Aggressive Chemotherapy and the Selection of Drug Resistant Pathogens

Drug resistant pathogens are one of the key public health challenges of the 21^st century. There is a widespread belief that resistance is best managed by using drugs to rapidly eliminate target pathogens from patients so as to minimize the probability that pathogens acquire resistance de novo. Yet strong drug pressure imposes intense selection in favor of resistance through alleviation of competition with wild-type populations. Aggressive chemotherapy thus generates opposing evolutionary forces which together determine the rate of drug resistance emergence. Identifying treatment regimens which best retard resistance evolution while maximizing health gains and minimizing disease transmission requires empirical analysis of resistance evolution in vivo in conjunction with measures of clinical outcomes and infectiousness. Using rodent malaria in laboratory mice, we found that less aggressive chemotherapeutic regimens substantially reduced the probability of onward transmission of resistance (by >150-fold), without compromising health outcomes. Our experiments suggest that there may be cases where resistance evolution can be managed more effectively with treatment regimens other than those which reduce pathogen burdens as fast as possible.     

A Reservoir of Drug-Resistant Pathogenic Bacteria in Asymptomatic Hosts

The population genetics of pathogenic bacteria has been intensively studied in order to understand the spread of disease and the evolution of virulence and drug resistance. However, much less attention has been paid to bacterial carriage populations, which inhabit hosts without producing disease. Since new virulent strains that cause disease can be recruited from the carriage population of bacteria, our understanding of infectious disease is seriously incomplete without knowledge on the population structure of pathogenic bacteria living in an asymptomatic host. We report the first extensive survey of the abundance and diversity of a human pathogen in asymptomatic animal hosts. We have found that asymptomatic swine from livestock productions frequently carry populations of Salmonella enterica with a broad range of drug-resistant strains and genetic diversity greatly exceeding that previously described. This study shows how agricultural practice and human intervention may lead and influence the evolution of a hidden reservoir of pathogens, with important implications for human health.     

At Least Two Origins of Fungicide Resistance in Grapevine Downy Mildew Populations

Quinone outside inhibiting (QoI) fungicides represent one of the most widely used groups of fungicides used to control agriculturally important fungal pathogens. They inhibit the cytochrome bc₁ complex of mitochondrial respiration. Soon after their introduction onto the market in 1996, QoI fungicide-resistant isolates were detected in field plant pathogen populations of a large range of species. However, there is still little understanding of the processes driving the development of QoI fungicide resistance in plant pathogens. In particular, it is unknown whether fungicide resistance occurs independently in isolated populations or if it appears once and then spreads globally by migration. Here, we provide the first case study of the evolutionary processes that lead to the emergence of QoI fungicide resistance in the plant pathogen Plasmopara viticola. Sequence analysis of the complete cytochrome b gene showed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Phylogenetic analysis of a large mitochondrial DNA fragment including the cytochrome b gene (2,281 bp) across a wide range of European P. viticola isolates allowed the detection of four major haplotypes belonging to two distinct clades, each of which contains a different QoI fungicide resistance allele. This is the first demonstration that a selected substitution conferring resistance to a fungicide has occurred several times in a plant-pathogen system. Finally, a high population structure was found when the frequency of QoI fungicide resistance haplotypes was assessed in 17 French vineyards, indicating that pathogen populations might be under strong directional selection for local adaptation to fungicide pressure.     

