Spatial Variation in Frequency and Intensity of Antibiotic Interactions among Streptomycetes from Prairie Soil

Antibiotic interactions are believed to be significant to microbial fitness in soil, yet little is known of the frequency, intensity, and diversity of antibiotic inhibition and resistance among indigenous microbes. To begin to address these issues, we studied the abilities of streptomycete isolates from prairie soil to inhibit growth and display resistance to antibiotics produced by a test collection of 10 streptomycete isolates. Wide variations in antibiotic inhibition and resistance for prairie isolates among three locations and four soil depths within a 1-m² plot were revealed. Fewer than 10% of 153 prairie isolates inhibited all 10 test isolates, while more than 40% of the isolates did not inhibit any of the test isolates. No field isolate was resistant to all of the test isolates, nor was any isolate susceptible to all of the test isolates. No correlation between inhibition and resistance phenotypes was found, suggesting that inhibition and resistance are under independent selection. The significant spatial variation in the frequency and intensity of antibiotic inhibition implies that the fitness benefits of antibiotic production are not the same among locations in soil. In contrast, the consistency of resistance over space indicates that its significance to fitness across locations is stable or the costs of maintaining resistance in the absence of selection are small or nonexistent. The spatial clustering of antibiotic inhibitory activity suggests a variable matrix of selection pressures and microbial responses across the soil landscape.     

Genetic and phenotypic traits of streptomycetes used to characterize antibiotic activities of field-collected microbes

Although antibiotic production may contribute significantly to microbial fitness, there is limited information on the ecology of antibiotic-producing microbial populations in soil. Indeed, quantitative information on the variation in frequency and intensity of specific antibiotic inhibitory and resistance abilities within soil microbial communities is lacking. Among the streptomycetes, antibiotic production is highly variable and resistance to antibiotics is highly specific to individual microbial strains. The objective of this work was to genetically and phenotypically characterize a reference collection of streptomycetes for use in distinguishing inhibition and resistance phenotypes of field-collected microbes. Specifically, we examined inhibition and resistance abilities of all isolates in all possible pairwise combinations, genetic relatedness using BOX-PCR and 16S rDNA sequence analyses, nutrient utilization profiles, and antibiotic induction among all possible three-way combinations of isolates. Each streptomycete isolate possessed a unique set of phenotypic and genetic characteristics. However, there was little correspondence between phenotypic and genetic traits. This collection of reference isolates provides the potential for distinguishing 1024 inhibition and resistance phenotypes in field-collected microbes. Relationships between the genetic and phenotypic characteristics examined may provide preliminary insight into the distinct strategies that microbes use in optimizing their fitness in natural environments.     

Lack of correspondence between genetic and phenotypic groups amongst soil-borne streptomycetes

Correspondence between two distinct genetic traits, 16S rRNA gene sequences and repetitive element-sequence-based BOX-PCR DNA fingerprints, and antibiotic inhibition and resistance phenotypes was explored for a spatially explicit sample of Streptomyces from a prairie soil. There was no correspondence between 16S rRNA gene sequence groups and antibiotic phenotypes. However, 16S rRNA gene sequence groups differed significantly in mean inhibition zone sizes. Specific antibiotic phenotypes may reflect local selection pressures, as suggested by the significant differences in mean inhibition zone sizes against specific test isolates by Streptomyces from the same 16S rRNA gene sequence group but from different locations in soil. Significant correlations between antibiotic phenotypes and BOX-PCR fingerprints were found, but were small (r=0.19-0.22). Although genetic characterizations alone were not predictive of specific antibiotic phenotypes, 16S rRNA gene sequence analyses may identify isolates that are most or least likely to possess substantial inhibitory potential, providing insight into the broad ecological strategy for individual isolates.     

A differential drug screen for compounds that select against antibiotic resistance

Antibiotics increase the frequency of resistant bacteria by providing them a competitive advantage over sensitive strains. Here, we develop a versatile assay for differential chemical inhibition of competing microbial strains, and use it to identify compounds that preferentially inhibit tetracycline-resistant relative to sensitive bacteria, thus "inverting" selection for resistance. Our assay distinguishes compounds selecting directly against specific resistance mechanisms and compounds whose selection against resistance is based on their physiological interaction with tetracycline and is more general with respect to resistance mechanism. A pilot screen indicates that both types of selection-inverting compounds are secreted by soil microbes, suggesting that nature has evolved a repertoire of chemicals that counteracts antibiotic resistance. Finally, we show that our assay can more generally permit simple, direct screening for drugs based on their differential activity against different strains or targets.     

