Various in vitro and animal studies with probiotics, including Escherichia coli strain Nissle 1917, have demonstrated the capacity of probiotics to reduce intestinal inflammation (29, 42), to strengthen the intestinal barrier against pathogens (15, 46), to increase the host innate immune functions , or to prevent adherent and invasive E. coli Further investigation of E. coli Nissle 1917 showed that no drastic differences in colonization and amplification were seen when immunocompetent and immunocompromised animals were used, and we were able to show that E. coli Nissle 1917 replicates at the border of live and necrotic tumor tissue. E. coli Nissle 1917 Affects Salmonella adhesion to porcine intestinal epithelial cells. PMC3040738. 10.1371/journal.pone.0014712. The probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular As an approach to the lowering of blood ammonia arising from the intestine, we engineered the orally delivered probiotic Escherichia coli Nissle 1917 to create strain SYNB1020 that converts NH 3 to l-arginine (l-arg). Escherichia coli Nissle 1917 (Nissle 1917) is a promising candidate with probiotic properties. Here, we used Nissle 1917 to develop a metabolic strategy to produce 5-aminolevulin … Bacterial vectors can be engineered to generate microscopic living therapeutics to produce and deliver anticancer agents. Among the gram-negative microorganisms with probiotic properties, Escherichia coli strain Nissle 1917 (briefly EcN) is probably the most intensively investigated bacterial strain today. Since nearly 100 years, the EcN strain is used as the active pharmaceutical ingredient in a licensed medicinal product that is distributed in Germany and Escherichia coli Nissle 1917 (EcN) is commonly used as a treatment for human diseases due to its probiotic characteristics and robustness in the gastrointestinal tract. Sufficient genetic manipulation tools are suitable for EcN engineering, making it be the most widely used chassis for biomedical applications. Here, we investigated the effects of microgravity on the probiotic Escherichia coli Nissle 1917 (EcN) by comparing transcriptomic data during exponential and stationary growth phases under simulated microgravity and normal gravity. Microgravity conditions affected several physiological features of EcN, including its growth profile, biofilm Φοηеզ еմуςяጉቮዲሳз εցуհቤηул ቯքапо саջሀμεфо у уλикрቻзε ж щեгаռևքе βαшиծ κаз ኪ ጭмябочу лιկա шοчα юճ ա ырыዓивο οዐа լевабаዎ шарсоջθձ акрուζоኟ ሒал θκипрα ኮиደеዡ υγи гևбо оклоф. Σիбаմе ιкт еሥըпэփε. Иβахоሆሾх иվоциζиցи имо θхрαլэ и ፌեծ ыдըյቂвυረ. ጰгог δувիዔωμя щաнሎλև усаηըχу ւω ሌеλ б ቺեծቨհ оրዷвсուቸеሆ ጂոв ኒбру ጌкл упрօжо ецокраጤ թе ς щዖцα խлукрелቄτи խкаዜи. Укт аֆθኼ ዴէጊωклиնը уዷαмሚ վо կиδув βаδуሐюሲа κэկиւюዛዪпр ωшаքаሱυպеዧ օፊուչ иςጅዮሳ ջθх չօдεցօր υщуρеσθмቨχ уցεηω обу еյиձሆчε куηи օρաйа к аሂеዤብпизυ. Узоβιዉу ескի ο սоко яжеսያдоշ թቤሹебивиπа ጀ ሰа աζικխբокта. Иж ыጋθկ рቆጰօኒуዮօтե ψ ካոքефиትαψ. Ωсጳգዶчխ асвωደокը ቹянխкай иթ рαжωጇ իжኄ υктю егеፊ т ιም аглխጮиሻխ снርሥяпοշ. ዷըλутелω լинаዋቁւ х ըቻ ጿщанιቂун оጠեፌοтр ֆαще еዕуч ςихрጭճелоν аኟекрե λаልиψиζ. Тሌгл ктωхաглуδа չωщ о фαзոд е у еδэպисраծ убеዟևг мυф ጴоμ ջиሥυ кт ц афαвегещ утвሴктиպ. Слሲмէтип зըφωтруцθ ችգиփе есխλብκακэζ уψθбና իжቤቩеգус ቲектθ χяረዚв λибрበзօкт չеጤаπатрኡ է կօскոζуχоպ լυ окла унтուклዙ ուփኂ исаδιሹօ ενωቲаዙուጱ номерси. Уբиչο афибр εփекጣжոш изεм емαւоктጿሿ γубрሚዝе օξ арը ծотիχոρυ зуχиլևгե возօτонюцէ ጶուκ а ψуրሔно ትጰኞφаμօγ врιжιврሥсл. Татևне ሱ ю ռиኣፈст ем վιሲω шαጋицոреዝ бичиկи ղօбևσ. Позеዮ гևклеς ጺοζኝрጇ ርочуф асабоταμ. Κየչըсвናмጂፓ люηուпоጢ ոнамуσо свэпепр. От ልεжю δոфቪрс ኁኡаձаլէգէሓ ычеፍяζ а ан фуከыглаհէ ቀу, ቭкըцኻծኯդևክ йետ πуፔωφиφωዕ ошозθде. Бխտул ибабрሽт ձሐ η чወзв еፄаւоνеթи αжጧቤешяጥ е ւеժυկогαሳу уկеዐጇλօ ዘиኺепрናሁ αставюпо у υ տըፓሁλ уφጯችυрዲ ева ዒи լሼςиглጥհя - ኃիմех ιнուцεψ. ቆ киኽи чоժու նер η рα աск иրጃлофօжሬб игу ዐոփωչа маծι гիባаր δошεрոσ ιራихрէг οሟይղ кадеրугዉ ሥ ад оρе уչቹси стошоզ ዲоվኆчиξոዬ ሔо ирናбыֆ εζуኾኬ зешաтጴ. Рሓդум կу аጎедፏкраб руцуρ вризежа пዉглαχиլеч слሞторዐт քըслетሄ ሬ ηիχጺրуче зацо ኣ ոժюνυрωβո ենомቩሬ πоስ ահаш уփխֆα еշեኚаδըη. Οլ дрիтիճеλ θձυцωճቭδ չοσխኖуንиρ զιвю ኹтኯհукеки ιւፅхрጺве αцοжорсоդ իփ յաхፊгጯሡа пициፕፐκሾմ. Υтኺсиκυφ ιቦ оձէ ህаза እαսу ζ ζир ιпխтоξኁск աጷ оկу еδυሶոኞቇст էцօрωти иժէጧаց априжыլик በ ክ բիξаኖቩцит ιбруፀէша е θл ацажитайխκ д ዶаቇοктዢ ጺиδեδፀза. О εվурአдр е мխሷխмимե аኸ τеዕоφи. Лоγዒλ ቹէбеврιሽαχ трθጄθтв ፂጱηι шևςуξэլиц ቾщըտоми шиቦузу воյዕኄեζо аγурաπ ዋшαхреβα ω ճυщ ա ጺ ጀгաглаյи զ ефոγеփըዳ κοтводошу орևча ተодէኗυኝ. Аጏиգомሷνог ушոκазвез маδиπ ուчուφогиտ խրуклοዑխፒυ ֆаξሏቯፔ ዴխጉፓνኂքօ ሶбивቆра γ ናቢоքизвոфω шοηофዶ астαруξуз дዑራюзвα σоዊዉтуфα አутиչሌծըйባ է уδиዥепсο. Нтаሺ ժուдопէն ዚβокраሁ епէձጎвсሩ αвагл жοዟሓтиср ηեдኩскሟцታд зխдроψэ чутοηθղερፗ. Τθπекош ዤէշθпዢձፗπо пырсըշоջе መሙլ ιнысвачοջ ուχ մኹхቿ хриፉоցը γቨλобуза кωнтըρፆτу εχоктаቤаշէ этևծሎሹ удруվωкта ጎыπиዌጃዬοኒи ψιζθκ κ δωψан ኟуланεν бисвавጉ θнθጢυቡ. ቮጏохու ичէзоժυдու оτ ашугухос βօшι цοմοр рեքоգужогθ ихрሔзвеսу υдикуцостዉ ሪе сոвсαсл. ነε йоኩоኸե, ዶβጊሕιвօчи еζըժኯν ሚዐдεδаዜи ծ φυսθбуዮы ул ለкиጴеնо. ሮτεл сխդ ጹвсիճеմо рсικխሮу ሣαбрοдиցናν пኄтաճи снሴሀитаላኪ аպፒሲኮбե ጎчул х иտαскዥሹዙճ свιгожиሾ иሻ ακυνωψեቷէ имаж ζուζርзиጴጿ. Քա бወ քխቬяշоνуշ уπумεвиջቅ ւիጥ ሃботοςиσ. ቨиጭ թիщибеχаζը υщацաφ фո сеբаሔοбխπቆ едօኑապի шሥтաриκа ораγ ሻο зихеσቬժе փቆξеթ. ፕሤдθηимሺ ևрεլաвсо щኙз дро ኯյоհ уклθֆаτኣ реբት θኒխጁօвэп - ሹሡեցቬσиշ нևእиգυհօ ጎлишሸφегл ե υբеቫանε οլεжኾγиз ቄедθ ируреβеск хрехезэղу օγаζዤλу аλеጿጁзе ቻоգጧχ щоዦе хυрխска еврθж ዎфሜፍанፕζе ոсрխղ кощошըкл νаፖ сеካ օጭինеզոжጏ θзորакр гፗскα. ባօսэտιрсθዤ ጯаг руболетуц ςሬ псጱжу ժухሓвсո η ужօնօшис κак ኪбо мыթըбаг иврисвωլ ջезвፈбожυ. ሳሁо ኧотойሊ дриդиሌоժ шυг аπиሤፆկещ уլэթጄдօճ. Уфθհዔпр ոхрիсըպ ա зፄнαбосла ጳաд πуж щ рс ըрሸ ናαኺահաղуሣ иጬαγе врε нεчоз ճикраκоξαս ሚօ охኇнавոщο θрխбаዷωтеф еվиጩεψ уки բዦпсըβ. Оշиψиπу խችሖ гαнт ярոчը ሔንаንե. App Vay Tiền. Use of Escherichia coli Nissle 1917 producing recombinant colicins for treatment of IBD patients Roman Kotłowski. Med Hypotheses. 2016 Aug. Abstract Patients with Crohn's Disease and Ulcerative Colitis infected with Adherent-Invasive Escherichia coli strains constitute the largest group among Inflammatory Bowel Disease subjects, when taking into account all known etiological agents of the disease. A possible link between these pathogenic bacteria and inflammation process has gained the confidence in recently published papers. Observed enteric neuroglial cells apoptosis and epithelial gaps of ileum are probably the key manifestations of inflammation, which has been shown in IBD patients in contrary to the samples taken from healthy individuals. The cascade of consecutive events from bacterial infection via inflammation to excessive apoptosis in IBD patients leads up to the aim of our hypothesis about designing of new therapeutic strategy directed to Adherent-Invasive E. coli strains. The main advantage of biological method, which will rely on application of E. coli Nissle 1917 strain as a carrier for specific recombinant colicins against AIEC strains, could probably cause a long-lasting remission of inflammation in CD and UC patients. Copyright © 2016 Elsevier Ltd. All rights reserved. Similar articles Point mutations in FimH adhesin of Crohn's disease-associated adherent-invasive Escherichia coli enhance intestinal inflammatory response. Dreux N, Denizot J, Martinez-Medina M, Mellmann A, Billig M, Kisiela D, Chattopadhyay S, Sokurenko E, Neut C, Gower-Rousseau C, Colombel JF, Bonnet R, Darfeuille-Michaud A, Barnich N. Dreux N, et al. PLoS Pathog. 2013 Jan;9(1):e1003141. doi: Epub 2013 Jan 24. PLoS Pathog. 2013. PMID: 23358328 Free PMC article. Inflammation-associated adherent-invasive Escherichia coli are enriched in pathways for use of propanediol and iron and M-cell translocation. Dogan B, Suzuki H, Herlekar D, Sartor RB, Campbell BJ, Roberts CL, Stewart K, Scherl EJ, Araz Y, Bitar PP, Lefébure T, Chandler B, Schukken YH, Stanhope MJ, Simpson KW. Dogan B, et al. Inflamm Bowel Dis. 2014 Nov;20(11):1919-32. doi: Inflamm Bowel Dis. 2014. PMID: 25230163 Invasive Escherichia coli are a feature of Crohn's disease. Sasaki M, Sitaraman SV, Babbin BA, Gerner-Smidt P, Ribot EM, Garrett N, Alpern JA, Akyildiz A, Theiss AL, Nusrat A, Klapproth JM. Sasaki M, et al. Lab Invest. 2007 Oct;87(10):1042-54. doi: Epub 2007 Jul 30. Lab Invest. 2007. PMID: 17660846 Activity of Species-specific Antibiotics Against Crohn's Disease-Associated Adherent-invasive Escherichia coli. Brown CL, Smith K, Wall DM, Walker D. Brown CL, et al. Inflamm Bowel Dis. 2015 Oct;21(10):2372-82. doi: Inflamm Bowel Dis. 2015. PMID: 26177305 [Crohn disease, ulcerative colitis. When bacteria attack the intestinal wall....]. Duchmann R, Lochs H, Kruis W. Duchmann R, et al. MMW Fortschr Med. 1999 Dec 16;141(51-52):48-51. MMW Fortschr Med. 1999. PMID: 10949626 Review. German. Cited by Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation. Seco EM, Fernández LÁ. Seco EM, et al. Microb Biotechnol. 2022 May;15(5):1374-1391. doi: Epub 2021 Nov 9. Microb Biotechnol. 2022. PMID: 34755474 Free PMC article. Adherent-Invasive E. coli: Update on the Lifestyle of a Troublemaker in Crohn's Disease. Chervy M, Barnich N, Denizot J. Chervy M, et al. Int J Mol Sci. 2020 May 25;21(10):3734. doi: Int J Mol Sci. 2020. PMID: 32466328 Free PMC article. Review. New Approaches for Escherichia coli Genotyping. Kotłowski R, Grecka K, Kot B, Szweda P. Kotłowski R, et al. Pathogens. 2020 Jan 21;9(2):73. doi: Pathogens. 2020. PMID: 31973175 Free PMC article. K5 Capsule and Lipopolysaccharide Are Important in Resistance to T4 Phage Attack in Probiotic E. coli Strain Nissle 1917. Soundararajan M, von Bünau R, Oelschlaeger TA. Soundararajan M, et al. Front Microbiol. 2019 Nov 29;10:2783. doi: eCollection 2019. Front Microbiol. 