AbstractBackgroundGenetically modified probiotics have potential for use as a novel approach to express bioactive molecules for the treatment of obesity. The objective of the present study was to investigate the beneficial effect of genetically modified Escherichia coli Nissle 1917 (EcN-GM) in obese C57BL/6J an obesity model in C57BL/6J mice was successfully established. Then, the obese mice were randomly assigned into three groups: obese mice (OB), obese mice + EcN-GM (OB + EcN-GM), and obese mice + orlistat (OB + orlistat) (n = 10 in each group). The three groups were gavaged with ml of 1010 CFU/ml control EcN, EcN-GM (genetically engineered EcN) and 10 ml/kg orlistat. Body weight, food consumption, fat pad and organ weight, hepatic biochemistry and hepatic histopathological alterations were measured. The effects of EcN-GM on the levels of endocrine peptides and the intestinal microbiota were also supplementation for 8 weeks, EcN-GM was associated with decreases in body weight gain, food intake, fat pad and liver weight, and alleviation hepatocyte steatosis in obese mice. EcN-GM also increased the level of GLP-1 in serum and alleviated leptin and insulin resistance. Moreover, supplementation with EcN-GM increased the α-diversity of the intestinal microbiota but did not significantly influence the relative abundance of Firmicutes and results indicated that EcN-GM, a genetically modified E. coli strain, may be a potential therapeutic approach to treat obesity. The beneficial effect of EcN-GM may be independent of the alteration of the diversity and composition of the intestinal microbiota in obese mice. This is a preview of subscription content Access options Subscribe to JournalGet full journal access for 1 year111,22 €only 9,27 € per issueAll prices are NET prices. 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Am J Physiol Regul Integr Comp Physiol. 2016;310:R885– PubMed Central Google Scholar Download referencesAuthor informationAuthors and AffiliationsDepartment of Research and Development, Weichuang Tianyi Biotechnology Co., Ltd, Chengdu, Sichuan, PR ChinaJie MaDepartment of Research and Development, LiTong Bio-Medical Science, Chengdu, Sichuan, PR ChinaJie Ma & Lu XuSavaid Medical School, University of Chinese Academy of Sciences, Beijing, PR ChinaJunrui WangDepartment of Orthopaedics, Chengdu Second People’s Hospital, Chengdu, Sichuan, PR ChinaJunrui WangCollege of Comprehensive Health Management, Xihua University, Chengdu, Sichuan, PR ChinaYuanqi LiuDepartment of Neurosurgery, PLA Strategic Support Force Characteristic Medical Center, Beijing, PR ChinaJianwen GuAuthorsJie MaYou can also search for this author in PubMed Google ScholarJunrui WangYou can also search for this author in PubMed Google ScholarLu XuYou can also search for this author in PubMed Google ScholarYuanqi LiuYou can also search for this author in PubMed Google ScholarJianwen GuYou can also search for this author in PubMed Google ScholarContributionsAll authors contributed to this work. JM, JW, and JG designed the experiment. JM and JW performed the experiment. LX and YL analyzed the data. JM and JW drafted the manuscript. JM, LX, and YL prepared the figures. JM, JW, LX, and JG critically revised the manuscript. All the listed authors reviewed and approved the submitted authorsCorrespondence to Jie Ma or Jianwen declarations Competing interests The authors declare no competing interests. Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional and permissionsAbout this articleCite this articleMa, J., Wang, J., Xu, L. et al. The beneficial effects of genetically engineered Escherichia coli Nissle 1917 in obese C57BL/6J mice. Int J Obes 46, 1002–1008 (2022). citationReceived: 17 June 2021Revised: 07 January 2022Accepted: 12 January 2022Published: 25 January 2022Issue Date: May 2022DOI:

The E. coli Nissle 1917 strain containing envZP41L also became more resistant to bile salts and colicin V and grew 50% slower in vitro in mucus and 15% to 30% slower on several sugars present in mucus, yet it was a 10-fold better colonizer than E. coli Nissle 1917.

