Pesquisa de genes de resistência a quinolonas em bacilos Gram negativos de origem clínica e ambiental / Research for genes of quinolone resistance in Gram negative bacilli from clinical and environmental origin

Introdução. Quinolonas são antimicrobianos sintéticos que inibem as enzimas DNA-girase e topoisomerase IV resultando na morte bacteriana. São altamente eficazes no tratamento de infecções bacterianas, especialmente causadas por bactérias Gram negativas, e portanto amplamente utilizados na medicina humana e veterinária, na qual também são empregados como profiláticos. Porém, o uso indiscriminado e inadequado levou ao aumento de bactérias resistentes a estes compostos. Esta resistência pode ocorrer devido a mutações nas enzimas DNA-girase e topoisomerase IV, e também por genes contidos em plasmídeos. Estes últimos são os principais responsáveis pela disseminação e circulação da resistência entre o meio ambiente e o ambiente hospitalar. Objetivos. Pesquisar genes de resistência a antimicrobianos do grupo das quinolonas em bactérias Gram negativas de origem clínica e ambiental que apresentam resistência fenotípica a este grupo. Material e Métodos. 73 cepas de Enterobacteriaceae e Aeromonas sp. de origem clínica e ambiental foram selecionadas para o estudo, e avaliadas quanto à sensibilidade aos antimicrobianos do grupo das quinolonas e à pesquisa de genes de resistência a este mesmo grupo e mutações no gene que codifica a enzima DNA-girase por meio de PCR e sequenciamento. Resultados. Das 73 cepas previamente selecionadas para compor o estudo, 65 foram utilizadas, devido à exclusão de perfis clonais similares. Nestas, foram observados os genes, qnrS1 (1,5 por cento ), qnrS2 (26,2 por cento ), qnrB1 (3,1 por cento ), qnrB19 (12,3 por cento ), qnrD1 (1,5 por cento ), aac(6)-Ib-cr (10,8 por cento ), oqxA (43,1 por cento ) e oqxB (41,5 por cento ), e duas variantes determinadas qnrB-like (3,1 por cento ) e qnrB69-like (1,5 por cento ). Os genes qnrA, qnrC e qepA não foram identificados. Mutações na enzima DNA-girase foram observadas em 97,9 por cento das cepas positivas para algum dos genes pesquisados...

Introduction. Quinolones are synthetic antimicrobial agents that inhibit DNA gyrase and topoisomerase IV enzymes resulting in bacterial death. They are highly effective in the treatment of bacterial infections, especially the ones caused by Gram negative bacteria, as well as for prophylaxy. Therefore they are widely used in human and veterinary medicine. However, indiscriminate and improper use led to an increase of bacteria resistance to these compounds. This resistance can be due to mutations in DNA gyrase and topoisomerase IV enzymes and also by genes contained in plasmids, which are mainly responsible for the spread and transmission of resistance between the environment and the hospital set. Objectives. To search for genes of resistance to quinolone antimicrobial agents in Gram-negative bacteria from clinical and environmental strains that present phenotypic resistance to this group. Material and Methods. 73 strains of Enterobacteriaceae and Aeromonas spp., from clinical and environmental origin, were selected for this study, and evaluated for antimicrobial susceptibility of quinolone and search of resistance genes in this same group and also for mutations in the gene encoding the enzyme DNA gyrase by PCR and sequencing. Results. Of the 73 strains previously selected to compose this study, 65 were used, due to the exclusion of similar clonal profiles. In these, genes qnrS1 (1.5 per cent ), qnrS2 (26.2 per cent ) qnrB1 (3.1 per cent ), qnrB19 (12.3 per cent ) qnrD1 (1.5 per cent ) aac(6')-Ib-cr (10.8 per cent ) oqxA (43.1 per cent ) and oqxB (41.5 per cent ) were observed, and two variants were named as qnrB-like (3.1 per cent ) and qnrB69-like (1.5 per cent ). The qnrA, qnrC and qepA genes were not identified. Mutations in DNA gyrase enzyme were observed in 97.9 per cent of the positive strains for at least one of the genes studied. It was possible to establish the association of aac(6')-Ib-cr with class 1 integron gene in four strains...

