First national reference of microplastic contamination of French soils.

The recent emergence of studies on plastic contamination of terrestrial environments has revealed the presence of microplastics (MP) in a variety of soil types, from the most densely populated areas to the most remote ones. However, the concentrations and chemical natures of MP in soils vary between studies, and only a few ones have focused on this issue in France. The MICROSOF project aimed to establish the first national references for French soil contamination by microplastics. 33 soil samples randomly chosen on the French soil quality-monitoring network were analyzed. The study collected data on the abundance of microplastics in the [315-5000] µm range, their chemical nature and size, as well as mass abundance estimates and other relevant information. Results demonstrated that 76 % of the soil samples contained microplastics, in concentrations ranging from <6.7 to 80 MP.kg-1 (dry soil). Most samples from croplands, grasslands and vineyards and orchards were contaminated, whereas only one sample from forest contained MP, suggesting an increased risk of microplastic contamination in soils exposed to agricultural practices. The MP abundances are not statistically different from similar studies, indicating an intermediate level of contamination in French soils. Despite intervention reports and surveys, the sources remain unclear at this stage. For the first time, an overview of the state of soil contamination in France, as well as the potential risks is provided.

The epoxiconazole and tebuconazole fungicides impair granulosa cells functions partly through the aryl hydrocarbon receptor (AHR) signalling with contrasted effects in obese, normo-weight and polycystic ovarian syndrome (PCOS) patients.

Polycystic ovarian syndrome (PCOS), frequently associated to obesity, is the main reproductive disorder in women in age to procreate. Some evidence suggests that pesticides can result in alterations of the female reproductive system, including polycystic ovary syndrome (PCOS). Here, we detected two fungicides, Tebuconazole (Tb) and Epoxiconazole (Epox) in the soils and waters of French area. Our hypothesis is that these two triazoles could be associated to the etiology of PCOS. We used the human KGN cell line and primary human granulosa cells (hGCs) from different group of patients: normal weight non PCOS (NW), normal weight PCOS (PCOS NW), obese (obese) and obese PCOS (PCOS obese). We exposed in vitro these cells to Tb and Epox from 0 up to 10 mM for 24 and 48 h and analysed cell viability and steroidogenesis. In hGCs NW, cell viability was reduced from 12.5 µM for Tb and 75 µM for Epox. In hGCs NW, Epox decreased progesterone (Pg) and estradiol (E2) secretions and inhibited STAR, HSD3B and CYP19A1 mRNA expressions from 25 µM and increased AHR mRNA expression from 75 µM. Tb exposure also reduced steroid secretion and STAR and CYP19A1 mRNA expressions and increased AHR mRNA expression but at cytotoxic concentrations. Silencing of AHR in KGN cells reduced inhibitory effects of Tb and Epox on steroid secretion. Tb and Epox exposure decreased more steroid secretion in hGCs from obese, PCOS NW and PCOS obese groups than in NW group. Moreover, we found a higher gene expression of AHR within these three groups. Taken together, both Epox and Tb reduced steroidogenesis in hGCs through partly AHR and Tb was more cytotoxic than Epox. These triazoles alter more strongly PCOS and/or obese hGCs suggesting that human with reproductive disorders are more sensitive to triazoles exposure.

In vitro exposure to triazoles used as fungicides impairs human granulosa cells steroidogenesis.

Triazoles are the main components of fungicides used in conventional agriculture. Some data suggests that they may be endocrine disruptors. Here, we found five triazoles, prothioconazole, metconazole, difenoconazole, tetraconazole, and cyproconazole, in soil or water from the Centre-Val de Loire region of France. We then studied their effects from 0.001 µM to 1000 µM for 48 h on the steroidogenesis and cytotoxicity of ovarian cells from patients in this region and the human granulosa line KGN. In addition, the expression of the aryl hydrocarbon receptor (AHR) nuclear receptor in KGN cells was studied. Overall, all triazoles reduced the secretion of progesterone, estradiol, or both at doses that were non-cytotoxic but higher than those found in the environment. This was mainly associated, depending on the triazole, with a decrease in the expression of CYP51, STAR, CYP11A1, CYP19A1, or HSD3B proteins, or a combination thereof, in hGCs and KGN cells and an increase in AHR in KGN cells.

Biogeographical patterns of the soil fungal:bacterial ratio across France.

