Coli Nissle Research Articles

Engineering Adhesion of the Probiotic Strain Escherichia coli Nissle to the Fungal Pathogen Candida albicans.

Engineering live biotherapeutic products against fungal pathogens such as Candida albicans has been suggested as a means to tackle the increasing threat of fungal infections and the development of resistance to classical antifungal treatments. One important challenge in the design of live therapeutics is to control their localization inside the human body. The specific binding capability to target organisms or tissues would greatly increase their effectiveness by increasing the local concentration of effector molecules at the site of infection. In this study, we utilized surface display of carbohydrate binding domains to enable the probiotic E. coli Nissle 1917 to adhere specifically to the pathogenic yeast Candida albicans. Binding was quantified using a newly developed method based on the automated analysis of microscopic images. In addition to a rationally selected chitin binding domain, a synthetic peptide of identical length but distinct sequence also conferred binding. Efficient binding was specific to fungal hyphae, the invasive form of C. albicans, while the yeast form, as well as abiotic cellulose and PET particles, was only weakly recognized.

Extracellular Vesicles of the Probiotic Escherichia coli Nissle 1917 Reduce PepT1 Levels in IL-1β-Treated Caco-2 Cells via Upregulation of miR-193a-3p.

PepT1, a proton-coupled oligopeptide transporter, is crucial for intestinal homeostasis. It is mainly expressed in small intestine enterocytes, facilitating the absorption of di/tri-peptides from dietary proteins. In the colon, PepT1 expression is minimal to prevent excessive responses to proinflammatory peptides from the gut microbiota. However, increased colonic PepT1 is linked to chronic inflammatory diseases and colitis-associated cancer. Despite promising results from animal studies on the benefits of extracellular vesicles (EVs) from beneficial gut commensals in treating IBD, applying probiotic EVs as a postbiotic strategy in humans requires a thorough understanding of their mechanisms. Here, we investigate the potential of EVs of the probiotic Nissle 1917 (EcN) and the commensal EcoR12 in preventing altered PepT1 expression under inflammatory conditions, using an interleukin (IL)-1-induced inflammation model in Caco-2 cells. The effects are evaluated by analyzing the expression of PepT1 (mRNA and protein) and miR-193a-3p and miR-92b, which regulate, respectively, PepT1 mRNA translation and degradation. The influence of microbiota EVs on PepT1 expression is also analyzed in the presence of bacterial peptides that are natural substrates of colonic PepT1 to clarify how the regulatory mechanisms function under both physiological and pathological conditions. The main finding is that EcN EVs significantly decreases PepT1 protein via upregulation of miR-193a-3p. Importantly, this regulatory effect is strain-specific and only activates in cells exposed to IL-1β, suggesting that EcN EVs does not control PepT1 expression under basal conditions but can play a pivotal role in response to inflammation as a stressor. By this mechanism, EcN EVs may reduce inflammation in response to microbiota in chronic intestinal disorders by limiting the uptake of bacterial proinflammatory peptides.

Engineered Bacteria for Visualization and Localization of Gastrointestinal Bleeding: A Promising Application.

Gastrointestinal bleeding, especially obscure gastrointestinal bleeding (OGIB), is a common and serious clinical emergency with a notable incidence rate. However, the current diagnostic method, gastroscopy, is invasive and often struggles to efficiently detect microhemorrhagic lesions, leading to diagnostic challenges and potential misdiagnoses. Here, we developed an intelligently engineered bacterium utilizing synthetic biology techniques for in vivo localization detection of gastrointestinal bleeding. By constructing three gene circuit modules within E. coli Nissle 1917 for heme recognition, response, and output generation, we have successfully enabled specific heme sensing and real-time optical signal production in vivo. This innovative strategy overcomes the limitations of the existing diagnostic methods, offering a noninvasive and precise means of detecting gastrointestinal bleeding. These advancements hold promise for enhancing diagnostic accuracy and treatment efficacy in future clinical settings within the realm of gastroenterology.

Priority order of neonatal colonization by a probiotic or pathogenic Escherichia coli strain dictates the host response to experimental colitis.

