Environmental Growth of Enterococci and Escherichia coli in Feedlot

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Environmental growth of enterococci and Escherichia coli in feedlot catch basins and a constructed wetland in the absence of fecal input Lisa Tymensen, Calvin W Booker, Sherry J Hannon, Shaun R Cook, Rahat Zaheer, Ron Read, and Tim A. McAllister Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 21 Apr 2017 Downloaded from http://pubs.acs.org on April 22, 2017

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Environmental Science & Technology

Environmental growth of enterococci and Escherichia coli in feedlot catch basins and a constructed wetland in the absence of fecal input

Lisa Tymensen,*,† Calvin W. Booker,‡ Sherry J. Hannon,‡ Shaun R. Cook,†,§ Rahat Zaheer,§ Ron Read, and Tim A. McAllister§

†

Irrigation and Farm Water Branch, Alberta Agriculture and Forestry, Lethbridge, Alberta, Canada, T1J 4V6.

‡

Feedlot Health Management Services, Ltd., Okotoks, Alberta, Canada, T1S 2A2.

§

Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada, T1J 4B1.

Microbiology, Immunology and Infectious Diseases, University of Calgary, Alberta, Canada, T1Y 6J4.

Submitted to: Environmental Science & Technology

*Corresponding author Irrigation and Farm Water Branch Alberta Agriculture and Forestry 100, 5401 1st Avenue South Lethbridge, Alberta, Canada TIJ 4V6 Phone: (+1) 403 381 5165; fax: (+1) 403 081 5765; e-mail: [email protected]

ACS Paragon Plus Environment


ABSTRACT: Population structures of fecal indicator bacteria (FIB) isolated from catch basins, a constructed wetland, and feces from a beef cattle feedlot were compared over a two-year period. Enterococcus hirae accounted for 92% of the fecal isolates, whereas secondary environments were characterized by greater relative abundance of environmentally adapted species including Enterococcus casseliflavus. While enterococci densities in the catch basins and wetland were similar under wet and drought conditions, E. hirae predominated during rainy periods while E. casseliflavus predominated during drought conditions. Environmentally adapted species accounted for almost half of the erythromycin resistant enterococci isolated from the wetland. Densities of Escherichia coli were also comparable during wet versus drought conditions, and the relative abundance of strains from environmentally adapted clades was greater in secondary environments compared to feces. Unlike enterococci, fewer environmentally adapted E. coli strains were isolated on selective media containing ceftriaxone from the wetland compared to feces, suggesting resistance to this antibiotic may not be well maintained in the absence of selective pressure. Overall, these findings suggest that secondary environments select for environmentally adapted FIB. While these species and clades tend to be of limited clinical relevance, they could potentially serve as reservoirs of antimicrobial resistance.


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INTRODUCTION Bacteria commonly found in human, livestock, and wildlife feces such as Escherichia coli or enterococci have traditionally been used as indicators of fecal contamination for surface water. While most fecal indicator bacteria (FIB) strains are commensal, some have been implicated as pathogens in nosocomial and community-acquired infections. Enteropathogenic E. coli are one of the leading bacterial causes of diarrhea,1 while extra-intestinal pathogenic E. coli, mainly from the B2 phylogroup,2 and enterococci, in particular Enterococcus faecalis and Enterococcus faecium, are primary causes of urinary tract infections and sepsis.3 Treatment for these bacterial infections is becoming increasingly challenging due to rising prevalence of antimicrobial resistant strains. Metaphylactic and therapeutic antimicrobial use in livestock production has been posited as one of the contributing factors; however, transmission routes from cattle to humans, particularly environmental transmission routes, are poorly understood.4 Intensive cattle feeding operations typically capture and store pen runoff in catch basins, which can then be used for irrigating crops. Occasionally, constructed wetlands have been incorporated into waste treatment processes to remove nutrients or pathogens prior to effluent use.5-7 Such catchments represent important secondary environments which impose selective pressures on bacterial populations, eliminating strains not adapted for survival and promoting persistence of those with environmentally adaptive traits.8 Environmental stressors (e.g., nutrient limitation, low temperature, solar radiation) may impact the potential for strain dissemination in the environment, and thus have important implications for bacterial transmission, including antimicrobial resistant strains, from feedlots to the environmental continuum.9-11 Research to elucidate bacterial strain dynamics in feedlots and surrounding environments is thus important for furthering our understanding of environmental pathogen transmission.



