Graduation Year
2025
Document Type
Dissertation
Degree
Ph.D.
Degree Name
Doctor of Philosophy (Ph.D.)
Degree Granting Department
Integrative Biology
Major Professor
Valerie J. Harwood, Ph.D.
Committee Member
Kathleen Scott, Ph.D.
Committee Member
Mark Margres, Ph.D.
Committee Member
Amy Pruden, Ph.D.
Keywords
Antimicrobial Resistance Monitoring, Enterococcus spp., Escherichia coli, Meta-Analysis, Method Comparison, Surface Water
Abstract
More than 2.8 million infections due to antibiotic-resistant bacteria occur annually in the United States, and more than 35,000 people die as a result. Domestic wastewater (sewage), which contains antibiotic-resistant bacteria and antibiotic resistance genes, is collected at treatment plants for removal of contaminants, e.g. pathogenic microorganisms and nutrients. While wastewater treatment is designed to remove antibiotic-resistant bacteria and other microorganisms through treatment and disinfection, some bacteria may escape treatment. Treated wastewater effluent is generally discharged into surface waters; however, some fraction of the effluent may be used as recycled water. Compromised wastewater collection infrastructure and sewage spills can disseminate antibiotic-resistant bacteria to the environment. This leads to the water environment being a recipient and source of antibiotic-resistant bacteria. Monitoring antibiotic-resistant bacteria and antibiotic resistance genes in the environment is useful for understanding the contributions of the water environment to the spread of antibiotic resistance, which is listed as a #1 priority by the United Nations Environment Program 2017 Frontiers Report on Emerging Issues of Environmental Concern report.
Humans may be exposed to antibiotic-resistant bacteria by interacting with environmental waters contaminated with wastewater, therefore the efficacy of wastewater treatment for removal of antibiotic-resistant bacteria, the characterization of antibiotic-resistant bacteria present in wastewater, and standardization of methods used in monitoring for surveillance of antibiotic-resistant bacteria are important to safeguard public health. Two candidate bacteria, cefotaxime-resistant Escherichia coli and vancomycin-resistant Enterococcus spp., were identified as strong candidates for use in monitoring antibiotic-resistant bacteria in the environment. Cefotaxime is a third-generation cephalosporin antibiotic used to treat conditions such as urinary tract infections, meningitis, and pneumonia. Vancomycin is a glycopeptide antibiotic used primarily to treat serious Gram-positive bacterial infections such as bone infections, endocarditis, and septicemia. Monitoring of cefotaxime-resistant E. coli or vancomycin-resistant Enterococcus spp. could provide a measure of human health threat in the water environment and can identify environmental reservoirs of antibiotic-resistant bacteria and hotspots for evolution and dissemination.
This dissertation aims to narrow the following knowledge gaps: i) effectiveness of wastewater treatment on the reduction of vancomycin-resistant Enterococcus spp., ii) phenotypic and genotypic characteristics of vancomycin-resistant Enterococcus spp. isolated throughout wastewater treatment, iii) comparability and effectiveness of methods for isolating cefotaxime-resistant E. coli in environmental waters, and iv) identification of potential predictors of cephalosporin resistance in E. coli through meta-analysis. In Chapter Two, I isolated and characterized vancomycin-resistant Enterococcus spp. throughout wastewater treatment by phenotypic methods (antibiotic resistance profiling) and genotypic methods (identification of antibiotic resistance genes and virulence factors). Multidrug-resistant (resistance to three or more antibiotic classes) phenotypes were found in 88.9% of vancomycin-resistant Enterococcus spp.. Thirteen of 18 (83.3%) vancomycin-resistant Enterococcus spp. strains displayed an identical multidrug resistance pattern, with resistance to ampicillin, ciprofloxacin, erythromycin, nitrofurantoin, and tetracycline and susceptibility to fosfomycin, linezolid, and quinupristin-dalfopristin. Whole genome sequencing focused on these 13 strains, all of which were identified as Ent. faecium. Whole genome sequence analysis could not determine a single common genetic factor such as a mobile genetic element, set of antibiotic resistance genes, or single sequence type across our isolates that results in this multidrug resistance pattern, and instead identified a combination of features strongly associated with clonal complex 17 strains that best explains the phenotype.