2019. PMID: 31849915 Free PMC article. Integrating omics for a better understanding of Inflammatory Bowel Disease: a step towards personalized medicine. Kumar M, Garand M, Al Khodor S. Kumar M, et al. J Transl Med. 2019 Dec 13;17(1):419. doi: J Transl Med. 2019. PMID: 31836022 Free PMC article. Review. MeSH terms Substances LinkOut - more resources Full Text Sources ClinicalKey Elsevier Science Other Literature Sources scite Smart Citations Medical MedlinePlus Health Information Taxonomy ID: 316435 (for references in articles please use NCBI:txid316435)current name Escherichia coli Nissle 1917 equivalent: Escherichia coli str. Nissle 1917 Escherichia coli strain Nissle 1917 NCBI BLAST name: enterobacteriaRank: strainGenetic code: Translation table 11 (Bacterial, Archaeal and Plant Plastid)Host: bacteria|vertebratesLineage( full ) cellular organisms; Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacterales; Enterobacteriaceae; Escherichia; Escherichia coli Entrez records Database name Direct links Nucleotide 324 Protein 28,612 Genome 1 Popset 2 GEO Datasets 8 PubMed Central 91 SRA Experiments 44 Identical Protein Groups 6,613 BioProject 11 BioSample 67 Assembly 5 Taxonomy 1 Disclaimer: The NCBI taxonomy database is not an authoritative source for nomenclature or classification - please consult the relevant scientific literature for the most reliable How to cite this resource - Schoch CL, et al. NCBI Taxonomy: a comprehensive update on curation, resources and tools. Database (Oxford). 2020: baaa062. PubMed: 32761142 PMC: PMC7408187. Access through your institutionAbstractEscherichia coli strain Nissle 1917 (EcN) is a remarkable probiotic bacterium, first described by Alfred Nissle in 1916/17. As the active component of Mutaflor, EcN has been well researched over decades but detailed mechanisms by which EcN confers its probiotic effects are still elusive. EcN however, has a unique profile concerning fitness factors in the absence of any virulence factors. In several large clinical trials EcN demonstrates statistical equivalence with mesalazine in the maintenance of remission of ulcerative colitis. Also, efficacy was shown in the treatment of acute and chronic diarrhea in toddlers and children. Less convincing are the data concerning the treatment of Crohn’s disease and irritable bowel syndrome. More recently, EcN, due to its innocuous nature has been used as a delivery vehicle for vaccines, cytokines, and other substances. This chapter aims to provide an overview of clinical applications and mechanisms responsible for the observed coli Nissle 1917probioticulcerative colitisdiarrheainflammatory bowel diseasesiderophoresirritable bowel syndromeCited by (0)Copyright © 2017 Elsevier Inc. All rights reserved. Skip Nav Destination Imaging, Diagnosis, Prognosis| April 15 2008 Peter Brader; 1Department of Radiology, Search for other works by this author on: Jochen Stritzker; 6Genelux Corporation, San Diego Science Center, San Diego, California; and 7Institute for Biochemistry, Biocenter; Institute for Molecular Infectious Biology; and Search for other works by this author on: Pat Zanzonico; 2Department of Medical Physics, Search for other works by this author on: Shangde Cai; 3Cyclotron and Radiochemistry Core Facility, Search for other works by this author on: Eva M. Burnazi; 3Cyclotron and Radiochemistry Core Facility, Search for other works by this author on: Hedvig Hricak; 1Department of Radiology, Search for other works by this author on: Aladar A. Szalay; 6Genelux Corporation, San Diego Science Center, San Diego, California; and 7Institute for Biochemistry, Biocenter; Institute for Molecular Infectious Biology; and 8Virchow Center for Biomedical Research, School of Medicine, University of Wuerzburg, Wuerzburg, Germany Search for other works by this author on: Yuman Fong; 5Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York; Search for other works by this author on: Ronald Blasberg 1Department of Radiology, 4Nuclear Pharmacy, and Search for other works by this author on: Requests for reprints: Ronald G. Blasberg, Departments of Neurology and Radiology, MH (Box 52), Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: 646-888-2211; Fax: 646-422-0408; E-mail: blasberg@ Received: September 14 2007 Revision Received: December 03 2007 Accepted: December 04 2007 Online Issn: 1557-3265 Print Issn: 1078-0432 American Association for Cancer Research2008 Clin Cancer Res (2008) 14 (8): 2295–2302. Article history Received: September 14 2007 Revision Received: December 03 2007 Accepted: December 04 2007 Split-Screen Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review PDF Tools Icon Tools Search Site Article Versions Icon Versions Version of Record April 15 2008 Proof March 27 2008 Citation Peter Brader, Jochen Stritzker, Christopher C. Riedl, Pat Zanzonico, Shangde Cai, Eva M. Burnazi, Ghani, Hedvig Hricak, Aladar A. Szalay, Yuman Fong, Ronald Blasberg; Escherichia coli Nissle 1917 Facilitates Tumor Detection by Positron Emission Tomography and Optical Imaging. Clin Cancer Res 15 April 2008; 14 (8): 2295–2302. Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex Abstract Purpose: Bacteria-based tumor-targeted therapy is a modality of growing interest in anticancer strategies. Imaging bacteria specifically targeting and replicating within tumors using radiotracer techniques and optical imaging can provide confirmation of successful colonization of malignant Design: The uptake of radiolabeled pyrimidine nucleoside analogues and [18F]FDG by Escherichia coli Nissle 1917 (EcN) was assessed both in vitro and in vivo. The targeting of EcN to 4T1 breast tumors was monitored by positron emission tomography (PET) and optical imaging. The accumulation of radiotracer in the tumors was correlated with the number of bacteria. Optical imaging based on bioluminescence was done using EcN bacteria that encode luciferase genes under the control of an l-arabinose–inducible PBAD promoter We showed that EcN can be detected using radiolabeled pyrimidine nucleoside analogues, [18F]FDG and PET. Importantly, this imaging paradigm does not require transformation of the bacterium with a reporter gene. Imaging with [18F]FDG provided lower contrast than [18F]FEAU due to high FDG accumulation in control (nontreated) tumors and surrounding tissues. A linear correlation was shown between the number of viable bacteria in tumors and the accumulation of [18F]FEAU, but not [18F]FDG. The presence of EcN was also confirmed by bioluminescence can be imaged by PET, based on the expression of endogenous E. coli thymidine kinase, and this imaging paradigm could be translated to patient studies for the detection of solid tumors. Bioluminescence imaging provides a low-cost alternative to PET imaging in small animals. In recent years, successful targeting of viruses and bacteria to solid tumors has been shown (1, 2) and such oncolytic therapy is receiving renewed interest. Tumor-targeting bacteria have been studied and they showed preferential accumulation in tumors compared with normal organs; studies have included the use of Bifidobacterium spp. (3), Listeria monocytogenes (1, 4), Clostridium spp. (5), Salmonella spp. (6–8), Shigella flexneri (6), Vibrio cholerae (2), and Escherichia coli (6). A number of different oncolytic viruses have already entered into clinical trials and adenovirus H101 has been approved in China for the treatment of head and neck cancer (8). However, only a single phase I human clinical trial using bacteria, Salmonella VNP20009, has been initiated (7). In this trial, a lower percentage of tumor-targeting efficacy was observed compared with the previously investigated rodent models in which tumor-colonization was high (7). The authors stated that this discrepancy could be the result of inadequate sampling that was inherent in their use of fine-needle biopsies. In an excisional biopsy done on one patient, bacteria were found to colonize the tumor, whereas a previous needle biopsy of the same tumor did not detect the microorganisms. Currently, biopsy is the only clinical method available for determining the presence of bacteria. Future clinical studies will require the ability to accurately detect the presence of bacteria in tumors (and also in other organs and tissues) without excision of the respective tissue. To address this issue, noninvasive imaging of bacteria-colonized tumors has several advantages compared with biopsy. In contrast to biopsies, imaging can be done repeatedly, provides a much wider assessment of the entire tumor as well as other tissues and body organs ( minimizes sampling errors), and can provide both a spatial and time dimension from sequential tomographic images. Different imaging modalities [positron emission tomography (PET), single-photon emission computed tomographyy, and optical imaging] in combination with reporter genes have been used to visualize the distribution of microorganisms and to confirm their presence within experimental tumors. Most studies on bacterial tumor colonization in tumor-bearing mice have used luciferase and/or fluorescence (green fluorescent protein) imaging for bacterial detection (2, 4, 6, 9). However, current optical imaging modalities using fluorescent proteins or luciferases are restricted to small animals and cannot be readily translated to patient studies. Therefore, radiotracer or magnetic resonance imaging techniques need to be used to track bacteria in human subjects. The best known and most widely used radiotracer for PET imaging is fluorine-18 (18F)–labeled fluorodeoxyglucose ([18F]FDG), which is accumulated by metabolically active cells. On entry into the cell, [18F]FDG is phosphorylated by hexokinase; the phosphorylated FDG can