Beta lactam resistance producing isolates of Enterobacteriacea and non-Enterobacteriacea have emerged since more than seventy years ago (Abraham and Chain, 1940). They are known to cause both community and hospital-acquired infections. Resistance against carbapenem is primarily mediated by the production of enzymes that destroy the beta lactam antimicrobials, which are produced by these isolates involving the expression of serine and metalobetalactamase genes KPC, IMP, VIM, NDM-1 and OXA-48. Quinolone resistance is predominanty mediated by mutations in the qnrA, qnrB, qnrS, and aac-6-Ib genes. Carbapenemase-producing organisms especially Klebsiella pneumoniae carbapenemases (KPCs) emerged as important pathogens especially among critically ill patients causing significant morbidity and mortality. This study aims to determine the prevalence and types of 15 quinolone resistance and carbapenemases genes among different isolates from patients admitted to the University Hospital of Leipzig over a period of ten months. During the period from January 2011 through October 2011, a total of 50 carbapenemases isolates were recovered from patients of the University Hospital of Leipzig/ Germany. The isolates were identified by biochemical tests and their susceptibility to antimicrobials was determined by the microbroth dilution method according to ISO standard. The KPC, IMP, VIM, OXA-48, NDM-1, and aac-6-Ib genes as well as qnrA, qnrB, and qnrS genes were detected by multiplex PCR, respectively. Results showed that KPC gene was detected in 82% of the isolates while 8% were KPC negative. The qnrA, qnrS, IMP, NDM-1, and OXA-48 genes were not detected in any of the isolates while qnrB and VIM genes were found in 2%. On the other hand, aac-6-Ib gene was the most prevalent gene among the study isolates and composed a percentage of 96%. Results also showed that KPC, and aac-6-Ib genes were detected in isolates collected from urine, blood, wounds, swabs, sputum, tracheal secretions, biopsies, and anal smears, while VIM gene was detected in one isolate collected from blood. The qnrB gene was found in one isolate collected from urine specimen. The wide spread of carbapenem and quinolone resistance-producing organisms is a critical problem that complicates the treatment of infections resulting from these organisms. Necessary measures must, therefore, be taken to limit their spread, which include appropriate antibiotic treatment, control of hospital infections, observe of personal hygiene, and the use of appropriate methods of sterilization and disinfection to prevent the dissemination of these organisms. Keywords: Resistance, carbapenemases, QRDR, multiplex PCR, antimicrobials

Beta lactam resistance producing isolates of Enterobacteriacea and non-Enterobacteriacea have emerged since more than seventy years ago (Abraham and Chain, 1940). They are known to cause both community and hospital-acquired infections. Resistance against carbapenem is primarily mediated by the production of enzymes that destroy the beta lactam antimicrobials, which are produced by these isolates involving the expression of serine and metalobetalactamase genes KPC, IMP, VIM, NDM-1 and OXA-48. Quinolone resistance is predominanty mediated by mutations in the qnrA, qnrB, qnrS, and aac-6-Ib genes. Carbapenemase-producing organisms especially Klebsiella pneumoniae carbapenemases (KPCs) emerged as important pathogens especially among critically ill patients causing significant morbidity and mortality. This study aims to determine the prevalence and types of 15 quinolone resistance and carbapenemases genes among different isolates from patients admitted to the University Hospital of Leipzig over a period of ten months. During the period from January 2011 through October 2011, a total of 50 carbapenemases isolates were recovered from patients of the University Hospital of Leipzig/ Germany. The isolates were identified by biochemical tests and their susceptibility to antimicrobials was determined by the microbroth dilution method according to ISO standard. The KPC, IMP, VIM, OXA-48, NDM-1, and aac-6-Ib genes as well as qnrA, qnrB, and qnrS genes were detected by multiplex PCR, respectively. Results showed that KPC gene was detected in 82% of the isolates while 8% were KPC negative. The qnrA, qnrS, IMP, NDM-1, and OXA-48 genes were not detected in any of the isolates while qnrB and VIM genes were found in 2%. On the other hand, aac-6-Ib gene was the most prevalent gene among the study isolates and composed a percentage of 96%. Results also showed that KPC, and aac-6-Ib genes were detected in isolates collected from urine, blood, wounds, swabs, sputum, tracheal secretions, biopsies, and anal smears, while VIM gene was detected in one isolate collected from blood. The qnrB gene was found in one isolate collected from urine specimen. The wide spread of carbapenem and quinolone resistance-producing organisms is a critical problem that complicates the treatment of infections resulting from these organisms. Necessary measures must, therefore, be taken to limit their spread, which include appropriate antibiotic treatment, control of hospital infections, observe of personal hygiene, and the use of appropriate methods of sterilization and disinfection to prevent the dissemination of these organisms. Keywords: Resistance, carbapenemases, QRDR, multiplex PCR, antimicrobials

