RESUMEN
An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosa isolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding ß-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosa isolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and ß-lactamase production were responsible for the multidrug resistance in the isolates analysed.
Asunto(s)
Carbapenémicos/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Mutación , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Resistencia betalactámica/genética , beta-Lactamasas/metabolismo , Aminoglicósidos/metabolismo , Anfotericina B/análogos & derivados , Anfotericina B/metabolismo , Antifúngicos/metabolismo , Brasil , Cefalosporinasa/clasificación , Cefalosporinasa/metabolismo , Codón sin Sentido/metabolismo , Activación Enzimática/genética , Mutación del Sistema de Lectura/genética , Regulación Bacteriana de la Expresión Génica/genética , Humanos , Proteínas de Transporte de Membrana/metabolismo , Metiltransferasas/metabolismo , Nucleotidiltransferasas/metabolismo , Mutación Puntual/genética , Porinas/metabolismo , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/aislamiento & purificación , Secuencias Repetitivas de Ácidos Nucleicos , beta-Lactamasas/genéticaRESUMEN
An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.
Asunto(s)
Humanos , Carbapenémicos/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Mutación , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Resistencia betalactámica/genética , beta-Lactamasas/metabolismo , Aminoglicósidos/metabolismo , Anfotericina B/análogos & derivados , Anfotericina B/metabolismo , Antifúngicos/metabolismo , Brasil , Cefalosporinasa/clasificación , Cefalosporinasa/metabolismo , Codón sin Sentido/metabolismo , Activación Enzimática/genética , Mutación del Sistema de Lectura/genética , Regulación Bacteriana de la Expresión Génica/genética , Proteínas de Transporte de Membrana/metabolismo , Metiltransferasas/metabolismo , Nucleotidiltransferasas/metabolismo , Mutación Puntual/genética , Porinas/metabolismo , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/aislamiento & purificación , Secuencias Repetitivas de Ácidos Nucleicos , beta-Lactamasas/genéticaRESUMEN
Extended-spectrum cephalosporinase resistance is currently the fastest emerging antimicrobial resistance problem worldwide; however, evidence documenting the effect of potential risk factors is limited. The main objective of this study was to investigate the effect of using third and fourth generation cephalosporins on the occurrence of extended-spectrum cephalosporinase-producing Escherichia coli (ESC-Ec) in Danish pig herds. Conventional, integrated, medium to large herds were selected based on information from the Danish Central Husbandry Register and two groups were formed based on the use of third and fourth generation cephalosporins within a specified period, namely, 20 herds with no cephalosporin use (non-exposed) and 19 herds with frequent use (exposed). Data on prescribed antimicrobials were obtained from the National database (VetStat). Management data were obtained through a questionnaire. At the herd level, three pooled faecal samples were collected from sows with their piglets (farrowing pens), weaners, and finishers. ESC-Ec were then identified using selective enrichment. Because several of the herds only had a low number of weaners and/or finishers, analysis was only performed on samples from the farrowing pens. Logistic regression showed a significant effect of using cephalosporins-III/IV on the occurrence of ESC-Ec in the farrowing pens, even when adjusted for use of other antimicrobials 1 year prior to sampling. No confounding effect was identified in relation to management data. The relative risk ESC-Ec in exposed compared to non-exposed was 4.7 (95% confidence interval 2.0-11.5), confirming that regular use of cephalosporins-III/IV was a significant risk factor for the occurrence of ESC-Ec.
