Charles University in Prague, Faculty of Science Department of Genetics and Microbiology Ph.D. study program: Molecular and Cellular Biology, Genetics and Virology Genetic basis of multidrug resistance in Acinetobacter baumannii Lenka Křížová Supervisor: Doc. RNDr. Alexandr Nemec, Ph.D. Supervisor-consultant: RNDr. Lubomír Janda, Ph.D. Prague 2014 SUMMARY Acinetobacter baumannii has emerged as a significant bacterial pathogen pre-eminently associated with hospital-acquired infections. Strains of this species may currently exhibit resistance to nearly all or even all clinically relevant drugs. The vast majority of epidemic and multidrug-resistant A. baumannii strains belong to a few globally spread lineages, in particular to the so-called European (EU) clones I, II, and III. Complex resistance patterns displayed by these strains result from their marked capacity to develop, acquire, and combine secondary resistance mechanisms against originally effective agents. The aim of this thesis was to broaden our knowledge on the genetic basis and epidemiology of multidrug resistance in A. baumannii. The obtained results have been published in the form of six studies which are part of this thesis. In the first study, we analysed the epidemiology of carbapenem resistance among hospital strains of Acinetobacter in the...

My dissertation encompasses five different studies that are linked by a common theme the investigation of transposable element (TE) contributions to eukaryotic gene sequences. A detailed analysis of exonization events of LTR elements in the human genome shows the preference towards the fixation of LTR elements in gene untranslated regions, which supports the existing concept of a major role of LTR elements as a natural source of regulatory sequences. The ability of different classes of sequence similarity search methods to detect TE-derived sequences was evaluated. In general, the different search methods are found to be complementary, and combined search approaches are needed to systematically check any data set for all potential TE-associated coding sequences. On average, TE-derived exon sequences have low protein coding potential. In particular, non-coding TEs, are frequently exonized but unlikely to encode protein sequences. Many of these non-coding exonized TEs may be actually involved in gene regulation via the formation of double stranded RNA complexes with complementary TE-derived exons. The investigation of the relationship between human miRNAs and TEs shows that 55 experimentally verified human miRNA genes (~12%) originated from TEs. Overall, TE-derived miRNA genes are less conserved than non TE-derived miRNAs. The potential regulatory and functional significance of TE-derived miRNAs was explored. An ab initio prediction algorithm I developed was used to discover putative cases of novel TE-derived miRNA genes. A miRNA gene family, hsa-mir-548, was found to be derived from Made1 family of MITEs. The palindromic structure of the Made1 elements, and MITEs in general, points to a specific mechanism by which these sequences can be recognized and processed by the miRNA biogenesis pathway. MITEs may also represent an evolutionary link between siRNAs and miRNAs. An original model for a siRNA-to-miRNA evolutionary transition mediated by DNA-type TEs is proposed. This model is supported by the presence of evolutionary intermediate TE sequences that encode both siRNAs and miRNAs in the Arabidopsis and rice genomes. The siRNA-to-miRNA evolutionary transition is representative of a number of other regulatory mechanisms that evolved to silence TEs and were later co-opted to serve as regulators of host gene expression.

Thesis (Ph. D.)--Carleton University, 1992. / Also available in electronic format on the Internet.

Selfish genetic elements can distort the sex ratios of their hosts by increasing their own transmission to the next generation in a non-mendelian fashion. These elements can be either nuclear genes on a sex chromosome or cytoplasmically inherited microbes, and achieve an increased transmission by manipulating gametogenesis or host reproduction. Often these selfish elements benefit from a female biased population (for example heritable microbes are passed on maternally in the egg cytoplasm), while non-selfish, autosomal genes are selected to produce a balanced sex ratio. These differing reproductive strategies cause a genetic conflict that results in an “evolutionary arms race” that can promote the evolutionary change of sex determination systems. In this thesis, I investigate an extreme sex ratio distortion in a species of booklouse, Liposcelis sp. This species contains two distinct female types, one of which carries a maternally transmitted selfish genetic element that results in exclusively female offspring being produced. Recently, a candidate for the sex ratio distortion was identified as a horizontally transferred bacterial gene, that we have called Odile, and that is present in the genome of the (distorter) female carrying the distorting element. The gene originates from the endosymbiotic bacterium Wolbachia that is well known for its ability to distort the sex ratio of its hosts. I investigated this horizontal gene transfer event and attempt to characterize Odile. I provide evidence that this Wolbachia gene has been integrated into the genome of the distorter females and is not a bacterial contaminant. I found that the Odile gene has been duplicated and may have been horizontally transferred from Wolbachia independently to at least three other insect genomes. Additionally, I found that Odile is transcribed at low levels in a life-stage specific manner that is suggestive of a role in development. Additionally, I looked into male mate choice in this species as one aspect of the persistence of the distorting element. I found that male Liposcelis sp. do not discriminate between the two female types and do not spend more time mating with one female type over the other. These results contribute to ongoing research into the extreme sex ratio distortion found in this species and the candidate gene that may be the cause. Selfish genetic elements are an important driver of sex determination evolution, and Liposcelis sp. provides a unique and exciting system to investigate the implications of selfish elements in a genome further. / Graduate / 2019-12-17

