RESUMEN
Class C ß-lactamases or cephalosporinases can be classified into two functional groups (1, 1e) with considerable molecular variability (≤20% sequence identity). These enzymes are mostly encoded by chromosomal and inducible genes and are widespread among bacteria, including Proteobacteria in particular. Molecular identification is based principally on three catalytic motifs (64SXSK, 150YXN, 315KTG), but more than 70 conserved amino-acid residues (≥90%) have been identified, many close to these catalytic motifs. Nevertheless, the identification of a tiny, phylogenetically distant cluster (including enzymes from the genera Legionella, Bradyrhizobium, and Parachlamydia) has raised questions about the possible existence of a C2 subclass of ß-lactamases, previously identified as serine hydrolases. In a context of the clinical emergence of extended-spectrum AmpC ß-lactamases (ESACs), the genetic modifications observed in vivo and in vitro (point mutations, insertions, or deletions) during the evolution of these enzymes have mostly involved the Ω- and H-10/R2-loops, which vary considerably between genera, and, in some cases, the conserved triplet 150YXN. Furthermore, the conserved deletion of several amino-acid residues in opportunistic pathogenic species of Acinetobacter, such as A. baumannii, A. calcoaceticus, A. pittii and A. nosocomialis (deletion of residues 304-306), and in Hafnia alvei and H. paralvei (deletion of residues 289-290), provides support for the notion of natural ESACs. The emergence of higher levels of resistance to ß-lactams, including carbapenems, and to inhibitors such as avibactam is a reality, as the enzymes responsible are subject to complex regulation encompassing several other genes (ampR, ampD, ampG, etc.). Combinations of resistance mechanisms may therefore be at work, including overproduction or change in permeability, with the loss of porins and/or activation of efflux systems.
Asunto(s)
beta-Lactamasas , beta-Lactamas , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbapenémicos , Pruebas de Sensibilidad Microbiana , Porinas , Serina , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , beta-Lactamas/farmacologíaRESUMEN
For medical biologists, sequencing has become a commonplace technique to support diagnosis. Rapid changes in this field have led to the generation of large amounts of data, which are not always correctly listed in databases. This is particularly true for data concerning class A ß-lactamases, a group of key antibiotic resistance enzymes produced by bacteria. Many genomes have been reported to contain putative ß-lactamase genes, which can be compared with representative types. We analyzed several hundred amino acid sequences of class A ß-lactamase enzymes for phylogenic relationships, the presence of specific residues, and cluster patterns. A clear distinction was first made between dd-peptidases and class A enzymes based on a small number of residues (S70, K73, P107, 130SDN132, G144, E166, 234K/R, 235T/S, and 236G [Ambler numbering]). Other residues clearly separated two main branches, which we named subclasses A1 and A2. Various clusters were identified on the major branch (subclass A1) on the basis of signature residues associated with catalytic properties (e.g., limited-spectrum ß-lactamases, extended-spectrum ß-lactamases, and carbapenemases). For subclass A2 enzymes (e.g., CfxA, CIA-1, CME-1, PER-1, and VEB-1), 43 conserved residues were characterized, and several significant insertions were detected. This diversity in the amino acid sequences of ß-lactamases must be taken into account to ensure that new enzymes are accurately identified. However, with the exception of PER types, this diversity is poorly represented in existing X-ray crystallographic data.
Asunto(s)
Bacterias/enzimología , Variación Genética , Genotipo , Filogenia , beta-Lactamasas/clasificación , beta-Lactamasas/genética , Cristalografía por Rayos X , Conformación Proteica , Relación Estructura-Actividad , beta-Lactamasas/química , beta-Lactamasas/metabolismoRESUMEN
OBJECTIVES: So far, two types of mechanism are known to be involved in carbapenem non-susceptibility of Escherichia coli clinical isolates: reduced outer membrane permeability associated with production of ESBLs and/or overproduction of class C ß-lactamases; and production of carbapenemases. Non-susceptibility to only imipenem observed in two clinical isolates suggested a new mechanism, described in the present study. METHODS: The ST was determined for the two isolates of E. coli (strains LSNy and VSBj), and their chromosomal region encoding the penicillin-binding domain of PBP2 was amplified, sequenced and then used for recombination experiments in E. coli K12 C600. Antibiotic MICs were determined using the Etest method. RESULTS: Strains LSNy and VSBj, which displayed ST23 and ST345, respectively, showed amino acid substitutions in their PBP2 penicillin-binding domain. Substitution Ala388Ser located in motif 2 (SXD) was common to the two strains. Two additional substitutions (Ala488Thr and Leu573Val) located outside the two other motifs were identified in strain LSNy, whereas another one (Thr331Pro) located in motif 1 was identified in strain VSBj. Recombination experiments to reproduce non-susceptibility to imipenem in E. coli K12 C600 were not successful when only the common substitution was transferred, whereas recombination with DNA fragments including either the three substitutions (strain LSNy) or the two substitutions (strain VSBj) were successful. CONCLUSIONS: Substitution of amino acids in the penicillin-binding domain of PBP2 is a new mechanism by which E. coli clinical isolates specifically resist imipenem.
