Opções terapêuticas para tratar infecções por Pseudomonas aeruginosa são limitadas por seus diversos mecanismos de resistência, que podem ou não ser detectados no laboratório clínico. Um fenótipo observado na rotina laboratorial é o surgimento de subpopulações resistentes a partir de uma população sensível aos antimicrobianos – heteroresistência. Em P. aeruginosa esse fenômeno já foi investigado para carbapenêmicos, porém, em relação à polimixina B, não há dados literários. Objetivamos avaliar a heteroresistência à polimixina B em dois grupos de P. aeruginosa, um sensível e outro resistente aos carbapenêmicos. Cento e vinte e quatro isolados de P. aeruginosa foram obtidos, aleatoriamente, no Hospital de Clínicas de Porto Alegre em 2011. O perfil de susceptibilidade aos antimicrobianos, disco difusão e microdiluição em caldo – CIM (determinação da concentração inibitória mínima) para polimixina B, foi realizada conforme o Clinical Laboratory Standard Institute (CLSI) 2011. Isolados resistentes aos carbapenêmicos foram avaliados para CIM dos carbapenêmicos e cefalosporinas, e para pesquisa fenotípica e genotípica de metalo-β-lactamase (MβL). Um total de 24/124 isolados foi separado em dois grupos, um sensível (grupo S) e outro resistente (grupo R) aos carbapenêmicos (imipenem e/ou meropenem) para investigação da heteroresistência a polimixina B. Realizou-se ensaio de heteroresistência em duplicata através de diluições seriadas, partindo de uma suspensão de 0,5 de MacFarland e inoculadas em Agar Mueller Hinton com concentrações crescentes de polimixina B (0; 0,5; 1; 2; 4 e 8μg/mL). Após 5 dias de passagem em meio sem antibiótico, foi determinada a CIM dos isolados que cresceram na concentração mais alta de polimixina B. O perfil de análise populacional (PAP) foi definido pela razão do número de unidades formadoras de colônia (UFC) da placa com maior concentração de polimixina B onde houve crescimento bacteriano, pelo número de UFC da placa sem antibiótico. Foram consideradas heteroresistentes amostras que apresentaram subpopulações com crescimento em concentração de polimixina B ≥ 2 μg/mL. Amostras com subpopulações com crescimento em concentração de polimixina B superiores duas vezes ao CIM original, mas < 2 μg/mL, foram classificadas como heterogêneas. O resultado do disco difusão indicou heterogeneidade de suscetibilidade, sendo que gentamicina e imipenem foram os antibióticos com maior percentual de resistência e aztreonam e ciprofloxacino apresentaram os maiores perfis de sensibilidade. Todos os isolados foram sensíveis à polimixina B, com CIM50 e CIM90 de 1μg/mL e 2μg/mL, respectivamente. Trinta e sete isolados (30%; 37/124) apresentaram resistência aos carbapenêmicos. Quatro amostras foram positivas para MβL no teste fenotípico, sendo que o gene blaIMP foi idenficado nestas amostras. O grupo S não apresentou subpopulação heteroresistente, porém 3 isolados apresentaram subpopulação heterogênea. A freqüência do PAP no grupo S variou entre 2,1x10-4 a 4,0x10-7. O grupo R apresentou uma amostra heteroresistente e 6 isolados apresentaram subpopulação heterogênea, a freqüência do PAP variou entre 2,6x10-4 a 2,0x10-7. Os resultados deste estudo indicam baixa ocorrência de heteroresistência à polimixina B em amostras de P. aeruginosa tanto resistentes quanto sensíveis aos carbapenêmicos. No entanto, diversas amostras apresentaram subpopulações heterogêneas (CIM aumentada para a polimixina B), o que poderia