Introduction of Plasmid to the Murine Gut via Consumption of an Escherichia coli Carrier and Examining the Impact of Bacterial Dosing and Antibiotics on Persistence

Abstract

Purpose We examine the impacts of dosing strategies of plasmids on bacterial communities in the murine gut by measuring the quantity of plasmids in mouse feces. Methods We fed mice carrier bacteria, E. coli, that contain plasmids with both a reporter gene and an antibiotic resistant gene. We varied the quantity of the plasmid-carrying bacteria and the length of time the mice consumed the bacteria. We also pretreated the gut with broad-spectrum antibiotics and used continuous antibiotic treatment to investigate selection pressure. We collected bacteria from fecal pellets to quantify the number of plasmid-carrying bacteria via plate assay. Results Dosing regimens with plasmid-carrying bacteria resulted in a significantly increased duration of persistence of the plasmid within the gut when supplemented continuously with kanamycin during as well as after completion of bacterial dosing. The carrier bacteria concentration influenced the short-term abundance of carrier bacteria. Conclusion We evaluated the persistence of plasmid-carrying bacteria in the murine gut over time using varying dosage strategies. In future work, we will study how bacterial diversity in the gut impacts the degree of plasmid transfer and the prevalence of plasmid-carrying bacteria over time. Lay Summary Observing how plasmids persist within the gut can help us understand how newly introduced genes, including antibiotic resistance, are transmitted within the gut microbiome. In our experiments, mice were given bacteria containing a genetically engineered plasmid and were examined for the persistence of the plasmid in the gut. We found long-term persistence of the plasmid in the gut when administering antibiotics during and following dosing of the mice with bacteria carrying the plasmid. The use of higher concentrations of carrier bacteria influenced the short-term abundance of the plasmid-carrying bacteria in the gut. Description of Future Works Building on evidence from these initial studies that persistence of plasmids within the gut can be regulated by the dosage strategy, we will explore future studies and models of gene uptake in the context of spatial and taxonomic control and further determine if dosing strategies alter the compositional diversity of the gut microbiome. Graphical abstract

Engineering Escherichia coli for Conversion of Dietary Isoflavones in the Gut

“…As previously reported, ad lib consumption of recombinant E. coli Nissle 1917 supplied in the drinking water of mice could ensure persistence of the recombinant bacteria within the gut by controlling the duration and concentration of the bacterial supplement. 20 After 1 week, blood collection was again performed in the same manner and the serum frozen until analysis. Because the bacterial strains were provided continuously within the drinking water for the given groups, the bacteria could persist as previously demonstrated at a level of approximately 10 7 CFU per fecal pellet within the gut during the course of the experiments.…”

“…Because the bacterial strains were provided continuously within the drinking water for the given groups, the bacteria could persist as previously demonstrated at a level of approximately 10 7 CFU per fecal pellet within the gut during the course of the experiments. 20 A positive serum equol control group of 3 mice did not receive Nissle P1 or Nissle P2 but were instead provided regular water along with a 60 g hydrogel containing 14 mg of equol (replaced at two day intervals) for 1 week of ad lib consumption, and blood collection was performed as described above. All gels and water bottles were weighed every 2 days in order to determine the average amount of hydrogel or liquid consumed per mouse.…”

“…After blood collection, the mice remained on their phytoestrogen free diet and were provided the same category of hydrogel for an additional week along with replacement of their water bottle with water containing a 1-to-1 mixture of Nissle P1 and Nissle P2 each at a concentration of 10 8 CFU/mL. As previously reported, ad lib consumption of recombinant E. coli Nissle 1917 supplied in the drinking water of mice could ensure persistence of the recombinant bacteria within the gut by controlling the duration and concentration of the bacterial supplement . After 1 week, blood collection was again performed in the same manner and the serum frozen until analysis.…”

“…Our prior studies examining the persistence of E. coli Nissle 1917 within the murine gut suggest a twice weekly supplement of bacteria at 10 8 CFU/mL to be sufficient for maintaining the presence of the engineered E. coli Nissle without the use of selection pressure. 20 Thus, we examined the ability of the combination of engineered E. coli Nissle strains harboring P1-DZNR-DDRC or P2-DHDR-THDR, herein referred to as Nissle P1 and Nissle P2, respectively, to provide production of equol within the gut when cosupplemented along with dietary daidzein in murine studies. As discussed below, we confirmed the conversion of daidzein to equol in cultures of Nissle P1 mixed with Nissle P2 as well as examined the individual wholecell biotransformation rates in separate cultures.…”

“…E. coli Nissle was implemented as this strain has been widely adopted as a probiotic and has been engineered in other studies for use as bacterial therapeutics of metabolic diseases; however, in this work we report the first expression of the enzymatic pathway for conversion of daidzein to equol in E. coli Nissle. Our prior studies examining the persistence of E. coli Nissle 1917 within the murine gut suggest a twice weekly supplement of bacteria at 10 8 CFU/mL to be sufficient for maintaining the presence of the engineered E. coli Nissle without the use of selection pressure . Thus, we examined the ability of the combination of engineered E. coli Nissle strains harboring P1-DZNR-DDRC or P2-DHDR-THDR, herein referred to as Nissle P1 and Nissle P2, respectively, to provide production of equol within the gut when cosupplemented along with dietary daidzein in murine studies.…”