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ABSTRACT Few antibiotics are left that are effective against methicillin-resistant Staphylococcus aureus (MRSA) and even strains resistant to these agents have been isolated. Previous studies have identified five distinct MRSA clonotypes, which are present globally. No comprehensive national study has previously been undertaken to investigate the MRSA types in South Africa, and this study was aimed at elucidating the genotypic population structure of South African MRSA isolates. SmaI digested genomic DNA, separated by pulsed-field gel electrophoresis, was used to characterise 349 S. aureus isolates, obtained from various state and private diagnostic laboratories. PFGE results were complemented with those of spa typing and staphylococcal cassette chromosome mec (SCCmec) typing results. Two-hundred-and-five different PFGE patterns were identified, which were grouped into twenty-four clusters. Three were major lineages, containing more than 20% of the isolates with a similarity cut-off of 70%. Only thirty-seven spa types were identified (fourteen novel spa types), which clustered into six spa-Clonal Complexes after BURP analysis. SCCmec types I-IV were identified, including variants of each type. Data suggest that the Archaic clone (RSA05), oldest of the epidemic clones, represents one of the major clones in South Africa. Strains that were part of this complex (n=98 (28.2%); t064; SCCmec type I-pls) clustered together with strain E2125/ATCC BAA-38 (t051; SCCmec type I). Another major complex, RSA16 (n=90 (25.7%); t012; SCCmec type II/IIB) possessed a single-locus variant (SLV) spa type and the same or a SLV SCCmec types as EMRSA-16 (t018; SCCmec type II). The third major complex, RSA03 (n=74 (21.2%); t037; SCCmec type III/IIIE), had similar spa and SCCmec types to control strainANS46 (t037; SCCmec type III). One MRSA and twelve MSSA isolates were also identified as carrying genes for the toxin Panton-Valentine leukocidin, which was confirmed by DNA nucleotide sequencing.

Technique of pulsed field gel electrophoresis (PFGE) has found widespread use in the analysis of the genome of all life organisms. It is applied to the separation of the large DNA molecules above thousands base pairs up to millions of base pairs in size, where using conventional gel electrophoresis techniques are not possible (for instance large bacterial, yeast, fungal or mammalian chromosome). Presented work was realized as a comparative analysis of genome of several carotenogenic yeasts. The conditions of isolation and analysis of chromosomal yeast DNA were optimized. A lysis of yeast cells and deproteination of DNA within agarose chops was shown as the most appropriate method for DNA isolation. Cultivation to late exponential phase (50 hours) is the most suitable to obtaining intact DNA in sufficient amount and quality. Carotenogenic yeasts undergo the random mutagenesis using alkylation reagent ethyl methanesulfonate (EMS). Genome of pigment overproducing mutants was analyzed by pulsed field gel electrophoresis and amount of carotenoids by high pressure liquid chromatography (HPLC). However, overproduction of beta-carotene was analyzed in mutant strains Rhodotorula glutinis (10.6 g/l of biomass enriched 0,34 mg/g of beta-carotene) and Cystofilobasidium capitatum (8.5 g/l of biomass enriched 0,23 mg/g of beta-carotene). Selection of mutant strains overproducing carotenoid pigments was in presented experiment series successful in almost all analyzed strains except in the case of the strain Rhodotorula aurantiaca.

