Resistance to Bacillus thuringiensis (Bt) cotton, Gossypium hirsutum, is associated with fitness costs in pink bollworm, Pectinophora gossypiella. Previous work has demonstrated that costs of resistance are induced by environmental factors including insecticidal cotton phytochemicals such as gossypol. This study (1) tested the hypothesis that the presence of toxic phytochemicals would increase the dominance and magnitude of fitness costs associated with genes conferring resistance to the Bacillus thuringiensis (Bt) toxin, (2) coupled a selection experiment and simulation modeling to evaluate the fitness cost of resistance to Bt in pink bollworm and (3) used laboratory strains containing susceptible and resistant genotypes to test the hypothesis that gossypol accumulates more readily in pink bollworm with Bt resistance alleles, and that this gossypol accumulation reduces fitness. To test hypothesis (1), larval survival and weight were measured in two independent strains of pink bollworm, Pectinophora gossypiella, reared on diet containing the cotton phytochemicals gossypol and cyclopropenoid fatty acid, alone, or in combination. Cotton phytochemicals increased the dominance and magnitude of fitness costs associated with cadherin-based resistance to Bacillus thuringiensis (Bt) toxin. Gossypol and cyclopropenoid fatty acid combined had a more detrimental effect on fitness of pink bollworm than either compound alone. To test hypothesis (2), two pink bollworm strains fed synthetic diet were monitored over 30 generations to test the hypothesis that costs associated with Bt resistance would result in a decline in the frequency of resistance. A decrease in resistance allele frequency did occur in both strains and costs affecting each resistant genotypes were estimated. To test hypothesis (3), two strains of pink bollworm were fed on diet containing gossypol and on diet without gossypol, and gossypol accumulation in tissues of genotypes was measured. In both strains, significantly more gossypol accumulated in genotypes containing at least one resistance allele and gossypol accumulation was additive to dominant. In both strains, an increase in the dominance or magnitude of costs affecting larval weight was observed on gossypol diet, and the change in the magnitude of costs was positively associated with gossypol absorption. In one strain, the presence of gossypol increased survival costs but only in the genotype with the highest gossypol absorption. The mutation conferring resistance to the Bt toxin Cry1Ac is found in the cadherin-encoding region in three lepidopeteran pests (Helicoverpa armigera, Heliothis virescens and Pectinophora gossypiella) including the pink bollworm. Cadherin proteins had been hypothesized to play a role in maintaining the integrity of the insect midgut epithelial tissue. Taken together, these results support the hypothesis that cadherin proteins do contribute to gut integrity, toxic plant phytochemicals accumulate more readily in pink bollworm with resistance alleles, and that such accumulation increases the dominance and magnitude of fitness costs.

Wolbachia are maternally inherited, obligate, intracellular bacteria inducing a form of sterility known as cytoplasmic incompatibility. Wolbachia based strategies have been proposed for the control of disease vectors. One example is to use a population replacement strategy to drive into natural population a novel Wolbachia that modifies the age structure of a vector population, reducing disease transmission. In this research, the effects of a life-shortening stain of Wolbachia (popcorn Wolbachia) are transferred into the mosquitoes Aedes albopictus (Chapter Two and Three) and A. aegypti (Chapter Four and Five). In Chapter Two, the Wolbachia symbiosis significantly reduced fecundity and egg hatches in A. albopictus, with Wolbachia being highly pathogenic in this mosquito species. In Chapter Three, the relationship between popcorn Wolbachia and its host (in a triple-infected mosquito strain) varied with the mosquito diet. Feeding on mouse blood was associated with the loss of infection, whereas the infection was maintained in human blood-fed mosquito lines. Egg viability of triple infected mosquito was reduced only with mouse blood. In Chapter Four, the reduced competitiveness (e.g., low survival and increased developmental time) of infected A. aegypti immatures was associated with popcorn Wolbachia, relative to uninfected individuals in low food condition. In Chapter Five, the decreased survival of immature A. aegypti was associated with popcorn Wolbachia in the presence of potential predators (i.e., older A. aegypti or A. albopictus larvae). Using a novel behavioral assay, a delayed larval reaction to light avoidance was observed to be associated with the infection, suggesting Wolbachia effects on immature host behaviors. In Chapter Six, popcorn Wolbachia and wAlbB infected A. aegypti showed similar reproduction potential. No reduced level of CI or mating competitiveness was observed in wAlbB infected males. The results suggest the wAlbB infection in A. aegypti can be an additional agent for Wolbachia-based control strategies. In Chapter Seven, a filtering system using commercially available sieves was able to separate immature mosquitoes from water, preventing escape of mosquitoes. In Chapter Eight, an inexpensive artificial blood feeding was designed for feeding multiple mosquito cages. The results support the use of these tools to facilitate mass rearing of mosquitoes.

