Isolation and Characterization of the Microbiota from the Left Ventricle in NIH Mice (Mus musculus): An In Vitro Study

Isolation and Characterization of the Microbiota from the Left Ventricle in NIH Mice (Mus musculus): An In Vitro Study | 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 Isolation and Characterization of the Microbiota from the Left Ventricle in NIH Mice ( Mus musculus ): An In Vitro Study Jose Alberto Domínguez López, Hanna Guevara-Rodríguez, Peggy Elizabeth Álvarez Gutiérrez, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6086827/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 Aim: The aim of this study was to characterize the hematic microbiota of the left ventricle of NIH strain mice via culture, Gram staining and catalase assays. Materials and Methods: An in vitro study was conducted in the Department of Pharmacology of the Faculty of Medicine at the Autonomous University of Chiapas and the Laboratory of Experimental Microbiology at the Technological Institute of Tuxtla Gutierrez, Mexico, between November 2019 and February 2020. Thirty NIH mice were used, distributed into groups and maintained under sterile conditions. Blood samples were obtained via puncture of the left ventricle and inoculated in different culture media (blood agar, Sabouraud agar and brain-heart agar) for isolation and characterization of the microbiota. Results: The results revealed the growth of the microorganisms on all the culture media. On blood agar, 22, 22 and 29 colony-forming units (CFUs) were recorded for the mice in groups A, B and C, respectively. On Sabouraud agar, 65, 44 and 78 CFUs were observed, and on brain-heart agar, 26, 131 and 10 CFUs were observed. Taxonomic characterization revealed the presence of gram-positive and gram-negative bacteria, as well as fungi, with a predominance of Streptococcus and Bacillus in different samples. Conclusion: This study revealed the existence of a native microbiota in the blood of NIH mice, which are predominantly gram-negative, with diverse fungi. General Microbiology Cardiothoracic Surgery Infectious Diseases Bacteriology Physiology gut microbiomes bloodstream infections bloodstream sepsis probiotics blood microbiota Figures Figure 1 Figure 2 Figure 3 Introduction The term “blood microbiome” refers to the set of all microorganisms present in the blood, i.e., bacteria, viruses and fungi, as well as genomes or genomic fragments and the DNA and/or RNA of these microorganisms [ 1 – 4 ]. Traditionally, human blood is considered a sterile environment, where the occasional entry and proliferation of microorganisms can trigger an abnormal and dysregulated physiological response in the host, resulting in severe clinical manifestations such as sepsis, septic shock or even death [ 5 – 10 ]. Recent studies have revealed the presence of multiple microbial species circulating in the blood of healthy humans. However, most of these studies have been performed in relatively small cohorts or lack rigorous controls to differentiate between true biological measurements and sources of contamination [ 11 , 12 ]. Therefore, the concept of a microbial community in the blood of healthy humans remains controversial. The gold standard for detecting live microorganisms in the bloodstream is blood culture. In addition, next-generation sequencing (NGS) technology has led to highly sensitive approaches, such as 16S RNA sequencing. These NGS-based methods have been employed to describe microbial populations in different niches of the body, including the blood, gut, respiratory tract, skin, and urogenital tract. This has significantly improved our understanding of the human microbiome and the host‒host relationship in both physiological and pathological contexts [ 7 – 9 ]. On the other hand, some studies have concluded that the presence of bacteria in human blood is an unusual event, as most healthy donors (78–84%) do not contain any bacterial species in their blood [ 11 , 12 ]. According to Tan et al. (2022) [ 12 ], from a total of 9770 healthy human donors, microbial DNA was present in only 16% of healthy individuals, with a median of one microbial species per individual. Furthermore, there is no consensus on the structure and diversity of a healthy human blood microbiome on the basis of existing studies. Staphylococcus spp. are common genera found in blood in addition to the Proteobacteria Division [ 13 – 16 ]. While the study of microbiotas in human cohorts is crucial, it is necessary to employ a rigorous analytical process through metagenomics and phylogenetic relationships. In addition to strict biosafety measures to avoid