La résistance aux fluoroquinolones (FQ) est le principal facteur d'aggravation du pronostic de la tuberculose multi-résistante. Il apparait donc essentiel de mieux comprendre le développement de la résistance aux FQ afin d'améliorer les outils permettant une détection précoce de cette résistance. Nous avons (i) évalué les performances du séquençage des gènes gyrA et gyrB dans la détection de la résistance aux FQ grâce à une étude prospective menée au CNR-MyRMA ; (ii) étudié l'histoire naturelle de l'émergence de la résistance aux FQ in vivo dans un modèle murin de tuberculose et (iii) identifié de nouveaux mécanismes de résistance aux FQ par génomique comparative. Nous avons montré que la méthode des proportions, désignée comme méthode de référence, n'est pas performante pour la détection des bas niveaux de résistance aux FQ et que ni les méthodes génotypiques ni les méthodes phénotypiques, ne permettent le diagnostic de la résistance hétérogène aux FQ. Une stratégie combinée reposant sur une détection phénotypique d'une proportion anormale de bactéries résistantes et une caractérisation génotypique de ces bactéries résistantes permettrait d'améliorer la détection de cette résistance hétérogène. Nous avons identifié des pistes pour de nouveaux mécanismes de résistance aux FQ. Il pourrait s'agir de mécanismes responsables d'une résistance de bas niveau facilitant la sélection d'une résistance de haut niveau due à une mutation dans les gènes codant l'ADN gyrase dans un deuxième temps. Cependant, leur implication dans la résistance aux FQ, ainsi que notre hypothèse quant au processus de sélection, reste à démontrer. / Fluoroquinolone (FQ) resistance is the main factor of worsened prognosis of multidrug resistant tuberculosis. Therefore to better understand the development of FQ resistance is essential in order to improve the tools for early detection of this resistance. We have (i) evaluated the performance of gyrA and gyrB sequencing in the detection of FQ resistance through a prospective study; (ii) studied the natural history of the emergence of FQ resistance in vivo using a murine model of tuberculosis; and (iii) identified tracks for new mechanisms of resistance to FQ by comparative genomics. We showed that the proportion method, designated as the reference method, is not effective in detecting low levels of FQ resistance and that, neither genotypic methods nor phenotypic methods, allow the diagnosis of FQ heterogeneous resistance. A combined strategy based on phenotypic detection of an abnormal proportion of resistant bacteria and genotypic characterization of these resistant bacteria would improve the detection of this heterogeneous resistance. We have identified hypotheses for new FQ resistance mechanisms. These new mechanisms could be responsible of a low-level resistance facilitating the selection of a high-level resistance due to mutations in genes encoding DNA gyrase in a second time. However, their involvement in FQ resistance and our assumption about the selection process remain to be demonstrated.

L'objectif était d'étudier l'histoire naturelle de la colonisation nasale par S. aureus, en particulier l'impact des antibiotiques sur l'acquisition et la persistance de S. aureus résistant (SARM) ou sensible à la méticilline (SASM). Ce travail s'est appuyé sur les données issues d'une cohorte prospective multicentrique incluant des patients hospitalisés dans des centres de médecine physique et réadaptation et non colonisés par S. aureus à l'admission. 1) Les facteurs de risque d'acquisition de SARM et de SASM ont été examinés à l'aide d'une analyse cas-cas-témoins nichée. L'utilisation de fluoroquinolones, le sexe masculin et une plus grande intensité des soins à l'admission étaient significativement associés à l'acquisition de SARM. L'aide à la toilette et l'utilisation d'un dispositif urinaire étaient significativement associées à l'acquisition de SASM. 2) Les facteurs influençant la perte de colonisation ont été examinés à l'aide de modèles de Cox à fragilité partagée. L'utilisation de fluoroquinolones et la présence d'une plaie positive pour une souche différente de celle du nez étaient significativement associées à la décolonisation de SASM. Le phénotype de résistance à la méticilline n'était pas associé à la durée de colonisation par S. aureus, les durées médianes estimées de colonisation par SARM et par SASM étant respectivement de 3 et 2 semaines. Ce travail offre un nouvel éclairage sur l'histoire naturelle de la colonisation nasale par S. aureus en discriminant les facteurs relatifs à la forme résistante et à la forme sensible de la bactérie. Ces résultats montrent un impact différentiel des fluoroquinolones sur l'acquisition et la persistance de SARM ou de SASM. / The objective was to study the natural history of nasal S. aureus colonization, particularly the impact of antibiotics on methicillin-resistant (MRSA) or methicillin-sensitive (MSSA) S. aureus acquisition and persistence. This work was based on data provided by a prospective multicenter cohort including patients hospitalized in long-term-care facilities and not colonized with S. aureus at admission. 