Progress 07/01/18 to 06/30/23
Outputs
Target Audience:The target audience for this reporting project includes the environmental engineering scientific community, the food science and technology scientific community, the wastewater treatment utilities' operators, engineers, and managers, Oregon and nationalproduce growers, andpolicymakers at the State and national levels. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Over the course of this project, two PhD students (one dissertation work and one research project work), threeMasters students (thesis work), two PhD students (one thesis work and one lab rotation), four graduate students in research rotations, two graduate students for research credits,ten undergraduate students, and seven high school studentshave been trained and involved in the project. Existing and new lab protocols have been developedand modified. Participating students have been trained and their efforts resulted in several presentations that have beenvaluable in their professional development including three invited talks, eight oral presentations withpeer-reviewed abstracts, two poster presentations with peer-reviewed abstracts, and nineteen non-peer-reviewed poster presentations. How have the results been disseminated to communities of interest?A report was sent to the 17 participating wastewater utilities in 2022 including copies ofthree publications from the project. In this report, a summary of each paper was described and findings and conclusions were discussed. In addition, to each utility,individual utility's data was shared privately. A final report will be shared with utilities once the remaining three papers are published. Seven female, under-represented minority high school students were involved in summer projects to understand the emergence and associated health risks of antibiotic resistance in environmental reservoirs. These students were mentored by graduate and undergrad students in the lab to quantify antibiotic-resistant E. coli in water and soil samples. They used several treatments (boiling, chlorine disinfection, and UV disinfection) to test and understand the impact of treatment processes on the reduction and removal of bacteria in water and soil samples. Their results were presented atposter symposiums onthe OSU campus. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Accomplishments under Research Objectives 1 and 2 are summarized in four (papers 1-4) and two parts (papers 5-6) respectively, each published or in preparation for publication in a peer-reviewed journal. 1) Major activities completed: Paper 1. Wastewater samples (influent, secondary effluent, final effluent, and biosolids) were collected from 17 wastewater utilities across Oregon (12 in the Willamette Valley and coastal region and 5 in the eastern high desert).Samples were collected in the Winter and Summers of 2019 and 2020. Paper 2. Treated wastewater effluent was collected from one utility weekly for ten weeks between February and April 2019. Papers 3&4. Eleven extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates were collected and confirmed from eight wastewater utilities across Oregon. Papers 5&6. Carrot seeds were planted in soil that received biosolids amendment and/or wastewatereffluent irrigation in a greenhouse setting. Soil samples were collected weekly and carrot rinse were collected at harvest. 2) Data collected Paper 1. 20 antibiotics andfour geneswere quantified. The samples' microbiomes were assessed through 16S amplicon sequencing. E. coliwas isolated from wastewater samples to determine the phenotypic resistance to common or clinically relevant antibiotics. Paper 2. Ten commonly reported antibiotic resistance genes (ARGs) and 20 antibiotics were quantified. Time variances of ARGs and antibiotics were assessed and associations between analytes were investigated. Paper 3. Whole genome sequencing was performed to identify the most abundant ARGs including ESBL-associated genes, virulence factors, as well as their sequence types. Moreover, the phenotypes of antibiotic resistance were characterized. Paper 4. The genome of ESBL-producing E. coli isolates were resolved. Using a hybrid sequencing and assembly strategy, ARGs and virulence factors harbored on plasmids and chromosomes were identified. The transferability of these plasmid-mediated ESBLs via conjugation was investigated. Using shotgun metagenomic analysis of the E. coli-originated wastewater samples, we described the microbial community, the resistome composition, and the potential associations with plasmid-mediated ESBL genes and other ARGs. Paper 5. Total and antibiotic-resistant fecal bacterial indicators (E. coli and enterococci) were quantified weekly in soil and at harvest (day 77) on carrots. Antibiotic susceptibility of 121 E. coli and 110 enterococci collected isolates were determined. Paper 6. Shotgun metagenomics was used to determine the impact of biosolids land application on the enrichment of the microbiome and resistome in soil. 3) Summary and discussion of results Paper 1. During the summer months, trimethoprim was found to be significantly more prevalent in influent samples as compared to winter samples. Lincomycin and roxithromycin were significantly more prevalent in influent during the winter months. Final effluent and biosolids samples in winter also demonstrateda considerably higher prevalence of multidrug resistance when compared to summer samples. When investigating regional variations, despite having relatively similar influent concentrations, higher removals of trimethoprim were observed in eastern Oregon compared to the coastal/valley region. Lincomycin was significantly more abundant in samples from eastern treatment utilities. Western Oregon was observed to have higher rates of phenotypicE. coliresistance to at least one antibiotic in every sample type. The prevalence of multidrug resistance was observed to be higher in western samples when compared to eastern in secondary, effluent, and biosolids samples. Paper 2. We determined that there was no significant temporal variation in the abundance of individual ARGs throughout the study. The most prevalent ARGs were sul1, tetM, and ermB. There were significant correlations between the prevalence of sul2 with intI1, qnrS, and sul1. The most prevalent antibiotic was trimethoprim. Paper 3. ESBL-associated genes were found in all but one of the isolates with five isolates carrying two of these genes. The ampC gene and virulence factors were present in all the E. coli isolates. Across all the isolates, 31 different ARGs were identified. All E. coli isolates harbored phenotypic resistance to beta-lactams, while 8 of the 11 isolates carried multidrug