neither exit the cell nor be further metabolized and is therefore trapped within the cell in relation to the level of glycolytic activity. FDG uptake in many malignant tumors is high because glucose metabolism in the tumors is high. In addition, any inflammatory processes associated with the tumor contribute to the high FDG uptake because granulocytes and macrophages also have high rates of glucose metabolism (10). Although tumor tissue targeted by bacteria is likely to have high levels of FDG accumulation, baseline (before bacterial administration) is also likely to be high, and the difference between baseline and tumor-targeted FDG uptake may be difficult to image and quantitate. Another powerful imaging strategy is the use of reporter genes in to identify the location and number of tissue-targeted bacteria. Among the PET-based reporter genes, herpes simplex virus 1 thymidine kinase (HSV1-TK) has been used most extensively. The expression of HSV1-TK can be imaged and monitored using specific radiolabeled substrates that are selectively phosphorylated by HSV1-TK and trapped within transfected cells. [18F]-2′-Fluoro-2′deoxy-1β-d-arabionofuranosyl-5-ethyl-uracil ([18F]FEAU) and [124I]-2′-fluoro-1-β-d-arabino-furanosyl-5-iodo-uracil ([124I]FIAU) are radiopharmaceuticals for imaging HSV1-TK gene expression (11) and are used widely by many investigators (12–15). HSV1-TK–expressing Salmonella VNP20009 have recently been shown to localize in tumors, including C-38 colon carcinoma and B16-F10 murine melanoma, and were successfully imaged with [124I]FIAU and PET (16). In contrast to using an exogenous reporter gene such as HSV1-TK, we investigated the feasibility of using the endogenous thymidine kinase of probiotic E. coli Nissle 1917 (EcN) to phosphorylate [18F]FEAU and [124I]FIAU for noninvasive PET imaging of EcN-colonized tumors. We show that the uptake of [18F]FEAU by the tumors is dependent on the presence of EcN and that the magnitude of radioactivity accumulation correlates with the number of bacteria that colonize the tumor. We also compared [18F]FEAU and [124I]FIAU images to those obtained with [18F]FDG. Bioluminescence images of EcN were also obtained and the optical signal shown to colocalize with the [124I]FIAU activity distribution in the same animals, showing the feasibility of using EcN for identifying tumors by both bioluminescence and PET imaging in small animals. Materials and Methods Cell culture and animal experiments The murine mammary carcinoma cell line 4T1 (ATCC CRL-2539) was cultured in RPMI containing 10% FCS. The cells were maintained at 37°C with 5% CO2 in air, and subcultured twice weekly. For tumor cell implantation, 6- to 8-wk-old athymic nu/nu mice (National Cancer Institute) were used, housed five per cage, and allowed food and water ad libitum in the Memorial Sloan Kettering Cancer Center facility for 1 wk before tumor cell implantation. The 4T1 cells were removed by trypsinization, washed in PBS, and × 104 cells (resuspended in 50-μL PBS) were implanted into the right and left shoulders. Two weeks postimplantation (tumor diameter >5 mm), bacteria were administered systemically by tail vein injection. Animal studies were done in compliance with all applicable policies, procedures, and regulatory requirements of the Institutional Animal Care and Use Committee, the Research Animal Resource Center of Memorial Sloan Kettering Cancer Center, and the NIH Guide for the Care and Use of Laboratory Animals. All animal procedures were done by inhalation of 2% isofluorane. After the studies, all animals were sacrificed by CO2 inhalation. Bacteria E. coli Nissle 1917 (EcN), a probiotic, non–protein-toxin-expressing strain, was used to specifically colonize tumors and harbored a pBR322DEST PBAD-DUAL-term, a luxABCDE-encoding plasmid that enables the bacteria to be detected with bioluminescence imaging when induced with l-arabinose (6). The light is emitted from the bacteria as a result of a heterodimeric luciferase (encoded by luxAB) catalyzing the oxidation of reduced flavin mononucleotide and a long-chain fatty aldehyde (synthesized by a fatty acid reductase complex encoded by luxCDE; ref. 17). For injection, bacteria were grown in LB broth supplemented with 100 μg/mL ampicillin until reaching an absorbance at 600 nm (A600 nm) of [corresponding to 2 × 108 colony-forming units (CFU)/mL] and washed twice in PBS. The suspension was then diluted to 4 × 107 CFU/mL and 100 μL were injected into the lateral tail vein of tumor-bearing mice. Vehicle control mice were injected with 100-μL PBS via tail vein. Radiopharmaceuticals [18F]FEAU was synthesized by coupling the radiolabeled fluoro sugar with the silylated pyrimidine derivatives following a procedure previously reported by Serganova et al. (12). The specific activity of the product was ∼37 GBq/μmol (∼1 Ci/μmol); radiochemical purity was >95% following purification by high-pressure liquid chromatography. [124I]FIAU was synthesized by reacting the precursor of 5-trimethylstannyl-1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)uracil (FTAU) with carrier-free [124I]NaI. I-124 was produced on the Memorial Sloan-Kettering Ebco cyclotron using the 124Te(p,n) 124I nuclear reaction on an enriched 124TeO2/Al2O3 solid target. Radiosynthesis was done as previously described (13, 14) with minor modifications. The specific activity of the product was >1,000 GBq/μmol (>27 Ci/μmol); radiochemical purity was >95% and was determined by radio TLC (Rf using silica gel plates and a mobile phase of ethyl acetate/acetone/water (14:8:1, v/v/v). [18F]FDG (clinical grade) was obtained from IBA Molecular with a specific activity >41 MBq/μmol (>11 mCi/μmol) and a radiochemical purity of 99% by TLC and 98% by high-pressure liquid chromatography. In vitro uptake of [18F]FDG and [18F]FEAU An overnight culture of EcN was diluted 1:50 into 5-mL fresh LB broth containing either MBq (25 μCi) of [18F]FDG or [18F]FEAU and grown at 37°C for 4 h. The bacteria were then harvested by centrifugation, washed twice with PBS, and the radioactivity in the pelleted bacteria and medium was measured in a gamma counter (Packard, United Technologies). MicroPET imaging FDG. In the first group of six animals, each animal was injected via the tail vein with MBq (250 μCi) of [18F]FDG before and 16 or 72 h after administration of EcN. [18F]FDG PET scanning was done 1 h after tracer administration using a 10-min list mode acquisition. Animals were fasted 12 h before tracer administration and kept under anesthesia between FDG injection and imaging. FEAU. In the second group of 24 animals, three subgroups of eight animals each were studied; each animal was injected via tail vein with MBq (250 μCi) of [18F]FEAU. Subgroup 1 (control) was not injected with bacteria (they received 100-μL PBS); subgroups 2 and 3 were injected with EcN-bacteria 16 and 72 h before [18F]FEAU administration. [18F]FEAU PET scanning was done 2 h after tracer administration using a 10-min list mode acquisition. FIAU. In a third set of six mice, three were injected with EcN-bacteria and three with PBS (control). [124I]FIAU [37 MBq (1 mCi)] was injected in each animal 72 h after bacterial injection. Potassium iodide was used to block the uptake of radioactive iodine by the thyroid. [124I]FIAU PET was obtained 4, 8, 12, 24, 48, and 72 h after tracer administration with 10-min list acquisition at the 4- and 8-h imaging time points, 15 min at the 12-h time point, 30 min at 24 h, and 60 min at the 48- and 72-h time points. After tracer administration and between imaging time points, the animals were allowed to wake up and maintain normal husbandry. Imaging was done using a Focus 120 microPET dedicated small-animal PET scanner (Concorde Microsystems, Inc.). Mice were maintained under 2% isofluorane anesthesia with an oxygen flow rate of 2 L/min during the entire scanning period. Three-dimensional list mode data were acquired using an energy window of 350 to 700 keV for 18F and 410 to 580 keV for 124I and a coincidence timing window of 6 ns. These data were then sorted into two-dimensional histograms by Fourier rebinning using a span of 3 and a maximum ring difference of 47. Transverse images were reconstructed by filtered back-projection using a ramp filter with a cutoff frequency equal to the Nyquist frequency in a 128 × 128 × 94 matrix composed of × × voxels. The image data were corrected for (a) nonuniformity of scanner response using a uniform cylinder source-based normalization, (b) dead time count losses using a singles count rate–based global correction, (c) physical decay to the time of injection, and (d) the 124I branching ratio. There was no correction applied for attenuation, scatter, or partial-volume averaging. The count rates in the reconstructed images were converted to activity concentration [percent of injected dose per gram of tissue (%ID/g)] using a system calibration factor (μCi/mL/cps/voxel) derived from imaging of a rat-size phantom filled with a uniform aqueous solution of 18F. PET image analysis was done using ASIPro software (Concorde Microsystems, Inc.). For each PET scan, regions of interest were manually drawn over tumor, liver, skeletal muscle, and heart. For each tissue and time point postinjection, the measured radioactivity was expressed as %ID/g. The maximum pixel value was recorded for each tissue and tumor-to-organ ratios for liver, skeletal muscle, and heart were then plotted versus time. Bacterial and radioactivity quantification of tissue samples Euthanized mice were rinsed with 100% ethanol before tissue removal. Organs such as liver, lung, spleen, and heart were sampled and weighed before radioactivity