The use of FIB for assessing recent surface water fecal contamination is predicated on the assumption that fecal bacteria are transient in the environment.12 However, transmission of FIB from the feedlot environment to humans would require prolonged environmental survival. Although often considered to primarily inhabit the intestine, E. coli actually have a biphasic life cycle.13 Their adaptability and persistence in the extra-enteric environment increases the probability of transmission and colonization of new hosts.14 Tremendous variability exists among different enterococci and E. coli strains with respect to environmental survival,9, 15 with some perishing within a day, and others able to establish autochthonous populations.16-18 Several studies have shown that E. coli phylogroup B1 strains,15, 19 including those from the ET-1 clade,20, 21 tend to predominate in aquatic environments, and are thus considered to be particularly well-adapted for survival outside of the intestine. Recent phylogenetic analyses of E. coli strains from diverse environments have also revealed divergent lineages, designated as Escherichia cryptic clades III, IV and V, which are primarily adapted to aquatic environments.22, 23

Likewise, there are several Enterococcus species that are predominantly associated with

aquatic environments. These species typically exhibit adaptations which aid in environmental survival, like the production of yellow pigments that protect against photoinactivation.24, 25 The aim of this study was to examine the temporal dynamics of FIB populations from various surface waters associated with a beef cattle feedlot. Enterococcus and E. coli isolates were characterized at the broad taxonomic level of species and phylogroups, respectively, with the rationale that particular taxonomic groups tend to be associated with different habitats and lifestyles.26-28 The first objective was to determine how FIB densities and population structures (i.e., taxon richness and evenness) changed as wastewater moved sequentially from a catch basin, to a constructed wetland, and an ephemeral creek that potentially received FIB by way of


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runoff from crop land that was irrigated with wastewater. It was expected that FIB densities would decrease, and that environmentally adapted taxa would become increasingly abundant as wastewater moved through the system. The study was conducted over a two-year period, in which the first year had high rainfall and large amounts of pen runoff followed by a drought in the second year. Given this scenario, the second objective was to compare FIB populations within each catchment between the two years to determine the effect of prolonged storage. Due to fewer FIB entering the catchments during the drought and the increasing exposure to environmental stress during prolonged storage, it was expected that overall FIB densities within each catchment would be lower during the second year and that environmentally adapted taxa would more abundant. Bacterial populations from two different catch basins and the constructed wetland were examined to determine if changes in population structure were consistent for catchments draining separate regions of the feedlot. The third objective was to examine the density and structure of FIB subpopulations isolated on selective media containing antibiotics, as survival of antimicrobial resistant strains may differ from antimicrobial sensitive populations due to the predicted metabolic cost of resistance determinants that may reduce environmental fitness of corresponding strains.29 We anticipated that selectively isolated FIB would decrease in density as wastewater moved through the system, and would be almost entirely eliminated during prolonged storage. The antibiotics considered were third-generation cephalosporins (3GC) and macrolides, which are categorized by the World Health Organization as critically important for use in human medicine and of highest priority for risk management.30 Specifically, 3GCs are used for treatment of invasive E. coli infections in humans. Ceftiofur, which is a 3GC used to treat severe illness in cattle including bovine respiratory disease (BRD), has been shown to transiently



increase the prevalence of 3GC resistant E. coli in treated cattle.31, 32 Erythromycin, an important drug used in human medicine, belongs to the broad class of macrolides that also includes tylosin, used to prevent liver abscesses, and tulathromycin, used to treat BRD in cattle. Since E. coli are intrinsically tylosin and erythromycin resistant, enterococci are often used to monitor macrolide resistance. Use of macrolides in cattle has been shown to increase the prevalence of erythromycin resistant enterococci.33, 34