1) Risk factors for MRSA or MSSA acquisition were investigated using a nested case-case-control analysis. Fluoroquinolone use, male sex and more intensive care at admission were significantly associated with MRSA acquisition. Body-washing assistance and use of a urination device were significantly associated with MSSA acquisition. 2) Factors influencing the time to loss of colonization were investigated using shared-frailty Cox models. Fluoroquinolone use and the presence of a wound positive for a non-nasal strain were significantly associated with MSSA decolonization. The methicillin resistance phenotype was not associated with S. aureus colonization duration, and estimated median time to MRSA or MSSA decolonization was 3 or 2 weeks respectively. This work provides new insights into the natural history of nasal S. aureus colonization discriminating factors for the methicillin-resistant or -sensitive phenotypes of the bacteria. These results strongly suggest a distinct impact of fluoroquinolones on MRSA or MSSA acquisition and persistence.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Os quimioterápicos antimicrobianos são considerados contaminantes emergentes, com a capacidade de criar resistência em bactérias. Têm sido o foco de inúmeras pesquisas relacionadas a impactos ambientais, mas no Brasil, as pesquisas sobre a sua ocorrência em ambientes aquáticos continentais e costeiros são escassas. A cidade de Ubatuba tem uma densidade demográfica cinco vezes maior que a média nacional, aumentada em até doze vezes no verão, pressionando ainda mais os ecossistemas da região. Os rios Acaraú, Lagoa-Tavares, Grande e Indaiá deságuam na baía de Ubatuba, comprometendo a qualidade das suas águas. Este trabalho investigou a contribuição da descarga desses rios para a ocorrência e distribuição de quimioterápicos antimicrobianos (fluoroquinolonas e sulfonamidas) na baía de Ubatuba. Foi analisado um total de 36 amostras de água superficial. As coletas foram realizadas no período seco/chuvoso de 2014/2015. Para a determinação e quantificação dos fármacos foi utilizada SPE como método de preparo de amostra e CLAE-MS/MS para a detecção e quantificação dos antimicrobianos estudados. As características físico-químicas pH, temperatura, salinidade, oxigênio dissolvido, potencial redox (ORP), além do Carbono Orgânico Dissolvido (COD) também foram determinados para caracterização das águas da região. A distribuição espaço-temporal dos fármacos e a possível associação com os demais dados investigados foi avaliada pela ferramenta quimiométrica CA, visando à extração da maior quantidade possível de informações. Os rios e, consequentemente a baía estavam impactados pelo despejo de esgoto doméstico e os seguintes quimioterápicos antimicrobianos foram encontrados: sulfametoxazol (SMX), sulfatiazol (STZ), sulfacloropiridiazina (SCP), sulfaquinoxalina (SQX) e norfloxacina (NOR). Observou-se uma variação espacial e temporal, nos perfis de contaminação revelados pela CA. / Antimicrobial chemotherapeutical agents are considered emerging contaminants capable of creating bacterial resistance. They have been the focus of numerous studies related to environmental impacts, but in Brazil, there is little research on their occurrence in continental and coastal aquatic environments. The city of Ubatuba has a population density five times higher than the national average, which may be increased up to twelve times during the summer, pushing further the region\'s ecosystems. The rivers Acaraú, Lagoa-Tavares, Grande and Indaiá flow into Ubatuba Bay, compromising the quality of its waters. This work investigated those rivers\' discharge contribution on the occurrence and distribution of antimicrobial chemotherapeutical agents (fluoroquinolones and sulfonamides) in Ubatuba Bay. Thirty-six samples of surface water were analyzed. The samples were withdrawn during the dry and rainy seasons of 2014 and 2015, respectively. For the determining and quantifying the antimicrobial chemotherapeutical agents, it was used SPE as a sample preparation method and HPLC?MS/MS for their detection and quantification. Physicochemical characteristics like pH, temperature, salinity, dissolved oxygen, and redox potential (ORP), as well as dissolved organic carbon (DOC) were also determined to characterize the waters of the region. The spatial-temporal distribution of the agents and their possible association with other investigated data was assessed by the chemometric tool CA, aiming at extracting the greatest possible amount of information. The rivers and, consequently, the bay were contaminated by domestic sewage discharges and the following antimicrobial chemotherapeutical agents were detected: sulfamethoxazole (SMX), sulfathiazole (STZ), sulfachloropyridiazine (SCP), sulfaquinoxaline (SQX), and norfloxacin (NOR). The contamination profiles revealed by the CA showed a spatial variation and a temporal one.

