Atividades antropogênicas relacionadas ao uso massivo de antibacterianos tem favorecido para que ambientes aquáticos sejam importantes locais para seleção e/ou disseminação de bactérias multirresistentes (MR). O objetivo do estudo foi monitorar a ocorrência de bactérias MRs em ambientes aquáticos públicos no estado de SP, caracterizando genótipos de resistência adquirida. Foram analisados 50 isolados de bactérias gram-negativas, recuperadas de amostras de água, sendo que 70% dos isolados apresentaram perfil de MR, identificando-se os genes blaCTX-M-2 (n= 5), blaCTX-M-9 (n= 1), blaCTX-M-15 (n= 2), blaKPC-2 (n= 3), qnrB (n= 1), oqxA (n= 3), oqxB (n= 3) e, aac(6)1b-cr (n= 7). Destaca-se a primeira detecção de A. calcoaceticus produtora de KPC-2. Ambientes aquáticos de acesso público podem ser importantes fontes para a disseminação e/ou transmissão de uma ampla variedade de espécies bacterianas MRs tanto para seres humanos como para ecossistemas associados, sendo que a presença de genótipos endêmicos sugere contaminação por esgoto doméstico e/ou hospitalar. / Anthropogenic activities related to the massive use of antibacterial has contributed to the selection and spread of multidrug-resistant (MDR) into the aquatic environment. This study aimed to monitor the occurrence of MDR bacteria in public aquatic environments, in the state of São Paulo, characterizing genotypes of acquired resistance. Of the 50 gram-negative isolates, recovered of water samples, 70% exhibited a MDR profile. Indeed, blaCTX-M-2 (n= 5), blaCTX-M-9 (n= 1), blaCTX-M-15 (n= 2)-, blaKPC-2 (n= 3)-, qnrB (n= 1)-, oqxA (n= 3)-, oqxB (n= 3)- and aac(6)-1b-cr (n = 7) genes were identified. Noteworthy is the first the detection of KPC-2-producing Acinetobacter calcoaceticus. Aquatic environments, with public access, may be important sources for the dissemination and/or transmission of a wide variety of MDR bacterial species to both humans and associated ecosystems. Moreover, the presence of endemic genotypes suggests contamination by domestic and/or hospital sewage.

Introdução: Bactérias do gênero Aeromonas estão presentes em ambientes de água doce, salgada e salobra. O isolamento destes microrganismos já foi relatado em água de abastecimento público e alimentos. Algumas espécies podem ser patogênicas ao homem, causando gastrenterites e outras infecções. Isolados de Aeromonas de fontes diversas expressam resistência a antimicrobianos, especialmente a -lactâmicos, devido à presença de enzimas -lactamases. A patogenicidade das espécies se deve à virulência multifatorial, que compreende a produção de enterotoxinas (Act, Alt e Ast), de elastase, presença de flagelo, entre outros. Objetivo: Isolar, identificar e quantificar Aeromonas hydrophila isoladas de esgoto e lodo tratado; pesquisar a ocorrência dos genes de virulência e resistência a -lactâmicos. Material e Métodos: A detecção e quantificação de Aeromonas hydrophila foram realizadas por meio da técnica de membrana filtrante e meio de cultura específico; a identificação foi realizada por meio da PCR utilizando um par de primers específicos para a espécie. Após a confirmação da espécie foi realizado o antibiograma para conhecer o perfil de resistência aos antibióticos; a pesquisa dos genes de virulência act, alt, ast, ela, lip e fla e genes de resistência a -lactâmicos foi realizada por meio da PCR e seqüenciamento. Resultados: Foram analisadas 15 amostras (seis de esgoto tratado e nove de lodo tratado). Destas, somente nove foram positivas para A. hydrophila, obtendo-se 441 colônias típicas, das quais 348 foram positivas, por PCR para identificação do gênero e 209 para identificação da espécie. Os 209 isolados, sendo 92 do esgoto tratado e 117 do lodo tratado, apresentaram os seguintes valores na pesquisa dos genes de virulência: 36 por cento (act), 40 por cento (ast), 78 por cento (alt), 82 por cento (fla), 86 por cento (lip) e 87 por cento (ela) e 100 por cento dos isolados apresentaram pelo menos um dos genes. Para os testes de sensibilidade aos antibióticos todos os isolados foram resistentes a pelo menos um dos antibióticos. A produção de enzimas MBL, ESBL e AmpC foi detectada em isolados. Também foram encontrados genes de resistência cphA, bla TEM e bla MOX, enquanto que os genes bla VIM, bla IMP, bla e bla não foram detectados. Conclusão: Os resultados sugerem que A. hydrophila pode resistir ao processo de tratamento de esgoto e lodo, além disso, pode apresentar diversos genes de virulência e resistência a antibióticos, motivos pelos quais A. hydrophila pode ser uma ameaça a Saúde Pública, uma vez que estas amostras são reutilizadas para fins urbanos ou agrícolas / Introduction: Bacteria of the genus Aeromonas are present in fresh, brackish and salty waters. The isolation of these microorganisms has been reported in public water supplies and foods. Some species can be pathogenic to humans, causing gastroenteritis and other infections. Aeromonas isolates from different sources express resistance to antimicrobials, especially -lactams, due to the presence of lactamase enzymes. The pathogenicity of the species is due to the multifactorial virulence, wich includes the production of enterotoxins (Act, Alt and Ast) of Elastase and presense of flagello, among others. Objectives: Identify and quantify Aeromonas hydrophila isolated from treated wastewater and