explicar eventuais falhas terapêuticas durante o tratamento. / Therapeutic options to treat infections caused by Pseudomonas aeruginosa are limited because of their different resistance mechanisms that can be or don't be detected in the clinical laboratory. A phenotype that has been observed in our laboratory is the emergence of resistant subpopulations from a population sensitive to antibiotics - a phenomenon named heteroresistance. In P. aeruginosa this phenomenon has been investigated for carbapenems, however, in relation to the polymyxin B no data in the literature. We investigate the heteroresistance and polymyxin B into two groups P. aeruginosa, one sensitive and second resistant to carbapenems. One hundred twenty-four strains of P. aeruginosa were obtained randomly at the Hospital de Clinicas de Porto Alegre in 2011. The Antimicrobial Susceptibility Testing (Disk-difusion and the microdiluition broth, with determination of minimum inhibitory concentration (MIC) for polymyxin B, was performed according the Clinical Laboratory Standards Institute (CLSI), 2011. Isolates resistant to carbapenems were evaluated for MIC of carbapenems and cephalosporins, also for phenotypic and genotypic metallo-β-lactamase (MβL). A total of 24/124 strains were separated in two groups, one sensitive (S group) and other resistant (R group) to carbapenems (imipenem and / or meropenem) for investigation of heteroresistance polymyxin B. The assay was performed in duplicate heteroresistance through serial dilutions, starting from a 0.5 MacFarland suspension and inoculated into Mueller Hinton Agar with increasing concentrations of polymyxin B (0; 0,5; 1; 2; 4 e 8μg/mL). After 5 days of passage in medium without antibiotics, was determined the MIC of the isolates that grew at the highest concentration of polymyxin B. The population analysis profile (PAP) was defined as the ratio of the number of colony forming units (CFU) on the card with the highest concentration of polymyxin B in which bacterial growth, the number of CFU plate without antibiotic. We considered heteroresistant samples that showed subpopulations with growth in concentration of polymyxin B ≥ 2 mg / mL. Samples with subpopulations growing at higher concentration of polymyxin B twice CIM original, but <2 mg / mL were classified as heterogeneous. The result of AST indicated heterogeneity of susceptibility, and gentamicin and imipenem were the highest percentage with antibiotic resistance and aztreonam and norfloxacin showed the highest sensitivity profiles. All isolates were susceptible to polymyxin B, with CIM50 and CIM90 of 1μg/mL and 2μg/mL, respectively. Thirty-seven isolates (30%; 37/124) were resistant to carbapenems. Four samples were positive for the phenotypic test to MβL and the blaIMP gene was indentificated in this samples. The S group showed no subpopulation heteroresistente, but 3 isolates showed heterogeneous subpopulation. The frequency of PAP in group S varied between 2,0x10-4 to 4,0x10-7. The group R provided a sample heteroresistant and 6 isolates showed heterogeneous subpopulation, the frequency of PAP varied between 2,6x10- 4 a 2,0x10-7. The results of this study indicate a low occurrence of heteroresistance to polymyxin B in samples of P. aeruginosa so resistant assensitive to carbapenems. However, several samples showed heterogeneous subpopulations (MIC increased to polymyxin B) which could explain possible treatment failure during treatment.