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MATERIALS AND METHODS Fecal and Water Sampling. The feedlot was located in Alberta, Canada and had an operating capacity of ~15,000 cattle during the study. Production conditions were typical for western Canadian commercial cattle feedlots, with animals housed in open-air, dirt-floor pens arranged side-by-side with central feed alleys. Detailed records of all antibiotics administered to cattle were maintained. Ceftiofur and macrolide use is summarized in Table S1. Fecal sampling was conducted according to a protocol approved by the University of Calgary’s Animal Care Committee (Protocol number AC14-0029). Fecal samples were collected in April, June, and August of both 2014 and 2015 from 20 different pens (100-300 cattle/pen), with the same 20 pens sampled throughout the study. Feces (~10 g) were sampled from 20 fecal pats and combined to generate a composite pen sample (n = 183) and transported to the lab in Cary-Blair enteric transport medium (BD Canada, Inc., Mississauga, ON). Samples were processed within 36 h. Approximately two-thirds of feedlot pens drained runoff into a large catch basin (designated ‘CBA’; Figure S1). The remaining pens drained runoff into a smaller catch basin (designated ‘CBB’). Accumulated runoff in CBB was periodically transferred into a constructed wetland or CBA. The constructed wetland was composed of two separate cells that were connected by adjoining channels and was designed to contain runoff rather than permit flow-through into the environment. The land adjacent to the feedlot was used for silage production. It received regular manure application, and was periodically irrigated with stored runoff from CBA. The land drained naturally into an adjacent ephemeral creek. Water samples were collected monthly from CBA (n = 12), CBB (n = 12), the wetland (n = 13), and the creek (n = 9) from April to October of 2014 and 2015. One liter of water (mid-depth) was collected using a polyethylene bottle



attached to a telescopic pole at four different locations per site, and combined to generate a composite sample. For the wetland, samples from four locations per cell were collected and combined into one sample. Sampling locations remained consistent throughout the study. Water samples were maintained on ice during transport and processed within 24 h. Although holding times exceeded those recommended, it has been shown that if samples are held below 10°C and not allowed to freeze, FIB data for samples held up to 48 h are generally comparable to those processed within 8 h.35 Historical weather data was retrieved from AgroClimatic Information Service (Government of Alberta). Average temperatures and precipitation were calculated from data collected at three separate weather stations located within 30 km of the feedlot. FIB Enumeration and Isolation. The isolation of FIB from feces and water is outlined in Figure S2. Briefly, fecal enterococci isolation was conducted by direct plating of fecal suspensions onto bile esculin agar (BEA, n = 366) or BEA containing 8 µg ml-1 erythromycin (n = 241).3 The erythromycin concentration was based on the breakpoint value established by the 2014 Clinical and Laboratory Standards Institute (M100-S24). E. coli were isolated by directly plating fecal suspensions onto MacConkey agar (n = 389), with selective enrichment in EC broth (BD Difco) containing 2 µg ml-1 cefotaxime followed by primary isolation on MacConkey agar containing 1 µg ml-1 ceftriaxone (n = 250).36 The ceftriaxone concentration was one quarter of the 2014 Clinical and Laboratory Standards Institute breakpoint for resistant E. coli. This concentration was selected based on a previous study of susceptibility patterns from a wellcharacterized collection of ESBL strains.36 Fecal enterococci and E. coli isolates were suspended in brain heart infusion broth containing 15% glycerol and stored at -80°C. Enterococci were enumerated and isolated from catch basin, wetland, and creek surface water using membrane


