Amy Pruden

Management options for reducing the release of antibiotics and antibiotic resistance genes to the environment

Background: There is growing concern worldwide about the role of polluted soil and water environments in the development and dissemination of antibiotic resistance. Objective: Our aim in this study was to identify management options for reducing the spread of antibiotics and antibiotic-resistance determinants via environmental pathways, with the ultimate goal of extending the useful life span of antibiotics. We also examined incentives and disincentives for action. Methods: We focused on management options with respect to limiting agricultural sources; treatment of domestic, hospital, and industrial wastewater; and aquaculture. Discussion: We identified several options, such as nutrient management, runoff control, and infrastructure upgrades. Where appropriate, a cross-section of examples from various regions of the world is provided. The importance of monitoring and validating effectiveness of management strategies is also highlighted. Finally, we describe a case study in Sweden that illustrates the critical role of communication to engage stakeholders and promote action. Conclusions: Environmental releases of antibiotics and antibiotic-resistant bacteria can in many cases be reduced at little or no cost. Some management options are synergistic with existing policies and goals. The anticipated benefit is an extended useful life span for current and future antibiotics. Although risk reductions are often difficult to quantify, the severity of accelerating worldwide morbidity and mortality rates associated with antibiotic resistance strongly indicate the need for action.

Using the class 1 integron-integrase gene as a proxy for anthropogenic pollution

Around all human activity, there are zones of pollution with pesticides, heavy metals, pharmaceuticals, personal care products and the microorganisms associated with human waste streams and agriculture. This diversity of pollutants, whose concentration varies spatially and temporally, is a major challenge for monitoring. Here, we suggest that the relative abundance of the clinical class 1 integron-integrase gene, intI1, is a good proxy for pollution because: (1) intI1 is linked to genes conferring resistance to antibiotics, disinfectants and heavy metals; (2) it is found in a wide variety of pathogenic and nonpathogenic bacteria; (3) its abundance can change rapidly because its host cells can have rapid generation times and it can move between bacteria by horizontal gene transfer; and (4) a single DNA sequence variant of intI1 is now found on a wide diversity of xenogenetic elements, these being complex mosaic DNA elements fixed through the agency of human selection. Here we review the literature examining the relationship between anthropogenic impacts and the abundance of intI1, and outline an approach by which intI1 could serve as a proxy for anthropogenic pollution.

Correlation Between Upstream Human Activities and Riverine Antibiotic Resistance Genes

Antimicrobial resistance remains a serious and growing human health challenge. The water environment may represent a key dissemination pathway of resistance elements to and from humans. However, quantitative relationships between landscape features and antibiotic resistance genes (ARGs) have not previously been identified. The objective of this study was to examine correlations between ARGs and putative upstream anthropogenic sources in the watershed. sul1 (sulfonamide) and tet(W) (tetracycline) were measured using quantitative polymerase chain reaction in bed and suspended sediment within the South Platte River Basin, which originates from a pristine region in the Rocky Mountains and runs through a gradient of human activities. A geospatial database was constructed to delineate surface water pathways from animal feeding operations, wastewater treatment plants, and fish hatchery and rearing units to river monitoring points. General linear regression models were compared. Riverine sul1 correlated with upstream capacities of animal feeding operations (R(2) = 0.35, p < 0.001) and wastewater treatment plants (R(2) = 0.34, p < 0.001). Weighting for the inverse distances from animal feeding operations along transport pathways strengthened the observed correlations (R(2) = 0.60-0.64, p < 0.001), suggesting the importance of these pathways in ARG dissemination. Correlations were upheld across the four sampling events during the year, and averaging sul1 measurements in bed and suspended sediments over all events yielded the strongest correlation (R(2) = 0.92, p < 0.001). Conversely, a significant relationship with landscape features was not evident for tet(W), which, in contrast to sul1, is broadly distributed in the pristine region and also relatively more prevalent in animal feeding operation lagoons. The findings highlight the need to focus attention on quantifying the contribution of water pathways to the antibiotic resistance disease burden in humans and offer insight into potential strategies to control the spread of ARGs.

tet and sul Antibiotic Resistance Genes in Livestock Lagoons of Various Operation Type, Configuration, and Antibiotic Occurrence

Although livestock operations are known to harbor elevated levels of antibiotic resistant bacteria, few studies have examined the potential of livestock waste lagoons to reduce antibiotic resistance genes (ARGs). The purpose of this study was to determine the prevalence and examine the behavior of tetracycline [tet(O) and tet(W)] and sulfonamide [sul(I) and sul(II)] ARGs in a broad cross-section of livestock lagoons within the same semiarid western watershed. ARGs were monitored for one year in the water and the settled solids of eight lagoon systems by quantitative polymerase chain reaction. In addition, antibiotic residues and various bulk water quality constituents were analyzed. It was found that the lagoons of the chicken layer operation had the lowest concentrations of both tet and sul ARGs and low total antibiotic concentrations, whereas sul ARGs were highest in the swine lagoons, which generally corresponded to the highest total antibiotic concentrations. A marginal benefit of organic and small dairy operations also was observed compared to conventional and large dairies, respectively. In all lagoons, sul ARGs were observed to be generally more recalcitrant than tet ARGs. Also, positive correlations of various bulk water quality constituents were identified with tet ARGs but not sul ARGs. Significant positive correlations were identified between several metals and tet ARGs, but Pearsons correlation coefficients were mostly lower than those determined between antibiotic residues and ARGs. This study represents a quantitative characterization of ARGs in lagoons across a variety of livestock operations and provides insight into potential options for managing antibiotic resistance emanating from agricultural activities.