bias due to sample contamination, these measures should be adopted in future research on the human blood microbiome. In the present in vitro study, the hematic microbiota of the left ventricle of mice of the NIH strain was characterized by culture, Gram staining and catalase tests. Materials & Methods Study design and sampling of experimental units An in vitro study was conducted at the Department of Pharmacology of the Faculty of Medicine at the Autonomous University of Chiapas and the Laboratory of Experimental Microbiology at the Technological Institute of Tuxtla Gutierrez (Fig. 1 ), Chiapas, Mexico. November 2019 to February 2020. The Faculty Research Ethics Committee approved this study (certificate number: FMH-180035-2019). In the present study, we used NIH strain mice (n = 30) acquired from the Animal Experimentation Unit (UNEXA-Harlan) of the National Autonomous University of Mexico, which were donated to the Department of Pharmacology of the Autonomous University of Chiapas. All procedures were approved by the institutional bioethics committee and in accordance with the Official Mexican Standard on Technical Specifications for the Production, Care and Use of Laboratory Animals (NOM-062-ZOO-1999). The regulatory guidelines guarantee working with experimental units free of viruses, bacteria and parasites listed in the recommendations of the Federation of European Associations for Laboratory Animal Science. We used 15 male and 15 female mice, 5–6 weeks of age and an average weight of 30 g, randomly distributed into 6 groups of 5 individuals each, which were used to separate 3 experimental groups of homogeneous characteristics, separated into acrylic cages (Soluciones MG, Mexico City), containing wood shavings previously sterilized in an autoclave. The mice were fed standard rodent chow (LabDiet 5001, St. Louis, MO, USA) and purified water. The cages were cleaned every 3 days. Thoracotomy and extraction of blood samples. Blood samples were obtained from the experimental unit via direct extraction via puncture of the apex of the left ventricle via a midthoracic axillary line approach. The samples were deposited in EDTA tubes (Vacutainer K2, BD, USA). Culture, isolation and morphological characterization of the hematic microbiota of NIH mice. From a total of 3 ml of blood from each experimental group, 83 µl was inoculated into each Petri dish. The inocula were distributed in 12 Petri dishes for each culture medium (blood agar, Sabouraud agar and brain-heart agar) and 4 for each experimental group (mice A, B and C, respectively). The inocula in the culture media were incubated at 38°C for 72 hours in an incubator (Kenton WS series). Colony-forming units (CFUs) were counted via a darkfield colony counter (SOL-BAT, Q20). For taxonomic characterization of the hematic microbiota of NIH mice, we used the Gram staining technique and the catalase test. Results Table 1 summarizes the findings found in the culture media used in this study. For the hematic inocula on blood agar, 22, 22 and 29 CFUs were detected in the experimental groups “Mice A”, “Mice B” and “Mice C”, respectively. Hematic inocula on Sabouraud agar yielded 65, 44 and 78 CFUs for the experimental groups “Mice A”, “Mice B” and “Mice C”, respectively. Hematic inocula on brain-heart agar produced 26 CFU, 131 CFU and 10 CFU for the experimental groups “Mice A”, “Mice B” and “Mice C”, respectively. Table 1 Colony-forming unit counts from bloodstream samples of the mouse ( Mus musculus ) NIH strain Experimental group Blood Agar (CFU) Sabouraud Agar (CFU) Brain Heart Infusion Agar (CFU) Total CFU per Mouse A 22 65 26 113 B 22 44 131 197 C 29 78 10 117 Total CFU per Treatment 73 187 167 - The sum of CFU per culture medium, considering the samples from mice A, B and C, was as follows: 73 for blood agar, 187 for Sabouraud agar and 167 for brain-heart agar. The sum of the CFUs for each group in the three culture media was as follows: 113 for “group A”, 197 for “group B” and 117 for “group C”. The photograph in Fig. 2 from left to right shows the morphology of CFU on Sabouraud agar, the morphology of CFU on blood agar, and the morphology of CFU on brain-heart agar, respectively. Table 2 summarizes the taxonomic characterization by Gram staining and catalase test for the strains grown on blood agar and brain heart agar media. Table 2 Morphological characterization of the blood microbiota of NIH mice via Gram staining and the catalase test. Experimental Group Blood Agar Brain Heart Infusion Agar A gram-negative cocci and streptococci, Catalase (+) gram-negative cocci, Catalase (+) B gram-positive cocci and