Comparative Genomic Analysis and Isolation of a Novel Burkholderia gladioli Strain Producing Lethal Bongkrekic Acid in China

Comparative Genomic Analysis and Isolation of a Novel Burkholderia gladioli Strain Producing Lethal Bongkrekic Acid in China | 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 Comparative Genomic Analysis and Isolation of a Novel Burkholderia gladioli Strain Producing Lethal Bongkrekic Acid in China RongGui Lv, Song Jiang, Hongwei Shen, Xi Chen, Ewnji Yoon, Xiaxia Yu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5794685/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 Background Bongkrekic acid (BKA) is a potent mitochondrial toxin secreted by certain strains of Burkholderia gladioli, previously known as Pseudomonas cocovenenans. It poses a significant risk to public health, particularly through contaminating fermented or spoiled food products. This study focuses on the isolation and genomic analysis of a novel strain of Burkholderia gladioli from a non-fermented rice noodle linked to a foodborne outbreak in China. Results The study employed a multifaceted approach including microbiological isolation, phenotypic characterization, biochemical assays, and molecular identification. Animal experiments were conducted to observe the effects of BKA on rats. Genomic DNA was extracted, sequenced, and analyzed using bioinformatics tools to determine the genetic makeup, gene function annotation, and comparative genomic analysis. The isolated strain, designated as Burkholderia gladioli 201, was found to produce BKA, which was confirmed through animal experiments showing characteristic symptoms and subsequent toxicological examinations. The complete genome sequence revealed a high degree of genetic similarity with other B. gladioli strains, with a distinct BA biosynthesis gene cluster. Comparative genomic analysis and phylogenetic trees constructed from single-copy core genes indicated a close relationship between Burkholderia gladioli 201 and other strains of the species. Conclusion This study successfully isolated and characterized a novel strain of Burkholderia gladioli capable of producing BKA from a non-fermented food source. The genomic analysis provided insights into the genetic basis of BKA production and the evolutionary relationships within the Burkholderia genus. The findings underscore the need for enhanced surveillance and preventive measures to mitigate the risk of BKA-associated food poisoning. Burkholderia gladioli Bongkrekic Acid Genomic Analysis Foodborne Outbreak Phylogenetic Relationship Figures Figure 1 Figure 2 Figure 3 Background Bongkrekic acid (BKA), a hazardous tricarboxylic fatty acid, is secreted by Burkholderia gladioli cocovenenans, previously recognized as Pseudomonas cocovenenans subsp. farinofermentans in the Chinese context [1]. Functioning as a mitochondrial toxin through inhibition of adenine nucleotide translocase [2, 3], BKA represents a severe threat, particularly contaminating four key food groups: fermented or semi-fermented corn and coconut products, spoiled Tremella fuciformis (also known as white fungi), and other decayed starchy foods such as glutinous rice, millet, sorghum, and potato flour. P. cocovenenans, initially recognized for its role in food poisoning, was found to generate both toxoflavin and BKA [4]. The 1970s witnessed sporadic occurrences of high mortality rates, exceeding 40%, from food poisoning in parts of China, linked to fermented corn and spoiled Tremella intake [5–7]. The implicated bacterium, a yellow-pigmented, gram-negative rod, was initially named "Flavobacterium farinofermentans" [5], later confirmed as P. cocovenenans [4]. Reclassification of P. cocovenenans into the Burkholderia genus as B. cocovenenans in 1995 was supported by 16S rRNA gene sequencing analysis [1, 4]. Coenye et al. employed comprehensive methodologies, including whole-cell protein profiling and DNA-DNA hybridization, to investigate the connection between B [8]. gladioli and B. cocovenenans, concluding that B. cocovenenans was a subspecies of B. gladioli. Nonetheless, the study's scope was limited, focusing on a single B. gladioli pathovar without fully addressing pathogenic variability. BKA-related poisoning incidents have been documented globally, notably in China, Indonesia, and Mozambique. China has reported over 9,000 BKA poisoning cases since the mid-20th century, with over 1,000 fatalities traced back to contaminated corn products [9]. Indonesia has seen foodborne illness outbreaks tied to fermented food consumption[3], while a 2015 event in Mozambique resulted in 75 deaths from a fermented cornmeal drink, affecting 230 people [10]. These incidents emphasized the high-risk nature of fermented or spoiled corn and coconut goods in relation to BKA poisoning. A 2018 poisoning case in Guangdong Province, China, involving BKA from seemingly unspoiled wet rice noodles, highlighted the potential for BKA contamination even in non-fermented foods[9]. Despite more similar cases recorded post-event [11], the complete molecular characterization of the causal strains remains elusive. This study presents the isolation and characterization of a new B. gladioli strain, designated as “Burkholderia gladioli 201”, which was recovered from a non-fermented rice noodle implicated in a Chinese foodborne outbreak, capable of producing lethal BKA. We conducted an exhaustive taxonomic assessment, incorporating genetic, chemotaxonomic, and phenotypic methods to define the isolate's characteristics, pathophysiology, and unique molecular profile. Furthermore, we offer an up-to-date comparative analysis between toxic and non-toxic B. gladioli strains and construct a genome-scale phylogenetic tree, involving 356 other Burkholderia strains, to establish the isolate's phylogenetic position. A species tree was also developed to explore the evolutionary patterns of gene families associated with this pathogen. Results Genomic Features of Burkholderia gladioli 201 The Burkholderia gladioli 201 genome was fully sequenced, revealing a length of 8,492,480 base pairs with a GC content of 68.38%. This species is noted for its ubiquitous presence across various environments, as evidenced by the global distribution of the 356 strains studied, which were sourced from a diverse range of origins including plants, human clinical samples, animal samples, and environmental samples. The most extensively studied strain, B. gladioli ATCC R 10248, was first isolated from Gladiolus sp. in the United States in 1913. The genomes of these strains varied in size from 5.49 to 13.08 Mbp, with a mean size of 8.13 Mbp and a median of 8.03 Mbp, and GC contents ranging from 55–68.6%, with a mean of 67.82% and a median of 66.45%. Animal Experiments In our experiments, rats were administered a solution derived from the culture of Burkholderia gladioli 201 via gavage. Within an hour, the rats developed symptoms of lethargy, general weakness, and an inability to stand or walk, along with rapid breathing and nodding respiration, signaling a critical state. Toxicological examinations post-mortem confirmed the presence of bongkrekic acid in multiple organs, with the highest concentration in the liver. ANI Analysis of B. gladioli Genetic similarity among B. gladioli genomes was assessed using Average Nucleotide Identity (ANI) values, which ranged from 95.00–100.00%, with a mean of 98.27%. This exceeds the species delineation threshold of 95% (Fig. 1 ). Burkholderia gladioli 201 showed the highest ANI (99.17%) with B. gladioli BCC1880 from the United Kingdom, and lower identities with B. gladioli SCCH61 Bgd92-3601 and B. gladioli AU35657 from Russia and the United States, respectively (Fig. 1 ). Core- and Pan-Genome of Burkholderia gladioli The pan-genome of B. gladioli, comprising 182,662 genes, included 141 core genes present in at least 99% of strains. The pan-genome also contained soft-core genes, shell genes, and cloud genes, numbered at 73, 7,843, and 174,605, respectively. BA Biosynthesis Gene Cluster Analysis A 15,807-bp sequence in Burkholderia gladioli 201, exhibiting 99.36% identity to the BA biosynthesis gene cluster bon of B. cocovenenans DMSZ11318, was identified. This bon gene cluster was also found in 46 other B. gladioli genomes with high sequence identity (Table 1 ). Table 1 The presence of the BA biosynthesis gene cluster bon in Burkholderia gladioli 201 and other genomes query acc.ver subject acc.ver % identity alignment length mismatches gap opens q. start q. end s. start s. end evalue bit score JX173632.1 201GL005572 99.361 15807 82 9 35177 50970 1 15801 0 28613 JX173632.1 201GL005571 99.415 14013 59 6 21132 35136 11299 25296 0 25401 JX173632.1 201GL005574 99.297 4551 17 3 54707 59251 4542 1 0 8213 JX173632.1 201GL005573 99.558 2262 10 0 51113 53374 2262 1 0 4122 JX173632.1 201GL000481 99.723 2166 6 0 61163 63328 1 2166 0 3967 JX173632.1 201GL000482 99.661 1476 5 0 63325 64800 1 1476 0 2699 JX173632.1 201GL005575 100 1455 0 0 59206 60660 1 1455 0 2687 JX173632.1 201GL005570 99.686 1275 4 0 9106 10380 1 1275 0 2333 JX173632.1 201GL005569 99.841 1260 2 0 7842 9101 1 1260 0 2316 JX173632.1 201GL005564 99.676 1233 4 0 3802 5034 1 1233 0 2255 JX173632.1 201GL005568 99.647 1134 4 0 6650 7783 1 1134 0 2073 JX173632.1 201GL006899 99.906 1065 1 0 1 1065 1 1065 0 1962 JX173632.1 201GL005565 99.803 1014 2 0 5153 6166 1 1014 0 1862 JX173632.1 201GL000483 