resistance phenotypes. Paper 4. Class A beta-lactamase genes on chromosomes were clustered with ARGs associated with other classes of antibiotics along with insertional elements. One novel IncF plasmid was identified which carried a multi-drug resistance genotype in addition to a mercury resistance operon, colicin, and aerobactin genes. The occurrence of additional ESBL-associated genes was identified in metagenomes and their occurrence was correlated with core microbial genera. Paper 5. E. coli or enterococci were not recovered from the soil without biosolids amendment regardless of the irrigation water source. After biosolids amendment, soil E. coli and enterococci concentrations increased significantly within the first week, declined slowly over time, but stayed above the detection limit over the entirety of the study. No statistical difference was found between quantified bacteria after effluent wastewater or water irrigation in soil or on carrots. Soil antibiotic-resistant E. coli and enterococci decayed significantly faster than total E. coli and enterococci. At harvest, E. coli were present on carrots; the majority of which were resistant to ampicillin. Paper 6. At the beginning of the study, a higher microbial richness was observed in biosolids-amended samples but it decreased over time to roughly equal levels with non-amended soil at harvest. A total of 198 and 318 unique ARGs were identified in pristine and amended soils, respectively. The relative abundance of multi-drug resistance genes increased with time in biosolids-amended samples, whereas they stayed comparatively stable in pristine soil. 4) Key outcomes Paper 1. While the presence of antibiotic resistance has been shown to have seasonal and regional influence, findings suggest that the differences may be attributed to the populations' access to and consumption of different antibiotics. Paper 2. The observations of ARG and antibiotic abundances can serve to support the existing literature in describing the nature of the prevalence of, and relationships within and between antibiotic and ARG abundances. Paper 3. Findings highlight the risks associated with the presence of ESBL-producing E. coli isolates in wastewater systems that have the potential to enter the environment and may pose direct or indirect risks to human health. Paper 4. This study's unique incorporation of hybrid whole-genome sequencing, culture-dependent methods, and metagenomics provides a holistic picture of ESBL-producing E. coli within Oregonian wastewater systems, and by proxy Oregonian communities. Paper 5. The survival of antibiotic-resistant enteric bacteria in soil and on harvested carrots indicates there are transmission risks associated with biosolids amendment use in root crops. Paper 6. The lasting increase in abundance and diversity of soil ARGs indicates that biosolids amendment contributed to the enrichment of the resistome. Papers 1-6 citations: Ste. Marie,et al. (in preparation) Seasonal and regional impacts on antibiotic pollution and antibiotic resistance in Oregon wastewater systems Ste. Marie, et al. (in preparation) Temporal variability of antibiotics and antibiotic resistance genes in wastewater effluent Easler, et al. (2022) Journal of Water and Health, 20 (4), 670-679. Cheney,et al. (in preparation) Resolved Genomes of ESBL-Producing Escherichia coli and Metagenomic Analysis of Source Wastewater Samples Mays, et al. (2021) Water Research X, X, 13, 100119. Ste. Marie, et al. (in preparation) Impact of biosolids amendment on the soil resistome and microbial community
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2019
Citation:
Mays, C. (2019) Antibiotic Resistance in Treated Wastewater Effluent and Biosolids and Fate after Agricultural Reuse, Oregon State University
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Khorshidi-Zadeh, M. (2021) Prevalence and Fate of Antibiotic-resistant E. coli in Municipal Wastewater Treatment Plants across Oregon, Oregon State University
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2022
Citation:
Cheney, C. (2022) Genomic Characterization of Extended-Spectrum Beta-Lactamase Producing Escherichia coli in Oregonian Wastewater, Oregon State University
- Type:
Theses/Dissertations
Status:
Awaiting Publication
Year Published:
2023
Citation:
Ste. Marie, J. (2023) Antibiotic Pollution and the Resistome: Antibiotic Resistance in Oregon Wastewater and Receiving Environments
- Type:
Journal Articles
Status:
Other
Year Published:
2023
Citation:
Ste. Marie, J., Mays, C, Jones, G., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (in preparation) Temporal variability of antibiotics and antibiotic resistance genes in wastewater effluent
- Type:
Journal Articles
Status:
Other
Year Published:
2023
Citation:
Ste. Marie, J., Mays, C., Guo, B., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (in preparation) Impact of biosolids amendment on the soil resistome and microbial community
- Type:
Journal Articles
Status:
Other
Year Published:
2023
Citation:
Ste. Marie, J., Khorshidi-Zadeh, M., Yiu, S.Y., Nguyen, J.N., Garza, G.L., Jones, G., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (in preparation) Seasonal and regional impacts on antibiotic pollution and antibiotic resistance in Oregon wastewater systems
- Type:
Journal Articles
Status:
Other
Year Published:
2023
Citation:
Cheney, C., Johnson, J.D., Ste Marie, J., Gacosta, K.Y.M., Denlinger Drumm, N.B., Jones, G., Waite-Cusic, J., Navab-Daneshmand, T. (in preparation) Resolved Genomes of ESBL-Producing Escherichia coli and Metagenomic Analysis of Source Wastewater Samples
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Ste. Marie, J., Khorshidi, M., Nguyen, J., Yiu, S.Y., Radniecki, T., Jones, G., Waite-Cusic, J., Navab-Daneshmand, T. (2023) Seasonal and geographical variations of antimicrobial resistance in Oregon wastewater, Association of Environmental Engineering and Science Professors Conference, Boston, MA (peer-reviewed conference presentation)
|
Progress 07/01/21 to 06/30/22
Outputs
