Location: Water Management and Conservation Research
Project Number: 2020-13000-004-001-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 3, 2015
End Date: Aug 1, 2019
Objective:
Determine the likelihood of significantly altering the number and type of antibiotic resistant soil bacteria as a consequence of continuous low-level exposure to human antibiotics from effluent irrigation of forested and cropped land.
This objective includes three main hypotheses: I. Environments that receive continuous influxes of low level human antibiotics will have altered numbers of antibiotic resistant bacteria compared to areas only exposed to naturally occurring antibiotics. II. The antibiotic resistant bacteria in areas exposed to low-level human antibiotics will possess resistance to different and/or multiple types of antibiotics compared to those resistant bacteria in areas exposed only to naturally occurring antibiotics. III. Environmental bacteria constantly exposed to low levels of human antibiotics will have novel antibiotic resistance genes compared to environmental bacteria not exposed to human antibiotics.
These hypotheses will be answered by three key research questions: 1. What numbers of antibiotic resistant bacteria are present in environments exposed continuously to low levels of human antibiotics? 2. What types of antibiotic resistance do these resistant bacteria exposed to human antibiotics possess? 3. How does the set of antibiotic resistance genes found in environmental bacteria (environmental resistome) exposed continuously to low levels of human antibiotics compare with resistance genes found in bacteria not exposed to human antibiotics?
Approach:
1. Quantify antibiotics in WWTP effluent spray-irrigated over cropped and forested lands and soil receiving WWTP effluent. Effluent and soil samples will be collected from a site, known as the Living Filter, that receives WWTP effluent year-round to irrigate cropped, grassed and forested lands. Control soil samples will be collected from a control site that has received neither WWTP effluent nor manure applications. Water and soil samples will be analyzed to extract antibiotics using SPE methods modified from EPA 1694 and quantified by LC-MS/MS.
2. Quantify resistance in cultivable soil bacteria. From the soil samples collected at the Living Filter and the control site, bacteria will be selectively cultured and plated using broths and agars for Gram-positive and Gram-negative bacteria. Single isolates will be grown in serial dilutions of select ß-lactams, quinolones, macrolides, sulfonamides, trimethoprim, and lincosamides. Growth at or above a threshold of susceptibility signifies the presence of antibiotic resistance. Although this technique only provides information about cultivable bacteria, it offers a baseline for the type and amount of resistance and allows assessment of multidrug resistance, while making comparisons between Gram-positive and Gram-negative bacteria (Partly answers KQ’s 1 & 2).
3. Quantify resistance genes in soil bacteria. DNA will be extracted from soil samples using a MoBIO PowerSoil DNA isolation kit. Isolated DNA will be analyzed via qPCR to quantify specific resistance genes commonly found in human pathogens. Quantification of genes based on DNA will provide information about the entire bacterial population. However, DNA analysis does not convey gene expression. (Answers KQ 1). The limitation of qPCR is that it only provides information about known ARGs and overlooks unknown ARGs (Partly answers KQ 2).
4. Isolate and identify ARGs in soil bacteria. Isolated DNA will be cloned into various vectors to produce multiple libraries of the total genome of environmental bacteria [10]. DNA will be digested by restriction enzymes, EcoRI or PstI, gel purified by electroelution, and inserted into vectors that do not confer resistance to antibiotics of interest. Vector libraries will be transformed into electrocompetent E. coli cells. To determine average insert size, a subset of plasmids will be isolated, digested, and sized by gel electrophoresis. Clones will then be plated on LB medium containing inhibitory concentrations of select antibiotics, specified previously in 2 of this approach. Colonies possessing antibiotic resistance will be isolated and digested to isolate inserts, which will be sent for DNA sequencing to determine possible ARG(s) (Answers KQ 2). ARG clones will represent an environmental resistome library that can be compared to resistance genes (resistomes) of control sites not receiving WWTP effluent (Answers KQ 3).