Asunto(s)
Cefalosporinasa/metabolismo , Cefalosporinas/farmacología , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/enzimología , Enfermedades de los Porcinos/microbiología , Animales , Cefalosporinasa/clasificación , Cefalosporinasa/genética , Cefalosporinas/administración & dosificación , Dinamarca/epidemiología , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Infecciones por Escherichia coli , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Porcinos , Enfermedades de los Porcinos/epidemiologíaRESUMEN
Whole-genome sequencing of a collection of 103 Acinetobacter strains belonging to 22 validly named species and another 16 putative species allowed detection of genes for 50 new class D ß-lactamases and 65 new Acinetobacter-derived cephalosporinases (ADC). All oxacillinases (OXA) contained the three typical motifs of class D ß-lactamases, STFK, (F/Y)GN, and K(S/T)G. The phylogenetic tree drawn from the OXA sequences led to an increase in the number of OXA groups from 7 to 18. The topologies of the OXA and RpoB phylogenetic trees were similar, supporting the ancient acquisition of blaOXA genes by Acinetobacter species. The class D ß-lactamase genes appeared to be intrinsic to several species, such as Acinetobacter baumannii, Acinetobacter pittii, Acinetobacter calcoaceticus, and Acinetobacter lwoffii. Neither blaOXA-40/143- nor blaOXA-58-like genes were detected, and their origin remains therefore unknown. The phylogenetic tree analysis based on the alignment of the sequences deduced from blaADC revealed five main clusters, one containing ADC belonging to species closely related to A. baumannii and the others composed of cephalosporinases from the remaining species. No indication of blaOXA or blaADC transfer was observed between distantly related species, except for blaOXA-279, possibly transferred from Acinetobacter genomic species 6 to Acinetobacter parvus. Analysis of ß-lactam susceptibility of seven strains harboring new oxacillinases and cloning of the corresponding genes in Escherichia coli and in a susceptible A. baumannii strain indicated very weak hydrolysis of carbapenems. Overall, this study reveals a large pool of ß-lactamases in different Acinetobacter spp., potentially transferable to pathogenic strains of the genus.
Asunto(s)
Acinetobacter/genética , Cefalosporinasa/clasificación , Cefalosporinasa/genética , Filogenia , Acinetobacter/clasificación , Acinetobacter/efectos de los fármacos , Acinetobacter/enzimología , Secuencias de Aminoácidos , Antibacterianos/metabolismo , Antibacterianos/farmacología , Cefalosporinasa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes/clasificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , beta-Lactamas/metabolismo , beta-Lactamas/farmacologíaRESUMEN
The characterization of AmpC-type beta-lactamases was performed in a collection of 32 clinical Pseudomonas aeruginosa isolates with intermediate susceptibility or resistance to imipenem and ceftazidime. Twenty-one out of those 32 isolates overexpressed AmpC beta-lactamase, and the MICs of ceftazidime and imipenem were reduced after cloxacillin addition. Cloning and sequencing identified 10 AmpC beta-lactamase variants. Reduced susceptibility to imipenem, ceftazidime, and cefepime was observed only with recombinant P. aeruginosa strains expressing an AmpC beta-lactamase that had an alanine residue at position 105. The catalytic efficiencies (k(cat)/K(m)) of the AmpC variants possessing this residue were increased against oxyiminocephalosporins and imipenem. In addition, we show here that those AmpC variants constitute a favorable background for the in vitro selection of imipenem-resistant strains. This report identified a novel resistance mechanism that may contribute to imipenem resistance in P. aeruginosa.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , beta-Lactamasas/metabolismo , beta-Lactamas/farmacología , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Ceftazidima/farmacología , Cefalosporinasa/clasificación , Cefalosporinasa/genética , Cefalosporinasa/metabolismo , Humanos , Imipenem/farmacología , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Pseudomonas aeruginosa/genética , Análisis de Secuencia de ADN , Resistencia betalactámica , beta-Lactamasas/clasificación , beta-Lactamasas/genéticaRESUMEN