Target Audience:The target audience for this reporting period includes the environmental engineering scientific community, the food science and technology scientific community, the wastewater treatment facility operators, Oregon produce growers, and policymakers at the state and national levels. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In this reporting period, one Masters student (thesis work), two PhD students (one thesis work and one lab rotation), and three undergraduate students have been trained and involved in the project. Existing and new lab protocols have been developed and modified. Participating students have been trained and their efforts resulted in several presentations which have been valuable in their professional development. Following is the list of presentations (the names of the students are underlined): Easler, M., Cheney, C., Johnson, J.D., Khorshidi-Zadeh, M., Nguyen, J.N., Yiu, S.Y., Waite-Cusic, J., Radniecki, T.R., Navab-Daneshmand, T. (2022) Resistome characterization of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from wastewater treatment utilities in Oregon, Journal of Water and Health, 20 (4), 670-679. Cheney, C., Johnson, J., Gacosta, K., Drumm, N.D., Jones, G., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (2022) Extended-spectrum beta-lactamase resistance spreads horizontally in Oregonian wastewater system, Association of Environmental Engineering and Science Professors Conference, Washington University, St. Louise, MO. Ste Marie, J., Mays, C., Radniecki, T., Waite-Cusic, J., Guo, B., Navab-Daneshmand, T. (2022) Impact of biosolids soil amendment on the resistome and microbial community structure in a greenhouse study, Association of Environmental Engineering and Science Professors Conference, Washington University, St. Louise, MO. Cheney, C., Easler, M., Johnson, J.D., Khorshidi-Zadeh, M., Nguyen, J.N., Yiu, S.Y., Waite-Cusic, J., Radniecki, T., Navab-Daneshmand, T. (2021) ESBL-producing Escherichia coli in Oregonian wastewater treatment systems, The Society of Environmental Toxicology and Chemistry (SETAC) North America Virtual Conference. Khorshidi-Zadeh, M., Yiu, S.Y., Nguyen, J.N., Garza, G.L., Waite-Cusic, J., Radniecki, T., Navab-Daneshmand, T. (2021) Seasonal trends of antibiotic-resistant E. coli in Oregon wastewater treatment utilities, The Society of Environmental Toxicology and Chemistry (SETAC) North America Virtual Conference. Ste Marie, J., Mays, C., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (2021) Time variance of antibiotics and antibiotic resistance genes in wastewater effluent from a conventional wastewater treatment facility, The Society of Environmental Toxicology and Chemistry (SETAC) North America Virtual Conference. Cheney, C., Easler, M., Johnson, J.D., Khorshidi-Zadeh, M., Nguyen, J.N., Yiu, S.Y., Waite-Cusic, J., Radniecki, T., Navab-Daneshmand, T. (2021) Multi-drug resistant and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from wastewater treatment facilities in Oregon, Microbial Ecology and Water Engineering Specialist Virtual Conference. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Summary of accomplishments under research objective 1: Extended-spectrum beta-lactamase (ESBL) producing Escherichia coli are a serious threat to human health because of their resistance to the most commonly prescribed antibiotics: penicillins and cephalosporins. In our study, we provide a genomic and metagenomic context for the determinant ESBL genes of E. coli isolated from various wastewater treatment utilities in Oregon, USA. Results showed that class A beta-lactamase genes on chromosomes (blaCTX-M, blaTEM) were clustered with antibiotic resistance genes associated with other classes of antibiotics (sulfonamides and aminoglycosides) along with insertional elements. ESBL genes such as blaCTX-M, blaTEM and blaSHV were also detected on conjugable plasmids of IncF and IncI incompatibility types. One IncF plasmid novel to this study carried a multi-drug resistance genotype in addition to a mer mercury resistance operon and colicin and aerobactin virulence factors. Shotgun metagenomic analysis of the E. coli-originating wastewater samples showed the presence of class A beta-lactamases, however, the specific ESBL genes in the E. coli genomes were below the detection limits. The occurrence of blaOXA.11, blaFOX.7, and blaGES.17 ESBL-associated genes were correlated with core microbial genera such as Paraprevotella. In both the E. coli genomes and the wastewater samples, tetracycline, aminoglycoside, and beta-lactam resistance determinants frequently co-occurred. The unique combination of whole-genome and metagenomic analysis provides a holistic description of ESBL-producing organisms and genes in the Oregonian wastewater system. The co-occurrence of antibiotics, pathogens, ARGs, mobile genetic elements, and already resistant bacteria make conventional wastewater treatment facilities a high-risk environment for the dissemination of resistance. However, there is no consensus on the abundance of antibiotics and ARGs in wastewater effluent. In our study, effluent from a conventional wastewater treatment facility in Oregon was sampled weekly for ten weeks to investigate variations in the abundance of ARGs and antibiotics in time. Presence/absence of 7 ARGs and one class 1 integron gene were determined. Relative abundance (normalized to 16S rRNA gene copies) of detectable genes was further quantified. Relative ARG abundance ranged from 4.2 × 10-1 to 2.3 × 10-5. Concentrations of 20 target antibiotics representing major drug classes will be determined by HPLC-MS/MS. Correlations between concentrations of antibiotics and ARGs will be statistically tested to determine co-occurrence. It is hypothesized that some antibiotics will be commonly detected in all samples due to their use in the serving population and incomplete removal during treatment. ARGs are hypothesized to remain prevalent in the effluent due to selective pressure from corresponding classes of antibiotics, resulting in their co-occurrence (e.g., sulfonamide antibiotics will co-occur with sulfonamide resistance). The key preliminary result from this study is that no outliers were detected from 10 sampling days regarding the relative abundance of each studied ARG. This suggests that ARG discharge from a facility without combined sewer systems may be relatively consistent over time. This work provides a better framework for interpreting variability in antibiotic and ARG data from wastewater treatment plants, as well as a broader understanding of the risk posed by antibiotics in wastewater. Summary of accomplishments under research objective 2: Biosolids generated during wastewater treatment are often land-applied to improve soil properties. Due to the presence of antibiotics, pathogens, and antibiotic-resistant bacteria, this amendment is thought to contribute to the proliferation and dissemination of antibiotic resistance genes (ARGs). However, the diversity of ARGs and the transience of the resistome and microbial community in amended soils are not well characterized. Here, we characterized the impact of biosolids amendment on the diversity of bacteria and ARGs in soil throughout the cultivation of carrots in a greenhouse study. Soil samples were collected from triplicate pots of pristine and biosolids-amended soil at the time of planting, halfway