Listeria monocytogenes the causative agent of foodborne listeriosis is a facultative pathogen that lives as a saprophyte in soil and as an intracellular parasite in host tissues. A regulatory protein, the transcriptional activator PrfA, plays a key role in the “saprotroph to parasite” conversion of L. monocytogenes by selectively activating key virulence genes essential for infection when the bacteria enter host cells. Central to this conversion is the plastic ability of PrfA allosterically shift between two states, weakly active (“ON-OFF”, outside in the environment) and strongly active (“ON”, intracellular compartment). In this thesis, I have used the PrfA “ON-OFF” virulence switch to understand the role of virulence regulators in the adaptability of facultative parasites to a wide range of niches. Using the PrfA model, I have also examined the trade-offs between the saprotroph and virulent states of facultative pathogens and the role of plasticity in maintaining adaptation to multiple environments. Using soil as a natural environment model, I have shown that overexpression of the PrfA-dependent virulence regulon has a negative impact on environmental survival of L. monocytogenes. Then I investigated the fitness consequences of losing PrfA switchability in non-host environments. The results in in-vitro growth conditions with isogenic strains with PrfA locked in the “ON” state and in which all the genes of the virulence regulon were deleted, showed that PrfA-dependent gene overexpression causes a reduction in fitness. Our data indicate this was directly attributable to the costs associated with the overproduction of an array of unneeded proteins and not to indirect effects of hyperactive PrfA in Listeria metabolism. Finally, I used experimental evolution studies in in-vitro only conditions and in alternate in-vitro and intracellular conditions with bacteria with wild-type or “ON-locked” PrfA alleles to visualize the selective pressures acting on the PrfA switch. The results of the selection experiments showed that adaptation to the different conditions involves a rapid evolution of PrfA with mutations changing its activity according to the specific environment in which selection occurred. The findings from this thesis highlight the importance of plastic ability, evolution of properly regulated genetic systems and the role of these genetic systems in enabling organisms to maximise their fitness during the adaptation process to a specific niche.

The rapid emergence of bacterial resistance to antibiotics has necessitated the development of alternative treatment strategies. Antimicrobial peptides (AMPs) are important immune system components that kill microbes rapidly and have broad activity-spectra, making them promising leads for new pharmaceuticals. Although the need for novel antimicrobials is great, we also need a better understanding of the mechanisms underlying resistance development to enable design of more efficient drugs and reduce the rate of resistance development. The focus of this thesis has been to examine development of bacterial resistance to AMPs and the resulting effects on bacterial physiology. The major model organism used was Salmonella enterica variant Typhimurium LT2. In Paper I, we observed that bacteria resistant to PR-39 appeared at a high rate, and that the underlying sbmA resistance mutations were low cost or even cost-free. Such mutants are more likely to rapidly appear in a population and, most importantly, will not disappear easily once the selective pressure is removed. In paper II, we isolated protamine-resistant hem- and cydC-mutants that had reduced growth rates and were cross-resistant to several other antimicrobials. These mutants were small colony variants (SCVs), a phenotype often associated with persistent infections. One SCV with a hemC-mutation reverted to faster growth when evolved in the absence of protamine. In paper III, the mechanism behind this fitness compensation was determined, and was found to occur through hemC gene amplification and subsequent point mutations. The study provides a novel mechanism for reversion of the SCV-phenotype and further evidence that gene amplification is a common adaptive mechanism in bacteria. In Paper IV, the antibacterial properties of cyclotides, cyclic mini-proteins from plants, were evaluated. Cycloviolacin O2 from violets was found to be bactericidal against Gram-negative bacteria. Cyclotides are very stable molecules and may be potential starting points for development of peptide antibiotics.