bacilli, Catalase (-) gram-negative cocci, Catalase (+) C gram-negative cocci and streptococci, Catalase (+) gram-negative cocci, Catalase (+) From the blood sample of experimental group “A” on both blood agar and brain heart agar, gram-negative and catalase-positive Streptococcus grew (Fig. 3 ). From the blood sample of experimental group “B”, Streptococcus and Bacillus gram-positive and catalase negative grew on blood agar, and gram-negative Streptococcus and catalase positive Streptococcus grew on brain heart agar (Fig. 3 ). From the blood sample of the experimental group “C” both on blood agar and brain-heart agar, gram-negative Streptococcus and catalase-positive Streptococcus grew (Fig. 3 ). Discussion All the culture media used showed growth of microorganisms typical of the native microbiota of the bloodstream in NIH mice. This microbiota is composed of diverse microorganisms, gram-positive and gram-negative bacteria, fungi and yeast. The general morphological characteristics of these bacteria correspond to the divisions of Proteobacteria, Bacteroides and Actinobacteria, in that order of abundance. These findings are consistent with the results of authors Panaiotov, S. 2019, Amar, J. 2019, Schierwagen, R. 2019 [ 17 – 19 ]. Dysbiosis and both compensatory and pathological physiological changes are closely related [ 20 – 23 ]. According to Wilmanski, T. 2019, at least 40 blood plasma metabolites vary depending on the diversity of the different apparatuses and systems of the human microbiota (digestive system, circulatory system and cardiovascular system) [ 14 ]. In an inflammatory process, which is typical of diseases involving the immune system, many cytokines, such as interleukins, interferons and neurotransmitters, are released. These compounds have been shown [ 21 – 26 ] to have greater reactivity and proinflammatory effects in mice that are free of microorganisms. A case‒control study performed at the Toulouse Teaching Hospital in France [ 19 ], with 103 patients at high cardiovascular risk without coronary artery disease compared with 99 patients with a history of acute myocardial infarction, analyzed the blood microbiota by 16S qPCR sequencing and revealed that bacterial diversity is decreased in patients with MI, with at least 6 known to include species capable of metabolizing cholesterol. The intestinal microbiota is involved in the programming of the T lymphocyte-mediated response and that when the microbial load is suppressed due to dysbiosis, which is present in septic patients, the immune response is altered [ 24 ]. The hematic microbiota of NIH mice is morphologically identical to the intestinal, skin and reproductive system microbiota populations; this identity allows us to suppose that, under septicemic conditions, dysbiosis can be expected in the bloodstream, which is likely correlated with a process of immune reactions mediated by lymphocytes, macrophages and neutrophils. The limitations of the present study were mainly due to institutional measures during the COVID-19 pandemic, which did not allow further genomic analysis of the isolated bacterial and fungal populations with NGS or 16S qPCR sequencing, as well as a larger sample size. Future research should consider an approach involving clinical samples from human patients diagnosed with septic shock. Conclusions We demonstrated the effectiveness of characterizing the mouse left ventricular microbiota in vitro; similar methods have been developed to understand the ecology of the human microbiome. The microbiota of the left ventricle of NIH mice is mainly gram-negative and gram-positive. The fungal population in the hematic microbiota is diverse; however, we were unable to determine the species. The present study provides evidence supporting the presence of microbiota in the blood of healthy organisms, as previously described in humans by other authors. Otherwise, we had limitations due to COVID-19 pandemic. References The HMP Consortium (2012) A Framework for Human Microbiome Research. Nature 486:215–221 Tsafarova B, Hodzhev Y, Yordanov G, Tolchkov V, Kalfin R, Panaiotov S (2023) Morphology of blood microbiota in healthy individuals assessed by light and electron microscopy. Front Cell Infect Microbiol 18:1091341 Castillo DJ, Rifkin RF, Cowan DA, Potgieter M (2019) The Healthy Human Blood Microbiome: Fact or Fiction? Front Cell Infect Microbiol 9:148 Païssé S, Valle C, Servant F, Courtney M, Burcelin R, Amar J (2016) Comprehensive description of blood microbiome from healthy donors assessed by 16S targeted metagenomic sequencing. Transfusion 56(5):1138–1147 Fleischmann C, Scherag