinsp;=\u0026thinsp;39). Analysis of the sequencing data showed high bacterial diversity and the predominance of \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eSalmonella enterica\u003c/em\u003e and \u003cem\u003eEscherichia marmotae\u003c/em\u003e in the samples, with a total number of reads of 65744 and 165325 for the two batches, respectively. The sample category \"Homemade mixture of food condiments (ready to use)\", which includes all \"Local Spices or Herbs\" samples, showed remarkable bacterial diversity. These were followed by Curcuma, a blend of 7 spices and thyme, respectively. These results highlight a highly diverse genomic profile of the microbial community, including pathogenic bacteria, in spice samples. This is the first metagenomic study to assess microbial diversity and analyze microbial community structures in spices in Senegal.\u003c/p\u003e","manuscriptTitle":"A metagenomic assessment of bacterial community in spices sold open-air markets in Saint-Louis, Senegal","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-08 17:28:53","doi":"10.21203/rs.3.rs-4202002/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a1d27abb-28e3-4459-88ea-70f3cd065277","owner":[],"postedDate":"April 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-10T18:45:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-08 17:28:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4202002","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4202002","identity":"rs-4202002","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}