Bacterial plasmid transfer in the gut: Regulation and inhibition
Date
2024-08
Authors
Ott, Logan Cain
Major Professor
Advisor
Mellata, Melha
Halverson, Larry
McNeill, Elizabeth
Schalinske, Kevin
Smith, Ryan
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Abstract
The emergence and spread of multidrug-resistant (MDR) bacteria are a global concern for human and animal health, with the World Bank estimating a predicted global economic impact of over one trillion dollars in additional healthcare costs by 2050. Furthermore, the Centers for Disease Control (CDC) estimates that 2.8 million antimicrobial-resistant (AMR) infections with an associated 35,000 deaths occurring every year in the United States alone. While the emergence of AMR genes in bacteria involves the relatively slow process of random mutation in genes associated with the mechanism of action of bactericidal chemicals, the spread of resistance genes from bacteria to bacteria is propelled mainly by the rapid plasmid-mediated horizontal gene transfer (HGT), or the process by which extrachromosomal DNA is transferred directly between bacteria through a physically mediated sex pili. This rapid transfer of whole genetic elements allows for the immediate expression and alteration of bacterial resistance, virulence, and metabolic phenotypes. Notably, the gut of humans and other animals plays a significant role as reservoirs for the dense storage and maintenance of complex microbial communities that can further serve as temporary hosts for bacterial plasmids.
However, studying the process of bacterial plasmid conjugation in the host is inherently limited due to the complex nature of the animal gut. Thus, the need for experimental animal models that offer both a simplified, yet representative gut environment is desired. Overall, the objectives of these studies are to evaluate both novel animal models for their potential as proxies for human and agricultural gastrointestinal tracts for gut-mediated plasmid conjugation, as well as identify host factors involved in regulating successful plasmid conjugation in the gut environment.
In Chapter Two, we reviewed the models utilized in the study of bacterial plasmid conjugation both in vitro and in vivo. We reported the limitations and gaps in knowledge of each model. In general, the models are broken into predictive (in silico), host-independent (in vitro), host tissue-associated (ex vivo), and living hosts (in vivo). In Chapter Three, we investigated the reduced complexity Altered Schadler Flora (ASF) murine model as a novel in vivo model for gut-mediated plasmid conjugation. Two genetic backgrounds of ASF mice were orally gavaged with donor (Salmonella Kentucky CVM29188) and recipient (E. coli HS-4) bacteria and monitored for the fecal shedding of donor, recipient, and newly formed transconjugants (E. coli HS-4(pCVM29188_146)). Furthermore, a gene knockout model for chronic inflammation (IL10-/-) and a chemically induced model for acute inflammation (dextran sodium sulfate) were used to demonstrate the effect of host inflammation on conjugation. The fecal shedding of donor, recipient, and transconjugant bacteria was monitored, and the abundance of each of the eight ASF members was determined from qPCR analysis of fecal total DNA. In our model, donor inoculum and host inflammation had a negligible effect on conjugation. However, significant differences in the levels of transconjugant bacteria were detected between mice from each genetic background. This indicates that host genetics provides a significant regulatory function over bacterial plasmid conjugation in a model where all other conditions are controlled for. Additionally, colonization and subsequent conjugation were associated with significant shifts in ASF taxon abundance and distribution in the feces, with increases in the majority of ASF taxon members at day 14 post-inoculation and persistence of elevated concentrations observed at day 28 for Lactobacillus sp. strain ASF360, and Mucispirillum schaedleri strain ASF457. These shifts in ASF taxon abundance indicate that colonization with donor and recipient bacteria results in the competitive exclusion of resident microbiota in the environmental niches of the murine gut.
In Chapter Four, to further determine if host genetics are a driving factor in regulating gut-mediated plasmid conjugation in additional animal models, adult Drosophila melanogaster (fruit flies) of either the CantonS or W1118 genetic background were segregated into male/female populations and challenged with a constant donor strain (E. coli strain K12 substrain MG1655) containing one of three plasmids; the broad host range pKJK5-GM (IncP1ε), or either of the narrow host range plasmids pCVM29188_146 (IncFIB), or pC20-GM (IncI1). The E. coli HS-4 human commensal isolate was used as the recipient in all conjugations. We observed only the transfer of plasmids pKJK5-GM and pC20-GM but not of the plasmid pCVM29188_146 in the gut of Drosophila, indicating that plasmid genetics is a driving force in regulating the incidence of bacterial plasmid transfer in the gut of the Drosophila model for the animal gut. Furthermore, neither host sex nor genetics demonstrated a regulatory role in conjugation in the Drosophila model. This indicates that the complex interactions between host and bacterial plasmid transfer are not universal between animals. However, the differences in the genetics of the two fly strains used in this study may not be divergent enough for a host genetic effect to be observable, and experiments need to be replicated with a greater number of Drosophila genetic backgrounds.