A, Adhikari NK, Hartog CS, Tsaganos T, Schlattmann P (2016) Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Current Estimates and Limitations. Am J Respir Crit Care Med 193(3):259–272 Raeisi J, Oloomi M, Zolfaghari M, Siadat SD, Zargar M, Pourramezan Z (2022) Bacterial DNA Detection in the Blood of Healthy Subjects. Iran Biomed J 26:230–239 Lawrence G, Midtervoll I, Samuelsen SO, Kristoffersen AK, Enersen M, Haheim LL (2022) The blood microbiome and its association to cardiovascular disease mortality: Case-cohort study. BMC Cardiovasc Disord 22:344 Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M (2016) The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 315(8):801–810 Julián-Jiménez A, Rubio-Díaz R, González D, Castillo J, Candel FJ (2022) Nuevos modelos predictivos de bacteriemia en el servicio de urgencias: un paso adelante. Rev Esp Quimioter 35:344–356 Cheng HS, Tan SP, Wong DMK, Koo WLY, Wong SH, Tan NS (2023) The Blood Microbiome and Health: Current Evidence, Controversies, and Challenges. Int J Mol Sci 15:5633 Grumaz S, Grumaz C, Vainshtein Y, Stevens P, Glanz K, Decker SO (2019) Enhanced Performance of Next-Generation Sequencing Diagnostics Compared With Standard of Care Microbiological Diagnostics in Patients Suffering From Septic Shock. Crit Care Med 47(5):e394–e402 Tan CCS, Ko KKK, Chen H, Liu J, Loh M, SG10K_Health Consortium (2023) No evidence for a common blood microbiome based on a population study of 9,770 healthy humans. Nat Microbiol 8(5):973–985 Sciarra F, Franceschini E, Campolo F, Venneri MA (2023) The Diagnostic Potential of the Human Blood Microbiome: Are We Dreaming or Awake? Int J Mol Sci 21:10422–10410 Cabrera-Perez J, Badovinac VP, Griffith TS (2017) Enteric immunity, the gut microbiome, and sepsis: Rethinking the germ theory of disease. Experimental Biology Med 242:127–139 Schierwagen R, Alvarez-Silva C, Madsen MSA, Kolbe CC, Meyer C, Thomas D (2019) Circulating microbiome in blood of different circulatory compartments. Gut 68(3):578–580 Rinninella E, Raoul P, Cintoni M, Franceschi F, Miggiano GAD, Gasbarrini A (2019) What is the Healthy Gut Microbiota Composition? A Changing Ecosystem across Age, Environment, Diet, and Diseases. Microorganisms 7(1):14 Panaiotov S, Hodzhev Y, Tsafarova B, Tolchkov V, Kalfin R (2021) Culturable and Non-Culturable Blood Microbiota of Healthy Individuals. Microorganisms 9(7):1464 Whittle E, Leonard MO, Harrison R, Gant TW, Tonge DP (2019) Multi-method characterization of the human circulating microbiome. Front Microbiol 10:1–12 Amar J, Lelouvier B, Servant F, Lluch J, Burcelin R, Bongard V (2019) Blood Microbiota Modification After Myocardial Infarction Depends Upon Low-Density Lipoprotein Cholesterol Levels. J Am Heart Assoc 8(19):e011797 Szabó BG, Kiss R, Makra N, Pénzes K, Vad E, Kamotsay K (2022) Composition and changes of blood microbiota in adult patients with community-acquired sepsis: A pilot study from bench to bedside. Front Cell Infect Microbiol 12:1067476 Bäumler AJ, Sperandio V (2016) Interactions between the microbiota and pathogenic bacteria in the gut. Nature 535(7610):85–93 Vrancken G, Gregory AC, Huys GRB, Faust K, Raes J (2019) Synthetic ecology of the human gut microbiota. Nat Rev Microbiol Haussner F, Chakraborty S, Halbgebauer R, Huber-Lang M (2019) Challenge to the intestinal mucosa during sepsis. Front Immunol Wilmanski T, Rappaport N, Earls JC, Magis AT, Manor O, Lovejoy J (2019) Blood metabolome predicts gut microbiome α-diversity in humans. Nat Biotechnol 37(10):1217–1228 Traykova D, Schneider B, Chojkier M, Buck M (2017) Blood Microbiome Quantity and the Hyperdynamic Circulation in Decompensated Cirrhotic Patients. PLoS ONE 12:0169310 Qiu J, Zhou H, Yang J, Dong C (2019) Association between blood microbiome and type 2 diabetes mellitus: A nested case-control study. J Clin Lab Anal 33:22842 Additional Declarations The authors declare no competing interests. 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6086827","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":419508259,"identity":"494e6bc7-32ce-4547-bda9-9bfbba0c0fbd","order_by":0,"name":"Jose Alberto Domínguez 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Staining.\u003c/p\u003e","description":"","filename":"Figura3.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6086827/v1/01ed0c11fada12387eef1a6b.jpg"},{"id":78417427,"identity":"5b019240-7848-4365-8b8d-2f2d2dc8b4f3","added_by":"auto","created_at":"2025-03-13 05:04:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7442151,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6086827/v1/5ce52719-d2c9-478a-b0a5-5a80d3dd602e.