Impact of Vegetative Treatment Systems on Multiple Measures of Antibiotic Resistance in Agricultural Wastewater

Wastewater is an important vector of antibiotic resistant bacteria and antibiotic resistance genes (ARB/G). While there is broad agreement that ARB/G from agricultural (ag) wastewaters can be transported through the environment and may contribute to untreatable infectious disease in humans and animals, there remain large knowledge gaps surrounding applied details on the types and amounts of ARB/G associated with different agricultural wastewater treatment options and different ag production systems. This study evaluates a vegetative treatment system (VTS) built to treat the wastewater from a beef cattle feedlot. Samples were collected for three years, and plated on multiple media types to enumerate tetracycline and cefotaxime-resistant bacteria. Enterobacteriaceae isolates (n = 822) were characterized for carriage of tetracycline resistance genes, and E. coli isolates (n = 673) were phenotyped to determine multi-drug resistance (MDR) profiles. Tetracycline resistance in feedlot runoff wastewater was 2-to-3 orders of magnitude higher compared to rainfall runoff from the VTS fields, indicating efficacy of the VTA for reducing ARB over time following wastewater application. Clear differences in MDR profiles were observed based on the specific media on which a sample was plated. This result highlights the importance of method, especially in the context of isolate-based surveillance and monitoring of ARB in agricultural wastewaters

Microbial Communities Associated With Stable Fly (Diptera: Muscidae) Larvae and Their Developmental Substrates

Bacteria are essential for stable fly (Stomoxys calcitrans (L.)) larval survival and development, but little is known about the innate microbial communities of stable flies, and it is not known if their varied dietary substrates influence their gut microbial communities. This investigation utilized 454 sequencing of 16S and 18S amplicons to characterize and compare the bacterial and eukaryotic microbial communities in stable fly larvae and their developmental substrates. The microbial community of the third-instar stable fly larvae is unambiguously distinct from the microbial community of the supporting substrate, with bacterial communities from larvae reared on different substrates more similar to each other than to the communities from their individual supporting substrates. Bacterial genera that were more abundant proportionally in larvae compared to their substrates were Erysipelothrix, Dysgonomonas, Ignatzschineria (Gammaproteobacteria), and Campylobacter (Epsilonprotobacteria). The alphaproteobacteria Devosia, Brevundimonas, Sphingopyxix, and Paracoccus were more abundant proportionally in field substrates compared to their larvae. The main genera responsible for differences between the positive and negative field substrates were Dysgonomonas and Proteiniphilum. In contrast to Dysgonomonas, Proteiniphilum was more abundant in substrate than in the larvae. A large number of sequences were assigned to an unclassified protest of the superphylum Alveolata in larvae and their substrate. Microscopy validated these findings and a previously undescribed gregarine (phylum Apicomplexa, class Conoidasida) was identified in stable fly larvae and adults

Distribution and Quantification of Antibiotic Resistant Genes and Bacteria across Agricultural and Non- Agricultural Metagenomes

There is concern that antibiotic resistance can potentially be transferred from animals to humans through the food chain. The relationship between specific antibiotic resistant bacteria and the genes they carry remains to be described. Few details are known about the ecology of antibiotic resistant genes and bacteria in food production systems, or how antibiotic resistance genes in food animals compare to antibiotic resistance genes in other ecosystems. Here we report the distribution of antibiotic resistant genes in publicly available agricultural and non-agricultural metagenomic samples and identify which bacteria are likely to be carrying those genes. Antibiotic resistance, as coded for in the genes used in this study, is a process that was associated with all natural, agricultural, and human-impacted ecosystems examined, with between 0.7 to 4.4% of all classified genes in each habitat coding for resistance to antibiotic and toxic compounds (RATC). Agricultural, human, and coastal-marine metagenomes have characteristic distributions of antibiotic resistance genes, and different bacteria that carry the genes. There is a larger percentage of the total genome associated with antibiotic resistance in gastrointestinal-associated and agricultural metagenomes compared to marine and Antarctic samples. Since antibiotic resistance genes are a natural part of both human-impacted and pristine habitats, presence of these resistance genes in any specific habitat is therefore not sufficient to indicate or determine impact of anthropogenic antibiotic use. We recommend that baseline studies and control samples be taken in order to determine natural background levels of antibiotic resistant bacteria and/or antibiotic resistance genes when investigating the impacts of veterinary use of antibiotics on human health. We raise questions regarding whether the underlying biology of each type of bacteria contributes to the likelihood of transfer via the food chain