Comparability across studies is important in global monitoring efforts of antibiotic-resistant bacteria in the environment. In Chapter Three, two proposed methods of culturing cefotaxime-resistant E. coli were tested for comparability. The media and incubation conditions of two protocols, The World Health Organization Tricycle Protocol and the U.S. Environmental Protection Agency Method 1603, were cross-tested and compared. Total and putative cefotaxime-resistant E. coli concentrations did not differ significantly between media or by incubation method; however, colonies isolated on mTEC (EPA Method 1603) were more frequently confirmed to species (97.1%) than those isolated on TBX (Tricycle Protocol) (92.5%). Resuscitation in a 35°C ± 0.5°C incubator followed by incubation in a 44.5°C ± 0.2°C water bath significantly decreased non-specific background growth and improved confirmation frequency on both media. Both media performed comparably using the resuscitation step and higher water bath incubation temperature of 44.5°C, suggesting that the TriCycle Protocol is adaptable to standard methods that may be used in different locales. These results also offer a means to improve specificity of more accessible media by decreasing the frequency of false-positive identification of cefotaxime-resistant E. coli by modifying incubation conditions.
Meta-analysis provides a means to synthesize data from several studies and assess the impact different variables have on effect size. In Chapter Four, a meta-analysis was conducted to evaluate the prevalence of cephalosporin-resistant E. coli in surface water, non-disinfected wastewater, and disinfected wastewater matrices. The objective was to identify significant and insignificant predictors of cephalosporin resistance in E. coli globally. Published and peer-reviewed articles between January 1, 2000 and December 31, 2024 were screened for inclusion in the meta-analysis. After excluding candidate articles that pre-enriched samples or did not confirm E. coli, 34 articles were included from 19 different countries. The following variables were examined: matrix sampled (surface water, non-disinfected wastewater, and disinfected wastewater), cephalosporin generation, continent, and sampling year. Of these variables, only continent and sampling year were identified as significant predictors of cephalosporin resistance in E. coli. A significantly higher proportion of cephalosporin-resistant E. coli was found in Africa (0.56, 95% CI [0.30; 0.81]) compared to Asia, Europe, and North America, and a significantly lower proportion of cephalosporin-resistant E. coli was found in Europe (0.09, 95% CI [0.01; 0.25]), (p=0.022). Studies were divided into two equal groups by year(s) when they were conducted to determine whether there were any trends over time. A significantly lower proportion of cephalosporin-resistant E. coli was found during sampling years 2000-2016 (0.18, 95% CI [0.08; 0.31]) than during in sampling years 2017-2024 (0.38, 95% CI [0.23; 0.53]), (p=0.039). This meta-analysis showed that the prevalence of cephalosporin-resistant E. coli in wastewater and environmental waters is influenced more by location and year than by matrix sampled or generation of cephalosporin tested. These findings can help to inform selection of which cephalosporin antibiotic(s) to use in future studies and can further support monitoring efforts.
This work will benefit public health by providing information and tools that could improve identification of antibiotic-resistant bacteria in wastewater and environmental waters. The occurrence of antibiotic-resistant bacteria in wastewater is likely to reflect the occurrence of antibiotic-resistant bacteria in the clinical setting. Identification of antibiotic-resistant strains with a shared multidrug resistance profile that is prevalent in wastewater can help inform treatment options for clinical infections. Monitoring antibiotic-resistant bacteria in wastewater and environmental waters allows for a better understanding of the threat to human health caused by exposure to surface waters contaminated with wastewater. As global surveillance of antibiotic-resistant bacteria increases, confidence in our ability to identify predictors of resistance will increase. Knowledge gained from this research can expand accessibility of methods to monitor antibiotic-resistant bacteria and assist in standardization to increase comparability of monitoring efforts globally.
Scholar Commons Citation
Calarco, Jeanette, "A Multi-Faceted Study of Antibiotic-Resistant, Opportunistic Pathogens in Wastewater and Environmental Water" (2025). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/11075