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filtration onto modified mEI (BD Difco) agar according to US EPA Method 160037 (n = 696), as well as mEI containing 8 µg ml-1 erythromycin (n = 257). E. coli were enumerated and isolated from surface water using membrane filtration onto modified mTEC (BD Difco) agar according to US EPA Method 160338 (n = 864), and on selective media containing 1 µg ml-1 ceftriaxone (n = 103). The limits of detection for enumeration of enterococci and E. coli were 10 CFU 100 ml-1, 5 CFU 100 ml-1, and 1 CFU 100 ml-1 for the catch basins, wetland, and creek, respectively. Enterococci were suspended in tryptic soy broth containing 30% glycerol and E. coli were suspended in Luria-Bertani broth containing 30% glycerol and stored at -80°C. All media were prepared by Dalynn Biologicals Inc. (Calgary, AB, Canada). Enterococci Speciation. Genomic DNA was prepared by suspending one Enterococcus colony in 125 µl of buffer containing 10 mM Tris plus 1mM EDTA, pH 7.4, and heating at 95°C for 1 min. DNA was stored at -20°C. For fecal enterococci, the hypervariable groES-EL spacer region was PCR amplified as previously described.39 Sanger sequencing of PCR products was conducted by Functional Biosciences (Madison, WI, USA), Bio Basic (Markham, ON, Canada), or Eurofins Genomics (Louisville, KY, USA). Species assignment was determined by BLAST comparison against a custom database of groES-EL spacer/intergenic region for Enterococcus species.39 Species identification of enterococci isolated from surface water was determined according to a series of species-specific PCR reactions based on the sodA gene.40, 41 Briefly, each 20 µl reaction contained 1x KAPA2G buffer B, 0.2 mM dNTPs, 0.5 µM each primer, and 0.4 units KAPA2G Robust HotStart DNA Polymerase (Kapa Biosystems, Wilmington, MA, USA), plus 1.5 µl of genomic DNA. The following primers were used in combination: HI1 and HI2B (Enterococcus hirae) plus CA1B and CA2 (Enterococcus casseliflavus) and FL1 and FL2 (Enterococcus



faecalis) plus FM1B and FM2B (Enterococcus faecium), and separately: MU1 and MU2 (Enterococcus mundtii) and DU1b and DU2B (Enterococcus durans). The PCR reaction consisted of a 3 min denaturation at 95°C, followed by 30 cycles of denaturation at 95°C for 15 s, annealing at previously specified temperatures40, 41 for 15 s, and elongation at 72°C for 20 s. Reactions were analysed via capillary gel electrophoresis using a QIAxcel (Qiagen Inc., Mississauga, ON, Canada) with a screening cartridge and 50 to 800 bp size markers. Enterococci that did not generate a species-specific PCR product were subject to DNA sequencing of the groES-EL spacer region as described above. Approximately 10% of enterococci that generated PCR products with species-specific primers were sequenced to ensure concordance between PCR-based speciation and groES-EL sequencing. E. coli Phylotyping. Genomic DNA was prepared as described above. E. coli phylotyping was conducted according to the previously described ‘Improved Clermont’ PCR assay, which targets the TspE4.C2, yjaA, chuA, and arpA genes.42 Phylogroup B1 isolates were also screened for the presence of the clpX6 allele (Whitman MLST scheme) as previously described.21 Isolates producing a PCR product were assigned to the environmentally adapted ET-1 clade.20 Statistical Analysis. FIB enumeration data were log-transformed prior to statistical analysis. Enumeration negative samples were assigned an arbitrary value based on 50% of the limit of detection.43 Accordingly, the assigned values were 0.70 log10 CFU 100 ml-1, 0.40 log10 CFU 100 ml-1, and -0.30 log10 CFU 100 ml-1 for the catch basin, wetland, and creek, respectively. The Students t-test (data distributed normally) or Mann-Whitney Rank Sum (data not distributed normally) was used to compare differences between means of two groups. Fisher’s exact test was used to test differences in frequencies of various taxa between two populations. Significance was established at P < 0.05. Genetic diversity was calculated according to the Shannon index.44