99.88 831 1 0 64785 65615 1 831 0 1530 JX173632.1 201GL006898 99.517 828 4 0 1132 1959 828 1 0 1507 JX173632.1 201GL000486 100 807 0 0 66740 67546 1 807 0 1491 JX173632.1 201GL000484 100 750 0 0 65617 66366 1 750 0 1386 JX173632.1 201GL006897 99.474 570 3 0 2005 2574 570 1 0 1037 JX173632.1 201GL000485 99.625 267 1 0 66414 66680 1 267 3.87E-136 488 JX173632.1 201GL006896 100 240 0 0 2787 3026 240 1 8.49E-123 444 JX173632.1 201GL005562 98.387 186 3 0 3412 3597 186 1 8.93E-88 327 JX173632.1 201GL005563 100 162 0 0 3594 3755 162 1 1.95E-79 300 JX173632.1 201GL005566 100 129 0 0 6200 6328 1 129 4.30E-61 239 JX173632.1 201GL005567 100 117 0 0 6433 6549 117 1 2.02E-54 217 Gene Tree and Species Tree Analysis The gene tree depicted 357 strains distributed across 10 clades, with the Burkholderia gladioli 201 strain part of the largest clade (Fig. 2 ). The species tree of 30 representative bacterial species demonstrated that Burkholderia gladioli 201 clustered with Burkholderia plantarii and Burkholderia glumae AU6208, indicating that the three species were closely related (Fig. 3 ). Furthermore, the genome of Burkholderia gladioli 201 assembled in this study was similar to the previously reported Burkholderia gladioli SCCH61 Bgd92-3601 (GCA 028462525.1). Notably, the genome of Burkholderia gladioli 201 had a higher number of annotated single-copy genes compared to Burkholderia gladioli SCCH61 Bgd92-3601, while having fewer multi-copy genes, indicating genetic diversity within the species. In the most differentiated branches of the species tree, an expansion of gene types, particularly single-copy genes, was observed, as exemplified by the increased number of single-copy genes in Burkholderia aenigmatica. Discussion This study investigates a comprehensive analysis of a novel Burkholderia gladioli strain, designated as Burkholderia gladioli 201 and isolated from a non-fermented rice noodle associated with a foodborne outbreak in China. The primary findings of this research include the successful isolation and characterization of this strain, which was confirmed to produce the lethal Bongkrekic Acid (BKA). The complete genome sequence of the strain was determined, revealing a high degree of genetic similarity with other B. gladioli strains, particularly in the presence of a distinct BA biosynthesis gene cluster responsible for BKA production. Furthermore, the study conducted a detailed comparative genomic analysis, which, through phylogenetic reconstruction, demonstrated the close evolutionary relationship of Burkholderia gladioli 201 with other strains of species. The findings from animal experiments, which involved the administration of a solution derived from the Burkholderia gladioli 201 culture to rats, corroborated the production of BKA, as evidenced by the observed symptoms and subsequent toxicological examinations. The ANI analysis in this study reveals the genomic similarity between Burkholderia gladioli 201 and other strains of the same species. The average ANI value of 98.27% is higher than the species delineation threshold of 95%, indicating a high level of genomic similarity between B. gladioli 201 and other strains of the same species at the genomic [12, 13]. This high level of genomic similarity may be related to the shared biological functions and ecological niches of these strains. However, it is important to note that although ANI values are commonly used to define species boundaries, there may still be significant phenotypic differences between different strains above this threshold. For example, B. gladioli 201 can produce the deadly Bongkrekic Acid (BKA), while other strains of the same species may not have this ability. This indicates that in addition to ANI analysis, it is also necessary to combine other phenotypic and genotypic characteristics to fully understand the differences between strains. Furthermore, the results of the ANI analysis also support the phylogenetic analysis of B. gladioli 201 conducted in this study. By constructing a phylogenetic tree based on single-copy core genes, we observed a close phylogenetic relationship between B. gladioli 201 and other strains such as B. plantarii and B. glumae AU6208. This is consistent with the results of the ANI analysis and further confirms the taxonomic position of B. gladioli 201 at the genomic level. These discoveries highlight the genetic basis for BKA production and emphasize the need for enhanced surveillance and preventive measures to prevent BKA-associated food poisoning incidents. The present study introduces several innovative elements to the existing body of research on Burkholderia gladioli and its production of the deadly Bongkrekic Acid (BKA). A significant contribution of this work is the isolation and detailed characterization of a novel strain, Burkholderia gladioli 201, from an atypical non-fermented food source–a rice noodle associated with a foodborne outbreak in China. This finding diverges from the majority of BKA poisoning incidents that have been predominantly linked to fermented or spoiled food items, as reported in the literature such as the cases documented by Zhao et al. and Gudo et al [1, 10]. The phylogenetic reconstruction and comparative genomic analysis presented here provide a robust framework for understanding the evolutionary relationships among Burkholderia gladioli strains. This study's approach extends beyond the taxonomic delineations made by Coenye et al. [8] by offering a more nuanced view of the genetic diversity and phylogenetic placement of the newly identified strain. Moreover, the integration of phenotypic, biochemical, and molecular identification methods in this study represents a comprehensive approach not commonly found in the literature. This multifaceted methodology stands in contrast to earlier studies that often relied on a limited set of identification techniques, thereby enhancing the accuracy and depth of the characterization of the Burkholderia gladioli 201 strain. The novelty of this study is underscored by the isolation of a new strain from an unusual food matrix, the in-depth genomic characterization that includes the identification of a BA biosynthesis gene cluster, the advanced comparative genomic analysis, and the multifaceted identification approach. These elements collectively contribute to a more refined comprehension of the Burkholderia gladioli species and its role in foodborne illness, offering a significant advancement over previous research in this field. Despite the valuable contributions of the study in isolating, characterizing, and performing genomic analysis on a novel Bongkrekic acid producing Burkholderia gladioli strain, the findings must be contextualized within certain limitations. Firstly, the reliance on a single isolate restricts the broad applicability of conclusions to other strains or ecological contexts, given the lack of representativeness in sample size. Secondly, the study's use of FastANI for genomic relatedness assessments may not fully elucidate the diversity among more distantly related isolates due to its inability to accurately calculate ANI values below 80%. This limitation could lead to an incomplete understanding of the strain's phylogenetic positioning. Additionally, the standard biochemical and phenotypic assays employed may inadequately reflect the full spectrum of metabolic activities and virulence attributes inherent to B. gladioli, potentially missing strain-specific characteristics. Lastly, while the comparative genomic analysis was extensive, a deeper exploration could offer enhanced insights into the pathogenic variability and species diversity within Burkholderia. Collectively, these limitations underscore the necessity for more comprehensive sampling strategies, advanced detection techniques, and a deeper genomic and biological exploration to comprehensively elucidate the epidemiology and virulence mechanisms of B. gladioli strains. Conclusions A novel strain of Burkholderia gladioli that can produce bongkrekic acid (BKA) from non-fermented rice noodles connected to a foodborne outbreak was successfully isolated and described in this study. A unique BKA biosynthesis gene cluster was identified by the genomic analysis, which shed light on the evolutionary relationships within the Burkholderia genus as well as the genetic underpinnings of toxin production. These results underline the need for improved food safety protocols and systematic surveillance, as well as the growing risk of BKA contamination outside fermented foods. The relationships between toxic and non-toxic strains are further elucidated by the phylogenetic analysis, which also provides useful markers for prevention and monitoring. In order to better reduce the risks of foodborne illness linked to BKA, this work emphasizes the significance of ongoing research into strain diversity and virulence mechanisms. Materials & Methods Isolation and Identification of Burkholderia gladioli from Rice Noodles Samples were obtained from a rice noodle factory located in Shenzhen City. Employing the protocols specified in "Microbiological Examination of Food Hygiene—Examination of Pseudomonas cocovenenans subsp. farinofermentans" (GB/T 4789.29–2003), we successfully isolated a strain of Burkholderia gladioli, designated as Burkholderia gladioli 201, from the collected noodle samples. The samples were cultured on Tryptic Soy Agar (TSA) and