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

Negli ultimi decenni l'utilizzo degli antibiotici a scopo terapeutico o come promotori della crescita nell'allevamento animale ha portato alla comparsa e alla diffusione di microrganismi resistenti. In questo contesto, la presenza di Lattobacilli (LAB) antibiotico resistenti non rappresentano di per sé un rischio clinico. Tuttavia la possibilità che essi ma possono essere veicolo di geni codificanti l'antibiotico-resistenza verso batteri patogeni presenti negli alimenti o nel tratto gastro-intestinale umano (inclusi enterococchi, streptococchi e listeria), costituisce un possibile rischio per la salute umana che deve essere attentamente valutato. Obiettivo di questo lavoro è stato quello di valutare attraverso metodi di indagine fenotipica con le tecniche delle microdiluizioni in brodo, Etest e disc-diffusion, i livelli di antibiotico resistenza per le specie S. thermophilus e L. plantarum verso gli antibiotici tetraciclina, eritromicina, clindamicina, streptomicina, gentamicina, ampicillina. Ceppi atipici appartenenti alla specie S. thermophilus sono stati sottoposti ad analisi genetiche con lo scopo di caratterizzare e localizzare i geni responsabili della resistenza. E' stato inoltre testato il possibile trasferimento orizzontale dei geni di antibiotico resistenza nativi da S. thermophilus verso i batteri Gram-positivi E. faecalis e Listeria monocytogenes. In alcuni ceppi di S. thermophilus resistenti si sono infine osservati e studiati particolari caratteri fenotipici ( fitness ) correlati alla presenza delle determinanti genetiche di antibiotico resistenza nell'ospite batterico. / In the last decades, the use of antibiotics in human therapy or in animal husbandry as growth promoters has induced the development and the diffusion in antibiotic resistant micro-organisms. In this context antibiotic resistant Lactic Acid Bacteria (LAB) do not represent a clinical risk in themselves. However, the possibility that S. thermophilus cultures might transfer antibiotic resistance genes to pathogenic species either present in food or in the gastrointestinal tract (including enterococci, streptococci and listeria) represents a potential clinical risk that needs to be carefully evaluated. The aim of this study was to evaluate by means of phenotypic methods (microdilution, E-test, disc-diffusion) the levels of antibiotic resistance for S. thermophilus and L. plantarum species against the antibiotic tetracycline, erythromycin, clyndamicin, streptomycin, gentamycin and ampicillin. The atypical resistant S. thermophilus strains were subjected to genetic analyses in order to characterise and to localise the antibiotic resistance determinants. Furthermore the ability of the resistant S. thermophilus strains in transferring the antibiotic resistant determinant was assessed in mating experiments using as recipients the Gram-positive bacteria E. faecalis and Listeria monocytogenes. In same resistant S. thermophilus strains, special bacterial fitness related with the presence of the antibiotic resistance determinants in the bacterial hosts were observed and studied.

