The Sociality and Evolution of Plasmid-Mediated Antimicrobial Resistance

Overuse and misuse of antibiotics has led to the global spread of antimicrobial resistance, threatening our ability to treat bacterial infections. The horizontal acquisition of multidrug resistance (MDR) plasmids, from other bacterial lineages, has been instrumental in spreading resistance. Newly acquired plasmids are often poorly adapted to hosts causing intragenomic conflicts, reducing the competitiveness of plasmid-carrying strains. Costs can be overcome by positive selection for plasmid-encoded adaptive traits in the short-term, or ameliorated by compensatory evolution in the long-term. How the selection and adaptation of MDR plasmids varies with antibiotic treatment remains unclear. First, I demonstrate that the selective conditions for the maintenance of an MDR plasmid are dependent upon the sociality of resistance it encodes. Selection for efflux of antibiotics, a selfish trait, occurred at very low concentrations of antibiotic, far below the minimum inhibitory concentration of sensitive plasmid-free strain. In contrast, selection for inactivation of antibiotics, a cooperative trait, increased the amount of antibiotic required to select for the MDR plasmid, allowing sensitive plasmid-free bacteria to survive high levels of antibiotic. These selection dynamics were only accurately predicted when mathematical models included the mechanistic details of antibiotic resistance. Secondly, I show that the trajectory of evolution following MDR plasmid acquisition varies with antibiotic treatment. Tetracycline treatment favoured a distinct coevolutionary trajectory of chromosomal resistance mutations coupled with plasmid mutations impairing plasmid-borne resistance. This led to high-level, low-cost antibiotic resistance, but also produced an integrated genome of co-dependent replicons that may limit the onward spread of co-adapted MGEs to other lineages. This evolutionary trajectory was strikingly repeatable across independently evolving populations despite the emergence of multiple competing lineages within populations. The results presented here demonstrate that the interaction between positive selection and compensatory evolution can help to explain the persistence of MDR plasmids in the clinic and the environment

Ecology and evolution of antimicrobial resistance in bacterial communities

Accumulating evidence suggests that the response of bacteria to antibiotics is significantly affected by the presence of other interacting microbes. These interactions are not typically accounted for when determining pathogen sensitivity to antibiotics. In this perspective, we argue that resistance and evolutionary responses to antibiotic treatments should not be considered only a trait of an individual bacteria species but also an emergent property of the microbial community in which pathogens are embedded. We outline how interspecies interactions can affect the responses of individual species and communities to antibiotic treatment, and how these responses could affect the strength of selection, potentially changing the trajectory of resistance evolution. Finally, we identify key areas of future research which will allow for a more complete understanding of antibiotic resistance in bacterial communities. We emphasise that acknowledging the ecological context, i.e. the interactions that occur between pathogens and within communities, could help the development of more efficient and effective antibiotic treatments

Non-antibiotic pharmaceuticals are toxic against <i>Escherichia coli</i> with no evolution of cross-resistance to antibiotics

Antimicrobial resistance can arise in the natural environment via prolonged exposure to the effluent released by manufacturing facilities. In addition to antibiotics, pharmaceutical plants also produce non-antibiotic pharmaceuticals, both the active ingredients and other components of the formulations. The effect of these on the surrounding microbial communities is less clear. We aimed to assess whether non-antibiotic pharmaceuticals and other compounds produced by pharmaceutical plants have inherent toxicity, and whether long-term exposure might result in significant genetic changes or select for cross-resistance to antibiotics. To this end, we screened four non-antibiotic pharmaceuticals (acetaminophen, ibuprofen, propranolol, metformin) and titanium dioxide for toxicity against Escherichia coli K-12 MG1655 and conducted a 30 day selection experiment to assess the effect of long-term exposure. All compounds reduced the maximum optical density reached by E. coli at a range of concentrations including one of environmental relevance, with transcriptome analysis identifying upregulated genes related to stress response and multidrug efflux in response ibuprofen treatment. The compounds did not select for significant genetic changes following a 30 day exposure, and no evidence of selection for cross-resistance to antibiotics was observed for population evolved in the presence of ibuprofen in spite of the differential gene expression after exposure to this compound. This work suggests that these compounds, at environmental concentrations, do not select for cross-resistance to antibiotics in E. coli

A Tale of Three Species: Adaptation of Sodalis glossinidius to Tsetse Biology, Wigglesworthia Metabolism, and Host Diet.

