RESUMEN
Objective High-throughout sequencing of all plasmid of 206 strains of Klebsiella pneu-moniae using Solexa/Illumina sequencing technology in order to investigate the resistance for plasmids in Klebsiella pneumoniae. Methods Bacterial isolates were obtained over the years 2002-2008. Solexa/Illumi-na sequencing technology was used to sequence both samples (S1 and S2) to a depth of between 10-560 fold coverage. We used SOAP provided by BGI to find SNPS and use velvet package to assemble these sequences and gained some long sequences, and MAQ programs developed in the laboratory were used to annotate SNPs and compare lineage-specific mutations in SHV-ESBLs. Results The Metagenome of plasmid encodes a 13 variety of resistance-related genes with exceptionally high copy numbers, including ABC-type efflux pumps and 4 variety of β-lactamases, SHV-ESBLs is abroad presence. We systematically investigated single nucleo-tide substitutions in plasmids metagenome, and found an amount of nonsynonymous mutations in the SHV-ESBLs genes. Conclusion Probabily in press of positive selection, we can clearly see these nonsynonymous changes predominantly occurred in plasmid SHV-ESBLs genes. And our findings indicate a unspecial low-level resistance contribute to antimicrobial efflux in the metagenome of plasmid in Klebsiella pneumoniae.
RESUMEN
Las BLEEs son β-lactamasas producidas por una variedad de bacterias gramnegativas, que confieren resistencia a cefalosporinas de tercera y cuarta generación y aztreonam. Son principalmente producidas por K. pneumoniae y E. coli, aunque la naturaleza plasmídica de los genes que las codifican ha permitido una amplia diseminación a otros géneros bacterianos. Se realizó la detección fenotípica y molecular de BLEE a 224 aislados de Enterobacterias provenientes de ocho Centros de Salud de Caracas. El 91,1% de las cepas analizadas mediante el método de doble disco y el NCCLS 2004, fueron productoras de BLEE. El análisis de CIM para ceftazidime, cefotaxime, cefepime y aztreonam mostró una mayor proporción de cepas BLEE con actividad ceftazidimasa, compatibles con las familias SHV y/o TEM, y en menor proporción pertenecientes a la familia CTX-M. Los resultados del PCR basados en la amplificación de los genes blaSHV, blaTEM y blaCTXM, confirmaron el predominio de SHV-BLEE (72%) y CTX-M-BLEE (21,1%). Los carbapenemos con un 100% de sensibilidad. constituyen la mejor opción terapéutica. Esta investigación permitió detectar el tipo de BLEE circulante en ocho centros hospitalarios y es el primer reporte de la determinación de BLEE del tipo CTX-M, en nuestro país.
The extended-spectrum ß-lactamases (ESBLs) are ß-lactamases produced by a variety of gram-negative bacteria that confers resistance to cephalosporins of third and quarter generation and aztreonam. They are most commonly produced by isolates of K. pneumoniae and E. coli, although the plasmidic nature of the genes that encode them has allowed a wide dissemination to other bacterial genera. A phenotypical and molecular detection of ESBLs were carried out to 224 Enterobacteria isolates from eight Health Centers of Caracas . A 91.1% of stumps analyzed by double disk method and the NCCLS 2004, were ESBLs' producers. The MIC analysis for ceftazidime, cefotaxime, cefepime and aztreonam showed a bigger proportion of stumps ESBL with ceftazidimase activity compatible to the SHV families and/or TEM, and in a smaller proportion to the CTX-M family. The PCR results based on genes blaSHV, blaTEM and blaCTX-M, amplification confirmed a predominance of SHV-ESBL (72%) and CTX-M-ESBL (21.1%). Therefore, carbapenems with 100% of sensibility constitute the best therapeutic option. This investigation has allowed the detection of circulating ESBLs type in eight hospitals, and is also the first report that determines the CTX-M type ESBLs in our country.