Soils are one of the major reservoirs of biological diversity on our planet because they host a huge richness of microorganisms. The fungal:bacterial (F:B) ratio targets two major functional groups of organisms in soils and can improve our understanding of their importance and efficiency for soil functioning. To better decipher the variability of this ratio and rank the environmental parameters involved, we used the French Soil Quality Monitoring Network (RMQS)-one of the most extensive and a priori-free soil sampling surveys, based on a systematic 16 km × 16 km grid and including more than 2,100 samples. F:B ratios, measured by quantitative PCR targeting the 18S and 16S rDNA genes, turned out to be heterogenously distributed and spatially structured in geographical patterns across France. These distribution patterns differed from bacterial or fungal densities taken separately, supporting the hypothesis that the F:B ratio is not the mere addition of each density but rather results from the complex interactions of the two functional groups. The F:B ratios were mainly influenced by soil characteristics and land management. Among soil characteristics, the pH and, to a lesser extent, the organic carbon content and the carbon:nitrogen (C:N) ratio were the main drivers. These results improved our understanding of soil microbial communities, and from an operational point of view, they suggested that the F:B ratio should be a useful new bioindicator of soil status. The resulting dataset can be considered as a first step toward building up a robust repository essential to any bioindicator and aimed at guiding and helping decision making. IMPORTANCE In the face of human disturbances, microbial activity can be impacted and, e.g., can result in the release of large amounts of soil carbon into the atmosphere, with global impacts on temperature. Therefore, the development and the regular use of soil bioindicators are essential to (i) improve our knowledge of soil microbial communities and (ii) guide and help decision makers define suitable soil management strategies. Bacterial and fungal communities are key players in soil organic matter turnover, but with distinct physiological and ecological characteristics. The fungal:bacterial ratio targets these two major functional groups by investigating their presence and their equilibrium. The aim of our study is to characterize this ratio at a territorial scale and rank the environmental parameters involved so as to further develop a robust repository essential to the interpretation of any bioindicator of soil quality.

Pesticide Residues in French Soils: Occurrence, Risks, and Persistence.

Contamination of the environment by pesticide residues is a growing concern given their widespread presence in the environment and their effects on ecosystems. Only a few studies have addressed the occurrence of pesticides in soils, and their results highlighted the need for further research on the persistence and risks induced by those substances. We monitored 111 pesticide residues (48 fungicides, 36 herbicides, 25 insecticides and/or acaricides, and two safeners) in 47 soils sampled across France under various land uses (arable lands, vineyards, orchards, forests, grasslands, and brownfields). Pesticides were found in 98% of the sites (46 of the 47 sampled), including untreated areas such as organic fields, forests, grasslands, and brownfields, with up to 33 different substances detected in one sample, mostly fungicides and herbicides. The concentrations of herbicides were the highest in soils with glyphosate, and its transformation product, AMPA, contributed 70% of the cumulative herbicides. Risk assessment underlined a moderate to high risk for earthworms in arable soils mostly attributed to insecticides and/or acaricides. Finally, the comparison with pesticide application by farmers underlines the presence of some residues long after their supposed 90% degradation and at concentrations higher than predicted environmental concentrations, leading to questions their real persistence in soils.

Correction: Mapping and predictive variations of soil bacterial richness across France.

[This corrects the article DOI: 10.1371/journal.pone.0186766.].

Biogeography of Soil Bacterial Networks along a Gradient of Cropping Intensity.

Although land use drives soil bacterial diversity and community structure, little information about the bacterial interaction networks is available. Here, we investigated bacterial co-occurrence networks in soils under different types of land use (forests, grasslands, crops and vineyards) by sampling 1798 sites in the French Soil Quality Monitoring Network covering all of France. An increase in bacterial richness was observed from forests to vineyards, whereas network complexity respectively decreased from 16,430 links to 2,046. However, the ratio of positive to negative links within the bacterial networks ranged from 2.9 in forests to 5.5 in vineyards. Networks structure was centered on the most connected genera (called hub), which belonged to Bacteroidetes in forest and grassland soils, but to Actinobacteria in vineyard soils. Overall, our study revealed that soil perturbation due to intensive cropping reduces strongly the complexity of bacterial network although the richness is increased. Moreover, the hub genera within the bacterial community shifted from copiotrophic taxa in forest soils to more oligotrophic taxa in agricultural soils.

