However, Escherichia coli Nissle 1917 (EcN) is another promising candidate with probiotic properties. EcN offers increased applicability for cancer treatment with the development of new molecular biology and complete genome sequencing techniques. Probiotic Escherichia coli Nissle 1917 (EcN) has been widely studied for the treatment of intestinal inflammatory diseases and infectious diarrhea, but the mechanisms by which they communicate with the host are not well-known. Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and deliver microbial molecules to distant target Background: Mesalamine and the probiotic E. coli Nissle 1917 (EcN) are both effective agents for the treatment of ulcerative colitis. A combined therapy may have more than additive efficacy. A combined therapy may have more than additive efficacy. There have been extensive genome sequencing studies for Escherichia coli strains, particularly for pathogenic isolates, because fast determination of pathogenic potential and/or drug resistance and their propagation routes is crucial. For laboratory E. coli strains, however, genome sequence information is limited except for several well-known strains. We determined the complete genome sequence Lactobacillus rhamnosus GG (LGG), a gram-positive lactic acid bacterium, is one of the most widely used probiotics; while fewer gram-negative probiotics including Escherichia coli Nissle 1917 (EcN) are characterized. A mechanistic understanding of their individual and interactive effects on human rotavirus (HRV) and immunity is lacking. Here, we show that metabolic engineering can be used to increase spermidine production by the probiotic Escherichia coli Nissle 1917 (EcN), used in humans. First, we found that increasing the expression of genes involved in polyamine biosynthesis, namely the S-adenosylmethionine synthase speD and the spermidine synthase speE , resulted in an Escherichia coli Nissle is known to secrete enzymes and antimicrobial compounds, such as microcins, to antagonize its competitors but experiments carried out with our H-diffusion cell suggest that E. coli Nissle’s effect on E. coli MG1655 derivatives recovering from Levo treatment is contact-dependent. Relapse rates were 11.3% under mesalazine and 16.0% under E. coli Nissle 1917 (N.S.). Life table analysis showed a relapse-free time of 103 +/- 4 days for mesalazine and 106 +/- 5 days for E. coli Nissle 1917 (N.S.). Global assessment was similar for both groups. Tolerability to the treatment was excellent and did not differ. The probiotic bacterium Escherichia coli Nissle 1917 (EcN) constitutes a prospective vector for delivering heterologous therapeutic molecules to treat several human disorders. To add versatility to this carrier system, bacteria should be equipped with expression modules that can be regulated deliberately in a temporal and quantitative manner. Escherichia coli strain Nissle 1917 has been widely used as a probiotic for the treatment of inflammatory bowel disorders and shown to have immunomodulatory effects. Nissle 1917 expresses a K5 capsule, the expression of which often is associated with extraintestinal and urinary tract isolates of E. … yl1GDmg. Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine Free W Kruis1, P Frič2, J Pokrotnieks3, M Lukáš4, B Fixa5, M Kaščák6, M A Kamm7, J Weismueller8, C Beglinger9, M Stolte10, C Wolff11, J Schulze111Evangelisches Krankenhaus Kalk, University of Cologne, Germany2Ustředná vojenská nemocnice, II interní oddělení, Praha, Czech Republic3Paula Stradina Clinical University Hospital, Riga, Latvia4IV Interni Klinika, Charles University, Praha, Czech Republic52nd Department of Medicine, Charles University Prague, Medical Faculty, Hradec Kralove, Czech Republic6Interné oddelenie NsP, Trenčín, Slovak Republic7St Mark’s Hospital, London, UK8Private Practice, Koblenz, Germany9Division of Gastroenterology, University Hospital, Basel, Switzerland10Institut für Pathologie, Klinikum Bayreuth, Germany11Ardeypharm, Herdecke, GermanyCorrespondence to: Dr W Kruis Evangelisches Krankenhaus Kalk, Buchforststr 2, 51103 Cologne, Germany; Abstract Background and aim: Evidence exists for the pathogenic role of the enteric flora in inflammatory bowel disease. Probiotics contain living microorganisms which exert health effects on the host. We compared the efficacy in maintaining remission of the probiotic preparation Escherichia coli Nissle 1917 and established therapy with mesalazine in patients with ulcerative colitis. Patients and methods: In total, 327 patients were recruited and assigned to a double blind, double dummy trial to receive either the probiotic drug 200 mg once daily (n = 162) or mesalazine 500 mg three times daily (n = 165). The study lasted for 12 months and patients were assessed by clinical and endoscopic activity indices (Rachmilewitz) as well as by histology. The primary aim of the study was to confirm equivalent efficacy of the two drugs in the prevention of relapses. Results: The per protocol analysis revealed relapses in 40/110 ( patients in the E coli Nissle 1917 group and 38/112 ( in the mesalazine group (significant equivalence p = Subgroup analyses showed no differences between the treatment groups in terms of duration and localisation of disease or pretrial treatment. Safety profile and tolerability were very good for both groups and were not different. Conclusions: The probiotic drug E coli Nissle 1917 shows efficacy and safety in maintaining remission equivalent to the gold standard mesalazine in patients with ulcerative colitis. The effectiveness of probiotic treatment further underlines the pathogenetic significance of the enteric flora. UC, ulcerative colitisIBD, inflammatory bowel diseaseEcN, Escherichia coli Nissle 1917GCP, good clinical practiceCAI, clinical activity indexEI, endoscopic indexITT, intention to treat populationPP, per protocol population5-ASA, 5-aminosalicylic acidulcerative colitismaintenance therapyprobioticsEscherichia coli Nissle Statistics from Request Permissions If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways. UC, ulcerative colitisIBD, inflammatory bowel diseaseEcN, Escherichia coli Nissle 1917GCP, good clinical practiceCAI, clinical activity indexEI, endoscopic indexITT, intention to treat populationPP, per protocol population5-ASA, 5-aminosalicylic acidulcerative colitismaintenance therapyprobioticsEscherichia coli Nissle Ulcerative colitis (UC) is a chronic relapsing disease. The aims of treatment are induction of remission and prevention of relapses. Guidelines1,2 recommend aminosalicylates for maintenance treatment. Aminosalicylates exert various effects on leukotrienes, cytokines, and oxygen Their mode of action in UC remains unclear. It is suggested that the sum of their anti-inflammatory activities constitutes their therapeutic principle. Thus maintenance treatment with aminosalicylates is only effective when inflammation starts, but not in the non-inflamed gut. Growing evidence exists for a role of the intestinal microflora in the pathogenesis of inflammatory bowel disease (IBD). Findings from genetically engineered animal models as well as clinical observations have elucidated the importance of commensal Antibacterial treatment showed some beneficial effects7,8 but the use of antibiotics is limited. Therefore, treatment with probiotics has been proposed. Probiotics are viable