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eIsolation and Characterization of the Microbiota from the Left Ventricle in NIH Mice (\u003cem\u003eMus musculus\u003c/em\u003e): An In Vitro Study\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe term “blood microbiome” refers to the set of all microorganisms present in the blood, i.e., bacteria, viruses and fungi, as well as genomes or genomic fragments and the DNA and/or RNA of these microorganisms [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e–\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTraditionally, human blood is considered a sterile environment, where the occasional entry and proliferation of microorganisms can trigger an abnormal and dysregulated physiological response in the host, resulting in severe clinical manifestations such as sepsis, septic shock or even death [\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e–\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies have revealed the presence of multiple microbial species circulating in the blood of healthy humans. However, most of these studies have been performed in relatively small cohorts or lack rigorous controls to differentiate between true biological measurements and sources of contamination [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, the concept of a microbial community in the blood of healthy humans remains controversial.\u003c/p\u003e \u003cp\u003eThe gold standard for detecting live microorganisms in the bloodstream is blood culture. In addition, next-generation sequencing (NGS) technology has led to highly sensitive approaches, such as 16S RNA sequencing. These NGS-based methods have been employed to describe microbial populations in different niches of the body, including the blood, gut, respiratory tract, skin, and urogenital tract. This has significantly improved our understanding of the human microbiome and the host‒host relationship in both physiological and pathological contexts [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e–\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, some studies have concluded that the presence of bacteria in human blood is an unusual event, as most healthy donors (78–84%) do not contain any bacterial species in their blood [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. According to Tan et al. (2022) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], from a total of 9770 healthy human donors, microbial DNA was present in only 16% of healthy individuals, with a median of one microbial species per individual.\u003c/p\u003e \u003cp\u003eFurthermore, there is no consensus on the structure and diversity of a healthy human blood microbiome on the basis of existing studies. Staphylococcus spp. are common genera found in blood in addition to the Proteobacteria Division [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e–\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. While the study of microbiotas in human cohorts is crucial, it is necessary to employ a rigorous analytical process through metagenomics and phylogenetic relationships. In addition to strict biosafety measures to avoid bias due to sample contamination, these measures should be adopted in future research on the human blood microbiome.\u003c/p\u003e \u003cp\u003eIn the present in vitro study, the hematic microbiota of the left ventricle of mice of the NIH strain was characterized by culture, Gram staining and catalase tests.\u003c/p\u003e "},{"header":"Materials \u0026 Methods","content":"\u003cp\u003eStudy design and sampling of experimental units\u003c/p\u003e\u003cp\u003eAn in vitro study was conducted at the Department of Pharmacology of the Faculty of Medicine at the Autonomous University of Chiapas and the Laboratory of Experimental Microbiology at the Technological Institute of Tuxtla Gutierrez (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), Chiapas, Mexico. November 2019 to February 2020. The Faculty Research Ethics Committee approved this study (certificate number: FMH-180035-2019).