Asunto(s)
Antibacterianos/farmacología , Compuestos de Azabiciclo/farmacología , Aztreonam/farmacología , Enterobacteriaceae Resistentes a los Carbapenémicos/efectos de los fármacos , Ceftazidima/farmacología , Sinergismo Farmacológico , Klebsiella pneumoniae/efectos de los fármacos , Inhibidores de beta-Lactamasas/farmacología , Enterobacteriaceae Resistentes a los Carbapenémicos/aislamiento & purificación , Colistina/farmacología , Colombia , Combinación de Medicamentos , Farmacorresistencia Bacteriana , Francia , Humanos , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , TurquíaRESUMEN
Gene modifications in the PmrAB and PhoPQ two-component regulatory systems, as well as inactivation of the mgrB gene, are known to be causes of colistin resistance in Klebsiella pneumoniae. The objective of this study was to characterise the mechanism involved in colistin resistance in a Klebsiella oxytoca isolate. A K. oxytoca clinical isolate showing resistance to colistin was recovered in Cali, Colombia. The pmrA, pmrB, phoP, phoQ and mgrB genes were amplified and sequenced. Wild-type mgrB genes from K. pneumoniae and K. oxytoca were cloned, and corresponding recombinant plasmids were used for complementation assays. By analysing the mgrB gene of the K. oxytoca isolate and its flanking sequences, an insertion sequence (IS) of 1196bp was identified in its promoter region. The insertion was located between nucleotides -39 and -38 when referring to the start codon of the mgrB gene, thus negatively interfering with expression of the mgrB gene by modifying its promoter structure. This IS was very similar to ISKpn26 (99% nucleotide identity) belonging to the IS5 family. Complementation assays with mgrB genes from wild-type K. pneumoniae or K. oxytoca restored full susceptibility to colistin. In conclusion, here we identified the mechanism involved in colistin resistance in a K. oxytoca isolate. Modulation of mgrB gene expression was the key factor for this acquired resistance to colistin.
Asunto(s)
Antibacterianos/farmacología , Colistina/farmacología , Farmacorresistencia Bacteriana , Expresión Génica , Klebsiella oxytoca/efectos de los fármacos , Klebsiella oxytoca/genética , Proteínas de la Membrana/metabolismo , Anciano , Clonación Molecular , Colombia , Elementos Transponibles de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Prueba de Complementación Genética , Humanos , Infecciones por Klebsiella/microbiología , Klebsiella oxytoca/aislamiento & purificación , Klebsiella oxytoca/metabolismo , Masculino , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Análisis de Secuencia de ADNRESUMEN
A series of colistin-resistant Klebsiella pneumoniae isolates recovered from different countries was investigated in order to evaluate the involvement of the PmrA/PmrB two-component system in this resistance. Six isolates possessed a mutated PmrB protein, which is encoded by the pmrB gene, part of the pmrCAB operon involved in lipopolysaccharide modification. The same amino acid substitution (Thr157Pro) in PmrB was identified in the six isolates. The six isolates belonged to four distinct clonal groups, recovered in South Africa (sequence type 14 [ST14]), Turkey (ST101), and Colombia (ST258 and ST15). Three out of the four clones produced a carbapenemase, OXA-181, OXA-48, or KPC-3, while a single isolate did not produce any carbapenemase. Expression assays revealed an overexpression of the pmrA (70-fold), pmrB (70-fold), pmrC (170-fold), and pmrK (40-fold) genes in the pmrB-mutated isolate compared to expression of the pmrB wild-type isogenic K. pneumoniae isolate, confirming that the PmrB substitution was responsible for increased expression levels of those genes. Complementation assays leading to the expression of a wild-type PmrB protein restored the susceptibility to colistin in all isolates, confirming that the substitution in PmrB was responsible for the resistance phenotype. This study identified a key amino acid located in the PmrB protein as being responsible for the overexpression of pmrCAB and pmrHFIJKLM operons, leading to resistance to colistin.