Approval Year Name Type Language ESCHERICHIA COLI STRAIN NISSLE 1917 Source: Common Name English MUTAFLOR Source: Common Name English E. COLI NISSLE 1917 Source: Common Name English ESCHERICHIA COLI NISSLE 1917 Source: Common Name English DSM-6601 Source: Code English ESCHERICHIA COLI STRAIN NISSLE 1917 WHOLE Source: Common Name English Code System Code Type Description EVMPD Source: SUB76233 Created by admin on Sun Jun 27 00:47:46 UTC 2021 , Edited by admin on Sun Jun 27 00:47:46 UTC 2021 PRIMARY SUBSTANCE RECORD

Results. Whole genome expression analysis revealed 126 genes specifically regulated after treatment of confluent Caco-2 cells with E. coli Nissle 1917.Among others, expression of genes encoding the proinflammatory molecules monocyte chemoattractant protein-1 ligand 2 (MCP-1), macrophage inflammatory protein-2 alpha (MIP-2α) and macrophage inflammatory protein-2 beta (MIP-2β) was increased up ^{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.} 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. ^{Loading metrics Open Access Peer-reviewed Research Article Sandeep Kumar, Lesley A. Ogilvie, Bhavik A. Patel, Cinzia Dedi, Wendy M. Macfarlane, Brian V. Jones Disruption of Escherichia coli Nissle 1917 K5 Capsule Biosynthesis, through Loss of Distinct kfi genes, Modulates Interaction with Intestinal Epithelial Cells and Impact on Cell Health Jonathan Nzakizwanayo, Sandeep Kumar, Lesley A. Ogilvie, Bhavik A. Patel, Cinzia Dedi, Wendy M. Macfarlane, Brian V. Jones x Published: March 19, 2015 Figures AbstractEscherichia coli Nissle 1917 (EcN) is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants), prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB), or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC), were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health. Citation: Nzakizwanayo J, Kumar S, Ogilvie LA, Patel BA, Dedi C, Macfarlane WM, et al. (2015) Disruption of Escherichia coli Nissle 1917 K5 Capsule Biosynthesis, through Loss of Distinct kfi genes, Modulates Interaction with Intestinal Epithelial Cells and Impact on Cell Health. PLoS ONE 10(3): e0120430. Editor: Markus M. Heimesaat, Charité, Campus Benjamin Franklin, GERMANYReceived: December 9, 2014; Accepted: January 22, 2015; Published: March 19, 2015Copyright: © 2015 Nzakizwanayo 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 creditedData Availability: All relevant data are within the paper and its Supporting Information Support is provided by the Medical Research Council (G0901553) awarded to BVJ; University of Brighton Studentship to JN; Society of Applied Microbiology; BVJ is also supported by the Queen Victoria Hospital Charitable Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the interests: The authors have declared that no competing interests exist. IntroductionDue to the intimate role of the gut microbiome in human health and disease processes, this predominantly bacterial community is increasingly viewed as an important target for the development of novel approaches to diagnose, prevent, or treat a wide range of disorders [1–4]. In this context, probiotics are among the most promising tools for manipulation of the gut microbiome, and have been defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” [5]. The majority of probiotics are Gram-positive bacterial species, and considerable evidence is accumulating regarding the efficacy of these organisms in treating or preventing a variety of gastrointestinal (GI) diseases, and potentially also extra-intestinal disorders [1–4]. Among the probiotics currently available, Escherichia coli Nissle 1917 (EcN; serotype O6:K5:H1) is of particular interest. Not only is this one of the most extensively characterized probiotic organisms (in terms of phenotype, genotype, and clinical efficacy), but is currently the only Gram-negative species in use [6]. EcN was first isolated from the faeces of a World War I soldier who, in contrast to comrades in his trench, was not affected by an outbreak of dysentery [7]. This gastroprotective strain is the active component of Mutaflor (Ardeypharm GmbH, Herdecke, Germany), a microbial drug that is marketed and used in several countries. Clinical trials have shown EcN to be effective for maintaining remission of ulcerative colitis (UC) [8–11], stimulation of the immune system in premature infants [12], treatment