The alarming prevalence of inflammatory bowel disease (IBD) in early childhood is associated with imbalances in the microbiome, the immune response, and environmental factors. Some pathogenic Escherichia coli (E. coli) strains have been found in IBD patients, where they may influence disease progression. Therefore, the discovery of new harmful bacterial strains that have the potential to drive the inflammatory response is of great importance. In this study, we compared the immunomodulatory properties of two E. coli strains of serotype O6: the probiotic E. coli Nissle 1917 and the uropathogenic E. coli O6:K13:H1. Using the epithelial Caco-2 cell line, we investigated the different abilities of the strains to adhere to and invade epithelial cells. We confirmed the potential of E. coli Nissle 1917 to modulate the Th1 immune response in a specific manner in an in vitro setting by stimulating mouse bone marrow-derived dendritic cells (BM-DCs). In gnotobiotic in vivo experiments, we demonstrated that neonatal colonization with E. coli Nissle 1917 achieves a stable high concentration in the intestine and protects mice from the progressive effect of E. coli O6:K13:H1 in developing ulcerative colitis in an experimental model. In contrast, a single-dose treatment with E. coli Nissle 1917 is ineffective in achieving such high concentrations and does not protect against DSS-induced ulcerative colitis in mice neonatally colonized with pathobiont E. coli O6:K13:H1. Despite the stable coexistence of both E. coli strains in the intestinal environment of the mice, we demonstrated a beneficial competitive interaction between the early colonizing E. coli Nissle 1917 and the late-arriving strain O6:K13:H1, suggesting its anti-inflammatory potential for the host. This study highlights the importance of the sequence of bacterial colonization, which influences the development of the immune response in the host gut and potentially impacts future quality of life.

Engineered probiotic cocktail with two cascade metabolic Escherichia coli for the treatment of hyperlysinemia.

Engineering probiotics have emerged as a potential strategy for the treatment of metabolic diseases. However, due to the exceptional complexity of these metabolic disorders and the intricate relationship between gut microbes, it is difficult to achieve an ideal therapeutic effect in a specific metabolic disorder using only a single engineered strain. In this work, we proposed a probiotic cocktail strategy by engineering two cascade metabolic bacteria to treat hyperlysinemia, an inherited lysine metabolic disorder with loss of α-aminoadipate semialdehyde synthase (AASS) activity. A probiotic E. coli Nissle 1917 strain EcNT (pTLS) with a heterologous enzyme pathway in Saccharomyces cerevisiae was engineered to metabolize the excess lysine. Another one EcNT (pK25) was engineered to consume the products of lysine metabolism. The bacterial cocktail enables the maintenance of a metabolic cascade with AASS-like functional activity to maintain the blood lysine concentrations and downstream metabolites. In vitro experimental results showed that the cocktail bacteria had a better metabolic capacity and metabolites balance at a ratio of EcNT (pTLS) and EcNT (pK25) of 1:2. Feeding of the cocktail bacteria to the mouse model effectively reduced the concentration of lysine and balanced saccharopine in the plasma of hyperlysinemia-like mice. These findings not only provide a promising strategy of probiotic stains for the treatment of hyperlysinemia but also highlight the potential of engineered cascade cocktails to intervene and even cure other inherited metabolic diseases.

Modulation of Serotonin-Related Genes by Extracellular Vesicles of the Probiotic Escherichia coli Nissle 1917 in the Interleukin-1β-Induced Inflammation Model of Intestinal Epithelial Cells.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition involving dysregulated immune responses and imbalances in the gut microbiota in genetically susceptible individuals. Current therapies for IBD often have significant side-effects and limited success, prompting the search for novel therapeutic strategies. Microbiome-based approaches aim to restore the gut microbiota balance towards anti-inflammatory and mucosa-healing profiles. Extracellular vesicles (EVs) from beneficial gut microbes are emerging as potential postbiotics. Serotonin plays a crucial role in intestinal homeostasis, and its dysregulation is associated with IBD severity. Our study investigated the impact of EVs from the probiotic Nissle 1917 (EcN) and commensal E. coli on intestinal serotonin metabolism under inflammatory conditions using an IL-1β-induced inflammation model in Caco-2 cells. We found strain-specific effects. Specifically, EcN EVs reduced free serotonin levels by upregulating SERT expression through the downregulation of miR-24, miR-200a, TLR4, and NOD1. Additionally, EcN EVs mitigated IL-1β-induced changes in tight junction proteins and oxidative stress markers. These findings underscore the potential of postbiotic interventions as a therapeutic approach for IBD and related pathologies, with EcN EVs exhibiting promise in modulating serotonin metabolism and preserving intestinal barrier integrity. This study is the first to demonstrate the regulation of miR-24 and miR-200a by probiotic-derived EVs.