non-pathogenic microorganisms that confer health benefits to the host by improving the microbial balance of the indigenous Apart from anecdotal experience, two controlled studies with the probiotic bacterial strain Escherichia coli Nissle 1917 (EcN) in UC already These trials showed no difference between the relapse preventing effects of EcN and standard mesalazine. However, some criticism was raised as to the validity of these The present study was undertaken to confirm that the relapse preventing effects of probiotic therapy with EcN and standard mesalazine are equivalent. MATERIALS AND METHODS The study was conducted according to the Helsinki Declaration (revised version of Hong Kong) and adhered to good clinical practice (GCP) guidelines. The study was approved by the Ethikkommission der Ärztekammer Nordrhein, Germany, as well as by the local ethics committees of the participating centres. All patients received material in their own language and gave written informed consent. Patients were included in the study if aged 18–70 years and diagnosed with UC in remission (clinical activity index (CAI) ⩽4, endoscopic index (EI) ⩽4, and no signs of acute inflammation on histological examination). In addition, inclusion criteria comprised at least two acute attacks of UC prior to the study and a duration of the current remission of no longer than 12 months. Exclusion criteria were: active UC; proctitis with up to 10 cm proximal spread; Crohn’s disease; infectious colitis; severe accompanying illnesses or major colonic surgery; use of antibiotics, sulphonamides, steroids, or other therapies for UC at entry into the trial; administration of EcN within the previous six months before trial entry; as well as known intolerance to salicylates. Study medication The investigational drug was a bacterial preparation for oral use containing non-pathogenic Escherichia coli of strain Nissle 1917 (serotype O6:K5:H1). Capsules were enteric coated to protect the microorganisms from gastric juice and contained viable bacteria (Mutaflor 100 mg; Ardeypharm GmbH, Herdecke, Germany). The control preparation was mesalazine, consisting of eudragit L coated 5-aminosalicylic acid (Salofalk500 mg; Dr Falk Pharma GmbH, Freiburg, Germany). The test group received one capsule of Mutaflor 100 mg once daily and one tablet of placebo three times daily from day 1 to day 4, and two capsules of Mutaflor 100 mg once daily and one tablet of placebo three times daily from day 5 to the end of the study. The control group received one capsule of placebo once daily and one tablet of Salofalk 500 mg three times daily from day 1 to day 4, and two capsules of placebo once daily and one tablet of Salofalk 500 mg three times daily from day 5 to the end of the study. No concomitant medication for UC was allowed throughout the study. Study design This was a randomised, double blind, double dummy trial comparing the relapse preventing effects and safety of a bacterial preparation containing viable EcN and mesalazine for 12 months in patients with UC in remission. The study was conducted in 60 hospitals and private settings in 10 European countries (see list of participating investigators in the appendix). Randomisation was carried out in a double blind manner in blocks of four patients using 1:1 allocation to the two treatment groups. Only complete blocks of random numbers were used for each centre. If patients were eligible for study entry, they were assigned to random numbers ( = patient numbers) in ascending order within each centre according to the chronological order of their randomisation and were given the corresponding study medication. Evaluation Clinic visits were required at the start and end of the study as well as after 1, 2, 3, 6, and 9 months of treatment. The primary objective of the study was to compare the number of patients experiencing a relapse of UC during the 12 month observation period between the two treatment groups. Patients were classified as suffering a relapse when all three of the following criteria were met: CAI >6 or an increase in CAI of at least 3 points with CAI = 4 being exceeded at the same time; EI >4; and histological signs of acute inflammation. CAI was defined according to At trial entry and at the end of the study, patients underwent colonoscopy where biopsies were taken. Endoscopic activity was assessed using a four point index14: granularity, vascular pattern, vulnerability of mucosa, and mucosal damage. All biopsies were examined by a single pathologist using a four point Secondary efficacy variables were the physician’s and patient’s assessment of general well being and calculation of a quality of life Additionally, time to relapse, CAI, EI, and histological findings were documented. Laboratory assessments, including erythrocyte sedimentation rate, C reactive protein, orosomucoids, blood counts, liver enzymes, creatinine, serum iron, and serum albumin were performed at trial entry and at the end of the study. Incidence and severity of adverse events were reported according to GCP for clinical trials of medication in the European Community (91/507/EWG, CPMP/ICH/135/95). Tolerance of the study medication was assessed on a four point scale (very good, good, fair, poor), and patient compliance was ascertained by pill counting. Statistical analysis The aim of the study was to statistically confirm one sided equivalent efficacy of EcN and mesalazine in preventing relapses of UC. Relapse rates were compared using the one sided test of Farrington and Manning17: this tests the null hypothesis that the difference between treatment groups is greater than or equal to the upper equivalence margin Δ of 20% versus the alternative that the true difference is less than 20% (α = upper confidence limit 95%). Assuming a 12 month relapse rate of 30% under mesalazine treatment and 35% under EcN treatment, to reach a statistical power of 80% at least n = 127 patients were required in each treatment group according to the sample size term for comparative binomial trials with the null hypothesis of non-zero risk Two sets of patients were analysed: an intention to treat population (ITT), including all patients who took at least one dose of the study medication, and a per protocol population (PP). According to generally accepted standards for equivalence and non-inferiority trials,18 primary analysis of the main objective (difference in relapse rates) was based on the PP population. Assuming 25% protocol violators, a total number of 160 patients in each treatment group was therefore planned. Baseline comparability and statistical analysis of secondary objectives was assessed using Fisher’s exact test (two sided; α = In addition, Kaplan-Meier curves were plotted. If no CAI or other parameter was documented at the individual study end, the “last observation carried forward” method was applied. Results are given as mean (SD). Statistical tests were executed using SPSS software package version under the Microsoft Windows NT operating system. For exploratory comparisons (tables 2, 3), the Student’s t test was used. RESULTS Patient characteristics In total, 327 patients were enrolled and randomised to either the EcN preparation (n = 162) or mesalazine (n = 165). The two patient groups were matched with regard to demographic, clinical, and pretreatment characteristics (table 1). The time gap between