Diferentes métodos de caracterização e inferências filogenéticas baseadas na seqüência de alguns genes nucleares indicam que Typanosoma cruzi pode ser dividido em dois grupos principais, denominados T. cruzi I e T. cruzi II. Outros subgrupos de isolados foram descritos, tais como grupo de rDNA 1/2 e zimodema 3 (Z3) cuja relação filogenética com os grupos T.cruzi I e T.cruzi II ainda não está clara. O cariótipo molecular é uma ferramenta que permite abordar diferentes aspectos do genoma de organismos. Neste trabalho, caracterizamos o cariótipo molecular de 22 isolados de T. cruzi a partir da separação de seus cromossomos por eletroforese de campo pulsado e hibridação com sondas que representam genes codificadores de proteína e de RNA. Verificamos extenso polimorfismo cromossômico entre os isolados e que isolados pertencentes aos grupos T. cruzi II, rDNA 1/2 e Z3 têm cromossomos de tamanho molecular maior (até 3,5 Mb) em relação a isolados de T. cruzi I (até 2,8 Mb). Os dados do cariótipo molecular também foram utilizados para averiguar o significado evolutivo do polimorfismo cromossômico. As análises fenéticas foram baseadas no índice de diferença absoluta de tamanho cromossômico (aCSDI) obtido a partir da hibridação dos cromossomos com um número variável de marcadores genéticos. Inicialmente analisamos nove isolados, classificados por diferentes abordagens moleculares nos gruposT. cruzi I, T. cruzi II e grupo de rDNA 1/2, este último considerado um grupo de isolados híbridos e incluído por alguns autores no grupo T. cruzi II. Dendrogramas de aCSDI obtidos a partir de 3 a 21 sondas definiram em todos os casos três grupos: dois correspondentes a T. cruzi I e T. cruzi II e um terceiro, ao grupo de rDNA 1/2. O isolado CL Brener - organismo de referência do Projeto Genoma deT. cruzi - ora agrupou com T. cruzi II ora com o grupo de rDNA 1/2, ilustrando seu caráter híbrido. Três grupos também foram observados no dendrograma construído com dados de polimorfismo de tamanho de fragmentos de restrição (RFLP) hibridados com dezoito sondas. A topologia dos dendrogramas de dados de cariótipo molecular e de RFLP é similar, com um coeficiente de correlação significante (r = 0.86062; P < 0.0001), corroborando uma forte estruturação dos grupos. Subseqüentemente, avaliamos a posição de isolados de Z3 em relação aos três grupos acima mencionados, uma vez que alguns autores situam Z3 no grupo T. cruzi II. Analisamos o padrão de hibridação de 9 sondas com os cromossomos de 19 isolados (oito de Z3, três de T. cruzi I, três de T. cruzi II e cinco de rDNA 1/2). A topologia dos dendrogramas mostrou quatro grupos correspondentes, respectivamente, a T. cruzi I, T. cruzi II, grupo de rDNA 1/2 + CL Brener e Z3 (oito dos nove isolados). Este estudo também revelou que isolados híbridos têm uma maior proporção de cromossomos homólogos de tamanhos diferentes em comparação com isolados não híbridos. De um modo geral, nossos resultados mostram que cromossomos são caracteres valiosos para a identificação de táxons em T. cruzi. Uma vez que isolados do grupo de rDNA 1/2 apresentam cistrons ribossômicos de tipo 1 e de tipo 2, caracterizamos a distribuição dos cistrons nas bandas cromossômicas destes isolados. Verificamos que a fração majoritária dos genes de rDNA do tipo 2 está localizada na banda de 1,5 Mb e que os cistrons do tipo 1 estão localizados principalmente na banda de 1,1 Mb. Também estimamos o número de cromossomos e tamanho do genoma de quatro isolados de T. cruzi , com base na análise densitométrica da intensidade de fluorescência dos cromossomos corados com SYBR Green I. Para esta finalidade idealizamos um modelo matemático que permitiu estimar 52, 65, 72 e 44 cromossomos (na célula 2N), respectivamente, para CL Brener, Esmeraldo cl3, SO3 cl5 e Silvio X10 cl1. O tamanho do genoma dos isolados foi calculado, respectivamente em, 70 Mb, 78 Mb, 94,7 Mb e 47 Mb. Os novos aspectos do cariótipo molecular de T. cruzi aqui apresentados contribuirão para a compreensão da organização do genoma deste parasita e auxiliarão na atribuição de arcabouços