through cultivation (week 6), and at harvest (week 11) and environmental DNA was shotgun sequenced. At the beginning of the study, a higher microbial richness was observed in biosolids-amended samples compared to pristine soils, but it decreased over time to roughly equal levels at harvest. The biosolids-amended samples harbored a higher relative abundance of proteobacteria, which increased over time while remaining relatively stable in pristine soil. Bacteroidetes were also enriched in amended soils but decreased over time to near the same level as pristine samples. A total of 198 and 318 unique ARGs were identified in pristine and amended soils, respectively. The most enriched ARGs in the amended soils confer resistance to sulfonamides, tetracyclines, and multi-drug resistance. The relative abundance of multi-drug resistance genes increased with time in biosolids-amended samples, whereas they stayed comparatively stable in pristine soil. ARG diversity in biosolids-amended pots remained considerably higher throughout cultivation indicating that biosolids-amendment may be associated with long-term increases in ARG diversity and multi-drug resistance. The results from this study illuminate the role that biosolids-amendment plays in ARG proliferation in soil and enable us to better foster informed decisions and actions in biosolids management.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Easler, M., Cheney, C., Johnson, J.D., Khorshidi-Zadeh, M., Nguyen, J.N., Yiu, S.Y., Waite-Cusic, J., Radniecki, T.R., Navab-Daneshmand, T. (2022) Resistome characterization of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from wastewater treatment utilities in Oregon, Journal of Water and Health, 20 (4), 670-679.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Cheney, C., Johnson, J., Gacosta, K., Drumm, N.D., Jones, G., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (2022) Extended-spectrum beta-lactamase resistance spreads horizontally in Oregonian wastewater system, Association of Environmental Engineering and Science Professors Conference, Washington University, St. Louise, MO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Ste Marie, J., Mays, C., Radniecki, T., Waite-Cusic, J., Guo, B., Navab-Daneshmand, T. (2022) Impact of biosolids soil amendment on the resistome and microbial community structure in a greenhouse study, Association of Environmental Engineering and Science Professors Conference, Washington University, St. Louise, MO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Cheney, C., Easler, M., Johnson, J.D., Khorshidi-Zadeh, M., Nguyen, J.N., Yiu, S.Y., Waite-Cusic, J., Radniecki, T., Navab-Daneshmand, T. (2021) ESBL-producing Escherichia coli in Oregonian wastewater treatment systems, The Society of Environmental Toxicology and Chemistry (SETAC) North America Virtual Conference.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Khorshidi-Zadeh, M., Yiu, S.Y., Nguyen, J.N., Garza, G.L., Waite-Cusic, J., Radniecki, T., Navab-Daneshmand, T. (2021) Seasonal trends of antibiotic-resistant E. coli in Oregon wastewater treatment utilities, The Society of Environmental Toxicology and Chemistry (SETAC) North America Virtual Conference.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Ste Marie, J., Mays, C., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (2021) Time variance of antibiotics and antibiotic resistance genes in wastewater effluent from a conventional wastewater treatment facility, The Society of Environmental Toxicology and Chemistry (SETAC) North America Virtual Conference.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Cheney, C., Easler, M., Johnson, J.D., Khorshidi-Zadeh, M., Nguyen, J.N., Yiu, S.Y., Waite-Cusic, J., Radniecki, T., Navab-Daneshmand, T. (2021) Multi-drug resistant and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from wastewater treatment facilities in Oregon, Microbial Ecology and Water Engineering Specialist Virtual Conference.
|
Progress 07/01/20 to 06/30/21
Outputs
Target Audience:The target audience for this reporting period includes the environmental engineering scientific commununity, the food science and technology scientific community, the wastewater treatment facility operators, Oregon produce growers, and policy makers at the state and national levels. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In this reporting period, two Masters students (thesis work), two PhD students (one thesis work and one lab rotation), and four undergraduate students have been trained and involved in the project. Existing and new lab protocols have been developed and modified. Participating students have been trained and their efforts resulted in several presentations which have been valuable in their professional development. Following is the list of presentations (the names of the students are underlined): Ste. Marie, J., Mays, C., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (2021) Changes in the microbial community and resistome in pristine and biosolids-amended soils during carrot cultivation, Center for Quantitative Life Science Virtual Conference, Oregon State University. Ste. Marie, J., Mays, C., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (2021) Time variance of antibiotics and antibiotic resistance genes in wastewater effluent from a conventional wastewater treatment facility, Clean Water Virtual Workshop, Oregon State University. Gacosta, K., Denlinger-Drumm, N., Cheney, C., Easler, M., Khorshidi-Zadeh, M., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (2021) Characterization and gene transfer rates of beta-lactam resistant Escherichia coli from Oregonian wastewater treatment plants, Clean Water Virtual Workshop, Oregon State University. Denlinger-Drumm, N., Ste. Marie, J., Navab-Daneshmand, T. (2021) Quantification of Antibiotic Concentrations within Wastewater, Celebrating Undergraduate Excellence, Oregon State University. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Summary of accomplishments under research objective 1: Wastewater treatment utilities are considered one of the main sources and reservoirs of antimicrobial resistance. The objective of this study was to determine the diversity and prevalence of antibiotic-resistant Escherichia coli in wastewater treatment systems across the state of Oregon. Influent, secondary effluent, final effluent, and biosolids were collected from 17 wastewater treatment utilities across Oregon during the winter and summer seasons of 2019 and 2020 (n = 246). E. coli strains were recovered from samples by culturing on mTEC, followed by confirmation with MacConkey with MUG agar plates. Antibiotic susceptibility of 1143 E. coli isolates against 8 antibiotics were determined, and resistance profiles and indices were analyzed between utilities, seasons, and flows. Antibiotic resistancephenotypes were detectedin 31.6% of the collected E. coli isolates. Among those antibiotic-resistant E. coli isolates, multi-drug resistance (i.e., resistance to three or more classes of antibiotics) was harbored by 27.7% with some strains showing resistance to up to six classes of antibiotics. The most prevalent resistance was to ampicillin (n = 207) and the most common combinations of multi-drug resistance included simultaneous resistances to ampicillin, streptomycin, and tetracycline (n = 49), followed by ampicillin, streptomycin, and sulfamethoxazole/trimethoprim (n = 46). Significant correlations were observed between resistance to sulfamethoxazole/trimethoprim and resistances to ampicillin, ciprofloxacin, and tetracycline (p < 0.001). A small percentage (1.1%) of the E. coli isolates displayed extended-spectrum beta lactamase (ESBL) activity and a single isolate carried resistance to imipenem. Compared to wastewater influent, ciprofloxacin resistance was significantly more prevalent in biosolids (p <0.05) and tetracycline resistance was significantly lower in effluent (p <0.05). Seasonal impact on antibiotic-resistant E. coli in wastewater influent was observed through significantly higher multiple antibiotic resistance (MAR) index, ampicillin resistance prevalence, and ciprofloxacin resistance prevalence in summer compared to winter (p < 0.05).This state-wide study confirms the widespread distribution of antibiotic-resistant, multi-drug resistant, and extended-spectrum beta lactamase-producingE. coli in wastewater systems across different flows and seasonal variations, making them the recipients, reservoirs, and sources of antimicrobial resistance. Moreover, infections resistant to broad spectrum antibiotics due to the emergence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is of global concern. This study characterizes the resistome (i.e., entire ecology of resistance determinants) of 11 ESBL-producing E. coli isolates collected from eight wastewater treatment utilities across Oregon. The collected isolates were sequenced using an Illumina MiSeq system and analyzed to identify the most abundant antibiotic resistance genes including ESBL-associated genes, virulence factors, as well as their sequence types. Moreover, the phenotypes of antibiotic resistance were characterized. ESBL-associated genes (i.e., blaCMY, blaCTX, blaSHV, blaTEM) were found in all but one of the isolates with five isolates carrying two of these genes (4 with blaCTX and blaTEM; 1 with blaCMY and blaTEM). The ampC gene and virulence factors were present in all the E. coli isolates. Across all the isolates, 31 different antibiotic resistance genes were identified. Additionally, all E. coli isolates harbored phenotypic resistance to beta-lactams (penicillins and cephalosporins), while eight of the 11 isolates carried multi-drug resistance phenotypes (resistance to three or more classes of antibiotics). Findings highlight the risks associated with the presence of these ESBL-producing E. coli isolates in wastewater systems that have the potential to enter the environment and may pose direct or indirect risks to human health. Summary of accomplishments under research objective 2: Reuse of wastewater effluent and biosolids in agriculture is essential to sustainable water and nutrient resource management practices. Wastewater and biosolids, however, are reportedly the recipients, reservoirs, and sources of antibiotic-resistant enteric pathogens. While decay rates of fecal bacterial indicators in soil are frequently studied, very few studies have reported on the persistence of the antibiotic-resistant sub-populations. Little is known about how multi-drug resistance phenotypes of enteric bacteria in agricultural soil change over time. In this study, germinated carrot seeds were planted in soil that received biosolids amendment and/or wastewater effluent irrigation in a greenhouse setting. We quantified total and antibiotic-resistant fecal bacterial indicators (E. coli and enterococci) weekly in soil and total E. coli at harvest (day 77) on carrots. Antibiotic susceptibility of 121 E. coli and 110 enterococci collected isolates were determined. E. coli or enterococci were not recovered from the soil without biosolids amendment regardless of the irrigation water source. After biosolids amendment, soil E. coli and enterococci concentrations increased more than 3 log10CFU/g-TS within the first week, declined slowly over time, but stayed above the detection limit (0.39 CFU/g-TS) over the entirety of the study. No statistical difference was found between effluent wastewater or water irrigation in soil total and antibiotic-resistant E. coli and enterococci concentrations or carrots E. coli levels. Soil antibiotic-resistant E. coli and enterococci decayed significantly faster than total E. coli and enterococci. Moreover, the prevalence of multi-drug resistant (resistance to three or more antibiotics) E. coli declined significantly over time, while almost all collected enterococci isolates showed multi-drug resistance phenotypes. At harvest, E. coli were present on carrots; the majority of which were resistant to ampicillin. The survival of antibiotic-resistant enteric bacteria in soil and on harvested carrots indicates there are transmission risks associated with biosolids amendment use in root crops.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Mays, C., Garza, G.L., Waite-Cusic, J., Radniecki, T.R., Navab-Daneshmand, T. (2021) Impact of biosolids amendment and wastewater effluent irrigation on enteric antibiotic-resistant bacteria ⿿ a greenhouse study, Water Research X, X, 13, 100119. https://doi.org/10.1016/j.wroa.2021.100119.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Khorshidi-Zadeh, M., Yiu, S.Y., Nguyen, J.N., Garza, G.L., Waite-Cusic, J., Radniecki, T.R., Navab-Daneshmand, T. (under review) Antibiotic resistance profile of E. coli isolates in 17 municipal wastewater utilities across Oregon.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Easler, M., Cheney, C., Johnson, J.D., Khorshidi-Zadeh, M., Nguyen, J.N., Yiu, S.Y., Waite-Cusic, J., Radniecki, T.R., Navab-Daneshmand, T. (under review) Resistome characterization of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from wastewater treatment utilities in Oregon.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Navab-Daneshmand, T. (2020) Antibiotic resistance in wastewater and biosolids, and fate after agricultural reuse, AEESP Converging COVID-19, Environment, Health, and Equity Virtual Conference. (invited conference presentation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Mays, C., Waite Cusic, J., Radniecki, T., Navab-Daneshmand, T. (2020) Antibiotic-resistant E. coli and enterococci in soil and on carrots after biosolids amendment and wastewater irrigation, Water and Health Virtual Conference. (peer-reviewed conference presentation)