the end of the last relapse before the study and entry into the study was not longer than four weeks in of patients receiving EcN and in receiving mesalazine, and not longer than three months in and of EcN and mesalazine patients, respectively. All 327 randomised patients received at least one dose of the study medication and thus were included in the ITT and safety analysis this table:View inline Table 1 Demographic data and prestudy clinical characteristics Before unblinding the study, a steering committee assessed protocol violations in 105/327 ( patients. Major protocol deviations comprised violation of inclusion criteria (CAI ⩽4, EI ⩽4, and no signs of acute inflammation on histological examination) (32 patients in both groups), premature discontinuation of the study without relapse (see below), and unknown or not unequivocally assessed end point (EcN 29 patients, mesalazine 24 patients). Accordingly, the PP analysis set comprised 222 patients (EcN 110, mesalazine 112). Mean duration of the study observation period was 250 (144) (median 357) days in the EcN group and 287 (125) (median 360) days in the mesalazine group. The number of patients in the study at the scheduled visits is shown in fig 1. Premature discontinuation of the study for reasons other than relapse of disease occurred in 39/327 ( patients (in 19/162 ( patients in the EcN group and in 20/165 ( patients in the mesalazine group) (table 2). Newly emerged exclusion criteria during the study were start of concomitant medication in four patients on EcN. One patient on mesalazine became afraid of 5-aminosalicylic acid (5-ASA) and another patient underwent cardiac this table:View inline Table 2 Reasons for premature discontinuation of the study Relapse (primary objective) PP analysis revealed relapse in 40/110 ( patients in the EcN group and in 38/112 ( patients in the mesalazine group (fig 2), resulting in significant equivalence between the two groups (p = The corresponding one sided upper 95% confidence limit for the difference in treatment was (that is, within the equivalence range of 20%). Figure 3 depicts the probability of remaining in remission by Kaplan-Meier curves. Median time to relapse in the EcN group could not be calculated due to the large number of late censorings. In the mesalazine group it was 386 days. ITT analysis confirmed these results, showing a relapse rate of in the EcN group and in the mesalazine group (significant equivalence p = The upper limit of the 95% confidence interval for the difference in treatment was Subgroup analyses (secondary objectives) All subgroup analyses were performed in the ITT population. CAI increased in all patients by ( points over the study period, showing a slightly larger increase in the EcN group ( ( than in the mesalazine group ( ( No differences were observed in EI or histology between the start and end of the study (fig 4). Table 3 lists relapse rates with regard to duration, localisation, and pretrial treatment. There were no significant differences between the treatment groups for any of these characteristics. Quality of life scores on admission were ( in the EcN group and ( in the mesalazine group. Respective values after 12 months were ( and ( No significant changes occurred during the 12 month observation this table:View inline Table 3 Relapse rates according to clinical characteristics (intention to treat population) Safety and tolerance As rated by the patients, overall tolerance was very good or good in the EcN group in and in the mesalazine group in According to the physician’s assessment, the respective values were and Discontinuation of the study medication due to adverse events (relapse included) occurred in 22 ( patients (11 ( in the EcN group and 11 ( in the mesalazine group). Most frequent reasons were gastrointestinal disorders such as bloody stools, nausea, diarrhoea, mucous secretion (EcN mesalazine and abdominal pain (EcN mesalazine Generally, no unexpected drug reactions occurred during the study. No deaths but 17 serious adverse events were reported in 13/327 (4%) patients (EcN 7, mesalazine 6). Each serious adverse event occurred only once. Adverse events were reported in 68/162 ( patients treated with EcN and in 58/165 ( patients treated with mesalazine. Many adverse events reflect symptoms common for active UC such as bloody stools ( diarrhoea ( and abdominal pain ( The most frequent non-intestinal adverse events were viral infections (EcN mesalazine nausea ( and headache ( Laboratory tests showed no significant alterations. DISCUSSION Most controlled trials are designed to test differences in efficacy. In contrast, our trial was aimed at proving equivalence. Indeed, we demonstrated that the probiotic EcN provides significantly equivalent efficacy in preventing relapses of UC and is not inferior to the established gold standard mesalazine. This result was not only confirmed by statistical analysis of the PP population, which is preferred in equivalence studies,18 but also by ITT analysis. Therapeutic efficacy is usually demonstrated by superiority in a placebo controlled trial. In serious disease however when effective therapy exists that has already been tested by comparison with placebo, additional placebo controlled trials may be considered A meta-analysis19 reviewed 16 studies of maintenance therapy involving 2341 patients with UC. In four of these 16 trials, preparations containing 5-ASA were compared with placebo; in the remaining 12 studies sulphasalazine was compared. 5-ASA was observed to be significantly more effective than placebo in all dosage subgroups (<1 g/day, 1– g/day, ⩾2 g/day). A dose dependent trend was not Indeed, some studies comparing at least two doses were performed showing mainly negative or conflicting results20: Pentasa 3 g/day was not superior to g/day; balsalazide 4 g/day was better than 2 g/day; balsalazide 6 g/day was better than 3 g/day in one study but in another trial was similarly effective; and two studies with olsalazine reached different conclusions. Thus superior efficacy of doses higher than g/day has not been It can be stated that mesalazine g/day presently reflects the standard in the prevention of UC relapses and thus it qualifies as a control in an equivalence trial. Previous studies on EcN were criticised12,13 for several reasons—for example, short observation period10 or heterogeneity of patients and outcome The present trial considered this critique and followed actual standards. The observation period was 12 months, only patients with UC in remission were included, and the clinical outcome was assessed by well established endoscopic and histological activity indices resulting in a low relapse rate for the mesalazine group comparable with previous A total of 327 patients were included to achieve a statistical power sufficient to test for equivalence in a one sided set. Most likely, IBD is caused by an unrestrained inflammatory response to as yet undefined agents. Although precise identification of the antigenic stimuli has not been determined, the intestinal microflora represents a likely To manipulate the resident gut bacteria therefore seems to offer a rational approach to maintaining remission in IBD. One way of doing this, which has gained credence over recent years, is by using Mechanisms which may account for probiotic activity include production of antimicrobial