The tsetse fly is the insect vector for the Trypanosoma brucei parasite, the causative agent of human African trypanosomiasis. The colonization and spread of the trypanosome correlate positively with the presence of a secondary symbiotic bacterium, Sodalis glossinidius The metabolic requirements and interactions of the bacterium with its host are poorly understood, and herein we describe a metabolic model of S. glossinidius metabolism. The model enabled the design and experimental verification of a defined medium that supports S. glossinidius growth ex vivo This has been used subsequently to analyze in vitro aspects of S. glossinidius metabolism, revealing multiple unique adaptations of the symbiont to its environment. Continued dependence on a sugar, and the importance of the chitin monomer N-acetyl-d-glucosamine as a carbon and energy source, suggests adaptation to host-derived molecules. Adaptation to the amino acid-rich blood diet is revealed by a strong dependence on l-glutamate as a source of carbon and nitrogen and by the ability to rescue a predicted l-arginine auxotrophy. Finally, the selective loss of thiamine biosynthesis, a vitamin provided to the host by the primary symbiont Wigglesworthia glossinidia, reveals an intersymbiont dependence. The reductive evolution of S. glossinidius to exploit environmentally derived metabolites has resulted in multiple weaknesses in the metabolic network. These weaknesses may become targets for reagents that inhibit S. glossinidius growth and aid the reduction of trypanosomal transmission.IMPORTANCE Human African trypanosomiasis is caused by the Trypanosoma brucei parasite. The tsetse fly vector is of interest for its potential to prevent disease spread, as it is essential for T. brucei life cycle progression and transmission. The tsetse's mutualistic endosymbiont Sodalis glossinidius has a link to trypanosome establishment, providing a disease control target. Here, we describe a new, experimentally verified model of S. glossinidius metabolism. This model has enabled the development of a defined growth medium that was used successfully to test aspects of S. glossinidius metabolism. We present S. glossinidius as uniquely adapted to life in the tsetse, through its reliance on the blood diet and host-derived sugars. Additionally, S. glossinidius has adapted to the tsetse's obligate symbiont Wigglesworthia glossinidia by scavenging a vitamin it produces for the insect. This work highlights the use of metabolic modeling to design defined growth media for symbiotic bacteria and may provide novel inhibitory targets to block trypanosome transmission

A Tale of Three Species: Adaptation of Sodalis glossinidius to Tsetse Biology, Wigglesworthia Metabolism, and Host Diet.

The tsetse fly is the insect vector for the Trypanosoma brucei parasite, the causative agent of human African trypanosomiasis. The colonization and spread of the trypanosome correlate positively with the presence of a secondary symbiotic bacterium, Sodalis glossinidius The metabolic requirements and interactions of the bacterium with its host are poorly understood, and herein we describe a metabolic model of S. glossinidius metabolism. The model enabled the design and experimental verification of a defined medium that supports S. glossinidius growth ex vivo This has been used subsequently to analyze in vitro aspects of S. glossinidius metabolism, revealing multiple unique adaptations of the symbiont to its environment. Continued dependence on a sugar, and the importance of the chitin monomer N-acetyl-d-glucosamine as a carbon and energy source, suggests adaptation to host-derived molecules. Adaptation to the amino acid-rich blood diet is revealed by a strong dependence on l-glutamate as a source of carbon and nitrogen and by the ability to rescue a predicted l-arginine auxotrophy. Finally, the selective loss of thiamine biosynthesis, a vitamin provided to the host by the primary symbiont Wigglesworthia glossinidia, reveals an intersymbiont dependence. The reductive evolution of S. glossinidius to exploit environmentally derived metabolites has resulted in multiple weaknesses in the metabolic network. These weaknesses may become targets for reagents that inhibit S. glossinidius growth and aid the reduction of trypanosomal transmission.IMPORTANCE Human African trypanosomiasis is caused by the Trypanosoma brucei parasite. The tsetse fly vector is of interest for its potential to prevent disease spread, as it is essential for T. brucei life cycle progression and transmission. The tsetse's mutualistic endosymbiont Sodalis glossinidius has a link to trypanosome establishment, providing a disease control target. Here, we describe a new, experimentally verified model of S. glossinidius metabolism. This model has enabled the development of a defined growth medium that was used successfully to test aspects of S. glossinidius metabolism. We present S. glossinidius as uniquely adapted to life in the tsetse, through its reliance on the blood diet and host-derived sugars. Additionally, S. glossinidius has adapted to the tsetse's obligate symbiont Wigglesworthia glossinidia by scavenging a vitamin it produces for the insect. This work highlights the use of metabolic modeling to design defined growth media for symbiotic bacteria and may provide novel inhibitory targets to block trypanosome transmission

Resistance evolution can disrupt antibiotic exposure protection through competitive exclusion of the protective species