Opções terapêuticas para tratar infecções por Pseudomonas aeruginosa são limitadas por seus diversos mecanismos de resistência, que podem ou não ser detectados no laboratório clínico. Um fenótipo observado na rotina laboratorial é o surgimento de subpopulações resistentes a partir de uma população sensível aos antimicrobianos – heteroresistência. Em P. aeruginosa esse fenômeno já foi investigado para carbapenêmicos, porém, em relação à polimixina B, não há dados literários. Objetivamos avaliar a heteroresistência à polimixina B em dois grupos de P. aeruginosa, um sensível e outro resistente aos carbapenêmicos. Cento e vinte e quatro isolados de P. aeruginosa foram obtidos, aleatoriamente, no Hospital de Clínicas de Porto Alegre em 2011. O perfil de susceptibilidade aos antimicrobianos, disco difusão e microdiluição em caldo – CIM (determinação da concentração inibitória mínima) para polimixina B, foi realizada conforme o Clinical Laboratory Standard Institute (CLSI) 2011. Isolados resistentes aos carbapenêmicos foram avaliados para CIM dos carbapenêmicos e cefalosporinas, e para pesquisa fenotípica e genotípica de metalo-β-lactamase (MβL). Um total de 24/124 isolados foi separado em dois grupos, um sensível (grupo S) e outro resistente (grupo R) aos carbapenêmicos (imipenem e/ou meropenem) para investigação da heteroresistência a polimixina B. Realizou-se ensaio de heteroresistência em duplicata através de diluições seriadas, partindo de uma suspensão de 0,5 de MacFarland e inoculadas em Agar Mueller Hinton com concentrações crescentes de polimixina B (0; 0,5; 1; 2; 4 e 8μg/mL). Após 5 dias de passagem em meio sem antibiótico, foi determinada a CIM dos isolados que cresceram na concentração mais alta de polimixina B. O perfil de análise populacional (PAP) foi definido pela razão do número de unidades formadoras de colônia (UFC) da placa com maior concentração de polimixina B onde houve crescimento bacteriano, pelo número de UFC da placa sem antibiótico. Foram consideradas heteroresistentes amostras que apresentaram subpopulações com crescimento em concentração de polimixina B ≥ 2 μg/mL. Amostras com subpopulações com crescimento em concentração de polimixina B superiores duas vezes ao CIM original, mas < 2 μg/mL, foram classificadas como heterogêneas. O resultado do disco difusão indicou heterogeneidade de suscetibilidade, sendo que gentamicina e imipenem foram os antibióticos com maior percentual de resistência e aztreonam e ciprofloxacino apresentaram os maiores perfis de sensibilidade. Todos os isolados foram sensíveis à polimixina B, com CIM50 e CIM90 de 1μg/mL e 2μg/mL, respectivamente. Trinta e sete isolados (30%; 37/124) apresentaram resistência aos carbapenêmicos. Quatro amostras foram positivas para MβL no teste fenotípico, sendo que o gene blaIMP foi idenficado nestas amostras. O grupo S não apresentou subpopulação heteroresistente, porém 3 isolados apresentaram subpopulação heterogênea. A freqüência do PAP no grupo S variou entre 2,1x10-4 a 4,0x10-7. O grupo R apresentou uma amostra heteroresistente e 6 isolados apresentaram subpopulação heterogênea, a freqüência do PAP variou entre 2,6x10-4 a 2,0x10-7. Os resultados deste estudo indicam baixa ocorrência de heteroresistência à polimixina B em amostras de P. aeruginosa tanto resistentes quanto sensíveis aos carbapenêmicos. No entanto, diversas amostras apresentaram subpopulações heterogêneas (CIM aumentada para a polimixina B), o que poderia explicar eventuais falhas terapêuticas durante o tratamento. / Therapeutic options to treat infections caused by Pseudomonas aeruginosa are limited because of their different resistance mechanisms that can be or don't be detected in the clinical laboratory. A phenotype that has been observed in our laboratory is the emergence of resistant subpopulations from a population sensitive to antibiotics - a phenomenon named heteroresistance. In P. aeruginosa this phenomenon has been investigated for carbapenems, however, in relation to the polymyxin B no data in the literature. We investigate the heteroresistance and polymyxin B into two groups P. aeruginosa, one sensitive and second resistant to carbapenems. One hundred twenty-four strains of P. aeruginosa were obtained randomly at the Hospital de Clinicas de Porto Alegre in 2011. The Antimicrobial Susceptibility Testing (Disk-difusion and the microdiluition broth, with determination of minimum inhibitory concentration (MIC) for polymyxin B, was performed according the Clinical Laboratory Standards Institute (CLSI), 2011. Isolates resistant to carbapenems were evaluated for MIC of carbapenems and cephalosporins, also for phenotypic and