agents, inhibition of adhesion of pathogens, and influence on mucosal barrier It was reported that inhibition of nuclear factor κB could be mediated by probiotic The properties of EcN are well characterised25 and its genome has been extensively It carries non-pathogenic adhesion molecules. A specific lipopolysaccharide renders it immunogenic without showing any immunotoxic Immunomodulating activity was demonstrated for specific immune responses as well as for induction of non-specific natural immunity in preterm EcN develops antagonistic activity against enterobacteria such as Salmonella enteritidis, Shigella dysenteriae, Yersinia enterocolitica, and Vibrio It prevents invasion of Salmonella typhimurium into intestinal cells,31 inhibits adhesion and invasion of adherent invasive E coli,32 and reduces concentrations of mucosa associated colonic microflora constituents in EcN is safe. Molecular genetics as well as functional analyses have revealed that EcN does not produce any virulence factors or carry any genes for pathogenicity It does not bear genes for antibiotic resistance, transferable genes or plasmids, and does not take up foreign pathogenic DNA. No formation of enterotoxins, cytotoxins, or haemolysins has been observed and there is no serum Clinical studies have demonstrated a favourable safety profile for EcN compared with placebo,35,36 mesalazine,10,11 and Our study confirms this excellent safety and tolerance record. There are other controlled studies with different probiotics. Relapse prevention with Lactobacillus GG tested negatively for maintenance therapy in surgically induced remission of Crohn’s disease38 but a small study showed positive results when Saccharomyces boulardii was added to Inflammation of the ileal pouch constructed after proctocolectomy and ileoanal anastomosis in patients with UC is of particular interest because bacterial growth seems to be of pivotal pathophysiological significance. Cases successfully treated with EcN have been A formulation comprising eight different probiotic bacteria demonstrated convincing therapeutic effects in primary prevention41 and chronic In an uncontrolled study, this preparation was able to colonise the gut and maintain remission in patients with In conclusion, the use of probiotics in IBD is in accordance with its pathogenesis. They may prevent induction of inflammatory reactions. EcN shows therapeutic efficacy and safety in maintaining remission in UC. It can be considered as an alternative to mesalazine. APPENDIX The following institutions, local principal investigators, and local coordinators participated in this study: Austria: University Hospital, Graz: W Petritsch. Czech Republic: Nemocnice Milosrdnych sester sv Karla Boromejského, Prague: J Dosedel; University Hospital, Hradec Kralove: B Fixa; Central Military Hospital, Prague: P Frič; University Hospitals, Prague: M Kment, M Lukáš; University Hospital Plzen: J Koželuhová; University Hospital Brno: H Simonová; Masaryk Hospital, Ústí nad Labem: K Mareš, J Stehlík. Estonia: Central Hospital, Tallin: B Margus; University Hospital, Tartu: R Salupere. Germany: Private Practice, Essen: A Boekstegers; University Hospital, Jena: H Bosseckert; University Hospital, Regensburg: V Gross; DRK-Kliniken Westend, Berlin: R Büchsel; Charité-Campus Virchow, Berlin: A Dignass; Private Practice, Rottenburg aN: F Dreher; Private Practice, Frankenberg: R Engelhard; Private Practice, Bad Homburg: G Ermert; Private Practice, Karlsruhe: U Farack; Private Practice, Marburg: J Hein; Kreisklinik München-Pasing, München: J Heinkelein; Mittelrhein-Klinik Bad Salzig, Boppard: R Herz; Private Practice, Bautzen: I König; Ev Krankenhaus Kalk, Köln: W Kruis; Private Practice, Münster: Th Krummenerl; Private Practice, Cottbus: A Kühn; Israelitisches Krankenhaus, Hamburg: P Layer; University Hospital, Dresden: G Lobeck; Charité-Humboldt-University, Berlin: H Lochs; Private Practice, Neuenkirchen: R Moellmann; Private Practice, Cottbus: E Muehlberg; University Hospital Großhadern, München: Th Ochsenkühn; Städtisches Klinikum Friedrichstadt, Dresden: H Porst; Krankenhaus Tabea, Hamburg: A Raedler; University Hospital, Erlangen: M Raithel; Krankenhaus Nordwest, Frankfurt: W Rösch; University Hospital, Bonn: Ch Scheurlen; Private Practice, Gera: U Schindler; Private Practice, Reutlingen: W Schmeißer; Private Practice, Regensburg: E Schütz; Krankenhaus Speyerer, Heidelberg: R Singer; University Hospital Benjamin Franklin, Berlin: R Stange; University Hospital, Frankfurt: J Stein; Klinikum der RWTH, Aachen: Th Schönfelder; University Hospital, Mainz: R Wanitschke; Private Practice, Koblenz: A Lütke, J Weismüller; St Michael Krankenhaus, Völklingen: D Woerdehoff; Private Practice, Erlangen: J Zeus. Latvia: Paula Stradina Clinical University Hospital, Riga: J Pokrotnieks. Lithuania: University Hospital, Vilnius: A Irnius; Kauno Medicinos Akademija, Kaunas: L Kupcinskas. Slovak Republic: Comenius University Hospital, Bratislava: M Huorka; City Hospital, Trencíne: M Kaščák; University Hospital, Košice: T Hildebrand. Sweden: Sabbatsberg Naersjukhuset, Stockholm: P Benno; Karolinska Institutet: A Uribe. Switzerland: Kantonsspital-University, Basel: Ch Beglinger. UK: Leeds General Infirmary, Leeds: ATR Axon; St Mark’s Hospital, London: MA Kamm. REFERENCES↵ ↵ Stange EF, Riemann J, von Herbay A, et al. Diagnosis and therapy of ulcerative colitis—results of an evidence-based consensus conference of the German Society of Digestive and Metabolic Diseases. Z Gastroenterol2001;39:19–20. ↵ Travis SP, Jewell DP. Salicylates for ulcerative colitis—their mode of action. Pharmacol Ther1994;63:135–61. ↵ Shanahan F . Probiotics and inflammatory bowel disease: is there a scientific rationale? Inflamm Bowel Dis2000;6:107–15. D’Haens GR, Geboes K, Peeters M, et al. Early lesions of recurrent Crohn’s disease caused by infusion of intestinal contents in excluded ileum. Gastroenterology1998;114:262–7. ↵ Sartor RB. Postoperative recurrence of Crohn’s disease: the enemy is within the fecal stream. Gastroenterology1998;114:398–400. ↵ Rutgeerts P , Hiele M, Geboes K, et al. Controlled trial of metronidazole treatment for prevention of Crohn’s recurrence after ileal resection. Gastroenterology1995;108:1617–21. ↵ Hulten K , Almashhrawi A, El Zaatari FA, et al. Antibacterial therapy for Crohn’s disease: a review emphasizing therapy directed against mycobacteria. Dig Dis Sci2000;45:445–56. ↵ Hart AL, Stagg AJ, Kamm MA. Related articles, links use of probiotics in the treatment of inflammatory bowel disease. J Clin Gastroenterol2003;36:111–19. ↵ Kruis W , Schütz E, Fric P, et al. Double-blind comparison of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. Aliment Pharmacol Ther1997;11:853–8. ↵ Rembacken BJ, Snelling AM, Hawkey PM, et al. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet1999;354:635–9. ↵ Folwaczny C . Probiotics for prevention of ulcerative colitis recurrence: alternative medicine added to standard treatment? Z Gastroenterol2000;38:547–50. ↵ Faubion WA, Sandborn WJ. Probiotic therapy with E. coli for ulcerative colitis: take the good with the bad, Gastroenterology2000;118:630–1. ↵ Rachmilewitz D . Coated mesalazine (5-aminosalicylic acid) versus sulphasalazine in the treatment of active ulcerative colitis: a randomised trial. BMJ1989;298:82–6. ↵ Riley SA, Mani V, Goodman MJ, et al. Microscopic activity in ulcerative colitis: what does it mean? Gut1991;32:174–8. ↵ Guyatt G , Mitchell A, Irvine EJ, et al. A new measure of health status for clinical trials in inflammatory bowel disease. Gastroenterology1989;96:804–10. ↵ Farrington CP, Manning G. Test statistics and sample size formulae for comparative binomial trials with null hypothesis of non-zero risk difference of non-unity relative risk. Stat Med1990;9:1447–54. ↵ ↵ Sutherland LR, Roth DE, Beck PL. Alternatives to sulfasalazine: A meta-analysis of 5-ASA in the treatment of ulcerative colitis. Inflamm Bowel Dis1997;3:65–78. ↵ Riley SA. What dose of 5-aminosalicylic acid (mesalazine) in ulcerative colitis? Gut1998;42:761–3. ↵ Sartor RB. Enteric microflora in IBD: pathogens or commensals? Inflamm Bowel Dis1997;3:230–5. ↵ Campieri M , Gionchetti P. Bacteria as the cause of ulcerative colitis. Gut2001;48:132–5. ↵ Hamilton-Miller JMT. A review of clinical trials of probiotics in the management of inflammatory bowel disease. Infect Dis Rev2001;3:83–7. ↵ Neish AS, Gewirtz AT, Zeng H, et al. Prokaryotic regulation of epithelial responses by inhibition of IkappaB-alpha ubiquitination. Science2000;289:1560–3. ↵ Blum G , Marre R, Hacker J. Properties of Escherichia coli strains of serotype O6. Infection1995;23:234–6. ↵ Blum-Oehler G . The scanned bacterium: Analysis of the microbial genome. 4th Interdisciplinary Symposium: Internal microflora in symbiosis and pathogenicity, Berlin 2000. Hagen: Alfred-Nissle-Gesellschaft eV 2001:23–31. ↵ Grozdanov L , Zaehringer U, Blum-Oehler G, et al. A single-nucleotide exchange in the wzy gene is responsible for the semi-rough O6 LPS phenotype and serum sensitivity of Escherichia coli strain Nissle 1917. J Bacteriol2002;184:5912–25. ↵ Cukrowska B , Lodinova-Zadnikova R, Enders C, et al. Specific proliferative and antibody responses of premature infants to intestinal colonization with nonpathogenic probiotic E. coli strain Nissle 1917. Scand J Immunol2002;55:204–9. ↵ Schulze J , Sonnenborn U. Oral administration of a certain strain of live Escherichia coli for intestinal disorders? Infection1995;23:184–6. ↵ Schulze J , Lorenz A, Mandel L. Colonisation of Escherichia coli in different gnotobiotic animal models. Microb Ecol Health Dis1992;5:iv–v. ↵ Oelschläger TA, Altenhoefer A, Hacker J. Inhibition of Salmonella typhimurium invasion into intestinal cells by the probiotic E. coli strain Nissle 1917. Gastroenterology2001;120 (suppl) :A326. ↵ Boudeau J , Rich C, France CF, et al. Escherichia coli strain Nissle 1917 inhibits adhesion to and invasion of intestinal epithelial cells by adherent-invasive E. coli isolated from a Crohn’s disease patient. Gastroenterology2001;120 (suppl) :A190. ↵ Swidsinski A , Swidsinski S, Godzun A, et al. Therapy with E. coli Nissle reduces concentrations of mucosa associated colonic flora in patients with ulcerative colitis. Gastroenterology2000;118 (suppl) :A1138. ↵ Schulze J , Sonnenborn U. The role of the gut flora in inflammatory bowel diseases. In: Shimoyama T, Axon A, Lee A, et al, eds. In: Helicobacter meets inflammatory bowel disease. Tokyo: Medical Tribune Inc, 2002:393–417. ↵ Möllenbrink M , Bruckschen E. Treatment of chronic constipation with physiologic Escherichia coli bacteria. Results of a clinical study of the effectiveness and tolerance of microbiological therapy with the E. coli Nissle 1917 strain (Mutaflor). Med Klin1994;89:587–93. ↵ Malchow HA. Crohn’s disease and Escherichia coli. A new approach in therapy to maintain remission of colonic Crohn’s disease? J Clin Gastroenterol1997;25:653–8. ↵ Bruckschen E , Horosiewicz H. Chronic constipation. Comparision of microbiological therapy and lactulose. MMW1994;136:241–5. ↵ Prantera C , Scribano ML, Falasco G, et al. Ineffectiveness of probiotics in preventing recurrence after curative resection for Crohn’s disease: a randomised controlled trial with Lactobacillus GG. Gut2002;51:405–9. ↵ Guslandi M , Mezzi G, Sorghi M, et al. Saccharomyces boulardii in maintenance treatment of Crohn’s disease. Dig Dis Sci2000;45:1462–4. ↵ Kuzela L , Kaščák M, Vavrecka A. Induction and maintenance of remission with nonpathogenic Escherichia coli in patients with pouchitis. Am J Gastroenterol2001;96:3218–19. ↵ Gionchetti P , Rizzello F, Helwig U, et al. Prophylaxis of pouchitis onset with probiotic therapy: a double-blind, placebo-controlled trial. Gastroenterology2003;124:1202–9. ↵ Gionchetti P , Rizzello F, Venturi A, et al. Oral bacteriotherapy as maintenance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial. Gastroenterology2000;119:305–9. ↵ Venturi A , Gionchetti P, Rizzello F, et al. Impact on the composition of the faecal flora by a new probiotic preparation: preliminary data on maintenance treatment of patients with ulcerative colitis. Aliment Pharmacol Ther1999;13:1103–8. Read the full text or download the PDF: Log in using your username and password Loading metrics Open Access Peer-reviewed Research Article Francine C. Paim, Ayako Miyazaki, Stephanie N. Langel, David D. Fischer, Juliet Chepngeno, Steven D. Goodman, Gireesh Rajashekara, Linda J. 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. Similar studies are warranted in children to moderate the symptoms of other gastrointestinal infections and disorders including gastritis and chronic inflammatory bowel disease. Supporting information Acknowledgments We thank Marcia Lee and Rosario Candelero-Rueda for their technical assistance and Dr. Juliette Hanson, Ronna Wood, Jeffery Ogg, Megan Strother and Sara Tallmadge for animal care assistance. References1. Tate JE, Burton AH, Boschi-Pinto C, Steele AD, Duque J, et al. (2012) 2008 estimate of worldwide rotavirus-associated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis. Lancet Infect Dis 12: 136–141. pmid:22030330 View Article PubMed/NCBI Google Scholar 2. Clark A, Black R, Tate J, Roose A, Kotloff K, et al. (2017) Estimating global, regional and national rotavirus deaths in children aged <5 years: Current approaches, new analyses and proposed improvements. PLoS One 12: e0183392. pmid:28892480 View Article PubMed/NCBI Google Scholar 3. Nelson EA, Glass RI (2010) Rotavirus: realising the potential of a promising vaccine. Lancet 376: 568–570. pmid:20692032 View Article PubMed/NCBI Google Scholar 4. UNICEF W (2017) World Bank Group Joint Child Malnutrition Estimates Levels and trends in child malnutrition. 