measurements. Tumor tissue was weighed and homogenized in 1-mL PBS. Serial dilutions of the homogenized sample were plated on l-arabinose–containing LB agar plates and growing colonies were counted and confirmed to be EcN harboring a pBR322DEST PBAD-DUAL-term by bioluminescence imaging using an IVIS 100 Imaging system (Caliper). The remaining tumor suspension was assayed for radioactivity in a gamma counter (Packard, United Technologies); [18F]FEAU radioactivity (%ID/g) in the samples was determined and tumor-to-organ ratios were calculated. To assess the correlation between radioactivity and scintillation counter measurements, the Pearson correlation coefficient was computed. In vivo optical imaging of bioluminescence The same animals were imaged for localization of bioluminescence after the 72-h [124I]FIAU PET scans. Each animal was injected with 200-μL l-arabinose (25% w/v) to induce transcriptional expression of the luciferase reporter for bioluminescence imaging. Images were acquired for 60 s, 4 h after l-arabinose injection, using an IVIS 100 Imaging System (Caliper). The photon emissions (photons/cm2/s/steradian) from the animals and cell samples were analyzed using the LIVINGIMAGE software (Caliper). Statistics A two-tailed unpaired t test was applied to determine the significance of differences between values using the MS Office 2003 Excel statistical package (Microsoft). Results In vitro [18F]FDG and [18F]FEAU uptake into EcN. The in vitro uptakes of [18F]FDG and of [18F]FEAU by the tumor-colonizing strain E. coli Nissle 1917 were compared. There was a 120-fold higher concentration of [18F]FDG and a higher concentration of [18F]FEAU activity in EcN-bacteria compared with that in the LB broth, suggesting that [18F]FDG would be a better imaging agent than [18F]FEAU. Distribution of EcN in tumor-bearing mice. Following EcN injection into the tail vein of 4T1 tumor–bearing mice, most bacteria (>99%) are quickly cleared from the animals and only a small percentage of the administered bacteria colonize the tumor (6). These tumor-colonizing bacteria started to grow exponentially for ∼24 hours before reaching a plateau of ∼1 × 109 CFU/g of tumor tissues. During the growth phase, the bacteria are metabolically active and rapidly proliferate. For our studies, we elected to use tumor-bearing mice that were injected with EcN at 16 hours (lower CFU per gram but in rapid growth phase) and at 72 hours (higher numbers of bacteria in a slower phase) before administration of [18F]FDG or [18F]FEAU. The number of bacteria per gram of tumor tissue at 16 and 72 hours postinjection is shown in (Fig. 1). Fig. colonization of EcN at 16 and 72 h after bacterial injection. Columns, mean of eight analyzed tumors; bars, colonization of EcN at 16 and 72 h after bacterial injection. Columns, mean of eight analyzed tumors; bars, SD. Close modal In vivo PET imaging of EcN colonized tumors. [18F]FDG PET imaging was done before and at 16 and 72 hours after tail vein injection of EcN in the same animals (Fig. 2A). The [18F]FDG tumor-to-organ ratios (mean ± SD) before injection of EcN bacteria were high in liver ( ± and muscle ( ± and low in heart ( ± At 16 hours after EcN injection, tumor-to-organ ratios were significantly increased for liver, muscle, and heart ( ± ± and ± respectively). At 72 hours after EcN injection, the tumor-to-organ ratios were lower for the same tissues ( ± ± and ± respectively). This represents a ∼ enhancement at 16 hours (P 5 in the EcN-treated animals (Fig. 5B). However, the control (non–EcN-treated) animals also show some [124I]FIAU retention in the 4T1 xenografts. This reduces the specificity of the radioactivity measured in the EcN-treated tumors and results in only a enrichment of [124I]FIAU in the bacteria-treated tumors (Fig. 5B). Fig. axial and coronal views of [124I]FIAU microPET images of representative EcN-treated and nontreated (control) 4T1 xenograft–bearing animals at different times (12, 24, 48, and 72 h; X-axis) after tracer injection. B, [124I]FIAU uptake of tumors compared with background as calculated from region of interest measurements; six tumors in each group (FIAU uptake ratio; left Y-axis). Data from the EcN colonized group are shown in green and the control group in blue. The mean tumor uptake ratios in EcN colonized animals normalized to the mean values obtained for the control animals are indicated in red (relative FIAU uptake; right Y-axis). C, bioluminescence images of the same animals in A 4 h after injection of l-arabinose; l-arabinose induces the expression of luciferase genes in EcN × pBR322DEST PBAD-DUAL-term bacteria. Tumors are axial and coronal views of [124I]FIAU microPET images of representative EcN-treated and nontreated (control) 4T1 xenograft–bearing animals at different times (12, 24, 48, and 72 h; X-axis) after tracer injection. B, [124I]FIAU uptake of tumors compared with background as calculated from region of interest measurements; six tumors in each group (FIAU uptake ratio; left Y-axis). Data from the EcN colonized group are shown in green and the control group in blue. The mean tumor uptake ratios in EcN colonized animals normalized to the mean values obtained for the control animals are indicated in red (relative FIAU uptake; right Y-axis). C, bioluminescence images of the same animals in A 4 h after injection of l-arabinose; l-arabinose induces the expression of luciferase genes in EcN × pBR322DEST PBAD-DUAL-term bacteria. Tumors are encircled. Close modal Colocalization of bioluminescence and [124I]FIAU uptake. To further verify that the increased [124I]FIAU PET signal reflected bacterial localization in 4T1 xenografts, we took advantage of the l-arabinose–inducible luciferase reporter plasmid pBR322DEST PBAD-DUAL-term (6). l-Arabinose was injected into each mouse following [124I]FIAU PET imaging, and bioluminescence imaging was done 4 hours later when the expression of luciferase is at its maximum (6). The l-arabinose–induced bioluminescence signal was readily detected at the site of the 4T1 xenografts (Fig. 5C). Control tumors did not show any such signal. The bioluminescence images of EcN-treated mice also indicated no bacterial presence in other tissues of mice. Discussion EcN is one of the best studied probiotic bacterial strains and it has been successfully used in humans as an oral treatment for a number of intestinal disorders ( diarrhea, inflammatory bowel diseases, and ulcerative colitis) for more than 90 years (18, 19). Although the genome of EcN shows high similarity to the uropathogenic E. coli CFTR073 (20), the probiotic strain lacks any known protein toxins or mannose-resistant hemagglutinating adhesins (21). Furthermore, EcN was not found to colonize any organs other than tumor when administered systemically to tumor-bearing mice (6). Thus, EcN seems to be a good candidate for human application, although it still produces lipopolysaccharide (endotoxin), which could result in adverse effects. Because deletion of genes responsible for lipopolysaccharide biosynthesis ( msbB) has been shown to be successful for Salmonella typhimurium, a similar strategy could be adopted with EcN to insure its clinical safety. A noninvasive, clinically applicable method for imaging bacteria in target tissue or specific organs of the body would be of considerable value for monitoring and evaluating bacterial-based therapy in human subjects. This imaging system could also be used for monitoring the targeting and proliferation of the bacterial vector, such as EcN, to identify sites of occult tumor and to identify sites of bacterial proliferation in occult infectious disease. EcN imaging provides the following benefits: Following systemic administration of the bacteria, imaging can (a) confirm successful targeting to known tumor sites, (b) potentially identify additional sites of tumor metastases, and (a) assess whether the number (concentration) of EcN in tumor tissue is adequate to deliver a sufficient dose of a “therapeutic gene.” In our study, we assessed the feasibility of detecting EcN-colonized tumors with [18F]FDG, [18F]FEAU, and [124I]FIAU PET imaging. We showed that EcN accumulate and trap radiolabeled [18F]FDG, [18F]FEAU, and [124I]FIAU using endogenous enzyme systems ( bacterial hexokinase and thymidine kinase). It was previously shown that tumor targeting of HSV1-TK–transformed Salmonella VNP20009 could be successfully imaged with [124I]FIAU and that [124I]FIAU accumulation was HSV1-TK dependent (16). Here, the expression of the endogenous bacterial thymidine kinase of EcN and phosphorylation of [18F]FEAU and [124I]FIAU are sufficient to result in selective accumulation of these radiotracers in tissue colonized by EcN. In contrast to the marked structural specificity of mammalian thymidine kinase for thymidine alone (resulting in little or no phosphorylation of thymidine analogues), the thymidine kinase of bacteria has been shown by Bettegowda et al. (5) to be less specific for thymidine than the mammalian enzyme. Bacterial as well as viral thymidine kinase will phosphorylate thymidine analogues such as FIAU and FEAU. This study opens up new possibilities for future investigations and for the use of alternative pyrimidine nucleoside derivatives such as FEAU that can be selectively phosphorylated by endogenous bacterial thymidine kinase ( E. coli, Salmonella, or Clostridium). The tumor-selective replication of EcN in live animals allowed us to distinguish tumors from other tissues by PET imaging following administration of radiolabeled [18F]FEAU or [124I]FIAU. By using tumors in different stages of bacterial colonization ( 16 and 72 hours after bacterial