\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eIn the present study, we used NIH strain mice (n = 30) acquired from the Animal Experimentation Unit (UNEXA-Harlan) of the National Autonomous University of Mexico, which were donated to the Department of Pharmacology of the Autonomous University of Chiapas. All procedures were approved by the institutional bioethics committee and in accordance with the Official Mexican Standard on Technical Specifications for the Production, Care and Use of Laboratory Animals (NOM-062-ZOO-1999). The regulatory guidelines guarantee working with experimental units free of viruses, bacteria and parasites listed in the recommendations of the Federation of European Associations for Laboratory Animal Science. We used 15 male and 15 female mice, 5–6 weeks of age and an average weight of 30 g, randomly distributed into 6 groups of 5 individuals each, which were used to separate 3 experimental groups of homogeneous characteristics, separated into acrylic cages (Soluciones MG, Mexico City), containing wood shavings previously sterilized in an autoclave. The mice were fed standard rodent chow (LabDiet 5001, St. Louis, MO, USA) and purified water. The cages were cleaned every 3 days.\u003c/p\u003e\u003cp\u003eThoracotomy and extraction of blood samples.\u003c/p\u003e\u003cp\u003eBlood samples were obtained from the experimental unit via direct extraction via puncture of the apex of the left ventricle via a midthoracic axillary line approach. The samples were deposited in EDTA tubes (Vacutainer K2, BD, USA).\u003c/p\u003e\u003cp\u003eCulture, isolation and morphological characterization of the hematic microbiota of NIH mice.\u003c/p\u003e\u003cp\u003eFrom a total of 3 ml of blood from each experimental group, 83 µl was inoculated into each Petri dish. The inocula were distributed in 12 Petri dishes for each culture medium (blood agar, Sabouraud agar and brain-heart agar) and 4 for each experimental group (mice A, B and C, respectively). The inocula in the culture media were incubated at 38°C for 72 hours in an incubator (Kenton WS series).\u003c/p\u003e\u003cp\u003eColony-forming units (CFUs) were counted via a darkfield colony counter (SOL-BAT, Q20).\u003c/p\u003e\u003cp\u003eFor taxonomic characterization of the hematic microbiota of NIH mice, we used the Gram staining technique and the catalase test.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the findings found in the culture media used in this study. For the hematic inocula on blood agar, 22, 22 and 29 CFUs were detected in the experimental groups \u0026ldquo;Mice A\u0026rdquo;, \u0026ldquo;Mice B\u0026rdquo; and \u0026ldquo;Mice C\u0026rdquo;, respectively. Hematic inocula on Sabouraud agar yielded 65, 44 and 78 CFUs for the experimental groups \u0026ldquo;Mice A\u0026rdquo;, \u0026ldquo;Mice B\u0026rdquo; and \u0026ldquo;Mice C\u0026rdquo;, respectively. Hematic inocula on brain-heart agar produced 26 CFU, 131 CFU and 10 CFU for the experimental groups \u0026ldquo;Mice A\u0026rdquo;, \u0026ldquo;Mice B\u0026rdquo; and \u0026ldquo;Mice C\u0026rdquo;, respectively.\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\u003eColony-forming unit counts from bloodstream samples of the mouse (\u003cem\u003eMus musculus\u003c/em\u003e) NIH strain\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBlood Agar (CFU)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSabouraud Agar (CFU)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBrain Heart Infusion Agar (CFU)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal CFU per Mouse\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e113\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e197\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e117\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal CFU per Treatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\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 \u003cp\u003eThe sum of CFU per culture medium, considering the samples from mice A, B and C, was as follows: 73 for blood agar, 187 for Sabouraud agar and 167 for brain-heart agar.\u003c/p\u003e \u003cp\u003eThe sum of the CFUs for each group in the three culture media was as follows: 113 for \u0026ldquo;group A\u0026rdquo;, 197 for \u0026ldquo;group B\u0026rdquo; and 117 for \u0026ldquo;group C\u0026rdquo;.\u003c/p\u003e \u003cp\u003eThe photograph in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e from left to right shows the morphology of CFU on Sabouraud agar, the morphology of CFU on blood agar, and the morphology of CFU on brain-heart agar, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes the taxonomic characterization by Gram staining and catalase test for the strains grown on blood agar and brain heart agar media.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMorphological characterization of the blood microbiota of NIH mice via Gram staining and the catalase test.