incubated at 28°C for 48 hours. Bacterial colonies were then streaked onto fresh TSA plates to ensure the isolation of single colonies for use in this study. Isolated colonies were stored at 4°C for immediate use or preserved in a glycerol stock at -78°C for long-term storage in an ultra-low temperature freezer. Control reference strains of Burkholderia gladioli were sourced from a reputable culture collection for comparative analysis during the experiments. Phenotypic Characterization Colony morphology was examined on nutrient agar containing meat extract, peptone, and NaCl, following incubation at 28°C for 24 to 48 hours. Biochemical characterization was performed using the miniaturized BIOLOG system, which assessed the utilization of 95 different carbon substrates with BIOLOG GN2 microplates, strictly following the manufacturer's instructions. The identification of the strain was achieved through a comprehensive approach involving selective media, conventional biochemical tests, commercial diagnostic systems, and PCR-based assays, in line with the guidelines of "Microbiological Examination of Food Hygiene." Animal Experiments Burkholderia gladioli 201 was cultured at 26°C for seven days. The culture medium was processed through extraction and separation using ether to obtain a solution, which was administered to rats via gavage. The rats' symptoms were monitored, and necropsies were performed post-mortem to conduct toxicological examinations. Molecular Characterization of Bacteria PCR amplification with species-specific primers was conducted using a programmable thermal cycler. The PCR reaction mixture contained PCR buffer, MgCl2, primers, dNTP mix, Taq DNA polymerase, and bacterial genomic DNA extracted using a specific kit. Amplification was followed by gel electrophoresis, staining, and visualization under ultraviolet light. PCR products were cloned and sequenced, with nucleotide sequences aligned using specific software and homology determined using the NCBI Blast Network Server. Genomic DNA extraction was performed using a genomic DNA kit, with DNA quality and quantity assessed using Nanodrop and Qubit, respectively. Library construction for sequencing involved the use of specific instruments and kits, and sequencing was conducted using a particular technology. Genomic Sequence Analysis Genome assembly and annotation were performed using various software to predict coding genes, tRNA, rRNA, and other ncRNAs, and to identify CRISPRs and gene islands. Gene function annotation was conducted using Interproscan and BlastP to identify protein domains and corresponding GO annotations, and to find matches in KEGG and RefSeq databases. Gene clusters, including the BA biosynthesis gene cluster, were identified using BLAST, and core genes were analyzed using CD-HIT. The phylogenetic tree was constructed based on single-copy core genes using MUSCLE and PhyML, and the Average Nucleotide Identity (ANI) was calculated using JSpeciesWS. Average Nucleotide Identity (ANI) Analysis ANI values were calculated between the genomes of Burkholderia gladioli 201 and other strains using FastANI. Hierarchical clustering and visualization were performed using the heatmap package in R (version 4.2.2). BA Biosynthetic Gene Cluster bon Analysis The BA biosynthesis gene cluster bon from B. cocovenenans was used as a reference sequence to analyze the presence of similar gene clusters in Burkholderia gladioli 201 and other genomes using BLASTn (version 2.15.0+). Comparative Genomic Analysis The complete genome sequence of Burkholderia gladioli 201 was deposited in GenBank under the accession number(To be submitted). Comparative analysis was performed using the genomic sequences of other 356 Burkholderia strains downloaded from the NCBI database. OrthoFinder (version 2.5.5) was used for gene clustering[12], and a species tree was constructed using single copy genes characterized from selected subgroups. Declarations Data availability All data generated or analysed during this study are included in this article and can be found in Additional file. Acknowledgments The work was supported by Medical Scientific Research Foundation of Guangdong Province of China (B2023180, B2023371). Authors and Affiliations Department of Critical Care Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China RongGui Lv & Zhanguo Liu Department of Critical Care Medicine, Shenzhen Hospital, Southern Medical University, Shenzhen, Guangdong, China RongGui Lv, Song Jiang, Ewnji Yoon & Yong Liu Department of Clinical Laboratory, Shenzhen Hospital, Southern Medical University, Shenzhen, Guangdong, China Hongwei Shen Department of Pharmacy, Shenzhen Hospital, Southern Medical University, Shenzhen, Guangdong, China Xi Chen School of Nursing, Hong Kong Polytechnic University, Hong Kong SAR, China Xiaxia Yue School of Biomedical Engineering, Shenzhen University Medical School, Shenzhen University, Shenzhen, Guangdong, 518055, China Kin Cheung Shenzhen Prevention and Treatment Center for Occupational Diseases, 518020 Shenzhen, China Bai Jiao Center for Disease Prevention and Control of Baoan District, Guangdong, Shenzhen, China Lingguo Zhao Contributions Z.L., Y.L., and L.Z. designed the study, provided financial support, supervised the research, and revised the first draft. R.L. and S.J. wrote the first draft and participated in the visualization. H.S., X.C., and E.Y. contributed to the study design and data analysis. X.Y., K.C., and B.J. supported data collection and experimental validation. All authors have read and agreed to the published version of the manuscript. Corresponding authors Lingguo Zhao, Center for Disease Prevention and Control of Baoan District, Guangdong, Shenzhen, 518101, China, E-mail: [email protected] ; Yong Liu, Department of Critical Care Medicine, Shenzhen Hospital, Southern Medical University, No 1333, Xinhu Avenue, Baoan District, Shenzhen, Guangdong Province, 518055, China,.E-mail: [email protected] ; Zhanguo Liu, Department of Critical Care Medicine, Zhujiang Hospital, Southern Medical University, 253 Gongye Middle Avenue, Haizhu District, Guangzhou, 510280, Guangdong, China, E-mail: [email protected] . Ethics declarations Ethics approval and consent to participate The samples used in this study, including rice noodles implicated in the foodborne outbreak, were obtained from a local rice noodle factory in Shenzhen. Permission for sampling and related analyses was granted by the relevant local health and food safety authorities. The animal experiments conducted in this study were approved by the Ethics Committee of Zhujiang Hospital, Southern Medical University (approval number:2024-0127). All experimental procedures strictly adhered to institutional, national, and international guidelines and regulations for the ethical treatment of animals. The authors confirm that all experimental research and field studies comply with relevant ethical standards and legislation. Consent for publication Not applicable. Competing interests The authors declare no competing interests. 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Li J, Zhou LU, Long C, Fang L, Chen Q, Chen Q, Liang J, Yang Y, Zhu H, Chen Z et al : An Investigation of Bongkrekic Acid Poisoning Caused by Consumption of a Nonfermented Rice Noodle Product without Noticeable Signs of Spoilage . J Food Prot 2019, 82 (10):1650-1654. Gudo ES, Cook K, Kasper AM, Vergara A, Salomão C, Oliveira F, Ismael H, Saeze C, Mosse C, Fernandes Q et al : Description of a Mass Poisoning in a Rural District in Mozambique: The First Documented Bongkrekic Acid Poisoning in Africa . Clin Infect Dis 2018, 66 (9):1400-1406. Zhang H, Guo Y, Chen L, Liu Z, Liang J, Shi M, Gao F, Song Y, Chen J, Fu P: Epidemiology of foodborne bongkrekic acid poisoning outbreaks in China, 2010 to 2020 . PLoS One 2023, 18 (1):e0279957. Castillo-Ramírez S: On the road to genomically defining bacterial intra-species units . mSystems 2024, 9 (7):e0058424. Rodriguez RL, Conrad RE, Viver T, Feistel DJ, Lindner BG, Venter SN, Orellana LH, Amann R, Rossello-Mora R, Konstantinidis KT: An ANI gap within bacterial species that advances the definitions of intra-species units . mBio 2024, 15 (1):e0269623. Additional Declarations No competing interests reported. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5794685","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":400533502,"identity":"aaad9e22-b576-4840-9788-11397553a965","order_by":0,"name":"RongGui Lv","email":"","orcid":"","institution":"Southern Medical University Shenzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"RongGui","middleName":"","lastName":"Lv","suffix":""},{"id":400533503,"identity":"edfb6853-3cdf-4853-b8f2-f167f09d53d0","order_by":1,"name":"Song Jiang","email":"","orcid":"","institution":"Southern Medical University Shenzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Song","middleName":"","lastName":"Jiang","suffix":""},{"id":400533504,"identity":"21ede04d-074b-4944-a1fb-13ba1f9937f5","order_by":2,"name":"Hongwei Shen","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongwei","middleName":"","lastName":"Shen","suffix":""},{"id":400533505,"identity":"d92d6239-cfc7-4166-98d6-7b86538b08ea","order_by":3,"name":"Xi Chen","email":"","orcid":"","institution":"Southern Medical University Shenzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Chen","suffix":""},{"id":400533506,"identity":"b831e801-0cd5-4209-bdb0-0effe76321e3","order_by":4,"name":"Ewnji Yoon","email":"","orcid":"","institution":"Southern Medical University Shenzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ewnji","middleName":"","lastName":"Yoon","suffix":""},{"id":400533507,"identity":"ee1dd524-3c15-4ffc-ae44-09d468658c5e","order_by":5,"name":"Xiaxia Yu","email":"","orcid":"","institution":"Shenzhen University Medical School, Shenzhen University","correspondingAuthor":false,"prefix":"","firstName":"Xiaxia","middleName":"","lastName":"Yu","suffix":""},{"id":400533508,"identity":"258f4f89-85a4-46b9-954d-a269fbac0f5b","order_by":6,"name":"Kin Cheung","email":"","orcid":"","institution":"Hong Kong Polytechnic University","correspondingAuthor":false,"prefix":"","firstName":"Kin","middleName":"","lastName":"Cheung","suffix":""},{"id":400533509,"identity":"bbd5f0a0-f0ec-4378-9af7-527d51cb2dad","order_by":7,"name":"Bai Jiao","email":"","orcid":"","institution":"Shenzhen Prevention and Treatment Center for Occupational Diseases,","correspondingAuthor":false,"prefix":"","firstName":"Bai","middleName":"","lastName":"Jiao","suffix":""},{"id":400533510,"identity":"72bed2b9-9b85-4bbf-a0bc-f22b51dba24e","order_by":8,"name":"Lingguo Zhao","email":"","orcid":"","institution":"Center for Disease Prevention and Control of Baoan District","correspondingAuthor":false,"prefix":"","firstName":"Lingguo","middleName":"","lastName":"Zhao","suffix":""},{"id":400533511,"identity":"7e7dfc61-3529-4d97-8bb1-8d6743b24da2","order_by":9,"name":"Yong Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYBACA2YIDaSYDwBpCwYGHuK1sCUwMCRIEKEFweQxIFILO4+ZxMcdtez8EjkfH/P+kJDj5znA+OFjDj6H8ZhJzjxznFlyRu5mY54ECWPJ3gZmyZnb8GuR5m07xmxwI3ebNFBL4obzDGzMvMRpyXlGkpYakBY2iJazDYS0sBVbzmw7wCzZ88zYcE4a0C89B5vx+sW+//DGGx/b6pL52ZMfPnhjYwMMseSDHz7i0QIELMC4OJzMIJAAE2BswKseCJg/MDDU2THwHyCkcBSMglEwCkYqAACDi0XB6bAhRgAAAABJRU5ErkJggg==","orcid":"","institution":"Southern Medical University Shenzhen Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yong","middleName":"","lastName":"Liu","suffix":""},{"id":400533512,"identity":"b82c6aac-a325-4341-ba08-c631939da53a","order_by":10,"name":"Zhanguo Liu","email":"","orcid":"","institution":"Zhujiang Hospital, Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhanguo","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-01-09 08:38:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5794685/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5794685/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73632356,"identity":"ada06a8a-41b1-4e40-b794-f0801e8249a7","added_by":"auto","created_at":"2025-01-13 06:41:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":421804,"visible":true,"origin":"","legend":"\u003cp\u003eBurkholderia gladioli 201 shared the greatest ANI (99.17%) with B. gladioli BCC1880 (isolated from United Kingdom), followed by B. gladioli BCC1661, BCC1686 and B. gladioli 1697 (all isolated from clinical samples, United States). In contrast, Burkholderia gladioli 201 only shared 96.84% and 97.37% identity with B. gladioli SCCH61 Bgd92-3601 and B. gladioli AU35657 (isolated from clinical samples in Russia and United States, respectively).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5794685/v1/fc10163198694a8484f9fe46.png"},{"id":73632361,"identity":"f7688b38-4b4a-4f3f-92fa-6f4dc6834f27","added_by":"auto","created_at":"2025-01-13 06:41:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":763686,"visible":true,"origin":"","legend":"\u003cp\u003eThe gene tree, which includes 357 strains divided into 10 clades, with the organism information for each strain mapped onto the tree. The largest clade in the tree, clade 10, constitutes 268 strains, including the Burkholderia gladioli 201 strain\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5794685/v1/c685ef60a320cc69219433ce.png"},{"id":73632441,"identity":"7db0deb1-fb25-4c3f-a4a4-fb3ef75ed359","added_by":"auto","created_at":"2025-01-13 06:49:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":283968,"visible":true,"origin":"","legend":"\u003cp\u003eThe species tree demonstrated that Burkholderia gladioli 201 is closely related to Burkholderia plantarii and Burkholderia glumae AU6208.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5794685/v1/6531ed47dcaa8555c2f63776.png"},{"id":80009867,"identity":"6d31ed23-801a-47b7-926f-c106ec6cb68e","added_by":"auto","created_at":"2025-04-07 00:46:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3173199,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5794685/v1/415f5cef-a7bc-4921-a0d2-1eba5a2ea8d7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Genomic Analysis and Isolation of a Novel Burkholderia gladioli Strain Producing Lethal Bongkrekic Acid in China","fulltext":[{"header":"Background","content":"\u003cp\u003eBongkrekic acid (BKA), a hazardous tricarboxylic fatty acid, is secreted by Burkholderia gladioli cocovenenans, previously recognized as Pseudomonas cocovenenans subsp. farinofermentans in the Chinese context [1]. Functioning as a mitochondrial toxin through inhibition of adenine nucleotide translocase [2, 3], BKA represents a severe threat, particularly contaminating four key food groups: fermented or semi-fermented corn and coconut products, spoiled Tremella fuciformis (also known as white fungi), and other decayed starchy foods such as glutinous rice, millet, sorghum, and potato flour. P. cocovenenans, initially recognized for its role in food poisoning, was found to generate both toxoflavin and BKA [4]. The 1970s witnessed sporadic occurrences of high mortality rates, exceeding 40%, from food poisoning in parts of China, linked to fermented corn and spoiled Tremella intake [5\u0026ndash;7]. The implicated bacterium, a yellow-pigmented, gram-negative rod, was initially named \"Flavobacterium farinofermentans\" [5], later confirmed as P. cocovenenans [4]. Reclassification of P. cocovenenans into the Burkholderia genus as B. cocovenenans in 1995 was supported by 16S rRNA gene sequencing analysis [1, 4].\u003c/p\u003e \u003cp\u003eCoenye et al. employed comprehensive methodologies, including whole-cell protein profiling and DNA-DNA hybridization, to investigate the connection between B [8]. gladioli and B. cocovenenans, concluding that B. cocovenenans was a subspecies of B. gladioli. Nonetheless, the study's scope was limited, focusing on a single B. gladioli pathovar without fully addressing pathogenic variability.\u003c/p\u003e \u003cp\u003eBKA-related poisoning incidents have been documented globally, notably in China, Indonesia, and Mozambique. China has reported over 9,000 BKA poisoning cases since the mid-20th century, with over 1,000 fatalities traced back to contaminated corn products [9]. Indonesia has seen foodborne illness outbreaks tied to fermented food consumption[3], while a 2015 event in Mozambique resulted in 75 deaths from a fermented cornmeal drink, affecting 230 people [10]. These incidents emphasized the high-risk nature of fermented or spoiled corn and coconut goods in relation to BKA poisoning.\u003c/p\u003e \u003cp\u003eA 2018 poisoning case in Guangdong Province, China, involving BKA from seemingly unspoiled wet rice noodles, highlighted the potential for BKA contamination even in non-fermented foods[9]. Despite more similar cases recorded post-event [11], the complete molecular characterization of the causal strains remains elusive.\u003c/p\u003e \u003cp\u003eThis study presents the isolation and characterization of a new B. gladioli strain, designated as \u0026ldquo;Burkholderia gladioli 201\u0026rdquo;, which was recovered from a non-fermented rice noodle implicated in a Chinese foodborne outbreak, capable of producing lethal BKA. We conducted an exhaustive taxonomic assessment, incorporating genetic, chemotaxonomic, and phenotypic methods to define the isolate's characteristics, pathophysiology, and unique molecular profile. Furthermore, we offer an up-to-date comparative analysis between toxic and non-toxic B. gladioli strains and construct a genome-scale phylogenetic tree, involving 356 other Burkholderia strains, to establish the isolate's phylogenetic position. A species tree was also developed to explore the evolutionary patterns of gene families associated with this pathogen.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGenomic Features of Burkholderia gladioli 201\u003c/h2\u003e \u003cp\u003eThe Burkholderia gladioli 201 genome was fully sequenced, revealing a length of 8,492,480 base pairs with a GC content of 68.38%. This species is noted for its ubiquitous presence across various environments, as evidenced by the global distribution of the 356 strains studied, which were sourced from a diverse range of origins including plants, human clinical samples, animal samples, and environmental samples. The most extensively studied strain, B. gladioli ATCC R 10248, was first isolated from Gladiolus sp. in the United States in 1913. The genomes of these strains varied in size from 5.49 to 13.08 Mbp, with a mean size of 8.13 Mbp and a median of 8.03 Mbp, and GC contents ranging from 55\u0026ndash;68.6%, with a mean of 67.82% and a median of 66.45%.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal Experiments\u003c/h3\u003e\n\u003cp\u003eIn our experiments, rats were administered a solution derived from the culture of Burkholderia gladioli 201 via gavage. Within an hour, the rats developed symptoms of lethargy, general weakness, and an inability to stand or walk, along with rapid breathing and nodding respiration, signaling a critical state. Toxicological examinations post-mortem confirmed the presence of bongkrekic acid in multiple organs, with the highest concentration in the liver.