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Flow cytometric evaluation of antibiotic effects on viability and mitochondrial function of refrigerated spermatozoa of Nile tilapia

Improved techniques for storage and evaluation of fish sperm would enhance breeding programs around the world. The goal of this study was to test the effect of antibiotics on refrigerated sperm from Nile tilapia (Oreochromis niloticus) by use of flow cytometry with 2 dual-staining protocols for objective assessment of sperm quality. Concentrations of 1 x 109 sperm/mL were suspended in Ringer's buffer at 318 mOsmol/kg (pH 8.0). The fluorescent stains Sybr 14 (10 ??M), propidium iodide (2.4 mM), and rhodamine 123 (0.13 ??M) were used to assess cell viability and mitochondrial function. Three concentrations of ampicillin, gentamicin, and an antibiotic/antimycotic solution were added to fresh spermatozoa. Motility estimates and flow cytometry measurements were made daily during 7 d of refrigerated storage (4 ??C). The highest concentrations of gentamicin and antibiotic/antimycotic and all 3 concentrations of ampicillin significantly reduced sperm viability. The highest of each of the 3 antibiotic concentrations significantly reduced mitochondrial function. This study demonstrates that objective sperm quality assessments can be made using flow cytometry and that addition of antibiotics at appropriate concentrations can lengthen refrigerated storage time for tilapia spermatozoa. With minor modifications, these protocols can be adapted for use with sperm from other species and with other tissue types.

Theriogenology↗

Occurrence of antibiotics in water from 13 fish hatcheries, 2001-2003

A 2-year study of extensive and intensive fish hatcheries was conducted to assess the general temporal occurrence of antibiotics in aquaculture. Antibiotics were detected in 15% of the water samples collected during the 2001-2002 collection period and in 31% of the samples during the 2003 collection period. Antibiotics were detected more frequently in samples from the intensive hatcheries (17 and 39%) than in samples from the extensive hatcheries (14 and 4%) during the 2001-2002 and 2003 collection periods, respectively. The maximum ormetoprim, oxytetracycline, and sulphadimethoxine concentrations were higher in samples from the intensive hatcheries (12, 10, and 36 &micro;g L-1), respectively, than in samples from the extensive hatcheries (<0.05, 0.31, and 1.2 &micro;g L-1), respectively. Sulphadimethoxine persisted for a longer period of time (up to 48 days) than ormetoprim (up to 28 days) and oxytetracycline (less than 20 days).

International Journal of Environmental Analytical ↗

Enhancing hatch rate and survival in laboratory-reared hybrid Devils Hole Pupfish through application of antibiotics to eggs and larvae

We evaluated the effectiveness of four antibiotics in enhancing the hatch rate, larval survival, and adult survival of hybrid Devils Hole Pupfish Cyprinodon diabolis (hybridized with Ash Meadows Amargosa Pupfish C. nevadensis mionectes ). Cephalexin (CEX; concentration = 6.6 mg/L of water), chloramphenicol (CAM; 50 mg/L), erythromycin (ERY; 12.5 mg/L), and trimethoprim sulfamethoxazole (TMP-SMX; 25 mg/L) were applied as a constant bath either to incubating eggs or to larvae that hatched from untreated eggs. Hatch rate was roughly doubled by incubation in the presence of CAM (68% hatch) and TMP-SMX (66%) relative to the control (28%). Cephalexin and ERY conferred no benefit upon the hatch rate. Among fry that hatched from treated eggs, there was no increase in 15-d larval survival. However, fish that hatched from eggs treated with CAM, ERY, and TMP-SMX demonstrated enhanced survival at 360 d (51.2, 38.4, and 43.6%, respectively) and at 540 d (22.6, 6.8, and 20.2%, respectively); the untreated control had no survivors to those time points. All groups of eggs treated with antibiotics showed reductions in bacterial colony-forming units (CFUs) at 24 h posttreatment. At 120 h posttreatment, CEX-treated eggs had CFU counts similar to those of the control, whereas the TMP-SMX-treated eggs had the lowest CFU counts. Eggs treated with CAM and ERY had similar CFU counts, which were significantly reduced from the control counts. Larvae that were treated with CAM and TMP-SMX within 12 h posthatch showed enhanced 15-d survival (74% and 72%, respectively) in comparison with the control (56%). For pupfish rearing efforts in which antibiotic use is appropriate, CAM and TMP-SMX appear to provide the greatest benefit, particularly when applied to incubating eggs rather than to hatched larvae.