5. Vlasova AN, Paim FC, Kandasamy S, Alhamo MA, Fischer DD, et al. (2017) Protein Malnutrition Modifies Innate Immunity and Gene Expression by Intestinal Epithelial Cells and Human Rotavirus Infection in Neonatal Gnotobiotic Pigs. mSphere 2. pmid:28261667 View Article PubMed/NCBI Google Scholar 6. Fischer DD, Kandasamy S, Paim FC, Langel SN, Alhamo MA, et al. (2017) Protein Malnutrition Alters Tryptophan and Angiotensin-Converting Enzyme 2 Homeostasis and Adaptive Immune Responses in Human Rotavirus-Infected Gnotobiotic Pigs with Human Infant Fecal Microbiota Transplant. Clin Vaccine Immunol 24. pmid:28637803 View Article PubMed/NCBI Google Scholar 7. Liu J, Bolick DT, Kolling GL, Fu Z, Guerrant RL (2016) Protein Malnutrition Impairs Intestinal Epithelial Cell Turnover, a Potential Mechanism of Increased Cryptosporidiosis in a Murine Model. Infect Immun 84: 3542–3549. pmid:27736783 View Article PubMed/NCBI Google Scholar 8. Hughes SM, Amadi B, Mwiya M, Nkamba H, Tomkins A, et al. (2009) Dendritic cell anergy results from endotoxemia in severe malnutrition. J Immunol 183: 2818–2826. pmid:19625645 View Article PubMed/NCBI Google Scholar 9. Iyer SS, Chatraw JH, Tan WG, Wherry EJ, Becker TC, et al. (2012) Protein energy malnutrition impairs homeostatic proliferation of memory CD8 T cells. J Immunol 188: 77–84. pmid:22116826 View Article PubMed/NCBI Google Scholar 10. Rytter MJ, Kolte L, Briend A, Friis H, Christensen VB (2014) The immune system in children with malnutrition—a systematic review. PLoS One 9: e105017. pmid:25153531 View Article PubMed/NCBI Google Scholar 11. Szajewska H, Mrukowicz JZ (2001) Probiotics in the treatment and prevention of acute infectious diarrhea in infants and children: a systematic review of published randomized, double-blind, placebo-controlled trials. J Pediatr Gastroenterol Nutr 33 Suppl 2: S17–25. pmid:11698781 View Article PubMed/NCBI Google Scholar 12. Sanders ME, Guarner F, Guerrant R, Holt PR, Quigley EM, et al. (2013) An update on the use and investigation of probiotics in health and disease. Gut 62: 787–796. pmid:23474420 View Article PubMed/NCBI Google Scholar 13. Kruis W, Fric P, Pokrotnieks J, Lukas M, Fixa B, et al. (2004) Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Gut 53: 1617–1623. pmid:15479682 View Article PubMed/NCBI Google Scholar 14. Schultz M (2008) Clinical use of E. coli Nissle 1917 in inflammatory bowel disease Clinical use of E. coli Nissle 1917 in inflammatory bowel disease. Inflamm Bowel Dis 14: 1012–1018. pmid:18240278 View Article PubMed/NCBI Google Scholar 15. Kleta S, Steinrück H., Breves G., Duncker S., Laturnus C., Wieler Schierack P. (2006) Detection and distribution of probiotic Escherichia coli Nissle 1917 clones in swine herds in Germany. J Appl Microbiol 101:1357–66 101 1357–1366. pmid:17105567 View Article PubMed/NCBI Google Scholar 16. Vlasova AN, Shao L, Kandasamy S, Fischer DD, Rauf A, et al. (2016) Escherichia coli Nissle 1917 protects gnotobiotic pigs against human rotavirus by modulating pDC and NK-cell responses. Eur J Immunol 46: 2426–2437. pmid:27457183 View Article PubMed/NCBI Google Scholar 17. Kandasamy S, Vlasova AN, Fischer D, Kumar A, Chattha KS, et al. (2016) Differential Effects of Escherichia coli Nissle and Lactobacillus rhamnosus Strain GG on Human Rotavirus Binding, Infection, and B Cell Immunity. J Immunol 196: 1780–1789. pmid:26800875 View Article PubMed/NCBI Google Scholar 18. Paim FC, Langel SN, Fischer DD, Kandasamy S, Shao L, et al. (2016) Effects of Escherichia coli Nissle 1917 and Ciprofloxacin on small intestinal epithelial cell mRNA expression in the neonatal piglet model of human rotavirus infection. Gut Pathog 8: 66. pmid:27999620 View Article PubMed/NCBI Google Scholar 19. Ding WK, Shah NP (2007) Acid, bile, and heat tolerance of free and microencapsulated probiotic bacteria. J Food Sci 72: M446–450. pmid:18034741 View Article PubMed/NCBI Google Scholar 20. Li XGC Sun Park Cha (2011) Preparation of alginate/chitosan/carboxymethyl chitosan complex microcapsules and application in Lactobacillus casei ATCC 393. Carbohydr Polym: 1479–1485. View Article Google Scholar 21. Muthukumarasamy PA-W P., Holley (2006) Stability of Lactobacillus reuteri in different types of microcapsules. J Food Sci 71: 20–24. View Article Google Scholar 22. Cook MT, Tzortzis G, Charalampopoulos D, Khutoryanskiy VV (2012) Microencapsulation of probiotics for gastrointestinal delivery. J Control Release 162: 56–67. pmid:22698940 View Article PubMed/NCBI Google Scholar 23. Kailasapathy K (2014) Microencapsulation for gastrointestinal delivery of probiotic bacteria,” in Nano Microencapsulation Foods. WileyBlackwell: 167–197. View Article Google Scholar 24. Navarro JB, Mashburn-Warren L, Bakaletz LO, Bailey MT, Goodman SD (2017) Enhanced Probiotic Potential of Lactobacillus reuteri When Delivered as a Biofilm on Dextranomer Microspheres That Contain Beneficial Cargo. Front Microbiol 8: 489. pmid:28396655 View Article PubMed/NCBI Google Scholar 25. Underwood MA, Arriola J, Gerber CW, Kaveti A, Kalanetra KM, et al. (2014) Bifidobacterium longum subsp. infantis in experimental necrotizing enterocolitis: alterations in inflammation, innate immune response, and the microbiota. Pediatr Res 76: 326–333. pmid:25000347 View Article PubMed/NCBI Google Scholar 26. Bahadoria PS, Mahapatra (2001) Prospects, technological aspects, and limitations of probiotics- a world wide review. Eur J Food Res Rev 1: 23–42. View Article Google Scholar 27. Ng EW, Yeung M, Tong PS (2011) Effects of yogurt starter cultures on the survival of Lactobacillus acidophilus. Int J Food Microbiol 145: 169–175. pmid:21196060 View Article PubMed/NCBI Google Scholar 28. Cogan TM, Beresford TP, Steele J, Broadbent J, Shah NP, et al. (2007) Invited review: Advances in starter cultures and cultured foods. J Dairy Sci 90: 4005–4021. pmid:17699017 View Article PubMed/NCBI Google Scholar 29. Guerrant RL, Oria RB, Moore SR, Oria MO, Lima AA (2008) Malnutrition as an enteric infectious disease with long-term effects on child development. Nutr Rev 66: 487–505. pmid:18752473 View Article PubMed/NCBI Google Scholar 30. Prendergast AJ, Kelly P (2016) Interactions between intestinal pathogens, enteropathy and malnutrition in developing countries. Curr Opin Infect Dis 29: 229–236. pmid:26967147 View Article PubMed/NCBI Google Scholar 31. Ibrahim MK, Zambruni M, Melby CL, Melby PC (2017) Impact of Childhood Malnutrition on Host Defense and Infection. Clin Microbiol Rev 30: 919–971. pmid:28768707 View Article PubMed/NCBI Google Scholar 32. Nahaisi MH RS, Noratto GD (2014) Probiotics as a Strategy to Improve Overall Human Health in Developing Countries. J Prob Health 2: 1–9. View Article Google Scholar 33. Maldonado Galdeano C, Cazorla SI, Lemme Dumit JM, Velez E, Perdigon G (2019) Beneficial Effects of Probiotic Consumption on the Immune System. Ann Nutr Metab 74: 115–124. pmid:30673668 View Article PubMed/NCBI Google Scholar 34. Kandasamy S, Chattha KS, Vlasova AN, Rajashekara G, Saif LJ (2014) Lactobacilli and Bifidobacteria enhance mucosal B cell responses and differentially modulate systemic antibody responses to an oral human rotavirus vaccine in a neonatal gnotobiotic pig disease model. Gut Microbes 5: 639–651. pmid:25483333 View Article PubMed/NCBI Google Scholar 35. Chattha KS, Vlasova AN, Kandasamy S, Esseili MA, Siegismund C, et al. (2013) Probiotics and colostrum/milk differentially affect neonatal humoral immune responses to oral rotavirus vaccine. Vaccine 31: 1916–1923. pmid:23453730 View Article PubMed/NCBI Google Scholar 36. Chattha KS, Vlasova AN, Kandasamy S, Rajashekara G, Saif LJ (2013) Divergent immunomodulating effects of probiotics on T cell responses to oral attenuated human rotavirus vaccine and virulent human rotavirus infection in a neonatal gnotobiotic piglet disease model. J Immunol 191: 2446–2456. pmid:23918983 View Article PubMed/NCBI Google Scholar 37. Vlasova AN, Chattha KS, Kandasamy S, Liu Z, Esseili M, et al. (2013) Lactobacilli and bifidobacteria promote immune homeostasis by modulating innate immune responses to human rotavirus in neonatal gnotobiotic pigs. PLoS One 8: e76962. pmid:24098572 View Article PubMed/NCBI Google Scholar 38. Meyer RC, Bohl EH, Kohler EM (1964) Procurement and Maintenance of Germ-Free Seine for Microbiological Investigations. Appl Microbiol 12: 