Leaching of antibiotic resistance genes and microbial assemblages following poultry litter applications in karst and non-karst landscapes.

Antibiotic resistance is increasingly recognized as a critical challenge affecting human, animal, and environmental health. Yet, environmental dynamics and transport of antibiotic resistance genes (ARGs) and microbial communities in karst and non-karst leachate following poultry litter land applications are not well understood. This study investigates impacts of broiler poultry litter application on the proliferation of ARGs (tetW, qnrS, ermB, sulI, and blaCTX-M-32), class 1 integron (intI1 i), and alterations in microbial communities (16S rRNA) within karst derived soils, which are crucial and under-researched systems in the global hydrological cycle, and non-karst landscapes. Using large, intact soil columns (45 cm diam. × 100 cm depth) from karst and non-karst landscapes, the role of preferential flow and ARG transport in leachate was enumerated following surface application of poultry litter and simulated rain events. This research demonstrated that in poultry litter amended karst soils, ARG (i.e., ermB and tetW) abundance in leachate increased 1.5 times compared to non-karst systems (p < 0.05), highlighting the influence of geological factors on ARG proliferation. Notably, microbial communities in karst soil leachate exhibited increased diversity and abundance, suggesting a potential linkage between microbial composition and ARG presence. Further, our correlation and network analyses identified relationships between leachate ARGs, microbial taxa, and physicochemical properties, underscoring the complex interplay in these environmentally sensitive areas. These findings illuminate the critical role of karst systems in shaping ARG abundance and pollutant dispersal and microbial community dynamics, thus emphasizing the need for landscape-specific approaches in managing ARG dissemination to the environment. This study provides a deeper understanding of hydrogeological ARG dynamics but also lays the groundwork for future research and strategies to mitigate ARG dissemination through targeted manure applications across agricultural landscapes.

Long-term impacts of conservation pasture management in manuresheds on system-level microbiome and antibiotic resistance genes.

Animal manure improves soil fertility and organic carbon, but long-term deposition may contribute to antibiotic resistance genes (ARGs) entering the soil-water environment. Additionally, long-term impacts of applying animal manure to soil on the soil-water microbiome, a crucial factor in soil health and fertility, are not well understood. The aim of this study is to assess: (1) impacts of long-term conservation practices on the distribution of ARGs and microbial dynamics in soil, and runoff; and (2) associations between bacterial taxa, heavy metals, soil health indicators, and ARGs in manures, soils, and surface runoff in a study following 15 years of continuous management. This management strategy consists of two conventional and three conservation systems, all receiving annual poultry litter. High throughput sequencing of the 16S ribosomal RNA was carried out on samples of cattle manure, poultry litter, soil, and runoff collected from each manureshed. In addition, four representative ARGs (intl1, sul1, ermB, and blactx-m-32) were quantified from manures, soil, and runoff using quantitative PCR. Results revealed that conventional practice increased soil ARGs, and microbial diversity compared to conservation systems. Further, ARGs were strongly correlated with each other in cattle manure and soil, but not in runoff. After 15-years of conservation practices, relationships existed between heavy metals and ARGs. In the soil, Cu, Fe and Mn were positively linked to intl1, sul1, and ermB, but trends varied in runoff. These findings were further supported by network analyses that indicated complex co-occurrence patterns between bacteria taxa, ARGs, and physicochemical parameters. Overall, this study provides system-level linkages of microbial communities, ARGs, and physicochemical conditions based on long-term conservation practices at the soil-water-animal nexus.

Do Long-Term Conservation Pasture Management Practices Influence Microbial Diversity and Antimicrobial Resistant Genes in Runoff?