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Progress 07/01/19 to 06/30/20
Outputs
Target Audience: The target audience for this reporting period includes the environmental engineering scientific commununity, the food science and technology scientific community, the wastewater treatment facility operators, Oregon produce growers, and policy makers at State and national levels. Changes/Problems:For research objective 1, 17 wastewater treatment facilities across Oregon were enrolled in the project. The plan included collecting wastewaste influent, secondary, and effleunt, as well as biosolids from each facility over winters and summers 2019 and 2020. In summer 2020 we were not able to collect samples from four of the 17 facilities due to the facilities limitations during the COVID-19 pandemic and the Oregon wildfires. The sampling from other three seasons as well as the other 13 facilities in summer 2020 are complete and therefore, we do not nticipate significant impact of the missed samples from these four facilities on the overall results and conclusions. What opportunities for training and professional development has the project provided? In this reporting period, two Masters students (thesis work), three PhD students (one thesis work and two lab rotations), five undergraduate students, and three high school students have been trained and involved in the project. Existing and new lab protocols have been developed and modified. Participating students have been trained and their efforts resulted in several presentations which have been valuable in their professional development. Following is the list of presentations (the names of the students are underlined): Invited talk: Navab-Daneshmand, T. (2019) Antibiotic resistance in environmental reservoirs and associated human health risks, Clean Water Workshop, Oregon State University. Poster presentations (non-peer-reviewed): Khorshidi-Zadeh, M., Ste Marie, J., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (2020) Characterizing the fate of antibiotic resistant E. coli in Oregon wastewater treatment plants, Virtual Engineering Showcase, Oregon State University. Yiu, S., Nguyen. J., Khorshidi Zadeh, M., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (2020) The effects of seasonal and geographical variations on the physical and chemical properties of wastewaterin Oregon, Virtual Engineering Showcase, Oregon State University. Nguyen, J., Khorshidi-Zadeh, M., Yiu, S., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (2019) Antibiotic resistance trends in wastewater, Summer Undergraduate Research Symposium, Oregon State University. Khorshidi-Zadeh, M., Yiu, S., Nguyen, J., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (2019) Association between heavy metals and antibiotic-resistant bacteria in wastewater treatment facilities across Oregon, Clean Water Workshop, Oregon State University. How have the results been disseminated to communities of interest? Three female, under-represented minority high school students were involved in a week-long project to understand the emergence and associated health risks of antibiotic resistance in environmental reservoirs. These students were mentored by grad and undergrad students in the lab to quantify antibiotic-resistance E. coli in water and soil samples. They used several treatments (boiling, chlorine disinfection, and UV disinfection) to test and understand the impact of treatment processes on the reduction and removal of bacteria in water and soil samples. Their results were presented at a poster symposium at OSU campus: Gallardo, C.V.,Nguyen, E., Ayala, G. (2019) Antibiotic resistance: a public health issue, Summer Experience in Science and Engineering for Youth, Oregon State University. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The overall objective of this project is to identify the abundance and diversity of antibiotic resistance in wastewater and biosolids across different seasonal and regional variations, and further determine the impact of the application of wastewater and biosolids on growth and dissemination of antibiotic-resistant bacteria and genes in soils and on vegetable crops. The central hypothesis underlying this research effort is that the abundance and diversity of antibiotic-resistant bacteria and genes and the mobile genetic elements in wastewater and biosolids, when used in agricultural practices, impact their persistence in soils and on food crops. To test our central hypothesis objectively, we are pursuing the following two specific aims: Research objective 1: Identify the variability of antibiotics and antibiotic resistance in wastewater treatment facilities in Oregon. Our working hypothesis is that seasonal (winter or summer) and geographical (coastal/valley or high desert, and urban or rural) variations impact the abundance and diversity of antibiotics and antibiotic-resistant bacteria and genes in wastewater influent, as well as the treatment efficiency, hence the variability of antibiotics and antibiotic-resistant bacteria/genes in wastewater effluent and biosolids. To test this hypothesis in this reporting period, 17 wastewater treatment facilities from across Oregon were enrolled in the study. Of these wastewater treatment facilities, 2 are located in the coastal region, 10 are in the Willamette Valley, and 5 are in eastern Oregon (high desert). We have collected wastewater influent, secondary wastewater (after biological treatment - if applicable - and before disinfection), wastewater effluent, and biosolids in winter and summer 2020. It should be noted that in summer 2020, due to the COVID-19 pandemic and the Oregon wildfires, we were able to collect and process samples from only 13 of the 17 enrolled facilities. Data was collected on facilities' treatment processes, serving population, and flow characteristics. For all samples, pH, conductivity, solids, and ammonia were measured. In addition, up to six E. coli colonies were isolated from each sample for further antibiotic susciptibility testing. Samples were processed and stored for further analyses of antibiotics, antibiotic- resistant genes, and metagenomics. Research objective 2: Determine the impact of irrigation with wastewater effluent and fertilization with biosolids on the prevalence and dissemination of antibiotic resistance in soil. Our working hypothesis is that irrigating with wastewater effluent or amendment of