Biogeography of soil bacteria and archaea across France.

Over the last two decades, a considerable effort has been made to decipher the biogeography of soil microbial communities as a whole, from small to broad scales. In contrast, few studies have focused on the taxonomic groups constituting these communities; thus, our knowledge of their ecological attributes and the drivers determining their composition and distribution is limited. We applied a pyrosequencing approach targeting 16S ribosomal RNA (rRNA) genes in soil DNA to a set of 2173 soil samples from France to reach a comprehensive understanding of the spatial distribution of bacteria and archaea and to identify the ecological processes and environmental drivers involved. Taxonomic assignment of the soil 16S rRNA sequences indicated the presence of 32 bacterial phyla or subphyla and 3 archaeal phyla. Twenty of these 35 phyla were cosmopolitan and abundant, with heterogeneous spatial distributions structured in patches ranging from a 43- to 260-km radius. The hierarchy of the main environmental drivers of phyla distribution was soil pH > land management > soil texture > soil nutrients > climate. At a lower taxonomic level, 47 dominant genera belonging to 12 phyla aggregated 62.1% of the sequences. We also showed that the phylum-level distribution can be determined largely by the distribution of the dominant genus or, alternatively, reflect the combined distribution of all of the phylum members. Together, our study demonstrated that soil bacteria and archaea present highly diverse biogeographical patterns on a nationwide scale and that studies based on intensive and systematic sampling on a wide spatial scale provide a promising contribution for elucidating soil biodiversity determinism.

Correction: Mapping and predictive variations of soil bacterial richness across France.

[This corrects the article DOI: 10.1371/journal.pone.0186766.].

Mapping and predictive variations of soil bacterial richness across France.

Although numerous studies have demonstrated the key role of bacterial diversity in soil functions and ecosystem services, little is known about the variations and determinants of such diversity on a nationwide scale. The overall objectives of this study were i) to describe the bacterial taxonomic richness variations across France, ii) to identify the ecological processes (i.e. selection by the environment and dispersal limitation) influencing this distribution, and iii) to develop a statistical predictive model of soil bacterial richness. We used the French Soil Quality Monitoring Network (RMQS), which covers all of France with 2,173 sites. The soil bacterial richness (i.e. OTU number) was determined by pyrosequencing 16S rRNA genes and related to the soil characteristics, climatic conditions, geomorphology, land use and space. Mapping of bacterial richness revealed a heterogeneous spatial distribution, structured into patches of about 111km, where the main drivers were the soil physico-chemical properties (18% of explained variance), the spatial descriptors (5.25%, 1.89% and 1.02% for the fine, medium and coarse scales, respectively), and the land use (1.4%). Based on these drivers, a predictive model was developed, which allows a good prediction of the bacterial richness (R2adj of 0.56) and provides a reference value for a given pedoclimatic condition.

Microbial Diversity Indexes Can Explain Soil Carbon Dynamics as a Function of Carbon Source.

Mathematical models do not explicitly represent the influence of soil microbial diversity on soil organic carbon (SOC) dynamics despite recent evidence of relationships between them. The objective of the present study was to statistically investigate relationships between bacterial and fungal diversity indexes (richness, evenness, Shannon index, inverse Simpson index) and decomposition of different pools of soil organic carbon by measuring dynamics of CO2 emissions under controlled conditions. To this end, 20 soils from two different land uses (cropland and grassland) were incubated with or without incorporation of 13C-labelled wheat-straw residue. 13C-labelling allowed us to study residue mineralisation, basal respiration and the priming effect independently. An innovative data-mining approach was applied, based on generalized additive models and a predictive criterion. Results showed that microbial diversity indexes can be good covariates to integrate in SOC dynamics models, depending on the C source and the processes considered (native soil organic carbon vs. fresh wheat residue). Specifically, microbial diversity indexes were good candidates to help explain mineralisation of native soil organic carbon, while priming effect processes seemed to be explained much more by microbial composition, and no microbial diversity indexes were found associated with residue mineralisation. Investigation of relationships between diversity and mineralisation showed that higher diversity, as measured by the microbial diversity indexes, seemed to be related to decreased CO2 emissions in the control soil. We suggest that this relationship can be explained by an increase in carbon yield assimilation as microbial diversity increases. Thus, the parameter for carbon yield assimilation in mathematical models could be calculated as a function of microbial diversity indexes. Nonetheless, given limitations of the methods used, these observations should be considered with caution and confirmed with more experimental studies. Overall, along with other studies on relationships between microbial community composition and SOM dynamics, this study suggests that overall measures of microbial diversity may constitute relevant ways to include microbial diversity in models of SOM dynamics.

