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.     

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.     

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.     

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.     

Distribution of phenotypes among Bacillus thuringiensis strains

An extensive collection of Bacillus thuringiensis isolates from around the world were phenotypically profiled using standard biochemical tests. Six phenotypic traits occurred in 20–86% of the isolates and were useful in distinguishing isolates: production of urease (U; 20.5% of isolates), hydrolysis of esculin (E; 32.3% of isolates), acid production from salicin (A; 37.4% of isolates), acid production from sucrose (S; 34.0% of isolates), production of phospholipase C or lecithinase (L; 79.7% of isolates), and hydrolysis of starch (T; 85.8% of isolates). With the exception of acid production from salicin and hydrolysis of esculin, which were associated, the traits assorted independently. Of the 64 possible combinations of these six phenotypic characteristics, 15 combinations accounted for ca. 80% of all isolates, with the most common phenotype being TL (23.6% of isolates). Surprisingly, while the biochemical traits generally assorted independently, certain phenotypic traits associated with the parasporal crystal were correlated with certain combinations of biochemical traits. Crystals that remained attached to spores (which tended to be non-toxic to insects) were highly correlated with the phenotypes that included both L and S. Among the 15 most abundant phenotypes characterizing B. thuringiensis strains, amorphous crystals were associated with TLE, TL, T, and Ø (the absence of positive tested biochemical traits). Amorphous crystal types displayed a distinct bias toward toxicity to dipteran insects. Although all common phenotypes included B. thuringiensis isolates producing bipyramidal crystals toxic to lepidopteran insects, those with the highest abundance of these toxic crystals displayed phenotypes TLU, TLUA, TLUAE, and TLAE.     

A new look at antibiotic resistance

Abstract Since the discovery of antibiotic resistance in clinically important microbes, attention has focused properly on the profound medical aspects of this problem. However, studies of antibiotic resistance are of great interest in their own right for studies of gene regulation, evolution, chromosome structure, etc.; several resistance genes in clinical isolates are components of classical 'operon' structures. The construction of cloning vectors and gene transfer systems, particularly for interspecies studies, rely heavily on the use of antibiotic resistance genes, since these phenotypes can be used to select for DNA transfer between microbes, plants, and animals. Studies of the role of resistance mechanisms and their genetic determinants in antibiotic-producing organisms have shown that these functions play important roles in biosynthetic pathways and can provide important genetic and biochemical tools for the rational analysis of antibiotic production.     

The Culturable Soil Antibiotic Resistome: A Community of Multi-Drug Resistant Bacteria

Understanding the soil bacterial resistome is essential to understanding the evolution and development of antibiotic resistance, and its spread between species and biomes. We have identified and characterized multi-drug resistance (MDR) mechanisms in the culturable soil antibiotic resistome and linked the resistance profiles to bacterial species. We isolated 412 antibiotic resistant bacteria from agricultural, urban and pristine soils. All isolates were multi-drug resistant, of which greater than 80% were resistant to 16–23 antibiotics, comprising almost all classes of antibiotic. The mobile resistance genes investigated, (ESBL, bla _NDM-1, and plasmid mediated quinolone resistance (PMQR) resistance genes) were not responsible for the respective resistance phenotypes nor were they present in the extracted soil DNA. Efflux was demonstrated to play an important role in MDR and many resistance phenotypes. Clinically relevant Burkholderia species are intrinsically resistant to ciprofloxacin but the soil Burkholderia species were not intrinsically resistant to ciprofloxacin. Using a phenotypic enzyme assay we identified the antibiotic specific inactivation of trimethoprim in 21 bacteria from different soils. The results of this study identified the importance of the efflux mechanism in the soil resistome and variations between the intrinsic resistance profiles of clinical and soil bacteria of the same family.     