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental Group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBlood Agar\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrain Heart Infusion Agar\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003egram-negative cocci and streptococci, Catalase (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003egram-negative cocci, Catalase (+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003egram-positive cocci and bacilli, Catalase (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003egram-negative cocci, Catalase (+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003egram-negative cocci and streptococci, Catalase (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003egram-negative cocci, Catalase (+)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFrom the blood sample of experimental group \u0026ldquo;A\u0026rdquo; on both blood agar and brain heart agar, gram-negative and catalase-positive Streptococcus grew (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom the blood sample of experimental group \u0026ldquo;B\u0026rdquo;, Streptococcus and Bacillus gram-positive and catalase negative grew on blood agar, and gram-negative Streptococcus and catalase positive Streptococcus grew on brain heart agar (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFrom the blood sample of the experimental group \u0026ldquo;C\u0026rdquo; both on blood agar and brain-heart agar, gram-negative Streptococcus and catalase-positive Streptococcus grew (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAll the culture media used showed growth of microorganisms typical of the native microbiota of the bloodstream in NIH mice. This microbiota is composed of diverse microorganisms, gram-positive and gram-negative bacteria, fungi and yeast. The general morphological characteristics of these bacteria correspond to the divisions of Proteobacteria, Bacteroides and Actinobacteria, in that order of abundance. These findings are consistent with the results of authors Panaiotov, S. 2019, Amar, J. 2019, Schierwagen, R. 2019 [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDysbiosis and both compensatory and pathological physiological changes are closely related [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. According to Wilmanski, T. 2019, at least 40 blood plasma metabolites vary depending on the diversity of the different apparatuses and systems of the human microbiota (digestive system, circulatory system and cardiovascular system) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn an inflammatory process, which is typical of diseases involving the immune system, many cytokines, such as interleukins, interferons and neurotransmitters, are released. These compounds have been shown [\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] to have greater reactivity and proinflammatory effects in mice that are free of microorganisms.\u003c/p\u003e \u003cp\u003eA case‒control study performed at the Toulouse Teaching Hospital in France [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], with 103 patients at high cardiovascular risk without coronary artery disease compared with 99 patients with a history of acute myocardial infarction, analyzed the blood microbiota by 16S qPCR sequencing and revealed that bacterial diversity is decreased in patients with MI, with at least 6 known to include species capable of metabolizing cholesterol.\u003c/p\u003e \u003cp\u003eThe intestinal microbiota is involved in the programming of the T lymphocyte-mediated response and that when the microbial load is suppressed due to dysbiosis, which is present in septic patients, the immune response is altered [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe hematic microbiota of NIH mice is morphologically identical to the intestinal, skin and reproductive system microbiota populations; this identity allows us to suppose that, under septicemic conditions, dysbiosis can be expected in the bloodstream, which is likely correlated with a process of immune reactions mediated by lymphocytes, macrophages and neutrophils.\u003c/p\u003e \u003cp\u003eThe limitations of the present study were mainly due to institutional measures during the COVID-19 pandemic, which did not allow further genomic analysis of the isolated bacterial and fungal populations with NGS or 16S qPCR sequencing, as well as a larger sample size. Future research should consider an approach involving clinical samples from human patients diagnosed with septic shock.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe demonstrated the effectiveness of characterizing the mouse left ventricular microbiota in vitro; similar methods have been developed to understand the ecology of the human microbiome.\u003c/p\u003e \u003cp\u003eThe microbiota of the left ventricle of NIH mice is mainly gram-negative and gram-positive. The fungal population in the hematic microbiota is diverse; however, we were unable to determine the species.