North American Journal of Aquaculture↗

Effects of antenna placement and antibiotic treatment on loss of simulated transmitters and mortality in hybrid striped bass

We compared the effects of two antenna placements (trailing and nontrailing) and antibiotic treatments (treated and nontreated) on mortality and transmitter loss in hybrid striped bass Morone saxatilis ?? M. chrysops (364 ?? 28 mm total length, 645 ?? 129 g [mean ?? SD]) implanted with simulated transmitters and held in the laboratory for 90 d. Although antibiotic treatment significantly increased the time to first mortality in fish surgically implanted with simulated transmitters (by an average of 14 d), we did not detect an effect on cumulative mortality. We also did not detect an effect of antenna type on the time to first mortality, but cumulative mortality was higher in the trailing antenna groups (50%) than in the nontrailing antenna groups (12%). Three transmitters were expelled during the study, all from trailing-antenna treatment groups, indicating a significant effect of antenna placement on the level of transmitter expulsion. Antibiotic treatment appears to be effective in preventing initial postsurgical infection; however, the antenna may serve as a continuous source of irritation and route of infection into the body cavity. The potential for infection and mortality in experimental animals must be weighed against the improved performance of transmitters with trailing antennas.

North American Journal of Fisheries Management↗

Land-cover effects on the fate and transport of surface-applied antibiotics and 17-beta-estradiol on a sandy outwash plain, Anoka County, Minnesota, 2008–09

A plot-scale field experiment on a sandy outwash plain in Anoka County in east-central Minnesota was used to investigate the fate and transport of two antibiotics, sulfamethazine (SMZ) and sulfamethoxazole (SMX), and a hormone, 17-beta-estradiol (17BE), in four land-cover types: bare soil, corn, hay, and prairie. The SMZ, SMX, and 17BE were applied to the surface of five plots of each land-cover type in May 2008 and again in April 2009. The cumulative application rate was 16.8 milligrams per square meter (mg/m 2 ) for each antibiotic and 0.6 mg/m2 for 17BE. Concentrations of each chemical in plant-tissue, soil, soil-water, and groundwater samples were determined by using enzyme-linked immunosorbent assay (ELISA) kits. Soil-water and groundwater sampling events were scheduled to capture the transport of SMZ, SMX, and 17BE during two growing seasons. Soil and plant-tissue sampling events were scheduled to identify the fate of the parent chemicals of SMZ, SMX, and 17BE in these matrices after two chemical applications. Areal concentrations (mg/m 2 ) of SMZ and SMX in soil tended to decrease in prairie plots in the 8 weeks after the second chemical application, from April 2009 to June 2009, but not in other land-cover types. During these same 8 weeks, prairie plots produced more aboveground biomass and had extracted more water from the upper 125 centimeters of the soil profile compared to all other land-cover types. Areal concentrations of SMZ and SMX in prairie plant tissue did not explain the temporal changes in areal concentrations of these chemicals in soil. The areal concentrations of SMZ and SMX in the aboveground plant tissues in June 2009 and August 2009 were much lower, generally two to three orders of magnitude, than the areal concentrations of these chemicals in soil. Pooling all treatment plot data, the median areal concentration of SMZ and SMX in plant tissues was 0.01 and 0.10 percent of the applied chemical mass compared to 22 and 12 percent in soil, respectively. Furthermore, areal concentrations of SMZ and SMX in plant-tissue samples were variable, and did not differ significantly between control and treatment plots within each land-cover type. SMZ was detected in 23 percent of soil-water samples and in 16 percent of groundwater samples collected between October 2008 and October 2009 in treatment plots, indicating that surface-applied SMZ leached below the rooting zone and reached groundwater. SMX was detected in only 1 percent of soil-water and groundwater samples during this same time period. In contrast to the antibiotics, 17BE was not reliably detected in soil samples. Additionally, ELISA-determined 17BE concentrations in plant-tissue, soil-water, and groundwater samples indicated the presence of chemicals that were not applied as part of this experiment [17BE from an external source or other chemical(s) that interfered with the 17BE ELISA kits].