Occurrence of Stenotrophomonas maltophilia in agricultural soils and antibiotic resistance properties.

The occurrence of Stenotrophomonas maltophilia was monitored in organic amendments and agricultural soils from various sites in France and Tunisia. S. maltophilia was detected in horse and bovine manures, and its abundance ranged from 0.294 (±0.509) × 10(3) to 880 (±33.4) × 10(3) CFU (g drywt)(-1) of sample. S. maltophilia was recovered from most tested soil samples (104/124). Its abundance varied from 0.33 (±0.52) to 414 (±50) × 10(3) CFU (g drywt)(-1) of soil and was not related to soil characteristics. Antibiotic resistance properties of a set of environmental strains were compared to a clinical set, and revealed a high diversity of antibiotic resistance profiles, given both the numbers of resistance and the phenotypes. Manure strains showed resistance phenotypes, with most of the strains resisting between 7 and 9 antibiotics. While French soil strains were sensitive to most antibiotics tested, some Tunisian strains displayed resistance phenotypes close to those of clinical French strains. Screening for metal resistance among 66 soil strains showed a positive relationship between antibiotic and metal resistance. However, the prevalence of antibiotic resistance phenotypes in the studied sites was not related to the metal content in soil samples.

Vegetation ecology meets ecosystem science: Permanent grasslands as a functional biogeography case study.

The effect of biodiversity on ecosystem functioning has been widely acknowledged, and the importance of the functional roles of species, as well as their diversity, in the control of ecosystem processes has been emphasised recently. However, bridging biodiversity and ecosystem science to address issues at a biogeographic scale is still in its infancy. Bridging this gap is the primary goal of the emerging field of functional biogeography. While the rise of Big Data has catalysed functional biogeography studies in recent years, comprehensive evidence remains scarce. Here, we present the rationale and the first results of a country-wide initiative focused on the C3 permanent grasslands. We aimed to collate, integrate and process large databases of vegetation relevés, plant traits and environmental layers to provide a country-wide assessment of ecosystem properties and services which can be used to improve regional models of climate and land use changes. We outline the theoretical background, data availability, and ecoinformatics challenges associated with the approach and its feasibility. We provide a case study of upscaling of leaf dry matter content averaged at ecosystem level and country-wide predictions of forage digestibility. Our framework sets milestones for further hypothesis testing in functional biogeography and earth system modelling.

Similar processes but different environmental filters for soil bacterial and fungal community composition turnover on a broad spatial scale.

Spatial scaling of microorganisms has been demonstrated over the last decade. However, the processes and environmental filters shaping soil microbial community structure on a broad spatial scale still need to be refined and ranked. Here, we compared bacterial and fungal community composition turnovers through a biogeographical approach on the same soil sampling design at a broad spatial scale (area range: 13300 to 31000 km2): i) to examine their spatial structuring; ii) to investigate the relative importance of environmental selection and spatial autocorrelation in determining their community composition turnover; and iii) to identify and rank the relevant environmental filters and scales involved in their spatial variations. Molecular fingerprinting of soil bacterial and fungal communities was performed on 413 soils from four French regions of contrasting environmental heterogeneity (Landes

Low occurrence of Pseudomonas aeruginosa in agricultural soils with and without organic amendment.

The occurrence of Pseudomonas aeruginosa was monitored at a broad spatial scale in French agricultural soils, from various soil types and under various land uses to evaluate the ability of soil to be a natural habitat for that species. To appreciate the impact of agricultural practices on the potential dispersion of P. aeruginosa, we further investigated the impact of organic amendment at experimental sites in France and Burkina Faso. A real-time quantitative PCR (qPCR) approach was used to analyze a set of 380 samples selected within the French RMQS ("Réseau de Mesures de la Qualité des Sols") soil library. In parallel, a culture-dependent approach was tested on a subset of samples. The results showed that P. aeruginosa was very rarely detected suggesting a sporadic presence of this bacterium in soils from France and Burkina Faso, whatever the structural and physico-chemical characteristics or climate. When we analyzed the impact of organic amendment on the prevalence of P. aeruginosa, we found that even if it was detectable in various manures (at levels from 10(3) to 10(5) CFU or DNA targets (g drywt)(-1) of sample), it was hardly ever detected in the corresponding soils, which raises questions about its survival. The only case reports were from a vineyard soil amended with a compost of mushroom manure in Burgundy, and a few samples from two fields amended with raw urban wastes in the sub-urban area of Ouagadougou, Burkina Faso. In these soils the levels of culturable cells were below 10 CFU (g drywt)(-1).