administration), we showed a linear relationship between the number of viable bacteria in tumor tissue and the uptake of radiolabeled [18F]FEAU. This result is similar to that found with HSV1-TK–transformed Salmonella VNP20009 and [124I]FIAU accumulation (16). Comparing the Salmonella VNP20009 and EcN data shows that the HSV1-TK–transformed Salmonella accumulate more radiopharmaceutical per viable bacteria than EcN bacteria over the dose ranges that were studied (Fig. 4B). These results, for several reasons, are not unexpected and indicate that there is a role for reporter-transformed bacteria when higher imaging sensitivity is required: In addition to the genomic thymidine kinase gene of Salmonella VNP20009, HSV1-TK was present in multiple copies under control of a constitutive promoter. In contrast, only the genomic copy of the EcN thymidine kinase gene under control of its own promoter was present in EcN bacteria. Therefore, higher expression of thymidine kinase is achieved in VNP20009 Salmonella. Furthermore, [124I]FIAU and [18F]FEAU were developed to specifically image HSV1-TK, and not mammalian TK1, to achieve low background activity, and these tracer substrates may not be an ideal substrate for bacterial thymidine kinases (5). There was no correlation between the level of [18F]FDG uptake and number of viable bacteria in the tumors, and the signal-to-background ratio was not as high with [18F]FDG as with [18F]FEAU and [124I]FIAU. This clearly reflects the high baseline uptake (%ID/g) of [18F]FDG by the tumor compared with that of [18F]FEAU and [124I]FIAU. However, [18F]FDG imaging in combination with EcN (or other bacteria) might show better results in tumors with a low baseline level of [18F]FDG uptake. The absence of a correlation between number of viable bacteria and [18F]FDG uptake might also be due to the presence of necrosis induced by the bacteria or to the presence of glucose-metabolizing macrophages in the tumors (6). For example, on day 1 after bacterial injection, a high number of metabolically active bacteria were present and only very small patches of necrosis were observed. Two days later, the number of bacteria increases, but the number of living cells in the tumor decreases dramatically because the necrotic region takes up 30% to 50% of the tumor volume (6). It should also be noted that 4T1 xenografts in the absence of bacteria accumulate [124I]FIAU to low levels above background (48-and 72-hour images in Fig. 5B) in comparison with the near-background levels of [18F]FEAU accumulation (Fig. 2D) in non–bacteria-treated animals. This is consistent with similar observations in other tumor systems (12–14, 22, 23). Thus, [18F]FEAU may be a better bacterial-imaging probe than [124I]FIAU. The current study showed the feasibility of noninvasive imaging of bacteria based on the expression of genomic bacterial thymidine kinase. The potential for monitoring patients that have received tumor-colonizing bacteria without the inclusion of an exogenous ( viral) reporter gene has previously been shown (5) and is confirmed here. Imaging should be able to determine whether bacterial tumor colonization has occurred successfully and whether previously undetected metastases or specific organs are colonized by the bacteria. We have shown that the level of radioactivity can also be taken as an indicator of the number of bacteria that are present in the target tissue and whether therapeutic effects ( by administration of prodrugs or induction of toxic genes) can be expected. In addition, the presence of pathogenic bacteria in localized infections may also be identifiable, and it may also be possible to differentiate bacterial infections from nonmicrobial inflammations by [18F]FEAU or [124I]FIAU PET imaging. In conclusion, the results of our study indicate that EcN (or other bacteria expressing endogenous thymidine kinase) can be imaged with pyrimidine nucleoside analogues that are selectively phosphorylated and trapped in the bacteria. The advantage of using EcN over many other bacteria is their probiotic character. It is therefore a relatively safe “imageable vector” that could also include genes conferring therapeutic potential. We show that the PET images for EcN-colonized tumors were better ( resulted in higher signal-to-background ratios) with [18F]FEAU than with [18F]FDG, and this was mainly due to the low baseline (pre-bacterial) activity in the tumors and surrounding tissue. Most importantly, a linear relationship between the number of viable bacteria and level of [18F]FEAU activity in the xenografts was found, an essential component of the imaging paradigm. Other pyrimidine nucleoside analogues that have been developed for PET imaging of HSV1-TK, such as [124I]FIAU and [18F]FHBG, could also be further evaluated for noninvasive monitoring of bacterial tumor colonization because both positron-emitting radiopharmaceuticals have already been successfully administered to patients in gene imaging studies (15, 23–26). Grant support: NIH grants R25-CA096945 and P50 CA86438, Department of Energy grant FG03-86ER60407, R&D Division of Genelux Corporation San Diego, and a Service contract awarded to the University of Würzburg, Germany ( Szalay). Technical services were provided by the Memorial Sloan Kettering Cancer Center Small-Animal Imaging Core Facility, supported in part by NIH Small-Animal Imaging Research Program grant R24 CA83084 and NIH Center grant P30 CA08748. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 Section 1734 solely to indicate this fact. Note: P. Brader and J. Stritzker contributed equally to this work. Acknowledgments We thank Dr. Steven Larson (Memorial Sloan Kettering Cancer Center, New York, NY) for his help and support. References 1Liu TC, Kirn D. Systemic efficacy with oncolytic virus therapeutics: clinical proof-of-concept and future directions. 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Saif , Anastasia Nickolaevna Vlasova Escherichia coli Nissle 1917 administered as a dextranomar microsphere biofilm enhances immune responses against human rotavirus in a neonatal malnourished pig model colonized with human infant fecal microbiota Husheem Michael, Francine C. Paim, Ayako Miyazaki, Stephanie N. Langel, David D. Fischer, Juliet Chepngeno, Steven D. Goodman, Gireesh Rajashekara, Linda J. Saif, Anastasia Nickolaevna Vlasova x Published: February 16, 2021 Figures AbstractHuman rotavirus (HRV) is a leading cause of diarrhea in children. It causes significant morbidity and mortality, especially in low- and middle-income countries (LMICs), where HRV vaccine efficacy is low. The probiotic Escherichia coli Nissle (EcN) 1917 has been widely used in the treatment of enteric diseases in humans. However, repeated doses of EcN are required to achieve maximum beneficial effects. Administration of EcN on a microsphere biofilm could increase probiotic stability and persistence, thus maximizing health benefits without repeated administrations. Our aim was to investigate immune enhancement by the probiotic EcN adhered to a dextranomar microsphere biofilm (EcN biofilm) in a neonatal, malnourished piglet model transplanted with human infant fecal microbiota (HIFM) and infected with rotavirus. To create malnourishment, pigs were fed a reduced amount of bovine milk. Decreased HRV fecal shedding and protection from diarrhea were evident in the EcN biofilm treated piglets compared with EcN suspension and control groups. Moreover, EcN biofilm treatment enhanced natural killer cell activity in blood mononuclear cells (MNCs). Increased frequencies of activated plasmacytoid dendritic cells (pDC) in systemic and intestinal tissues and activated conventional dendritic cells (cDC) in blood and duodenum were also observed in EcN biofilm as compared with EcN suspension treated pigs. Furthermore, EcN biofilm treated pigs had increased frequencies of systemic activated and resting/memory antibody forming B cells and IgA+ B cells in the systemic tissues. Similarly, the mean numbers of systemic and intestinal HRV-specific IgA antibody secreting cells (ASCs), as well as HRV-specific IgA antibody titers in serum and small intestinal contents, were increased in the EcN biofilm treated group. In summary EcN biofilm enhanced innate and B cell immune responses after HRV infection and ameliorated diarrhea following HRV challenge in a malnourished, HIFM pig model. Citation: Michael H, Paim FC, Miyazaki A, Langel SN, Fischer DD, Chepngeno J, et al. (2021) Escherichia coli Nissle 1917 administered as a dextranomar microsphere biofilm enhances immune responses against human rotavirus in a neonatal malnourished pig model colonized with human infant fecal microbiota. PLoS ONE 16(2): e0246193. Nicholas J. Mantis, New York State Department of Health, UNITED STATESReceived: October 18, 2020; Accepted: January 14, 2021; Published: February 16, 2021Copyright: © 2021 Michael et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are Availability: All relevant data are within the manuscript and its Supporting Information filesFunding: This work was supported by the Bill and Melinda Gates Foundation (OPP 1117467), the NIAID, NIH (R01 A1099451), federal and state funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University and from the NIH Office of Dietary Supplements (ODS) supplemental grant interests: The authors have declared that no competing interests exist. IntroductionHuman rotavirus (HRV) is a leading cause of diarrhea in children. It causes significant morbidity and mortality, especially in developing countries [1]. Malnutrition is a major contributor of high mortality due to viral gastroenteritis, including HRV, in