de genes (scaffolds) aos cromossomos de CL Brener. / Different typing methods and phylogenetic inference based on the nucleotide sequence of few nuclear genes indicate that Trypanosoma cruzi can be divided into two major groups named as T. cruzi I and T. cruzi II. Additional subgroups of isolates have been described, such as group of rDNA 1/2 and zymodeme 3 (Z3), whose phylogenetic relationships with the T. cruzi I and T. cruzi II groups is not clear. The molecular karyotype is a tool that allows investigation of particular aspects of the genome of organisms. In this study, we have characterized the molecular karyotype of 22 isolates following the separation of chromosomes by pulsed-field gel electrophoresis and hybridization with probes that represent genes coding for proteins or RNA species. We observed an extensive chromosome polymorphism between the isolates and that isolates typed as T. cruzi II, rDNA 1/2 and Z3 have chromosome of larger molecular size (up to 3.5 Mb) in relation to T. cruzi I isolates (up to 2.8 Mb). Data from molecular karyotype have been also used to verify the evolutionary meaning of chromosome polymorphism. The phenetic analyses were based on the absolute chromosomal size difference index (aCSDI), calculated from the hybridization of a variable number of genetic markers. Initially, we analyzed nine isolates, classified by different molecular approaches into T. cruzi I,T. cruzi II and rDNA 1/2 groups. This latter group has been considered of hybrid genotypes and has been included by some authors in the T. cruzi II group. aCSDI-based dendrograms obtained from 3 to 21 probes defined in all the cases three clusters: two corresponding, respectively, to T. cruzi I and T. cruzi II groups; and a third one, to rDNA group 1/2. CL Brener - the reference organism of the T. cruzi Genome Project - was alternatively positioned in T. cruzi II or rDNA 1/2 group clusters, illustrating the hybrid nature of this isolate. Three clusters were also observed in the dendrogram constructed with restriction fragment length polymorphism (RFLP) data from 18 probes. The topology of the chromosome and RFLP dendrograms is similar, with a significant correlation coefficient (r = 0.86062; P < 0.0001), supporting a strong structuring of the clusters. Subsequently we evaluated the position of Z3 isolates in relation to the above-mentioned groups, because some authors clustered Z3 with T. cruzi II group. For this purpose we determined the hybridization pattern of 9 probes with the chromosomes of 19 isolates (eight of Z3; three of T. cruzi I; three of T. cruzi II and five of rDNA 1/2). The topology of the aCSDI-based dendrograms showed four clusters corresponding, respectively, to T. cruzi I, T. cruzi II, rDNA 1/2 + CL Brener and Zymodeme 3 (eight out of nine isolates). This study also revealed that hybrid stocks have a larger proportion of two different-sized homologous chromosomes, as compared with non-hybrid. Overall, our results show that chromosomes are valuable characters for identification of evolutionary groups in T. cruzi. Because rDNA 1/2 isolates have ribosomal cistrons of type 1 and type 2, we have characterized the distribution of both cistrons in the chromosomal bands of these isolates. We verified that the majority of type-2 rDNA genes are localized in a 1.5 Mb band, whereas type-1 cistrons are mostly concentrated in a 1.1 Mb band. We have also estimated the number of chromosomes and genome size of four T. cruzi isolates, based on the densitometric analysis of the fluorescence intensity of chromosomes stained with SYBR Green I. For this purpose, we devised a mathematical model that allowed estimating 52, 65, 72 and 44 chromosomes (2N cell), respectively, in CL Brener, Esmeraldo cl3, SO3 cl5 and Silvio X10 cl1. The genome size in these isolates has been calculated, respectively, as 70 Mb, 78 Mb, 94,7 Mb and 47 Mb. The novel aspects of T. cruzi karyotype here presented contribute to the comprehension of the genome organization of this parasite and will assist the assignment of scaffold to the CL Brener chromosomes.