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RESULTS Field Observations. Precipitation was greater and average temperatures were lower from May to mid-August of 2014 compared to 2015 (Figure S3). Catch basin A remained relatively full throughout 2014, while runoff that accumulated in the much smaller CBB was transferred to the constructed wetland on two occasions prior to sampling in May and September (Table S2). In 2015, CBA was used for irrigation, while runoff from CBB was transferred to CBA to augment irrigation. The wetland did not receive any transfer of runoff from CBB during 2015, and by mid-July was dry in elevated areas (Figure S4). Changes in FIB Populations as Wastewater Moved Sequentially from CBB to the Constructed Wetland and Creek. Fewer enterococci were isolated from the wetland compared to CBB, which was the input source for the wetland (3.1 ± 1.1 log10 CFU 100 ml-1 versus 4.3 ± 0.9 log10 CFU 100 ml-1, respectively; P = 0.01, Table 1 and Figure S5). Likewise, fewer E. coli were isolated from the wetland compared to CBB (2.5 ± 1.4 log10 CFU 100 ml-1 versus 3.8 ± 1.0 log10 CFU 100 ml-1, respectively; P = 0.02). Enterococci were isolated from the ephemeral creek on four occasions in 2014, and two occasions in 2015, while E. coli were isolated from the creek on only two occasions in 2014 (Figure S6). Densities were lower than observed for the catchments and generally did not exceed 2.0 log10 CFU 100 ml-1. Of the presumptive enterococci isolated on media without antibiotics, 96% (1023/1062) were confirmed to be enterococci based on species-specific PCR or groES-EL sequencing. The enterococci populations varied across environments, with the lowest species diversity observed in cattle feces and the highest in the constructed wetland (Table S3). Enterococcus hirae represented 92% of the enterococci isolated from cattle feces, but was less abundant in the catchments, and accounted for 64% and 33% of isolates from CBB and the wetland, respectively.



The second most abundant species isolated from CBB and the wetland was E. casseliflavus, which comprised 21% and 29% of enterococci isolates, respectively. This species, along with Enterococcus gallinarum and E. durans, were isolated from CBB and the wetland, but not cattle feces. Although E. faecalis and E. faecium were rarely isolated from cattle feces (0.05). The species composition of the enterococci populations from the catch basins changed dynamically between sampling times (Figure 1), and alternated between populations that were predominated by either E. hirae or E. casseliflavus. In particular, E. hirae was the predominant species isolated (often exceeding 70% of the population) from both catch basins throughout 2014 and the spring of 2015. These sampling times corresponded with periods of relatively high precipitation. Conversely, E. casseliflavus predominated during periods of low precipitation from June to October of 2015. Comparatively, enterococci populations from the constructed wetland exhibited less temporal variability of species composition. There were only three occasions where E. hirae exceeded more than 50% of the population, namely May, June, and Sept of 2014, which corresponded to the transfer of waste from CBB into the wetland. Rather, populations



tended to have greater diversity regardless of wet versus drought conditions, and were generally composed of three or more similarly abundant species. In contrast to enterococci populations, dramatic temporal shifts in phylotype composition of the E. coli populations were not observed in any of the catchments (Figure 2). Selectively Isolated FIB Populations. Although fewer enterococci and E. coli were isolated on selective media compared to media without antibiotics (Table 1), overall densities exhibited similar temporal trends compared to those isolated on non-selective media (Figure S5). Significantly fewer enterococci were isolated from the wetland compared to CBB (1.8 ± 1.1 log10 CFU 100 ml-1 versus 2.9 ± 1.1 log10 CFU 100 ml-1, respectively; P = 0.03), while E. coli densities were not different between the two catchments (P > 0.05; Table 1). Erythromycin resistant enterococci were isolated from the creek only once, while E. coli were isolated twice (in 2014) on media containing ceftriaxone. Within the same catchment (i.e., CBA, CBB or wetland), enterococci densities were not different (P > 0.05) between wet and dry years (2014 and 2015, respectively; Table 1). Densities of E.coli did not differ between 2014 and 2015 in CBA or the wetland; however, they were lower in 2015 in CBB compared to 2014 (1.0 ± 0.4 log10 CFU 100 ml-1 versus 3.2 ± 1.1 log10 CFU 100 ml-1, respectively; P = 0.002). Of the erythromycin resistant enterococci, 98% (486/498) were confirmed to be enterococci. The species composition (i.e., types of species and relative species abundance) of resistant populations was generally similar to the composition of populations that were isolated using media without antibiotics for each respective environment (Table S3). For example, E. casseliflavus, which accounted for 29% of the non-selectively isolated enterococci population from the wetland, also represented 27% of erythromycin resistant enterococci.


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Of the presumptive E. coli isolated on selective media, almost 100% (352/353) were confirmed as Escherichia. Phylogroups A and B1 were the most frequent phylogroups isolated on selective media (Table S4). Fisher’s exact test indicated that while clade ET-1 strains were isolated at similar frequency from feces using non-selective versus selective media (i.e., 17% versus 21% E. coli, respectively; P >0.05, Table S4), these strains represented 27% of E. coli in the wetland (on non-selective media), but only accounted for 7% of E. coli obtained from selective media (P = 0.01).