of infectious diarrhoea [13], and protection of human intestinal epithelial cells (IECs) against pathogens [14, 15]. These benefits are largely attributed to the immuno-modulatory effects elicited by EcN, which encompass both innate and adaptive elements of the immune system. For example, colonisation with EcN has been indicated to alter the host cytokine profile, and also chemokine production in cultured IECs [16–19]; stimulate the production of mucosal peptide based defences [20]; influence the clonal expansion of T-Cell populations, and modulate antibody responses [12, 21, 22]. Notably, the modulation of T-cell functions mediated by EcN may also extend to γδ T-cells, potentially enabling EcN to coordinate modulation of both innate and adaptive responses [22]. EcN has also been indicated to alter COX-2 expression in intestinal epithelial cells [23], which is an important target in the treatment or prevention of several GI diseases including IBD and colorectal cancer [24–27]. Although most closely related to uropathogenic strains of E. coli (UPEC), EcN is considered non-pathogenic. Genomic characterisation has highlighted the absence of genes encoding the typical UPEC virulence factors, but the retention or accumulation of factors proposed to facilitate general adaptability, colonisation of the GI tract, and the probiotic effects of EcN [28, 29]. These include a range of surface associated structures that are likely to provide the primary interface between host and microbe in the GI tract, such as flagella, fimbriae, a special truncated lipopolysaccharide (LPS) variant, and a K5 type polysaccharide capsule [6, 29–31]. In particular, structures such as flagellin, peptidoglycan and LPS, are recognised by immune regulating Toll-like receptors (TLRS) expressed by IECs, which have been established as key routes of host-microbe communication in the gut, with TLR signalling integral to epithelial homoeostasis and defence [32–34]. Signaling by several TLRs is known to be modulated either directly or indirectly by EcN derived ligands [6, 17–20, 30, 35], which include surface associated structures absent in most or all other probiotic organisms. The K5 capsule produced by EcN in particular is notable in this context, and although not a ligand for known TLRs, the EcN capsule has been implicated in the interaction of this organism with IECs, and impact on chemokine expression and TLR signalling [18,19]. Nevertheless, as with other probiotics, the detailed mechanisms underlying the clinical effectiveness of EcN remain poorly understood overall, with a greater comprehension required to fully realise the potential of this important probiotic species. Here we describe the application of random transposon mutagenesis to identify genes and surface structures involved in the interaction of EcN with human intestinal epithelial cells, and provide new insight into the mechanisms through which EcN interacts with epithelial cells. Results Isolation and genetic characterisation of EcN mutants with disruptions in genes related to cell surface structures Because cell surface structures are a primary point of contact between EcN and IECs, and processes such as biofilm formation and attachment to abiotic surfaces also depends on many of the same structures, we reasoned that selection of mutants with alterations in biofilm formation would enrich for those defective in cell surface associated features also likely to be involved in EcN-IEC interaction. Therefore, we initially subjected a total of 4,116 EcN mini-Tn5 mutants to a preliminary high throughput screen for alterations in biofilm formation (both enhancements and reductions), in order to enrich for mutants attenuated in cell surface features. In this precursor biofilm screen 21 mutants were found to be significantly different in their ability to form biofilms as compared to the EcN wild-type (EcN WT), but unaltered in general growth rate. The majority of these (n = 15) exhibited a biofilm formation enhanced (BFE) phenotype, whereas six exhibited biofilm formation deficient (BFD) phenotype as compared to the WT (Table 1). Identities of genes disrupted in these mutants indicated that the majority were associated with synthesis of cell surface structures, or aspects of cell envelope biogenesis, previously linked to host-IEC interaction or intestinal colonisation (Table 1; [18, 35, 37–40]). A subset of 6 mutants disrupted in genes predicted to encode for cell surface structures, and encompassing both BFD and BFE phenotypes, were subsequently selected for further characterisation of their interaction with cultured IECs. Fig 1. Adherence of EcN mini-Tn5 mutants to Caco-2 cells. A subset of mutants recovered from biofilm screens with disruptions in genes predicted to be involved in generation of surface tstructures, were assessed for their ability to attach to Caco-2 cells in in vitro co-culture models. Caco-2 cell monolayers (~80% confluence) were exposed to bacterial suspensions from mid-log-phase cultures at an MOI of 1:1 for 4 h at 37°C, 5% CO2. Genes disrupted in mutants tested are noted in parentheses and details can be found in Table 1. Data are expressed as the mean of three replicates, and error bars show SE of the mean. Significant differences between attachment of EcN WT and mutants is indicated by ** (P ≤ or **** (P were confirmed biofilm altered mutants and defined as biofilm enhanced (BFE) or biofilm deficient (BFD) mutants. Mutants biofilm formation index was calculated as the percentage of CV (OD595) measured in the EcN WT. Genetic characterisation of biofilm-altered mutants Genes disrupted in mutants of interest were identified using a “cloning free” arbitrary PCR-based approach to amplify DNA segments flanking the transposon insertion, as described by Manoil [55] using primers listed in S2 Table. The resulting amplicons were sequenced by GATC Biotech Ltd. (London, UK) using transposon end primer pLR27Primer 3. The putative function of disrupted genes was assigned by mapping sequence data flanking the mini-Tn5 insert site to the E. coli Nissle Draft genomes sequence [28], and the previously published genomic islands [29]. Sequence reads from mutants were trimmed to remove the 5’ low quality regions (typically ~30–50 nt), and the immediate ~40 nt flanking sections correlated with the EcN genome. Where EcN genome annotations did not provide any clear indication of putative function wider searches of the nr dataset using BlastX and/or the conserved domain database were employed. Construction of kfiB and kfiC deletion mutants Deletion mutants EcNΔkfiB and EcNΔkfiC were constructed by homologous recombination using the Xer-ciseTM chromosomal modification system (Cobra Biologics, Keele, UK) according to manufacturer’s instructions and protocols described by Bloor and Cranenburgh [56]. The system comprises plasmids pTOPO-DifCAT and pLGBE, for construction of target gene specific integration cassette and provision of the Red λ recombination functions, respectively. Briefly, kfiB or kfiC integration cassettes consisting of the difE. coli-cat-difE. coli region from pTOPO-DifCAT plasmid flanked by 50 nt regions homologous to the 3’ and 5' ends of the target gene, were generated by PCR using 70-nt primers, or (listed in S2 Table). EcN WT was first transformed with the Tc-selectable plasmid pLGBE and transformants EcN-pLGBE were used to generate electrocompetent cells, which were subsequently transformed with the PCR product of the difE. coli-cat-difE. coli integration cassette constructs. Integrants were selected on LB agar supplemented with 20 μg ml–1 Chloramphenicol. Loss of pLGBE and generation of chloramphenicol-sensitive clones, indicating resolution of difE. coli-cat-difE. coli marker genes by native recombinases and generation of markerless deletion mutants (mutants EcNΔkfiB and EcNΔkfiC) was achieved by sub-culturing the integrants in LB broth in the absence of antibiotics. Loss of pLGBE was verified by plasmid extraction, and by PCR for marker cassettes kfiB or kfiC specific primers EcNkfiB _F/R or EcNkfiC _F/R, respectively, and confirmed by PCR. Examination of polar effects in EcNΔkfiB and EcNΔkfiC mutants The effect of gene deletion or disruptions in kfiB and kfiC mutants, on the expression of downstream genes (polar effects) was assessed using RT-PCR. Total RNA was extracted from mid-log-phase bacterial cells using the RNeasy Protect Cell Mini Kit (Qiagen) according to manufacturer’s instructions, and treated using the Ambion TURBO DNA-free system (Ambion-Life technologies, Paisley, UK) to remove any potential DNA contamination. The treated RNA was used to generate cDNA using the One Step RT-PCR kit (Qiagen) according to the