Journal or Publishing Institution: Duke University
Study: https://dukespace.lib.duke.edu/dspace/handle/10161/14394
Author(s): Gardner, C.M.
Article Type: Study
Record ID: 751
Abstract: Antibiotic resistance rates have increased in both clinical and environmental bacteria over the past several decades. While the causes underlying these trends have been investigated in a clinical setting, little work has been done to estimate the contribution of antibiotic resistance genes (ARGs) derived from non-traditional sources like transgenic (GM) crops. The cultivation and consumption of transgenic crops continues to be a widely debated topic, as the potential ecological impacts are not fully understood. In particular, because ARGs have historically been used as selectable markers in the genetic engineering of transgenic crops, it is important to determine if the genetic constructs found in decomposing or consumed transgenic crops persist long enough in the environment and if they can be transferred horizontally to indigenous microorganisms.
The first Objective of this dissertation addresses the question of persistence. Others have also estimated the DNA adsorption capacity of various clays, but have done so by manipulating the surface charge and size of particles tested which may overestimate sorption and underestimate the DNA available for horizontal transfer. In the present study, isotherms were generated using model Calf Thymus DNA and transgenic maize DNA without surface modification. Montmorillonite, kaolinite, and 3 soil mixtures with varying clay content were used in this study. The adsorption capacity of pure montmorillonite and kaolinite minerals was found to be one to two orders of magnitude less than previously estimated likely due to the distribution of clay particle sizes and heteroionic particle surface charge. However, it appears that a substantial amount of DNA is still able to adsorb onto these matrices (up to 200 mg DNA per gram of clay) suggesting the potential availability of free transgenic DNA in the environment may still be significant.
In addition to the soil environments described above, the persistence of these genes should be investigated in agricultural soils. Two important tools exist for understanding the potential contribution of transgenic crop-derived ARGs to environmental antibiotic resistance: (A) the microbiomes associated with transgenic maize and (B) decomposition behavior of transgenic crop biomass. The purpose of Objective Two is to characterize GM maize microbiomes and biomass decomposition to better how transgenic maize may be affecting antibiotic resistance rates among soil bacteria. To investigate this, bulk soil, rhizosphere soil, and internal endophyte prokaryotic microbiomes associated with conventional and transgenic maize were compared using lllumina MiSeq 16S amplicon sequencing. Internal endophytes were significantly influenced by time and location within the maize, but not by maize type. Nitrogen-cycling bacteria in the rhizosphere of transgenic pest-resistant (BT) maize experienced a decrease in diversity at day 56 of maize cultivation that was moderately correlated with the level of Cry1Ab protein exudates in soils surrounding transgenic BT maize. Secondly, bla ARG expression was tracked across 40 weeks of maize biomass decomposition in soils associated with conventional and transgenic BT maize. Bla expression significantly increased in soils associated with BT maize relative to conventional maize soils after 32 weeks or decomposition.
In addition to agricultural environments, wastewater treatment plants (WWTPs) may also contain genes derived from transgenic crops. Consumption of transgenic crops and foods containing them is common in the United States. Once ingested, DNA within transgenic crops can conceivably behave in three ways: 1) DNA will be degraded by acids or DNase I and II enzymes found within the digestive system, 2) DNA will be taken up by cells in the gut (host or bacterial), or 3) DNA will pass through the digestive system and excreted partially or wholly intact. In addition to degradation or uptake, transgenic DNA may be excreted as solid waste by the host. As free DNA contained within food matrices is protected from both enzymatic digestion and acid hydrolysis, the genes contained within foods containing GMOs may be allowed to pass through the host’s digestive tract intact and enter into WWTP environments. Objective 3 assessed this possibility by screening activated sludge and digester sludge obtained from France and the United States for the following four possible transgenes: p35 promoter gene, nos terminator gene, nptII ARG, and bla ARG. All wastewater samples were first screened using end-point PCR and all positive results were further analyzed with qRT-PCR to determine the relative concentration of each gene in wastewater. All domestic activated and digester sludge samples were found to contain at least one transgene, with the majority of samples containing all four. However, wastewater from France, where transgenic crops are neither cultivated nor consumed, contained significantly fewer copies of p35, nos, and nptII genes. Based on these findings, it is possible that some of these genes are derived from partially digested transgenic crop material present in human waste. This is supported in part by the significant differences in p35, nos, and nptII concentrations in French WWTP systems relative to domestic WWTP systems, as well as additional screening for Cauliflower Mosaic Virus (CaMV) ORF VII genes and larger fragments of possible transformation plasmids. Overall it is possible for genes contained within transgenic crops and processed foods containing their byproducts to survive the human digestive process and enter into WWTP environments, though the source of the detected genes could not be definitely confirmed.
The fate and behavior of these for genes in WWTPs remains largely uncharacterized. Further investigation of these trends in WWTP anaerobic digesters is of paramount importance, especially in the case of the bla and nptII ARGs, as a large fraction of WWTP-generated biosolids is used for land application. In addition, because of the relatively high background levels of antibiotic contaminants in wastewaters there is the further possibility that free ARGs may be horizontally transferred to WWTP bacteria, thus increasing antibiotic resistance in the environment. The ability of WWTP bacteria to take up these transgenes in this environment is a factor of (1) the observed rates of horizontal gene transfer (HGT) in anaerobic environments, (2) the abundance of genes of interest within WWTP anaerobic digesters, and (3) the presence of a selective pressure. Like most other topics related to HGT, observing and quantifying the rates of these event in anaerobic environments has proved to be a difficult task. To investigate the behavior of free ARG DNA in WWTP environments, anaerobic digesters were constructed using anaerobic feed sludge from the North Durham (NC) WWTP. Digesters belonged to one of the following treatments and were operated at 47oC in quadruplicate for a period of 60 days: 102 added bla and nptII transgene copies/mL, 104 added bla and nptII transgene copies/mL, or 106 added bla and nptII transgene copies/mL. Control digesters were also constructed. Across 60 days of operation, persistence of free DNA in anaerobic digester wastewater was significantly influenced by DNA fragment length. LUG genes significantly decreased after day 7, but nptII and bla ARGs persisted past 30 days of operation. Bla ARGs were only detected in iDNA within 106 transgene copies/mL treatments at day 60 of incubation. It is possible that this increase is due to bacterial uptake of free bla in eDNA as a result of an antibiotic-driven selective pressure, inducing the integration of bla into bacterial cells. NptII genes were below detection in iDNA at no time points after digester construction. Finally, the naturally occurring concentrations of bla and nptII ARGs in anaerobic digesters are consistent with the findings presented in Chapter 5. However, the existence of these ARGs as primarily eDNA is novel and presently unreported in the literature.
Keywords: Environmental engineering, Antibiotic resistance, DNA adsorption, DNA fate, Horizontal gene transfer, Transgenic crops
Citation: Gardner, C.M., 2017. Microbial Communities and Transgenic Crops: Understanding the Role Transgenic Crops May Play in the Rise of Antibiotic Resistance (Doctoral dissertation). Duke University.