Runoff from land-applied manure and poultry litter is one mechanism by which manure-borne bacteria are transported over large distances in the environment. There is a global concern that antimicrobial resistant (AMR) genes may be transmitted through the food chain from animal manures to soil to surface water. However, details are lacking on the ecology of AMR genes in water runoff as well as how conservation management practices may affect the runoff microbiome or minimize the movement of AMR genes. The aim of this study was to identify microbial community structure and diversity in water runoff following 14-years of poultry litter and cattle manure deposition and to evaluate the amount of AMR genes under five conventional and conservation pasture management strategies. Since 2004, all watersheds received annual poultry litter at a rate of 5.6 Mg ha-1 and were consistently managed. Surface runoff samples were collected from each watershed from 2018 to 2019, characterized using Illumina 16S rRNA gene amplicon sequencing and enumerated for four AMR-associated genes (ermB, sulI, intlI, and blactx-m-32 ) using quantitative PCR. Overall, long-term pasture management influenced water microbial community structure, with effects differing by year (p < 0.05). Bacterial richness (Chao1 index) was influenced by pasture management, with the lowest richness occurring in the control (nearby non-agricultural water source) and the greatest under fields that were hayed (no cattle presence). Runoff bacterial richness in watersheds increased following poultry litter applications, indicating poultry litter is a possible source of bacteria and altered runoff community structure. The blactx-m-32 gene was not detected in any surface water sample. The remaining three AMR genes were absent in the non-agricultural control, but present in agricultural samples. However, there was no impact (p > 0.05) from pasture management on the abundance of these genes, indicating both conventional and conservation practices have similar ecologies for these targets; however, there was a greater detection of sulI genes from runoff in continuously grazed systems in 2019, with hay being lowest in 2019. Results illustrate that the edge of field buffer strips may increase bacterial richness in water runoff, but these changes in richness do not greatly impact target AMR genes in the United States largest land-use category.

Variations in bacterial community structure and antimicrobial resistance gene abundance in cattle manure and poultry litter.

Cattle manure and poultry litter are widely used as fertilizers as they are excellent sources of nutrients; however, potential adverse environmental effects exist during land applications, due to the release of zoonotic bacteria and antimicrobial resistance (AMR) genes. This study was conducted to understand linkages between physiochemical composition, bacterial diversity, and AMR gene presence of cattle manure and poultry litter using quantitative polymerase chain reaction to enumerate four AMR genes (ermB, sulI, intlI, and blactx-m-32), Illumina sequencing of the 16 S region, and analysis of physical and chemical properties. Principal coordinate analysis of Bray-Curtis distance revealed distinct bacterial community structures between the two manure sources. Greater alpha diversity occurred in cattle manure compared to poultry litter (P < 0.05). Redundancy analysis showed a strong relationship between manure physiochemical and composition and bacterial abundance, with positive relationships occurring among electrical conductivity and carbon/nitrogen, and negative associations for total solids and soluble fractions of heavy metals. Cattle manure exhibited greater abundance of macrolide (ermB) and sulfonamide (sulI) resistant genes. Consequently, fresh cattle manure applications may result in greater potential spread of AMR genes to the soil-water environment (relative to poultry litter) and novel best management strategies (such as composting) may reduce the release of AMR genes to the soil-water environment.

Year, Location, and Variety Impact on Grape-, Soil-, and Leaf-Associated Fungal Microbiota of Arkansas-Grown Table Grapes.

With the recent advancement of next-generation sequencing methods, there has been an increase in studies on identification of vineyard microbiota, winery-associated microbiota, and microbiota in wine fermentation. However, there have been few studies investigating the fungal microbiota of table grapes which present distinct spoilage and food safety challenges. The aims of this study were to identify and compare the impact of year, variety, and vineyard location on grape, leaf, and soil fungal communities of two varieties of table grapes, Faith and Gratitude, grown in two open-air vineyards and one high tunnel vineyard. The grape, leaf, and soil mycobiota were analyzed using high throughput amplicon sequencing of the ITS region. The sampling year and location of table grapes had an impact on grape, leaf, and soil mycobiota. Fungal diversity of grape, leaf, and soil was greater in 2017 than in 2016. Grape and leaf samples presented strong similarities in fungal communities with abundance of Sporidiobolaceae and Filobasidium in two vineyards and Cladosporium in another one. The high tunnel structure had distinct grape and leaf fungal communities compared to the two other vineyard locations. Mortierella was the predominant genus (27%) in soil samples for the three locations; however, genera of lower abundance varied between locations. These results provide extensive description of fungal communities in less-studied table grape vineyards and high tunnels, providing useful insight of potential threats and preventive strategies to help improve the production and marketability of table grapes.