Made available in DSpace on 2014-07-29T15:26:25Z (GMT). No. of bitstreams: 1 AlessandraMarques2009.pdf: 692808 bytes, checksum: 9a7b1bde8039039ba579f8e25c59ead5 (MD5) Previous issue date: 2009-12-03 / In recent years the number of infections caused by microbacteria non-tuberculous mycobacteria (NTM) has increased mainly due to opportunistic infections in individuals imunocompormetidos and improvement of farming techniques and identification of MTN. Mycobacterium massilienese is an emerging body associated with wound infections, abscesses and pneumonia. An outbreak of infection after videoscopy occurred between 2005 and 2007 in seven hospitals in Goiânia-GO, in central Brazil. The objective of this study was to identify NTM isolated from patients with infection after arthroscopy and lararoscopia by PCR followed by analysis of fragment length polymorphism restrção (PRA-hsp65), compared by gel electrophoresis pulsed-field gel (PFGE), sequencing of the partial rpoB gene and determination of antimicrobial susceptibility in vitro. NTM were recovered from samples (exudate abscess subcutâneio) of 18 patients involved in the outbreak. In the period leading up to this study there was no reported case of infection after videoscopy caused by MTN in Goiania. The 18 isolates were identified as M, massiliene and genotyped as a single clone, indicating that they had a common origin, suggesting a common source of infection for the patients involved in the outbreak. The epidemic isolates were susceptible to amikacin (MIC90 4 micrograms / ml) and clarithromycin (MIC90 <1 ug / ml), but resistance to ciprofloxacin (MIC90 <128g/ml), tobramycin (MIC90 32 micrograms / ml) and intermediate susceptibility to cefoxitin (MIC90 64 ug / ml). In conclusion this study demonstrated the clonality of strains of M. massiliense involved in infections after procedures videoscopes and that they are susceptible to drugs indicated for the treatment / Durante os últimosd anos o número de infecções causadas por microbactérias não-tuberculosas (MNT) tem aumentado principalmente devido às infecções oportunistas em indivíduos imunocompormetidos e ao aprimoramento das técnicas de cultura e identificação das MTN. Mycobacterium massilienese é um organismo emergente, associado a infecções de feridas, formação de abscessos e pneumonias. Um surto de infecção após videoscopias ocorreu entre 2005 e 2007 em sete hospitais privados de Goiânia-GO, na região central do Brasil. O objetivo deste estudo foi identificar MNT isoladas de amostras de pacientes com infecção após artroscopia e lararoscopia por PCR seguida de análise de polimorfismo de fragmentos de restrção (PRA-hsp65), comparação por eletroforese em gel em campo pulsado (PFGE), sequenciamento parcial do gene rpoB e determinação da suscetibilidade antimicrobiana in vitro. MNT foram recuperadas das amostras (exsudato de abscesso subcutâneio) de 18 pacientes envolvidos no surto. No período antecedente a esse estudo não houve nenhum relato de caso de infecção após videoscopias causada por MTN em Goiânia. Os 18 isolados foram identificados como M, massiliene e genotipados como um único clone, indicando que tiveram uma origem em comum, o que sugere uma fonte comum de infecção para os pacientes envolvidos no surto. Os isolados epidêmicos apresentaram sensibilidade a amicacina (CIM90 4 ug/ml) e claritromicina (CIM90 < 1 ug/ml), porém resistência a ciprofloxacina (CIM90 < 128g/ml), tobramicina (CIM90 32 ug/ml), e sensibilidade intermediária a cefoxitina (CIM90 64 ug/ml). Em conclusão este estudo evidenciou a clonalidade de cepas de M. massiliense envolvidas em infecções após procedimentos de videoscopia e que as mesmas são suscetíveis às drogas indicadas para o tratamento