DISCUSSION Limited research has been conducted to decipher the impact of secondary environments on the genetic diversity of FIB populations in runoff from beef cattle feedlots. In the current study, the temporal dynamics of enterococci and E. coli populations isolated from two separate catch basins and a constructed wetland were characterized. As well, FIB populations from an ephemeral creek that drained land irrigated with wastewater were examined. However, no evidence was found to support that FIB from the creek originated from the feedlot, as populations were predominated by species not isolated from cattle feces, such as E. mundtii, while taxa commonly isolated from cattle feces, namely E. hirae and phylogroup A E. coli, were rarely isolated (Table S3). A key observation was that FIB densities in the catchments were similar during both years of the study, despite substantial differences in precipitation and pen runoff. The relatively high FIB densities during drought conditions in the second year were not attributable to other sources such as visiting or resident birds, since fecal input from an improbably large number of birds would be required to generate the high bacterial densities observed in the catchments. Furthermore, bird feces typically contain at least 10-fold more E. coli than enterococci,46 while densities of both indicators were similar. A more plausible explanation is that environmental growth of FIB within these nutrient-rich catchments maintained the high FIB densities in the absence of waste input from feedlot pens. Environmental growth of E. coli in cattle fecal pats following deposition has been well documented,47, 48 with moisture and temperature described as major driving factors.48 Enterococci and E. coli have also been observed to propagate in various aquatic environments including wastewater treatment plants, constructed wetlands, mesocosms, and recreational waters.46, 49-51


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Consistent with previous reports, E. hirae was the predominant Enterococcus species isolated from beef cattle feces (Table S3).33, 34 Abundance of this species was relatively diminished within the catch basins and wetland, suggesting that E. hirae may not be as well-adapted for secondary aquatic environments as other species. Conversely, the increased relative abundances of E. durans and the yellow-pigmented species, E. casseliflavus and E. gallinarum, in the catchments, indicates that secondary environments were likely more conducive to the growth of these species. Enterococcus casseliflavus is an environmentally adapted species primarily associated with vegetation.52 It has also been previously isolated from cattle feces,53 particularly from pastured animals, although it is not clear whether this species is a bonafide enteric resident of cattle or primarily acquired from the environment through ingestion of contaminated water. Nevertheless, given that E. casseliflavus was not isolated from cattle feces, it is likely this species represents an environmental taxon. Similarly, E. durans and E. gallinarum were isolated only from secondary environments. These species are ubiquitous among various hosts including cattle,16, 34 although the lack of isolation from cattle feces in the current study indicates they may also represent environmentally adapted species. E. mundtii, which was unique to the wetland, is also a yellow-pigmented species that is primarily plant-associated.28 Curiously, E. faecalis and E. faecium, which are the main species of relevance to human health, were relatively more abundant in the wetland compared to the catch basins or feces (Table S3). Similar increases in E. faecalis and E. faecium densities during the movement of human waste through a constructed wetland have been reported previously.6 Although the source of these species in the feedlot wetland remains unknown, it is possible they may represent plantassociated strains.28 Further studies to determine the origin and potential risk to human health of these strains are warranted.



The enterococci population structure in the catch basins changed dynamically, where populations composed almost exclusively of E. hirae and resembling cattle fecal populations during wet conditions, shifted to populations that were predominated by E. casseliflavus during the prolonged drought in 2015 (Figure 1). This suggests that the postulated bacterial growth that occurred in the catch basins under drought conditions was due primarily to environmentally adapted species. Importantly, similar changes in bacterial population structure were observed in both catch basins draining separate regions of the feedlot, making it unlikely that results were confounded by inadequate sampling depth or an inability to capture sufficient spatial variability (inherent short-comings associated with sampling large waste storages).54 Compared to enterococci populations from the catch basins, those from the constructed wetland exhibited greater temporal stability with respect to species composition (Figure 1). This stability may be indicative of the increased complexity of spatial factors that influenced niche partitioning in the wetland compared to the catch basins. For example, the shallow but broader surface area coupled with abundant vegetation may lead to more spatial variability and allow various taxa to occupy different niches, thereby decreasing dominance of a single species. Environmentally adapted E. coli strains were also relatively more abundant in secondary environments compared to cattle feces. In particular, strains from the ET-1 clade, originally described as a phylogenetically distinct subgroup of environmentally adapted phylogroup B1 strains from aquatic environments,20 were relatively more abundant in CBB and the constructed wetland (Table S4). Unlike the environmentally adapted enterococci species that were restricted to secondary environments, clade ET-1 strains were also isolated from cattle feces. It thus appears that cattle feces could be a source of these strains in the environment, where they undergo positive selection. These findings are in keeping with previous studies which have