Antibiotic degrading bacteria can reduce the efficacy of drug treatments by providing antibiotic exposure protection to pathogens. While this has been demonstrated at the ecological timescale, it is unclear how exposure protection might alter and be affected by pathogen antibiotic resistance evolution. Here, we utilised a two-species model cystic fibrosis (CF) community where we evolved the bacterial pathogen Pseudomonas aeruginosa in a range of imipenem concentrations in the absence or presence of Stenotrophomonas maltophilia, which can detoxify the environment by hydrolysing β-lactam antibiotics. We found that P. aeruginosa quickly evolved resistance to imipenem via parallel loss of function mutations in the oprD porin gene. While the level of resistance did not differ between mono- and co-culture treatments, the presence of S. maltophilia increased the rate of imipenem resistance evolution in the four μg/ml imipenem concentration. Unexpectedly, imipenem resistance evolution coincided with the extinction of S. maltophilia due to increased production of pyocyanin, which was cytotoxic to S. maltophilia. Together, our results show that pathogen resistance evolution can disrupt antibiotic exposure protection due to competitive exclusion of the protective species. Such eco-evolutionary feedbacks may help explain changes in the relative abundance of bacterial species within CF communities despite intrinsic resistance to anti-pseudomonal drugs

Aspergillus fumigatus strains that evolve resistance to the agrochemical fungicide ipflufenoquin in vitro are also resistant to olorofim

Widespread use of azole antifungals in agriculture has been linked to resistance in the pathogenic fungus Aspergillus fumigatus. We show that exposure of A. fumigatus to the agrochemical fungicide, ipflufenoquin, in vitro can select for strains that are resistant to olorofim, a first-in-class clinical antifungal with the same mechanism of action. Resistance is caused by non-synonymous mutations within the target of ipflufenoquin/olorofim activity, dihydroorotate dehydrogenase (DHODH), and these variants have no overt growth defects.</p

Disease Epidemiology in Arthropods Is Altered by the Presence of Nonprotective Symbionts

Inherited microbial symbionts can modulate host susceptibility to natural enemy attack. A wider range of symbionts influence host population demography without altering individual susceptibility, and it has been suggested that these may modify host disease risk through altering the rate of exposure to natural enemies. We present the first test of this thesis, specifically testing whether male-killing symbionts alter the epidemiology of a sexually transmitted infection (STI) carried by its host. STIs are typically expected to show female-biased epidemics, and we first present a simple model which indicates that male-biased STI epidemics may occur where symbionts create female-biased population sex ratios. We then examined the dynamics of a STI in the ladybird beetle Adalia bipunctata, which is also host to a male-killing bacterium. We present evidence that male-biased epidemics of the STI are observed in natural populations when the male-killer is common. Laboratory experiments did not support a role for differential susceptibility of male and female hosts to the STI, nor a protective role for the symbiont, in creating this bias. We conclude that the range of symbionts likely to alter parasite epidemiology will be much wider than previously envisaged, because it will additionally include those that impact host demography alone

Elevated mutation rates in the multi-azole resistant Aspergillus fumigatus clade drives rapid evolution of antifungal resistance

The evolution of antifungal resistance is an emerging global threat. Particularly concerning is the widespread occurrence of azole resistance within Aspergillus fumigatus, a globally ubiquitous environmental mould that causes over 1 million life-threatening invasive infections in humans each year. It is increasingly evident that the environmental use of azoles has led to selective sweeps across multiple genomic loci resulting in the rapid expansion of a genetically distinct cluster of genotypes (clade A) that results in resistance to clinically deployed azoles. Isolates within this cluster are more likely to be cross resistant to agricultural antifungals with unrelated modes of action suggesting they may be adapting rapidly to antifungal challenge. Here we show that this cluster is not only multi-azole resistant but has increased propensity to develop resistance to new antifungals because of variants in the DNA mismatch repair system. A variant in msh6 is found almost exclusively within clade A, occurs in 88% of multi-azole resistant isolates harbouring the canonical cyp51A azole resistance allelic variant TR34/L98H, and is globally distributed. Naturally occurring isolates with this msh6 variant display a 4 to 9-times higher rate of mutation, leading to an increased propensity to evolve resistance to current and next generation antifungals. We argue that pervasive environmental use of fungicides creates selective arenas whereby genotypes of A. fumigatus with increased adaptive capability thrive in the face of strong directional selection, leading to the genesis and amplification of antifungal resistance. These results help explain the pronounced clustering of multiple independent resistance mechanisms within the mutable clade A. Our findings further suggest that resistance to next generation antifungals is more likely to emerge within organisms that are already multi-azole resistant, posing a major problem due to the prospect of dual use of novel antifungals in clinical and agricultural settings

Raw_epidemic_data

Data supporting figure 4 in the journal. Prevalence of C. hippodamiae on A. bipunctata beetles in a series of epidemics, partitioned by A. bipunctata sex