The emergence of extended spectrum β-lactamase (ESBL) producing Enterobacteriaceae worldwide has led to an increased use of carbapenems and may drive the development of carbapenem resistance. Existing mechanisms are mainly due to acquired carbapenemases or the combination of ESBL-production and reduced outer membrane permeability. The focus of this thesis was to study the development of carbapenem resistance in Escherichia coli in the presence and absence of acquired β-lactamases. To this end we used the resistance plasmid pUUH239.2 that caused the first major outbreak of ESBL-producing Enterobacteriaceae in Scandinavia. Spontaneous carbapenem resistance was strongly favoured by the presence of the ESBL-encoding plasmid and different mutational spectra and resistance levels arose for different carbapenems. Mainly, loss of function mutations in the regulators of porin expression caused reduced influx of antibiotic into the cell and in combination with amplification of β-lactamase genes on the plasmid this led to high resistance levels. We further used a pharmacokinetic model, mimicking antibiotic concentrations found in patients during treatment, to test whether ertapenem resistant populations could be selected even at these concentrations. We found that resistant mutants only arose for the ESBL-producing strain and that an increased dosage of ertapenem could not prevent selection of these resistant subpopulations. In another study we saw that carbapenem resistance can even develop in the absence of ESBL-production. We found mutants in export pumps and the antibiotic targets to give high level resistance albeit with high fitness costs in the absence of antibiotics. In the last study, we used selective amplification of β-lactamases on the pUUH239.2 plasmid by carbapenems to determine the cost and stability of gene amplifications. Using mathematical modelling we determined the likelihood of evolution of new gene functions in this region. The high cost and instability of the amplified state makes de novo evolution very improbable, but constant selection of the amplified state may balance these factors until rare mutations can establish a new function. In my studies I observed the influence of β-lactamases on carbapenem resistance and saw that amplification of these genes would further contribute to resistance. The rapid disappearance of amplified arrays of resistance genes in the absence of antibiotic selection may lead to the underestimation of gene amplification as clinical resistance mechanism. Amplification of β-lactamase genes is an important stepping-stone and might lead to the evolution of new resistance genes.