A survey of multiple candidate probiotic bacteria reveals specificity in the ability to modify the effects of key wound pathogens.

We have evaluated the inhibitory effects of supernatants and lysates derived from several candidate probiotics, on the growth and biofilm formation of wound pathogens, and their ability to protect human primary epidermal keratinocytes from the toxic effects of pathogens. Supernatants (neutralized and non-neutralized) and lysates (via sonication) from Lactiplantibacillus plantarum, Limosilactobacillus reuteri, Bifidobacterium longum, Lacticaseibacillus rhamnosus GG, and Escherichia coli Nissle 1917 were tested for their inhibitory effects against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumanni. The supernatants of L. plantarum, L. rhamnosus, B. longum, and L. rhamnosus GG reduced the growth of S. aureus, E. coli, and A. baumanni. B. longum additionally inhibited P. aeruginosa growth. However, neutralized Lactobacillus supernatants did not inhibit growth and in some cases were stimulatory. Lysates of L. plantarum and L. reuteri inhibited S. pyogenes while B. longum lysates inhibited E. coli and S. aureus growth. E. coli Nissle 1917 lysates enhanced the growth of S. pyogenes and P. aeruginosa. Biofilm formation by E. coli was reduced by lysates of L. reuteri and neutralized supernatants of all candidate probiotics. P. aeruginosa biofilm formation was reduced by E. coli Nissle supernatant but increased by L. plantarum, L. reuteri, and Bifidobacterium longum lysates. L. reuteri decreased the toxic effects of S. aureus on keratinocytes while E. coli Nissle 1917 lysates protected keratinocytes from S. pyogenes toxicity. In conclusion, lactobacilli and E. coli Nissle lysates confer inhibitory effects on pathogenic growth independently of acidification and may beneficially alter the outcome of interactions between host cell-pathogen in a species-specific manner.IMPORTANCEOne of the attributes of probiotics is their ability to inhibit pathogens. For this reason, many lactobacilli have been investigated for their effects as potential topical therapeutics against skin pathogens. However, this field is in its infancy. Even though probiotics are known to be safe when taken orally, the potential safety concerns when applied to potentially compromised skin are unknown. For this reason, we believe that extracts of probiotics will offer advantages over the use of live bacteria. In this study, we have surveyed five candidate probiotics, when used as extracts, in terms of their effects against common wound pathogens. Our data demonstrate that some probiotic extracts promote the growth of pathogens and highlight the need for careful selection of species and strains when probiotics are to be used topically.

Acinetobacter species secrete compounds that promote intestinal inflammation

Background: The gut microbiota is known to impact multiple aspects of human health, including immune modulation, nutrient uptake, cell metabolism, and defense against pathogens. The composition and diversity of the gut microbiota is known to be altered in the setting of inflammatory bowel disease (IBD). IBD, which encompasses the subsets Crohn’s Disease and Ulcerative Colitis, is a life-long condition characterized by chronic inflammation of the gastrointestinal tract. It has long been speculated that gut bacteria drive or exacerbate inflammation in IBD patients, but few studies have identified bacterial candidates. Analysis of 3,853 publicly available RNA-Seq samples revealed that Acinetobacter calcoaceticus was one of the top 10 highest elevated bacteria in Crohn's Disease patients. Acinetobacter are well-characterized by their antibiotic resistance, but little is known about its relationship to the intestine. We hypothesize that A. calcoaceticus stimulates intestinal inflammation and exacerbates colitis. Materials & Results: To identify if A. calcoaceticus could stimulate inflammation, we generated inside out mouse jejunal and colonic organoids and added live A. calcoaceticus bacteria to the cultures. As a positive control, we added another Acinetobacter species A. baumannii and as a negative control, we added E. coli Nissle. After 6 hrs, we observed significant expression of pro-inflammatory cytokines in response to A. calcoaceticus and A. baumannii, while E. coli Nissle did not generate the same responses. To further investigate the compounds responsible to inflammation, we grew our bacterial strains and exposed Caenorhabditis elegans to the bacterial secreted compounds. Similar to our organoid experiments, A. calcoaceticus and A.baumannii were lethal to worms, while E. coli had no effect on C. elegans viability. Next, we sought to address the effects of Acinetoacter on the gut in vivo. We oral gavaged adult BALB/c mice with A. calcoaceticus, A. baumannii and E. coli Nissle and found that in the setting of a complex gut microbiota, these bacteria alone did not stimulation inflammation. However, supplementation of anbitioics prior to introduction of our bacteria resulted in a loss of protection and enhanced inflammation. To model IBD, we administered 2,4,6-trinitrobenzene sulfonic acid (TNBS) and induced colitis and then treated mice with our bacterial strains. We found that TNBS treated mice receiving A. calcoaceticus and A. baumannii lost more weight and had worse histological scores than mice treated with vehicle control and TNBS; indicating A. calcoaceticus worsens intestinal inflammation. Conclusions: These data show that A. calcoaceticus can promote intestinal inflammation and may be a contributing factor to the inflammation experienced by IBD patients. Funding Sources for this project include: 1T32GM132055-01, The Histochemical Society’s Cornerstone Grant and the Porter Physiology Development Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

