RESUMEN
Clinical isolates of carbapenem-resistant Klebsiella pneumoniae (K. pneumoniae) strains are being increased worldwide. Five pan-resistant K. pneumoniae strains have been isolated from respiratory and ICU wards in a Chinese hospital, and reveal strong resistance to all β-lactams, fluoroquinolones and aminoglycosides. Totally 27 β-lactamase genes and 2 membrane pore protein (porin) genes in 5 K. pneumoniae strains were screened by polymerase chain reaction (PCR). The results indicated that all of 5 K. pneumoniae strains carried blaTEM-1 and blaDHA-1 genes, as well as base deletion and mutation of OmpK35 or OmpK36 genes. Compared with carbapenem-sensitive isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the resistant isolates markedly lacked the protein band of 34-40 kDa, which might be the outer membrane proteins of OmpK36 according to the electrophoresis mobility. In addition, the conjugation test was confirmed that blaDHA-1 mediated by plasmids could be transferred between resistant and sensitive strains. When reserpine (30 µg/mL) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 µg/mL) were added in imipenem and meropenem, the MICs had no change against K. pneumoniae strains. These results suggest that both DHA-1 β-lactamase and loss or deficiency of porin OmpK36 may be the main reason for the cefoxitin and carbapenem resistance in K. pneumoniae strains in our hospital.
Asunto(s)
Humanos , Antibacterianos/farmacología , Carbapenémicos/farmacología , Cefoxitina/farmacología , Farmacorresistencia Bacteriana , Klebsiella pneumoniae/efectos de los fármacos , Porinas/deficiencia , beta-Lactamasas , Proteínas Bacterianas/análisis , China , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Hospitales , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/química , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/genética , Pruebas de Sensibilidad Microbiana , Peso Molecular , Reacción en Cadena de la Polimerasa , beta-Lactamasas/genéticaRESUMEN
Objetivo. Identificar la proteína de membrana externa ausente en los aislamientos resistentes y determinar tanto las causas de su ausencia en la membrana, como la presencia de otros mecanismos de resistencia a carbapenemes en aislamientos clínicos de Pseudomonas aeruginosa. Métodos. Se estudió un brote de 20 aislamientos de P. aeruginosa previamente caracterizados como productores de la metalobetalactamasa IMP-13. Estos aislamientos presentaron igual expresión de la enzima IMP-13, pero solo cinco de ellos fueron resistentes acarbapenemes. En esos cinco aislamientos resistentes se confirmó la ausencia de una proteína de membrana externa. Se secuenciaron oprD y ampC; se identificaron las proteínas de membrana externa por desorción/ionización láser asistida por matriz/espectometría de masa tiempo de vuelo (MALDI-TOF); se determinó el nivel de expresión de OprD, de AmpC y de los sistemas de eflujo tipo Mex, por reacción en cadena de polimerasa en tiempo real, y por último, se determinó la contribución del déficit de OprD a la resistencia a carbapenemes. Resultados. La proteína de la membrana externa ausente en el grupo R (resistentes a ambos carbapenemes) fue identificada como OprD-TS, pero no se observaron variaciones en suexpresión. El gen oprD presentó mutaciones en los cinco aislamientos resistentes. Se observó la misma producción de la enzima tipo AmpC PDC-5 y del sistema de eflujo Mex AB-OprM entre los aislamientos sensibles y resistentes a carbapenemes. Se analizó cómo la presencia conjunta de IMP-13 y el déficit de OprD contribuyen al aumento de la resistencia.Conclusiones. Distintos mecanismos contribuyen a la resistencia de aislamientos productores de IMP-13 a carbapenemes. La posibilidad de no detectar estos aislamientos productores de IMP-13 representa un riesgo latente de selección de mutantes con mecanismos de resistencia que se suman para aumentar la resistencia a carbapenemes.
Objective. To identify the outer membrane protein absent in the resistant isolates and to determine both the causes of its absence in the membrane and the presence of othermechanisms of carbapenem resistance in clinical isolates of Pseudomonas aeruginosa. Methods. Twenty isolates from an outbreak of P. aeruginosa previously characterized as metallo-beta-lactamase IMP-13 producers were studied. All the isolates exhibitedequal expression of the IMP-13 enzyme, but only five of them were carbapenemresistant. It was found that the five resistant isolates lacked a outer membrane protein. The oprD and ampC genes were sequenced; the outer membrane proteins were identifiedusing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry; the OprD and AmpC expressions, as well as the Mex efflux system, were assessed by real-time polymerase chain reaction; and finally, the contribution of reduced OprD to carbapenem resistance was determined. Results. The absent outer membrane protein in group R was identified as OprD-TS; however, no variations in its expression were observed. The oprD gene presentedmutations in the five resistant isolates. The production of AmpC PDC-5-type enzyme and the MexAB-OprM efflux system was the same in both carbapenem-sensitive and‑resistant isolates. The contribution of the combined presence of IMP-13 and reducedOprD to increased resistance was examined. Conclusions. Different mechanisms contribute to carbapenem resistance in IMP-13-producing isolates. The possibility that these IMP-13-producing isolates could go undetected poses a latent risk when selecting mutants with added resistancemechanisms in order to enhance carbapenem resistance.
Asunto(s)
Humanos , Proteínas Bacterianas/fisiología , Carbapenémicos/farmacología , Farmacorresistencia Bacteriana Múltiple/fisiología , Porinas/genética , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Resistencia betalactámica/fisiología , beta-Lactamasas/fisiología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/fisiología , Proteínas Bacterianas/genética , Análisis Mutacional de ADN , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple/genética , Electroforesis en Gel de Campo Pulsado , Genes Bacterianos , Imipenem/metabolismo , Imipenem/farmacología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/fisiología , Mutación , Porinas/deficiencia , Porinas/fisiología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Estudios Retrospectivos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tienamicinas/metabolismo , Tienamicinas/farmacología , Resistencia betalactámica/genética , beta-Lactamasas/genéticaRESUMEN
PURPOSE: Porins are outer membrane protein (OMP) that form water filled channels that permit the diffusion of small hydrophilic solutes like beta-lactam antibiotics across the outer membrane. Two major porins that facilitate diffusion of antimicrobials have been described in Klebsiella spp. and Escherichia coli. The present study was carried out to examine the role of porins among Extended Spectrum beta-Lactamase (ESBL) and AmpC beta-Lactamase positive strains of Klebsiella spp. and E.coli. METHODS: Preparation of OMP from phenotypically characterized clinical isolates K.pneumoniae and E.coli and the separation of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were performed as per a previously described procedure. RESULTS: OMP analysis revealed that cefoxitin and ceftazidime resistance was mediated by loss of a porin Omp K35 in the isolates of K.pneumoniae and E.coli. CONCLUSIONS: Loss of porin mediated resistance mechanism against cefoxitin was observed among the multidrug resistant K.pneumoniae and E.coli.