The potential for rust infection to cause natural selection in apomictic Arabis holboellii (Brassicaceae)

Few studies have examined the potential for pathogens with complex life cycles to cause selection on their required alternate (=intermediate) hosts. Here we examine the effects of two fungal pathogens on an herbaceous mustard, Arabis holboellii. One pathogen species uses A. holboellii as a primary host, the other uses it as an alternate host. This plant-pathogen system is especially interesting because the host, A. holboellii, is apomictic; thus individuals reproduce exact copies of themselves. Despite this mode of reproduction, A. holboellii populations are surprisingly genetically diverse. Could frequency dependent selection by pathogens be maintaining clonal diversity? This study assesses the potential for selection by pathogens. In a controlled greehouse experiment we show that there is heritable variation in A. holboellii's resistance to the rust, Puccinia monoica, and that host fitness is severely reduced by P. monoica infection in both the greenhouse and under natural conditions. Field observations indicate that host clones are also differentially susceptible to the short-cycled rust, P. thlaspeos, and that host fitness is reduced by infection to this pathogen as well. Although the preconditions for pathogen-mediated selection are present, frequency-dependent selection by pathogens is unlikely to be important in structuring populations of Arabis holboellii because multiple host genotypes are susceptible to the same inoculum and the pathogen has a long generation time.     

Fast drug rotation reduces bacterial resistance evolution in a microcosm experiment

Drug rotation (cycling), in which multiple drugs are administrated alternatively, has the potential for limiting resistance evolution in pathogens. The frequency of drug alternation could be a major factor to determine the effectiveness of drug rotation. Drug rotation practices often have low frequency of drug alternation, with an expectation of resistance reversion. Here we, based on evolutionary rescue and compensatory evolution theories, suggest that fast drug rotation can limit resistance evolution in the first place. This is because fast drug rotation would give little time for the evolutionarily rescued populations to recover in population size and genetic diversity, and thus decrease the chance of future evolutionary rescue under alternate environmental stresses. We experimentally tested this hypothesis using the bacterium Pseudomonas fluorescens and two antibiotics (chloramphenicol and rifampin). Increasing drug rotation frequency reduced the chance of evolutionary rescue, and most of the finally surviving bacterial populations were resistant to both drugs. Drug resistance incurred significant fitness costs, which did not differ among the drug treatment histories. A link between population sizes during the early stages of drug treatment and the end-point fates of populations (extinction vs survival) suggested that population size recovery and compensatory evolution before drug shift increase the chance of population survival. Our results therefore advocate fast drug rotation as a promising approach to reduce bacterial resistance evolution, which in particular could be a substitute for drug combination when the latter has safety risks.     

Evolution of antifungal-drug resistance: mechanisms and pathogen fitness

Like other microorganisms, fungi exist in populations that are adaptable. Under the selection imposed by antifungal drugs, drug-sensitive fungal pathogens frequently evolve resistance. Although the molecular mechanisms of resistance are well-characterized, there are few measurements of the impact of these mechanisms on pathogen fitness in different environments. To predict resistance before a new drug is prescribed in the clinic, the full spectrum of potential resistance mutations and the interactions among combinations of divergent mechanisms can be determined in evolution experiments. In the search for new strategies to manage drug resistance, measuring the limits of adaptation might reveal methods for trapping fungal pathogens in evolutionary dead ends.     

Population studies of the wild tomato species Solanum chilense reveal geographically structured major gene-mediated pathogen resistance

Natural plant populations encounter strong pathogen pressure and defence-associated genes are known to be under selection dependent on the pressure by the pathogens. Here, we use populations of the wild tomato Solanum chilense to investigate natural resistance against Cladosporium fulvum, a well-known ascomycete pathogen of domesticated tomatoes. Host populations used are from distinct geographical origins and share a defined evolutionary history. We show that distinct populations of S. chilense differ in resistance against the pathogen. Screening for major resistance gene-mediated pathogen recognition throughout the whole species showed clear geographical differences between populations and complete loss of pathogen recognition in the south of the species range. In addition, we observed high complexity in a homologues of Cladosporium resistance (Hcr) locus, underlying the recognition of C. fulvum, in central and northern populations. Our findings show that major gene-mediated recognition specificity is diverse in a natural plant-pathosystem. We place major gene resistance in a geographical context that also defined the evolutionary history of that species. Data suggest that the underlying loci are more complex than previously anticipated, with small-scale gene recombination being possibly responsible for maintaining balanced polymorphisms in the populations that experience pathogen pressure.     