Inhibitory and nutrient use phenotypes among coexisting Fusarium and Streptomyces populations suggest local coevolutionary interactions in soil

Bacteria and fungi are key components of virtually all natural habitats, yet the significance of fungal-bacterial inhibitory interactions for the ecological and evolutionary dynamics of specific bacterial and fungal populations in natural habitats have been overlooked. More specifically, despite the broad consensus that antibiotics play a key role in providing a fitness advantage to competing microbes, the significance of antibiotic production in mediating cross-kingdom coevolutionary interactions has received relatively little attention. Here, we characterize reciprocal inhibition among Streptomyces and Fusarium populations from prairie soil, and explore antibiotic inhibition in relation to niche overlap among sympatric and allopatric populations. We found evidence for local adaptation between Fusarium and Streptomyces populations as indicated by significantly greater inhibition among sympatric than allopatric populations. Additionally, for both taxa, there was a significant positive correlation between the strength of inhibition against the other taxon and the intensity of resource competition from that taxon among sympatric but not allopatric populations. These data suggest that coevolutionary antagonistic interactions between Fusarium and Streptomyces are driven by resource competition, and support the hypothesis that antibiotics act as weapons in mediating bacterial–fungal interactions in soil.     

Competition and antibiosis in the biological control of potato scab

Nonpathogenic, antibiotic-producing streptomycetes have been shown to reduce potato scab when added to disease-conducive soil. Spontaneous mutants of the pathogenic Streptomyces scabies RB4 that are resistant to at least one antibiotic activity produced by the nonpathogenic suppressive isolates Streptomyces diastatochromogenes strain PonSSII and S. scabies PonR have been isolated. To determine the importance of antibiosis in this biocontrol system, these mutants were investigated for their ability to cause disease in the presence of the two pathogen antagonists in a greenhouse assay. Disease caused by one of the mutant strains was reduced in the presence of both suppressive isolates, whereas disease caused by the other five mutants was not significantly reduced by either suppressive strain. In addition, a nonpathogenic mutant of S. scabies RB4 was isolated, which produced no detectable in vitro antibiotic activity and reduced disease caused by its pathogenic parent strain when the pathogen and mutant were coinoculated into soil. Population densities of the pathogen were consistently lower than those of the suppressive strains when individual strains were inoculated into soil. When a pathogen was coinoculated with a suppressive strain, the total streptomycete population density in the pot was always less than that observed when the suppressive isolate was inoculated alone. When the pathogens were inoculated individually into soil, a positive correlation was seen between population density and disease severity. In coinoculation experiments with pathogen and suppressive strains, higher total streptomycete population densities were correlated with lower amounts of disease.     

Variations in antibiotic resistance profile in Enterobacteriaceae isolated from wild Australian mammals

We carried out a retrospective analysis of 946 strains of Enterobacteriaceae isolated from wild Australian mammals between 1993 and 1997. The prevalence of resistance to fixed concentrations of 32 antimicrobial agents was determined, and the respective roles that taxonomic family of the host, state of origin and bacterial species play in defining prevalence and range of resistance were investigated. Our results demonstrated a low but widespread prevalence of antimicrobial resistance in wild isolates. Only amikacin, ciprofloxacin, meropenem and gentamicin inhibited growth in all 946 samples. There was extensive variation in the combination of antibiotics to which isolates were resistant, and multiple antibiotic resistance was common. Geographical location and host group significantly influenced the antibiotic resistance profile of an isolate, whereas bacterial species influenced both the resistance profile of an isolate and the number of antibiotics it was resistant to. The role of these factors in determining observed antibiotic resistance profiles suggests that any study measuring resistance in wild isolates should include the broadest possible range of bacterial species, host species and sampling locations. As such, this study provides an important new baseline for future measurements of antibiotic resistance in the Australian environment.     