Os quimioterápicos antimicrobianos são considerados contaminantes emergentes, com a capacidade de criar resistência em bactérias. Têm sido o foco de inúmeras pesquisas relacionadas a impactos ambientais, mas no Brasil, as pesquisas sobre a sua ocorrência em ambientes aquáticos continentais e costeiros são escassas. A cidade de Ubatuba tem uma densidade demográfica cinco vezes maior que a média nacional, aumentada em até doze vezes no verão, pressionando ainda mais os ecossistemas da região. Os rios Acaraú, Lagoa-Tavares, Grande e Indaiá deságuam na baía de Ubatuba, comprometendo a qualidade das suas águas. Este trabalho investigou a contribuição da descarga desses rios para a ocorrência e distribuição de quimioterápicos antimicrobianos (fluoroquinolonas e sulfonamidas) na baía de Ubatuba. Foi analisado um total de 36 amostras de água superficial. As coletas foram realizadas no período seco/chuvoso de 2014/2015. Para a determinação e quantificação dos fármacos foi utilizada SPE como método de preparo de amostra e CLAE-MS/MS para a detecção e quantificação dos antimicrobianos estudados. As características físico-químicas pH, temperatura, salinidade, oxigênio dissolvido, potencial redox (ORP), além do Carbono Orgânico Dissolvido (COD) também foram determinados para caracterização das águas da região. A distribuição espaço-temporal dos fármacos e a possível associação com os demais dados investigados foi avaliada pela ferramenta quimiométrica CA, visando à extração da maior quantidade possível de informações. Os rios e, consequentemente a baía estavam impactados pelo despejo de esgoto doméstico e os seguintes quimioterápicos antimicrobianos foram encontrados: sulfametoxazol (SMX), sulfatiazol (STZ), sulfacloropiridiazina (SCP), sulfaquinoxalina (SQX) e norfloxacina (NOR). Observou-se uma variação espacial e temporal, nos perfis de contaminação revelados pela CA. / Antimicrobial chemotherapeutical agents are considered emerging contaminants capable of creating bacterial resistance. They have been the focus of numerous studies related to environmental impacts, but in Brazil, there is little research on their occurrence in continental and coastal aquatic environments. The city of Ubatuba has a population density five times higher than the national average, which may be increased up to twelve times during the summer, pushing further the region\'s ecosystems. The rivers Acaraú, Lagoa-Tavares, Grande and Indaiá flow into Ubatuba Bay, compromising the quality of its waters. This work investigated those rivers\' discharge contribution on the occurrence and distribution of antimicrobial chemotherapeutical agents (fluoroquinolones and sulfonamides) in Ubatuba Bay. Thirty-six samples of surface water were analyzed. The samples were withdrawn during the dry and rainy seasons of 2014 and 2015, respectively. For the determining and quantifying the antimicrobial chemotherapeutical agents, it was used SPE as a sample preparation method and HPLC?MS/MS for their detection and quantification. Physicochemical characteristics like pH, temperature, salinity, dissolved oxygen, and redox potential (ORP), as well as dissolved organic carbon (DOC) were also determined to characterize the waters of the region. The spatial-temporal distribution of the agents and their possible association with other investigated data was assessed by the chemometric tool CA, aiming at extracting the greatest possible amount of information. The rivers and, consequently, the bay were contaminated by domestic sewage discharges and the following antimicrobial chemotherapeutical agents were detected: sulfamethoxazole (SMX), sulfathiazole (STZ), sulfachloropyridiazine (SCP), sulfaquinoxaline (SQX), and norfloxacin (NOR). The contamination profiles revealed by the CA showed a spatial variation and a temporal one.