Molecular mechanisms of drug resistance in natural Leishmania populations vary with genetic background

The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L.) donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread applicability.     

Ecological genetics of the Bromus tectorum (Poaceae)-Ustilago bullata (Ustilaginaceae) pathosystem: A role for frequency-dependent selection?

? Premise of the study: Evolutionary processes that maintain genetic diversity in plants are likely to include selection imposed by pathogens. Negative frequency-dependent selection is a mechanism for maintenance of resistance polymorphism in plant-pathogen interactions. We explored whether such selection operates in the Bromus tectorum-Ustilago bullata pathosystem. Gene-for-gene relationships between resistance and avirulence loci have been demonstrated for this pathosystem. ? Methods: We used molecular markers and cross-inoculation trials to learn whether the SSR genotypes of the host exhibited resistance to co-occurring pathogen races, whether host genotypes within a population had equal disease probability, and whether a common resistance locus and its corresponding avirulence locus exhibited predicted allele frequency changes during an epidemic. ? Key results: Five of six putative resistance loci that conferred resistance to co-occurring pathogen races occurred in common host SSR genotypes. Some common genotypes within populations were more likely to be diseased than others, and genotype frequencies sometimes changed across years in patterns consistent with frequency-dependent selection. Observed changes in frequency of resistance and virulence alleles during an epidemic provided further support, but evidence was inconclusive. ? Conclusions: Frequency-dependent selection may operate at endemic disease levels in this pathosystem, but is difficult to detect because many susceptible plants escape infection. Most pathogen isolates were virulent on most host genotypes, minimizing the apparent importance of frequency-dependent selection even during epidemics.     

The impact of refugia on the development of thiabendazole resistance in Haemonchus contortus

Martin P. J., Le Jambre L. F. and Claxton J. H. 1981. The impact of refugia on the development of thiabendazole resistance in Haemonchus contortus. International Journal for Parasitology11: 35–41. Beginning with a mixture of 5% thiabendazole-resistant and 95% non thiabendazole-resistant Haemonchus contortus larvae, worm free sheep were artificially infected with either 10,000, 9000, 7000, 2500 or zero larvae. Four weeks later, the infected sheep were treated with 44 mg/kg of thiabendazole. Seven days after anthelmintic treatment the same sheep were infected with zero, 1000, 3000, 7500 or 10,000 larvae respectively so that each sheep was given a total of 10,000 larvae. The larvae given after thiabendazole treatment are referred to as being in refugia (defined as an area where individual members of a population can escape exposure to a drug). This was repeated for six generations of parasites, maintaining the same proportion of larvae in refugia in each generation. The egg hatch assay for resistance indicated that refugia delayed the development of resistance in Haemonchus contortus. Where none or a small proportion of larvae were in refugia, a rapid increase in resistance occurred. With an increased proportion of larvae in refugia, resistance was slower to develop. Population size, as estimated by faecal egg counts done after anthelmintic treatment and corrected for the number of larvae given in each treatment, was lower at the higher levels of refugia. This was due to a lower level of resistance in the higher refugia lines and consequently a greater effect of the drug. However, the estimate of population size, from egg counts done after the larvae introduced from refugia had matured, were similar in all lines.     

Clonal Expansion during Staphylococcus aureus Infection Dynamics Reveals the Effect of Antibiotic Intervention

To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population “bottleneck” whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where inappropriate usage leading to resistance may reduce the efficacy of life-saving drugs.     

