Environmental Hot Spots and Resistance-Associated Application Practices for Azole-Resistant Aspergillus fumigatus, Denmark, 2020-2023.

Azole-resistant Aspergillus fumigatus (ARAf) fungi have been found inconsistently in the environment in Denmark since 2010. During 2018-2020, nationwide surveillance of clinical A. fumigatus fungi reported environmental TR34/L98H or TR46/Y121F/T289A resistance mutations in 3.6% of isolates, prompting environmental sampling for ARAf and azole fungicides and investigation for selection of ARAf in field and microcosmos experiments. ARAf was ubiquitous (20% of 366 samples; 16% TR34/L98H- and 4% TR46/Y121F/T289A-related mechanisms), constituting 4.2% of 4,538 A. fumigatus isolates. The highest proportions were in flower- and compost-related samples but were not correlated with azole-fungicide application concentrations. Genotyping showed clustering of tandem repeat-related ARAf and overlaps with clinical isolates in Denmark. A. fumigatus fungi grew poorly in the field experiment with no postapplication change in ARAf proportions. However, in microcosmos experiments, a sustained complete (tebuconazole) or partial (prothioconazole) inhibition against wild-type A. fumigatus but not ARAf indicated that, under some conditions, azole fungicides may favor growth of ARAf in soil.

Adhesion of and to soil in runoff as influenced by polyacrylamide.

Polyacrylamide (PAM) is used in agriculture to reduce soil erosion and has been reported to reduce turbidity, nutrients, and pollutants in surface runoff water. The objective of this work was to determine the effect of PAM on the concentration of enteric bacteria in surface runoff by comparing four enteric bacteria representing phenotypically different motility and hydrophobicity from three soils. Results demonstrated that bacterial surface runoff was differentially influenced by the PAM treatment. Polyacrylamide treatment increased surface runoff for adhered and planktonic cells from a clay soil; significantly decreased surface runoff of adhered bacteria, while no difference was observed for planktonic bacteria from the sandy loam; and significantly decreased the surface runoff of planktonic cells, while no difference was observed for adhered bacteria from the clay loam. Comparing strains from a final water sample collected after 48 h showed a greater loss of while serovar Poona was almost not detected. Thus, (i) the PAM efficiency in reducing the concentration of enteric bacteria in surface runoff was influenced by soil type and (ii) variation in the loss of enteric bacteria highlights the importance of strain-specific properties that may not be captured with general fecal indicator bacteria.

Linking denitrification and pesticide transformation potentials with community ecology and groundwater discharge in hyporheic sediments in a lowland stream.

Contamination of rivers by nitrate and pesticides poses a risk for aquatic ecosystems in lowland catchments that are often intensively used for agriculture. Here, the hyporheic zone, the streambed underneath the stream, plays a vital role due to its efficient self-purification capacity. The present study aims to evaluate the denitrification and transformation potential of 14 pesticides and three transformation products in the hyporheic sediment from a lowland stream with a high N load and by comparing an agricultural straightened section to a natural meandering part of the stream influenced by different groundwater discharges. Batch experiments were set up to evaluate the denitrification and pesticide transformation potentials in hyporheic sediment from two depths (5-15 cm (a) and 15-25 cm (b)). Our results revealed that (i) differences between the agricultural and natural sections of the river did not influence pollutant attenuation, (ii) both the nitrate and pesticide attenuation processes were more rapid in the upper "a" layer compared to the "b" layer due to higher microbial abundance, (iii) high groundwater discharge reduced the denitrification potential while pesticide transformation was unaffected, (iv) denitrification correlated with denitrifier abundance (nirK) in the "b" layer, while this correlation was not seen in the "a" layer, and (v) a microbial community with low diversity can explain limited transformation for the majority of tested pesticides. Overall, our results suggest that high groundwater discharge zones with reduced residence time in the hyporheic zone can be an important source of pesticides and nitrate to surface water.

Impact of surface-applied liquid manure on the drainage resistance profile of an agricultural tile-drained clay till field.