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shown that E. coli from cattle feces exhibit extended survival in aquatic environments,9, 15 and that persistent strains predominantly belong to phylogroup B1.15 While it is presumed that the clade ET-1 strains in the catch basins and wetland originated from cattle feces, it is possible that cattle may have different clade ET-1 strains compared to those in the environment. Clade ET-1 is a genetically heterogeneous group represented by diverse MLST profiles.20, 21 Consequently, it remains to be determined if higher resolution typing methods can distinguish between fecal and environmental strains. Nonetheless, the current study provides insight into the potential nature of the environmentally persistent population from cattle, and adds to the growing body of evidence suggesting that clade ET-1 strains have superior fitness in secondary environments.20, 21, 55 The relative abundance of clade ET-1 strains was not increased in all secondary environments, as the abundance of these strains in CBA was similar to that of feces. Initially, it was considered whether clade ET-1 strains were more abundant in cattle feces from specific pens which drained into CBB compared to those that drained into CBA. However, this did not appear to be the case as clade ET-1 strains accounted for 12.3% of fecal E. coli from pens that drained into CBB and 19.8% of fecal strains from pens that drained into CBA. Instead, it is suspected that this finding may be related to differences between the environments. Most obviously, both CBB and the wetland contained vegetation (although it was relatively sparse in the CBB compared to the wetland), while CBA did not. Interestingly, many phylogroup B1 strains exhibit adaptive traits linked to plant colonization.56 It has also been observed that vegetated zones of constructed wetlands promote E. coli growth, while open zones do not.5 We cautiously speculate that the greater relative abundance of clade ET-1 strains in the CBB and the wetland could be a result of environmental adaptations that facilitate plant colonization.



Given that carriage of antimicrobial resistant (AMR) determinants may result in a competitive fitness disadvantage for host bacteria due to increased metabolic burden,29 it was anticipated that such strains would be almost entirely eliminated during prolonged waste storage. Contrary to our expectations, substantial numbers of enterococci and E. coli were selectively isolated despite prolonged storage during drought conditions (Table 1). Erythromycin resistance was widespread among Enterococcus species, including fecal (E. hirae) as well as environmentally adapted species. While the nature and origin of erythromycin resistance among the enterococci has yet to be determined, such findings suggest that erythromycin resistant enterococci can readily persist among environmentally adapted strains within catchments. In contrast, AMR determinants conferring reduced susceptibility to ceftriaxone do not seem to be well maintained among environmentally adapted E. coli strains. For instance, while clade ET-1 strains represented 27% of the total E. coli from the wetland, they only accounted for 7% of the selectively isolated strains. It appears that E. coli strains carrying AMR determinants may have reduced fitness in secondary environments, or that AMR determinants may not be readily maintained among clade ET-1 strains in in the absence of selective pressure. Nonetheless, isolation of enterococci and E. coli on selective media long after the input of wastewater from cattle pens and selection pressure had ceased, is similar to previous findings showing that resistant FIB can be isolated from cattle that have not been treated with antibiotics or where treatment has long ended.31, 32, 34 Ultimately, the fate of FIB in secondary environments is complex, with population diversity reflecting the balance between selective pressures that culminate in the elimination of strains not adapted to secondary environments, and selection of those that are. Strains that adapt to secondary environments may be more likely to be broadly disseminated, while still differing in their potential risk of transmission to humans. Although environmentally adapted species like E.