The age and evolution of an antiviral resistance mutation in Drosophila melanogaster

What selective processes underlie the evolution of parasites and their hosts? Arms-race models propose that new host-resistance mutations or parasite counter-adaptations arise and sweep to fixation. Frequency-dependent models propose that selection favours pathogens adapted to the most common host genotypes, conferring an advantage to rare host genotypes. Distinguishing between these models is empirically difficult. The maintenance of disease-resistance polymorphisms has been studied in detail in plants, but less so in animals, and rarely in natural populations. We have made a detailed study of genetic variation in host resistance in a natural animal population, Drosophila melanogaster, and its natural pathogen, the sigma virus. We confirm previous findings that a single (albeit complex) mutation in the gene ref(2)P confers resistance against sigma and show that this mutation has increased in frequency under positive selection. Previous studies suggested that ref(2)P polymorphism reflects the progress of a very recent selective sweep, and that in Europe during the 1980s, this was followed by a sweep of a sigma virus strain able to infect flies carrying this mutation. We find that the ref(2)P resistance mutation is considerably older than the recent spread of this viral strain and suggest that—possibly because it is recessive—the initial spread of the resistance mutation was very slow.     

Novel Inhibitors of Staphylococcus aureus Virulence Gene Expression and Biofilm Formation

Staphylococcus aureus is a major human pathogen and one of the more prominent pathogens causing biofilm related infections in clinic. Antibiotic resistance in S. aureus such as methicillin resistance is approaching an epidemic level. Antibiotic resistance is widespread among major human pathogens and poses a serious problem for public health. Conventional antibiotics are either bacteriostatic or bacteriocidal, leading to strong selection for antibiotic resistant pathogens. An alternative approach of inhibiting pathogen virulence without inhibiting bacterial growth may minimize the selection pressure for resistance. In previous studies, we identified a chemical series of low molecular weight compounds capable of inhibiting group A streptococcus virulence following this alternative anti-microbial approach. In the current study, we demonstrated that two analogs of this class of novel anti-virulence compounds also inhibited virulence gene expression of S. aureus and exhibited an inhibitory effect on S. aureus biofilm formation. This class of anti-virulence compounds could be a starting point for development of novel anti-microbial agents against S. aureus.     

Using Agent-Based Modelling to Predict the Role of Wild Refugia in the Evolution of Resistance of Sea Lice to Chemotherapeutants

A major challenge for Atlantic salmon farming in the northern hemisphere is infestation by the sea louse parasite Lepeophtheirus salmonis. The most frequent method of controlling these sea louse infestations is through the use of chemical treatments. However, most major salmon farming areas have observed resistance to common chemotherapeutants. In terrestrial environments, many strategies employed to manage the evolution of resistance involve the use of refugia, where a portion of the population is left untreated to maintain susceptibility. While refugia have not been deliberately used in Atlantic salmon farming, wild salmon populations that migrate close to salmon farms may act as natural refugia. In this paper we describe an agent-based model that explores the influence of different sizes of wild salmon populations on resistance evolution in sea lice on a salmon farm. Using the model, we demonstrate that wild salmon populations can act as refugia that limit the evolution of resistance in the sea louse populations. Additionally, we demonstrate that an increase in the size of the population of wild salmon results in an increased effect in slowing the evolution of resistance. We explore the effect of a population fitness cost associated with resistance, finding that in some cases it substantially reduces the speed of evolution to chemical treatments.     

Variation in resistance to multiple pathogen species: anther smuts of Silene uniflora

The occurrence of multiple pathogen species on a shared host species is unexpected when they exploit the same micro‐niche within the host individual. One explanation for such observations is the presence of pathogen‐specific resistances segregating within the host population into sites that are differentially occupied by the competing pathogens. This study used experimental inoculations to test whether specific resistances may contribute to the maintenance of two species of anther‐smut fungi, Microbotryum silenes‐inflatae and Microbotryum lagerheimii, in natural populations of Silene uniflora in England and Wales. Overall, resistance to the two pathogens was strongly positively correlated among host populations and to a lesser degree among host families within populations. A few instances of specific resistance were also observed and confirmed by replicated inoculations. The results suggest that selection for resistance to one pathogen may protect the host from the emergence via host shifts of related pathogen species, and conversely that co‐occurrence of two species of pathogens may be dependent on the presence of host genotypes susceptible to both.     