Dissemination of antibiotic resistance genes (ARGs) in aquatic environments is a concern due to human and animal health. Application of liquid manure on agricultural land is an important source of ARGs, where pathogens, antibiotic-resistant bacteria, and selective agents are released. To improve our understanding of ARGs spreading through soils, our main objective was to evaluate the effectiveness of the soil as a barrier protecting water resources. Over the course of a year, profiles and abundances of ARGs and mobile genetic elements in soil and drainage from an agricultural tile-drained clay till field were investigated upon liquid pig manure application by applying high-throughput quantitative polymerase chain reaction targeting 143 genes. The findings were as follows: (a) 97 genes were detected, where only the transposon gene tnpA-03/ IS6 was shared between the genes detected in drainage and those in acidified liquid manure or fertilized soils, indicating that liquid manure application had a limited impact on the drainage resistance profile; (b) intI1 gene was present in ∼60% of drainage samples in concentrations up to 1,634 intI1 ml-1 ; and (c) evapotranspiration from barley (Hordeum vulgare L., 'KWS Irina') and a low groundwater table appeared to reduce preferential transport to drainage during the first 3 mo of liquid manure application. Interestingly, the first preferential transport to drainage was observed immediately after the harvest of spring barley. Overall, during the monitoring year we found the soil to be an effective barrier against the spread of fecal ARGs even though the occurrence of the intI1 gene questions the barrier effect from previous years.

Degradation potential of MCPA, metolachlor and propiconazole in the hyporheic sediments of an agriculturally impacted river.

Hyporheic sediments are influenced by physical, biological, and chemical processes due to the interactions with river water and has been shown to play an important role in the environmental fate of pesticides. Therefore, this study evaluated the bacterial degradation potential of MCPA, metolachlor and propiconazole in hyporheic sediments sampled along a 20 km long stretch of an agriculturally impacted river dominated primarily by water losing conditions. Water physicochemical parameters in the river and nearby groundwater wells were assessed along with pesticide sorption to sediments and bacterial community composition. Degradation and mineralisation batch experiments were set up from six locations (five water losing, one water gaining) using environmentally relevant concentrations of pesticides (10 µg kg-1). Highly variable DT50 values from 11 to 44 days for MCPA, 11-27 days for metolachlor (MTC) and 60-147 days for propiconazole were calculated based on ~140 day studies. Degradation of MTC led to accumulation of the transformation products MOA and MESA in batch experiments. Noteworthy, MESA was detected in the groundwater wells adjacent to the part of the river impacted by losing conditions suggesting that degradation processes in hyporheic sediments may lead to the formation of transformation products (TP) leaching towards groundwater. Further, from propiconazole was identified a persistent transformation product being different from 1,2,4-triazole. Specific calculated DT50 values could not the linked to bacterial diversity. However, generally all sediment samples were characterised by high bacterial diversity, where approximately 80% of the relative sequence abundances were < 1%, which may increase the likelihood of finding contaminant-degrading genes, thereby explaining the general high contaminant-degrading activity. The studied sediments revealed a high potential to degrade pesticides despite only being exposed to low diffuse pollutant concentrations that is similar to calculated DT50 values in agricultural soils.

Leaching of Cryptosporidium parvum oocysts, Escherichia coli, and a Salmonella enterica serovar Typhimurium bacteriophage through intact soil cores following surface application and injection of slurry.

Increasing amounts of livestock manure are being applied to agricultural soil, but it is unknown to what extent this may be associated with contamination of aquatic recipients and groundwater if microorganisms are transported through the soil under natural weather conditions. The objective of this study was therefore to evaluate how injection and surface application of pig slurry on intact sandy clay loam soil cores influenced the leaching of Salmonella enterica serovar Typhimurium bacteriophage 28B, Escherichia coli, and Cryptosporidium parvum oocysts. All three microbial tracers were detected in the leachate on day 1, and the highest relative concentration was detected on the fourth day (0.1 pore volume). Although the concentration of the phage 28B declined over time, the phage was still found in leachate at day 148. C. parvum oocysts and chloride had an additional rise in the relative concentration at a 0.5 pore volume, corresponding to the exchange of the total pore volume. The leaching of E. coli was delayed compared with that of the added microbial tracers, indicating a stronger attachment to slurry particles, but E. coli could be detected up to 3 months. Significantly enhanced leaching of phage 28B and oocysts by the injection method was seen, whereas leaching of the indigenous E. coli was not affected by the application method. Preferential flow was the primary transport vehicle, and the diameter of the fractures in the intact soil cores facilitated transport of all sizes of microbial tracers under natural weather conditions.

Transport and distribution of Salmonella enterica serovar Typhimurium in loamy and sandy soil monoliths with applied liquid manure.

A leaching experiment, where liquid manure spiked with Salmonella enterica serovar Typhimurium (Tet(+)) DSM554 was applied to soil surfaces, was conducted on intact soil monoliths (60 cm in diameter and 100 cm long). A total of 6.5 x 10(10) CFU was applied to each column. We found that Salmonella serovar Typhimurium could be transported to a 1-m depth in loamy soil at concentrations reaching 1.3 x 10(5) CFU/ml of leachate. The test strain was found in concentrations ranging from 300 to 1.3(5) cells/ml in loamy soil throughout the 27 days of the experiment, while concentrations below 20 cells/ml were sporadically detected in the leachates from sandy monoliths. Real-time PCR targeting invA DNA showed a clear correspondence between the total and culturable numbers of cells in the leachate, indicating that most cells leached were viable. On day 28, distribution of Salmonella serovar Typhimurium at five depths in the four monoliths was determined. The highest recovery rate, ranging from 1.5% to 3.8% of the total applied inoculum, was found in the top 0.2 m.