Selfish genes that bias their own transmission during gametogenesis can spread rapidly in populations, even if they contribute negatively to the fitness of their host. Driving X chromosomes provide a clear example of this type of selfish propagation. These chromosomes, which are found in a broad range of taxa including plants, mammals, and insects, can have important evolutionary and ecological consequences. In this thesis, I report a new case of X chromosome drive (X drive) in a widespread woodland fly, Drosophila testacea. I show that males carrying the driving X (SR males) sire 80-100% female offspring, and that the majority of sons produced by SR males are sterile and appear to lack a Y chromosome. This suggests that meiotic defects involving the Y chromosome may underlie X drive in this species. Abnormalities in sperm cysts of SR males reflect that some spermatids are failing to develop properly, confirming that drive is acting during gametogenesis. Further, I show that SR males possess a diagnostic X chromosome haplotype that is perfectly associated with the sex ratio distortion phenotype. Phylogenetic analysis of X-linked sequences from D. testacea and related species strongly suggests that the driving X arose prior to the split of D. testacea and its sister species, D. neotestacea and D. orientacea. Suppressed recombination between the XST and XSR due to inversions on the XSR likely explains their disparate evolutionary histories. By screening wild-caught flies using progeny sex ratios and a diagnostic X-linked marker, I demonstrate that the driving X is present in wild populations at a frequency of ~10% and that autosomal suppressors of drive are segregating in the same population. Both SR males and homozygous females for the driving X have reduced fertility, which helps to explain the persistence of the driving X over evolutionary timescales. The testacea species group appears to be a hotspot for X drive, and D. testacea is a promising model to compare driving X chromosomes in closely related species, some of which may even be younger than the chromosomes themselves. / Graduate / 2019-04-16

Antibiotic resistance (AR) is a pervasive crisis that is intricately woven into social and environmental systems. Its escalation is fueled by factors such overuse, poverty, climate change, and the heightened interconnectedness characteristic of our era of globalization. In this dissertation, the impact of antibiotic usage is addressed from the perspective of wastewater-based surveillance (WBS) at the wastewater treatment plant (WWTP) and microbial ecology. Antibiotic usage and contamination was found to influence the prevalence of antibiotic resistance genes (ARGs) and resistant bacteria in both lab-scale and full-scale wastewater treatment settings. Through application of novel bioinformatic approaches developed herein, metagenomics revealed associations between sewage-associated microbes and community antibiotic use that were in part mediated by microbial ecological processes and horizontal gene transfer (HGT). In sum, this dissertation increases the arsenal of bioinformatic tools for AR surveillance in wastewater environments and advances knowledge with respect to the contribution of antibiotic use to the spread of antibiotic resistance at the community-scale. Three studies served to evaluate and/or develop bioinformatic resources for molecular characterization of AR in wastewater. Hybrid assembly combining emerging long read DNA sequencing and short read sequencing was evaluated and found to improve accuracy relative to assembly of long or short reads alone. A novel database of mobile genetic element (MGE) marker genes, mobileOG-db, was compiled in order to address short-comings with pre-existing resources. A pipeline for detecting HGT in metagenomes, Kairos, was created in order to facilitate the detection of HGT in metagenome assemblies which greatly amplified coverage of ARGs. In Chapter 5, a lab-scale study of WWTP bioreactors revealed that elevated antibiotic contamination was correlated with increased prevalence of corresponding ARGs. In addition, multiple in situ HGT events of ARGs encoding resistance to the elevated antibiotics were predicted, including one HGT event likely mediated by a novel bacteriophage. In Chapter 6, influent and effluent from a full-scale municipal WWTP were collected twice-weekly for one year and subjected to deep shotgun metagenomic sequencing. In parallel, collaboration with clinicians enabled statistical modeling of antibiotic usage and resistance, revealing associations between antibiotic prescriptions patterns in the region and resistance at the WWTP. Finally, Chapter 7 details bioinformatic recovery of diverse extended spectrum beta-lactamase gene recovery from the influent and effluent metagenomes, shedding light on the dynamics of circulating resistance genes. In sum, this dissertation identifies bioinformatic evidence for the selection of AR in wastewater environments as a result of antibiotic use in the community and advances hypotheses for explaining the mechanisms of the observed phenomena. / Doctor of Philosophy / Antibiotics are key lifesaving drugs that have dramatically improved life expectancy throughout the 20th and 21st centuries. However, there has been an increased incidence of resistance among many important bacterial pathogens in recent decades. The more antibiotics are used, the more chance that resistant bacteria can evolve, survive, and spread. Outpatient care accounts for the vast majority of therapeutic antibiotic use, with more than 200 million prescriptions written for antibiotics in 2021 in the United States. While performing a vital function in combatting disease, oral antibiotics can inadvertently harm the resident microbes of the intestinal tract (i.e., the gut microbiome) by decreasing the diversity of the microbes present and increasing the number of resistant bacteria. At a community level, antibiotic usage also has the potential to induce increased prevalence of antibiotics and antibiotic resistant bacteria in the environment as well, primarily via human excreta (urine and feces). Wastewater represents a key interface between human-derived contaminants and the environment. In regions with centralized wastewater management, antibiotics- and resistant bacteria-containing excreta are typically transported via sewage conveyance systems to a wastewater treatment plant (WWTP). At the WWTP, diverse microbes interact with and degrade various organic contaminants in a series of processes combining physical, chemical, and biological treatments. Due to the intermingling of environmental microbes, antibiotics, and antibiotic resistant bacteria, wastewater is increasingly being recognized as an important venue for antibiotic resistance surveillance and for potential interventions. Awareness of wastewater-based surveillance and epidemiology has surged as a result of the COVID-19 pandemic and such efforts are enshrined in the National COVID-19 Preparedness Plan. However, such a task is fundamentally more challenging for antibiotic resistance than for SARS-CoV-2, as it comprises multiple bacterial strains, antibiotic resistance genes, and resistance mechanisms. In this respect, DNA sequencing of wastewater, i.e., "metagenomics," holds promise as a broad monitoring tool with an unprecedented degree of biological granularity. In this dissertation, we address the impact of antibiotic usage at the WWTP from the perspective of wastewater-based surveillance. We evaluate antibiotic usage at the community-scale as a selective force among bacteria inhabiting WWTPs and identify microbial interactions that influence the escape of resistant bacteria in the effluent. A field-study of wastewater entering the WWTP and cleaned effluent water discharged by the WWTP revealed certain antibiotics and corresponding forms of antibiotic resistance were particularly prone to proliferation in the WWTP. Novel bioinformatic tools were developed and applied to the study of wastewater to reveal these associations. In sum, this dissertation advances knowledge of wastewater as both a mediator of environmental health and as a reflection of community-health in the form of antibiotic resistance.

The antimicrobial resistance (AMR) crisis, where new antibiotic discovery is not keeping pace with the emergence of resistant pathogens, is driven by mobile genetic elements (MGEs). MGEs can autonomously transfer between bacteria, along with AMR genes. The widespread use of antibiotics in the clinic, in agriculture, and animal husbandry, has accelerated the MGE-mediated transfer of AMR genes in the environment. However, despite playing such an important role in the AMR crisis, the dynamics and mechanisms behind the transmission of genes are poorly understood. Furthermore, which natural and man-made compounds inhibit or promote their movement in these environments is unknown. One method to combat the rise in AMR is to identify small molecules as probes to understand the molecular basis of transmission and apply this information to prevent MGE-mediated resistance dissemination. Since conjugation is the main mechanism for AMR gene transfer, targeting MGEs that use conjugation, such as conjugative plasmids (e.g. Tn1549) and conjugative transposons (e.g. F plasmid), has the potential to prevent the emergence of multi-drug resistant pathogens. In this work, a high throughput assay modeled after Tn1549 excision was screened against a library of known bioactive compounds to find modulators of the integrase and excisionase activity. Several fluoroquinolone antibiotics including ciprofloxacin were identified as dose-dependent inhibitors of excision, which acted by changing supercoiling levels in the cell. Ciprofloxacin enhanced conjugation frequency of Tn1549 at sub-MIC concentrations relative to an untreated control and inhibited conjugation frequency at higher concentrations. A second project was focused on a high throughput conjugation assay based on the separation of the lux operon between a donor and recipient cell, such that only transconjugants produce luminescence to reflect active gene transfer. This work furthers our understanding of the development of assays to target MGEs and screening for inhibitors of their movement. / Thesis / Master of Science (MSc) / Antibiotics are small molecules that cure bacterial infections. However, their efficacy is fading as a result of the ability of mobile genetic elements (MGEs) to spread antimicrobial resistance genes between bacteria. Conjugative plasmids (CPs) and conjugative transposons (CTns) are two of the major types of MGEs that contribute to the dissemination of antimicrobial resistance in pathogens. The goal of this research is to search for inhibitors of CTns and CPs in order to prevent the emergence of multi-drug resistant bacteria. High throughput assays were designed to model both a CTn (Tn1549) and a CP (F plasmid) to find small molecules targeting their movement. A screen of the Tn1549 excision assay identified fluoroquinolone antibiotics that inhibit excision in a dose-dependent manner and indirectly inhibit the integrase used to excise the CTn. Ciprofloxacin, a fluoroquinolone, inhibited the conjugation frequency of Tn1549. Future work will focus on identifying new inhibitors of these MGEs and their characterization.

O reconhecimento da resistência antimicrobiana como um fenômeno emergente em saúde pública, tem constituído um problema em nível mundial. O abuso na utilização de antibióticos na medicina humana e veterinária, e na agricultura, tem originado incremento na diversidade de micro-organismos resistentes, refletindo em falha terapêutica. Os mecanismos de resistência a antibióticos em micro-organismos são mediados principalmente por genes adquiridos de DNA exógeno. A dinâmica da transferência horizontal é realizada por meio de elementos genéticos móveis que carregam genes de resistência. A ampla distribuição deste tipo de estruturas, como o elemento SXT, isolado inicialmente em V. cholerae, tem contribuído para a disseminação de complexos específicos clonais em determinadas áreas geográficas. Este estudo pioneiro no Brasil pesquisou a presença de elementos SXT, em espécies bacterianas do grupo das gama proteobactérias em espécies ambientais, determinou suas características estruturais e funcionais, incluindo genes de resistência a antibióticos, bem como a sensibilidade aos antibióticos dentre os isolados bacterianos que os abrigam. O resultado foi a classificação de 43 elementos SXT obtidos no Brasil, através da comparação com aqueles descritos na literatura. Dentre os elementos SXT obtidos, quatro são albergados por Morganella morganii, fato inédito na literatura. O conhecimento da evolução bacteriana constitui importante ferramenta para estabelecer estratégias eficazes de controle e tratamento de infecções, sem aumentar a pressão seletiva sobre os micro-organismos, bem como instrumento preciso e de grande importância para subsidiar estudos epidemiológicos. / Recognition of antimicrobial resistance as an emerging phenomenon in public health has been a problem worldwide. The abuse in the use of antibiotics in human and veterinary medicine, and agriculture, has caused an increase in the diversity of resistant microorganisms, reflecting in treatment failure. The mechanisms of antibiotic resistance in microorganisms are primarily mediated by genes acquired from exogenous DNA. The dynamics of the horizontal transfer is performed by mobile genetic elements which carry resistance genes. The wide distribution of these structures, such as the SXT element originally isolated from V. cholerae, has contributed to the spread of specific clonal complexes in certain geographical areas. This pioneering study in Brazil researched the presence of SXT elements in the group of bacterial species in environmental gamma-proteobacteria species, determined their structural and functional characteristics, including genes for resistance to antibiotics and the antibiotic susceptibility among bacterial isolates that harbor them. The result was the classification of 43 SXT elements found in Brazil, by comparison with those found in the literature. Among the SXT elements found, four are sheltered by Morganella morganii, unprecedented in the literature. Knowledge of bacterial evolution is an important to establish effective strategies to control and treat infections without increasing the selective pressure on microorganisms, as well as a precise instrument and very important tool to support epidemiological studies.

O reconhecimento da resistência antimicrobiana como um fenômeno emergente em saúde pública, tem constituído um problema em nível mundial. O abuso na utilização de antibióticos na medicina humana e veterinária, e na agricultura, tem originado incremento na diversidade de micro-organismos resistentes, refletindo em falha terapêutica. Os mecanismos de resistência a antibióticos em micro-organismos são mediados principalmente por genes adquiridos de DNA exógeno. A dinâmica da transferência horizontal é realizada por meio de elementos genéticos móveis que carregam genes de resistência. A ampla distribuição deste tipo de estruturas, como o elemento SXT, isolado inicialmente em V. cholerae, tem contribuído para a disseminação de complexos específicos clonais em determinadas áreas geográficas. Este estudo pioneiro no Brasil pesquisou a presença de elementos SXT, em espécies bacterianas do grupo das gama proteobactérias em espécies ambientais, determinou suas características estruturais e funcionais, incluindo genes de resistência a antibióticos, bem como a sensibilidade aos antibióticos dentre os isolados bacterianos que os abrigam. O resultado foi a classificação de 43 elementos SXT obtidos no Brasil, através da comparação com aqueles descritos na literatura. Dentre os elementos SXT obtidos, quatro são albergados por Morganella morganii, fato inédito na literatura. O conhecimento da evolução bacteriana constitui importante ferramenta para estabelecer estratégias eficazes de controle e tratamento de infecções, sem aumentar a pressão seletiva sobre os micro-organismos, bem como instrumento preciso e de grande importância para subsidiar estudos epidemiológicos. / Recognition of antimicrobial resistance as an emerging phenomenon in public health has been a problem worldwide. The abuse in the use of antibiotics in human and veterinary medicine, and agriculture, has caused an increase in the diversity of resistant microorganisms, reflecting in treatment failure. The mechanisms of antibiotic resistance in microorganisms are primarily mediated by genes acquired from exogenous DNA. The dynamics of the horizontal transfer is performed by mobile genetic elements which carry resistance genes. The wide distribution of these structures, such as the SXT element originally isolated from V. cholerae, has contributed to the spread of specific clonal complexes in certain geographical areas. This pioneering study in Brazil researched the presence of SXT elements in the group of bacterial species in environmental gamma-proteobacteria species, determined their structural and functional characteristics, including genes for resistance to antibiotics and the antibiotic susceptibility among bacterial isolates that harbor them. The result was the classification of 43 SXT elements found in Brazil, by comparison with those found in the literature. Among the SXT elements found, four are sheltered by Morganella morganii, unprecedented in the literature. Knowledge of bacterial evolution is an important to establish effective strategies to control and treat infections without increasing the selective pressure on microorganisms, as well as a precise instrument and very important tool to support epidemiological studies.

Bacteria are social organisms which participate in multiple cooperative and group behaviours. They moreover have peculiar genetic systems, as they often bear mobile genetic elements like plasmids, molecular symbionts that are the cause of widespread horizontal gene transfer and play a large role in bacterial evolution. Both cooperation and horizontal transfer have consequences for human health: cooperative behaviours are very often involved in the virulence of pathogens, and horizontal gene transfer leads to the spread of antibiotic resistance. The evolution of plasmid transfer has mainly been analyzed in terms of infectious benefits for selfish mobile elements. However, chromosomal genes can also modulate horizontal transfer. A huge diversity in transfer rates is observed among bacterial isolates, suggesting a complex co-evolution between plasmids and hosts. Moreover, plasmids are enriched in genes involved in social behaviours, and so could play a key role in bacterial cooperative behaviours. We study here the coevolution of gene mobility and sociality in bacteria. To investigate the selective pressures acting on plasmid transfer and public good production, we use both mathematical modelling and a synthetic system that we constructed where we can independently control public good cooperation and plasmid conjugation in Escherichia coli. We first show experimentally that horizontal transfer allows the specific maintenance of public good alleles in a structured population by increasing relatedness at the gene-level. We further demonstrate experimentally and theoretically that this in turn allows for second-order selection of transfer ability: when cooperation is needed, alleles promoting donor and recipient abilities for public good traits can be selected both on the plasmid and on the chromosome in structured populations. Moreover, donor ability for private good traits can also be selected on the chromosome, provided that transfer happens towards kin. The interactions between transfer and cooperation can finally lead to an association between transfer and public good production alleles, explaining the high frequency of genes related to cooperation that are located on plasmids. Globally, these results provide insight into the mechanisms maintaining cooperation in bacteria, and may suggest ways to target cooperative virulence.