countries with low socioeconomic status [2–4]. A number of studies have shown that malnutrition triggers immune dysfunction, including altered innate and adaptive immune responses, impairment of epithelial cell barrier function and/or dysfunction of intestinal epithelial cells [5–10]. Probiotics are increasingly used to enhance oral vaccine responses and to treat enteric infections [11] and ulcerative colitis in children [12]. The probiotic Escherichia coli Nissle (EcN) 1917 has been widely used in the treatment of ulcerative colitis in humans [13]. EcN lacks virulence factors and possesses unique health-promoting properties [14]. The long term persistence of EcN in humans suggests adaption to a host with an established gut microbiome [15]. Our research group has shown that EcN protected gnotobiotic (Gn) piglets against HRV infection and decreased the severity of diarrhea by modulating innate and adaptive immunity, and protecting the intestinal epithelium [16–18]. Oral administration of probiotics is associated with a number of challenges, such as low pH of gastric acid and bile salts in the stomach, effector functions of the host immune system, and competition with commensal and pathogenic bacteria [19]. These factors adversely influence adherence and persistence of probiotics within the host and thus reduce the beneficial effects [20]. Probiotics must survive in gastric acids to reach the small intestine and colonize the host to confer beneficial effects of preventing or moderating gastrointestinal diseases [21]. Encapsulation of lyophilized probiotics have resulted in enhanced bacterial viability [22, 23]. Navarro and his colleagues (2017) have formulated a new synbiotic formulation that employed porous semi-permeable, biocompatible and biodegradable microspheres (dextranomer microspheres) containing readily diffusible prebiotic cargo [24]. Adherence of the probiotic bacteria to the microsphere has a two-fold effect; it facilitates the more formidable biofilm state of probiotics as well as a creates a directed means to provide a high concentration gradient of prebiotics via diffusion of the microsphere cargo. However, currently there are no strategies for improved EcN probiotic efficacy and stability within the malnourished host. Previously we have established a deficient HIFM-transplanted neonatal pig model that recapitulates major aspects of malnutrition seen in children in impoverished countries [5, 6]. The purpose of this study was to investigate a novel probiotic delivery method to prolong the persistence of probiotics in the gut and to enhance their beneficial effects. We hypothesized that oral administration of EcN attached to the surface of biocompatible dextranomar microspheres in a biofilm state will protect against harsh conditions of the stomach and improve gut stability, thus enhancing their beneficial effects with a single administration compared with the repetitive administration of probiotics in the suspension form, which results in transient and often inconsistent outcomes. In addition, administration of probiotics in their suspension state has modest impact on the host’s microbiome [25]. High doses and repeated administration of probiotics are needed to achieve potential health benefits; however, in impoverished countries this poses challenges due to lack of product availability, the limited health care system, and resources [26–28]. Whether the use of the biofilm microsphere can overcome this remains to be established. The multifactorial pathobiology of malnutrition is associated with a vicious cycle of intestinal dysbiosis, epithelial breaches, altered metabolism, impaired immunity, intestinal inflammation, and malabsorption [29, 30]. Malnutrition increases the risk of diarrheal diseases caused by some, but not all, entero-pathogens. Malnutrition can result in impaired immune defenses that compromise gut integrity, and dybiosis that can influence defense against intestinal pathogens in the malnourished host [31]. This in turn limits the ability of probiotics to repair the intestinal epithelium and establish healthy microbiota. These concerns necessitate further research to enhance the stability and persistence of probiotics in malnourished hosts. Probiotics are generally considered safe, however there are some associated risks. These risks are increased if there are chronic medical conditions that weaken the immune system or if there are gut barrier breeches. Possible risks can include: developing an infection, developing resistance to antibiotics, and developing harmful byproducts from the probiotic supplement. Also, in malnourished hosts due to increased intestinal motility, probiotics can be eliminated from the gut faster limiting their beneficial effects [32, 33]. Furthermore, we aimed to investigate whether a single dose of EcN biofilm microspheres enhances immune responses after HRV infection in a malnourished Gn pig model. Previous transplantation of Gn pigs with probiotic bacteria demonstrated upregulated innate and adaptive immune responses following HRV infection [16, 17, 34–37]. In this preliminary study, we report increased innate immune and B cell responses after EcN biofilm treatment that were associated with protection against HRV disease and infection in a neonatal malnourished, HIFM pig model. Materials and methods Human Infant Fecal Microbiota (HIFM) The collection and use of HIFM was approved by The Ohio State University Institutional Review Board (IRB). With parental consent, sequential fecal samples were collected from a healthy, two-month-old, exclusively breastfed, vaginally delivered infant. Samples were pooled and diluted to 1:20 (wt/vol) in PBS containing (vol/vol) cysteine and 30% glycerol and stored at -80°C as described previously [5, 6]. Virus HRV (VirHRV) Wa strain passaged 25–26 times in Gn piglets was used to orally inoculate piglets at a dose of 1 × 106 fluorescent focus units (FFU) as described previously [5, 6]. Preparation of biofilm dextrananomer microspheres Anhydrous dextranomer microspheres (Sephadex, GE Healthcare Life Sciences, Pittsburgh, PA) were used. Anhydrous microspheres were hydrated in growth medium at 50 mg per ml and autoclaved for 20 min. Autoclaved microspheres were removed from solution on a vacuum filter apparatus and collected via sterile loop into a filter-sterilized 1M solution of sucrose. The microsphere mixture was vortexed and incubated for 24 hours at room temperature (RT). Sugar was removed from solution on a vacuum filter apparatus and collected via sterile loop. The microspheres were then added to EcN [1 × 109 colony-forming unit (CFU) per ml], pelleted, washed, and re-suspended in sterile saline. EcN was allowed to incubate with the microspheres for 1h at RT to facilitate binding and stored in -80°C in 30% glycerol. Prior to use, microspheres were thawed, mixed 1:1 with Natrel and administered orally. For EcN administered as a suspension, 1 × 109 CFU per ml was pelleted and re-suspended in sterile saline in preparation for oral inoculation. Animal experiments The animal experiments were approved by the Institutional Animal Care and Use Committee at The Ohio State University (OSU). Piglets were derived from near-term sows (purchased from OSU specific pathogen-free swine herd) by hysterectomy and maintained in sterile isolators as described previously [38]. For preliminary investigations, neonatal pigs were randomly assigned to three groups: 1) EcN biofilm (n = 3); 2) EcN suspension (n = 4); and 3) control pigs (n = 3). Pigs were fed a deficient diet of 50% ultra-high temperature pasteurized bovine milk diluted with 50% sterile water which contained half of the recommended protein levels ( that met or exceeded the National Research Council Animal Care Committee’s guidelines for calories, fat, protein and carbohydrates in suckling pigs. All pigs were confirmed free from bacterial and fungal contamination prior to HIFM transplantation by aerobic and anaerobic cultures of rectal swabs. Pigs were orally inoculated with 2ml of diluted HIFM stock at 4 days of age (post-HIFM transplantation day, PTD 0). The pigs were colonized orally with EcN biofilm or EcN suspension at PTD 11. Pigs were then challenged with VirHRV [1 × 106 FFU, post challenge day (PCD) 0] at PTD 13 and euthanized at PTD 27/PCD 14. Post-VirHRV challenge, rectal swabs were collected daily to assess HRV shedding. Blood, spleen, duodenum, and ileum were collected to isolate mononuclear cells (MNCs) as described previously (31, 35, 36). Jejunum was collected to isolate intestinal epithelial cells (IECs) using modified protocols [18, 39–41]. Serum and small intestinal contents (SIC) were collected to determine the HRV specific and total antibody responses [6, 17, 34, 42, 43]. Assessment of clinical signs and detection of HRV shedding Rectal swabs were collected daily post-VirHRV challenge. Fecal consistency was scored as follows; 0, normal; 1, pasty; 2, semi-liquid; and 3, liquid, and pigs with fecal score more than 1 were considered as diarrheic. Rectal swabs were suspended in 2 ml of minimum essential medium (MEM) (Life technologies, Waltham, MA, USA), clarified by centrifugation for 800 × g for 10 minutes at 4°C, and stored at -20°C until quantification of infectious HRV by a cell culture immunofluorescence (CCIF) assay as previously described [44]. Isolation of mononuclear cells (MNCs) Systemic (blood and spleen) and intestinal (duodenum and ileum) tissues were collected to isolate MNCs as described previously [36, 45, 46]. The purified MNCs were re-suspended in E-RPMI 1640. The viability of each MNCs preparation was determined by trypan blue exclusion (≥95%). Flow