Subinhibitory Antibiotic Concentrations Mediate Nutrient Use and Competition among Soil Streptomyces

Though traditionally perceived as weapons, antibiotics are also hypothesized to act as microbial signals in natural habitats. However, while subinhibitory concentrations of antibiotics (SICA) are known to shift bacterial gene expression, specific hypotheses as to how SICA influence the ecology of natural populations are scarce. We explored whether antibiotic ‘signals’, or SICA, have the potential to alter nutrient utilization, niche overlap, and competitive species interactions among Streptomyces populations in soil. For nine diverse Streptomyces isolates, we evaluated nutrient utilization patterns on 95 different nutrient sources in the presence and absence of subinhibitory concentrations of five antibiotics. There were significant changes in nutrient use among Streptomyces isolates, including both increases and decreases in the capacity to use individual nutrients in the presence vs. in the absence of SICA. Isolates varied in their responses to SICA and antibiotics varied in their effects on isolates. Furthermore, for some isolate-isolate-antibiotic combinations, competition-free growth (growth for an isolate on all nutrients that were not utilized by a competing isolate), was increased in the presence of SICA, reducing the potential fitness cost of nutrient competition among those competitors. This suggests that antibiotics may provide a mechanism for bacteria to actively minimize niche overlap among competitors in soil. Thus, in contrast to antagonistic coevolutionary dynamics, antibiotics as signals may mediate coevolutionary displacement among coexisting Streptomyces, thereby hindering the emergence of antibiotic resistant phenotypes. These results contribute to our broad understanding of the ecology and evolutionary biology of antibiotics and microbial signals in nature.     

Determination of metabolic activity of streptomycetes in soil microcosms

Two Streptomyces griseus strains were isolated from different soil types. S. griseus CAG17 strain was isolated from an agricultural area with low organic matter but rich in phosphorus content and S. griseus 26K strain was isolated from a forest area rich in organic matter with a low phosphorus content. The survival and metabolic activity of these isolates were studied in dynamic sterile soil microcosm systems. The fitness of each isolate was studied by re-inoculation in a soil type different from its origin. Maximum percentage of germination and respiration rates occurred within the first 48 h after each soil turnover (removal and addition of certain soil volumes). Data suggested that S. griseus CAG17 survived better independently of the soil type in comparison with S. griseus 26K which sporulated within the first 12 h after inoculation. Incubation temperatures did affect the lifecycles in relation to soil type. For example, the lowest temperature tested, 22 degrees C, was more favourable for extended germination and adaptation in general but revealed lesser spore numbers in the 'foreign' soil environment. Monitoring metabolic activity by estimation of urease, phosphatases and dehydrogenase-specific activities, between 18 and 35 degrees C incubation temperatures, was a reliable method for studying the survival and growth of streptomycete populations in soil. Results also confirmed that respiration rate and enzyme-specific activity corresponded with spore counts in long-term experiments which were designed for the investigation of survival and growth of S. griseus CAG17. Under selective pressure by heavy metals, in soil microcosm systems, metabolic activity proved a useful tool for the investigation of streptomycete activity. These methods could also be applied in agricultural field studies for monitoring microbial populations under conditions where various 'pollutants' are present in soil samples.     

Spatial Variation in <Emphasis Type="Italic">Streptomyces</Emphasis> Genetic Composition and Diversity in a Prairie Soil

Understanding how microbial genotypes are arrayed in space is crucial for identifying local factors that may influence the spatial distribution of genetic diversity. In this study we investigated variation in 16S rDNA sequences and rep-PCR fingerprints of Streptomyces stains isolated from prairie soil among three locations and four soil depths. Substantial variation in Streptomyces OTU (operational taxonomic unit) and BOX-PCR fingerprint diversity was found among locations within a limited spatial area (1 m²). Further, phylogenetic lineages at each location were distinct. However, there was little variation in genetic diversity among isolates from different soil depths and similar phylogenetic lineages were found at each depth. Some clones were found at a localized scale while other clones had a relatively widespread distribution. There was poor correspondence between 16S rDNA groupings and rep-PCR fingerprint groupings. The finding of distinct phylogenetic lineages and the variation in spatial distribution of clones suggests that selection pressures may vary over the soil landscape.     