\u003c/p\u003e \u003cp\u003eThe present study provides evidence supporting the presence of microbiota in the blood of healthy organisms, as previously described in humans by other authors. 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J Am Heart Assoc 8(19):e011797\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzab\u0026oacute; BG, Kiss R, Makra N, P\u0026eacute;nzes K, Vad E, Kamotsay K (2022) Composition and changes of blood microbiota in adult patients with community-acquired sepsis: A pilot study from bench to bedside. Front Cell Infect Microbiol 12:1067476\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB\u0026auml;umler AJ, Sperandio V (2016) Interactions between the microbiota and pathogenic bacteria in the gut. Nature 535(7610):85\u0026ndash;93\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVrancken G, Gregory AC, Huys GRB, Faust K, Raes J (2019) Synthetic ecology of the human gut microbiota. Nat Rev Microbiol\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaussner F, Chakraborty S, Halbgebauer R, Huber-Lang M (2019) Challenge to the intestinal mucosa during sepsis. Front Immunol\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilmanski T, Rappaport N, Earls JC, Magis AT, Manor O, Lovejoy J (2019) Blood metabolome predicts gut microbiome α-diversity in humans. Nat Biotechnol 37(10):1217\u0026ndash;1228\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTraykova D, Schneider B, Chojkier M, Buck M (2017) Blood Microbiome Quantity and the Hyperdynamic Circulation in Decompensated Cirrhotic Patients. PLoS ONE 12:0169310\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiu J, Zhou H, Yang J, Dong C (2019) Association between blood microbiome and type 2 diabetes mellitus: A nested case-control study. J Clin Lab Anal 33:22842\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"gut microbiomes, bloodstream infections, bloodstream, sepsis, probiotics, blood microbiota","lastPublishedDoi":"10.21203/rs.3.rs-6086827/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6086827/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAim: The aim of this study was to characterize the hematic microbiota of the left ventricle of NIH strain mice via culture, Gram staining and catalase assays.\u003c/p\u003e\n\u003cp\u003eMaterials and Methods: An in vitro study was conducted in the Department of Pharmacology of the Faculty of Medicine at the Autonomous University of Chiapas and the Laboratory of Experimental Microbiology at the Technological Institute of Tuxtla Gutierrez, Mexico, between November 2019 and February 2020. Thirty NIH mice were used, distributed into groups and maintained under sterile conditions. Blood samples were obtained via puncture of the left ventricle and inoculated in different culture media (blood agar, Sabouraud agar and brain-heart agar) for isolation and characterization of the microbiota.\u003c/p\u003e\n\u003cp\u003eResults: The results revealed the growth of the microorganisms on all the culture media. On blood agar, 22, 22 and 29 colony-forming units (CFUs) were recorded for the mice in groups A, B and C, respectively. On Sabouraud agar, 65, 44 and 78 CFUs were observed, and on brain-heart agar, 26, 131 and 10 CFUs were observed. Taxonomic characterization revealed the presence of gram-positive and gram-negative bacteria, as well as fungi, with a predominance of Streptococcus and Bacillus in different samples.\u003c/p\u003e\n\u003cp\u003eConclusion: This study revealed the existence of a native microbiota in the blood of NIH mice, which are predominantly gram-negative, with diverse fungi.\u003c/p\u003e","manuscriptTitle":"Isolation and Characterization of the Microbiota from the Left Ventricle in NIH Mice (Mus musculus): An In Vitro Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-13 04:40:25","doi":"10.21203/rs.3.rs-6086827/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":"b4fa7ed6-5be5-45b9-8d54-c2edd7b1c681","owner":[],"postedDate":"March 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44726989,"name":"General Microbiology"},{"id":44726990,"name":"Cardiothoracic Surgery"},{"id":44726991,"name":"Infectious Diseases"},{"id":44726992,"name":"Bacteriology"},{"id":44726993,"name":"Physiology"}],"tags":[],"updatedAt":"2025-03-13T04:40:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-13 04:40:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6086827","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6086827","identity":"rs-6086827","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}