La scoperta e il successivo uso di antibiotici hanno reso resistenti molte specie batteriche sia di origine animale sia umana. I geni di resistenza agli antibiotici possono essere trasferiti tramite la catena alimentare, a partire dagli animali e alimenti, fino al tratto gastrointestinale degli esseri umani. Il presente studio descrive la proprietà coniugativa di alcuni nuovi plasmidi, in particolare di uno identificato in un ceppo di Lactococcus lactis spp. lactis, isolato dall'intestino di pesce, e di altri plasmidi individuati in ceppi di Lactobacillus brevis, Lb. plantarum e Lb. reuteri, isolati da salame. La trasferibilità dei plasmidi che portano i geni di resistenza per l’eritromicina o tetraciclina è stata valutata con metodi di elettroporazione e coniugazione in vitro. Nello specifico è riportato il trasferimento di tali plasmidi a specie batteriche patogene per l’uomo come Listeria monocytogenes e Staphylococcus spp. e a un agente responsabile di Lactococcosi nei pesci come Lc. garvieae. Dopo lo studio sulle proprietà coniugative si è proceduto alla caratterizzazione di questi elementi extracromosomici con esperimenti di comobilizzazione e stabilità. I dati ottenuti suggeriscono come i LAB possano essere un serbatoio di diffusione dei geni per l’antibiotico resistenza, con gravi rischi per l’allevamento di prodotti ittici e salute umana. / The discovery and subsequent widespread use of antibiotics have rendered many bacterial species of human and animal origin resistant to some antibiotics. Antibiotic resistance gene may be transferred via food chain, from animals into fermented and other food or in the human gastrointestinal tract. The transferability of some plasmids that harbor the tetracycline or erythromycin resistance genes to animal and human pathogens was assessed using electrotrasformation and conjugation. The present study describes the proprieties of some new plasmids, originally isolated from fish intestinal Lactococcus lactis ssp. lactis and from fermented sausage Lactobacillus brevis, Lb. plantarum and Lb. reuteri. In particular, here I report the potentially of transferable antibiotic resistance determinants to human pathogenic bacterial like Listeria monocytogenes and Staphylococcus spp. and to an etiologic agent of Lactococcus infection like Lc. garvieae. The possibility of transferring natural Lactococcus and Lactobacillus plasmids into pathogenic bacterial strains involved the characterization of these elements, like comobilization and plasmid stability. These data suggest that lactic acid bacteria (LAB) might be reservoir organism for acquired resistance genes that can be spread both to fish and human pathogens, posing a risk to aquaculture and human health.

The persistence and propagation of bacterial antibiotic resistance presents significant challenges to the treatment of drug resistant bacteria with current antimicrobial chemotherapies, while a dearth in replacements for these drugs persists. The thiopeptide family of antibiotics may represent a potential source for new drugs and thiostrepton, the prototypical member of this antibiotic class, is the primary subject under study in this thesis. Using a facile semi-synthetic approach novel, regioselectively-modified thiostrepton derivatives with improved aqueous solubility were prepared. In vivo assessments found these derivatives to retain significant antibacterial ability which was determined by cell free assays to be due to the inhibition of protein synthesis. Moreover, structure-function studies for these derivatives highlighted structural elements of the thiostrepton molecule that are important for antibacterial activity. Organisms that produce thiostrepton become insensitive to the antibiotic by producing a resistance enzyme that transfers a methyl group from the co-factor S-adenosyl-L-methionine (AdoMet) to an adenosine residue at the thiostrepton binding site on 23S rRNA, thus preventing binding of the antibiotic. Extensive site-directed mutagenesis was performed on this enzyme to generate point mutations at key active site residues. Ensuing biochemical assays and co-factor binding studies on these variants identified amino acid residues in the active site that are essential to the formation of the AdoMet binding pocket and provided direct evidence for the involvement of an active site arginine in the catalytic mechanism of the enzyme. Certain bacteria that produce neither thiostrepton nor the resistance methyltransferase express the thiostrepton binding proteins TIP-AL and TIP-AS, that irreversibly bind to the antibiotic, thereby conferring resistance by sequestration. Here, it was found that the point mutation of the previously identified reactive amino acid in TIP-AS did not affect covalent binding to the antibiotic, which was immediately suggestive of a specific, high affinity non-covalent interaction. This was confirmed in binding studies using chemically synthesized thiostrepton derivatives. These studies further revealed structural features from thiostrepton important in this non-covalent interaction. Together, these results indicate that thiostrepton binding by TIP-AS begins with a specific non-covalent interaction, which is necessary to properly orient the thiostrepton molecule for covalent binding to the protein. Finally, the synthesis of a novel AdoMet analogue is reported. The methyl group of AdoMet was successfully replaced with a trifluoromethyl ketone moiety, however, the hydrated form (germinal diol) of this compound was found to predominate in solution. Nevertheless, the transfer of this trifluoroketone/ trifluoropropane diol group was demonstrated with the thiopurine methyltransferase.