Microarray analysis of erythromycin resistance determinants

AIMS: To develop a DNA microarray for analysis of genes encoding resistance determinants to erythromycin and the related macrolide, lincosamide and streptogramin B (MLS) compounds. METHODS AND RESULTS: We developed an oligonucleotide microarray containing seven oligonucleotide probes (oligoprobes) for each of the six genes (ermA, ermB, ermC, ereA, ereB and msrA/B) that account for more than 98% of MLS resistance in Staphylococcus aureus clinical isolates. The microarray was used to test reference and clinical S. aureus and Streptococcus pyrogenes strains. Target genes from clinical strains were amplified and fluorescently labelled using multiplex PCR target amplification. The microarray assay correctly identified the MLS resistance genes in the reference strains and clinical isolates of S. aureus, and the results were confirmed by direct DNA sequence analysis. Of 18 S. aureus clinical strains tested, 11 isolates carry MLS determinants. One gene (ermC) was found in all 11 clinical isolates tested, and two others, ermA and msrA/B, were found in five or more isolates. Indeed, eight (72%) of 11 clinical isolate strains contained two or three MLS resistance genes, in one of the three combinations (ermA with ermC, ermC with msrA/B, ermA with ermC and msrA/B). CONCLUSIONS: Oligonucleotide microarray can detect and identify the six MLS resistance determinants analysed in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that microarray-based detection of microbial antibiotic resistance genes might be a useful tool for identifying antibiotic resistance determinants in a wide range of bacterial strains, given the high homology among microbial MLS resistance genes.     

Sympatric inhibition and niche differentiation suggest alternative coevolutionary trajectories among Streptomycetes

Soil bacteria produce a diverse array of antibiotics, yet our understanding of the specific roles of antibiotics in the ecological and evolutionary dynamics of microbial interactions in natural habitats remains limited. Here, we show a significant role for antibiotics in mediating antagonistic interactions and nutrient competition among locally coexisting Streptomycete populations from soil. We found that antibiotic inhibition is significantly more intense among sympatric than allopatric Streptomycete populations, indicating local selection for inhibitory phenotypes. For sympatric but not allopatric populations, antibiotic inhibition is significantly positively correlated with niche overlap, indicating that inhibition is targeted toward bacteria that pose the greatest competitive threat. Our results support the hypothesis that antibiotics serve as weapons in mediating local microbial interactions in soil and suggest that coevolutionary niche displacement may reduce the likelihood of an antibiotic arms race. Further insight into the diverse roles of antibiotics in microbial ecology and evolution has significant implications for understanding the persistence of antibiotic inhibitory and resistance phenotypes in environmental microbes, optimizing antibiotic drug discovery and developing strategies for managing microbial coevolutionary dynamics to enhance inhibitory phenotypes.     

土霉素暴露对小麦根际抗生素抗性细菌及土壤酶活性的影响

通过培养的方法研究了土霉素暴露和小麦根际抗性细菌的数量、种类、分布特征及土壤酶活性之间的剂量效应关系。结果表明,土霉素暴露下小麦根际单一抗生素抗性细菌数量和抗土霉素—链霉素双重抗性细菌数都明显增加,且与暴露剂量呈正效应关系;同时,土壤磷酸酶、脱氢酶活性下降,但与土霉素的剂量效应关系不明显。从土霉素暴露的土壤中分离到50株抗性细菌,经形态观察、RFLP分组和16S rDNA序列测定与分析,将它们聚集在Actinobacteria、Bacilli、Alphaproteobacteria、Gammaproteobacteria 和Sphingobacteria类群。其中放线菌最多（15株）,占抗性菌总数的30 %;其次是Bacillus属细菌（9株）和Pseudomonas属细菌（8株）,分别占18 %和16 %。同时,具有抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌在土霉素暴露的样品中均被分离到,分别占抗性菌株总数的16 %、8 %和4 %。值得注意的是,随着土霉素暴露剂量的增加,小麦根际优势促生菌Bacillus属细菌的抗性检出率逐步降低;但具有抗生素抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌的检出率却明显增加,提示可能会进一步增大其机会致病性。     