295–300. pmid:14199016 View Article PubMed/NCBI Google Scholar 39. Gonzalez LM, Williamson I, Piedrahita JA, Blikslager AT, Magness ST (2013) Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration. PLoS One 8: e66465. pmid:23840480 View Article PubMed/NCBI Google Scholar 40. Nossol C, Diesing AK, Walk N, Faber-Zuschratter H, Hartig R, et al. (2011) Air-liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC). Histochem Cell Biol 136: 103–115. pmid:21681518 View Article PubMed/NCBI Google Scholar 41. Collado-Romero M, Arce C, Ramirez-Boo M, Carvajal A, Garrido JJ (2010) Quantitative analysis of the immune response upon Salmonella typhimurium infection along the porcine intestinal gut. Vet Res 41: 23. pmid:19941811 View Article PubMed/NCBI Google Scholar 42. Kandasamy S, Chattha KS, Vlasova AN, Saif LJ (2014) Prenatal vitamin A deficiency impairs adaptive immune responses to pentavalent rotavirus vaccine (RotaTeq(R)) in a neonatal gnotobiotic pig model. Vaccine 32: 816–824. pmid:24380684 View Article PubMed/NCBI Google Scholar 43. Chattha KS, Kandasamy S, Vlasova AN, Saif LJ (2013) Vitamin A deficiency impairs adaptive B and T cell responses to a prototype monovalent attenuated human rotavirus vaccine and virulent human rotavirus challenge in a gnotobiotic piglet model. PLoS One 8: e82966. pmid:24312675 View Article PubMed/NCBI Google Scholar 44. Ward LA, Yuan L, Rosen BI, To TL, Saif LJ (1996) Development of mucosal and systemic lymphoproliferative responses and protective immunity to human group A rotaviruses in a gnotobiotic pig model. Clin Diagn Lab Immunol 3: 342–350. pmid:8705681 View Article PubMed/NCBI Google Scholar 45. Zhang W, Azevedo MS, Wen K, Gonzalez A, Saif LJ, et al. (2008) Probiotic Lactobacillus acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs. Vaccine 26: 3655–3661. pmid:18524434 View Article PubMed/NCBI Google Scholar 46. Yuan L, Ward LA, Rosen BI, To TL, Saif LJ (1996) Systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease. J Virol 70: 3075–3083. pmid:8627786 View Article PubMed/NCBI Google Scholar 47. Annamalai T, Saif LJ, Lu Z, Jung K (2015) Age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs. Vet Immunol Immunopathol 168: 193–202. pmid:26433606 View Article PubMed/NCBI Google Scholar 48. Cario E (2010) Toll-like receptors in inflammatory bowel diseases: a decade later. Inflamm Bowel Dis 16: 1583–1597. pmid:20803699 View Article PubMed/NCBI Google Scholar 49. Pan D, Das A, Liu D, Veazey RS, Pahar B (2012) Isolation and characterization of intestinal epithelial cells from normal and SIV-infected rhesus macaques. PLoS One 7: e30247. pmid:22291924 View Article PubMed/NCBI Google Scholar 50. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3: 1101–1108. pmid:18546601 View Article PubMed/NCBI Google Scholar 51. Azevedo MS, Yuan L, Iosef C, Chang KO, Kim Y, et al. (2004) Magnitude of serum and intestinal antibody responses induced by sequential replicating and nonreplicating rotavirus vaccines in gnotobiotic pigs and correlation with protection. Clin Diagn Lab Immunol 11: 12–20. pmid:14715539 View Article PubMed/NCBI Google Scholar 52. To TL, Ward LA, Yuan L, Saif LJ (1998) Serum and intestinal isotype antibody responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease. J Gen Virol 79 (Pt 11): 2661–2672. View Article Google Scholar 53. Louis P, O’Byrne CP (2010) Life in the gut: microbial responses to stress in the gastrointestinal tract. Sci Prog 93: 7–36. pmid:20222354 View Article PubMed/NCBI Google Scholar 54. Kamada N, Chen GY, Inohara N, Nunez G (2013) Control of pathogens and pathobionts by the gut microbiota. Nat Immunol 14: 685–690. pmid:23778796 View Article PubMed/NCBI Google Scholar 55. Holloway G, Coulson BS (2013) Innate cellular responses to rotavirus infection. J Gen Virol 94: 1151–1160. pmid:23486667 View Article PubMed/NCBI Google Scholar 56. Foligne B, Zoumpopoulou G, Dewulf J, Ben Younes A, Chareyre F, et al. (2007) A key role of dendritic cells in probiotic functionality. PLoS One 2: e313. pmid:17375199 View Article PubMed/NCBI Google Scholar 57. Sugimura T, Takahashi H, Jounai K, Ohshio K, Kanayama M, et al. (2015) Effects of oral intake of plasmacytoid dendritic cells-stimulative lactic acid bacterial strain on pathogenesis of influenza-like illness and immunological response to influenza virus. Br J Nutr 114: 727–733. pmid:26234407 View Article PubMed/NCBI Google Scholar 58. Kruis W, Chrubasik S, Boehm S, Stange C, Schulze J (2012) A double-blind placebo-controlled trial to study therapeutic effects of probiotic Escherichia coli Nissle 1917 in subgroups of patients with irritable bowel syndrome. Int J Colorectal Dis 27: 467–474. pmid:22130826 View Article PubMed/NCBI Google Scholar 59. Winzler C, Rovere P, Rescigno M, Granucci F, Penna G, et al. (1997) Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J Exp Med 185: 317–328. pmid:9016880 View Article PubMed/NCBI Google Scholar 60. Deal EM, Lahl K, Narvaez CF, Butcher EC, Greenberg HB (2013) Plasmacytoid dendritic cells promote rotavirus-induced human and murine B cell responses. J Clin Invest 123: 2464–2474. pmid:23635775 View Article PubMed/NCBI Google Scholar 61. Tezuka H, Abe Y, Asano J, Sato T, Liu J, et al. (2011) Prominent role for plasmacytoid dendritic cells in mucosal T cell-independent IgA induction. Immunity 34: 247–257. pmid:21333555 View Article PubMed/NCBI Google Scholar 62. Schlickum S, Sennefelder H, Friedrich M, Harms G, Lohse MJ, et al. (2008) Integrin alpha E(CD103)beta 7 influences cellular shape and motility in a ligand-dependent fashion. Blood 112: 619–625. pmid:18492951 View Article PubMed/NCBI Google Scholar 63. Hajela N, Nair GB, Abraham P, Ganguly NK (2012) Health impact of probiotics—vision and opportunities. Gut Pathog 4: 1. pmid:22410274 View Article PubMed/NCBI Google Scholar 64. Skipper M, Lewis J (2000) Getting to the guts of enteroendocrine differentiation. Nat Genet 24: 3–4. pmid:10615112 View Article PubMed/NCBI Google Scholar 65. Carulli AJ, Samuelson LC, Schnell S (2014) Unraveling intestinal stem cell behavior with models of crypt dynamics. Integr Biol (Camb) 6: 243–257. pmid:24480852 View Article PubMed/NCBI Google Scholar 66. Olson JK, Rager TM, Navarro JB, Mashburn-Warren L, Goodman SD, et al. (2016) Harvesting the benefits of biofilms: A novel probiotic delivery system for the prevention of necrotizing enterocolitis. J Pediatr Surg 51: 936–941. pmid:27032609 View Article PubMed/NCBI Google Scholar 67. Shelby RD, Janzow GE, Mashburn-Warren L, Galley J, Tengberg N, et al. (2020) A novel probiotic therapeutic in a murine model of Clostridioides difficile colitis. Gut Microbes 12: 1814119. pmid:32954922 View Article PubMed/NCBI Google Scholar 68. Olson JK, Navarro JB, Allen JM, McCulloh CJ, Mashburn-Warren L, et al. (2018) An enhanced Lactobacillus reuteri biofilm formulation that increases protection against experimental necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 315: G408–G419. pmid:29848024 View Article PubMed/NCBI Google Scholar 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 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. VAT will be added later in the calculation will be finalised during articleGet time limited or full article access on ReadCube.