Biological Chemistry and Crop Protection
CYP51 Paralogues and Azole Sensitivity in Rhynchosporium commune
Author(s): Hawkins, NJ, Cools, HJ, Fraaije, BA
The azoles, inhibiting sterol 14-α demethylase encoded by CYP51, are widely used both as clinical antifungal drugs and as agricultural fungicides. In contrast to some fungicide classes, no single mutation has arisen that confers broad, qualitative resistance to all azoles, but quantitative sensitivity shifts have arisen through a range of mechanisms. In some cases, sensitivity shifts may be due to enhanced efflux of the drug, or altered sterol metabolism, but other mechanisms involve the fungicide target site: CYP51 mutations confer reduced sensitivity in the clinical pathogen Candida albicans and the wheat pathogen Mycosphaerella graminicola, and CYP51 over-expression confers reduced sensitivity in the fruit pathogen Venturia inaequalis. Genome sequencing has revealed that some filamentous ascomycetes possess multiple CYP51 paralogues, with CYP51B present in all sequenced species, CYP51A in around half, and CYP51C only in Fusarium species. Presence of CYP51A reduces intrinsic azole sensitivity, with CYP51A mutations and over-expression resulting in further reductions in sensitivity in clinical isolates of Aspergillus fumigatus. However, in the barley pathogen Rhynchosporium commune, CYP51A is only present in some isolates, and acquired reductions in azole sensitivity in the last fifteen years are linked to the re-emergence of CYP51A in the R. commune population. Historical and functional studies provide evidence for the role of CYP51A in reduced azole sensitivity in R. commune.
Department of Genetics
Accurate exome sequencing in a microscopic invertebrate
Author(s): Chevalier, FD, LoVerde, PT, Anderson, TJC
Small parasites and larval stages pose a problem for population genomic analyses because limited amounts of DNA template are available, while the large size of many parasite genomes makes sequencing complete genomes prohibitively expensive. For example, schistosome adults live in human blood vessels and only microscopic larval stages (miracidia) are available for molecular analysis. Furthermore, sequencing of the complete 363Mb genome is impractical for population samples. We evaluate the accuracy of exome sequencing of single miracidia following whole genome amplification and exome capture using a custom Agilent SureSelect array designed to capture 92% of the 15.3Mb exome. Because the miracidial exomes sequenced were obtained from a cross between a male and female worm of known sequence, we were able to predict the SNP alleles present in the progeny and quantify the genotyping error rate. We were also able to evaluate bias in capture of alleles recovered from genome regions showing high levels of polymorphism compared with the reference genome from which the capture array was designed. In initial work we robustly scored 18,993 SNPs, while judicious multiplexing of samples prior to genome capture reduced costs while maintaining accuracy. We conclude that scoring of genome wide exomic SNPs and indels from exome sequence is feasible, economical and extremely accurate and has many advantages over other methods for generating reduced representation libraries such as RAD-seq. This approach will allow research on schistosomes (and other parasite species) to progress from population genetics using small numbers of markers, to population genomics utilizing genome-wide marker information.
Adaptation of Pseudomonas aeruginosa to different antibiotics
Author(s): Barbosa, C, Schulenburg, H, Jansen, G
Pseudomonas aeruginosa is a widespread Gram-negative bacterium found in water, soil, plants and animals. Its diverse array of virulence factors allows it to establish and proliferate in environments ranging from plant roots to the human respiratory tract. It plays an important role in chronic infections such as in cystic fibrosis (CF), and is the most commonly isolated nosocomial bacterium. Pseudomonads naturally possess multiple response mechanisms against antimicrobial treatments granting it resistance against the most commonly used antibiotics. They range from efflux pump systems and complex genetic regulation to intricate social behaviours like biofilm formation or swarming. However, to date, we lack detailed understanding of the relative importance of each of these mechanisms and their interplay during resistance evolution. Thus, our study uses controlled evolution experiments to evaluate in how far different resistance mechanisms are selectively favored throughout P.aeruginosa adaptation to different types of antibiotics.
Chair of Pediatrics
An adaptive evolution of Helicobacter pylori (H.pylori): what is a role Cag-A loss in the era of antibacterial treatment?