genotypic metallo-β-lactamase (MβL). A total of 24/124 strains were separated in two groups, one sensitive (S group) and other resistant (R group) to carbapenems (imipenem and / or meropenem) for investigation of heteroresistance polymyxin B. The assay was performed in duplicate heteroresistance through serial dilutions, starting from a 0.5 MacFarland suspension and inoculated into Mueller Hinton Agar with increasing concentrations of polymyxin B (0; 0,5; 1; 2; 4 e 8μg/mL). After 5 days of passage in medium without antibiotics, was determined the MIC of the isolates that grew at the highest concentration of polymyxin B. The population analysis profile (PAP) was defined as the ratio of the number of colony forming units (CFU) on the card with the highest concentration of polymyxin B in which bacterial growth, the number of CFU plate without antibiotic. We considered heteroresistant samples that showed subpopulations with growth in concentration of polymyxin B ≥ 2 mg / mL. Samples with subpopulations growing at higher concentration of polymyxin B twice CIM original, but <2 mg / mL were classified as heterogeneous. The result of AST indicated heterogeneity of susceptibility, and gentamicin and imipenem were the highest percentage with antibiotic resistance and aztreonam and norfloxacin showed the highest sensitivity profiles. All isolates were susceptible to polymyxin B, with CIM50 and CIM90 of 1μg/mL and 2μg/mL, respectively. Thirty-seven isolates (30%; 37/124) were resistant to carbapenems. Four samples were positive for the phenotypic test to MβL and the blaIMP gene was indentificated in this samples. The S group showed no subpopulation heteroresistente, but 3 isolates showed heterogeneous subpopulation. The frequency of PAP in group S varied between 2,0x10-4 to 4,0x10-7. The group R provided a sample heteroresistant and 6 isolates showed heterogeneous subpopulation, the frequency of PAP varied between 2,6x10- 4 a 2,0x10-7. The results of this study indicate a low occurrence of heteroresistance to polymyxin B in samples of P. aeruginosa so resistant assensitive to carbapenems. However, several samples showed heterogeneous subpopulations (MIC increased to polymyxin B) which could explain possible treatment failure during treatment.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2018-08-03T12:16:33Z No. of bitstreams: 1 gabriellafreitasferreira.pdf: 1111733 bytes, checksum: 4b8a7e3965e36b834dcb1890165ad008 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-08-03T12:21:08Z (GMT) No. of bitstreams: 1 gabriellafreitasferreira.pdf: 1111733 bytes, checksum: 4b8a7e3965e36b834dcb1890165ad008 (MD5) / Made available in DSpace on 2018-08-03T12:21:08Z (GMT). No. of bitstreams: 1 gabriellafreitasferreira.pdf: 1111733 bytes, checksum: 4b8a7e3965e36b834dcb1890165ad008 (MD5) Previous issue date: 2015-07-03 / PROQUALI (UFJF) / Embora os principais mecanismos de ação do fluconazol, itraconazol e anfotericina B estejam relacionados ao egosterol, é possível que essas drogas tenham outros efeitos nas células fúngicas. Além dos mais, a heterorresistência é considerada um mecanismo de adaptação frente a um estresse induzido por concentrações crescentes de antifúngicos no ambiente. Sendo o itraconazol um dos azólicos usados no tratamento da criptococose, o objetivo desse estudo foi avaliar a influência do estresse oxidativo e nitrosativo gerado pelos antifúngicos em células de C. gattii e sua influência no surgimento de clones heterorresistentes. Foram estudados distintos parâmetros para avaliar os estresses oxidativo e nitrosativo induzidos pelo fluconazol, itraconazol e anfotericina B em C. gattii. Já os efeitos da heterorresistência ao itraconazol foram estudados por meio de ensaios in vitro e em modelo murino. O itraconazol simultaneamente reduziu o conteúdo de ergosterol das células de C. gattii e induziu a produção de espécies reativas de oxigênio no início do tratamento, o que levou ao aumento da atividade das enzimas peroxidase e superoxidodismutase. O mesmo não aconteceu com o fluconazol. Já a anfotericina B promoveu grande estresse oxidativo e nitrosativo nas células de C. gattii, o que levou a uma elevada peroxidação lipídica e ineficiente ativação do sistema antioxidante celular. A heterorresistência ao itraconazol foi intrínseca para todas as linhagens testaddas, alterou parâmetros farmacodinâmicos, diminuiu o diâmetro celular e o tamanho da cápsula e ativação do sistema antioxidante celular. Observou-se uma correlação positiva entre a razão superfície/volume e o nível de heterorresistência ao itraconazol. Além do mais, a heterorresistência levou a maior internalização das células