The mutant-prevention concentration (MPC) is a novel susceptibility measurement defined by a concentration threshold that would require cells to contain two concurrent resistance mutations for growth. Pneuococcal pneumonia, infections caused by <i> Pseudomonas aeruginosa</i>, and urinary tract infections caused by Gram-negative bacilli represent three distinct clinical situations for which fluoroquinolone-resistance occurs. MPC results were defined and measured for fluoroquinolones against clinical isolates of <i>Citrobacter freundii, Enterobacter cloacae, Escherichia. coli, Klebsiella pneumoniae, P. aeruginosa,</i> and <i> Streptococus pneumoniae</i>. Against clinical isolates of <i>S. pneumoniae</i>, MPC results for six fluoroquinolones were measured. Based on their potential for restricting the selection of resistant mutants, the six fluoroquinolones, in descending order, were found to be gemifloxacin > moxifloxacin > trovafloxacin > gatifloxacin > grepafloxacin > levofloxacin. For several compounds, 90% of clinical isolates that lacked a known resistance mutation had a MPC value that was close to or below the serum levels that could be attained with a dosing regimen recommended by the manufacturers. These data identify gemifloxacin, moxifloxacin and gatifloxacin as good candidates for determining whether MPC can be used as a guide for choosing and eventually administering fluoroquinolones to significantly reduce the development of fluoroquinolone ¡Vresistant <i>S. pneumoniae</i>. MPC90 results for 155 clinical isolates of <i>P. aeruginosa </i>against ciprofloxacin and levofloxacin were 4 and 16 Ýg/ml, respectively. Serum drug concentrations reported previously for standard doses were above MPC90 for 5.5 hr for ciprofloxacin and 0 hr for levofloxacin. These data suggest that superior clinical performance of ciprofloxacin correlates with activity against resistant mutant subpopulations measured in vitro. MPC results were compared with minimum inhibitory concentrations (MIC) measurements preformed by agar dilution, and microbroth dilution and minimal inhibitory concentrations (MBC) for 100 clinical isolates of <i>C. freundii </i> (n=20), <i>E. cloacae</i> (n=20), <i>E. coli</i> (n=20), <i>K. pneumoniae</i> (n=20), and <i>P. aeruginosa</i> (n=20) for ciprofloxacin, levofloxacin and garenoxacin. MPC results were 2-to-8 fold higher than MIC or MBC results. Ciprofloxacin MPC results for <i>E.coli, C. freundii, E. cloacae, K. pneumoniae</i>, and <i>P. aeruginosa</i> were 0.5, 2, 1, 1, and 4 Ýg/ml, respectively. Levofloxacin, MPC results were were 1, 2, 4, 1, and 16 Ýg/ml, respectively. Garenoxacin, MPC were 1, 8, >8, 4, and >32 Ýg/ml, respectively. Garenoxacin had the highest MIC and MPC results and was the least active compound tested against isolates of <i>C. freundii, E. cloacae</i>, and <i>P. aeruginosa</i>. These data support the rational use of quinolones in the treatments of urinary tract infections and suppression of resistance. Incorporation of the MPC measurement into dosing strategies may preserve the longevity of antimicrobial compounds for future infectious diseases.