Minnesota↗

Fecal indicator and pathogenic bacteria and their antibiotic resistance in alluvial groundwater of an irrigated agricultural region with dairies

Surveys of microbiological groundwater quality were conducted in a region with intensive animal agriculture in California, USA. The survey included monitoring and domestic wells in eight concentrated animal feeding operations (CAFOs) and 200 small (domestic and community supply district) supply wells across the region. Campylobacter was not detected in groundwater, whereas Escherichia coli O157:H7 and Salmonella were each detected in 2 of 190 CAFO monitoring well samples. Nonpathogenic generic E. coli and Enterococcus spp. were detected in 24.2% (46/190) and 97.4% (185/190) groundwater samples from CAFO monitoring wells and in 4.2% (1/24) and 87.5% (21/24) of CAFO domestic wells, respectively. Concentrations of both generic E. coli and Enterococcus spp. were significantly associated with well depth, season, and the type of adjacent land use in the CAFO. No pathogenic bacteria were detected in groundwater from 200 small supply wells in the extended survey. However, 4.5 to 10.3% groundwater samples were positive for generic E. coli and Enterococcus. Concentrations of generic E. coli were not significantly associated with any factors, but concentrations of Enterococcus were significantly associated with proximity to CAFOs, seasons, and concentrations of potassium in water. Among a subset of E. coli and Enterococcus isolates from both surveys, the majority of E. coli (63.6%) and Enterococcus (86.1%) isolates exhibited resistance to multiple (≥3) antibiotics. Findings confirm significant microbial and antibiotic resistance loading to CAFO groundwater. Results also demonstrate significant attenuative capacity of the unconfined alluvial aquifer system with respect to microbial transport.

California↗

Work plan for determining the occurrence of glyphosate, its transformation product AMPA, other herbicide compounds, and antibiotics in midwestern United States streams, 2002

Changes in herbicide use in the Midwestern United States have been substantial over the last 5 years. Most significant is a tripling in the use of glyphosate (N-[phosphonomethyl]glycin). Over this same time period (1997&shy;2001), atrazine use increased by 20 percent and acetochlor use increased by 10 percent, while cyanazine use decreased by 99 percent, alachlor use decreased by 70 percent, and metolachlor use decreased by 55 percent. Previous studies have documented that herbicide flushes occur in midwestern streams during runoff events for several weeks to several months following application, and that herbicide concentrations in midwestern streams during flushing events are related to rates of herbicide use. The objective of this study is to determine the distribution of glyphosate and its primary transformation product aminomethylphosphonic acid (AMPA) in midwestern streams during post-application and harvest-season runoff events. Water samples will be collected in 2002 during two post-herbicide-application runoff events and one harvest-season runoff event from 53 sites on streams in the Midwestern United States. All samples will be analyzed at the U.S. Geological Survey Organic Geochemistry Research Laboratory in Lawrence, Kansas, for glyphosate and 20 other herbicides. Samples will also be analyzed for a glyphosate transformation product (AMPA) and 26 other herbicide transformation products, using GC/MS or HPLC/MS. Selected samples will be analyzed for 36 antibiotics or antibiotic transformational products. Results from this study will represent the first broad-scale investigation of glyphosate and AMPA in U.S. water resources.