biosolids promotes the persistence of antibiotic-resistant bacteria and determinant genes in soils and food crops. To test this hypothesis, we investigated the variability of antibiotic-resistant genes (ARGs) in wastewater effluent over time. Moreover, we examined the impact of biosolids amendment and wastewater effluent irrigation on the persistence and decay rates of antibiotic-resistant enteric bacterial indicators and their phenotypic characteristics in agricultural soil. Abundance of eight ARGs and intI1 (an indicator gene for multiple resistance and horizontal gene transfer) were quantified weekly in wastewater effluent from a facility serving 55,000 people over a 10-week period from February to April 2019. In a greenhouse setting, biosolids and wastewater irrigation were applied to soil during the growth season of carrots from seeds (eleven weeks). Cultivatable total and antibiotic-resistant presumptive E. coli and enterococci were quantified in biosolids, wastewater effluent, soil, and carrots. Decay rates of antibiotic resistance versus susceptible counterparts were determined. Finally, the phenotypes of antibiotic susceptibility of presumptive E. coli (n = 118) and enterococci (n = 110) colonies and the prevalence of multi-drug resistance during the growth period were identified. The relative abundance of ARGs ranged from 2.3 × 10-5 to 4.2 × 10-1 copies/16S rRNA in wastewater effluent. Strong correlations were observed between intI1 and sul1, sul2, and blaCTX-m, (p < 0.01) demonstrating the prominence of these ARGs as indicators of multiple resistances. Associations seen between tetM and ermB (p = 0.03) suggest the perceived linkage in genetic elements and, as a result, likelihood of co-transfer. Results suggest no statistical difference between the abundance of ARGs over time (p > 0.05). This information, coinciding with the lack of dilution from stormwater in this treatment facility, suggests that discharge of ARGs in this facility remains relatively consistent. In the greenhouse, no E. coli or enterococci were detected in soil without biosolids amendment under either effluent wastewater or water irrigation. After biosolids amendment, E. coli and enterococci concentrations in soil were at 3.6 ± 0.2 log10CFU/g-TS, declined slowly with time, and stayed above the detection limit over the entirety of the study. Almost all E. coli detected in soil were resistant to ampicillin for the first four weeks of the study but declined quickly to below the limit of detection by week seven. Resistance of E. coli to tetracycline and ciprofloxacin was present at ~2 log10CFU/g-TS in soil after biosolids applications and reached the detection limit by week two. Ampicillin- and tetracycline-resistant enterococci were ~3 log10CFU/g-TS in biosolids-amended soil and declined to the detection limit by week five. No statistical significance were observed between total and antibiotic-resistant E. coli and enterococci levels in soil or on carrots after effluent wastewater or water irrigation (p > 0.05). Antibiotic-resistant E. coli and enterococci decayed at significantly faster rates than total E. coli and enterococci (p < 0.01). In biosolids-amended soil, E. coli were present on carrots at harvest, majority of which were resistant to ampicillin. Prevalence of multi-drug resistant E. coli declined significantly over time (p < 0.001), while the multi-drug resistant enterococci persisted during the study (p > 0.05). This work is the first to investigate variations of wastewater effluent ARGs over time, to directly compare decay rate constants of total and antibiotic-resistant E. coli and enterococci, and to demonstrate the persistence of multi-drug resistance phenotypes of these enteric bacterial indicators in soils after biosolids amendment and wastewater irrigation. Findings allow for cautious optimism that even though ARGs and multi-drug resistance is present in wastewater, the use of wastewater effluent would not significantly increase antibiotic-resistant enteric bacteria in soil. The survival of fecal indicator bacteria in soil and on harvested carrots, however, indicates the transmission risks associated with biosolids amendment to harvested root crops.
Publications
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Progress 07/01/18 to 06/30/19
Outputs
Target Audience:The target audience for this reporting period includes the environmental engineering scientific commununity, the food science and technology scientific community, the wastewater treatment facility operators, Oregon produce growers, and policy makers at State and national levels. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In this reporting period, two Masters students (thesis work), two PhD students (research for credits), and 3 undergraduate students have been trained and involved in the project. Existing and new lab protocols have been developed and modified. Participating students have been trained and their efforts resulted in several presentations which have been valuable in their professional development. Following is the list of presentations (the names of the students are underlined): Peer-reviewed poster presentations: Mays, C., Waite Cusic, J., Radniecki, T., Navab-Daneshmand, T. (2019) Impact of biosolids amendment and wastewater irrigation on the persistence of antibiotic resistance in agricultural soil and prevalence on food crops, Association of Environmental Engineering and Science Professors Conference, Arizona State University, Tempe, AZ. Other poster presentations: Lippman, L., Mays, C., Garza, G., Hugie, C., Yiu, S., Navab-Daneshmand, T. (2018) Persistence of E. coli in Agricultural Soil after Wastewater Irrigation and Biosolids Soil Amendment, American Institute of Chemical Engineers Conference Undergraduate Student Poster Session, Pittsburg, PA. Garza, G., Hugie, C., Mays, C., Waite-Cusic, J., Radniecki, T., Navab-Daneshmand, T. (2018) The contribution of biosolids soil amendment and wastewater irrigation to antibiotic-resistant enteric bacteria in agricultural soil and food crops, Summer Undergraduate Research Symposium, Oregon State University. Yiu, S., Mays, C., Reed, H., Navab-Daneshmand, T. (2018) Variability of physical and chemical parameters of Oregon wastewater, Summer Undergraduate Research Fellowship Symposium, Oregon State University. Hugie, C., Waite-Cusic, J. Radniecki, T., Navab-Daneshmand, T. (2018) Risk assessment of food crops in biosolids amended soil with wastewater irrigation, Summer Undergraduate Research Fellowship Symposium, Oregon State University. Garza, G., Hugie, C., Waite-Cusic, J. Radniecki, T., Navab-Daneshmand, T. (2018) Prevalence of antibiotic-resistant enteric bacteria in agricultural soil and food