manufacturer’s instructions, utilising 15 ng RNA per reaction as template. Resulting cDNA was used as template in standard PCRs for detection of gene transcripts with specific primers detailed in S2 Table. Confirmation of K5 capsule absence in EcNΔkfiB and EcNΔkfiC mutants The K5 capsule-specific bacteriophage (ΦK5) [57] was used in this study to determine if the K5 capsule was expressed by EcN WT and deletion mutants. The bacteriophage was diluted and maintained in phage dilution buffer (PDB) (100 mM NaCl, 8 mM MgSO4, gelatine, 50 mM Tris pH Cultures of mutants EcNΔkfiB and EcNΔkfiC, controls EcN WT and MG1655 were grown in LB with shaking at 37°C to an OD600 of then pelleted by centrifugation (10,000 × g for 10 min) and resuspended in ice-cold 10 mM MgSO4. Aliquots of cell suspension (100 μl) were mixed with 100 μl of the appropriate bacteriophage dilution (ranging from 101 to 109 PFU ml–1 from stock suspension of × 109 PFU ml–1) in sterile mL Eppendorf tube then incubated at RT for 30 min, statically. The phage-bacteria mixture was added to a volume of 3 ml of soft agar (1% NaCl, yeast extract, 1% tryptone, agar) held at 42°C in 15 ml sterile glass tube, and the content of the tubes were mixed gently by swirling. The inoculated soft agar was poured on top of LB agar and incubated for 16 h at 37°C to allow formation of plaques. Intestinal epithelial cell culture and co-culture conditions Caco-2 cells (passage 51–79) were grown at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM, g glucose l–1) supplemented with 10% fetal bovine serum and 1× non-essential amino acids (PAA Laboratories, Somerset, UK). Cells were seeded into 6-well or 96-well plates, grown up to ~ 60–80% confluence, and used in co-culture experiments with bacteria. Mid-log-phase bacteria (OD600 of were washed with PBS and suspended in DMEM to the required final count, corresponding to the appropriate multiplicity of infection (MOI) and added to Caco-2 monolayers before plates were incubated at 37°C and 5% CO2. Bacterial adherence to Caco-2 cells Adherence was calculated according to the strategy employed by Hafez et al. [18]. Mid-log phase bacteria cultures were suspended in DMEM then added to monolayers of Caco-2 grown in 6-well plates (80% confluence) at an MOI of 1:1 and incubated at 37°C and 5% CO2 for 4 h. The monolayers were washed 3 times with PBS to remove non-adherent cells then treated with lysis solution, 1% wt / vol saponin (Sigma Aldrich) in trypsin-EDTA (PAA Laboratories, Somerset, UK) for 10 min to allow permeabilisation of Caco-2 cells and recovery of total cell-associated bacteria. Cells were mixed gently by pipetting, serially diluted in sterile PBS, plated onto LB agar, and incubated at 37°C overnight. The obtained viable count represented the total number of cell associated bacteria (adherent and internalised). Internalised bacteria were calculated using the same protocol but Caco-2 cells were treated with gentamicin for 2h (200 μg ml-1) to kill external bacteria prior to lysis and enumeration. The number of adherent bacteria was taken as the difference between total cell associated bacteria and internalised bacteria. The effect of EcN mutants on induction of apoptosis in Caco-2 cells The effect of EcN mutants on induction of apoptosis Caco-2 cells was assessed by measuring the activity of caspase 3/7 using the Caspase-Glo 3/7 kit (Promega, Southampton, UK), according to manufacturer’s instructions. Cells were seeded in 96-well plates with 5,000 cells/well and cultured to achieve ~ 60% confluence then treated with bacteria or bacterial supernatants in co-culture. Media was replaced with serum-free DMEM for 12 h prior to the treatment. Bacterial suspensions were prepared in serum-free DMEM from mid-log-phase cultures then added to Caco-2 cells at an MOI of 10:1 (bacteria:Caco-2) in a final volume of 100 μl/ well. The plates were incubated for 2 h at 37°C and 5% CO2 then media was replaced with fresh serum-free DMEM supplemented with gentamicin at 200 μg ml–1 to stop bacterial growth, and plates were incubated for another 10 h. Bacterial supernatants were obtained from cells grown in 5 mL serum-free DMEM at 37°C overnight, with shaking, and recovered by centrifugation (1,500 × g for 10 min), pH adjusted to and filter-sterilised ( The supernatants were diluted in fresh serum-free DMEM at a ratio of 