Microbial Responses to Environmentally Toxic Cadmium

Abstract We analyzed the soil microbial communities from one uncontaminated and two metal-impacted soils and found that while cadmium adversely affected the numbers of culturable bacteria in all soils, cadmium-resistant isolates were found from each of the soils. With exposure to 24 and 48 μg ml^-1 soluble cadmium, the metal-contaminated soil communities were more resistant than the uncontaminated soil community. In addition, in one metal-stressed soil, the resistant population became more resistant with increased cadmium levels. Ribosomal 16S DNA sequencing identified the isolates as Arthrobacter, Bacillus, or Pseudomonas spp. Further characterization demonstrated that two of the isolates were highly resistant to soluble cadmium with maximum resistance at 275 μg ml^-1 cadmium. These isolates were also resistant to a variety of antibiotics, namely ampicillin, gentamicin, penicillin, and streptomycin, but no overall correlation was found between enhanced antibiotic resistance and cadmium resistance. One Pseudomonas isolate H1 did become more resistant with increasing cadmium levels, suggesting a different resistance mechanism at high cadmium concentrations. Received: 29 April 1999; Accepted: 7 July 1999; Online Publication: 30 November 1999     

Soil microbes alter plant fitness under competition and drought

Plants exist across varying biotic and abiotic environments, including variation in the composition of soil microbial communities. The ecological effects of soil microbes on plant communities are well known, whereas less is known about their importance for plant evolutionary processes. In particular, the net effects of soil microbes on plant fitness may vary across environmental contexts and among plant genotypes, setting the stage for microbially mediated plant evolution. Here, we assess the effects of soil microbes on plant fitness and natural selection on flowering time in different environments. We performed two experiments in which we grew Arabidopsis thaliana genotypes replicated in either live or sterilized soil microbial treatments, and across varying levels of either competition (isolation, intraspecific competition or interspecific competition) or watering (well‐watered or drought). We found large effects of competition and watering on plant fitness as well as the expression and natural selection of flowering time. Soil microbes increased average plant fitness under interspecific competition and drought and shaped the response of individual plant genotypes to drought. Finally, plant tolerance to either competition or drought was uncorrelated between soil microbial treatments suggesting that the plant traits favoured under environmental stress may depend on the presence of soil microbes. In summary, our experiments demonstrate that soil microbes can have large effects on plant fitness, which depend on both the environment and individual plant genotype. Future work in natural systems is needed for a complete understanding of the evolutionary importance of interactions between plants and soil microorganisms.     

Effects of the soil microbiome on the demography of two annual prairie plants

1. Both mutualistic and pathogenic soil microbes are known to play important roles in shaping the fitness of plants, likely affecting plants at different life cycle stages.
2. In order to investigate the differential effects of native soil mutualists and pathogens on plant fitness, we compared survival and reproduction of two annual tallgrass prairie plant species (Chamaecrista fasciculata and Coreopsis tinctoria) in a field study using 3 soil inocula treatments containing different compositions of microbes. The soil inocula types included fresh native whole soil taken from a remnant prairie containing both native mutualists and pathogens, soil enhanced with arbuscular mycorrhizal (AM) fungi derived from remnant prairies, and uninoculated controls.
3. For both species, plants inoculated with native prairie AM fungi performed much better than those in uninoculated soil for all parts of the life cycle. Plants in the native whole prairie soil were either generally similar to plants in the uninoculated soil or had slightly higher survival or reproduction.
4. Overall, these results suggest that native prairie AM fungi can have important positive effects on the fitness of early successional plants. As inclusion of prairie AM fungi and pathogens decreased plant fitness relative to prairie AM fungi alone, we expect that native pathogens also can have large effects on fitness of these annuals. Our findings support the use of AM fungi to enhance plant establishment in prairie restorations.

     

Improved annotation of antibiotic resistance determinants reveals microbial resistomes cluster by ecology

Antibiotic resistance is a dire clinical problem with important ecological dimensions. While antibiotic resistance in human pathogens continues to rise at alarming rates, the impact of environmental resistance on human health is still unclear. To investigate the relationship between human-associated and environmental resistomes, we analyzed functional metagenomic selections for resistance against 18 clinically relevant antibiotics from soil and human gut microbiota as well as a set of multidrug-resistant cultured soil isolates. These analyses were enabled by Resfams, a new curated database of protein families and associated highly precise and accurate profile hidden Markov models, confirmed for antibiotic resistance function and organized by ontology. We demonstrate that the antibiotic resistance functions that give rise to the resistance profiles observed in environmental and human-associated microbial communities significantly differ between ecologies. Antibiotic resistance functions that most discriminate between ecologies provide resistance to β-lactams and tetracyclines, two of the most widely used classes of antibiotics in the clinic and agriculture. We also analyzed the antibiotic resistance gene composition of over 6000 sequenced microbial genomes, revealing significant enrichment of resistance functions by both ecology and phylogeny. Together, our results indicate that environmental and human-associated microbial communities harbor distinct resistance genes, suggesting that antibiotic resistance functions are largely constrained by ecology.     