Midwest region↗

Chapter A5. Section 6.1.F. Wastewater, Pharmaceutical, and Antibiotic Compounds

The USGS differentiates between samples collected for analysis of wastewater compounds and those collected for analysis of pharmaceutical and antibiotic compounds, based on the analytical schedule for the laboratory method. Currently, only the wastewater laboratory method for field-filtered samples (SH1433) is an approved, routine (production) method. (The unfiltered wastewater method LC 8033 also is available but requires a proposal for custom analysis.) At this time, analysis of samples for pharmaceutical and antibiotic compounds is confined to research studies and is available only on a custom basis.

Techniques of Water-Resources Investigations↗

Antibiotic resistance in marine microbial communities proximal to a Florida sewage outfall system

Water samples were collected at several wastewater treatment plants in southeast Florida, and water and sediment samples were collected along and around one outfall pipe, as well as along several transects extending both north and south of the respective outfall outlet. Two sets of samples were collected to address potential seasonal differences, including 38 in the wet season (June 2018) and 42 in the dry season (March 2019). Samples were screened for the presence/absence of 15 select antibiotic resistance gene targets using the polymerase chain reaction. A contrast between seasons was found, with a higher frequency of detections occurring in the wet season and fewer during the dry season. These data illustrate an anthropogenic influence on offshore microbial genetics and seasonal flux regarding associated health risks to recreational users and the regional ecosystem.

Florida↗

Vegetative buffer strips show limited effectiveness for reducing antibiotic transport in surface runoff

Vegetative buffer strips (VBS) have been demonstrated to effectively reduce loads of sediment, nutrients, and herbicides in surface runoff, but their effectiveness for reducing veterinary antibiotic (VA) loads in runoff has not been well documented. The objective of this study was to determine the effectiveness of VBS vegetation and width on surface runoff loads of the VAs sulfamethazine (SMZ) and lincomycin (LIN). Experimental design of the plots (1.5 × 25 m) was a two-way factorial with four vegetation treatments (tall fescue [ Festuca aruninacea Schreb.]; tall fescue with switchgrass [ Panicum virgatum L.] hedge; warm-season native grass mix; and continuous fallow control), and four buffer widths (0, 2, 5, and 9 m). Turkey litter spiked with SMZ and LIN was applied to the source area (upper 7 m) of each plot, and runoff was collected at each width. Runoff was generated with a rotating boom simulator. Results showed VA loads in runoff at the 0-m sampler ranged from 3.8 to 5.9% of applied, and overall VA transport in runoff was predominately in the dissolved phase (90% for SMZ and 99% for LIN). Among vegetation treatments, only tall fescue significantly reduced loads of SMZ and LIN compared with the control, with load reductions of ∼30% for both VAs. Estimated field-scale reductions in VA loads showed that source-to-buffer area ratios (SBARs) of 10:1 to 20:1 reduced VA loads by only 7 to 16%. Overall, the grass VBS tested here were less effective at reducing SMZ and LIN loads in surface runoff than has been previously demonstrated for sediment, nutrients, and herbicides.

Journal of Environmental Quality↗

Microbial source tracking and land use associations for antibiotic resistance genes in private wells influenced by human and livestock fecal sources

Antimicrobial resistance is a growing public health problem that requires an integrated approach among human, agricultural, and environmental sectors. However, few studies address all three components simultaneously. We investigated the occurrence of five antibiotic resistance genes (ARGs) and the class 1 integron gene ( intI1 ) in private wells drawing water from a vulnerable aquifer influenced by residential septic systems and land-applied dairy manure. Samples ( n = 138) were collected across four seasons from a randomized sample of private wells in Kewaunee County, Wisconsin. Measurements of ARGs and intI1 were related to microbial source tracking (MST) markers specific to human and bovine feces; they were also related to 54 risk factors for contamination representing land use, rainfall, hydrogeology, and well construction. ARGs and intI1 occurred in 5–40% of samples depending on target. Detection frequencies for ARGs and intI1 were lowest in the absence of human and bovine MST markers (1-30%), highest when co-occurring with human and bovine markers together (11-78%), and intermediate when co-occurring with just one type of MST marker (4-46%). Gene targets were associated with septic system density more often than agricultural land, potentially because of the variable presence of manure on the landscape. Determining ARG prevalence in a rural setting with mixed land use allowed an assessment of the relative contribution of human and bovine fecal sources. Because fecal sources co-occurred with ARGs at similar rates, interventions intended to reduce ARG occurrence may be most effective if both sources are considered.