criptocócicas pelos macrófagos, mas também a uma maior proliferação dentro dessa célula fagocítica, o que culminou com o aumento da virulência dos clones heterorresistentes e alta carga fúngica nos pulmões e cérebro dos camundongos. Diante desses resultados, concluiu-se que o estresse oxidativo possuiu um importante papel no mecanismo de ação do itraconazol e pode ser um dos mecanismos que levam a heterorresistência e o aumento da virulência das células de C. gattii. / Although the most accepted mechanisms of action of amphotericin B and azoles are related to ergosterol, it is possible that these drugs have other effects on the fungal cell. Moreover, heteroresistance is an adaptive mechanism developed by the microorganism to counteract the stress of increasing drug concentration in the environment. Since itraconazole is used in the therapy of cryptococcosis, the aim of this study was to evaluate the role of endogenous reactive oxygen species (ROS) and peroxynitrite produced by azoles and amphotericin B in the fungus C. gattii and its influence on emergence of heteroresistante clones to itraconazole. We studied distinct parameters to evaluate the effect of oxidative and nitrosative stresses induced by fluconazole, itraconazole and amphotericin B in C. gattii cells. The effects of the heteroresistance to itraconazole were studied by performing tests in vitro and in a murine model. Itraconazole reduces the level of ergosterol and led to ROS production in C. gattii cells in the early stages of the treatment, enhancing the antioxidant activity. The same did not happen with fluconazole. Amphotericin B caused lipid peroxidation in C. gattii cells through a greatly enhanced production of oxidative and nitrosative radicals with increased lipid peroxidation and inefficient ativaction of antioxidant cellular system. Heteroresistance to itraconazole was intrinsic in all strains tested and changed pharmacodynamics parameters, diminished cell and capsule sizes, reduced ergosterol content and enhanced the antioxidant system of heteroresistant clones. Indeed, heteroresistance to itraconazole led to the increased internalization of cryptococcal cells by macrophages, but also to a prominent proliferation inside these phagocytic cells, culminating in the higher virulence of heteroresistant clones. Based on these results, we conclude that oxidative bursts play an important role in the antifungal activity of itraconazole and may be one of the mechanisms that lead to heteroresistance and the increased virulence of C. gattii.

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.

This is a two-part project. Proton pump inhibitors (PPIs) have been associated with increased risk of C. difficile infections and increased toxin production when combined with antimicrobial therapy. The first part of this project involved characterization of a hypervirulent NAP1 C. difficile strain, including genome sequencing and assembly, and the development of methods to study its transcriptomics using RNA-Seq, which will enable future researchers to study different expression patterns when toxigenic C. difficile is challenged with PPIs and/or antimicrobials in vitro. The second part of this project involved characterizing a clinical isolate of a NAP1 C. difficile displaying a markedly elevated MIC to metronidazole (MIC = 16 mg/mL), which initially exhibited MIC of 32 mg/mL. A method of obtaining a metronidazole-susceptible revertant from this isolate was developed and a revertant was obtained. The genomes of both isolates were sequenced, assembled, and aligned, then compared to each other for polymorphisms.

This is a two-part project. Proton pump inhibitors (PPIs) have been associated with increased risk of C. difficile infections and increased toxin production when combined with antimicrobial therapy. The first part of this project involved characterization of a hypervirulent NAP1 C. difficile strain, including genome sequencing and assembly, and the development of methods to study its transcriptomics using RNA-Seq, which will enable future researchers to study different expression patterns when toxigenic C. difficile is challenged with PPIs and/or antimicrobials in vitro. The second part of this project involved characterizing a clinical isolate of a NAP1 C. difficile displaying a markedly elevated MIC to metronidazole (MIC = 16 mg/mL), which initially exhibited MIC of 32 mg/mL. A method of obtaining a metronidazole-susceptible revertant from this isolate was developed and a revertant was obtained. The genomes of both isolates were sequenced, assembled, and aligned, then compared to each other for polymorphisms.