Bulk soil and maize rhizosphere resistance genes, mobile genetic elements and microbial communities are differently impacted by organic and inorganic fertilization.

Organic soil fertilizers, such as livestock manure and biogas digestate, frequently contain bacteria carrying resistance genes (RGs) to antimicrobial substances and mobile genetic elements (MGEs). The effects of different fertilizers (inorganic, manure, digestate) on RG and MGE abundance and microbial community composition were investigated in a field plot experiment. The relative abundances of RGs [sul1, sul2, tet(A), tet(M), tet(Q), tet(W), qacEΔ1/qacE] and MGEs [intI1, intI2, IncP-1, IncP-1ε and LowGC plasmids] in total community (TC)-DNA from organic fertilizers, bulk soil and maize rhizosphere were quantified by qPCR before/after fertilization and prior to maize harvest. Microbial communities were analyzed via Illumina sequencing of 16S rRNA gene fragments amplified from TC-DNA. Compared to inorganic fertilization, manure treatments increased relative abundances of all RGs analyzed, integrons and few genera affiliated to Bacteroidetes and Firmicutes in bulk soil, while digestate increased sul2, tet(W) and intI2. At harvest, treatment effects vanished in bulk soil. However, organic fertilizer effects were still detectable in the rhizosphere for RGs [manure: intI1, sul1; digestate: tet(W)] and Clostridium related sequences (digestates) with increased relative abundance. Our data indicated transient organic fertilizer effects on RGs, MGEs and microbial community composition in bulk soil with long-term history of digestate or manure application.

Conservative tracer bromide inhibits pesticide mineralisation in soil.

Bromide is a conservative tracer that is often applied with non-conservative solutes such as pesticides to estimate their retardation in the soil. It has been applied in concentrations of up to 250 g Br L-1, levels at which the growth of single-celled organisms can be inhibited. Bromide applications may therefore affect the biodegradation of non-conservative solutes in soil. The present study investigated the effect of potassium bromide (KBr) on the mineralisation of three pesticides - glyphosate, MCPA and metribuzin - in four agricultural A-horizon soils. KBr was added to soil microcosms at concentrations of 0, 0.5, 2.5 and 5 g Br- L-1 in the soil solution. The study concluded that KBr had a negative effect on pesticide mineralisation. The inhibitory effect varied depending on the KBr concentration, the type of pesticide and the type of soil. Furthermore, 16 S amplicon sequencing revealed that the KBr treatment generally reduced the abundance of bacteroidetes and proteobacteria on both an RNA and DNA level. Therefore, in order to reduce the effect of KBr on the soil bacterial community and consequently also on xenobiotic degradation, it is recommended that KBr be applied in a concentration that does not exceed 0.5 g Br- L-1 in the soil water.

Redistribution and persistence of microorganisms and steroid hormones after soil-injection of swine slurry.

The redistribution and fate of contaminants in pig slurry after direct injection were investigated at two field sites, Silstrup (sandy clay loam) and Estrup (sandy loam), in Denmark. Intact soil samples were collected for up to seven weeks after slurry injection and concentrations of Salmonella Typhimurium Bacteriophage 28B (phage 28B), Escherichia coli, steroid hormones and other slurry components (water, volatile solids, chloride and mineral N) determined in and around the injection slit. The two experiments at Silstrup and Estrup differed with respect to slurry solid content (6.3 vs. 0.8%), as well as soil clay content (27 vs. 15%) and differed considerably with respect to the initial redistribution of slurry-borne contaminants in soil. The transport of microorganisms from the slurry injection slit to the surrounding soil was much lower than that of mineral N and chloride due to attachment and entrapment. The redistribution of E. coli was more affected by site-specific conditions compared to phage 28B, possibly due to the larger cell size of E. coli. The overall recovery of phage 28B was 0.8-4%, and of E. coli 0.0-1.3% in different samples, by the end of the study. Nine different steroid hormones were detected in the slurry slit, and a slow redistribution to the surrounding soil was observed. Overall recovery of estrogens was 0.0 to 6.6% in different samples. The study showed that the combination of soil and slurry properties determined the initial spreading of contaminants, and hence the potential for subsequent leaching.