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casseliflavus have occasionally been reported to be associated with disease,57 this is generally infrequent compared to E. faecalis and E. faecium. Likewise, environmentally adapted E. coli clade ET1 strains belong to phylogroup B1, a phylogroup not commonly associated with extraintestinal infections.2 Furthermore, enteric pathogenicity is unlikely as very few clade ET-1 strains are Shiga toxin producers, as evidenced by the lack of entries in the Shigatox.net database.58 In conclusion, the overall changes in FIB population dynamics within the catch basins, and particularly within the constructed wetland, appeared to limit environmental dissemination of clinically relevant FIB by selecting for environmentally adapted strains. While environmentally adapted Enterococcus and E. coli may still function as a potential reservoir for AMR determinants, results suggest that they may have impaired zoonotic potential due to their adaptation for survival and proliferation in these secondary environments.

ASSOCIATED CONTENT Supporting Information. Includes antimicrobial usage data (Table S1), field observations (Table S2), distribution of Enterococcus species and E. coli phylogroups (Tables S3 and S4), feedlot schematic (Figure S1), experimental methods (Figure S2), climate data (Figure S3), field sites photos (Figure S4), FIB enumeration (Figure S5), and FIB isolation from the creek (Figure S6).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; Phone: 1-403-381-5165; Fax: 1-403-381-5765. Notes



The authors declare no competing financial interest.

ACKNOWLEDGMENTS We are grateful for funding provided by the Beef Cattle Research Council (project FOS.10.13) and to the cooperating feedlot for allowing access to the feedlot.


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Table 1. Enumeration of Enterococci and E. coli from Secondary Environments log10 CFU 100 ml-1 (mean ± standard deviation) enterococci E. coli site

2014

2015

average (2014/15)

2014

2015

Non-selective media Catch basin A 3.9 ± 0.8 4.1 ± 0.8 n/a 4.0 ± 1.1 3.4 ± 0.8 (CBA) Catch basin B 4.0 ± 1.0 4.5 ± 0.8 4.3 ± 0.9 4.0 ± 1.0 3.6 ± 1.1 (CBB) Constructed 3.1 ± 1.2 3.2 ± 1.1 3.1 ± 1.1 2.6 ± 1.5 2.5 ± 1.3 Wetland Selective media containing 8 µg ml-1 erythromycin (enterococci) and 1 µg ml-1 ceftriaxone (E. coli) Catch basin A 2.1 ± 1.0 2.5 ± 1.2 n/a 2.3 ± 1.6 1.1 ± 0.7 (CBA) Catch basin B 2.4 ± 1.1 3.4 ± 1.0 2.9 ± 1.1 3.2 ± 1.1 1.0 ± 0.4 (CBB) Constructed 1.9 ± 1.3 1.6 ± 1.0 1.8 ± 1.1 1.4 ± 1.4 0.9 ± 0.7 Wetland


average (2014/15)

n/a 3.8 ± 1.0 2.5 ± 1.4

n/a 2.1 ± 1.4 1.2 ± 1.1


Figure 1. Relative abundance of Enterococcus species isolated from catch basin A (CBA), catch basin B (CBB) and a constructed wetland for each sampling event. (Left) Enterococci isolated via non-selective media; (Right) Enterococci isolated via selective media containing 8 µg ml-1 erythromycin. Note that there were no samples for catch basin A on April 14, 2014 or catch basin B on September 15, 2014. Figure 2. Relative abundance of E. coli phylogroups isolated from catch basin A (CBA), catch basin B (CBB) and a constructed wetland for each sampling event. (Left) E. coli isolated via non-selective media; (Right) E. coli isolated via selective media containing 1 µg ml-1 ceftriaxone. Abbreviation: cc, cryptic clades III, IV, V. Note that there were no samples for catch basin A on April 14, 2014 or catch basin B on September 15, 2014.


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84x43mm (300 x 300 DPI)



Non-selective isolation

Selective isolation

Catch basin A

Catch basin B

Constructed wetland

Figure 1.


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Non-selective isolation

Selective isolation

Catch basin A

Catch basin B

Constructed wetland

Figure 2.


Environmental Growth of Enterococci and Escherichia coli in Feedlot

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