crops following wastewater irrigation and biosolids soil amendment, Clean and Sustainable Water Technology Initiative Workshop, Oregon State University. Mays, C., Yiu, S., Reed, H., Radniecki, T., Waite-Cusic, J., Navab-Daneshmand, T. (2018) Antibiotic resistance in Oregon wastewater treatment facilities, Clean and Sustainable Water Technology Initiative Workshop, Oregon State University. How have the results been disseminated to communities of interest?Two female, under-represented minority high school students were involved in a week-long project to understand the emergence and associated health risks of antibiotic resistance in environmental reservoirs. These students were mentored by grad and undergrad students in the lab to quantify antibiotic-resistance E. coli in water and soil samples. They used several treatments (boiling, chlorine disinfection, and UV disinfection) to test and understand the impact of treatment processes on the reduction and removal of bacteria in water and soil samples. Their results were presented at a poster symposium at OSU campus: Luna, J., Croak, J., Garza, G., Navab-Daneshmand, T. (2018) Antibiotic resistant bacteria, Summer Experience in Science and Engineering for Youth, Oregon State University. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The overall objective of this project is to identify the abundance and diversity of antibiotic resistance in wastewater and biosolids across different seasonal and regional variations, and further determine the impact of the application of wastewater and biosolids on growth and dissemination of antibiotic-resistant bacteria and genes in soils and on vegetable crops. The central hypothesis underlying this research effort is that the abundance and diversity of antibiotic-resistant bacteria and genes and the mobile genetic elements in wastewater and biosolids, when used in agricultural practices, impact their persistence in soils and on food crops. To test our central hypothesis objectively, we are pursuing the following two specific aims: Research objective 1: Identify the variability of antibiotics and antibiotic resistance in wastewater treatment facilities in Oregon. Our working hypothesis is that seasonal (winter or summer) and geographical (coastal/valley or high desert, and urban or rural) variations impact the abundance and diversity of antibiotics and antibiotic-resistant bacteria and genes in wastewater influent, as well as the treatment efficiency, hence the variability of antibiotics and antibiotic-resistant bacteria/genes in wastewater effluent and biosolids. To test this hypothesis in this reporting period, 17 wastewater treatment facilities from across Oregon were enrolled in the study. Of these wastewater treatment facilities, 2 are located in the coastal region, 10 are in the Willamette Valley, and 5 are in eastern Oregon (high desert). We have collected wastewater influent, secondary wastewater (after biological treatment - if applicable - and before disinfection), wastewater effluent, and biosolids in winter and summer 2019. Data was collected on facilities' treatment processes, serving population, and flow characteristics. For all samples, pH, conductivity, solids, ammonia and COD were measured. In addition, up to six E. coli colonies were isolated from each sample for further antibiotic susciptibility testing. Samples were processed and stored for further testing of heavy metals, antibiotics, and antibiotic-resistant genes. Research objective 2: Determine the impact of irrigation with wastewater effluent and fertilization with biosolids on the prevalence and dissemination of antibiotic resistance in soil. Our working hypothesis is that irrigating with wastewater effluent or amendment of biosolids promotes the persistence of antibiotic-resistant bacteria and determinant genes in soils and food crops. To test this hypothesis in this reporting period, a pilot-scale study was performed in a greenhouse setting at Oregon State University. 4' × 4' growth trays were placed on mesh grid tables to collect and contain contaminated runoff. Polyvinyl chloride pipe cages with mosquito netting were constructed around each tray to prevent flying insects from accessing contaminated plants and soil. Carrots seeds were planted in 2-gallon pots in 4 treatment groups with 6 replicates for each treatment. Some soil pots were amended with biosolids in the start of the study. Some pots were irrigated with autoclaved DI water and others were irrigated with wastewater effluent. Soil cores were collected in triplicates and tested weekly for the presence of cultivatable total and antibiotic-resistant E. coli and Enterococci. Ampicillin, tetracycline, and ciprofloxacin were used to quantify antibiotic susceptibility. At harvest, carrots were rinsed and the rinsate were quantified for total and antibiotic-resistant E. coli and Enterococci. Soil core samples were appropriately processed and stored for the analyses of antibiotic-resistant genes and antibiotic compounds. Preliminary results demonstrate very little - if any - culturable E. coli and Enterococci throughout the study in unamended soils irrigated with water or wastewater. In biosolids amended soil, however, 2-4 logs total and antibiotic-resistant E. coli and Enterococci per dry gram of soil were observed in the beginning of the study. These concentrations were reduced to the detection limit before harvest (week 11). E. coli and Enterococci reached the detection limit by week 10. Ampicillin-, tetracyline-, and ciprofloxacin-resistant E. coli reached the detection limit by week 7, 2, and 2, respectively. Ampicillin- and tetracyline-resistant Enterococci reached the detection limit by week 4 and 7, respectively. These results suggest no apparent impact of wastewater irrigation on culturable total and antibiotic-resistant E. coli and Enterococci in soils during the growth of carrots. Application of biosolids, however, increased culturable total and antibiotic-resistant E. coli and Enterococci in soil. These counts were reduced to the limit of detection before harvest. Further testing and analyses are undergoing before we can make conclusions and recommendations to stakeholders (treatment facilities, farmers, and policy makers).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Mays, C., Waite Cusic, J., Radniecki, T., Navab-Daneshmand, T. (2019) Impact of biosolids amendment and wastewater irrigation on the persistence of antibiotic resistance in agricultural soil and prevalence on food crops, Association of Environmental Engineering and Science Professors Conference, Arizona State University, Tempe, AZ. (peer-reviewed poster presentation)
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