The mutant-prevention concentration (MPC) is a novel susceptibility measurement defined by a concentration threshold that would require cells to contain two concurrent resistance mutations for growth. Pneuococcal pneumonia, infections caused by <i> Pseudomonas aeruginosa</i>, and urinary tract infections caused by Gram-negative bacilli represent three distinct clinical situations for which fluoroquinolone-resistance occurs. MPC results were defined and measured for fluoroquinolones against clinical isolates of <i>Citrobacter freundii, Enterobacter cloacae, Escherichia. coli, Klebsiella pneumoniae, P. aeruginosa,</i> and <i> Streptococus pneumoniae</i>. Against clinical isolates of <i>S. pneumoniae</i>, MPC results for six fluoroquinolones were measured. Based on their potential for restricting the selection of resistant mutants, the six fluoroquinolones, in descending order, were found to be gemifloxacin > moxifloxacin > trovafloxacin > gatifloxacin > grepafloxacin > levofloxacin. For several compounds, 90% of clinical isolates that lacked a known resistance mutation had a MPC value that was close to or below the serum levels that could be attained with a dosing regimen recommended by the manufacturers. These data identify gemifloxacin, moxifloxacin and gatifloxacin as good candidates for determining whether MPC can be used as a guide for choosing and eventually administering fluoroquinolones to significantly reduce the development of fluoroquinolone ¡Vresistant <i>S. pneumoniae</i>. MPC90 results for 155 clinical isolates of <i>P. aeruginosa </i>against ciprofloxacin and levofloxacin were 4 and 16 Ýg/ml, respectively. Serum drug concentrations reported previously for standard doses were above MPC90 for 5.5 hr for ciprofloxacin and 0 hr for levofloxacin. These data suggest that superior clinical performance of ciprofloxacin correlates with activity against resistant mutant subpopulations measured in vitro. MPC results were compared with minimum inhibitory concentrations (MIC) measurements preformed by agar dilution, and microbroth dilution and minimal inhibitory concentrations (MBC) for 100 clinical isolates of <i>C. freundii </i> (n=20), <i>E. cloacae</i> (n=20), <i>E. coli</i> (n=20), <i>K. pneumoniae</i> (n=20), and <i>P. aeruginosa</i> (n=20) for ciprofloxacin, levofloxacin and garenoxacin. MPC results were 2-to-8 fold higher than MIC or MBC results. Ciprofloxacin MPC results for <i>E.coli, C. freundii, E. cloacae, K. pneumoniae</i>, and <i>P. aeruginosa</i> were 0.5, 2, 1, 1, and 4 Ýg/ml, respectively. Levofloxacin, MPC results were were 1, 2, 4, 1, and 16 Ýg/ml, respectively. Garenoxacin, MPC were 1, 8, >8, 4, and >32 Ýg/ml, respectively. Garenoxacin had the highest MIC and MPC results and was the least active compound tested against isolates of <i>C. freundii, E. cloacae</i>, and <i>P. aeruginosa</i>. These data support the rational use of quinolones in the treatments of urinary tract infections and suppression of resistance. Incorporation of the MPC measurement into dosing strategies may preserve the longevity of antimicrobial compounds for future infectious diseases.

Emergent multidrug resistant Escherichia coli increase clinical challenges. This thesis describes the resistance patterns, molecular epidemiology and mechanisms, for 315 E. coli from patients in the Vancouver Coastal Health Region for 2006/2007. Automated susceptibility testing was confirmed via E-test® for AmpC and/or ESBL production. PFGE, RFLP and PCR were used to assess genotypic relationships, and plasmid character. AmpC production was facilitated mainly by promoter mutations (54.5%). The principal ESBL detected was CTX-M-15 (49.5%). An unidentified ESBL-producer, with a pI near 8.3, was detected. A plasmid displayed variant resistance phenotypes dependent on selective growth media. A positive correlation between ST131 with CTX-M-15 and CIPR indicated the dissemination of companion phenotypes. Ciprofloxacin resistance resulted mainly (98.0%) from a double gyrA mutation. Overall fluoroquinolone resistance was not assessable due to exclusive selection parameters in this evaluation. Fluoroquinolone resistance factors require further examination to understand what causes resistant phenotypes exclusive of chromosomal mutations. ii

Emergent multidrug resistant Escherichia coli increase clinical challenges. This thesis describes the resistance patterns, molecular epidemiology and mechanisms, for 315 E. coli from patients in the Vancouver Coastal Health Region for 2006/2007. Automated susceptibility testing was confirmed via E-test® for AmpC and/or ESBL production. PFGE, RFLP and PCR were used to assess genotypic relationships, and plasmid character. AmpC production was facilitated mainly by promoter mutations (54.5%). The principal ESBL detected was CTX-M-15 (49.5%). An unidentified ESBL-producer, with a pI near 8.3, was detected. A plasmid displayed variant resistance phenotypes dependent on selective growth media. A positive correlation between ST131 with CTX-M-15 and CIPR indicated the dissemination of companion phenotypes. Ciprofloxacin resistance resulted mainly (98.0%) from a double gyrA mutation. Overall fluoroquinolone resistance was not assessable due to exclusive selection parameters in this evaluation. Fluoroquinolone resistance factors require further examination to understand what causes resistant phenotypes exclusive of chromosomal mutations. ii

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.