1:1, and used in place of cell suspensions as described above. Caspase 3/7 activity was measured as relative light units (RLUs) using a Synergy Multi-Mode Plate Reader (BioTek, Potton, UK) operated with BioTek software. Analysis of cytotoxicity The effect of EcN strains on induction of cytotoxicity in Caco-2 cells was assessed by measuring the amount of lactate dehydrogenase (LDH) released into the co-culture media, using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega). Caco-2 cells were treated with bacteria and controls as described for the analysis of apoptosis (above) and both assays were performed in parallel. After treatment of Caco-2 cells, supernatants were collected from plate wells using a multichannel pipette then transferred to fresh 96-well at 50 μl/well. The supernatant was diluted further in serum-free culture media then mixed with the CytoTox 96 substrate at a ration of 1:1. Plates were incubated in the dark at room temperature for 30 min and absorbance at 490 nm (OD490) was recorded. The percentage of cytotoxicity was calculated as LDH released in treated cells (OD490)/maximum LDH release (OD490) × 100. Maximum release was determined as the amount released by total lysis of untreated Caco-2 cells with the CytoTox 96 lysis Solution (10X). Analysis of cellular and nuclear morphology Membrane integrity and nuclear morphology of Caco-2 cells were analysed by fluorescent phalloidin (F-actin) and Dapi (DNA) stainings. Cells were grown on sterile glass cover slips in 6-well plates then treated with EcN strains and controls (MG1655 and mM camptothecin; Sigma) as described above (analysis of apoptosis). After the treatments, the cells on coverslips were washed with PBS then fixed with 4% formaldehyde (Sigma) in PBS for 20 min at RT. The fixed cells were washed three times with PBS and permeabilised with Triton X-100 (Sigma) in PBS for 5 min at RT. The cells were washed three times with PBS, 5 min per wash with gentle rocking, then treated with a μg ml–1 solution of fluorescein isothiocyanate-phalloidin (Sigma- Aldrich) in PBS for 1 h at RT in the dark. The cells were washed twice with PBS and were mounted with the Fluoroshield DAPI medium (Sigma) and examined under a Leica TCS SP5 Confocal Laser Scanning microscope (Leica Microsystems, Wetzlar, Germany). Analysis of COX-2 expression The expression of COX-2 protein in Caco-2 co-cultures was analysed by western blotting using standard methods. Briefly, Caco-2 cells were seeded in 6 wells plates, and at ~ 60% confluence, were treated with EcN K5 mutants and controls as described above (analysis of apoptosis). Lipopolysaccharide (LPS, final concentration, 5 μg ml–1) from Salmonella enterica (Sigma, UK) and human tumour necrosis factor alpha (TNF-α, 10 ng ml–1) (Sigma, UK) were used as pro-inflammatory stimulator positive controls. Treated Caco-2 cell monolayers were washed 3 times with PBS, trypsinised then resuspended in 100 μl of hypotonic buffer (10 mM HEPES, 10 mM KCl, mM EDTA, mM EGTA, 1 mM DTT in SDW, pH containing Sigma protease inhibitor cocktail (1:20), for 15 min at 4°C. Cells were lysed in 25 μl 10% Triton X-100 for 30 min and total protein obtained by centrifugation (10,000 g for 1 min at 4°C). Protein concentration was determined by the Bradford method (Bio-Rad) and equivalent amounts of protein lysates (10 μg) separated by electrophoresis on SDS—PAGE (10%), and then transferred onto a nitrocellulose membrane (GE Healthcare, Giles, UK). The blots were blocked at RT with 10% skimmed milk powder in TBST buffer (10 mM Tris, pH M NaCl, Tween 20), and incubated with primary antibody, anti-COX-2 rabbit polyclonal (Abcam, Cambridge, UK) 1:1,000 in TBST, overnight at 4°C. Blots were washed with TBST then incubated with anti-rabbit HRP-conjugated secondary antibody (Sigma, UK) 1:5,000 in TBST, for 1h at RT. Membranes were washed further then visualised by incubation with the ECL chemiluminescent reagent (Amersham, Little Chalfont, UK) and exposed to Kodak Image Station 440 for signal detection. Blots were then stripped and reprobed with loading control anti-GAPDH mouse monoclonal (Ambion, Cambridge, UK); anti-mouse IgG HRP-conjugated (Sigma, UK) as secondary antibody. The bands of COX-2 densitometry readings were normalized to the GAPDH control. Analysis of cell morphology and aggregation Bacteria were grown statically in 