Differential Survival of Non-O157 Shiga Toxigenic Escherichia coli in Simulated Cattle Feedlot Runoff

Environmental survival time is important when evaluating adverse health outcomes from foodborne pathogens. Although outbreaks associated with manure-impacted irrigation or runoff water are relatively infrequent, their broad scope, regulatory importance, and severe health outcomes highlight the need to better understand the environmental survival of manure-borne pathogens. Shiga toxigenic Escherichia coli (STEC) are excreted in feces and persist in the environment until they die or recolonize a new host. Surface waters contaminated with manure-borne STEC can infect humans through drinking and recreational water use or irrigated crops that are minimally cooked. In this study, manure-impacted water microcosms mimicking beef cattle feedlot runoff were used to assess survival of STEC strains representing seven STEC serotypes (O26, O45, O103, O111, O121, O145, and O157) and persistence of target O antigen genes. Microcosms were sampled over the course of 1 year, and the entire experiment was repeated in a second year. Culture and polymerase chain reaction (PCR)- based techniques were used for detection and enumeration. Serotype-specific survival results were observed. Both STEC O26 and O45 declined slowly and remained culturable at 24 months. In contrast, STEC O121 and O145 decreased rapidly (-0.84 and -1.99 log10 abundance per month, respectively) and were unculturable by months 4 and 5, but detectable by PCR for a mean of 4.5 and 8.3 months, respectively. STEC O103, O111, and O157 remained culturable for a mean of 11.6, 5.5, and 15 months and detectable by PCR for a mean of 12, 13.8, and 18.6 months after inoculation, respectively. Results document that some STEC serotypes have the biological potential to survive in manure-impacted waters for extended periods of time when competing microflora are eliminated. Serotype-specific differences in survival of target bacteria and persistence of target genes were observed in this sample set, with STEC O26 and O45 strains appearing the most robust in these microcosm studies

Impact of Vegetative Treatment Systems on Multiple Measures of Antibiotic Resistance in Agricultural Wastewater

Wastewater is an important vector of antibiotic resistant bacteria and antibiotic resistance genes (ARB/G). While there is broad agreement that ARB/G from agricultural (ag) wastewaters can be transported through the environment and may contribute to untreatable infectious disease in humans and animals, there remain large knowledge gaps surrounding applied details on the types and amounts of ARB/G associated with different agricultural wastewater treatment options and different ag production systems. This study evaluates a vegetative treatment system (VTS) built to treat the wastewater from a beef cattle feedlot. Samples were collected for three years, and plated on multiple media types to enumerate tetracycline and cefotaxime-resistant bacteria. Enterobacteriaceae isolates (n = 822) were characterized for carriage of tetracycline resistance genes, and E. coli isolates (n = 673) were phenotyped to determine multi-drug resistance (MDR) profiles. Tetracycline resistance in feedlot runoff wastewater was 2-to-3 orders of magnitude higher compared to rainfall runoff from the VTS fields, indicating efficacy of the VTA for reducing ARB over time following wastewater application. Clear differences in MDR profiles were observed based on the specific media on which a sample was plated. This result highlights the importance of method, especially in the context of isolate-based surveillance and monitoring of ARB in agricultural wastewaters