Antimicrobial resistant gene prevalence in soils due to animal manure deposition and long-term pasture management.

The persistence of antimicrobial resistant (AMR) genes in the soil-environment is a concern, yet practices that mitigate AMR are poorly understood, especially in grasslands. Animal manures are widely deposited on grasslands, which are the largest agricultural land-use in the United States. These nutrient-rich manures may contain AMR genes. The aim of this study was to enumerate AMR genes in grassland soils following 14-years of poultry litter and cattle manure deposition and evaluate if best management practices (rotationally grazed with a riparian (RBR) area and a fenced riparian buffer strip (RBS), which excluded cattle grazing and poultry litter applications) relative to standard pasture management (continuously grazed (CG) and hayed (H)) minimize the presence and amount of AMR genes. Quantitative PCR (Q-PCR) was performed to enumerate four AMR genes (ermB, sulI, intlI, and blactx-m-32 ) in soil, cattle manure, and poultry litter environments. Six soil samples were additionally subjected to metagenomic sequencing and resistance genes were identified from assembled sequences. Following 14-years of continuous management, ermB, sulI, and intlI genes in soil were greatest (P < 0.05) in samples collected under long-term continuous grazing (relative to conservation best management practices), under suggesting overgrazing and continuous cattle manure deposition may increase AMR gene presence. In general, AMR gene prevalence increased downslope, suggesting potential lateral movement and accumulation based on landscape position. Poultry litter had lower abundance of AMR genes (ermB, sulI, and intlI) relative to cattle manure. Long-term applications of poultry litter increased the abundance of sulI and intlI genes in soil (P < 0.05). Similarly, metagenomic shotgun sequencing revealed a greater total number of AMR genes under long-term CG, while fewer AMR genes were found in H (no cattle manure) and RBS (no animal manure or poultry litter). Results indicate long-term conservation pasture management practices (e.g., RBS and RBR) and select animal manure (poultry litter inputs) may minimize the presence and abundance of AMR genes in grassland soils.

Ecology and management of earthworm casting on sports turf.

Earthworm activity is beneficial in most natural and agricultural systems, but excessive earthworm casting is a problem on sports fields worldwide. Expulsion of soil-rich earthworm fecal matter, or casts, as mounds of soil on the turf canopy can muddy the surface, reduce photosynthesis, and lead to thinning, weed invasion and surface softening. Casts affect ball roll on sports fields, cause fouling of maintenance equipment, and dull mower blades. Build-up of cast material on reel mower units can affect height and quality of cut. Casting activity is dependent on environmental conditions such as soil texture, temperature, moisture, pH, and organic matter. Response to environmental conditions varies by species. Management options are limited, because no pesticides are specifically labeled for earthworm control at this time, and cultural control methods such as soil modification, turfgrass clipping removal, and sand topdressing have limited and inconsistent efficacy. Products containing plant-derived saponins and irritants show promise for earthworm management. Pest management practices to mitigate excessive earthworm casting will likely need to be species-specific, but limited knowledge of earthworm identification by end-users further inhibits the efficacy of control measures. © 2019 Society of Chemical Industry.

Transmissible Plasmids and Integrons Shift Escherichia coli Population Toward Larger Multiple Drug Resistance Numbers.

Transmissible plasmids and integrons may play important roles in the persistence and spread of antibiotic-resistant bacteria throughout aquatic environment by accumulating antibiotic resistance genes (ARG). Class 1 and class 2 integron (intI), mobilization (mob), sulfamethoxazole resistance (sul), and trimethoprim resistance (dfr) genes were PCR-amplified and confirmed through DNA sequencing following plasmid extraction from 139 antibiotic-resistant Escherichia coli. E. coli had previously been recovered from wastewater treatment plant effluent and receiving stream water in Northwest Arkansas and isolates had expressed resistance to one to six antibiotics. Almost half of the total isolates (47%) carried putatively transmissible plasmids with mobF12 gene as the most frequently detected mobilization gene. When two or three mob genes were detected per isolate, there was a significant shift in the population toward larger multiple drug resistance (MDR) number. Class 1 and/or 2 integrons were prevalent (46%), and the presence of integron significantly shifted the isolate population toward larger MDR number. More isolates carried single or coexistence of two or three sul genes (99.3%), and single or a combination up to five dfr genes (89.3%) than had exhibited in vitro resistance to the respective antibiotics. These findings indicate not only the role of the wastewater treatment effluent and the stream environment in coaccumulation of ARG with transmissible plasmids and integrons in multiple antibiotic-resistant E. coli populations but also suggest that density of sul and dfr resistance genes within an isolate may serve as a biomarker for mobile MDR in general.