Acinetobacter spp. are emerging as opportunistic hospital pathogens that demonstrate resistance to many classes of antibiotics. In a metropolitan hospital in Cleveland, a clinical isolate of Acinetobacter baumannii that tested resistant to cefepime and ceftazidime (MIC = 32 microg/ml) was identified. Herein, we sought to determine the molecular basis for the extended-spectrum-cephalosporin resistance. Using analytical isoelectric focusing, a beta-lactamase with a pI of > or = 9.2 was detected. PCR amplification with specific A. baumannii cephalosporinase primers yielded a 1,152-bp product which, when sequenced, identified a novel 383-amino-acid class C enzyme. Expressed in Escherichia coli DH10B, this beta-lactamase demonstrated greater resistance against ceftazidime and cefotaxime than cefepime (4.0 microg/ml versus 0.06 microg/ml). The kinetic characteristics of this beta-lactamase were similar to other cephalosporinases found in Acinetobacter spp. In addition, this cephalosporinase was inhibited by meropenem, imipenem, ertapenem, and sulopenem (K(i) < 40 microM). The amino acid compositions of this novel enzyme and other class C beta-lactamases thus far described for A. baumannii, Acinetobacter genomic species 3, and Oligella urethralis in Europe and South Africa suggest that this cephalosporinase defines a unique family of class C enzymes. We propose a uniform designation for this family of cephalosporinases (Acinetobacter-derived cephalosporinases [ADC]) found in Acinetobacter spp. and identify this enzyme as ADC-7 beta-lactamase. The coalescence of Acinetobacter ampC beta-lactamases into a single common ancestor and the substantial phylogenetic distance separating them from other ampC genes support the logical value of developing a system of nomenclature for these Acinetobacter cephalosporinase genes.
Asunto(s)
Acinetobacter baumannii/enzimología , Alelos , Proteínas Bacterianas/clasificación , Resistencia a las Cefalosporinas , Cefalosporinasa , Variación Genética , beta-Lactamasas/clasificación , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Secuencia de Aminoácidos , Antibacterianos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cefalosporinasa/química , Cefalosporinasa/clasificación , Cefalosporinasa/genética , Cefalosporinasa/metabolismo , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , beta-Lactamasas/química , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , beta-Lactamas/farmacologíaAsunto(s)
Acinetobacter/enzimología , Cefalosporinasa/biosíntesis , Cefalosporinasa/clasificación , Cromosomas Bacterianos/enzimología , Acinetobacter/efectos de los fármacos , Acinetobacter/genética , Infecciones por Acinetobacter/microbiología , Cefalosporinasa/genética , Inhibidores Enzimáticos/farmacología , Pruebas de Sensibilidad Microbiana , Resistencia betalactámica , beta-Lactamasas/biosíntesisAsunto(s)
beta-Lactamasas/clasificación , Cefuroxima/metabolismo , Cefalosporinasa/clasificación , Cromosomas Bacterianos , Bacterias Gramnegativas/enzimología , Bacterias Grampositivas/enzimología , Métodos , Penicilinasa/clasificación , Plásmidos , Especificidad por Sustrato , beta-Lactamasas/análisis , beta-Lactamasas/genéticaRESUMEN
A survey of 21 clinical isolates of Achromobacter species demonstrated a high level of beta-lactamase activity in all strains tested. The beta-lactamases were characterized by isoelectric focusing, purification by affinity chromatography, determination of molecular weight, immunological identity, and genetic analysis. At least three distinct patterns of beta-lactamases were found in 19 strains. The kinetic values Km and Vmax measured by a microacidimetric method showed that all three types of enzymes are cephalosporinases and did not hydrolyse oxacillin, cloxacillin, and methicillin. Two of the three types of cephalosporinases studied, namely MULB 901 (isoelectric point (pI)7.4) and MULB 905(pI 9.3) are enzymes mediated by genes of chromosomal origin. The MULB 906 (pI 8.1) enzyme, however, which has been previously shown to be mediated by an 8.2 MDal nonconjugative plasmid, showed hydrolysis of cefoxitime, cefotaxin, and moxalactam by the bioassay. In all cases, beta-lactamase synthesis appeared constitutive. This study confirms that beta-lactamase activity is commonly found in Achromobacter and that these enzymes are different and of clinical interest when compared with those observed in other Gram-negative bacteria.