sludge, to investigate the occurrence of virulence genes and resistance to -lactams. Material and methods: The detection and quantification of A. hydrophila were made through the membrane filter technique and specific culture medium, the identification was performed by PCR using a pair of primers specific for the species. After confirming the species sensitivity was performed to know the profile of antibiotic resistance, the survey of virulence genes act, alt, ast, ela, lip, fla and resistance to -lactams gene was performed by PCR and sequencing. Results: We analyzed 15 samples (six of nine treated wastewater and sludge). Of these only nine were positive for A. hydrophila, resulting in 441 typical colonies, of wich 348 were positive by PCR to identify the genus and 209 for species identification. The 209 isolates, being 92 and 117 of treated wastewater and treated sludge showed the following values in the study of the virulence genes: 36 per cent (act), 78 per cent (alt), 82 per cent (fla), 86 per cent (lip), 87 per cent (ela) and 100 per cent of the isolates had at least one of the genes. For antibiotic susceptibility testing all isolates were resistant to at least one antibiotic. The production of MBL, ESBL and AmpC enzyme was detected in isolates. It was also found resistance genes cphA, bla TEM and bla MOX, while genes bla VIM , bla IMP , bla and bla FOX CTX-M were not detected. Conclusion: The results suggest that A. hydrophila can resist the process of treating of wastewater and sludge, moreover, may have different virulence genes and antibiotic resistance, which is why A. hydrophila can be a threat to public health, since these samples are reused for agricultural or urban purposes. , bla SHV

Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this work, a summary is provided of our current understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria (Chapter 1). The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities. Although this thesis primarily explores structure and function, it also elucidates the hidden regulatory mechanism (post-translational) behind the association of a small protein called AcrZ with the tripartite complex AcrAB/TolC, in connection with the lipid environment, and the resulting changes in the latter's functionality (Chapter 2). A regulatory role of the native membrane lipid environment as well as of small proteins for efflux pump activity have previously been hypothesised. I present the first example of a function-regulating role of the lipid cardiolipin in combination with a small protein binding partner (AcrZ) for the substrate selectivity and transport activity of an efflux pump protein (AcrB). This regulation happens through induced structural changes which have remained unseen so far. Alongside with these results, a nanodisc reconstitution method was experimentally adapted for a structure-function investigation of an efflux pump (complex) using cryo-EM (Chapter 2). Beyond some fundamental regulatory insights, hidden intrinsic transport mechanisms for some transporters have also remained to be explored and studied. The discovery of a mechanism for active influx by a prominent efflux pump model system (Chapter 3) provides hope that this phenomenon is more common amongst multidrug transporters and that it could be utilised for drug discovery purposes. This novel feature explains the contradictory findings on this transporter in the past and raises new questions about the little-known physiological role and evolution of efflux pumps. The development and evolution of antimicrobial resistance has frequently shown to be a multifactorial and fast-moving process. One of these factors is the evolution of pumps itself towards an altered functionality (e.g. towards a broader or altered substrate spectrum or higher efflux rates). Against this background, the role of key carboxylate residues for efflux-energising proton trafficking was investigated for a prominent study model of a secondary-active transporter (Chapter 4). The re-allocation and/or addition of acidic residues was demonstrated to result in the preservation of wild type activity or the generation of hyper-efflux activity, respectively. These findings suggest that rapid emergence of antimicrobial resistance could be enhanced by the 'plasticity' in the location of key carboxylate residues with a role in proton coupling. It also demonstrates the necessity of antimicrobial drug design programmes to anticipate possible trajectories of an adaptive evolution of efflux pump. The 'cryo-EM revolution' has boosted the pace at which new structural and functional insights into multidrug efflux pumps are gained. Nevertheless, in order to derive the structure of individual pump components or of a full assembly, it is sometimes necessary to identify and characterise homologues and mutants, which would allow the application of cryo-EM for obtaining near-atomic maps. Functional analyses presented in this work helped to characterise a homologue and mutants of the MacAB/TolC tripartite complex to justify the obtained protein structures and strategies for further functional characterisation (Chapter 5). Given (1) the unusual stoichiometry of a MacB dimer in complex with a hexameric membrane-fusion protein (MacA), which leads to a seeming leakiness of the assembly, and (2) the fact that substrate has to pass through a narrow aperture in the membrane-fusion protein for extrusion, it is rather surprising that MacB was previously shown to transport an entire toxin. An experimental approach was developed that could enable the structure determination of a toxin-bound full assembly of MacAB/TolC (Chapter 5). Finally, the role of multidrug efflux pumps for the evolution of multidrug resistance is yet to be studied and better explored. For instance, evolutionary trajectories of pump overexpression, as compared to those of regular expression or no expression, are unknown yet could have the potential to reveal useful insights for spread prevention and drug design. The outline of an experimental design with some preliminary validating data is presented in Chapter 6.