\u003c/p\u003e\n\u003ch3\u003eANI Analysis of B. gladioli\u003c/h3\u003e\n\u003cp\u003eGenetic similarity among B. gladioli genomes was assessed using Average Nucleotide Identity (ANI) values, which ranged from 95.00\u0026ndash;100.00%, with a mean of 98.27%. This exceeds the species delineation threshold of 95% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Burkholderia gladioli 201 showed the highest ANI (99.17%) with B. gladioli BCC1880 from the United Kingdom, and lower identities with B. gladioli SCCH61 Bgd92-3601 and B. gladioli AU35657 from Russia and the United States, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCore- and Pan-Genome of Burkholderia gladioli\u003c/h3\u003e\n\u003cp\u003eThe pan-genome of B. gladioli, comprising 182,662 genes, included 141 core genes present in at least 99% of strains. The pan-genome also contained soft-core genes, shell genes, and cloud genes, numbered at 73, 7,843, and 174,605, respectively.\u003c/p\u003e\n\u003ch3\u003eBA Biosynthesis Gene Cluster Analysis\u003c/h3\u003e\n\u003cp\u003eA 15,807-bp sequence in Burkholderia gladioli 201, exhibiting 99.36% identity to the BA biosynthesis gene cluster bon of B. cocovenenans DMSZ11318, was identified. This bon gene cluster was also found in 46 other B. gladioli genomes with high sequence identity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\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\u003eThe presence of the BA biosynthesis gene cluster bon in Burkholderia gladioli 201 and other genomes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003equery acc.ver\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003esubject acc.ver\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e% identity\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ealignment length\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emismatches\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" 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class=\"Underline\" name=\"Emphasis\"\u003e15801\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e28613\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL005571\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.415\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e14013\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e59\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e6\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e21132\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e35136\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e11299\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e25296\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e25401\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL005574\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.297\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e4551\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e17\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e54707\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e59251\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e4542\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e8213\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL005573\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.558\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2262\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e10\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e51113\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e53374\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2262\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e4122\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL000481\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.723\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2166\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e6\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e61163\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e63328\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2166\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3967\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL000482\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.661\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1476\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e5\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e63325\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e64800\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1476\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2699\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL005575\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e100\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1455\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e59206\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e60660\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1455\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2687\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL005570\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.686\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1275\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e4\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e9106\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e10380\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1275\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2333\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL005569\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.841\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1260\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e7842\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e9101\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1260\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2316\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL005564\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.676\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1233\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e4\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3802\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e5034\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1233\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2255\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL005568\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.647\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1134\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e4\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e6650\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e7783\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1134\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2073\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL006899\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.906\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1065\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1065\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1065\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1962\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL005565\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.803\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1014\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e5153\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e6166\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1014\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1862\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL000483\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.88\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e831\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e64785\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e65615\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e831\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1530\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL006898\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.517\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e828\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e4\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1132\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1959\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e828\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1507\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL000486\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e100\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e807\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e66740\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e67546\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e807\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1491\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL000484\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e100\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e750\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e65617\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e66366\