E. Coli Nissle 1917 Based Carrier for DNA Delivery into Colorectal Carcinoma Cells

E. Coli Nissle 1917 Based Carrier for DNA Delivery into Colorectal Carcinoma Cells

Abstract 239: Establishing a murine intratumoral bacterial model

Abstract Background: Long thought to be sterile spaces, current evidence now suggests that host microbiota can be found within cancerous tissue. Emerging data has illuminated that microbes are integral components of tumor tissue itself, which may correlate with tumor progression, treatment responsiveness, and disease prognosis. A recent Cell article from Fu et al. demonstrated that tumor-resident intracellular microbiota can promote metastatic colonization in breast cancer. Others within this research landscape see therapeutic opportunity - harnessing the tropism of these microbes to deliver TME modifying payloads directly to cancerous lesions. Purpose: Before studying the mechanism underlying their tropism to solid tumors, it is necessary to first establish a murine model that would allow for an oncotropic bacteria to be detected and quantified within tissues of interest. My research entailed the design and execution of pilot experiments developing xenograft melanoma and genetic colorectal models for future research projects. Methods: In all mouse studies, an E. coli Nissle 1917 strain called “NisLux” was used, who’s integration with a luxCDABE cassette allowed it to be selectively identified via luminescence using traditional exposure photography or endpoint luminescence detection using a 96-well plate reader. A xenograft melanoma model was developed using C57BL/6J mice and a B16-F10 murine melanoma line. A genetics model was developed using C57BL/6J-Apcmin mice which spontaneously develop colorectal adenomas as they age. Tumors, spleens, and healthy tissue were harvested and processed following tail vein injections of either NisLux or PBS solution. Harvested tissue was normalized by weight and concentration of resident NisLux was calculated from serially diluted samples plated on LB agar. Results: In both the xenograft and genetics murine models, NisLux exhibited tropism to tumor sites, enriching at a concentration above that of healthy background tissue. NisLux dosing correlated with splenomegaly without changes to the mass of the primary lesion compared to control. Conclusions: NisLux exhibits tropism to tumor sites in our xenograft and genetics murine models. Both models should undergo further procedural and control optimization. Two possible modifications to future melanoma xenograft experiments include the collection of non-cancerous skin negative control samples and a further delayed takedown day for tissue processing. In future C57BL/6J-Apcmin mice experiments, healthy colon sections from NisLux dosed C57BL/6J mice should be used as negative controls. Overall, the results from both models were promising and encourage future exploration of the mechanism underlying NisLux’s tropism to solid tumors. Subsequent experiments could involve comparing the tropism of engineered KO strains to WT NisLux or performing comparative sequencing of isolated tumor resident NisLux to the initial dosing population. Citation Format: Ryan A. Hannon, Dongqing Xu, Fengyi Wan. Establishing a murine intratumoral bacterial model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 239.

Intranasal administration of Escherichia coli Nissle expressing the spike protein of SARS-CoV-2 induces long-term immunization and prevents spike protein-mediated lung injury in mice