Distribution and speciation of ambient selenium in contrasted soils, from mineral to organic rich.

Selenium adsorption onto oxy-hydroxides mainly controls its mobility in volcanic soils, red earths and soils poor in organic matter (OM) while the influence of OM was emphasized in podzol and peat soils. This work aims at deciphering how those solid phases influence ambient Se mobility and speciation under less contrasted conditions in 26 soils spanning extensive ranges of OM (1-32%), Fe/Al oxy-hydroxides (0.3-6.1%) contents and pH (4.0-8.3). The soil collection included agriculture, meadow and forest soils to assess the influence of OM quality as well. Trace concentrations of six ambient Se species (Se(IV), Se(VI) and 4 organo-Se compounds) were analyzed by HPLC-ICP-MS in three extractants (ultrapure water, phosphate and sodium hydroxide) targeting Se associated to different soil phases. The Kd values determined from ultrapure water extraction were higher than those reported in commonly used short-term experiments after Se-spiking. Correlations of ambient Se content and distribution with soil parameters explained this difference by an involvement of slow processes in Se retention in soils. The 26 Kd values determined here for a wide variety of soils thus represent a relevant database for long-term prediction of Se mobility. For soils containing less than 20% OM, ambient Se solubility is primarily controlled by its adsorption onto crystalline oxy-hydroxides. However, OM plays an important role in Se mobility by forming organo-mineral associations that may protect adsorbed Se from leaching and/or create anoxic zones (aggregates) where Se is immobilized after its reduction. Although for the first time, inorganic Se(IV), Se(VI) and organo-Se compounds were simultaneously investigated in a large soil collection, high Se proportions remain unidentified in each soil extract, most probably due to Se incorporation and/or binding to colloidal-sized OM. Variations of environmental factors regulating the extent of OM-mineral associations/aggregation may thus lead to changes in Se mobility and bio-availability.

Biotic and abiotic soil properties influence survival of Listeria monocytogenes in soil.

Listeria monocytogenes is a food-borne pathogen responsible for the potentially fatal disease listeriosis and terrestrial ecosystems have been hypothesized to be its natural reservoir. Therefore, identifying the key edaphic factors that influence its survival in soil is critical. We measured the survival of L. monocytogenes in a set of 100 soil samples belonging to the French Soil Quality Monitoring Network. This soil collection is meant to be representative of the pedology and land use of the whole French territory. The population of L. monocytogenes in inoculated microcosms was enumerated by plate count after 7, 14 and 84 days of incubation. Analysis of survival profiles showed that L. monocytogenes was able to survive up to 84 days in 71% of the soils tested, in the other soils (29%) only a short-term survival (up to 7 to 14 days) was observed. Using variance partitioning techniques, we showed that about 65% of the short-term survival ratio of L. monocytogenes in soils was explained by the soil chemical properties, amongst which the basic cation saturation ratio seems to be the main driver. On the other hand, while explaining a lower amount of survival ratio variance (11%), soil texture and especially clay content was the main driver of long-term survival of L. monocytogenes in soils. In order to assess the effect of the endogenous soils microbiota on L. monocytogenes survival, sterilized versus non-sterilized soils microcosms were compared in a subset of 9 soils. We found that the endogenous soil microbiota could limit L. monocytogenes survival especially when soil pH was greater than 7, whereas in acidic soils, survival ratios in sterilized and unsterilized microcosms were not statistically different. These results point out the critical role played by both the endogenous microbiota and the soil physic-chemical properties in determining the survival of L. monocytogenes in soils.

Nation-wide study of the occurrence of Listeria monocytogenes in French soils using culture-based and molecular detection methods.