Carbon substrate utilization, antibiotic sensitivity, and numerical taxonomy of bacterial isolates from the Great Salt Plains of Oklahoma

The current work extends the phenotypic characterization of a bacterial culture collection from the Great Salt Plains of Oklahoma. This barren expanse of mud flats is typically crusted with thalassohaline salt evaporites. The initial account of the aerobic heterotrophic bacteria from the Great Salt Plains described 105 halotolerant isolates that represented 47 phylotypes. Extensive phenotypic analyses were performed on 76 isolates representing 37 unique phylotypes. The current report extends these observations for 60 of the isolates by measuring a wider set of phenotypic characteristics. Utilization patterns for 45 carbon substrates were used to assign the isolates into seven coherent phenons, along with several singletons and a group of isolates that did not grow on single carbon substrates. Most of the isolates were able to utilize nearly all of the nitrogen sources tested, with nitrate being the least utilized. Little antibiotic resistance was seen in the collection as a whole; however, certain phenons were enriched for antibiotic-resistant organisms. A total of 81 phenotypic characteristics were used to generate dendrograms. The numerical taxonomy trees essentially agreed with those generated using 16S rRNA gene sequences. The pattern of carbon substrate utilization showed substantial changes at different salinities that may have relevance to the variable salinities microbes experience at the Salt Plains over time.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Friends and foes: streptomycetes as modulators of plant disease and symbiosis

The ecological role of soil streptomycetes within the plant root environment is currently gaining increased attention. This review describes our recent advances in elucidating the complex interactions between streptomycetes, plants, pathogenic and symbiotic microorganisms. Streptomycetes play diverse roles in plant-associated microbial communities. Some act as biocontrol agents, inhibiting plant interactions with pathogenic organisms. Owing to the antagonistic properties of streptomycetes, they exert a selective pressure on soil microbes, which may not always be for plant benefit. Others promote the formation of symbioses between plant roots and microbes, and this is in part due to their direct positive influence on the symbiotic partner, expressed as, e.g., promotion of hyphal elongation of symbiotic fungi. Recently, streptomycetes have been identified as modulators of plant defence. By repressing plant responses to pathogens they facilitate root colonisation with pathogenic fungi. In contrast, other strains induce local and systemic resistance against pathogens or enhance plant growth. In conclusion, while streptomycetes have a clear potential of acting as biocontrol agents, care has to be taken to avoid strains that select for virulent pathogens or enhance disease development. We argue towards the use of an integrated screening approach in the search for efficient biocontrol agents, including assays on in vitro antagonism, plant growth, and disease suppression.     

Integrating Culture-based Antibiotic Resistance Profiles with Whole-genome Sequencing Data for 11,087 Clinical Isolates

Emerging antibiotic resistance is a major global health threat. The analysis of nucleic acid sequences linked to susceptibility phenotypes facilitates the study of genetic antibiotic resistance determinants to inform molecular diagnostics and drug development. We collected genetic data(11,087 newly-sequenced whole genomes) and culture-based resistance profiles(10,991 out of the11,087 isolates comprehensively tested against 22 antibiotics in total) of clinical isolates including18 main species spanning a time period of 30 years. Species and drug specific resistance patterns were observed including increased resistance rates for Acinetobacter baumannii to carbapenems and for Escherichia coli to fluoroquinolones. Species-level pan-genomes were constructed to reflect the genetic repertoire of the respective species, including conserved essential genes and known resistance factors. Integrating phenotypes and genotypes through species-level pan-genomes allowed to infer gene–drug resistance associations using statistical testing. The isolate collection and the analysis results have been integrated into GEAR-base, a resource available for academic research use free of charge at https://gear-base.com.     

The diversity of microbes: resurgence of the phenotype

The introduction of molecular genetic methods has caused confusion about the nature of microbial species. Environmental DNA extraction has indicated the existence of a vast diversity of genotypes, but how this relates to functional and phenotypic diversity has not been sufficiently explored. It has been implied that genetic distance per se correlates with phenotypic differentiation and thus reflects subtle (but undiscovered) adaptive fine-tuning to the environment, and that microbes may show biogeographic patterns at the genetic level. Here, we argue that no theoretically based species concept exists; species represent only the basic unit in the taxonomic hierarchy. The significance of naming species is that it organizes biological information. The reason why microbial species collectively represent large genetic differences is owing to huge absolute population sizes, absence of allopatric speciation and low extinction rates. Microbial phenotypes are, therefore, ancient in terms of the geological time-scale and have been maintained through stabilizing selection. These problems are discussed with special reference to eukaryotic micro-organisms.     