While current anti-Spike protein (SP) vaccines have been pivotal in managing the pandemic, their limitations in delivery, storage, and the inability to provide mucosal immunization (preventing infections) highlight the ongoing necessity for research and innovation. To tackle these constraints, our research group developed a bacterial-based vaccine using a non-pathogenic E. coli Nissle 1917 (EcN) strain genetically modified to express the SARS-CoV-2 spike protein on its surface (EcN-pAIDA1-SP). We intranasally delivered the EcN-pAIDA1-SP in two doses and checked specific IgG/IgA production as well as the key immune mediators involved in the process. Moreover, following the initial and booster vaccine doses, we exposed both immunized and non-immunized mice to intranasal delivery of SARS-CoV-2 SP to assess the effectiveness of EcN-pAIDA1-SP in protecting lung tissue from the inflammation damage. We observed detectable levels of anti-SARS-CoV-2 spike IgG in serum samples and IgA in bronchoalveolar lavage fluid two weeks after the initial treatment, with peak concentrations in the respective samples on the 35th day. Moreover, immunoglobulins displayed a progressively enhanced avidity index, suggesting a selective binding to the spike protein. Finally, the pre-immunized group displayed a decrease in proinflammatory markers (TLR4, NLRP3, ILs) following SP challenge, compared to the non-immunized groups, along with better preservation of tissue morphology. Our probiotic-based technology provides an effective immunobiotic tool to protect individuals against disease and control infection spread.

Conquering homocystinuria with engineered probiotics

Pyridoxine-unresponsive homocystinuria has lifelong implications for health. In this issue, Perreault and colleagues present evidence that orally delivered engineered probiotic Escherichia Coli Nissle SYNB1353 is a promising candidate in reducing homocysteine, with successful trials in mice, monkeys, and humans. However, further probiotic optimization and safety assessments are required.

Stable expression of HIV-1 MPER extended epitope on the surface of the recombinant probiotic bacteria Escherichia Coli Nissle 1917 using CRISPR/Cas9

BackgroundMucosal vaccines have the potential to induce protective immune responses at the sites of infection. Applying CRISPR/Cas9 editing, we aimed to develop a probiotic-based vaccine candidate expressing the HIV-1 envelope membrane-proximal external region (MPER) on the surface of E. coli Nissle 1917.ResultsThe HIV-1 MPER epitope was successfully introduced in the porin OmpF of the E. coli Nissle 1917 (EcN-MPER) and the modification was stable over 30 passages of the recombinant bacteria on the DNA and protein level. Furthermore, the introduced epitope was recognized by a human anti-HIV-1 gp41 (2F5) antibody using both live and heat-killed EcN-MPER, and this antigenicity was also retained over 30 passages. Whole-cell dot blot suggested a stronger binding of anti-HIV-1 gp41 (2F5) to heat-killed EcN-MPER than their live counterpart. An outer membrane vesicle (OMV) – rich extract from EcN-MPER culture supernatant was equally antigenic to anti-HIV-1 gp41 antibody which suggests that the MPER antigen could be harboured in EcN-MPER OMVs. Using quantitative ELISA, we determined the amount of MPER produced by the modified EcN to be 14.3 µg/108 cfu.ConclusionsThe CRISPR/Cas9 technology was an effective method for establishment of recombinant EcN-MPER bacteria that was stable over many passages. The developed EcN-MPER clone was devoid of extraneous plasmids and antibiotic resistance genes which eliminates the risk of plasmid transfer to animal hosts, should this clone be used as a vaccine. Also, the EcN-MPER clone was recognised by anti-HIV-1 gp41 (2F5) both as live and heat-killed bacteria making it suitable for pre-clinical evaluation. Expression of OmpF on bacterial surfaces and released OMVs identifies it as a compelling candidate for recombinant epitope modification, enabling surface epitope presentation on both bacteria and OMVs. By applying the methods described in this study, we present a potential platform for cost-effective and rational vaccine antigen expression and administration, offering promising prospects for further research in the field of vaccine development.

Enhancing tumor-specific recognition of programmable synthetic bacterial consortium for precision therapy of colorectal cancer

Probiotics hold promise as a potential therapy for colorectal cancer (CRC), but encounter obstacles related to tumor specificity, drug penetration, and dosage adjustability. In this study, genetic circuits based on the E. coli Nissle 1917 (EcN) chassis were developed to sense indicators of tumor microenvironment and control the expression of therapeutic payloads. Integration of XOR gate amplify gene switch into EcN biosensors resulted in a 1.8-2.3-fold increase in signal output, as confirmed by mathematical model fitting. Co-culturing programmable EcNs with CRC cells demonstrated a significant reduction in cellular viability ranging from 30% to 50%. This approach was further validated in a mouse subcutaneous tumor model, revealing 47%-52% inhibition of tumor growth upon administration of therapeutic strains. Additionally, in a mouse tumorigenesis model induced by AOM and DSS, the use of synthetic bacterial consortium (SynCon) equipped with multiple sensing modules led to approximately 1.2-fold increased colon length and 2.4-fold decreased polyp count. Gut microbiota analysis suggested that SynCon maintained the abundance of butyrate-producing bacteria Lactobacillaceae NK4A136, whereas reducing the level of gut inflammation-related bacteria Bacteroides. Taken together, engineered EcNs confer the advantage of specific recognition of CRC, while SynCon serves to augment the synergistic effect of this approach.