Instead of planktonic growth in nature, many species of bacteria form biofilm to survive in harsh conditions. Although many chronic bacterial infections are caused by bacterial species in a biofilm lifestyle, previous research has focused on studying antibiotic resistance in planktonic growth. Here we used a modified MBEC assay, i.e. biofilm growth on pegs, to determine Escherichia coli biofilm inhibitory concentrations (BIC) of ciprofloxacin, streptomycin and rifampicin and to study the minimal selective concentration (MSC) for ciprofloxacin in E. coli biofilm. We could observe high inhibitory concentrations for all antibiotics in the biofilm pre-formed in media without antibiotics compared to the biofilm formed in antibiotics. We also show preliminary result indicating that sublethal concentrations of ciprofloxacin lead to the selection of ciprofloxacin resistant mutants in biofilm and that the selection level is lower than what was observed in planktonic growing E. coli. With more knowledge in how the biofilm formation precedes in different antibiotic settings, the treatment for chronic biofilm infections used today could be evaluated and changed so that the infections could be eradicated.

Dans une perspective comparative entre la France et les États-Unis, ce travail analyse le processus de publicisation des liens entre l’antibiorésistance et l’alimentation, ainsi que ses implications en termes de contribution au développement de la production appelée sans antibiotique et de l’approche One Health. En partant de la prise de conscience des conséquences de l’usage des antibiotiques dans l’élevage, la recherche s’inscrit dans une réflexion pragmatiste de constitution des problèmes publics et s’appuie sur un corpus hybride composé de documents publiés entre 1980 et 2016 (presse écrite, littérature institutionnelle et entretiens semi-directifs). La méthode développée s’enrichit des outils de textométrie issus de l’analyse de discours et s’intéresse à l’émergence des dénominations et des formules qui nomment le problème, ses causes et ses solutions. La comparaison montre que le processus de publicisation de liens entre l’antibiorésistance et l’alimentation dévoile une trajectoire opposée dans les deux pays. Dans le cas français, ce processus s’inscrit dans un schéma top-down et se caractérise par une publicisation tardive faisant suite aux démarches des instances sanitaires européennes et internationales. L’appropriation du problème par des associations de consommateurs, ainsi que l’investissement des acteurs agroalimentaires dans le développement de la production sans antibiotique, n’émergent que récemment. En revanche, aux États-Unis, ce processus s’inscrit dans un modèle bottom-up suite à la constitution d’un public d’organisations non gouvernementales autour du problème. Leur mobilisation a contribué significativement au développement de programmes d’élevage sans antibiotique ainsi qu’à la mise à l’agenda gouvernemental du problème et le lancement d’un plan national dans une approche One Health. / In a cross-country perspective between France and the United States, this research analyses the process of publicizing the links between antibiotic resistance and food, as well as its contribution to the development of the antibiotic free production and the implementation of the One Health approach. Starting with the awareness of the antibiotic use in livestock consequences, the study relies on the pragmatist approach of the constitution of the public problems. It is based on wide corpora composed by documents published between 1980 and 2016 (written press, institutional literature and semi-directive interviews). The analysis method uses textometric tools derived from discourse analysis and focuses on the emergence of formulas that name the problem, its causes and its solutions. The comparison uncovers an opposite process between the two countries. In France, this process is part of a top-down approach and is characterized by a late publicization following the European and international health authorities’ initiatives. The consumer associations taking over the problem, as well as the agri-food actors’ commitment to the antibiotic free production, is very recent. In the United States, this process reveals a bottom-up model following a non-governmental organizations public constitution taking over the problem. Their mobilization has contributed to the development of the antibiotic free breeding programs, as well as to place the problem on the government agenda that launched a national plan in a One Health approach.