Tracking Bacteria through the Entire Gastrointestinal Tract of a Beef Steer

Biological processes within beef cattle gastrointestinal tracts (GIT) influence animal health, and the output (feces) is an important vector for zoonotic pathogens and antibiotic resistant bacteria. Historically, outside of the rumen, little attention was paid to the bacteria along the GIT, despite their essential role in catabolizing feed into feces. Here we characterize bacteria from 15 GIT sites within a beef steer and examine the proportion of bacteria contributed by upstream compartments. This animal displayed characteristic differences between tissue and digesta communities in gastric and large-intestine, but not small-intestine, samples. The GIT sites shared between 50 and 80% of the operational taxonomic units (OTUs) with their neighboring upstream compartment, with the exception of the ileum. The ileum shared only 11% with the jejunum but displayed a similar phylum-level taxonomic distribution with the jejunum. It is unclear whether the observed changes between compartments represent a nonrandom decrease in bacterial number or rapid multiplication of certain taxa

Simulated Winter Incubation of Soil With Swine Manure Differentially Affects Multiple Antimicrobial Resistance Elements

Gastrointestinal bacteria that harbor antibiotic resistance genes (ARG) become enriched with antibiotic use. Livestock manure application to cropland for soil fertility presents a concern that ARG and bacteria may proliferate and be transported in the environment. In the United States, manure applications typically occur during autumn with slow mineralization until spring planting season. A laboratory soil incubation study was conducted mimicking autumn swine manure application to soils with concentrations of selected ARG monitored during simulated 120-day winter incubation with multiple freeze-thaw events. Additionally, the effects of two soil moistures [10 and 30% water holding capacity (WHC)] and two manure treatments [raw versus hydrated lime alkaline stabilization (HLAS)] were assessed. Fourteen tetracycline resistance genes were evaluated; tet(D), tet(G), and tet(L) were detected in background soil while swine manure contained tet(A), tet(B), tet(C), tet(G), tet(M), tet(O), tet(Q), and tet(X). By day 120, the manure-borne tet(M) and tet(O) were still detected while tet(C), tet(D), tet(L), and tet(X) genes were detected less frequently. Other tet resistance genes were detected rarely, if at all. The sum of unique tet resistance genes among all treatments decreased during the incubation from an average of 8.9 to 3.8 unique tet resistance genes. Four resistance elements, intI1, blactx−m−32, sul(I), erm(B), and 16s rRNA genes were measured using quantitative PCR. ARG abundances relative to 16S abundance were initially greater in the raw manure compared to background soil (−1.53 to −3.92 log abundance in manure; −4.02 to \u3c−6.7 log abundance in soil). In the mixed manure/soil, relative abundance of the four resistance elements decreased (0.87 to 1.94 log abundance) during the incubation largely because 16S rRNA genes increased by 1.21 log abundance. Throughout the incubation, the abundance of intI1, blactx−m−32, sul(I), and erm(B) per gram in soil amended with HLAS-treated manure was lower than in soil amended with raw manure. Under low initial soil moisture conditions, HLAS treatment reduced the abundance of intI1 and resulted in loss of blactx−m−32, sul(I), and erm(B)] compared to other treatment-moisture combinations. Although one might expect antibiotic resistance to be relatively unchanged after simulated winter manure application to soil, a variety of changes in diversity and relative abundance can be expected

Narrow grass hedge effects on microbial transport following variable applications of beef cattle manure

The effectiveness of a 1.4 m wide grass hedge in reducing microbial transport following manure application was examined in this study. Beef cattle manure was applied to 0.75 m wide by 4.0 m long plots established on an Aksarben silty clay loam located in southeast Nebraska. Manure was added at rates required to meet none or the 1-, 2-, or 4-year nitrogen requirements for corn. The transport of phages, total coliforms, E. coli, and enterococci was measured for three 30 min simulated rainfall events, which were separated by approximately 24 h intervals. The narrow grass hedge reduced total counts of phages, E. coli, and enterococci from 10.8 to 9.01 log PFU ha-1, from 12.4 to 11.9 log CFU ha-1, and from 11.8 to 11.2 log CFU ha-1, respectively. For the plots that received manure, no significant differences in transport of phages or enterococci were found among the three manure application rates. Rainfall simulation run significantly affected measurements of phages, total coliforms, and enterococci, with measurements during the three runs varying from 8.91 to 10.5 log PFU ha-1, from 12.7 to 13.3 log CFU ha-1, and from 11.2 to 11.7 log CFU ha-1, respectively. Counts for phages, total coliforms, and enterococci were significantly less for the first than the second and third rainfall simulation runs. All four of the microbial constituents were significantly correlated to dissolved P, particulate P, total P, and total N. A narrow grass hedge placed on the contour significantly reduced microbial transport following variable applications of beef cattle manure