Interestingly, during our studies on the Drosophila model, we identified the potential for one of the preservatives (propionic acid) used in Drosophila media as a potent conjugation inhibitor (COIN). In Chapter Five, we further evaluate the role of propionic acid and the other seven standard short-chain fatty acids (SCFA) as novel conjugation inhibitors. Using in vitro liquid broth conjugation models for the animal gut, we evaluated the effect of supplementation with formate, acetate, propionate, butyrate, valerate, isovalerate, iso-butyrate, and 2-Methylbutanoic acid on the incidence of plasmid transfer. Furthermore, we developed and implemented an in vitro ceca explant model to demonstrate the regulation of conjugation by SCFAs in a host tissue-associated environment. Broth conjugations revealed a universal inhibitory effect of all eight short-chain fatty acids on the transfer of the pathogen-associated pAPEC-O2-211 MDR plasmid from the pathogenic E. coli APEC-O2-211 strain to the human commensal E. coli HS-4 strain at either physiological or elevated concentrations of SCFAs. Furthermore, the same effect was observed when conjugations were repeated in a pH-buffered explant conjugation co-culture assay. Conjugation reactions in the presence of viable chicken cecal tissue and SCFAs demonstrated significant reductions in transconjugants compared to water-treated controls for all SCFAs tested. Finally, to identify if the regulation on conjugation was plasmid specific, in vitro broth conjugation using the broad host range pKJK5-GM (IncP1ε) plasmid or either of the narrow host range plasmids pCVM29188_146 (IncFIB), or pC20-GM (IncI1) were conducted. SCFA supplementation significantly reduced plasmid transfer measured as conjugation frequency for all plasmids tested.
In addition to supplementing SCFAs, we evaluated the efficacy of other dietary supplements with potential COIN activity. Chapter Six shows the application of dietary supplement-derived zinc gluconate and reagent-grade zinc gluconate as novel COINs. In vitro, broth conjugation assays between the E. coli APEC-O2-211 strain and the human commensal E. coli HS-4 strain were supplemented with either supplement-derived or reagent-derived zinc gluconate at varying concentrations. Total ablation of bacterial conjugation and significant bactericidal effects were observed in reactions supplemented with 1 mg/mL of zinc gluconate. Decreased inhibition was associated with stepwise decreases in the final concentration of zinc gluconate. Furthermore, the expression of the replication (rep) and transfer (tra) genes in conjugation supplemented with or without zinc gluconate were evaluated by reverse transcription qPCR. We identified a significant increase in the expression of the repA gene associated with plasmid replication, as well as a significant increase in the expression of tra genes (M, J, E, K, B, P, C, W, U, N, F, Q, D, I, and X) associated with the construction and function of the type four secretion system (T4SS) used by the incFIB plasmid pAPEC-O2-211A-ColV. Additionally, upon exposure to zinc, we observed a down-regulation of the expression of mRNA for the conjugal pilus gene traA and the entry exclusion gene traS, both of which are required for fertile plasmid conjugation and survival of recipient cells. Alteration in the expression of genes critical for both replication and the transfer of plasmid DNA during bacterial conjugation may cause the significant inhibition of plasmid conjugation observed. Further evaluation of both animal models and novel COINs is ongoing and warrants more research focus.
The studies described in this dissertation identify two novel in vivo models for conjugal plasmid transfer in the gut and two compounds with novel COIN activity. Continued studies are needed to fully elucidate the molecular mechanisms for host and plasmid genetic modulation of conjugation in the gut identified herein. Additionally, in vivo studies are required to confirm the efficacy of SCFA and zinc supplementation to mitigate plasmid-mediated MDR emergence and spread in human and animal gut microbiota.
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