Soil is a potential reservoir of human pathogens and a possible source of contamination of animals, crops and water. In order to study the distribution of Listeria monocytogenes in French soils, a real-time PCR TaqMan assay targeting the phosphoribosylpyrophosphate synthetase (prs) gene of L. monocytogenes was developed for the specific detection and quantification of this bacterium within a collection of 1315 soil DNAs originated from the French Soil Quality Monitoring Network. The prs real-time PCR TaqMan assay was specific for L. monocytogenes and could quantify accurately down to 10(4)L. monocytogenes per gram of dry soil. Among the 1315 soil DNAs, prs was not detected. This suggested that the level of L. monocytogenes in French soils is generally less than 10(4)L. monocytogenes per gram of dry soil. In order to confirm this hypothesis, we investigated the occurrence of L. monocytogenes in samples collected in the Burgundy region by culture-based and molecular detection methods on the same samples. By using cultivation-based detection, 17% of samples were positive for the presence of L. monocytogenes while only 2% were found positive by the molecular detection method. L. monocytogenes was repeatedly isolated from cow pasture soils but not from cultivated soils, meadows or forest soils. Isolates were grouped in the serovar 1/2a or 3a and 4b or 4d or 4e. Taken as a whole, molecular detection results globally demonstrate that the level of L. monocytogenes in French soils does not exceed 10(4)CFU per gram of dry soil. However, in comparison with culture-based method, PCR-based detection underestimates the occurrence of L. monocytogenes in soils. Soil sampling procedure also appears critical and may also lead to the underestimation of the incidence of L. monocytogenes.

Analyzing the spatial distribution of PCB concentrations in soils using below-quantification limit data.

Polychlorinated biphenyls (PCBs) are highly toxic environmental pollutants that can accumulate in soils. We consider the problem of explaining and mapping the spatial distribution of PCBs using a spatial data set of 105 PCB-187 measurements from a region in the north of France. A large proportion of our data (35%) fell below a quantification limit (QL), meaning that their concentrations could not be determined to a sufficient degree of precision. Where a measurement fell below this QL, the inequality information was all that we were presented with. In this work, we demonstrate a full geostatistical analysis-bringing together the various components, including model selection, cross-validation, and mapping-using censored data to represent the uncertainty that results from below-QL observations. We implement a Monte Carlo maximum likelihood approach to estimate the geostatistical model parameters. To select the best set of explanatory variables for explaining and mapping the spatial distribution of PCB-187 concentrations, we apply the Akaike Information Criterion (AIC). The AIC provides a trade-off between the goodness-of-fit of a model and its complexity (i.e., the number of covariates). We then use the best set of explanatory variables to help interpolate the measurements via a Bayesian approach, and produce maps of the predictions. We calculate predictions of the probability of exceeding a concentration threshold, above which the land could be considered as contaminated. The work demonstrates some differences between approaches based on censored data and on imputed data (in which the below-QL data are replaced by a value of half of the QL). Cross-validation results demonstrate better predictions based on the censored data approach, and we should therefore have confidence in the information provided by predictions from this method.

Occurrence of CTX-M Producing Escherichia coli in Soils, Cattle, and Farm Environment in France (Burgundy Region).

CTX-M [a major type of extended-spectrum beta-lactamase (ESBL)] producing Escherichia coli are increasingly involved in human infections worldwide. The aim of this study was to investigate potential reservoirs for such strains: soils, cattle, and farm environment. The prevalence of bla(CTX-M) genes was determined directly from soil DNA extracts obtained from 120 sites in Burgundy (France) using real-time PCR. bla(CTX-M) targets were found in 20% of the DNA extracts tested. Samples of cattle feces (n = 271) were collected from 182 farms in Burgundy. Thirteen ESBL-producing isolates were obtained from 12 farms and further characterized for the presence of bla genes. Of the 13 strains, five and eight strains carried bla(TEM-71) genes and bla(CTX-M-1) genes respectively. Ten strains of CTX-M-1 producing E. coli were isolated from cultivated and pasture soils as well as from composted manure within two of these farms. The genotypic analysis revealed that environmental and animal strains were clonally related. Our study confirms the occurrence of CTX-M producing E. coli in cattle and reports for the first time the occurrence of such strains in cultivated soils. The environmental competence of such strains has to be determined and might explain their long term survival since CTX-M isolates were recovered from a soil that was last amended with manure 1 year before sampling.