Chez les bactéries à Gram-négatif, l’imperméabilité de la membrane externe est un facteur majeur contribuant au développement de la résistance. Chez Escherichia coli, les porines OmpF et OmpC sont des protéines de la membrane externe qui forment des canaux pour la diffusion de petites molécules hydrophiles tels que les antibiotiques. L’expression des porines est soumise à une régulation fine, et des petits ARN non-codants (sRNAs, small RNAs) jouent un rôle important au niveau post-transcriptionnel. Dans ce cadre, et en utilisant E. coli comme bactérie modèle, les objectifs de mon travail de thèse étaient : (1) de caractériser la régulation du sRNA MicC et la co-régulation putative de la porine quiescente OmpN; (2) d’examiner l'effet global de MicC sur le transcriptome; (3) d’analyser l'impact de l'expression de MicC sur la sensibilité aux antibiotiques. Les résultats obtenus montrent l’induction de MicC en présence d'antibiotiques de la famille des β-lactamines, ou en l’absence du facteur sigma de réponse au stress de l’enveloppe sigmaE. Ces mêmes conditions activent aussi l'activité d'une fusion ompN-lacZ, indiquant une régulation transcriptionnelle commune de micC et ompN. Etant donnée la conservation de MicC chez les entérobactéries, nous avons effectué une étude par RNASeq pour déterminer l'impact de la surexpression de MicC sur le transcriptome d’E. coli et identifié 60 ARNm régulés par MicC en plus de sa cible initiale ompC. L'identification des spectres cibles globaux des sRNAs est importante pour comprendre leur importance dans la physiologie bactérienne, ici celui de MicC dans la résistance aux antibiotiques. / A major factor contributing to antimicrobial resistance is the inability of antibiotics to penetrate the bacterial outer membrane to reach their target. In Escherichia coli, the two abundantly expressed porins OmpF and OmpC form channels for diffusion of small hydrophilic molecules including antibiotics. The expression of porins is under complex regulation and the small regulatory RNAs (sRNAs) fine tune the porin expression level at post-transcriptional level. MicF and MicC are the two major sRNAs that negatively regulate expression of OmpF and OmpC, respectively. Interestingly, these two sRNAs are encoded next to porin gene, i.e. micF-ompC and micC-ompN, suggesting a dual regulation. Our goals in this work were: (1) to characterize the regulation of the sRNA MicC and the putative co-regulation of the quiescent porin OmpN in E. coli; (2) to examine the global effect of MicC on the E. coli transcriptome; (3) to analyze the impact of MicC expression on antibiotic susceptibility. Our work shows that the expression of micC was increased in the presence of carbapenems and cephalosporins and in an rpoE depleted mutant. The same conditions enhanced the expression of OmpN, suggesting a dual regulation of micC and ompN. We also performed RNA sequencing to determine the impact of MicC overexpression on E. coli transcriptome. This identified 60 mRNA targets negatively regulated by MicC apart from its original target. Identification of the global target spectra of MicC is of importance to understand its importance on the overall bacterial physiology, and more specifically on AMR.