Heterologous expression of protein-glutaminase in Escherichia coli Nissle 1917 for improved flavor properties of soy protein and its implications on high-moisture extrudates

Beany flavor affects sensory experience of soy protein-based food. Deamidation is an effective approach to improve the flavor of soy protein, but excessive hydrolysis has a negative effect on its molecular structure and processing property. In this study, protein-glutaminase (PG; EC 3.5.1.44) was heterologous expressed in E. coli Nissle 1917 (EcN) and used to specifically modify soy protein isolate (SPI) for improved flavor properties of textured protein. A fluorescence-based enzyme activity screening method was developed and optimized for fermentation conditions. After screening for optimal fermentation conditions based on cell fluorescence intensity, 3.01 U/mL of PG activity was achieved. SPI was treated with PG (0–24 h), and the degree of deamidation (DD) rapidly increased to 47.4% within 0–2 h. There was no significant decrease in the molecular weight of deamidated SPI (DSPI) after specific deamidation of PG within 0–10 h. The contents of main beany flavor substances (e.g., hexanal, benzaldehyde, and 2-pentylfuran) in DSPI decreased significantly with increasing DD. Compared with soy protein, moderate deamidation did not affect the processing properties and fibrous structure of DSPI in preparing high-moisture extrudates, while the structure of the extrudate decreased after a deamidation time of more than 6 h. This finding developed a safe expression system for PG and provided a promising method to address beany flavor in soy-based meat analogues produced by high moisture extrusion.

Reduction of Fecal Calprotectin Levels Induced by a Short Course of Escherichia Coli Nissle is Associated with a Lower Likelihood of Disease Flares in Patients with Ulcerative Colitis in Clinical Remission.

Fecal calprotectin (FC) is a biomarker of gut inflammation, and Escherichia coli Nissle 1917 (EcN) is a probiotic strain able to reduce gut inflammation and maintain disease remission in patients with inflammatory bowel disease (IBD). The aim is to assess the effects of EcN administration in patients with IBD in clinical remission and altered FC values. We prospectively included 82 patients with ulcerative colitis (UC) (n=49) and Crohn's disease (CD) (n=33) in clinical remission and with FC values above 250 mcg/g (T0) who were treated with EcN alone for 2 months. FC values were assessed at the end of EcN treatment (T1) and clinical disease activity at 3 months (T2). At T1 median FC values were significantly lower compared to T0 both in patients with CD (312 mcg/g vs 626 mcg/g, p<0.0001) and UC (100 mcg/g vs 584 mcg/g; p<0.0001). Patients with UC who experienced disease relapse at T2 had lesser reduction in median FC values at T1 (-229 mcg/g, vs -397 mcg/g, p=0.049), while in patients with CD we observed no statistically significant difference (-358 mcg/g, vs -427; p=0.568). In patients with UC, a reduction of at least 532 mcg/g in FC had an accuracy of 69.7% and a positive predictive value of 65.7% in predicting maintenance of remission. A short course of EcN was associated with a reduction of FC values in patients with IBD in clinical remission and baseline altered FC values, and in patients with UC this decrease was associated with maintenance of clinical remission.

Construction of an autolytic system in Escherichia coli Nissle 1917 for efficient release of intracellular recombinant proteins

BackgroundEscherichia coli has emerged as the preferred option in the field of recombinant protein production. However, this necessitates a cell lysis process to extract substantial quantities of recombinant protein. The E. coli Nissle 1917 (EcN) autolysis system was engineered to enhance convenience in the recovery process. This concept would simplify the procedure for retrieving recombinant proteins, streamlining the overall production process. MethodsPrevious studies showed that the fusion of cell-penetrating peptides (Pa) with T4 lysozyme (T4L) to generate 2PaT4L enables E. coli to undergo autolysis, releasing intracellular proteins into the cultivation medium. In this study, the expression plasmid of 2PaT4L was constructed and transformed into ENL6P, the ECN T7 expression system host cell. The induction of this autolysis process is achieved through the induction of IPTG. Significant FindingsAfter IPTG induction, the autolysis of cells gradually reached a dynamic equilibrium with the growth at a low cell concentration. After 24 h of cell autolysis induction, the yields of green fluorescent protein and trehalose synthase could reach 177.27 ± 7.40 and 84.59 ± 4.39 mg/L, respectively. The results show that the ECN autolytic expression system can overexpress recombinant proteins and simplify the recovery process.