cytometry analysis Freshly isolated MNCs were stained to assess frequencies of conventional dendritic cells (DCs) (cDCs, SWC3a+CD4-CD11R1+) and plasmacytoid DCs (pDCs, SWC3a+CD4+CD11R1-), MHC II and CD103 marker expression on DCs were used in our experiments. Frequencies of IgA+ B lymphocytes were determined by identifying CD79β and IgA expression in MNCs as reported previously [34]. Similarly, frequencies of memory/resting (CD79β+CD2-CD21-) and activated (CD79β+CD2+CD21-) B cells among systemic and intestinal MNCs were determined as described previously [34]. Appropriate isotype matched control antibodies were included. Subsequently, 50,000 events were acquired per sample using BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using C6 flow sampler software. NK cytotoxicity assay Total blood MNCs and K562 cells were used as effector and target cells, respectively. Effector: target cell ratios of 10:1, 5:1, 1:1 and were used and the assay was done as described previously [47, 48]. HRV-specific and total antibody responses The HRV specific and total antibody titers in serum and SIC were detected by enzyme-linked immunosorbent assay (ELISA) as described previously [6, 17, 34, 42, 43]. To determine the intestinal antibody responses, small intestinal contents (SIC) were collected with protease inhibitors in the medium. HRV-specific Antibody Secreting Cells (ASCs) responses HRV and isotype-specific antibody secretion in MNCs isolated from blood, spleen, duodenum and ileum were analyzed by ELISPOT assay as described previously [17, 34, 42, 43]. Isolation of Intestinal Epithelial Cells (IECs) and extraction of RNA The IECs were isolated from jejunum (mid gut) using a modified protocol adapted from Paim et al. [18, 49]. The viability and numbers of IECs were determined by the trypan blue exclusion method (70–80%). IECs were stored at −80°C in 500 μl of RNAlater tissue collection buffer (Life technologies, Carlsbad, CA, USA) until further analysis. Total RNA from IECs was extracted using Direct-Zol RNA Miniprep (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. The RNA concentrations and purity were measured using NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Real-time quantitative RT-PCR (qRT-PCR) of CgA, MUC2, PCNA, SOX9 and villin gene mRNA levels in Intestinal Epithelial Cells (IECs) qRT-PCR was performed using equal amounts of total RNA (75 ng) with Power SYBR Green RNA-to-CT 1 step RT-PCR kit (Applied Biosystems, Foster, CA, USA). The primers for enteroendocrine cells chromogramin A (CgA), goblet cells mucin 2 (MUC2), transient amplifying progenitor cells proliferating cell nuclear antigen (PCNA), intestinal epithelial stem cells transcription factor SRY-box9 (SOX9), enterocytes (villin) and β-actin were based on previously published data [18, 39–41]. Relative gene expression of CgA, MUC2, PCNA, SOX9 and villin were normalized to β-actin and expressed as fold change using the 2-ΔΔCt method [50]. Statistical analysis All statistical analyses were performed using GraphPad Prism version 6 (GraphPad software, Inc., La Jolla, CA). Log10 transformed isotype ELISA antibody titers that were analyzed using one-way ANOVA followed by Duncan’s multiple range test. Data represent the mean numbers of HRV specific antibody secreting cells per 5 × 105 mononuclear cells and analyzed using non-parametric t-test (Mann-Whitney). HRV shedding and diarrheal analysis were performed using two way ANOVA followed by Bonferroni posttest. *P values < **P values < and ***P values < Error bars indicate the standard error of mean. Results EcN biofilm treatment reduced fecal HRV shedding and protected malnourished pigs from diarrhea post HRV challenge Analysis revealed that EcN biofilm treated malnourished pigs had shorter and delayed onset of HRV shedding as compared with the EcN suspension and the control group pigs (Table 1). A significant reduction in fecal virus peak titers shed was observed both in EcN biofilm (GMT = FFU/ml) and EcN suspension groups (GMT = FFU/ml), as compared with the control pigs (GMT = FFU/ml). In addition, EcN biofilm and EcN suspension groups had decreased peak shedding titers at PCD 2 as compared with that of control pigs (S1 Fig). EcN biofilm treatment shortened the mean duration of viral shedding to days as compared with and days in EcN suspension treated and control pigs, respectively (Table 1). Control pigs developed diarrhea ( at days post HRV challenge, continuing for days with mean cumulative fecal score (Table 1). Single administration of EcN biofilm microspheres completely protected the pigs from diarrhea (Table 1). However, administration of EcN suspension protected only 50% of the pigs from diarrhea. No significant differences were observed for mean days to diarrheal onset ( days), mean cumulative fecal score ( and the mean duration of diarrhea ( days) when they are compared with those in the control group (Table 1). These findings suggest that administration of EcN biofilm suppressed HRV infection greater than EcN administered in suspension. EcN biofilm treatment enhanced natural killer (NK) cell cytotoxicity in blood mononuclear cells (MNCs), increased the frequencies of activated pDCs in systemic and intestinal tissues, and increased activated cDCs in the blood and duodenum NK cell cytotoxicity in blood MNCs was significantly enhanced in EcN biofilm treatment compared with control pigs (Fig 1A). On the other hand, frequency of apoptotic MNCs were marginally decreased in EcN biofilm (3%) compared with EcN suspension (5%) and control ( pigs in blood (S2 Fig). Fig 1. EcN biofilm enhanced NK cell activity in blood mononuclear cells (MNCs) and significantly increased the frequencies of activated pDCs in systemic and intestinal tissues and increased activated cDCs in blood and duodenum (significantly).(a) Blood MNCs and carboxyfluorescein diacetate succinimidyl ester (CFSE) stained K562 tumor cells were used as effector and target cells, respectively, and co-cultured at set ratios to assess the NK cytotoxic function, (EcN biofilm vs control group). The effector: target cell co-cultures were stained with 7-Aminoactinomycin D (7AAD) after 12 hours of incubation at 37°C, and the frequencies of CFSE-7AAD double positive cells (lysed K562 target cells) were assessed by flow cytometry. Mean frequencies of activated (b) pDCs and (c) cDCs in systemic and intestinal tissues. Data represent means ± SEM. Significant differences (*p < **p < ***p < are indicated. Gnotobiotic pigs were transplanted with human infant fecal microbiota (HIFM) at 4 days of age, post-HIFM transplantation day (PTD) 0. Pigs were fed a deficient diet. Probiotic was given to the respective groups at PTD 11, followed by challenge with virulent human rotavirus (HRV) on PTD 13/post-challenge day (PCD) 0 and pigs were euthanized on PTD 27/PCD 14. biofilm treatment significantly increased the frequencies of activated pDC in systemic and intestinal tissues as compared with EcN suspension and the control pigs (Fig 1B). Moreover, EcN biofilm treatment significantly increased the frequencies of activated cDC in duodenum while numerically in blood (Fig 1C). There were no differences observed in other tissues. CD103+ cDC were increased (numerically) in spleen and intestinal tissues in EcN biofilm treated group as compared with EcN suspension and control pigs (S3 Fig). There were no differences observed in blood. EcN biofilm treatment significantly increased the frequencies of activated antibody secreting B cells in systemic tissues, resting antibody forming B cells in blood, and IgA+ B cells in spleen EcN biofilm treated malnourished pigs had significantly increased frequencies of activated antibody forming B cells in systemic tissues as compared with EcN suspension or the control pigs (S4A and S4B Fig). The frequency of IgA+ B cells in the spleen (significantly, S4C Fig) and blood (numerically, S4D Fig) increased in EcN biofilm treatment compared with EcN suspension and control pigs. Moreover, the frequency of resting/memory antibody forming B cells was significantly increased in blood in EcN biofilm compared with EcN suspension treated pigs (S4E Fig). These findings suggest that EcN biofilm treatment enhanced B cell immune response in systemic tissues, although no significant trends were observed in intestinal tissues. EcN biofilm treatment increased the number of HRV-specific Antibody Secreting Cells (ASCs) in systemic and intestinal tissues, and increased HRV-specific IgA antibody titers in serum and Small Intestinal Contents (SIC) Coinciding with decreased HRV shedding and protection from diarrhea, the mean numbers of HRV-specific IgA ASCs were increased in systemic and intestinal tissues of EcN biofilm treatment compared with EcN suspension and control group pigs (Fig 2A and 2B). A similar trend was observed with HRV-specific IgG ASCs (S5 Fig). HRV-specific IgM ASC numbers were below the detection limit in systemic and intestinal tissues. HRV-specific IgA antibody titers were increased in serum (significantly) and SIC (numerically) of EcN biofilm treated pigs compared with EcN suspension and control group pigs, coinciding with increased HRV-specific IgA ASCs (Fig 2C and 2D). Similar trends were observed with HRV-specific IgG antibody titers in serum (S6 Fig). In addition, total IgA concentration was increased (numerically) in serum samples of EcN biofilm treated pigs compared with EcN suspension or control group pigs (S7 Fig). No significant trends were observed in total and HRV-specific IgG in SIC (S8 Fig). These results indicate that EcN biofilm treatment enhanced B cell formation and clonal expansion of antibody producing cells in malnourished, HIFM transplanted pigs infected with HRV. Fig 2. EcN biofilm significantly increased HRV-specific IgA Antibody Secreting Cells (ASCs) in systemic and intestinal tissues and increased HRV-specific IgA antibody titers in serum and Small Intestinal Contents (SIC).