Microbial Communities Associated With Stable Fly (Diptera: Muscidae) Larvae and Their Developmental Substrates

Bacteria are essential for stable fly (Stomoxys calcitrans (L.)) larval survival and development, but little is known about the innate microbial communities of stable flies, and it is not known if their varied dietary substrates influence their gut microbial communities. This investigation utilized 454 sequencing of 16S and 18S amplicons to characterize and compare the bacterial and eukaryotic microbial communities in stable fly larvae and their developmental substrates. The microbial community of the third-instar stable fly larvae is unambiguously distinct from the microbial community of the supporting substrate, with bacterial communities from larvae reared on different substrates more similar to each other than to the communities from their individual supporting substrates. Bacterial genera that were more abundant proportionally in larvae compared to their substrates were Erysipelothrix, Dysgonomonas, Ignatzschineria (Gammaproteobacteria), and Campylobacter (Epsilonprotobacteria). The alphaproteobacteria Devosia, Brevundimonas, Sphingopyxix, and Paracoccus were more abundant proportionally in field substrates compared to their larvae. The main genera responsible for differences between the positive and negative field substrates were Dysgonomonas and Proteiniphilum. In contrast to Dysgonomonas, Proteiniphilum was more abundant in substrate than in the larvae. A large number of sequences were assigned to an unclassified protest of the superphylum Alveolata in larvae and their substrate. Microscopy validated these findings and a previously undescribed gregarine (phylum Apicomplexa, class Conoidasida) was identified in stable fly larvae and adults

Management-intensive grazing impacts on total \u3ci\u3eEscherichia coli, E. coli\u3c/i\u3e O157: H7, and antibiotic resistance genes in a riparian stream

The impacts of management-intensive grazing (MIG) of cattle on concentrations of total Escherichia coli, total suspended solids (TSS), and nitrate-nitrite nitrogen (NO3+NO2-N), and occurrence of E. coli O157:H7 and selected antibiotic resistance genes (ARGs) in stream water and/or sediments were evaluated. Cattle were grazed for twoweek periods in May in each of three years. Overall, grazing increased total E. coli in downstream water by 0.89 log10 MPN/100 mL (p \u3c 0.0001), and downstream total E. coli concentrations were higher than upstream over all sampling intervals. Downstream TSS levels also increased (p ≤ 0.0294) during grazing. In contrast, there was a main effect of treatment for downstream NO3 + NO2-N to be lower than upstream (3.59 versus 3.70 mg/L; p = 0.0323). Overwintering mallard ducks increased total E. coli and TSS concentrations in January and February (p \u3c 0.05). For precipitation events during the 24 h before sampling, each increase of 1.00 cm of rainfall increased total E. coli by 0.49 log10 MPN/100 mL (p=0.0005). In contrast, there was no association of previous 24 h precipitation volume on TSS (p=0.1540), and there was a negative linear effect on NO3+NO2-N (p=0.0002). E. coli O157: H7 prevalence was low, but the pathogen was detected downstream up to 2½ months after grazing. Examination of ARGs sul1, ermB, blactx-m-32, and intI1 identified the need for additional research to understand the impact of grazing on the ecology of these resistance determinants in pasture-based cattle production. While E. coli remained higher in downstream water compared to upstream, MIG may reduce the magnitude of the downstream E. coli concentrations. Likewise, the MIG strategy may prevent large increases in TSS and NO3+NO2-N concentrations during heavy rain events. Results indicate that MIG can limit the negative effects of cattle grazing on stream water quality