<p>Antibiotic treatment causes an ecological disturbance on the human microflora. Four commensal bacteria: E. coli, enterococci, a-streptococci and coagulase-negative staphylococci, from patients with extensive, high antibiotic usage were investigated with regard to resistance pattern and mutation frequency. Among 193 investigated strains it was found that high antibiotic usage selected for resistant bacteria and enriched for bacteria with a small but significantly increased mutation frequency. </p><p>The relative biological fitness cost of resistance in <i>Staphylococcus epidermidis</i> was assessed in a human in vivo model where the indigenous flora was present. In vitro data of the bacterial growth rate correlated well to in vivo fitness assayed in the competition experiments on skin. </p><p>An in vitro kinetic model was shown to be a useful tool to establish the pharmacokinetic and pharmacodynamic (PK/PD) indices for efficacy of antibiotics. It was confirmed that the time, when the concentration exceeds the minimal inhibitory concentration (MIC), correlates with efficacy for b-lactam antibiotics. To achieve maximal killing for penicillin-resistant pneumococci, with an MIC of 2 mg/L, the peak concentration was also of importance. </p><p>Suboptimal dosing regimen facilitates selection of resistance. Penicillin-resistant pneumococci were easily selected in a mixed population with penicillin-sensitive, -intermediate and -resistant pneumococci in an in vitro kinetic model. The selection of the resistant strain was prevented when the benzylpenicillin concentration exceeded the MIC for approximately 50% of 24 h.</p>

<p>Antibiotic resistance is of current concern. Bacteria have become increasingly resistant to commonly used antibiotics and we are facing a growing resistance problem. The present thesis was aimed at studying the impact of antibiotic treatment on pathogenic bacteria as well as on the normal human microbiota, with focus on resistance development.</p><p>Among the factors that affect the appearance of acquired antibiotic resistance, the mutation frequency and biological cost of resistance are of special importance. Our work shows that the mutation frequency in clinical isolates of <i>Helicobacter pylori</i> was generally higher than for other studied bacteria such as <i>Enterobacteriaceae; </i>¼ of the isolates displayed a mutation frequency higher than<i> Enterobacteriaceae </i>defective<i> </i>mismatch repair mutants and could be regarded as mutator strains.</p><p>In <i>H. pylori</i>, clarithromycin resistance confers a biological cost, as measured by decreased competitive ability of the resistant mutants in mice. In clinical isolates, this cost could be reduced, consistent with compensatory evolution stabilizing the presence of the resistant phenotype in the population. Thus, compensation is a clinically relevant phenomenon that can occur in vivo.</p><p>Furthermore, our results show that clinical use of antibiotics selects for stable resistance in the human microbiota. This is important for several reasons. First, many commensals occasionally can cause severe disease, even though they are part of the normal microbiota. Therefore, stably resistant populations increase the risk of unsuccessful treatment of such infections. Second, resistance in the normal microbiota might contribute to increased resistance development among pathogens by interspecies transfer of resistant determinants.</p>

Antibiotic resistance is of current concern. Bacteria have become increasingly resistant to commonly used antibiotics and we are facing a growing resistance problem. The present thesis was aimed at studying the impact of antibiotic treatment on pathogenic bacteria as well as on the normal human microbiota, with focus on resistance development. Among the factors that affect the appearance of acquired antibiotic resistance, the mutation frequency and biological cost of resistance are of special importance. Our work shows that the mutation frequency in clinical isolates of Helicobacter pylori was generally higher than for other studied bacteria such as Enterobacteriaceae; ¼ of the isolates displayed a mutation frequency higher than Enterobacteriaceae defective mismatch repair mutants and could be regarded as mutator strains. In H. pylori, clarithromycin resistance confers a biological cost, as measured by decreased competitive ability of the resistant mutants in mice. In clinical isolates, this cost could be reduced, consistent with compensatory evolution stabilizing the presence of the resistant phenotype in the population. Thus, compensation is a clinically relevant phenomenon that can occur in vivo. Furthermore, our results show that clinical use of antibiotics selects for stable resistance in the human microbiota. This is important for several reasons. First, many commensals occasionally can cause severe disease, even though they are part of the normal microbiota. Therefore, stably resistant populations increase the risk of unsuccessful treatment of such infections. Second, resistance in the normal microbiota might contribute to increased resistance development among pathogens by interspecies transfer of resistant determinants.