5 mL LB in 50 mL sterile polystyrene tube at 37°C for 16 h. The cultures were mix gently by swirling and 3 μL of each was directly transferred onto glass slide, allowed to rest for 1 min then covered with a cover slip and visualised using ×40 magnification phase contrast microscopy. For each culture 10 randomly selected fields of view across each slide were captured using the Olympus Cell Sense software, and subsequently reviewed. Representative images were selected and adjusted only for brightness and contrast. Statistical analysis All statistical analysis was performed using Prism For Mac OS X (Graphpad Software inc. USA; Data was analysed using either Student’s t-test, or ANOVA with the Bonferroni correction for multiple comparisons. Supporting InformationS1 Fig. Overview of K5 capsule biosynthesis in E. coli, and associated genes disrupted in this show the genetic organisation of the K5 gene cluster in E. coli Nissle 1917 based on data from Cress et al. [28]; Grozdanov et al. [29], and an overview of the current model for K5 capsule biosynthesis and assembly adapted from Griffiths et al. [36]; Whitfield [41]; Petit et al. [42]; Bliss et al. [43]; Hodson et al. [44]; Corbett and Roberts [45]; Whitfield and Roberts [46]; Rigg et al. [47]; Whitfield and Willis [58]. A) Physical map of the EcN K5 capsular polysaccharide gene cluster. Region I (kpsF,E,D,U,C,S) and Region III (kpsM,T) encode elements of synthesis and export machinery, and are conserved among E. coli strains generating Group 2 polysaccharide capsules. Region II encodes K5 specific polysaccharide synthesis machinery (kfiA,B,C,D). Genes disrupted by transposon mutagenesis (kfiB, kpsT) and/or subject to gene knockout (kfiB,C) in this study are identified. HP—denote hypothetical proteins of unknown function B) Representation of main stages and associated K5 biosynthetic machinery (stages 1–3). K5 assembly is localised to the cytoplasmic face of the inner membrane, and is underpinned by the formation of a biosynthetic complex which catalyses synthesis and export polysaccharide precursors for incorporation in the maturing capsule on the cell surface. During K5 assembly it is believed that a unified biosynthetic complex is developed which progressively catalyses main stages [1–3]. However, for clarity here we have separated each main stage of K5 synthesis and associated membrane complexes. Stage 1) Proteins encoded by kpsF,U,C,S are believed to be responsible for the initial generation of the phospatyidyl acceptor and Kdo linker (keto-3-deoxy-manno-2-octulosonic acid), upon which the polysaccharide chain is synthesised. Stage 2) Proteins encoded by kfiA-D are responsible for synthesis of the polysaccharide chain through addition of alternating units of GlcA (glucuronic acid) and GlcNAc (N-acetyl-glucosamine) from UDP-sugar precursors. Stage 3) Proteins generated by kpsD,E,M,T form an ABC transporter complex that translocates completed polysaccharide chains to the cell surface, in an energy dependant process. Acknowledgments We wish to thank Prof Jun Zhu (University of Pennsylvania, School of Medicine) and Prof Ian Roberts (University of Manchester, Faculty of Life Sciences) for gifts of pRL27::mini-Tn5 system and ΦK5 bacteriophage, respectively. 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Ultrastructure of Proteus mirabilis swarmer cell rafts and role of swarming in catheter-associated urinary tract infection. Infect Immun. 2004; 72: 3941–3950. View Article Google Scholar Despite its myriad benefits, the safety of E. coli Nissle 1917 as a probiotic has been questioned. Gronback et al showed that when both the host gut microbiota and adaptive immunity are defective in mice, E. coli Nissle 1917 was able to translocate through the epithelial layer, leading to dissemination, septicemia, and death of the animals .} 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.

Escherichia coli Nissle 1917 (EcN; O6: K5: H1), as a probiotic, is attributed to a non-pathogenic and commensal E. coli. EcN is used to treat several inflammatory diseases like ulcerative colitis (UC) and Crohn’s disease (CD). The wide use of this strain is because probiotic treatment can promote an anti-inflammatory response.

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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escherichia coli nissle 1917