Es erfolgte die Re-Evaluierung der Empfindlichkeitstestung von Tuberkuloseerregern mit dem BacT/Alert-3D-System (bioMérieux) unter besonderer Berücksichtigung des Materialwechsels der Kulturflasche von Glas auf Plastik. Dazu wurden insgesamt 61 vergleichende MHK-Bestimmungen mit diesen beiden Flaschentypen durchgeführt, wobei sechs Antituberkulotika (INH, RMP, EMB, SM, PTH und MOX) sowie zwölf Mykobakterienstämme (davon neun Mtb.) untersucht wurden. Dabei findet sich kein Unterschied in den MHK-Werten zwischen Glas- und Plastikflaschen. Die Plastikflaschen sind folglich ebenso wie die Glasflaschen zur Empfindlichkeitstestung von Tuberkuloseerregern mit dem BacT/Alert-3D-System geeignet. Bei der bisherigen Methode wurde bei der PZA-Testung auf die Verwendung der Kontrollflasche zur Festlegung der Untersuchungszeit verzichtet, weil der erniedrigte pH-Wert ein unverhältnismäßig langsames oder gar fehlendes Wachstum in dieser Flasche bewirkt. Durch Optimierung von pH-Wert (pH 5,9) und PZA-Testkonzentration (200 mg/l) wurde erreicht, dass die PZA-Testung nach der gleichen Methode wie auch bei den anderen Antituberkulotika praktiziert, durchgeführt werden kann. Im weiteren wurde untersucht, wie hoch der Anteil resistenter Mutanten in Mischkulturen sein muss, um erkannt zu werden. Dazu wurden dem sensiblen Stamm H37Rv jeweils isogene Mutanten mit Monoresistenzen gegen INH, RMP oder SM bzw. EMB- und PZA-resistente Stämme in verschiedenen Anteilen zugesetzt. Es zeigte sich, dass ein Anteil von 1% resistenter Stämme noch nicht sicher detektiert wird. / The purpose of this paper is to optimize and reevaluate the methods of susceptibility testing of M.tuberculosis with the BacT/Alert 3D System (bioMérieux). The key aspect was the comparison between culture bottles from glass between those made out of plastic material. Therefore 61 comparative MIC-tests were carried out, with six antituberculous drugs (INH, RMP; EMB, SM, PTH and MOX) and twelve mycobacterial strains (nine Mtb.) in order to compare the MIC in the two different bottle types. The MIC of 40 comparative tests between plastic culture bottles were identical to those in glass material. Only 18 test results deviated between the two bottle types. In each of those tests the discrepancy was only one degree of dilution, which is considered as not relevant. Resistant strains were found in three tests, with no difference between glass and plastic bottles. The deviant twelve results of Mtb. strains show a six times higher MIC-value in plastic bottles and a six times lower MIC in plastic bottles. In the case of the substance INH all four discrepant results had a higher MIC in the plastic bottle. Since these results were known by the Institut für Medizinische Mikrobiologie und Infektionsepidemiologie of the University Leipzig, it was consequently used with good results in the laboratory routine, as well as in external quality control (INSTAND-Ringversuche 2009 to 2011). The second topic of this study was to optimize the current method of susceptibility testing of Mtb. concerning pyrazinamide (PZA). In the current method the low pH of the dilution (5.5) with the resulting weak growth of the strains forced the laboratory assistant to abandon the 1% control bottle as endpoint of measurement. The new and optimized pH of 5.9 and a concentration of PZA of 200 mg/l allows the susceptibility testing of PZA with the same method as practiced for the other antituberculous drugs. Yet another focus of this study was the sensitivity of the BacT/Alert 3D System to detect heteroresistant clones in the mycobacterial broth. Resistant clones and fully susceptible Mtb. strain (H37Rv) were mixed in different percentages and susceptibility testing was performed. It could be shown, that a secure detection of resistant clones is not possible at a percentage 1% of resistant clones in the culture.