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e750\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1386\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL006897\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.474\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e570\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2005\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2574\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e570\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1037\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL000485\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e99.625\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e267\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e66414\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e66680\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e267\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3.87E-136\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e488\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL006896\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e100\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e240\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2787\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3026\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e240\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e8.49E-123\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e444\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL005562\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e98.387\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e186\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3412\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3597\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e186\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e8.93E-88\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e327\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL005563\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e100\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e162\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3594\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3755\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e162\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1.95E-79\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e300\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL005566\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e100\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e129\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e6200\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e6328\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e129\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e4.30E-61\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e239\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eJX173632.1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e201GL005567\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e100\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e117\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e6433\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e6549\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e117\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2.02E-54\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e217\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGene Tree and Species Tree Analysis\u003c/h2\u003e \u003cp\u003eThe gene tree depicted 357 strains distributed across 10 clades, with the Burkholderia gladioli 201 strain part of the largest clade (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The species tree of 30 representative bacterial species demonstrated that Burkholderia gladioli 201 clustered with Burkholderia plantarii and Burkholderia glumae AU6208, indicating that the three species were closely related (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, the genome of Burkholderia gladioli 201 assembled in this study was similar to the previously reported Burkholderia gladioli SCCH61 Bgd92-3601 (GCA 028462525.1). Notably, the genome of Burkholderia gladioli 201 had a higher number of annotated single-copy genes compared to Burkholderia gladioli SCCH61 Bgd92-3601, while having fewer multi-copy genes, indicating genetic diversity within the species. In the most differentiated branches of the species tree, an expansion of gene types, particularly single-copy genes, was observed, as exemplified by the increased number of single-copy genes in Burkholderia aenigmatica.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigates a comprehensive analysis of a novel Burkholderia gladioli strain, designated as Burkholderia gladioli 201 and isolated from a non-fermented rice noodle associated with a foodborne outbreak in China. The primary findings of this research include the successful isolation and characterization of this strain, which was confirmed to produce the lethal Bongkrekic Acid (BKA). The complete genome sequence of the strain was determined, revealing a high degree of genetic similarity with other B. gladioli strains, particularly in the presence of a distinct BA biosynthesis gene cluster responsible for BKA production. Furthermore, the study conducted a detailed comparative genomic analysis, which, through phylogenetic reconstruction, demonstrated the close evolutionary relationship of Burkholderia gladioli 201 with other strains of species. The findings from animal experiments, which involved the administration of a solution derived from the Burkholderia gladioli 201 culture to rats, corroborated the production of BKA, as evidenced by the observed symptoms and subsequent toxicological examinations.\u003c/p\u003e \u003cp\u003eThe ANI analysis in this study reveals the genomic similarity between Burkholderia gladioli 201 and other strains of the same species. The average ANI value of 98.27% is higher than the species delineation threshold of 95%, indicating a high level of genomic similarity between B. gladioli 201 and other strains of the same species at the genomic [12, 13]. This high level of genomic similarity may be related to the shared biological functions and ecological niches of these strains.\u003c/p\u003e \u003cp\u003eHowever, it is important to note that although ANI values are commonly used to define species boundaries, there may still be significant phenotypic differences between different strains above this threshold. For example, B. gladioli 201 can produce the deadly Bongkrekic Acid (BKA), while other strains of the same species may not have this ability. This indicates that in addition to ANI analysis, it is also necessary to combine other phenotypic and genotypic characteristics to fully understand the differences between strains.\u003c/p\u003e \u003cp\u003eFurthermore, the results of the ANI analysis also support the phylogenetic analysis of B. gladioli 201 conducted in this study. By constructing a phylogenetic tree based on single-copy core genes, we observed a close phylogenetic relationship between B. gladioli 201 and other strains such as B. plantarii and B. glumae AU6208. This is consistent with the results of the ANI analysis and further confirms the taxonomic position of B. gladioli 201 at the genomic level.\u003c/p\u003e \u003cp\u003eThese discoveries highlight the genetic basis for BKA production and emphasize the need for enhanced surveillance and preventive measures to prevent BKA-associated food poisoning incidents.\u003c/p\u003e \u003cp\u003eThe present study introduces several innovative elements to the existing body of research on Burkholderia gladioli and its production of the deadly Bongkrekic Acid (BKA). A significant contribution of this work is the isolation and detailed characterization of a novel strain, Burkholderia gladioli 201, from an atypical non-fermented food source\u0026ndash;a rice noodle associated with a foodborne outbreak in China. This finding diverges from the majority of BKA poisoning incidents that have been predominantly linked to fermented or spoiled food items, as reported in the literature such as the cases documented by Zhao et al. and Gudo et al [1, 10].\u003c/p\u003e \u003cp\u003eThe phylogenetic reconstruction and comparative genomic analysis presented here provide a robust framework for understanding the evolutionary relationships among Burkholderia gladioli strains. This study's approach extends beyond the taxonomic delineations made by Coenye et al. [8] by offering a more nuanced view of the genetic diversity and phylogenetic placement of the newly identified strain.\u003c/p\u003e \u003cp\u003eMoreover, the integration of phenotypic, biochemical, and molecular identification methods in this study represents a comprehensive approach not commonly found in the literature. This multifaceted methodology stands in contrast to earlier studies that often relied on a limited set of identification techniques, thereby enhancing the accuracy and depth of the characterization of the Burkholderia gladioli 201 strain.\u003c/p\u003e \u003cp\u003eThe novelty of this study is underscored by the isolation of a new strain from an unusual food matrix, the in-depth genomic characterization that includes the identification of a BA biosynthesis gene cluster, the advanced comparative genomic analysis, and the multifaceted identification approach. These elements collectively contribute to a more refined comprehension of the Burkholderia gladioli species and its role in foodborne illness, offering a significant advancement over previous research in this field.