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Em diversos estados do Brasil, foram relatadas epidemias de infecções causadas por micobactérias de crescimento rápido (MCR) desde o ano 2000. A maioria dos casos foi principalmente associada ao clone BRA100 de Mycobacterium massiliense, recentemente renomeada para Mycobacterium abscessus subsp. bolletii, isolado de pacientes submetidos a procedimentos invasivos nos quais os instrumentos médicos não foram adequadamente esterilizados e/ou desinfetados. Sendo as quinolonas uma opção no tratamento de infecções por MCR e sugerida para esquemas terapêuticos para esses surtos, foram avaliadas nesse trabalho as atividades in vitro de quatro gerações de quinolonas para cepas clinicas e de referência de MCR através da microdiluição em caldo. Também foram analisadas as sequências peptídicas das regiões determinantes da resistência a quinolonas (RDRQ) das subunidades A e B da DNA gyrase (GyrA e GyrB) após o seqüenciamento de DNA seguido pela tradução da sequência de aminoácidos. Cinquenta e quatro cepas de M. abscessus subsp bolletii, incluindo o clone BRA100, isoladas em diferentes estados do Brasil, e 19 cepas de referência de MCR foram caracterizadas. Todas as 54 cepas clínicas de M. abscessus subsp. bolletii foram resistentes a todas as gerações de quinolonas e mostraram o mesmo resíduo nas RDRQ, incluindo Ala-83 em GyrA, Arg-447 e Asp-464 em GyrB, descritos como sendo responsáveis por gerar um baixo nível de resistência a quinolonas em micobactérias. Porém, outras espécies de MCR apresentaram diferentes susceptibilidade e padrões de mutações contrários aos classicamente já definidos, sugerindo que outros mecanismos de resistência, diferentes de mutações em gyrA e gyrB também possam estar envolvidos na alta resistência a quinolonas. / Several outbreaks of infections caused by rapidly growing mycobacteria (RGM) have been reported in many Brazilian states since 2000. Most of the cases were mainly associated to Mycobacterium massiliense, recently renamed as Mycobacterium abscessus subsp. bolletii, BRA100 clone recovered from patients who had undergone invasive procedures, in which medical instruments have not been properly sterilized and / or disinfected. Since quinolones have represented an option for the treatment of general RGM infections and suggested for therapeutic schemes for these outbreaks, we evaluated the in vitro activities of four generations of quinolones for clinical and reference RGM by broth microdilution, and analysis of peptide sequences of the quinolone resistance determining regions (QRDR) of GyrA and GyrB after DNA sequencing followed by amino acid translation. Fifty four isolates of M. abscessus subsp bolletii, including clone BRA100, recovered in different states of Brazil, and 19 reference strains of RGM species were characterized. All 54 M. abscessus subsp. bolletii isolates were resistant to all generations of quinolones and showed the same amino acids in the QRDR including the Ala-83 in GyrA, Arg-447 and Asp-464 in GyrB, described as responsible for an intrinsic low level of resistance to quinolones in mycobacteria. But other RGM species presented distinct susceptibilities to this class of antimicrobials and patterns of mutations contrary to what has been traditionally defined, suggesting that other mechanisms of resistance, different from gyrA or gyrB mutations, may also be involved in resistance to high levels of quinolones.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Em diversos estados do Brasil, foram relatadas epidemias de infecções causadas por micobactérias de crescimento rápido (MCR) desde o ano 2000. A maioria dos casos foi principalmente associada ao clone BRA100 de Mycobacterium massiliense, recentemente renomeada para Mycobacterium abscessus subsp. bolletii, isolado de pacientes submetidos a procedimentos invasivos nos quais os instrumentos médicos não foram adequadamente esterilizados e/ou desinfetados. Sendo as quinolonas uma opção no tratamento de infecções por MCR e sugerida para esquemas terapêuticos para esses surtos, foram avaliadas nesse trabalho as atividades in vitro de quatro gerações de quinolonas para cepas clinicas e de referência de MCR através da microdiluição em caldo. Também foram analisadas as sequências peptídicas das regiões determinantes da resistência a quinolonas (RDRQ) das subunidades A e B da DNA gyrase (GyrA e GyrB) após o seqüenciamento de DNA seguido pela tradução da sequência de aminoácidos. Cinquenta e quatro cepas de M. abscessus subsp bolletii, incluindo o clone BRA100, isoladas em diferentes estados do Brasil, e 19 cepas de referência de MCR foram caracterizadas. Todas as 54 cepas clínicas de M. abscessus subsp. bolletii foram resistentes a todas as gerações de quinolonas e mostraram o mesmo resíduo nas RDRQ, incluindo Ala-83 em GyrA, Arg-447 e Asp-464 em GyrB, descritos como sendo responsáveis por gerar um baixo nível de resistência a quinolonas em micobactérias. Porém, outras espécies de MCR apresentaram diferentes susceptibilidade e padrões de mutações contrários aos classicamente já definidos, sugerindo que outros mecanismos de resistência, diferentes de mutações em gyrA e gyrB também possam estar envolvidos na alta resistência a quinolonas. / Several outbreaks of infections caused by rapidly growing mycobacteria (RGM) have been reported in many Brazilian states since 2000. Most of the cases were mainly associated to Mycobacterium massiliense, recently renamed as Mycobacterium abscessus subsp. bolletii, BRA100 clone recovered from patients who had undergone invasive procedures, in which medical instruments have not been properly sterilized and / or disinfected. Since quinolones have represented an option for the treatment of general RGM infections and suggested for therapeutic schemes for these outbreaks, we evaluated the in vitro activities of four generations of quinolones for clinical and reference RGM by broth microdilution, and analysis of peptide sequences of the quinolone resistance determining regions (QRDR) of GyrA and GyrB after DNA sequencing followed by amino acid translation. Fifty four isolates of M. abscessus subsp bolletii, including clone BRA100, recovered in different states of Brazil, and 19 reference strains of RGM species were characterized. All 54 M. abscessus subsp. bolletii isolates were resistant to all generations of quinolones and showed the same amino acids in the QRDR including the Ala-83 in GyrA, Arg-447 and Asp-464 in GyrB, described as responsible for an intrinsic low level of resistance to quinolones in mycobacteria. But other RGM species presented distinct susceptibilities to this class of antimicrobials and patterns of mutations contrary to what has been traditionally defined, suggesting that other mechanisms of resistance, different from gyrA or gyrB mutations, may also be involved in resistance to high levels of quinolones.