Les intégrons de multirésistance sont des plateformes génétiques permettant aux bactéries de s’adapter à des pressions antibiotiques . Ils leur permettent de capturer et d’exprimer de s gènes de résistance sous forme de cassettes. La capture et le réarrangement des cassettes sont réalisés par une intégrase dont l’expression est régulée par la réponse SOS chez E. coli . L’expression des cassettes est quant à elle assurée par le promoteur Pc. Les travaux présentés dans ce manuscrit visent à préciser deux aspects liés à la dynamique d’évolution des intégrons . L’étude du coût biologique des intégrons a montré que ceux - ci sont des structures génétiques très peu coûteuses pour E. coli . Ce faible coût est notamment lié à la répression du gène de l’intégrase par la réponse SOS. Dans ces conditions de répression, le coût d’un intégron dépend de l’expression du réseau de cassettes et de son contenu. Ainsi ce coût augmente avec la force du promoteur Pc et le nombre de cassettes dans le réseau. D’autre part, le coût lié à la nature des cassettes est variable. L’étude de la dynamique d’évolution du promoteur Pc visait à vérifier l’hypothèse selon laquelle des pressions antibiotiques auraient conduit à l’émergence de promoteurs forts à partir d’un variant ancestral faible. L’évolution d’une souche de E. coli , contenant un intégron plasmidique portant un variant faible d e Pc, a été réalisée en chemostat sur 200 générations . L’analyse des populations évoluées par deep - sequencing n’a pas permis de mettre en évidence l’émergence de variants forts de Pc . Néanmoins, l ’ étude de ce s populations évoluées révèle une part majoritaire d’évolution chromosomique. Dans ces conditions, l’ absence d’évolution du Pc pourrait atteste r soit d’une réalité biologique ou soit d’un protocole expérimental d’évolution à optimiser . / Resistance integrons are genetic platforms able to catch and express resistance genes embedded within gene cassettes. Capture and reshuffling of gene cassettes are mediated by the integrase whose expression is regulated by the SOS response in E. coli. Gene cassettes are then expressed from the Pc promoter.This work aims to clarify the evolution dynamic of integrons.In a first part, the fitness cost of class 1 integron was assessed in E. coli. Results reveal that integrons are low cost structures and that their cost is reduced by the SOS-mediated repression system. While repressed, the cost of an integron mostly depends on cassettes array expression. The cost of an integron therefore increases with Pc strength and the number of cassettes in the array. Furthermore, different cassettes exhibit different costs.In a second part, the evolution dynamic of Pc promoter was assessed in response to antibiotic pressures. An E. coli strain, carrying a plasmidic integron with a weak Pc promoter, was propagated in chemostat for 200 generations. The deep-sequencing of evolved populations did not reveal any mutations in the promoter region. On the other hand, evolved bacteria presented evidence of chromosomal adaptation. In these conditions, the lack of evolution within the Pc region could reveal either a biological reality or an experimental protocol to optimize.

No / BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is considered to be less transmissible due to the fitness cost associated with drug resistance-conferring mutations in essential genes. OBJECTIVE: To test the hypothesis that TB drug resistance-conferring mutations with fitness cost are more frequent among human immunodeficiency virus (HIV) positive than among HIV-negative patients. DESIGN: We analysed all strains from the two TB drug resistance surveys conducted in Uganda between 2008 and 2011. Strains phenotypically susceptible to rifampicin and/or isoniazid were assumed to be wild-type; in all other cases, we performed whole-genome sequencing. Mutations at the rpoB531 and katG315 codons were considered without fitness loss, whereas other rpoB codons and non-katG were considered with fitness loss. RESULTS: Of the 897 TB patients, 286 (32.1%) were HIV-positive. Mutations with fitness loss in HIV-positive and HIV-negative patients were respectively as follows: non-531 rpoB: 1.03% (n = 3), 0.71% (n = 4) (OR 1.46, 95%CI 0.58-3.68); non-katG: 0.40% (n = 1), 1.0% (n = 6) (OR 0.40, 95%CI 0.07-2.20); rpoB531: 1.49% (n = 4), 0.69% (n = 4) (OR 2.29, 95%CI 0.83-5.77); katG315: 3.86% (n = 11), 2.55% (n = 15) (OR 1.54, 95%CI 0.81-2.90). The odds of mutations with and without fitness cost were higher for patients with a history of previous anti-tuberculosis treatment. CONCLUSIONS: Our data do not support the hypothesis that resistance-conferring mutations with fitness cost are likely to be often present in HIV-positive individuals.

The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development. In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4. The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways. Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host. In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.

