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.
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 Genetics
Efficient linkage mapping in Schistosoma mansoni using extreme QTL methods and exome sequencing
Author(s): Chevalier, FD, Valentim, CLL, LoVerde, PT, Anderson, TJC
Classical linkage mapping (LM) can be labor intensive, logistically challenging, expensive and limited in power, because both phenotypes and genotypes must be measured in each of the individual progeny. Extreme QTL (X-QTL, or linkage group selection) methods promise to increase the power and simplify logistics of LM, because many 1000s of progeny can be analysed, and progeny pools rather than individuals are genotyped. In X-QTL analysis large pools of selected and unselected F2 progeny are quantitatively genotyped to identify selected genome regions. We applied this method to examine the basis of oxamniquine (OXA) resistance, a Mendelian trait showing recessive inheritance, in the parasitic fluke Schistosoma mansoni. These parasites have a complex lifecycle utilizing snail intermediate hosts and human definitive hosts (or rodents in laboratory maintenance). We staged a genetic cross between drug sensitive and resistant parasites, then between two F1 progeny, to generate multiple F2 progeny. One group of F2 progeny infecting hamsters were treated with OXA, while a second group was left untreated. We prepared DNA from pooled males and pooled females from treated and untreated F2 populations and then used exome capture to reduce the size of the genome (from 363Mb to 15Mb) for analysis. Exomes from the four groups of pooled progeny (treated and untreated males, treated and untreated females) and the two parents were sequenced to high read depth (>95x) and allele frequencies of 14,489 variants compared between treated and untreated parasites. In both treated male and female pools, we observed dramatic enrichment of alleles (Z=11.07, p=8.74.10^-29) from the resistant parent in a small region of chr. 6. This region contains SmSULT a gene recently implicated in OXA resistance using classical LM. These results validate use of X-QTL methods for an important human pathogen, and will greatly simplify genetic analysis of traits such as virulence and host specificity.
Institute of Hydrobiology
Heavy metal driven co-selection of antibiotic resistances in aquatic environments and an evolution based concept of risk assessment
Author(s): Seiler, C, Von Tümpling, W, Sachse, R, Berendonk, TU
Recently, it has been realized that an important part of the dissemination of antibiotics and the evolution of antibiotic resistant microbes depends on aquatic environments. In aquatic environments, bacteria from different origins (human, animal, environmental) are able to mix in the presence of antibiotics or other pollutants such as heavy metals. Thus, the objective of our study was to identify which heavy metals potentially induce co-selection for antibiotic resistances in aquatic environments. Therefore, the available data on heavy metal driven co-selection in aquatic environments was reviewed and an evaluation criterion named minimum co-selective concentration (MCC) was developed. The MCC describes the lowest heavy metal concentration which was detected in correlation with increased bacterial antibiotic resistances in the environment. Additionally, a case study at the Western Bug River (Ukraine) investigating heavy metal concentrations of river water and sediment as well as antibiotic resistance of isolated Pseudomonas and Aeromonas strains was conducted. The results of both, the literature and the case study, indicate that metals such as mercury (Hg), cadmium (Cd), copper (Cu), and zinc (Zn) reach aquatic environments and accumulate to critical concentrations that trigger co-selection for antibiotic resistance. Hence, future risk assessments for antibiotic resistance evolution should move from a clinical perspective towards an environmentally based assessment, considering sublethal concentrations of selective pollutants such as heavy metals. The here presented approach also considers evolutionary processes such as selection, co-selection and mutation. This approach towards a novel concept for risk assessment will be pivotal for detecting antibiotic resistance evolution in the environment.
Microbiology and Biotechnology Lab
Hormesis as a resistance enhancer in Stenotrophomonas maltophilia: which consequences for clinical therapy?
Author(s): Serra, J, Tenreiro, R, Dias, R
Opportunistic pathogens are exposed to antimicrobial subinhibitory concentrations in nature and human body compartments during antibiotherapy. According to hormesis concept, although high therapeutic antimicrobial levels inhibit pathogens growth, subinhibitory concentrations could have a stimulatory effect in growth. Hence, this work aimed to probe bacterial growth stimulation by antimicrobials and assess if such stimulatory concentrations could enhance resistance in intrinsically resistant opportunistic pathogens, environmentally sourced, as Stenotrophomonas maltophilia. Using S. maltophilia D457 strain and a derivative mini-Tn5-mutant library, minimal stimulatory concentrations (MSCs) and minimal inhibitory concentrations (MICs) were established and used in evolution experiments performed until 157 generations. In accordance with hormesis, bacterial growth was stimulated under subinhibitory concentrations, at MSCs 16, 160 and 64 fold lower than MICs of norfloxacin, chloramphenicol and carbenicillin, respectively. Continuous exposure to MSCs during 157 generations lead to MICs 4 fold higher for norfloxacin, and 3 fold higher for chloramphenicol and carbenicillin, in comparison with evolution in antibiotic absence, and MICs 8, 6 and 4 fold higher, in comparison with initial generation. Exposures to MSCs during 9 or 157 generations lead to similar resistance levels. Thus, our results suggest that stimulatory concentrations, present in nature or human body compartments, might lead to the swift enrichment or emergence of resistant mutants, contributing to therapy failure as well as to the rapidity of epidemiological spread of S. maltophilia resistance determinants in clinical environments. This work highlights the importance of hormesis to be included in pharmacokinetic-pharmacodynamic modelling of existing and newer antimicrobials, to predict the impact on the rapidity and potential development of antimicrobial resistance in such relevant opportunistic pathogen.
department of molecular biology and ecology of plants
Implications of stress-induced genetic variation for minimizing multidrug resistance in bacteria
Author(s): Obolski, U, Hadany, L
Antibiotic resistance in bacterial infections is a growing threat to public health. Recent evidence shows that when exposed to stressful conditions, some bacteria perform higher rates of mutation and horizontal gene transfer, and thus acquire antibiotic resistance more rapidly. We incorporate this new notion into a mathematical model of ordinary differential equations describing the emergence of antibiotic multi-resistance in a hospital setting. Results: We show that when stress has a considerable effect on genetic variation, the emergence of antibiotic resistance is dramatically affected. In contrast with classical models, a strategy in which patients receive a combination of antibiotics (combining) is expected to facilitate the emergence of multi-resistant bacteria when genetic variation is stress-induced. The preference between a strategy in which one of two effective drugs is assigned randomly to each patient (mixing), and a strategy where only one drug is administered for a specific period of time (cycling) is determined by the resistance acquisition mechanisms. We analyze several features of the mechanisms by which stress affects variation and predict the conditions for success of different antibiotic treatment strategies. These findings should encourage the research of stress-induced genetic variation mechanisms and establish the importance of incorporating data about these mechanisms when considering antibiotic treatment strategies.
Institute of Integrative Biology
Invasion of fungicide resistance: is spatial scale important?
Author(s): Mikaberidze, A, Bonhoeffer, S, McDonald, BA
Fungicide resistance is an urgent problem in agriculture. It makes the chemical control of fungal diseases of plants ineffective and leads to sever crop losses. In order to manage fungicide resistance, a thorough understanding of invasion, persistance and spread of fungicide-resistant pathogen strains is required. At the same time, the dynamics of fungal diseases of plants is strongly influenced by spatial scales of the host-pathogen interaction: spatial extension of the host population and the dispersal range of the pathogen. Here, we explore theoretically the effect of these characteristic spatial scales on the invasion of fungicide resistant pathogen strains using a population dynamics modeling framework.