Diferentes métodos de caracterização e inferências filogenéticas baseadas na seqüência de alguns genes nucleares indicam que Typanosoma cruzi pode ser dividido em dois grupos principais, denominados T. cruzi I e T. cruzi II. Outros subgrupos de isolados foram descritos, tais como grupo de rDNA 1/2 e zimodema 3 (Z3) cuja relação filogenética com os grupos T.cruzi I e T.cruzi II ainda não está clara. O cariótipo molecular é uma ferramenta que permite abordar diferentes aspectos do genoma de organismos. Neste trabalho, caracterizamos o cariótipo molecular de 22 isolados de T. cruzi a partir da separação de seus cromossomos por eletroforese de campo pulsado e hibridação com sondas que representam genes codificadores de proteína e de RNA. Verificamos extenso polimorfismo cromossômico entre os isolados e que isolados pertencentes aos grupos T. cruzi II, rDNA 1/2 e Z3 têm cromossomos de tamanho molecular maior (até 3,5 Mb) em relação a isolados de T. cruzi I (até 2,8 Mb). Os dados do cariótipo molecular também foram utilizados para averiguar o significado evolutivo do polimorfismo cromossômico. As análises fenéticas foram baseadas no índice de diferença absoluta de tamanho cromossômico (aCSDI) obtido a partir da hibridação dos cromossomos com um número variável de marcadores genéticos. Inicialmente analisamos nove isolados, classificados por diferentes abordagens moleculares nos gruposT. cruzi I, T. cruzi II e grupo de rDNA 1/2, este último considerado um grupo de isolados híbridos e incluído por alguns autores no grupo T. cruzi II. Dendrogramas de aCSDI obtidos a partir de 3 a 21 sondas definiram em todos os casos três grupos: dois correspondentes a T. cruzi I e T. cruzi II e um terceiro, ao grupo de rDNA 1/2. O isolado CL Brener - organismo de referência do Projeto Genoma deT. cruzi - ora agrupou com T. cruzi II ora com o grupo de rDNA 1/2, ilustrando seu caráter híbrido. Três grupos também foram observados no dendrograma construído com dados de polimorfismo de tamanho de fragmentos de restrição (RFLP) hibridados com dezoito sondas. A topologia dos dendrogramas de dados de cariótipo molecular e de RFLP é similar, com um coeficiente de correlação significante (r = 0.86062; P < 0.0001), corroborando uma forte estruturação dos grupos. Subseqüentemente, avaliamos a posição de isolados de Z3 em relação aos três grupos acima mencionados, uma vez que alguns autores situam Z3 no grupo T. cruzi II. Analisamos o padrão de hibridação de 9 sondas com os cromossomos de 19 isolados (oito de Z3, três de T. cruzi I, três de T. cruzi II e cinco de rDNA 1/2). A topologia dos dendrogramas mostrou quatro grupos correspondentes, respectivamente, a T. cruzi I, T. cruzi II, grupo de rDNA 1/2 + CL Brener e Z3 (oito dos nove isolados). Este estudo também revelou que isolados híbridos têm uma maior proporção de cromossomos homólogos de tamanhos diferentes em comparação com isolados não híbridos. De um modo geral, nossos resultados mostram que cromossomos são caracteres valiosos para a identificação de táxons em T. cruzi. Uma vez que isolados do grupo de rDNA 1/2 apresentam cistrons ribossômicos de tipo 1 e de tipo 2, caracterizamos a distribuição dos cistrons nas bandas cromossômicas destes isolados. Verificamos que a fração majoritária dos genes de rDNA do tipo 2 está localizada na banda de 1,5 Mb e que os cistrons do tipo 1 estão localizados principalmente na banda de 1,1 Mb. Também estimamos o número de cromossomos e tamanho do genoma de quatro isolados de T. cruzi , com base na análise densitométrica da intensidade de fluorescência dos cromossomos corados com SYBR Green I. Para esta finalidade idealizamos um modelo matemático que permitiu estimar 52, 65, 72 e 44 cromossomos (na célula 2N), respectivamente, para CL Brener, Esmeraldo cl3, SO3 cl5 e Silvio X10 cl1. O tamanho do genoma dos isolados foi calculado, respectivamente em, 70 Mb, 78 Mb, 94,7 Mb e 47 Mb. Os novos aspectos do cariótipo molecular de T. cruzi aqui apresentados contribuirão para a compreensão da organização do genoma deste parasita e auxiliarão na atribuição de arcabouços de genes (scaffolds) aos cromossomos de CL Brener. / Different typing methods and phylogenetic inference based on the