Host ecotype generates evolutionary and epidemiological divergence across a pathogen metapopulation

The extent and speed at which pathogens adapt to host resistance varies considerably. This presents a challenge for predicting when—and where—pathogen evolution may occur. While gene flow and spatially heterogeneous environments are recognized to be critical for the evolutionary potential of pathogen populations, we lack an understanding of how the two jointly shape coevolutionary trajectories between hosts and pathogens. The rust pathogen Melampsora lini infects two ecotypes of its host plant Linum marginale that occur in close proximity yet in distinct populations and habitats. In this study, we found that within-population epidemics were different between the two habitats. We then tested for pathogen local adaptation at host population and ecotype level in a reciprocal inoculation study. Even after controlling for the effect of spatial structure on infection outcome, we found strong evidence of pathogen adaptation at the host ecotype level. Moreover, sequence analysis of two pathogen infectivity loci revealed strong genetic differentiation by host ecotype but not by distance. Hence, environmental variation can be a key determinant of pathogen population genetic structure and coevolutionary dynamics and can generate strong asymmetry in infection risks through space.     

Evolution of host resistance: looking for coevolutionary hotspots at small spatial scales

Natural plant populations are often found to be extremely diverse in their resistance to pathogens. While the potential of pathogens in driving the evolution of resistance in hosts has been widely recognized, empirical evidence linking disease dynamics to host population genetic structure has remained scarce. Here I show that current coevolutionary selection for resistance can be divergent even on a very fine spatial scale. In a natural plant-pathogen metapopulation, disease occurrence patterns were highly aggregated over space and time within host populations. A laboratory inoculation experiment showed higher resistance within areas of the host populations where encounter rates with the pathogen have been high. Higher resistance to sympatric than to allopatric strains of the pathogen suggests that this change has taken place as a response to local selection. These results constitute evidence of adaptive microevolution of resistance resulting from disease epidemics in natural plant-pathogen associations, and highlight the importance of finding the relevant scale at which to address questions of current coevolutionary selection.     

Host genotype and genetic diversity shape the evolution of a novel bacterial infection

Pathogens continue to emerge from increased contact with novel host species. Whilst these hosts can represent distinct environments for pathogens, the impacts of host genetic background on how a pathogen evolves post-emergence are unclear. In a novel interaction, we experimentally evolved a pathogen (Staphylococcus aureus) in populations of wild nematodes (Caenorhabditis elegans) to test whether host genotype and genetic diversity affect pathogen evolution. After ten rounds of selection, we found that pathogen virulence evolved to vary across host genotypes, with differences in host metal ion acquisition detected as a possible driver of increased host exploitation. Diverse host populations selected for the highest levels of pathogen virulence, but infectivity was constrained, unlike in host monocultures. We hypothesise that population heterogeneity might pool together individuals that contribute disproportionately to the spread of infection or to enhanced virulence. The genomes of evolved populations were sequenced, and it was revealed that pathogens selected in distantly-related host genotypes diverged more than those in closely-related host genotypes. S. aureus nevertheless maintained a broad host range. Our study provides unique empirical insight into the evolutionary dynamics that could occur in other novel infections of wildlife and humans.Subject terms: Molecular evolution, Bacterial evolution, Bacterial genetics     

NONADAPTIVE EVOLUTION OF DISEASE RESISTANCE IN AN ANNUAL LEGUME

Local populations of the plant Amphicarpaea bracteata often contain genetically divergent lineages that differ strongly in disease resistance toward the specialist pathogen Synchytrium decipiens. In one population, lineages with disease resistance were observed to significantly decrease in frequency over a two-year period, despite the continued presence of pathogens. Extensive self-pollination in A. bracteata restricts the opportunity for recombination of alleles affecting separate traits, resulting in strong correlations between disease resistance and other ecologically important characters, including plant morphology, phenology, and patterns of reproductive allocation. Natural selection on these correlated characters may thus cause nonadaptive changes in disease resistance. These results imply that A. bracteata's mating system is a basic constraint interfering with its adaptation to pathogen attack.