Tracking Antibiotic Resistance Genes in the South Platte River Basin Using Molecular Signatures of Urban, Agricultural, And Pristine Sources

A novel approach utilizing antibiotic-resistance-gene (ARG) molecular signatures was applied to track the sources of ARGs at sites along the Cache la Poudre (Poudre) and South Platte Rivers in Colorado. Two lines of evidence were employed: (1) detection frequencies of 2 sulfonamide and 11 tetracycline ARGs and (2) tet(W) phylotype and phylogenetic analysis. A GIS database indicating the locations of wastewater treatment plants (WWTPs) and animal feeding operations (AFOs) in the watershed was also constructed to assess congruence of the surrounding landscape with the putative sources identified by ARG molecular signatures. Discriminant analysis was performed on detection frequencies of tetARG groups that were previously identified to be associated with either WWTPs or AFOs. All but one (South Platte River-3, just downstream from the confluence with the Poudre River) of the eight sites were classified as primarily WWTP-influenced based on discriminant analysis of ARG detection frequencies. tet(W) phylotype analysis also aligned South Platte River-3 with putative AFO sources, while phylogenetic analysis indicated that it was not significantly different from the AFOs or WWTPs investigated. South Platte River-3 is situated in an intense agricultural area, but the upstream portion of the South Platte River receives substantial loading from metropolitan Denver. By contrast, tet(W) phylotype and phylogenetics of site Poudre River-4, located 4 km downstream of a WWTP, was also characterized and found to be significantly different from the AFO lagoons (p < 0.05), as expected. In general, a good correspondence was found between classification of the impacted river sites and the surrounding landscape. While the overall approach could be extended to other watersheds, the general findings indicate that transport of ARGs from specific sources is likely the dominant mechanism for ARG proliferation in this riverine environment relative to selection of ARGs among native bacteria by antibiotics and other pollutants.

Effect of Various Sludge Digestion Conditions on Sulfonamide, Macrolide, and Tetracycline Resistance Genes and Class I Integrons

Wastewater treatment processes are of growing interest as a potential means to limit the dissemination of antibiotic resistance. This study examines the response of nine representative antibiotic resistance genes (ARGs) encoding resistance to sulfonamide (sulI, sulII), erythromycin (erm(B), erm(F)), and tetracycline (tet(O), tet(W), tet(C), tet(G), tet(X)) to various laboratory-scale sludge digestion processes. The class I integron gene (intI1) was also monitored as an indicator of horizontal gene transfer potential and multiple antibiotic resistance. Mesophilic anaerobic digestion at both 10 and 20 day solids retention times (SRTs) significantly reduced sulI, suII, tet(C), tet(G), and tet(X) with longer SRT exhibiting a greater extent of removal; however, tet(W), erm(B) and erm(F) genes increased relative to the feed. Thermophilic anaerobic digesters operating at 47 °C, 52 °C, and 59 °C performed similarly to each other and provided more effective reduction of erm(B), erm(F), tet(O), and tet(W) compared to mesophilic digestion. However, thermophilic digestion resulted in similar or poorer removal of all other ARGs and intI1. Thermal hydrolysis pretreatment drastically reduced all ARGs, but they generally rebounded during subsequent anaerobic and aerobic digestion treatments. To gain insight into potential mechanisms driving ARG behavior in the digesters, the dominant bacterial communities were compared by denaturing gradient gel electrophoresis. The overall results suggest that bacterial community composition of the sludge digestion process, as controlled by the physical operating characteristics, drives the distribution of ARGs present in the produced biosolids, more so than the influent ARG composition.

Ultraviolet Disinfection of Antibiotic Resistant Bacteria and Their Antibiotic Resistance Genes in Water and Wastewater