Pathogenic organisms are transmitted to the host organism through all possible connected pathways, and cause a myriad of diseases states. Commonly occurring curable infectious diseases still impose the greatest health impacts on a worldwide perspective. The Bill &amp; Melinda Gates Foundation partnered with RAND Corporation to form the Global Health Diagnostics Forum, with the goal of establishing and interpreting mathematical models for what effects a newly introduced point-of-care pathogen diagnostic would have in developing countries. The results were astonishing, with potentially millions of lives to be saved on an annual basis. Golden standard for diagnostics of pathogenic bacteria has long been cultureable medias. Environmental biologists have estimated that less than 1% of all bacteria are cultureable. Genomic-based approaches offer the potential to identify all microbes from all the biological kingdoms. Nucleic acid based pathogen diagnostics has evolved significantly over the past decades. Novel technologies offer increased potential in sensitivity, specificity, decreased costs and parallel sample management. However, most methods are confined to core laboratory facilities. To construct an ultimate nucleic acid based diagnostic for use in areas of need, potential frontline techniques need to be identified and combined. The research focus of this doctoral thesis work has been to develop and apply nucleic acid based methods for pathogen diagnostics. Methods and assays were applied to the two distinct systems i) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae, and ii) genotype determination of the cancer causative Human Papillomavirus (HPV). The first part of the study included development of rapid, direct and multiplex Pyrosequencing nucleic acid screenings. With improved methodology in the sample preparation process, we could detect an existence of multiple co-infecting HPV genotypes at greater sensitivities than previously described, when using the same type of methodology. The second part of the study focused on multiplex nucleic acid amplification strategies using Molecular Inversion Probes with end-step Pyrosequencing screening. The PathogenMip assay presents a complete detection schematic for virtually any known pathogenic organism. We also introduce the novel Connector Inversion Probe, a padlock probe capable of complete gap-fill reactions for multiplex nucleic acid amplifications. / Patogena organismer smittas till värd organismen genom alla möjliga kontaktnätverk och skapar en mångfald olika sjukdomstillstånd. Dock är det fortfarande vanligt förekommande behandlingsbara infektiösa sjukdomar som orsakar den största hälsoförlusten, sett från ett globalt perspektiv. Bill och Melinda Gates Stiftelsen samarbetade med RAND kooperation för att forma “The Global Health Diagnostics Forum”. Deras mål var att etablera och analysera matematiska modeller för vilka effekter en ny diagnostisk metod utrustat för fältarbete skulle ha i utvecklingsländer. Resultaten var häpnadsveckande, med potentiellt miljoner av liv som skulle kunna räddas på en årlig basis. Den etablerade standarden för diagnostik av patogena bakterier har länge varit kultiveringsmedia baserad. Miljö specialiserade biologer har estimerat att mindre än 1 % av alla bakterie arter går att kultivera. Dock erbjuder genetiska analyser potentialen att kunna identifiera alla mikrober från alla de biologiska rikena. Nukleinsyrebaserade diagnostiska metoder har märkbart förbättrats över de senaste årtionden. Nya tekniker erbjuder utökad sensitivitet, selektivitet, sänkta kostnader och parallella analyser av patient prover. Dock är de flesta metoderna begränsade till standardiserade laboratoriemiljöer. För att konstruera en väl fungerande diagnostisk fältutrustning för användning i problem områden, behöver världsledande tekniker identifieras och kombineras. Fokuseringsområdet för denna doktorsavhandling har varit att utveckla och utföra nukleinsyrebaserade metoder för patogen diagnostik. Metoder och experimentella utförande applicerades på två distinkta system i) sökning av antibiotika resistens relaterade mutationer i den patogena bakterien Neisseria gonorrhoeae och ii) genotypning av det cancer orsakande Humana Papillomaviruset (HPV). Den första delen av studien inriktade sig mot utveckling av snabba, direkta och multiplexa Pyrosekvenserings baserade nukleinsyreanalyser. Med förbättrad provprepareringsmetodologi kunde vi detektera multipla HPV infektioner med högre sensitivitet än vad tidigare beskrivits med liknande metodologi. Den andra delen av studien fokuserades på multiplexa nukleinsyre amplifikationer med “Molecular Inversion Probe” tekniken med sista steg Pyrosekvenserings analys. “PathogenMip assay” erbjuder ett komplett detektionsprotokoll för alla kända patogena organismer. Vi introducerar även den nya “Connector Inversion Probe”, en “Padlock Probe” kapabel att genomföra kompletta gap fyllningar för multiplex nukleinsyre amplifiering. / QC 20100624