Integrones y su relación con el fenotipo de resistencia en bacilos gramnegativos aislados en el Hospital Torres Galdames de Iquique, Chile / Integrons and their relationship with resistance phenotype in Gram negative bacilli isolated in the Hospital Torres Galdames, Iquique, Chile

La resistencia antimicrobiana es codificada por algunos elementos genéticos que generan un flujo horizontal, particularmente, en ambientes que están sometidos a una fuerte presión selectiva, como ocurre en el ambiente hospitalario. En tal sentido, los bacilos gramnegativos, en el último tiempo, han cobrado importancia como agentes de infección nosocomial. Objetivo. Investigar la presencia de integrones en aislados clínicos de bacilos gramnegativos y su relación con el fenotipo de resistencia, Material y Métodos. Se analizaron 88 aislados clínicos de distintos servicios del Hospital Torres Galdames, durante el período: junio a diciembre de 2004. Fueron identificadas de acuerdo con su perfil bioquímico y se determinó la susceptibilidad a antimicrobianos mediante el método de difusión en agar. La presencia de integrones se detectó mediante RPC. Se realizó un análisis de cluster para estudiar la relación entre el fenotipo de resistencia y la presencia de integrones. Las cepas fueron genotipificadas mediante ERIC-PCR. Resultados. Dieciocho por ciento de las cepas aisladas correspondió a Proteus mirabilis, 17 por ciento a Escherichia coli y 32 por ciento a bacilos gramnegativos no fermentadores. La mayoría de los aislados presentó una elevada resistencia a los antimicrobianos evaluados: ampicilina 83 por ciento, cefalotina 82 por ciento, ceftriaxona 82 por ciento, ciprofloxacina 81 por ciento, gentamicina 81 por ciento y cotrimoxazol 82 por ciento. De las 88 cepas, 75 por ciento presentó integrones, siendo más común la clase 2. Los resultados del análisis de cluster no revelaron una clara relación entre la presencia de éstos y el perfil de resistencia para los antimicrobianos ensayados. Con la información disponible no fue posible relacionar la presencia de integrones con un determinado patrón de resistencia. Los patrones de bandas obtenidos con la técnica de ERIC-PCR revelaron una gran variedad genética entre las cepas analizadas, definiendo...

The antimicrobial resistance is coded in genetic elements which generate a horizontal flow of information, particularly in conditions that are under strong selective pressure like the nosocomial environment. In that sense, in the last decades, gram negative bacilli have become important agents of nosocomial infection. In order to investigate the presence of integrons among clinical isolates of gram negative bacilli and their relationship with their resistance profile, we studied 88 strains isolated from clinical specimens of different wards of the Hospital Torres Galdames, during the June-December period, 2004. They were identified according to biochemical tests. The antimicrobial susceptibility was evaluated by agar diffusion method. The integron presence was investigated by polymerase chain reaction (PCR). A cluster analysis was carried out to study the relationship between the presence of integrons and the resistance profile. The genotyping of the isolates was carried out by ERIC-PCR technique. Results: Of the isolated strains, 18 percent corresponded to Proteus mirabilis, 17 percent to Escherichia coli, and 32 percent to Non Fermentative Gram Negative bacilli. Most isolates presented high resistance to the antibiotics studied: 83 percent to ampicillin, 85 percent to cephalotin, 82 percent to ceftriaxone, 82 percent to ciprofloxacin, 81 percent to gentamycin and 82 percent to cotrimoxazole. Seventy-five percent of the 88 strains presented integrons. Class 2 integrons were found to be the most common. The results of the cluster analysis did not show a clear relationship among the presence of the integrons and the resistance profile. With the available information it is not possible to relate the integron presence with a certain resistance pattern. The patterns of bands obtained with the technique ERIC-PCR revealed a great genetic variety among the analyzed isolations, defining diverse genotypes, distributed in the different services...

In silico comparison of bacterial strains using mutual information.

Fast-sequencing throughput methods have increased the number of completely sequenced bacterial genomes to about 400 by December 2006, with the number increasing rapidly. These include several strains. In silico methods of comparative genomics are of use in categorizing and phylogenetically sorting these bacteria. Various word-based tools have been used for quantifying the similarities and differences between entire genomes. The simple di-nucleotide frequency comparison, codon specificity and k-mer repeat detection are among some of the well-known methods. In this paper, we show that the Mutual Information function, which is a measure of correlations and a concept from Information Theory, is very effective in determining the similarities and differences among genome sequences of various strains of bacteria such as the plant pathogen Xylella fastidiosa, marine Cyanobacteria Prochlorococcus marinus or animal and human pathogens such as species of Ehrlichia and Legionella. The short-range three-base periodicity, small sequence repeats and long-range correlations taken together constitute a genome signature that can be used as a technique for identifying new bacterial strains with the help of strains already catalogued in the database. There have been several applications of using the Mutual Information function as a measure of correlations in genomics but this is the first whole genome analysis done to detect strain similarities and differences.

A rapid hypochlorite method for extraction of polyhydroxy alkanoates from bacterial cells.

A new method has been standardized for extraction of polyhydroxy alkanoates from the bacteria, using sodium hypochlorite. This method is simple and quick as compared to the existing methods. Statistical analysis has proved the method to be reliable and reproducible.