Disinfection of wastewater treatment plant effluent may be an important barrier for limiting the spread of antibiotic-resistant bacteria (ARBs) and antibiotic resistance genes (ARGs). While ideally disinfection should destroy ARGs, to prevent horizontal gene transfer to downstream bacteria, little is known about the effect of conventional water disinfection technologies on ARGs. This study examined the potential of UV disinfection to damage four ARGs, mec(A), van(A), tet(A), and amp(C), both in extracellular form and present within a host ARBs: methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), Escherichia coli SMS-3-5, and Pseudomonas aeruginosa 01, respectively. An extended amplicon-length quantitative polymerase chain reaction assay was developed to enhance capture of ARG damage events and also to normalize to an equivalent length of target DNA (∼1000 bp) for comparison. It was found that the two Gram-positive ARBs (MRSA and VRE) were more resistant to UV disinfection than the two Gram-negative ARBs (E. coli and P. aeruginosa). The two Gram-positive organisms also possessed smaller total genome sizes, which could also have reduced their susceptibility to UV because of fewer potential pyrimidine dimer targets. An effect of cell type on damage to ARGs was only observed in VRE and P. aeruginosa, the latter potentially because of extracellular polymeric substances. In general, damage of ARGs required much greater UV doses (200-400 mJ/cm² for 3- to 4-log reduction) than ARB inactivation (10-20 mJ/cm² for 4- to 5-log reduction). The proportion of amplifiable ARGs following UV treatment exhibited a strong negative correlation with the number of adjacent thymines (Pearson r < -0.9; p < 0.0001). ARBs surviving UV treatment were negatively correlated with total genome size (Pearson r < -0.9; p < 0.0001) and adjacent cytosines (Pearson r < -0.88; p < 0.0001) but positively correlated with adjacent thymines (Pearson r > 0.85; p < 0.0001). This suggests that formation of thymine dimers is not the sole mechanism of ARB inactivation. Overall, the results indicate that UV is limited in its potential to damage ARGs and other disinfection technologies should be explored.

Molecular Survey of the Occurrence of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa, and Amoeba Hosts in Two Chloraminated Drinking Water Distribution Systems

ABSTRACT The spread of opportunistic pathogens via public water systems is of growing concern. The purpose of this study was to identify patterns of occurrence among three opportunistic pathogens (Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa) relative to biotic and abiotic factors in two representative chloraminated drinking water distribution systems using culture-independent methods. Generally, a high occurrence of Legionella (≥69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (≤20%) and M. avium (≤33.3%), and rare detection of Pseudomonas aeruginosa (≤13.3%) were observed in both systems according to quantitative PCR. Also, Hartmanella vermiformis was more prevalent than Acanthamoeba, both of which are known hosts for opportunistic pathogen amplification, the latter itself containing pathogenic members. Three-minute flushing served to distinguish distribution system water from plumbing in buildings (i.e., premise plumbing water) and resulted in reduced numbers of copies of Legionella, mycobacteria, H. vermiformis, and 16S rRNA genes (P < 0.05) while yielding distinct terminal restriction fragment polymorphism (T-RFLP) profiles of 16S rRNA genes. Within certain subgroups of samples, some positive correlations, including correlations of numbers of mycobacteria and total bacteria (16S rRNA genes), H. vermiformis and total bacteria, mycobacteria and H. vermiformis, and Legionella and H. vermiformis, were noted, emphasizing potential microbial ecological relationships. Overall, the results provide insight into factors that may aid in controlling opportunistic pathogen proliferation in real-world water systems.

Balancing Water Sustainability and Public Health Goals in the Face of Growing Concerns about Antibiotic Resistance

Global initiatives are underway to advance the sustainability of urban water infrastructure through measures such as water reuse. However, there are growing concerns that wastewater effluents are enriched in antibiotics, antibiotic resistant bacteria, and antibiotic resistance genes, and thus could serve as a contributing factor to growing rates of antibiotic resistance in human infections. Evidence for the role of the water environment as a source and pathway for the spread of antimicrobial resistance is examined and key knowledge gaps are identified with respect to implications for sustainable water systems. Efforts on the part of engineers along with investment in research in epidemiology, risk assessment, water treatment and water delivery could advance current and future sustainable water strategies and help avoid unintended consequences.

Identification of antibiotic-resistance-gene molecular signatures suitable as tracers of pristine river, urban, and agricultural sources.

Animal feeding operations (AFOs) and wastewater treatment plants (WWTPs) are potential sources of antibiotic resistance genes (ARGs) in rivers and/or antibiotics that may select for ARGs in native river bacteria. This study aimed to identify ARG distribution patterns that unambiguously distinguish putative sources of ARG from a native river environment. Such molecular signatures may then be used as tracers of specific anthropogenic sources. Three WWTPs, six AFO lagoons, and three sites along a pristine region of the Cache la Poudre (Poudre) River were compared with respect to the frequency of detection (FOD) of 11 sulfonamide and tetracycline ARGs. Principle-component and correspondence analyses aided in identifying the association of tet(H), tet(Q), tet(S), and tet(T) (tet group HQST) with AFO environments and tet(C), tet(E), and tet(O) (tet group CEO) with WWTPs. Discriminant analysis indicated that both tet group HQST and tet group CEO correctly classified the environments, but only the tet group HQST provided a significant difference in FOD among the environments (p < 0.05). Sul(I) was detected in 100% of the source environments but just once in the pristine Poudre River, which was dominated by tet(M) and tet(W). Tet(W) libraries generated from the pristine Poudre River, WWTPs, and AFO lagoons were also discernible based on restriction fragment length polymorphism and phylogenetic analysis. Thus, a novel approach was developed and demonstrated to be effective for the model river system, taking an important step in advancing the fundamental understanding of ARG transport in the environment.