O inseticida spinosad tem sido um dos mais utilizados para o controle de Spodoptera frugiperda (J. E. Smith) no Brasil, devido à sua eficácia e ao seu mecanismo de ação único (modulador alostérico de receptores nicotínicos da acetilcolina). Para fornecer subsídios a um programa de manejo da resistência, foram realizados estudos para compreender as bases genéticas e moleculares da resistência de S. frugiperda a este inseticida. Inicialmente, foi selecionada uma linhagem de S. frugiperda resistente a spinosad (Spin-res) em laboratório por meio da técnica \"F2 screen\". A razão de resistência, baseada na CL50, foi de aproximadamente 890 vezes. A partir de cruzamentos recíprocos entre a linhagem suscetível (Sus) e Spin-res, constatou-se que o padrão de herança da resistência de S. frugiperda a spinosad é autossômica e incompletamente recessiva. Retrocruzamentos da progênie F1 de cruzamentos recíprocos com a linhagem Spin-res confirmaram a hipótese de herança poligênica da resistência, com número mínimo de segregações independentes variando de 1,86 a 2,45. Além disso, observou-se um elevado custo adaptativo associado à resistência de S. frugiperda a spinosad, baseado nos parâmetros da tabela de vida e fertilidade. A partir dos dados de seqüenciamento de quatro bibliotecas de cDNA de lagartas de quarto ínstar das linhagens Sus e Spin-res (expostas ou não a spinosad), utilizando a plataforma HiScan1000&reg; (Illumina&copy;), foi realizada a comparação do perfil de transcrição e expressão diferencial de genes entre as linhagens Sus e Spin-R. O transcritoma foi montado utilizando a estratégia de novo contendo cerca de 19 milhões de leituras single-end com qualidades de score acima de 30, gerando 42406 transcritos com o N50 de 598 pb. A busca por similaridade no banco de dados não-redundante (nr) do NCBI, possibilitou a anotação funcional de 24980 (59%) transcritos, alinhando-se a Bombyx mori L., Helicoverpa armigera (Hübner) e Spodoptera spp. com 22,5; 3,81 e 3,6% das sequências respectivamente. Foram identificados 2903 transcritos apresentando expressão diferencial (P <= 0,05, t-test; fold-change > 2) entre as linhagens Spin-res e Sus. Dentre os transcritos relacionados a enzimas do complexo metabólico, 23 P450 monooxigenases, 13 glutathiona S-transferases, uma carboxilesterase e uma esterase foram superexpressas na linhagem Spin-res. Além disso, foi observada a superexpressão de 15 genes relacionados à produção energética na linhagem Spin-res, o que pode estar relacionada ao elevado custo adaptativo associado à resistência. Análises de PCR quantitativo em tempo real confirmaram que os padrões de expressão foram consistentes com os resultados de RNA-seq. Bioensaios com os sinergistas PBO e DEM mostraram pouco envolvimento de enzimas P450 e nenhum envolvimento de glutationa S-transferases na resistência de S. frugiperda a spinosad. O sequenciamento da subunidade &alpha;6 do receptor nicotínico de acetilcolina de ambas linhagens demonstrou a existência de uma mutação sinônima entre as duas linhagens (G567A), indicando que a subunidade &alpha;6 não é a única relacionada à resistência de S. frugiperda a spinosad. / Spinosad has been one of the most used insecticides to manage Spodoptera frugiperda (J. E. Smith) in Brazil, due to its efficacy and unique mode of action (nicotinic acetylcholine receptor allosteric modulator). To support an insect resistance management program (IRM), we selected and characterized in laboratory a spinosad-resistant strain (Spin-res) of S. frugiperda using the F2 screen method. The resistance ratio, based on LC50, was &asymp; 890-fold. Based on reciprocal crosses between susceptible (Sus) and Spin-res, the inheritance of spinosad resistance in S. frugiperda was autosomal incompletely recessive. Backcrosses between the F1 from reciprocal crosses and the parental Spin-res revealed a polygenic resistance, with an estimation of at least 1.86 to 2.45 genes related to spinosad resistance. Furthermore, it was observed a strong fitness cost associated to spinosad-resistance in Spin-res strain, based on the life table and fertility parameters. The characterization of the transcriptional profile and the differential gene expression comparison between susceptible and spinosad-resistant strains of Spodoptera frugiperda were obtained from the sequencing of cDNA libraries from fourth instar larvae of Sus and Spin-res strains (exposed or not to spinosad) using a HiScan1000&reg; platform (Illumina&copy;). The transcriptome was de novo assembled using nearly 19 million single-end reads with quality score over 30, yielding 42,406 transcripts with a N50 of 598 bp. Based on similarity search in the non-redundant (nr) nucleotide database, 24,980 (59%) transcripts were annotated. Most of the transcripts aligned to Bombyx mori L., Helicoverpa armigera (Hübner) and Spodoptera spp., with 22.5%, 3.81, and 3.6, respectively. We identified 2,032 differentially expressed transcripts (P <= 0.05, t-test; fold-change > 2) between the susceptible and spinosad-resistant strains. Among metabolic enzyme transcripts, 23 P450 monooxigenases, 13 glutathione S-transferases, one carboxylesterase and one esterase were up-regulated in the spinosad-resistant strain. In addition, it was observed 15 genes superexpressed in spinosad-resistant strain related to energy production, which can be related to the high fitness cost associated with resistance. Quantitative real-time PCR analysis showed that patterns of gene expression were consistent with RNA-seq results. Synergistic bioassays using PBO and DEM showed little involvement of P450s in spinosad-resistance and lack of involvement regarding the glutathione Stransferases. Furthermore, we sequenced and compared the subunit &alpha;6 from the nicotinic acetylcholine receptor of S. frugiperda Spin-res and Sus strains. Only one synonymous mutation within the two strains (G567A) was found, showing that the &alpha;6 is not the only subunit involved in S. frugiperda resistance to spinosad.

<p>Becoming antibiotic resistant is often associated with fitness costs for the resistant bacteria. This is seen as a loss of competitiveness against the antibiotic-sensitive wild-type in an antibiotic-free environment. In this study, the physiological alterations associated with fitness cost of antibiotic resistance <i>in vitro</i> (in the laboratory medium), and <i>in vivo</i> (in a mouse infection model), are identified in the model system of fusidic acid resistant (Fus^R) <i>Salmonella</i> <i>enterica</i> serovar Typhimurium.</p><p>Fus^R mutants have mutations in <i>fusA</i>, the gene that encodes translation elongation factor G (EF-G). Fus^R EF-G has a slow rate of regeneration of active EF-G·GTP off the ribosome, resulting in a slow rate of protein synthesis. The low fitness of Fus^R mutants <i>in vitro</i>, and <i>in vivo</i>, can be explained in part by a slow rate of protein synthesis and resulting slow growth. However, some Fus^R mutants with normal rates of protein synthesis still suffer from reduced fitness <i>in vivo</i>. We observed that Fus^R mutants have perturbed levels of the global regulatory molecule ppGpp. One consequence of this is an inefficient induction of RpoS, a regulator of general stress reponse and an important virulence factor for <i>Salmonella</i>. In addition, we found that Fus^R mutants have reduced amounts of heme, a co-factor of catalases and cytochromes. As a consequence of the heme defect, Fus^R mutants have a reduced ability to withstand oxidative stress and a low rate of aerobic respiration.</p><p>The pleiotropic phenotypes of Fus^R mutants suggest that antibiotic resistance can be associated with broad changes in bacterial physiology. Knowledge of physiological alterations that reduce the fitness of antibiotic-resistant mutants can be useful in identifying novel targets for antimicrobial agents. Drugs that alter the levels of global transcriptional regulators such as ppGpp or RpoS deserve attention as potential antimicrobial agents. Finally, the observation that Fus^R mutants have increased sensitivity to several unrelated classes of antibiotics suggests that the identification of physiological cost of resistance can help in optimizing treatment of resistant bacterial populations.</p>