\u003c/p\u003e \u003cp\u003eDespite the valuable contributions of the study in isolating, characterizing, and performing genomic analysis on a novel Bongkrekic acid producing Burkholderia gladioli strain, the findings must be contextualized within certain limitations. Firstly, the reliance on a single isolate restricts the broad applicability of conclusions to other strains or ecological contexts, given the lack of representativeness in sample size. Secondly, the study's use of FastANI for genomic relatedness assessments may not fully elucidate the diversity among more distantly related isolates due to its inability to accurately calculate ANI values below 80%. This limitation could lead to an incomplete understanding of the strain's phylogenetic positioning. Additionally, the standard biochemical and phenotypic assays employed may inadequately reflect the full spectrum of metabolic activities and virulence attributes inherent to B. gladioli, potentially missing strain-specific characteristics. Lastly, while the comparative genomic analysis was extensive, a deeper exploration could offer enhanced insights into the pathogenic variability and species diversity within Burkholderia. Collectively, these limitations underscore the necessity for more comprehensive sampling strategies, advanced detection techniques, and a deeper genomic and biological exploration to comprehensively elucidate the epidemiology and virulence mechanisms of B. gladioli strains.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA novel strain of Burkholderia gladioli that can produce bongkrekic acid (BKA) from non-fermented rice noodles connected to a foodborne outbreak was successfully isolated and described in this study. A unique BKA biosynthesis gene cluster was identified by the genomic analysis, which shed light on the evolutionary relationships within the Burkholderia genus as well as the genetic underpinnings of toxin production. These results underline the need for improved food safety protocols and systematic surveillance, as well as the growing risk of BKA contamination outside fermented foods. The relationships between toxic and non-toxic strains are further elucidated by the phylogenetic analysis, which also provides useful markers for prevention and monitoring. In order to better reduce the risks of foodborne illness linked to BKA, this work emphasizes the significance of ongoing research into strain diversity and virulence mechanisms.\u003c/p\u003e "},{"header":"Materials \u0026 Methods","content":"\u003ch2\u003eIsolation and Identification of Burkholderia gladioli from Rice Noodles\u003c/h2\u003e\u003cp\u003eSamples were obtained from a rice noodle factory located in Shenzhen City. Employing the protocols specified in \"Microbiological Examination of Food Hygiene—Examination of Pseudomonas cocovenenans subsp. farinofermentans\" (GB/T 4789.29–2003), we successfully isolated a strain of Burkholderia gladioli, designated as Burkholderia gladioli 201, from the collected noodle samples.\u003c/p\u003e\u003cp\u003eThe samples were cultured on Tryptic Soy Agar (TSA) and incubated at 28°C for 48 hours. Bacterial colonies were then streaked onto fresh TSA plates to ensure the isolation of single colonies for use in this study. Isolated colonies were stored at 4°C for immediate use or preserved in a glycerol stock at -78°C for long-term storage in an ultra-low temperature freezer. Control reference strains of Burkholderia gladioli were sourced from a reputable culture collection for comparative analysis during the experiments.\u003c/p\u003e\u003ch2\u003ePhenotypic Characterization\u003c/h2\u003e\u003cp\u003eColony morphology was examined on nutrient agar containing meat extract, peptone, and NaCl, following incubation at 28°C for 24 to 48 hours. Biochemical characterization was performed using the miniaturized BIOLOG system, which assessed the utilization of 95 different carbon substrates with BIOLOG GN2 microplates, strictly following the manufacturer's instructions.\u003c/p\u003e\u003cp\u003e The identification of the strain was achieved through a comprehensive approach involving selective media, conventional biochemical tests, commercial diagnostic systems, and PCR-based assays, in line with the guidelines of \"Microbiological Examination of Food Hygiene.\"\u003c/p\u003e\u003ch2\u003eAnimal Experiments\u003c/h2\u003e\u003cp\u003eBurkholderia gladioli 201 was cultured at 26°C for seven days. The culture medium was processed through extraction and separation using ether to obtain a solution, which was administered to rats via gavage. The rats' symptoms were monitored, and necropsies were performed post-mortem to conduct toxicological examinations.\u003c/p\u003e\u003ch2\u003eMolecular Characterization of Bacteria\u003c/h2\u003e\u003cp\u003ePCR amplification with species-specific primers was conducted using a programmable thermal cycler. The PCR reaction mixture contained PCR buffer, MgCl2, primers, dNTP mix, Taq DNA polymerase, and bacterial genomic DNA extracted using a specific kit. Amplification was followed by gel electrophoresis, staining, and visualization under ultraviolet light. PCR products were cloned and sequenced, with nucleotide sequences aligned using specific software and homology determined using the NCBI Blast Network Server.\u003c/p\u003e\u003cp\u003eGenomic DNA extraction was performed using a genomic DNA kit, with DNA quality and quantity assessed using Nanodrop and Qubit, respectively. Library construction for sequencing involved the use of specific instruments and kits, and sequencing was conducted using a particular technology.\u003c/p\u003e\u003ch2\u003eGenomic Sequence Analysis\u003c/h2\u003e\u003cp\u003eGenome assembly and annotation were performed using various software to predict coding genes, tRNA, rRNA, and other ncRNAs, and to identify CRISPRs and gene islands. Gene function annotation was conducted using Interproscan and BlastP to identify protein domains and corresponding GO annotations, and to find matches in KEGG and RefSeq databases.\u003c/p\u003e\u003cp\u003eGene clusters, including the BA biosynthesis gene cluster, were identified using BLAST, and core genes were analyzed using CD-HIT. The phylogenetic tree was constructed based on single-copy core genes using MUSCLE and PhyML, and the Average Nucleotide Identity (ANI) was calculated using JSpeciesWS.\u003c/p\u003e\u003ch2\u003eAverage Nucleotide Identity (ANI) Analysis\u003c/h2\u003e\u003cp\u003eANI values were calculated between the genomes of Burkholderia gladioli 201 and other strains using FastANI. Hierarchical clustering and visualization were performed using the heatmap package in R (version 4.2.2).\u003c/p\u003e\u003ch2\u003eBA Biosynthetic Gene Cluster bon Analysis\u003c/h2\u003e\u003cp\u003eThe BA biosynthesis gene cluster bon from B. cocovenenans was used as a reference sequence to analyze the presence of similar gene clusters in Burkholderia gladioli 201 and other genomes using BLASTn (version 2.15.0+).\u003c/p\u003e\u003ch2\u003eComparative Genomic Analysis\u003c/h2\u003e\u003cp\u003eThe complete genome sequence of Burkholderia gladioli 201 was deposited in GenBank under the accession number(To be submitted). Comparative analysis was performed using the genomic sequences of other 356 Burkholderia strains downloaded from the NCBI database. OrthoFinder (version 2.5.5) was used for gene clustering[12], and a species tree was constructed using single copy genes characterized from selected subgroups.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article and can be found in Additional file.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by Medical Scientific Research Foundation of Guangdong Province of China (B2023180, B2023371).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Critical Care Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China\u003c/p\u003e\n\u003cp\u003eRongGui Lv \u0026amp; Zhanguo Liu\u003c/p\u003e\n\n\u003cp\u003eDepartment of Critical Care Medicine, Shenzhen Hospital, Southern Medical University, Shenzhen, Guangdong, China\u003c/p\u003e\n\u003cp\u003eRongGui Lv, Song Jiang, Ewnji Yoon \u0026amp; Yong Liu\u003c/p\u003e\n\n\u003cp\u003eDepartment of Clinical Laboratory, Shenzhen Hospital, Southern Medical University, Shenzhen, Guangdong, China\u003c/p\u003e\n\u003cp\u003eHongwei Shen\u003c/p\u003e\n\n\u003cp\u003eDepartment of Pharmacy, Shenzhen Hospital, Southern Medical University, Shenzhen, Guangdong, China\u003c/p\u003e\n\u003cp\u003eXi Chen\u003c/p\u003e\n\n\u003cp\u003eSchool of Nursing, Hong Kong Polytechnic University, Hong Kong SAR, China Xiaxia Yue\u003c/p\u003e\n\n\u003cp\u003eSchool of Biomedical Engineering, Shenzhen University Medical School, Shenzhen University, Shenzhen, Guangdong, 518055, China\u003c/p\u003e\n\u003cp\u003eKin Cheung\u003c/p\u003e\n\n\u003cp\u003eShenzhen Prevention and Treatment Center for Occupational Diseases, 518020 Shenzhen, China\u003c/p\u003e\n\u003cp\u003eBai Jiao\u003c/p\u003e\n\n\u003cp\u003eCenter for Disease Prevention and Control of Baoan District, Guangdong, Shenzhen, China\u003c/p\u003e\n\u003cp\u003eLingguo Zhao\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.L., Y.L., and L.Z. designed the study, provided financial support, supervised the research, and revised the first draft. R.L. and S.J. wrote the first draft and participated in the visualization. H.S., X.C., and E.Y. contributed to the study design and data analysis. X.Y., K.C., and B.J. supported data collection and experimental validation. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLingguo Zhao, Center for Disease Prevention and Control of Baoan District, Guangdong, Shenzhen, 518101, China, E-mail: [email protected]; Yong Liu, Department of Critical Care Medicine, Shenzhen Hospital, Southern Medical University, No 1333, Xinhu Avenue, Baoan District, Shenzhen, Guangdong Province, 518055, China,.E-mail: [email protected]; Zhanguo Liu, Department of Critical Care Medicine, Zhujiang Hospital, Southern Medical University, 253 Gongye Middle Avenue, Haizhu District, Guangzhou, 510280, Guangdong, China, E-mail: [email protected].\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe samples used in this study, including rice noodles implicated in the foodborne outbreak, were obtained from a local rice noodle factory in Shenzhen. Permission for sampling and related analyses was granted by the relevant local health and food safety authorities. The animal experiments conducted in this study were approved by the Ethics Committee of Zhujiang Hospital, Southern Medical University (approval number:2024-0127). All experimental procedures strictly adhered to institutional, national, and international guidelines and regulations for the ethical treatment of animals. The authors confirm that all experimental research and field studies comply with relevant ethical standards and legislation.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhao NX, Ma MS, Zhang YP, Xu DC: \u003cstrong\u003eComparative description of Pseudomonas cocovenenans (van Damme, Johannes, Cox, and Berends 1960) NCIB 9450T and strains isolated from cases of food poisoning caused by consumption of fermented corn flour in China\u003c/strong\u003e. \u003cem\u003eInt J Syst Bacteriol \u003c/em\u003e1990, \u003cstrong\u003e40\u003c/strong\u003e(4):452-455.\u003c/li\u003e\n\u003cli\u003eHenderson PJ, Lardy HA: \u003cstrong\u003eBongkrekic acid. An inhibitor of the adenine nucleotide translocase of mitochondria\u003c/strong\u003e. \u003cem\u003eJ Biol Chem \u003c/em\u003e1970, \u003cstrong\u003e245\u003c/strong\u003e(6):1319-1326.\u003c/li\u003e\n\u003cli\u003eAnwar M, Kasper A, Steck AR, Schier JG: \u003cstrong\u003eBongkrekic Acid-a Review of a Lesser-Known Mitochondrial Toxin\u003c/strong\u003e. \u003cem\u003eJ Med Toxicol \u003c/em\u003e2017, \u003cstrong\u003e13\u003c/strong\u003e(2):173-179.\u003c/li\u003e\n\u003cli\u003eZhao N, Qu C, Wang E, Chen W: \u003cstrong\u003ePhylogenetic evidence for the transfer of Pseudomonas cocovenenans (van Damme et al. 1960) to the genus Burkholderia as Burkholderia cocovenenans (van Damme et al. 1960) comb. nov\u003c/strong\u003e. \u003cem\u003eInt J Syst Bacteriol \u003c/em\u003e1995, \u003cstrong\u003e45\u003c/strong\u003e(3):600-603.\u003c/li\u003e\n\u003cli\u003eMeng ZH, Wang X: \u003cstrong\u003e[Laboratory studies on the factors affecting the toxigenicity of Flavobacterium farinofermentans nov. sp]\u003c/strong\u003e. \u003cem\u003eZhonghua Yu Fang Yi Xue Za Zhi \u003c/em\u003e1987, \u003cstrong\u003e21\u003c/strong\u003e(2):80-82.\u003c/li\u003e\n\u003cli\u003eMeng Z, Wang D, Li Z, Jin J, Bai J, Zhang Y, Liu X, Cai L, Li X, Ren H\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eStudies on fermented corn flour food poisoning in rural areas of China. II. Isolation and identification of causal microorganisms\u003c/strong\u003e. \u003cem\u003eBiomed Environ Sci \u003c/em\u003e1988, \u003cstrong\u003e1\u003c/strong\u003e(1):105-114.\u003c/li\u003e\n\u003cli\u003eMeng Z, Li Z, Jin J, Zhang Y, Liu X, Yiang X, Ren H: \u003cstrong\u003eStudies on fermented corn flour poisoning in rural areas of China. I. Epidemiology, Clinical Manifestations, and Pathology\u003c/strong\u003e. \u003cem\u003eBiomed Environ Sci \u003c/em\u003e1988, \u003cstrong\u003e1\u003c/strong\u003e(1):101-104.\u003c/li\u003e\n\u003cli\u003eCoenye T, Holmes B, Kersters K, Govan JR, Vandamme P: \u003cstrong\u003eBurkholderia cocovenenans (van Damme et al. 1960) Gillis et al. 1995 and Burkholderia vandii Urakami et al. 1994 are junior synonyms of Burkholderia gladioli (Severini 1913) Yabuuchi et al. 1993 and Burkholderia plantarii (Azegami et al. 1987) Urakami et al. 1994, respectively\u003c/strong\u003e. \u003cem\u003eInt J Syst Bacteriol \u003c/em\u003e1999, \u003cstrong\u003e49 Pt 1\u003c/strong\u003e:37-42.\u003c/li\u003e\n\u003cli\u003eLi J, Zhou LU, Long C, Fang L, Chen Q, Chen Q, Liang J, Yang Y, Zhu H, Chen Z\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eAn Investigation of Bongkrekic Acid Poisoning Caused by Consumption of a Nonfermented Rice Noodle Product without Noticeable Signs of Spoilage\u003c/strong\u003e. \u003cem\u003eJ Food Prot \u003c/em\u003e2019, \u003cstrong\u003e82\u003c/strong\u003e(10):1650-1654.\u003c/li\u003e\n\u003cli\u003eGudo ES, Cook K, Kasper AM, Vergara A, Salom\u0026atilde;o C, Oliveira F, Ismael H, Saeze C, Mosse C, Fernandes Q\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eDescription of a Mass Poisoning in a Rural District in Mozambique: The First Documented Bongkrekic Acid Poisoning in Africa\u003c/strong\u003e. \u003cem\u003eClin Infect Dis \u003c/em\u003e2018, \u003cstrong\u003e66\u003c/strong\u003e(9):1400-1406.\u003c/li\u003e\n\u003cli\u003eZhang H, Guo Y, Chen L, Liu Z, Liang J, Shi M, Gao F, Song Y, Chen J, Fu P: \u003cstrong\u003eEpidemiology of foodborne bongkrekic acid poisoning outbreaks in China, 2010 to 2020\u003c/strong\u003e. \u003cem\u003ePLoS One \u003c/em\u003e2023, \u003cstrong\u003e18\u003c/strong\u003e(1):e0279957.\u003c/li\u003e\n\u003cli\u003eCastillo-Ram\u0026iacute;rez S: \u003cstrong\u003eOn the road to genomically defining bacterial intra-species units\u003c/strong\u003e. \u003cem\u003emSystems \u003c/em\u003e2024, \u003cstrong\u003e9\u003c/strong\u003e(7):e0058424.\u003c/li\u003e\n\u003cli\u003eRodriguez RL, Conrad RE, Viver T, Feistel DJ, Lindner BG, Venter SN, Orellana LH, Amann R, Rossello-Mora R, Konstantinidis KT: \u003cstrong\u003eAn ANI gap within bacterial species that advances the definitions of intra-species units\u003c/strong\u003e. \u003cem\u003emBio \u003c/em\u003e2024, \u003cstrong\u003e15\u003c/strong\u003e(1):e0269623.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Burkholderia gladioli, Bongkrekic Acid, Genomic Analysis, Foodborne Outbreak, Phylogenetic Relationship","lastPublishedDoi":"10.21203/rs.3.rs-5794685/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5794685/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBongkrekic acid (BKA) is a potent mitochondrial toxin secreted by certain strains of Burkholderia gladioli, previously known as Pseudomonas cocovenenans. It poses a significant risk to public health, particularly through contaminating fermented or spoiled food products. This study focuses on the isolation and genomic analysis of a novel strain of Burkholderia gladioli from a non-fermented rice noodle linked to a foodborne outbreak in China.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe study employed a multifaceted approach including microbiological isolation, phenotypic characterization, biochemical assays, and molecular identification. Animal experiments were conducted to observe the effects of BKA on rats. Genomic DNA was extracted, sequenced, and analyzed using bioinformatics tools to determine the genetic makeup, gene function annotation, and comparative genomic analysis. The isolated strain, designated as Burkholderia gladioli 201, was found to produce BKA, which was confirmed through animal experiments showing characteristic symptoms and subsequent toxicological examinations. The complete genome sequence revealed a high degree of genetic similarity with other B. gladioli strains, with a distinct BA biosynthesis gene cluster. Comparative genomic analysis and phylogenetic trees constructed from single-copy core genes indicated a close relationship between Burkholderia gladioli 201 and other strains of the species.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study successfully isolated and characterized a novel strain of Burkholderia gladioli capable of producing BKA from a non-fermented food source. The genomic analysis provided insights into the genetic basis of BKA production and the evolutionary relationships within the Burkholderia genus. The findings underscore the need for enhanced surveillance and preventive measures to mitigate the risk of BKA-associated food poisoning.\u003c/p\u003e","manuscriptTitle":"Comparative Genomic Analysis and Isolation of a Novel Burkholderia gladioli Strain Producing Lethal Bongkrekic Acid in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-13 06:41:40","doi":"10.21203/rs.3.rs-5794685/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":"56f8daf3-8906-407f-bf63-5e0343ab0daa","owner":[],"postedDate":"January 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-07T00:38:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-13 06:41:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5794685","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5794685","identity":"rs-5794685","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}