Reducing Phosphorus Runoff and Leaching from Poultry Litter with Alum: Twenty-Year Small Plot and Paired-Watershed Studies.

Treating poultry litter with alum has been shown to lower ammonia (NH) emissions and phosphorus (P) runoff losses. Two long-term studies were conducted to assess the effects of alum-treated poultry litter on P availability, leaching, and runoff under pasture conditions. From 1995 to 2015, litter was applied annually in a paired watershed study comparing alum-treated and untreated litter and in a small plot study comparing 13 treatments (an unfertilized control, four rates of alum-treated litter, four rates of untreated litter, and four rates of NHNO). In the paired watershed study, total P loads in runoff were 231% higher from pasture receiving untreated litter (1.96 kg P ha) than from that receiving alum-treated litter (0.85 kg P ha). In both studies, alum-treated litter resulted in significantly higher Mehlich III P (M3-P) and lower water-extractable P at the soil surface, reflecting greater retention of applied P and lesser availability of that P to runoff or leaching. In soils fertilized with alum-treated litter, M3-P was much higher when analyzed by inductively coupled argon plasma emission spectrometry than by colorimetry, possibly due to the formation of aluminum phytate. Indeed, alum-treated poultry litter leached less P over the 20-yr study: M3-P at 10 to 50 cm was 266% greater in plots fertilized with untreated litter (331 kg M3-P ha) than with alum-treated litter (124 kg M3-P ha). This research provides compelling evidence that treating poultry litter with alum provides short-term and long-term benefits to P conservation and water quality.

Genetic redundancy and persistence of plasmid-mediated trimethoprim/sulfamethoxazole resistant effluent and stream water Escherichia coli.

Antibiotic resistant bacteria may persist in effluent receiving surface water in the presence of low (sub-inhibitory) antibiotic concentrations if the bacteria possess multiple genes encoding resistance to the same antibiotic. This redundancy of antibiotic resistance genes may occur in plasmids harboring conjugation and mobilization (mob) and integrase (intI) genes. Plasmids extracted from 76 sulfamethoxazole-trimethoprim resistant Escherichia coli originally isolated from effluent and an effluent-receiving stream were used as DNA template to identify sulfamethoxazole (sul) and trimethoprim (dfr) resistances genes plus detect the presence of intI and mob genes using PCR. Sulfamethoxazole and trimethoprim resistance was plasmid-mediated with three sul (sul1, sul2 and sul3 genes) and four dfr genes (dfrA12, dfrA8, dfrA17, and dfrA1 gene) the most prevalently detected. Approximately half of the plasmids carried class 1 and/or 2 integron and, although unrelated, half were also transmissible. Sampling site in relationship to effluent input significantly affected the number of intI and mob but not the number of sul and dfr genes. In the presence of low (sub-inhibitory) sulfamethoxazole concentration, isolates persisted regardless of integron and mobilization gene designation, whereas in the presence of trimethoprim, the presence of both integron and mobilization genes made isolates less persistent than in the absence of both or the presence of a gene from either group individually. Regardless, isolates persisted in large concentrations throughout the experiment. Treated effluent containing antibiotic resistant bacteria may be an important source of integrase and mobilization genes into the stream environment. Sulfamethoxazole-trimethoprim resistant bacteria may have a high degree of genetic redundancy and diversity carrying resistance to each antibiotic, although the role of integrase and mobilization genes towards persistence is unclear.

Conjugative transmission of antibiotic-resistance from stream water Escherichia coli as related to number of sulfamethoxazole but not class 1 and 2 integrase genes.