Wisconsin↗

A universal method for the simultaneous determination of environmental pollutants in marine biological samples: Per- and polyfluoroalkyl substances and antibiotics as a case study

Conventional detection technologies for environmental contaminants have primarily focused on providing accurate qualitative and quantitative evaluations for single pollutant types, leading to increased costs and an inability to satisfy the growing demand for detecting a broader spectrum of pollutants. Here, we introduced a novel analytical method to simultaneously measure the concentration levels of diverse environmental pollutants, characterized by their distinct properties, across complex biological samples. Per- and polyfluoroalkyl substances (PFAS) and antibiotics were used as a case study due to their frequency of detection in the environment and known impacts Our method harnesses the salting-out effect of sodium chloride on proteins within the muscle tissues of 178 marine species, which significantly reduces the addition of extraneous substances, mitigates matrix interference, and avoids reliance on solid-phase extraction or dispersive extraction agents. The method provides a simultaneous pretreatment for the detection of several compounds, with detection limits from 0.002 to 0.41 ng/g dry weight, which are substantially lower than conventional methods. Overall, this method streamlines efficiency, decreases costs, lessens matrix effects, and sets a solid groundwork for future applications in the concurrent detection of a broader spectrum of environmentally pertinent pollutants with varied characteristics.

ACS ES&T Water↗

Quantitative microbial risk assessment for ingestion of antibiotic resistance genes from private wells contaminated by human and livestock fecal sources

We used quantitative microbial risk assessment to estimate ingestion risk for intI1 , erm (B), sul1 , tet (A), tet (W), and tet (X) in private wells contaminated by human and/or livestock feces. Genes were quantified with five human-specific and six bovine-specific microbial source-tracking (MST) markers in 138 well-water samples from a rural Wisconsin county. Daily ingestion risk (probability of swallowing ≥1 gene) was based on daily water consumption and a Poisson exposure model. Calculations were stratified by MST source and soil depth over the aquifer where wells were drilled. Relative ingestion risk was estimated using wells with no MST detections and >6.1 m soil depth as a referent category. Daily ingestion risk varied from 0 to 8.8 × 10 −1 by gene and fecal source (i.e., human or bovine). The estimated number of residents ingesting target genes from private wells varied from 910 ( tet (A)) to 1,500 ( intI1 and tet (X)) per day out of 12,000 total. Relative risk of tet (A) ingestion was significantly higher in wells with MST markers detected, including wells with ≤6.1 m soil depth contaminated by bovine markers (2.2 [90% CI: 1.1–4.7]), wells with >6.1 m soil depth contaminated by bovine markers (1.8 [1.002–3.9]), and wells with ≤6.1 m soil depth contaminated by bovine and human markers simultaneously (3.1 [1.7–6.5]). Antibiotic resistance genes (ARGs) were not necessarily present in viable microorganisms, and ingestion is not directly associated with infection. However, results illustrate relative contributions of human and livestock fecal sources to ARG exposure and highlight rural groundwater as a significant point of exposure.