nucleotide sequence of few nuclear genes indicate that Trypanosoma cruzi can be divided into two major groups named as T. cruzi I and T. cruzi II. Additional subgroups of isolates have been described, such as group of rDNA 1/2 and zymodeme 3 (Z3), whose phylogenetic relationships with the T. cruzi I and T. cruzi II groups is not clear. The molecular karyotype is a tool that allows investigation of particular aspects of the genome of organisms. In this study, we have characterized the molecular karyotype of 22 isolates following the separation of chromosomes by pulsed-field gel electrophoresis and hybridization with probes that represent genes coding for proteins or RNA species. We observed an extensive chromosome polymorphism between the isolates and that isolates typed as T. cruzi II, rDNA 1/2 and Z3 have chromosome of larger molecular size (up to 3.5 Mb) in relation to T. cruzi I isolates (up to 2.8 Mb). Data from molecular karyotype have been also used to verify the evolutionary meaning of chromosome polymorphism. The phenetic analyses were based on the absolute chromosomal size difference index (aCSDI), calculated from the hybridization of a variable number of genetic markers. Initially, we analyzed nine isolates, classified by different molecular approaches into T. cruzi I,T. cruzi II and rDNA 1/2 groups. This latter group has been considered of hybrid genotypes and has been included by some authors in the T. cruzi II group. aCSDI-based dendrograms obtained from 3 to 21 probes defined in all the cases three clusters: two corresponding, respectively, to T. cruzi I and T. cruzi II groups; and a third one, to rDNA group 1/2. CL Brener - the reference organism of the T. cruzi Genome Project - was alternatively positioned in T. cruzi II or rDNA 1/2 group clusters, illustrating the hybrid nature of this isolate. Three clusters were also observed in the dendrogram constructed with restriction fragment length polymorphism (RFLP) data from 18 probes. The topology of the chromosome and RFLP dendrograms is similar, with a significant correlation coefficient (r = 0.86062; P < 0.0001), supporting a strong structuring of the clusters. Subsequently we evaluated the position of Z3 isolates in relation to the above-mentioned groups, because some authors clustered Z3 with T. cruzi II group. For this purpose we determined the hybridization pattern of 9 probes with the chromosomes of 19 isolates (eight of Z3; three of T. cruzi I; three of T. cruzi II and five of rDNA 1/2). The topology of the aCSDI-based dendrograms showed four clusters corresponding, respectively, to T. cruzi I, T. cruzi II, rDNA 1/2 + CL Brener and Zymodeme 3 (eight out of nine isolates). This study also revealed that hybrid stocks have a larger proportion of two different-sized homologous chromosomes, as compared with non-hybrid. Overall, our results show that chromosomes are valuable characters for identification of evolutionary groups in T. cruzi. Because rDNA 1/2 isolates have ribosomal cistrons of type 1 and type 2, we have characterized the distribution of both cistrons in the chromosomal bands of these isolates. We verified that the majority of type-2 rDNA genes are localized in a 1.5 Mb band, whereas type-1 cistrons are mostly concentrated in a 1.1 Mb band. We have also estimated the number of chromosomes and genome size of four T. cruzi isolates, based on the densitometric analysis of the fluorescence intensity of chromosomes stained with SYBR Green I. For this purpose, we devised a mathematical model that allowed estimating 52, 65, 72 and 44 chromosomes (2N cell), respectively, in CL Brener, Esmeraldo cl3, SO3 cl5 and Silvio X10 cl1. The genome size in these isolates has been calculated, respectively, as 70 Mb, 78 Mb, 94,7 Mb and 47 Mb. The novel aspects of T. cruzi karyotype here presented contribute to the comprehension of the genome organization of this parasite and will assist the assignment of scaffold to the CL Brener chromosomes.