Engineered Bacteria: General Overview as Therapeutic Agent and a Novel Drug Delivery System.

Bacterial engineering modifies bacteria's genomic sequence using genetic engineering tools. These engineered bacteria can produce modified proteins, peptides, nucleic acids, and other biomolecules that can be used to treat various medical conditions. Engineered bacteria can target diseased tissues or organs, detect specific biomarkers in the diseased environment, and even induce specific conditions. Furthermore, a meticulously designed intracellular metabolic pathway can activate or inhibit the expression of related genes, synthesise biologically active therapeutic molecules, and precisely deliver drug payloads to diseased tissues or organs. Lactococcus (L. lactis), Salmonella (S. typhi)</i>, and E. coli (E. coli Nissle)</i> are the most studied engineered microorganisms used as drug carriers. These have been used in vaccines to treat multifactorial diseases such as cancer, autoimmune diseases, metabolic diseases, and inflammatory conditions. Other promising strains include Bifidobacterium animalis</i>, Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus lugdunensis</i>, and Clostridium sporogenes</i>. Despite the low reported risk, toxic effects associated with bacterial cells, limiting their efficacy and rapid clearance due to immune responses stimulated by high bacterial concentrations, remain major drawbacks. As a result, a better and more effective method of drug delivery must be developed by combining bacterial-based therapies with other available treatments, and more research in this area is also needed.

Engineered E. coli Nissle 1917 for delivery of bioactive IL-2 for cancer immunotherapy

In this study we performed a step-wise optimization of biologically active IL-2 for delivery using E. coli Nissle 1917. Engineering of the strain was coupled with an in vitro cell assay to measure the biological activity of microbially produced IL-2 (mi-IL2). Next, we assessed the immune modulatory potential of mi-IL2 using a 3D tumor spheroid model demonstrating a strong effect on immune cell activation. Finally, we evaluated the anticancer properties of the engineered strain in a murine CT26 tumor model. The engineered strain was injected intravenously and selectively colonized tumors. The treatment was well-tolerated, and tumors of treated mice showed a modest reduction in tumor growth rate, as well as significantly elevated levels of IL-2 in the tumor. This work demonstrates a workflow for researchers interested in engineering E. coli Nissle for a new class of microbial therapy against cancer.

Decolonizing drug-resistant E. coli with phage and probiotics: breaking the frequency-dependent dominance of residents.

Widespread antibiotic resistance in commensal bacteria creates a persistent challenge for human health. Resident drug-resistant microbes can prevent clinical interventions, colonize wounds post-surgery, pass resistance traits to pathogens or move to more harmful niches following routine interventions such as catheterization. Accelerating the removal of resistant bacteria or actively decolonizing particular lineages from hosts could therefore have a number of long-term benefits. However, removing resident bacteria via competition with probiotics, for example, poses a number of ecological challenges. Resident microbes are likely to have physiological and numerical advantages and competition based on bacteriocins or other secreted antagonists is expected to give advantages to the dominant partner, via positive frequency dependence. Since a narrow range of Escherichia coli genotypes (primarily those belonging to the clonal group ST131) cause a significant proportion of multidrug-resistant infections, this group presents a promising target for decolonization with bacteriophage, as narrow-host-range viral predation could lead to selective removal of particular genotypes. In this study we tested how a combination of an ST131-specific phage and competition from the well-known probiotic E. coli Nissle strain could displace E. coli ST131 under aerobic and anaerobic growth conditions in vitro. We showed that the addition of phage was able to break the frequency-dependent advantage of a numerically dominant ST131 isolate. Moreover, the addition of competing E. coli Nissle could improve the ability of phage to suppress ST131 by two orders of magnitude. Low-cost phage resistance evolved readily in these experiments and was not inhibited by the presence of a probiotic competitor. Nevertheless, combinations of phage and probiotic produced stable long-term suppression of ST131 over multiple transfers and under both aerobic and anaerobic growth conditions. Combinations of phage and probiotic therefore have real potential for accelerating the removal of drug-resistant commensal targets.