Author(s): Nijevitch, AA
A resistance to antibacterial drugs is a classic demonstration of natural selection in microorganisms during the period of host-bacterial interaction and high intensity of antibiotic exposure. The aim of the study was to determine the evolutionary role of the CagA pathogenicity island (PAI) presence in the outcome of eradication treatment of H.pylori. Sixty-seven pediatric patients with upper dyspepsia (mean age 13.7 years, range 5 to 17 years, male/female 24/43) undergoing endoscopy were investigated for H.pylori presence. Gastric antral biopsy specimens were taken for histology and/or culture and one biopsy fragment was used for CagA PCR determination. H.pylori+ (positive) patients were treated with a two-week course of amoxycilline (50 mg/kg/day), bismuth subcitrate (8 mg/kg/day), nifuratel (30 mg/kg/day) plus omeprazole (1 mg/kg, once daily). The criterion for eradication of H.pylori was the negative breath ammonia test. Forty one of 67 children (61.2%) were H.pylori+. Nineteen of 41 strains (46.3%) were CagA+ (group I) and 22 were CagA– (negative) (group II) (53.7%). H.pylori was eradicated in 33 patients (80.4%). Among the patients with successful eradication 19 children were CagA+, 14 - were CagA–. Thus, significantly more patients in group II than in group I had evidence of ongoing H.pylori infection (63.6% (14/22) versus 94.7% (18/19); χ2=4.08, p=0.0021). Eight children (19.6%) remained H.pylori+. Obviously, the carriage of CagA-lacking strain was strongly associated with unsuccessful eradication (OR=10.28; 95CI, 1.148 to 92.173). The strains of H.pylori lacking the cag PAI, or parts of it, have lost the island through transformation with an empty site or through deletion mutations. It is possible, that this particular phenomenon, associated with genome plasticity, not just allows the microbe to maintain balanced relationship with the host, but also to survive in antibacterial therapy conditions.
institute of biology
Antibiotic resistance among the killers
Author(s): Rozen, DE, Grimbergen, A
The discovery and development of antibiotics as part of the medical arsenal is one of the great triumphs in the effort to eradicate bacterial diseases. For more than 70 years these microbial products have worked with remarkable success, transforming the medical landscape and dramatically improving human health. However, despite their tremendous relevance for humans, there is a surprising lack of understanding of the role of antibiotics in nature for the organisms that produce them. Why are antibiotics produced and why do strains in nature evolve to resist them. Here we test the idea that antibiotic-mediated interference competition between coexisting bacterial species in soil drives reciprocal coevolutionary changes as strains evolve novel mechanisms of killing and resistance. Focusing on the prolific antibiotic producing genus, Streptomyces, and their coexisting competitors, we first characterize interaction networks between coexisting Streptomycetes and then ask whether antibiotic resistance shows evidence of local adaptation. Second, we are investigating the costs and in situ fitness of naturally resistant strains of Streptomyces in soil at different antibiotic concentrations and during co-cultivation with Streptomycetes that are natural antibiotic producers. Our work examines the natural context and population dynamics of resistance evolution, thereby providing insight into processes occurring in the clinical environment.
Antibiotic resistance and stress in the light of Fisher’s model
Author(s): Sousa, A, Trindade, S, Gordo, I
The role of mutations in evolution depends upon the distribution of their effects on fitness. This distribution is likely to depend on the environment. Indeed genotype-by-environment interactions are key for the process of local adaptation and ecological specialization. An important trait in bacterial evolution is antibiotic resistance, which presents a clear case of change in the direction of selection between environments with and without antibiotics. Here, we study the distribution of fitness effects of mutations, conferring antibiotic resistance to Escherichia coli, in benign and stressful environments without drugs.We interpret the distributions in the light of a fitness landscape model that assumes a single fitness peak. We find that mutation effects (s) arewell described by a shifted gamma distribution, with a shift parameter that reflects the distance to the fitness peak and varies across environments. Consistent with the theoretical predictions of Fisher’s geometrical model, with a Gaussian relationship between phenotype and fitness, we find that the main effect of stress is to increase the variance in s. Our findings are in agreement with the results of a recent meta-analysis, which suggest that a simple fitness landscape model may capture the variation of mutation effects across species and environments.
Azole resistance in Aspergillus fumigatus: condition-dependent mutations for resistance
Author(s): Zhang, J
Azole resistance in Aspergillus fumigatus: condition-dependent mutations for resistance and compensatory evolution Jianhua Zhang, Sijmen E. Schoustra, Marijke Slakhorst, Bertha Koopmanschap, Alfons J. M. Debets, Paul E. Verweij, Bas J. Zwaan Laboratory of Genetics, Wageningen University; Department of Medical Microbiology, Radboud University, Nijmegen. Abstract: Azole resistance is an emerging problem in the saprophytic mold Aspergillus fumigatus, which is involved in the vast majority of invasive infections in the human lung. Resistance is widespread in the Netherlands with a prevalence of 5.3%. The majority of highly resistant mutants have undergone two mutational events: an insert in the promoter region (tandem repeat TR34) and a point mutation in the structural region of the cyp51 gene (L98H). The occurrence of two mutational events at the same time is highly unlikely to happen by chance. To prevent and stop the spread of azole resistant strains, understanding the condition-dependent occurrence of TR34/L98H resistance and potential compensatory mutations for reducing the cost of resistance in the filamentous fungus A. fumigatus is crucial. We hypothesise that a combination of different modes of reproduction of the fungus facilitates the combination of the cyp51 point mutation with the tandem repeat into one background. In addition, mutagenic and recombinogenic environmental conditions may play an important role in the development of resistance. We present data of our study on the dynamics of TR34/L98H resistance by experimental evolution in the laboratory under various environmental conditions in combination with variation in the life.