Die Frage nach adäquaten mikrobiologischen und molekulargenetischen Methoden, um die Kolonisation des Respirationstrakts von Mukoviszidose-Patienten mit Pseudomonas aeruginosa nachzuweisen und zu charakterisieren, wird kontrovers diskutiert. Von 38 klinisch stabilen Patienten mit cystischer Fibrose (CF) wurden sequentiell im Abstand von 18 Monaten Proben aus Rachenabstrich, Sputum und Bronchiallavage (BAL) entnommen und bezüglich Pseudomonas-Nachweis untersucht. Die Pseudomonas-Stämme wurden mittels Random Amplified Polymorphic DNA (RAPD)-Analyse und Pulsfeld-Gelelektrophorese (PFGE) von DNA-Makrorestriktionsfragmenten typisiert und bezüglich der Frage nach genetisch divergierenden Isolaten innerhalb des selben Individuums sowie nach möglichen longitudinalen genetischen Veränderungen evaluiert. Sensitivität, negative und positive prädiktive Werte und Spezifität, um eine P. aeruginosa-Besiedlung zu erkennen, waren 36%, 74%, 83% und 96% im Falle der Kulturen aus dem Oropharynx von nicht-expektorierenden Patienten und 92%, 94%, 100% und 100% für Sputumkulturen von expektorierenden Probanden. RAPD-Analyse und PFGE waren in der Lage, zwischen unterschiedlichen Pseudomonas-Stämmen zu diskriminieren, wobei nur die DNA-Makrorestriktion zwischen Subtypen unterscheiden konnte. Die Genotypen der Pseudomonas-Isolate aus Rachenabstrich und Sputum divergierten in 55% und 40% zu den Isolaten der BAL. Longitudinale Variationen des Genotyps wurden in 62% der Fälle beobachtet, die Hälfte davon war nur mittels bronchoskopisch gewonnener Proben erkennbar. Zusammengefasst besitzen Sputumproben bezüglich des Pseudomonas-Nachweises dieselbe Wertigkeit wie Kulturen aus der BAL, während Rachenabstriche in einer frühen Krankheitsphase für die Charakterisierung der bakteriellen Flora des unteren Respirationstrakts wenig geeignet sind. Die Methode der DNA-Makrorestriktion kann als zuverlässige Technik für epidemiologische Untersuchungen empfohlen werden. Unterschiedliche Genotypen innerhalb desselben Individuums und longitudinale genetische Alterationen sind häufig, jedoch unter Umständen nur bronchoskopisch nachweisbar. / There is controversy about adequate specimen to detect and characterise colonisation of cystic fibrosis (CF) airways by Pseudomonas aeruginosa. Oropharyngeal, sputum and bronchoalveolar lavage (BAL) samples were evaluated sequentially from 38 stable CF patients for the detection of P. aeruginosa. Pseudomonas strains were typed by random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. The occurrence of genetically different isolates within the same host and longitudinal variations in the genotype during repeated examinations was assessed. Sensitivity, negative and positive predictive values and specificity to detect P. aeruginosa were 36%, 74%, 83% and 96% for oropharyngeal cultures in non-expectorating patients and 92%, 94%, 100% and 100% for sputum cultures from expectorating patients, respectively. RAPD analysis and PFGE were suitable to characterize P. aeruginosa CF isolates, although only DNA macrorestriction was able to distinguish between identical and closely related strains. Genotypes of Pseudomonas isolates recovered from oropharyngeal swabs and sputum differed to the strains recovered by bronchoscopy in 55% and 40%, respectively. In 62% longitudinal variations in the genotype occurred. Half of these alterations were only detectable from bronchoscopically obtained samples. In conclusion, sputum samples have the same value as specimens from BAL to detect P. aeruginosa colonisation, whereas cultures from the oropharynx are not suitable for characterising the bacterial conditions in the CF lungs in an early disease state. DNA macrorestriction is recommended as an excellent tool for epidemiological investigations. Different genotypes within the same host and longitudinal genetic alterations are common and may be detectable in the BAL fluid exclusively.