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.     

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.     

Natural soil microbes alter flowering phenology and the intensity of selection on flowering time in a wild Arabidopsis relative

Plant phenology is known to depend on many different environmental variables, but soil microbial communities have rarely been acknowledged as possible drivers of flowering time. Here, we tested separately the effects of four naturally occurring soil microbiomes and their constituent soil chemistries on flowering phenology and reproductive fitness of Boechera stricta, a wild relative of Arabidopsis. Flowering time was sensitive to both microbes and the abiotic properties of different soils; varying soil microbiota also altered patterns of selection on flowering time. Thus, soil microbes potentially contribute to phenotypic plasticity of flowering time and to differential selection observed between habitats. We also describe a method to dissect the microbiome into single axes of variation that can help identify candidate organisms whose abundance in soil correlates with flowering time. This approach is broadly applicable to search for microbial community members that alter biological characteristics of interest.     

Spatial structure maintains diversity of pyocin inhibition in household Pseudomonas aeruginosa

Nearly all bacteria produce narrow-spectrum antibiotics called bacteriocins. Studies have shown that bacteriocins can mediate microbial interactions, but the mechanisms underlying patterns of inhibition are less well understood. We assembled a spatially structured collection of isolates of Pseudomonas aeruginosa from bathroom and kitchen sink drains in nine households. Growth inhibition of these P. aeruginosa by bacteriocins, known as pyocins in this species, was measured using pairwise inhibition assays. Carbon source usage of these isolates was measured, and genetic distance was estimated using multilocus sequencing. We found that as the distance between sites of isolation increased, there was a significantly higher probability of inhibition, and that pyocin inhibition and susceptibility vary greatly among isolates collected from different houses. We also detected support for other mechanisms influencing diversity: inhibition outcomes were influenced by the type of drain from which isolates were collected, and while we found no indication that carbon source utilization influences inhibition, inhibition was favoured at an intermediate genetic distance. Overall, these results suggest that the combined effects of dispersal limitation among sites and competitive exclusion within them maintain diversity in pyocin inhibition and susceptibility phenotypes, and that additional processes such as local adaptation and effects of phylogenetic distance could further contribute to spatial variability.     

Comparison of genotypic and phenotypic techniques for assessing the variability of the fungus Epicoccum nigrum

AIMS: The diversity within a collection of worldwide isolates of Epicoccum nigrum has been studied using several phenotypic approaches. In addition, the abilities of phenotypic and genotypic techniques for the differentiation of a set of isolates are compared. METHODS AND RESULTS: The methodology used include the study of isozymes (acetyl esterase and alkaline phosphatase), HPLC profile of metabolites and antibiotic activities against a panel of bacteria, yeasts and filamentous fungi, and cytotoxicity against three mammalian cell lines. Two procedures for assessing the relationships within a collection of isolates, using a combination of the techniques, were evaluated, comparing the advantages and disadvantages of each method. CONCLUSIONS: The results showed that each individual technique allows differentiation of the isolates studied to some degree and that the information provided by each technique could be considered as complementary. Genotypic techniques were more powerful than the phenotypic ones to discriminate among the strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This work evaluates the predictive value of several phenotypic techniques on a collection of fungal isolates, and compares the results obtained with genotypic techniques performed on the same strains.     