$ prices are NET prices. Additional access options: Log in Learn about institutional subscriptions ReferencesKyle TK, Dhurandhar EJ, Allison DB. Regarding obesity as a disease: evolving policies and their implications. Endocrinol Metab Clin North Am. 2016;45:511– PubMed Central Google Scholar Obesity: preventing and managing the global epidemic. Report of a WHO consultation. World Health Organ Tech Rep Ser. 2000;894:1– GA, Kim KK, Wilding JPH, World Obesity F. Obesity: a chronic relapsing progressive disease process. A position statement of the World Obesity Federation. Obes Rev. 2017;18:715– Article Google Scholar O’Neil PM, Birkenfeld AL, McGowan B, Mosenzon O, Pedersen SD, Wharton S, et al. Efficacy and safety of semaglutide compared with liraglutide and placebo for weight loss in patients with obesity: a randomised, double-blind, placebo and active controlled, dose-ranging, phase 2 trial. Lancet. 2018;392:637– Google Scholar Apovian CM, Aronne LJ, Bessesen DH, McDonnell ME, Murad MH, Pagotto U, et al. Pharmacological management of obesity: an endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2015;100:342– Article Google Scholar Srivastava G, Apovian CM. Current pharmacotherapy for obesity. Nat Rev Endocrinol. 2018;14:12– Article Google Scholar Bessesen DH, Van Gaal LF. Progress and challenges in anti-obesity pharmacotherapy. Lancet Diabetes Endocrinol. 2018;6:237– Google Scholar Komaroff AL. The microbiome and risk for obesity and diabetes. JAMA. 2017;317:355– Google Scholar Heymsfield SB, Wadden TA. Mechanisms, pathophysiology, and management of obesity. N Engl J Med. 2017;376:254– Article Google Scholar Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature. 2006;444:1027– Google Scholar Pedrolli DB, Ribeiro NV, Squizato PN, de Jesus VN, Cozetto DA, Team AQAUai. Engineering microbial living therapeutics: the synthetic biology toolbox. Trends Biotechnol. 2019;37:100– Article Google Scholar Reardon S. Genetically modified bacteria enlisted in fight against disease. Nature. 2018;558:497– Article Google Scholar Hendrikx T, Duan Y, Wang Y, Oh JH, Alexander LM, Huang W, et al. Bacteria engineered to produce IL-22 in intestine induce expression of REG3G to reduce ethanol-induced liver disease in mice. Gut. 2019;68:1504– Article Google Scholar Chen Z, Guo L, Zhang Y, Walzem RL, Pendergast JS, Printz RL, et al. Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity. J Clin Invest. 2014;124:3391– Article PubMed Central Google Scholar Wassenaar TM. Insights from 100 years of research with probiotic E. Coli. Eur J Microbiol Immunol. 2016;6:147– Google Scholar Grozdanov L, Raasch C, Schulze J, Sonnenborn U, Gottschalk G, Hacker J, et al. Analysis of the genome structure of the nonpathogenic probiotic Escherichia coli strain Nissle 1917. J Bacteriol. 2004;186:5432– Article PubMed Central Google Scholar Hancock V, Dahl M, Klemm P. Probiotic Escherichia coli strain Nissle 1917 outcompetes intestinal pathogens during biofilm formation. J Med Microbiol. 2010;59:392– Google Scholar Kurtz CB, Millet YA, Puurunen MK, Perreault M, Charbonneau MR, Isabella VM. et al. An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans. Sci Transl Med. 2019;11: Google Scholar Isabella VM, Ha BN, Castillo MJ, Lubkowicz DJ, Rowe SE, Millet YA, et al. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat Biotechnol. 2018;36:857– Article Google Scholar Hwang IY, Koh E, Wong A, March JC, Bentley WE, Lee YS, et al. Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models. Nat Commun. 2017;8: Article PubMed Central Google Scholar Ma J, Li C, Wang J, Gu J. Genetically engineered Escherichia coli Nissle 1917 secreting GLP-1 analog exhibits potential antiobesity effect in high-fat diet-induced obesity mice. Obesity. 2020;28:315– Article Google Scholar Yumuk V, Tsigos C, Fried M, Schindler K, Busetto L, Micic D, et al. European guidelines for obesity management in adults. Obes Facts. 2015;8:402– PubMed Central Google Scholar Collaborators GBDO, Afshin A, Forouzanfar MH, Reitsma MB, Sur P, Estep K, et al. Health effects of overweight and obesity in 195 countries over 25 years. N Engl J Med. 2017;377:13– Google Scholar Reid G, Sanders ME, Gaskins HR, Gibson GR, Mercenier A, Rastall R, et al. New scientific paradigms for probiotics and prebiotics. J Clin Gastroenterol. 2003;37:105– Google Scholar Yadav H, Lee JH, Lloyd J, Walter P, Rane SG. Beneficial metabolic effects of a probiotic via butyrate-induced GLP-1 hormone secretion. J Biol Chem. 2013;288:25088– Article PubMed Central Google Scholar Kang JH, Yun SI, Park MH, Park JH, Jeong SY, Park HO. Anti-obesity effect of Lactobacillus gasseri BNR17 in high-sucrose diet-induced obese mice. PLoS ONE. 2013;8: Article PubMed Central Google Scholar Valsecchi C, Carlotta Tagliacarne S, Castellazzi A. Gut microbiota and obesity. J Clin Gastroenterol. 2016;50:S157–8. Suppl 2Proceedings from the 8th Probiotics, Prebiotics & New Foods for Microbiota and Human Health meeting held in Rome, Italy on September 13–15, Article Google Scholar Wei P, Yang Y, Li T, Ding Q, Sun H. A engineered Bifidobacterium longum secreting a bioative penetratin-Glucagon-like peptide 1 fusion protein enhances Glucagon-like peptide 1 absorption in the intestine. J Microbiol Biotechnol. 2015. Epub ahead of FF, Liu JH, March JC. Engineered commensal bacteria reprogram intestinal cells into glucose-responsive insulin-secreting cells for the treatment of diabetes. Diabetes. 2015;64:1794– Article PubMed Central Google Scholar Rao S, Hu S, McHugh L, Lueders K, Henry K, Zhao Q, et al. Toward a live microbial microbicide for HIV: commensal bacteria secreting an HIV fusion inhibitor peptide. Proc Natl Acad Sci USA. 2005;102:11993– Article PubMed Central Google Scholar Federico A, Dallio M, Rosa DIS, Giorgio V, Miele L. Gut microbiota, obesity and metabolic disorders. Minerva Gastroenterol Dietol. 2017;63:337– Google Scholar Bäckhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, et al. The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA. 2004;101:15718– PubMed Central Google Scholar Boulange CL, Neves AL, Chilloux J, Nicholson JK, Dumas ME. Impact of the gut microbiota on inflammation, obesity, and metabolic disease. Genome Med. 2016;8: PubMed Central Google Scholar Patterson E, Ryan PM, Cryan JF, Dinan TG, Ross RP, Fitzgerald GF, et al. Gut microbiota, obesity and diabetes. Postgrad Med J. 2016;92:286– Article Google Scholar Madsen MSA, Holm JB, Palleja A, Wismann P, Fabricius K, Rigbolt K, et al. Metabolic and gut microbiome changes following GLP-1 or dual GLP-1/GLP-2 receptor agonist treatment in diet-induced obese mice. Sci Rep. 2019;9: PubMed Central Google Scholar Zhang N, Tao J, Gao L, Bi Y, Li P, Wang H, et al. Liraglutide attenuates nonalcoholic fatty liver disease by modulating gut microbiota in rats administered a high-fat diet. Biomed Res Int. 2020;2020: PubMed Central Google Scholar Liu Q, Cai BY, Zhu LX, Xin X, Wang X, An ZM, et al. Liraglutide modulates gut microbiome and attenuates nonalcoholic fatty liver in db/db mice. Life Sci. 2020;261: Article Google Scholar Ehrlich SD. Probiotics—little evidence for a link to obesity. Nat Rev Microbiol. 2009;7: Article Google Scholar Daniel H, Gholami AM, Berry D, Desmarchelier C, Hahne H, Loh G, et al. High-fat diet alters gut microbiota physiology in mice. ISME J. 2014;8:295– Article Google Scholar Kanoski SE, Hayes MR, Skibicka KP. GLP-1 and weight loss: unraveling the diverse neural circuitry. 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: