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.

I lactobacilli sono considerati dei microorganismi non-patogeni. Molti di loro appartengono al gruppo batterico GRAS e/o sono nell’elenco QPS. Dal momento che i lactobacilli intenzionalmente aggiunti agli alimenti possono agire come reservoir di geni di resistenza, la valutazione del rischio deve essere continuamente aggiornata. Lo scopo di questa tesi era la valutazione di alcuni metodi usati per testare e caratterizzare le specie del genere Lactobacillus per quanto riguarda la sicurezza e la potenziale attività probiotica. Nella prima parte due metodi di micro diluzione, il metodo ISO e CLSI, soni stati comparati testando la resistenza agli antibiotici di 54 ceppi L. plantarum. Sulla base di risultati ottenuti il metodo ISO era più adatto per valutare la resistenza di questa specie. Il test del limite di sensibilità della PCR per 8 paia di primers specifici per il rilevamento dei lactobacilli e bifido batteri da feci ha confermato i loro diversi livelli di efficacia. La seconda parte della tesi descrive un progetto di ricerca mirato sulla identificazione di nuovi ceppi probiotici fra diversi ceppi di Lactobacillus paracasei e Lactobacillus rhamnosus identificando dei geni o loci responsabili della interazione con l’ospite, immunomodulazione, e l’inibizione della crescita dei patogeni. Le analisi fenotipiche dei 40 ceppi hanno confemato una grande variabilità fra di loro, che può servire per associare delle caratteristiche fenotipiche a quelle genotipiche. Tra i ceppi dello stesso progetto è stato individuato un ceppo di L. mucosae. Dal momento che questa è una specie relativamente nuova, le sue caratteristiche sono state analizzate comparandole con altri 3 ceppi appartenenti alla stessa specie. In questo modo sono state confermate alcune informazioni su L. mucosae, ma soprattutto sono stati forniti dei dati nuovi sulle proprietà di questa specie. / The species of the Lactobacillus genus are generally believed to be microorganisms with no pathogenic potential. Many of them have granted GRAS and QPS status. Non-pathogenic bacteria as lactobacilli-intentionally added or accidentally present in food-are under evaluation, as they could act as reservoir of resistant genes. This thesis was aimed to evaluate some methods used for testing and to characterize some Lactobacillus species, as regards their safety and potential probiotic activity. The first part of the research focused on the comparison of two broth microdilution methods: ISO and CLSI, in order to assess the resistance of 54 L. plantarum strains to antimicrobial agents. The results suggest better performances of the phenotypic assay developed by ISO, at least for strains belonging to L. plantarum species.Then the assessment of the PCR detection limit for 8 sets of primers for the detection of lactobacilli and bifidobacteria from infant faeces confirmed different levels of effectiveness for the primers. Next part of the thesis was the research project aimed at identifying genes or genetic loci of different strains of two Lactobacillus species (i.e. Lactobacillus paracasei and Lactobacillus rhamnosus) involved in the interaction with the host, immune-modulation of host cells and pathogen growth inhibition in order to find new probiotic strains. The phenotypic analysis of 40 selected strains demonstrated large variability between strains of these species, which could serve to the association of phenotypic differences to genome specificities. A strain of Lactobacillus mucosae was found within the framework of the same project. As it is a relatively new species, it was chosen to further investigate its properties, comparing it with three other L. mucosae strains. This study led to confirm some information but first and foremost it has provided new data on the examined species.

Humans are generally having a strong, widespread, and negative impact on nature. Given the many ways we are impacting nature and the many ways nature is responding, it is useful to study responses in an integrative context. My thesis is focused largely (two out of the three data chapters) on butterfly species’ range shifts consistent with modern climate change in Canada. I employed a macroecological approach to my research, drawing on methods and findings from evolutionary biology, phylogenetics, conservation biology, and natural history. I answered three main research questions. First, is there a trade-off between population growth rate (rmax) and carrying capacity (K) at the mutation scale (Chapter 2)? I found rmax and K to not trade off, but in fact to positively co-vary at the mutation scale. This suggests trade-offs between these traits only emerge after selection removes mutants with low resource acquisition rates (i.e., unhealthy genotypes), revealing trade-offs between remaining genotypes with varied resource allocation strategies. Second, did butterfly species shift their northern range boundaries northward over the 1900s, consistent with climate warming (Chapter 3)? Leading a team of collaborators, we found that most butterfly species’ northern range boundaries did indeed shift northward over the 1900s. But range shift rates were slower than those documented in the literature for more recent time periods, likely reflecting the weaker warming experienced in the time period of my study. Third, were species’ rates of range shift related to their phylogeny (Chapter 3) or traits (Chapter 4)? I found no compelling relationships between rates of range shift and phylogeny or traits. If certain traits make some species more successful at northern boundary range expansion than others, their effect was not strong enough to emerge from the background noise inherent in the broad scale data set I used.

Formål: Formålet med dette studie var at undersøge faktorer der kan have betydning for hvordan patienten magter at være isoleret under indlæggelse på hospital. Der søges afdækning af om der er baggrundsvariabler som køn, alder og tidligere erfaringer, som har betydning og hvilke konsekvenser det medfører. Formålet var endvidere på baggrund af en risikoanalyse af den enkelte patientat fokusere på at tilrettelægge organiseringen af pleje og behandling. Metode: Studiet er et kvalitativstudie, hvor det empiriske materiale blev indsamlet ved fempatientinterviews. Som analysemetode blev anvendt indholdsanalyse. Den konceptuelle ramme omfatter antibiotikaresistens i et folkesundhedsperspektiv, en beskrivelse af rammerne for infektionsforebyggelse i Danmark samt en teoretisk ramme af hvad det indebærer for patienter at være smittet med en multiresistent bakterie og oplevelse af at være isoleret. Resultater: Studiet viser, at lukket dør, mangel på kontakt og stimuli resulterer i følelsen af kedsomhed, monotoni og angst. Studiet viser endvidere at baggrundsvariabler synes at have betydning for hvordan isolationen opleves. Kvinder udviser større bekymringer omkring smitteforholdsregler, og er mere observante på personalets adfærd end mænd. Kvinder bekymrer sig mere om risikoen for smitteoverførsel til besøgende og familie. Kvinder er mere emotionelle under indlæggelsen og under isolationen. Mænd affinder sig udadtil med situationen og har ikke samme spekulationer omkring smitteforholdsregler. Mænd har en mere rationel tilgang, og der er en tendens til at mænd bedre magter at være isoleret på enestue. Yngre patienterser ud til at magte isolationen bedre og anser enestue som en fordel. De ældre bliver mere triste og føler sig ensomme. Erindringer fra tidligere indlæggelser kan lejres som negative oplevelser, og influere på nuværende indlæggelse. Forat patienterne kunne magte situationen, udviklede de selv strategier til egen hjælp og befandt sig således i en balance mellem stress og mestring. Konklusion: For at kunne forebygge de negative oplevelser det har for patienter som er isoleret, uden at kompromittere smitteforebyggelsen, vil et skærpet fokus på hele organiseringen, undervisning af personale, tilrettelæggelsen af isolationen med fokus på sengestuefaciliteter, tid til kontakt og grundig information være nødvendig. Her udover kan individuelle foranstaltninger på baggrund af en risikoanalyse overvejes. / Aim: This study explored and describedthe factors that may influence how patients react to source isolation from others during hospitalization. The study also sought to determine how background variables such as gender, age, and previous hospitalization affect source isolation. Based on individuals’ risk assessment, this study also focusedon how hospitalsplan and the organization of care and treatment. Method: This qualitative study used content analysisto reviewd ata collected from interviews with five patients. The conceptual framework describes antibiotic resistance and infection control from a public health perspective and exploredits prevention in Denmark. Thetheoretical framework describe show patients experiencean infection acquired by exposure to drug-resistant bacteria, as well assubsequent source isolation. Results: Thelimited space of an isolation room, including closed doors, lack of contact with people, and few sensory stimuli, resulted in patient boredom, monotony,and anxiety. Moreover, the data showed that background variables affected how patients experience source isolation. Compared with men, women showed greater concern about precautions against infection and greater awareness of staff behavior. Women also worried more about the risk of transmitting bacteria/disease to visitors and familymembers, and display more emotion during isolation. In contrast, men outwardly resigned themselves to the situation and didnot speculate about infection precautions. Men had more rational approach, and tended to cope better when isolated in a single room. Younger patients seemed to have a better coping strategy during isolation, and considered a single room an advantage compared to the ward. Elderly patients felt sad and lonely during source isolation. In addition, previous negative experiences from earlier hospitalization seemedto influence current isolation. Patients developed their own strategies for coping with source isolation and found themselves balanced between being stressed and coping. Conclusion: Hospitals need more alternatives (e.g., better training and improved treatment culture) to prevent negative psychological affects due to isolation without compromising infection prevention. Hospitals should update their personnel at all organizational levels, and focus on room facilities in the ward, contact time,and improved information and communication. Riskassessment should be individualizedfor each patient. / <p>ISBN 978-91-86739-98-0</p>

The extensive medical and agricultural use and misuse of antibiotics during the last 70 years has caused an enrichment of resistant pathogenic bacteria that now severely threatens our capacity to efficiently treat bacterial infections. While is has been known for a long time that high concentrations of antibiotics can select for resistant mutants, less is known about the lower limit at which antibiotics can be selective and enrich for resistant bacteria. In this thesis we investigated the role of low concentrations of antibiotics and heavy metals in the enrichment and evolution of antibiotic resistance. Selection was studied using Escherichia coli and Salmonella enterica serovar Typhimurium LT2 with different resistance mutations, different chromosomal resistance genes as well as large conjugative multidrug resistance plasmids. Using very sensitive competition experiments, we showed that antibiotic and heavy metal levels more than several hundred-fold below the minimal inhibitory concentration of susceptible bacteria can enrich for resistant bacteria. Additionally, we demonstrated that subinhibitory levels of antibiotics can select for de novo resistant mutants, and that these conditions can select for a new spectrum of low-cost resistance mutations. The combinatorial effects of antibiotics and heavy metals can cause an enrichment of a multidrug resistance plasmid, even if the concentration of each compound individually is not high enough to cause selection. These results indicate that environments contaminated with low levels of antibiotics and heavy metals such as, for example, sewage water or soil fertilized with sludge or manure, could provide a setting for selection, enrichment and transfer of antibiotic resistance genes. This selection could be a critical step in the transfer of resistance genes from environmental bacteria to human pathogens.

Benzalkonium chlorides (BACs) are widely used, broad-spectrum disinfectants and frequently detected in the environment, even at toxic levels for life. Since such disinfectants can induce broad resistance capabilities, BACs may fuel the emergence of antibiotic resistance in the environment. A substantial body of literature has reported that exposure to BACs causes antibiotic resistance; yet, other studies suggest that the resistance linkage is rare, unsystematic, and/or clinically insignificant. Accordingly, whether or not disinfectant exposure mediates antibiotic resistance and, if so, what molecular mechanisms underlie the resistance link remains to be clearly elucidated. Further, understanding how microbial communities degrade BACs is important not only for alleviating the possible occurrence of antibiotic resistance but also reducing the potential risks to environmental and public health. An integrated strategy that combines metagenomics, metatranscriptomics, genetics, and traditional culture-dependent approaches was employed to provide novel insights into these issues. The integrative approach showed that a microbial community exposed to BACs can acquire antibiotic resistance through two mechanisms: i) horizontal transfer of previously uncharacterized efflux pump genes conferring resistance to BACs and antibiotics, which were encoded on a conjugative plasmid and co-selected together upon BACs and ii) selective enrichment of intrinsically multi-drug resistant organisms. Further, a microbial community adapts to BAC exposure via a variety of mechanisms, including selective enrichment of BAC-degrading species and amino acid substitutions and horizontal transfer of genes related to BAC resistance and degradation. The metatranscriptomic data suggests that the BAC-adapted microbial community metabolized BACs by cooperative interactions among its members. More specifically, Pseudomonas nitroreducens cleaved (i.e., dealkylated) BACs, metabolized the alkyl chain (the dealkylated product of BACs), and released benzyldimethylamine (the other product of BACs), which was further metabolized by other community members (e.g., Pseudomonas putida). Collectively, this study demonstrates the role of BACs in promoting antibiotic resistance and advances current understanding of a microbial community degrading BACs. The results of this work have important implications for (appropriate) usage of disinfectants and for assessing, predicting, and optimizing biological engineering processes treating BAC-bearing waste streams.

Antibiotic resistance is a worsening threat to human health. Increasing our understanding of the mechanisms causing this resistance will be of great benefit in designing methods to evade resistance and in developing new classes of antibiotics. In this thesis, I have used the TetR Family Transcriptional Regulators (TFRs), which constitute one of the largest antibiotic resistance regulator families, as a model system to study the structure, function and evolution of antibiotic resistance determinants. I performed a thorough examination of the variation and conservation seen in TFR sequences and structures using computational approaches. Through structure comparison, I have identified the most conserved features shared by the TFR family that are crucial for their stability and function. Based on my findings on conserved TFR structural features, a quantitative assay of binding affinity determination was developed. Through sequence comparison and a residue contact map method, I discovered the existence of a conserved residue network that correlates well with the known allostery pathway of TetR. This predicted allosteric communication network was experimentally tested in TtgR. I have also developed methods to identify TFR operator sequences through genomic comparisons and validated my prediction through experiments. In addition, I have developed an in vivo system that can be used to identify and characterize proteins that mediate resistance to almost any antibiotic. This system is simple, fast, and scalable for high-throughput applications, and could be used to discover a wide range of novel antibiotic resistance mechanisms. The principles that I applied to the TFR family could also be applied to other protein families.

The worldwide increase in antibiotic resistance is a concern for public health. When the appropriate antibiotic dosage is determined, the priorities are efficacy and toxicity. The aim of this thesis was to gain knowledge about the most efficient dosing regimens in order to minimize the emergence and selection of antibiotic-resistant mutants. We also wanted to assess the impact of antibiotic selective pressure and host to host transmission for the dissemination of resistance. Escherichia coli bacteria with different levels of cefotaxime susceptibility were competed in an in vitro kinetic model, demonstrating a complex selection of low-level resistance influenced e.g. by the time duration of selective concentrations and the rise of new mutants. We also constructed a mathematical model incorporating biologically relevant parameters and showed its usefulness when assessing the risks of resistance development. When E. coli populations with pre-existing fluoroquinolone-resistant mutants were exposed to simulated serum concentrations, several currently used doses of fluoroquinolones clearly enhanced the development and selection of resistance. The mutant prevention concentration (MPC) was measured for several E. coli isolates with different fluoroquinolone susceptibilities, and because of fluctuating antibiotic concentrations in the human body, the pharmacokinetics was considered when evaluating MPC. Results indicate that the area under the serum concentration time curve in relation to the MPC may be a useful predictor for emergence of resistance. In the commensal flora of healthy human couples we noted a high frequency of trimethoprim-resistant E. coli. There was also an extensive sharing and transmission of E. coli clones. Treating the female with trimethoprim reduced the number of intestinal E. coli which might have facilitated the transmission from the male partner. These findings suggest that the rate of transmission is high and effectively contributes to the spread of both susceptible and antibiotic-resistant E. coli in intrafamilial settings.

Normally present in water, soil and waste water, Acinetobacter baumannii has become an important nosocomial pathogen, as causal agent of pneumonias, septicemias and urinary tract infections, among other complications in compromised patients from hospital’s intensive care units. One of its last acquired abilities is the resistance to colistin (polymixin E), the last therapeutic option for its infections. In this thesis, descriptive and quantitative differential expression proteomics is used in the study of acquired colistin resistance. As result of this research, 1,097 proteins belonging to the Acinetobacter genus have been identified by combined application of bidimensional gel electrophoresis (2DE), differential gel electrophoresis (DIGE), and peptide labeling with stable isobaric isotopes tags (iTRAQ). Analyses have been performed on the global expressed proteome of a reference, colistin-sensible strain (A. baumannii ATCC 19606) and, for comparative purposes, on a derived strain on which colistin resistance has been induced in vitro. The resistant phenotype shows reduced fitness, with significant differences in expression found in outer membrane proteins, membrane active transporters, diverse metabolic enzymes (fatty acids, citrate, phenylacetate, piruvate and nitrogen), proteins involved in stress response and biofilm formation, as well as in protein synthesis and folding pathways. The work has allowed to assess the strengths and weaknesses of the different techniques currently used in this type of proteomic analysis. / Acinetobacter baumannii, normalmente aislado en suelos y aguas (corrientes o residuales), se ha convertido en importante patógeno nosocomial, siendo agente causal de, entre otras complicaciones, neumonías, septicemias e infecciones del tracto urinario de pacientes comprometidos en unidades hospitalarias de cuidados intensivos. La más reciente de sus capacidades adquiridas es la resistencia a colistina (polimixina E), antibiótico peptídico considerado la última opción terapéutica en contextos clínicos. Esta tesis doctoral emplea la proteómica descriptiva y de expresión diferencial cuantitativa para investigar la resistencia adquirida por A. baumannii a dicho antibiótico. Los resultados han supuesto la identificación de 1.097 proteínas de Acinetobacter mediante el empleo combinado de electroforesis bidimensional convencional (2DE), 2DE diferencial (DIGE) y marcaje peptídico mediante isótopos isobáricos estables (iTRAQ). Los análisis se han realizado en el proteoma expresado por una cepa de referencia sensible a colistina (A. baumannii ATCC 19606), así como en una cepa derivada de ésta en la que se ha inducido, a efectos comparativos, resistencia a colistina in vitro. El fenotipo resistente manifestó reducida adaptabilidad biológica, encontrándose las principales diferencias en la estructura de la membrana externa, en la expresión de transportadores activos de membrana, en diversos enzimas metabólicos (ácidos grasos, citrato, fenilacetato, piruvato, nitrógeno) y de respuesta a condiciones de estrés, así como en la expresión de proteínas participantes en la formación de biopelículas y en el proceso de síntesis y plegamiento de proteínas. Además, el trabajo ha permitido evaluar los puntos fuertes y débiles de las técnicas empleadas actualmente en este tipo de análisis proteómicos.

The widespread use of β-lactams has undermined their effectiveness as chemotherapeutic agents by fueling the evolution and dissemination of multiple resistance mechanisms, including: (1) production of hydrolytic β-lactamase enzymes that inactivate β-­lactams, (2) expression of PBPs with low-affinity for β-­lactams and (3) overexpression of multidrug efflux pumps which actively expunge β-­lactams and other toxic substances. The overall goal of this thesis is the structural characterization of bacterial proteins involved in regulating β-lactam resistance. The notorious resistance of Staphylococcus aureus primarily stems from the production of β-lactamases and PBP2a, a low-affinity PBP which confers broad-spectrum β-­lactam resistance in methicillin-resistant S. aureus (MRSA) strains. Expression of these resistance determinants is controlled by a β-­lactam-inducible transmembrane receptor (BlaR1/MecR1) and repressor (BlaI/MecI). This dissertation presents the crystal structure of the BlaR1 sensor domain (BlaRs) from S. aureus, determined in its apo form and acylated with penicillin G. These structures reveal that acylation by β-lactams is not accompanied by a BlaRs conformational change. It is also shown that mutation of the BlaR1 L2 loop prevents induction of β-­lactamase expression in vivo, supporting that the L2 loop plays an important role in signal transduction. The intrinsic resistance of Pseudomonas aeruginosa to a variety of antibiotics (including β-lactams) is exacerbated in mutant strains that overexpress multidrug efflux pumps such as MexAB-OprM. Production of MexAB-OprM is controlled by the MarR family repressor, MexR, and several hyper-resistant strains of P. aeruginosa appear to involve mutations in either MexR or additional regulatory factors upstream of MexR. The allosteric effectors of MarR proteins are typically small lipophenolic compounds. This dissertation confirms that MexR is uniquely modulated by the 53 residue protein, ArmR. Electromobility gel shift assays and isothermal titration calorimetry demonstrate that a direct MexR-ArmR interaction is responsible for neutralizing the affinity of MexR for its DNA operator. The allosteric conformational change induced by ArmR-binding was assessed by determining the crystal structure of MexR double mutant Q106L/A110L (MexRLL) in complex with ArmR residues 29-53 (ArmRC). This structure shows that ArmR induces a dramatic conformational change which repositions the MexR DNA-binding lobes into an orientation that is incompatible with binding DNA.