A conjugation assay was used to determine the effects of phenotypic resistance to one to up to 5 antibiotics, sampling site of origin, presence or absence of class 1 and/or class 2 integrase (intI) genes (intI1 and intI2), and the number of sulfamethoxazole resistance (sul) and trimethoprim resistance (dfr) genes on the transfer frequencies of plasmids from environmental, antibiotic-resistant Escherichia coli. Of 51 sulfamethoxazole and trimethoprim-resistant E. coli isolates conferring at least one mob gene (mobP51, mobF11, mobF12, mobQ11, mobQ12 , or mobQu ), 38 produced transconjugants with an overall mean frequency of 1.60 × 10-3 transconjugants/ donors (T/D) or 5.89 × 10-3 transconjugants/recipients (T/R). The presence or absence of intI1 and intI2 and the presence or absence of different targeted dfr genes (dfrA1, dfrA8, dfrA12, dfrA14, dfrA17, and/or dfrB3) were not statistically related to plasmid transfer frequencies as determined by ANOVA (P ≥ 0.05). However, E. coli isolates recovered 2 km downstream of wastewater treatment plant effluent input, and those possessing resistance to 3 antibiotics had significantly greater plasmid transfer frequency than their counterparts when calculated as T/D (ANOVA followed by Fisher's least significant difference means comparison, P < 0.05). Greater plasmid transfer frequency calculated as T/D was also measured for E. coli possessing 3 compared to a single sul gene. The in-vitro frequency suggests that horizontal gene transfer of conjugative mediated-antibiotic (sul) resistance genes may be significant among resistant, stream bacteria.

Nematodes as bioindicators of ecosystem recovery during phytoremediation of crude oil contaminated soil.

Restoration of a weathered crude oil contaminated site undergoing phytoremediation was evaluated using nematodes as bioindicators. Samples were collected twice per year equating to spring and fall/winter. Mean annual total abundances ranged from 18-130 in the non-fertilized non-vegetated control (CTR) to 69-728 in tall fescue-ryegrass (FES) to 147-749 (100 g(-1)) in the fertilized bermudagrass-fescue (BER) treatment. Proportions of plant-parasitic (PP) and free-living (FL) nematodes were significantly impacted by treatment, but not year, with PP nematodes accounting for 27, 59, and 68% of CTR, FES, and BER communities, respectively. There was no significant year by season by treatment or treatment by year effect for total, PP, or FL nematode abundances. Diversity did not increase over time. The BER and FES treatments had more mature communities as indicated by higher plant-parasitic index (PPI) values. Phytoremediation accelerates petroleum degradation and alters the soil habitat which is reflected in the nematode community. However, low numbers and inconsistent presence of persister strategist omnivores and predators, and the lack in improvement over time in treatment effects for total and PP nematode abundances, PP and FL proportions, or PPI indicate the system is being rehabilitated but has not been restored after 69 months of phytoremediation.

Dead-end hollow-fiber ultrafiltration for concentration and enumeration of Escherichia coli and broad-host-range plasmid DNA from wastewater.

Broad-host-range plasmids can facilitate dissemination of antibiotic resistance determinants among diverse bacterial populations. We evaluated hollow-fiber ultrafiltration for increases in detection efficiency of broad-host-range plasmids and Escherichia coli DNA in wastewater. Ultrafiltration followed by PCR showed limited increases in DNA detection and quantification in effluent compared with membrane filtration alone.

Populations of antibiotic-resistant coliform bacteria change rapidly in a wastewater effluent dominated stream.