Department of Biological and Environmental Science
Deadly antibiotics and evolutionary rescue via horizontal gene transfer
Author(s): Jalasvuori, M, Ojala, V, Mattila, S
Evolution has caused one of the most concrete problems in the modern world by selecting for drug resistant bacteria. When bacteria are exposed to lethal levels of antibiotics, it has been assumed that there needs to be a pre-existing mutation for resistance within the population in order for the bacterial strain to survive the drug treatment. However and given that majority of resistance genes are carried by mobile genetic elements such as conjugative plasmids that can move between bacterial cells, it is possible that the resistance gene pre-exist in another bacterium which then rescues susceptible bacteria via horizontal gene transfer. We investigated whether bacteria may survive lethal antibiotic selection by acquiring resistance genes horizontally, and, if so, could this process be somehow prevented. Indeed and contradictory to previous paradigm, we demonstrate that the resistance does not need to pre-exist within the population as some bacteriosidic antibiotics cannot kill susceptible bacteria when the surrounding bacterial community carries mobile elements with resistance genes. Moreover, we show that viruses specifically infecting bacteria with mobile elements can both prevent the spread of resistances to other bacteria and to lead to the loss of resistance conferring elements from the population. Altogether these results suggest that the composition of bacterial community along with their viruses can play a defining role in the evolution antibiotic resistances.
Department of Zoology
Direct benefit of the SOS response vanishes after long term evolution in P. aeruginosa
Author(s): Torres-Barceló, C, Moxon, R, McWilliam, M, MacLean, R
Exposure to stressors, such as antibiotics, induces the expression of response pathways that could provide bacteria with a direct fitness benefit, stemming from increased protection against stress, and an indirect fitness benefit, stemming from increased evolvability due to elevated mutagenesis. We have quite a good understanding of the molecular mechanisms of stress response pathways, but quantitative measurements of the direct and indirect fitness benefits and costs associated with stress response pathway expression are lacking. By measuring the competitive fitness of wildtype and LexA mutant (SOS response blocked) strains of P. aeruginosa we show that the SOS response provides with a direct benefit across a range of sub-lethal concentrations of the antibiotic ciprofloxacin. Interestingly, in the absence of the antibiotic, the LexA mutation confers an advantage in the competitive ability and demonstrates the existence of a cost for the cells owning this complex pathway. We also analyse important parameters like cell viability, cell size and growth rate to explain the higher fitness of wildtype during selection with antibiotic. However, we find that the SOS pathway produces no indirect fitness benefit, as demonstrated by the fact that the LexA mutant shows a comparable ability to adapt to ciprofloxacin over a 600 generation selection experiment. Our study clearly demonstrates that natural selection favours the evolution of stress-induced mutagenesis pathways as a result of direct selection for increased stress tolerance and not indirect selection to favour evolvability. Altogether, we contribute with experimental evidence crucial to disentangle if bacterial stress responses play a role in accelerating adaptation to stressors such as antibiotics.
Institute of Integrative Biology
Effect of Phages on the Cost of Antibiotic Resistance
Author(s): Hall, A, Angst, D
Experimental studies of antibiotic resistance evolution typically employ a single bacterial species in pure culture. However, outside the laboratory, parasitism of bacteria by viruses (phages) is ubiquitous. I used experimental populations of Escherichia coli to show that phage parasitism altered the outcomes of competitions between antibiotic-resistant and sensitive genotypes. Phages caused rapid selective sweeps, associated with the emergence of phage-resistant mutants, and this was approximately equally likely to favour antibiotic-resistant or sensitive genotypes when their initial frequencies were similar. An elevated mutation rate was advantageous in the presence of phages, suggesting that a given antibiotic-resistant or sensitive genotype is more likely to fix under phage parasitism when it has a high mutation supply rate relative to other genotypes. Given their abundance in nature, phages probably modify selection on other traits in many different scenarios.