Wisconsin↗

Monitoring for early detection of Aeromonas salmonicida to enhance antibiotic therapy and control furunculosis in Atlantic salmon

Juvenile Atlantic salmon Salmo salar at the White River National Fish Hatchery were monitored quarterly for Aeromonas salmonicida , the etiologic agent of furunculosis. Samples were obtained in August, November, January, and March, within an 8‐month smolt production cycle during 1992–1993. Aeromonas salmonicida was isolated in August from external mucus and kidneys of fish within six production pools, each containing about 7,500 fish. Topical disinfection with a 60‐min bath of Chloramine‐T at 15 mg/L on three consecutive days did not control the prevalence of infection. Atlantic salmon were then fed 77.0 mg of oxytetracycline per kilogram of fish for 10 d. Mortality subsided in all tanks within 4 d after initiation of oral antibiotic therapy. Further examination failed to isolate the pathogen 21 d after cessation of treatment. In November, A. salmonicida was detected in the mucus of one fish from a single tank; treatment was not prescribed. Pathogens were not detected shortly after fish were marked with coded wire tags in January or prior to release in March. Monitoring programs allowed early detection of the pathogen; this led to treatment that permitted fish to be stocked in accordance with established fish health policy regulations for salmonids in New England.

Progressive Fish-Culturist↗

Identifying sources of antibiotic resistance genes in the environment using the microbial Find, Inform, and Test framework

Introduction: Antimicrobial resistance (AMR) is an increasing public health concern for humans, animals, and the environment. However, the contributions of spatially distributed sources of AMR in the environment are not well defined. Methods: To identify the sources of environmental AMR, the novel microbial Find, Inform, and Test (FIT) model was applied to a panel of five antibiotic resistance-associated genes (ARGs), namely, erm(B), tet(W), qnrA, sul1, and intI1, quantified from riverbed sediment and surface water from a mixed-use region. Results: A one standard deviation increase in the modeled contributions of elevated AMR from bovine sources or land-applied waste sources [land application of biosolids, sludge, and industrial wastewater (i.e., food processing) and domestic (i.e., municipal and septage)] was associated with 34–80% and 33–77% increases in the relative abundances of the ARGs in riverbed sediment and surface water, respectively. Sources influenced environmental AMR at overland distances of up to 13 km. Discussion: Our study corroborates previous evidence of offsite migration of microbial pollution from bovine sources and newly suggests offsite migration from land-applied waste. With FIT, we estimated the distance-based influence range overland and downstream around sources to model the impact these sources may have on AMR at unsampled sites. This modeling supports targeted monitoring of AMR from sources for future exposure and risk mitigation efforts.

Wisconsin↗

A multi-state assessment of per- and polyfluoroalkyl substances (PFAS), pesticides, and antibiotic resistance genes in white-tailed deer (Odocoileus virginianus) of the United States

Sentinel species provide a vital One Health perspective on ecosystem contamination driven by rising global chemical production. We evaluated white-tailed deer ( Odocoileus virginianus ; n = 54, 24 states, six matrices) as nationwide bioindicators for PFAS, pesticides, antimicrobial resistance (AMR) genes, and microbiome diversity. PFAS were detected in 100% of deer samples (26 compounds identified overall; non-detect to 12.3 ng/g ww), with preferential accumulation in the liver, indicating widespread systemic exposure. Neither region, landscape, nor source proximity predicted liver PFAS concentrations, whereas landscape had a significant, albeit weak, effect on spleen concentrations. This overall absence of strong spatial drivers suggests ubiquitous background contamination rather than localized point-source exposure. Conversely, 13 pesticides/transformation products (ranging from non-detect to 88.6 ng/g ww; highest in scat) were detected in only 28% of samples, despite widespread national use. Since not all possible transformation products were measured, these data reflect the accumulation of the target compound rather than total pesticide exposure. Driven by the rapid metabolism and excretion of herbicides, persistent insecticides dominated tissue samples. Additionally, biological threats were prevalent with eight distinct AMR genes identified in WTD scat, frequently featuring the florfenicol resistance gene (flo st, 33%). The presence of synthetic compounds and AMRs in game meat and scat pose a direct One Health risk to the human food chain. These findings highlight the value of using a single, widely distributed sentinel species as an early warning system for the ecological and public health threats posed by global chemical pollution.

contiguous United States↗