High phenotypic diversity in infecting but not in colonizing Staphylococcus aureus populations

In hostile environments diversity within a bacterial population may be beneficial for the fitness of the microbial community as a whole. Here we analysed the population diversity of Staphylococcus aureus in infecting and colonizing situations. In the study, performed independently in two German centres, the heterogeneity of the S. aureus population was determined by quantifying the occurrence of phenotypic variants (differences in haemolysis, pigmentation, colony morphology) in primary cultures from nose, oropharyngeal and sputum specimens from cystic fibrosis (CF) patients and in nose swabs from healthy S. aureus carriers. The proportion of heterogeneous samples, the number of clearly distinguishable isolates per sample and the qualitative differences between phenotypes was significantly higher in CF sputum specimens than in the other samples. The heterogeneity of the S. aureus population could be correlated with high bacterial densities in the sputum samples. In patients co-infected with Pseudomonas aeruginosa lower S. aureus bacterial loads and less heterogeneity in the S. aureus population were observed. Typing of all S. aureus isolates from heterogeneous samples by pulsed-field gel electrophoresis or spa typing revealed that the bacteria were polyclonal in 30%, monoclonal with minor genetic alterations in 25% or not distinguishable in 69% of the specimens. Some specimens harboured monoclonal and polyclonal variants simultaneously. Importantly, differences in antibiotic susceptibility were detected in phenotypic S. aureus variants within a single specimen. Diversification of a S. aureus population is highly favoured during chronic CF lung infection, supporting the general hypothesis that maintenance of intrahost diversity can be of adaptive value, increasing the fitness of the bacterial community.     

Restored fitness leads to long-term persistence of resistant Bacteroides strains in the human intestine

Acquired antibiotic resistance typically confers a cost to the bacteria, but these costs can be reduced by genetic compensation over time. The fitness of two Bacteroides thetaiotaomicron clones consecutively isolated in vivo was studied using an in vitro pair-wise competition method. The isolates derived from faecal samples of two clindamycin-exposed healthy volunteers and the two B. thetaiotaomicron clone types could be followed up to 18 months in these two subjects. The two clones were originally susceptible to clindamycin and lacked erm genes; however, after 7 days of clindamycin administration they carried the erm (erythromycin methylase)(G) or (F) gene, respectively, and expressed phenotypic clindamycin resistance. The initial cost of acquired resistance was high as seen in the in vitro pair-wise competition experiments. At 2 weeks post-administration, no growth disadvantage was detected for isolates of either of the two clones in the in vitro experiments and this regained fitness remained for isolates collected up to 18 months. Competition analysis of an in vitro isolated erm(G) positive transconjugant also demonstrated an initial reduction of fitness that was restored over time. The results indicate that the biological cost associated with a resistance gene can rapidly be compensated during in vivo growth. Thus, once the resistant clone has gained its resistance determinant it will be difficult to eliminate.     

The distribution of antibiotic resistance patterns within streptomycetes and their use in secondary metabolite screening

Summary A collection of 169 streptomycetes representing natural isolates and type strains were examined for resistance phenotypes to 11 antibiotics. A total of 84 profiles were obtained with 18 patterns being repeated in two or more strains. The most common pattern was resistance to penicillin in an otherwise sensitive phenotype and accounted for 51 strains. This data was used to cluster the strains and groups defined were examined for correlation with bioactivity. Antibiotic producers were found in clusters 1, 2, 4 and 5–10. Certain strains in these areas were highly bioactive and typically had multiple resistances. Almost half of the collection of strains examined grouped in cluster 3, and were characterized as having a sensitive phenotype and virtually no biological activity in agrochemical screens.     

On the possible ecological roles of antimicrobials

The Introduction of antibiotics into the clinical use in the middle of the 20th century had a profound impact on modern medicine and human wellbeing. The contribution of these wonder molecules to public health and science is hard to overestimate. Much research has informed our understanding of antibiotic mechanisms of action and resistance at inhibitory concentrations in the lab and in the clinic. Antibiotics, however, are not a human invention as most of them are either natural products produced by soil microorganisms or semisynthetic derivatives of natural products. Because we use antibiotics to inhibit the bacterial growth, it is generally assumed that growth inhibition is also their primary ecological function in the environment. Nevertheless, multiple studies point to diverse nonlethal effects that are exhibited at lower levels of antibiotics. Here we review accumulating evidence of antibiosis and of alternative functions of antibiotics exhibited at subinhibitory concentrations. We also speculate on how these effects might alter phenotypes, fitness, and community composition of microbes in the context of the environment and suggest directions for future research.