Kosinostatin, a major secondary metabolite isolated from the culture filtrate of Streptomyces violaceusniger strain HAL64

During a screening program, an actinomycete strain isolated from the Egyptian soil was investigated for its potential to show antimicrobial activity. The identification of this isolate was performed according to spore morphology and cell wall chemo-type, which suggested that this strain is a streptomycete. Further cultural, physiological characteristics and the analysis of the nucleotide sequence of the 16S rRNA gene (1480 bp) of this isolate indicated that this strain is identical to Streptomyces violaceusniger (accession number EF063682) and then designated S. violaceusniger strain HAL64. In its culture supernatant, this organism could produce one major compound strongly inhibits the growth of Gram-positive but the inhibition of Gram-negative indicator bacteria was lower. The antibiotic was separated by silica gel column chromatography and then purified on a sephadex LH-20 column and finally the purity was checked by HPLC. The chemical structure of the purified compound was determined using spectroscopic analyses (molecular formula of C33H32N2O10 and molecular weight of 617.21) and found to be identical to the kosinostatin, a quinocycline antibiotic which is known to be produced by Micromonspora sp. TP-A0468 (Igarashi et al., 2002) and to quinocycline B isolated from Streptomyces aureofaciens (Celmer et al., 1958). Although the antibiotic is known, the newly isolated strain was able to produce the antibiotic as a major product providing an important biotechnological downstream advantage.     

An experimental test of local adaptation in soil bacteria

Abstract.— We extracted bacterial isolates of similar colony morphology from spatially located soil samples within 1 ha of old-growth forest. The same soil samples were used to prepare growth medium. Each isolate was then cultured in each medium and its growth recorded. There was no overall tendency for isolates to grow more successfully in their home site (i.e., the medium derived from the soil sample from which they had been extracted). Most isolates grew very poorly, however, and when the analysis was restricted to the minority of vigorous isolates there was clear evidence of local adaptation: isolates tended to grow better at their home site than did isolates from elsewhere and grew better at their home site than they did at other sites. The variation of growth within the 1-ha plot made up a complex fitness landscape of peaks, ridges, and valleys. Most of the vigorous isolates were found at or near a local fitness (growth) peak, although seldom at a global peak. In consequence, there was a tendency for growth to diminish away from the home site. The home isolate was about 50% more fit than average at its home site; fitness diminished exponentially away from the home site at a rate of 0.0577 per meter. These figures are similar to those previously reported for plants. This selection gradient has matched the bacterial assemblage to the edaphic structure of the environment, although the fit is far from perfect.     

Copper amendment of agricultural soil selects for bacterial antibiotic resistance in the field

AIMS: The objective of this study was to determine whether Cu-amendment of field plots affects the frequency of Cu resistance, and antibiotic resistance patterns in indigenous soil bacteria. METHODS AND RESULTS: Soil bacteria were isolated from untreated and Cu-amended field plots. Cu-amendment significantly increased the frequency of Cu-resistant isolates. A panel of isolates were characterized by Gram-reaction, amplified ribosomal DNA restriction analysis and resistance profiling against seven antibiotics. More than 95% of the Cu-resistant isolates were Gram-negative. Cu-resistant Gram-negative isolates had significantly higher incidence of resistance to ampicillin, sulphanilamide and multiple (> or =3) antibiotics than Cu-sensitive Gram-negative isolates. Furthermore, Cu-resistant Gram-negative isolates from Cu-contaminated plots had significantly higher incidence of resistance to chloramphenicol and multiple (> or =2) antibiotics than corresponding isolates from control plots. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this field experiment show that introduction of Cu to agricultural soil selects for Cu resistance, but also indirectly selects for antibiotic resistance in the Cu-resistant bacteria. Hence, the widespread accumulation of Cu in agricultural soils worldwide could have a significant effect on the environmental selection of antibiotic resistance.