Incomplete elimination of bacteria and pharmaceutical drugs during wastewater treatment results in the entry of antibiotics and antibiotic-resistant bacteria into receiving streams with effluent inputs. In Mud Creek in Fayetteville, AR, ofloxacin, trimethoprim, and sulfamethoxazole have been detected in water and sediment, and tetracycline has been detected in sediment downstream of treated effluent input. These antibiotics have been measured repeatedly, but at low concentrations (<1µg/L) in the stream. To determine if effluent input results in detectable and stable changes in antibiotic resistances downstream of effluent input, antibiotic resistance in Escherichia coli and total coliform bacteria in Mud Creek stream water and sediment were determined using a culture-based method. Isolated E. coli colonies were characterized for multiple antibiotic resistance (MAR) patterns on solid media and to evaluate E. coli isolate richness by amplification of a partial uidA gene followed by denaturant gradient gel electrophoresis (DGGE). Despite temporal variability, proportions of antibiotic-resistant E. coli were generally high in effluent and 640m downstream. The MAR pattern ampicillin-trimethoprim-sulfamethoxazole was associated with a DGGE profile that was detected in effluent and downstream E. coli isolates, but not upstream. Percent resistance among coliform bacteria to trimethoprim and sulfamethoxazole was higher 640m downstream compared to upstream sediment and water (with one exception). Resistance to ofloxacin was too low to analyze statistically and tetracycline resistance was fairly constant across sites. Resistances changed from 640m to 2000m downstream, although dissolved nutrient concentrations within that stream stretch resembled effluent. Antibiotic resistant bacteria are entering the stream, but resistances change within a short distance of effluent inputs, more quickly than indicated based on chemical water properties. Results illustrate the difficulty in tracking the input and fate of antibiotic resistance and in relating the presence of low antibiotic concentrations to selection or persistence of antibiotic resistances.

Broad-host-range plasmids in treated wastewater effluent and receiving streams.

The occurrence of broad-host-range (BHR) plasmid amplicons belonging to incompatibility (Inc) groups IncA/C, IncN, IncP, and IncW in two wastewater treatment plant (WWTP) effluents and effluent-receiving streams in Northwest Arkansas, Mud Creek and Spring Creek, was determined. Community DNA captured on filter membranes and plasmid DNA extracted from antibiotic-resistant Escherichia coli isolated from Mud Creek was used for polymerase chain reaction at amplification of partial gene sequences specific to BHR plasmids. IncP plasmid amplicons were detected in effluent and downstream sites in both streams, while IncN and IncW plasmid amplicons were detected in Spring Creek in effluent and downstream but not upstream. IncA/C plasmid amplicons, in contrast, were detected at all sites, including upstream in most samples in Spring Creek and in one sample from Mud Creek. One IncP and two IncN were the only BHR plasmid amplicons found in 85 screened antibiotic-resistant E. coli isolates, and were detected only in isolates from effluent and downstream samples. Broad-host-range plasmids frequently carry antibiotic-resistance genes and can facilitate horizontal transfer of those genes. While BHR plasmids have been detected in WWTPs, WWTPs do not target these genetic elements for destruction. This study indicates that BHR plasmids are in WWTP effluent and are introducing BHR plasmids into streams. Additionally, species other than E. coli may be better targets as indicator bacteria for future studies of the impact of treated effluent on environmental dissemination of BHR plasmids.

Mesocosm-scale evaluation of faunal and microbial communities of aerated and unaerated leachfield soil.

Aeration improves the capacity of leachfields to decontaminate and reduce the nutrient load of wastewater. To gain a better understanding of the effects of aeration, we examined the faunal and microbial communities of septic system leachfield soil (0-4 and 4-13 cm) using replicated (n = 3) mesocosms that were actively aerated (AIR) or unaerated (LEACH). Protozoa were 40 to 140 times more abundant in AIR than in LEACH soil. No nematodes were found in LEACH soil, whereas AIR soil contained 5 to 14 x 10(3) nematodes (all bacteriovores) kg(-1). Active microbial biomass was four to five times higher in AIR than LEACH soil. Proteobacteria and actinomycetes/sulfate-reducing bacteria constituted a higher proportion of the community in AIR soil, whereas anaerobic Gram-negative bacteria/firmicutes were more prominent in LEACH soil. Ratios of prokaryotic to eukaryotic phospholipid fatty acids (PLFAs) were higher in LEACH soil, as were membrane stress index values, whereas the starvation index was higher in AIR soil. Community-level physiological profiles showed that 29 and 30 different substrates were used for growth by LEACH and AIR soil microorganisms, respectively. The AIR soil had more microorganisms capable of growing on 10 substrates, whereas growth on two substrates was higher in LEACH soil. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of 16S rRNA gene fragments revealed greater diversity of dominant phylotypes in AIR than LEACH soil, with communities separated by treatment. Aerated leachfield soil had a larger and more diverse faunal and microbial community than unaerated soil, possibly due to differences in the type and availability of electron acceptors.