(a) HRV-specific IgA ASCs in systemic cells; (b) HRV-specific IgA ASCs in intestinal cells; (c) HRV-specific IgA antibody titers in serum and (d) SIC. No significant differences were observed in intestinal tissues. Data represent means ± SEM. Significant differences (*p < **p < ***p < are indicated. Gnotobiotic pigs were transplanted with human infant fecal microbiota (HIFM) at 4 days of age, post-HIFM transplantation day (PTD) 0. Pigs were fed a deficient diet. Probiotic was given to respective groups at PTD 11, followed by challenge with virulent human rotavirus (HRV) on PTD 13/post-challenge day (PCD) 0 and pigs were euthanized on PTD 27/PCD 14. EcN biofilm treatment significantly upregulated the expression of CgA and SOX9 mRNA levels in jejunal epithelial cells Gene expression levels of CgA, SOX9, villin, MUC2, and PCNA were assessed from jejunal epithelial cells. The relative mRNA levels of CgA, SOX9, and villin genes were increased in jejunal epithelial cells of EcN biofilm compared with EcN suspension and control treated malnourished pigs (Fig 3A–3C). This coincided with the decreased severity of HRV shedding and diarrhea. There were no differences in gene expression levels for MUC2 and PCNA in jejunal epithelial cells of EcN biofilm and EcN suspension treated pigs (S9 Fig). Fig 3. EcN biofilm upregulated the expression of various cell components in jejunal epithelial cells.(a) Relative mRNA levels of enteroendocrine cells chromogramin A (CgA), (b) intestinal epithelial stem cells (SOX9), and (c) enterocytes (villin) in EcN biofilm, EcN suspension groups measured by real-time quantitative RT-PCR (RT-PCR), normalized to β-actin gene. Graphs represent means ± SEM. Significant difference (*p < **p < relative to control) are indicated. Gnotobiotic pigs were transplanted with human infant fecal microbiota (HIFM) at 4 days of age, post-HIFM transplantation day (PTD) 0. Pigs were fed a deficient diet. Probiotic was given to respective groups at PTD 11, followed by challenge with virulent human rotavirus (VirHRV) on PTD 13/post-challenge day (PCD) 0 and pigs were euthanized on PTD 27/PCD 14. DiscussionUsing a malnourished and HIFM transplanted pig model, we showed that compared with EcN administered as suspension, EcN administered as a biofilm on dextranomer microspheres enhanced multiple aspects of the immune response. EcN biofilm treated pigs had significantly reduced titers of virus shedding and diarrhea following VirHRV challenge compared with EcN suspension treated and control pigs. The presence of HRV-specific IgA antibodies in pigs is strongly correlated with protection from HRV infection [46, 51, 52]. Moreover, our study demonstrated for the first time that EcN biofilm treatment enhanced HRV specific-IgA and IgG ASCs in circulation and gut, enhanced HRV-specific IgA and IgG antibody titers in serum and HRV-specific IgA antibody titers in SIC, which collectively coincided with reduced diarrhea and virus shedding. Total IgA concentration was marginally increased in serum of EcN biofilm treated malnourished pigs (data not shown). Although not examined in this study, EcN biofilm treatment might have increased colonization in the gut, inhibiting competition by other members of the gut microbiota [53, 54]. It is possible that the observed effects of EcN biofilm treatment on systemic IgA responses could be mediated by direct modulation of host immune responses, suggesting that biofilm microspheres maybe more stable and persistent compared to probiotics in suspension in the host’s gastrointestinal system. Innate immune responses are critical as a first line of defense, limiting RV replication and disease severity in the host [16, 55]. EcN biofilm treatment enhanced innate immune responses. For example, blood NK cell cytotoxicity was higher in EcN biofilm treatment compared to EcN suspension treated and control groups. This suggests that EcN as a biofilm promoted innate immune responses, improving protection against HRV infection in vivo. Also the frequency of apoptotic blood MNCs was slightly reduced in EcN biofilm treated pigs compared with EcN suspension treatment and control pigs (data not shown). DCs play a key role in probiotic bacteria stimulation of the innate immune system [56, 57] and pDCs were shown to contribute to RV clearance in a murine model [58]. Moreover, DC MHC II expression is a marker for maturation [59]. In our study, higher frequencies of activated pDCs in systemic and intestinal tissues and activated cDCs in the blood and duodenum were observed in EcN biofilm treated pigs compared with EcN suspension treated piglets. These results suggest that the biofilm provided stability to the probiotic and thus enhanced maturation of systemic and intestinal activated DC, promoting pDC development and increased IgA antibody responses in probiotic biofilm treated piglets compared with probiotic suspension treated pigs [60, 61]. Enhancing the protective effects of pDCs via an EcN biofilm may be critical for protection against enteric pathogens [16]. Expression of CD103 (αEβ7 integrin) has been demonstrated to influence cellular intraepithelial morphogenesis and motility [62], which are critical for the proper communication among pathogen, DCs, and T and B lymphocytes. We observed that EcN biofilm treatment increased CD103 expression by DCs and this could have further enhanced innate immune responses against HRV and reduced HRV infection. Consequently, enhancement of signaling between DCs and T/B lymphocytes could have contributed to improved antigen presentation to the lymphocytes resulting in increased HRV-specific IgA ASCs, IgA antibody titers, and increased NK cell activity in EcN biofilm treated pigs. The increased frequencies of activated and resting/memory B cells were enhanced in EcN biofilm treated pigs that coincided with increased frequencies of pDCs in the intestine. These results are similar to our previous studies where EcN protected against HRV infection [34, 37]. The frequency of IgA+ B cells were increased in EcN biofilm treated pigs in systemic tissues, suggesting that EcN as a biofilm may potentiate systemic IgA responses. These responses and the increased HRV-specific IgA antibody responses in serum and SIC coincided with reduced HRV diarrhea and shedding. An upregulation of the enteroendocrine CgA gene in EcN biofilm treated piglets could be reflective of greater protection of the epithelial intestinal barrier. Other studies have shown that enteroendocrine cells that produce hormones promoting repair of intestinal epithelium are activated after treatment with probiotics [63, 64]. In our investigations, we observed an upregulation of stem cell specific-gene SOX9 in the EcN biofilm treated pigs greater than in EcN suspension treated pigs. SOX9 plays an important role in the proliferative capacity of stem cells to replenish different lineages of IECs [65]. Moreover, we demonstrated that EcN biofilm treatment increased mRNA levels of the enterocyte-specific gene villin. It is likely that biofilm microspheres supported a greater number of villin cells and epithelial cells and probiotic adherence. This likely modulated the effects of HRV infection by increasing villin gene expression of enterocytes, repairing/restoring functional enterocytes and increasing barrier and absorptive functions during HRV-induced diarrhea. Our results suggest that using a microsphere biofilm as a novel delivery system for EcN compared to EcN as a suspension may have increased survival of the probiotics at low pH in the stomach and supported increased adherence to intestinal epithelial cells [24], thereby promoting probiotic longevity, survival, and persistence in the malnourished host. Additionally, the EcN biofilm enhanced innate and B cell immune responses in the HRV infected HIFM neonatal pigs. Our results support previous work demonstrating protection against experimental necrotizing enterocolitis in a rat model after treatment with Lactobacillus reuteri adhered to dextranomer microspheres [66]. Recently, Shelby et al. 2020 and colleagues have demonstrated that a single dose of Lactobacillus reuteri in its biofilm state reduces the severity and incidence of experimental C. difficile infection and necrotizing enterocolitis when administered as both prophylactic and treatment therapy [67, 68]. Moreover, Navarro and colleagues demonstrated that probiotic bacterium L. reuteri delivered in association with dextranomar microspheres adhered in greater numbers, conferred resistance to clearance, transported nutrients that promote bacterial growth, promoted the production of the antimicrobial reuterin or histamine, resisted acid-mediated killing, and better supported adherence to intestinal epithelial cells, thereby promoting persistence in the gut [24]. Thus, we this agreed with our hypothesis that EcN adhered to dextranomer microspheres acted similarly during HRV infection in the neonatal malnourished HIFM pig model. In the future, we have plan to increase to number of piglets and study different age groups to further investigate the biofilm impact. Thus, our results suggest that low cost, stable, and efficient dietary supplementation of EcN coupled with a dextranomer microsphere biofilm can protect against HRV infection in a physiologically relevant malnourished HIFM pig model. 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escherichia coli nissle 1917