Asunto(s)
Sustitución de Aminoácidos , Antibacterianos/farmacología , Proteínas de Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Imipenem/farmacología , Mutación Missense , Proteínas de Unión a las Penicilinas/genética , ADN Bacteriano/genética , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Recombinación Genética , Genética InversaRESUMEN
Beta-lactamase (EC 3.5.2.6) synthesis, particularly in Gram-negative bacilli, is a major mechanism of natural and acquired resistance to beta-lactams, sometimes accompanied by impermeability and/or active efflux. These enzymes have been classified into four molecular classes (A-D). The serine enzymes of class A, which may be encoded by the bacterial chromosome or transferable elements and are susceptible to clinically available inhibitors (clavulanic acid, sulbactam, tazobactam, avibactam), are prevalent considering other molecular classes (B,C,D). The continual rapid development of genomic approaches and tremendous progress in automatic sequencer technology have resulted in the accumulation of massive amounts of data. A structure-based classification of class A beta-lactamases based on specific conserved motifs involved in catalytic mechanisms and/or substrate binding (S70XXK, S130DN, K234TG), together with E166 (Ambler numbering) and at least 24 other amino-acid residues or analogs such as G45, F66, V80, L81, L91, L101, P107, A134, L138, G143, G144, G156, L169, T181, T182, P183, was validated on 700 amino-acid sequences, including 132 representative types, but mostly probable enzyme sequences, many produced by environmental bacteria. Two subclasses (A1, A2), six major clusters or groups (e.g. natural limited-spectrum beta-lactamases (LSBL), wider spectrum beta-lactamases (WSBL), and various other clusters were identified on the basis of conserved (> 90%) and specific motifs, and residues such as S70TFKAL, S130DNTAANL, R164XEXXLN, V231GDKTG for subclass A1, S70VFKFH, S130DNNACDI,E166XXM, and V231AHKTG for subclass A2, a probable disulfide bridge C77-C123 and G236, A237, G238, and R244 for the LSBL group. This great diversity of primary structures was used as the basis for a structure-based and phylogenetic classification.
Asunto(s)
beta-Lactamasas/química , beta-Lactamasas/clasificación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Secuencia Conservada/genética , Farmacorresistencia Bacteriana/genética , Humanos , Modelos Moleculares , Unión Proteica/genética , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Inhibidores de beta-Lactamasas/química , Inhibidores de beta-Lactamasas/metabolismo , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
A series of 'retinoid-like chalcones' and diverse derivatives relative to licochalcone A were synthesized from a new enaminone synthon. These syntheses occurred via a new aromatic annelation. These new derivatives have been tested in vitro as potential antimalarial agents. The 4-hydroxy-chalcone-like (compound 6a, derived from beta-ionone) exhibits a good and reproducible inhibitory effect on the in vitro culture of Plasmodium falciparum, with an IC 50 lower than 10 microM for inhibition of 3H-hypoxanthine uptake by parasites (respectively, 4.93 and 8.47 microM for strains K1 and Thaï).
Asunto(s)
Antimaláricos/síntesis química , Chalcona/síntesis química , Eritrocitos/parasitología , Plasmodium falciparum/efectos de los fármacos , Retinoides/síntesis química , Animales , Antimaláricos/química , Antimaláricos/farmacología , Chalcona/análogos & derivados , Chalcona/farmacología , Humanos , Concentración 50 Inhibidora , Malaria/sangre , Malaria/tratamiento farmacológico , Malaria/parasitología , Parasitemia/tratamiento farmacológico , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Retinoides/química , Retinoides/farmacologíaRESUMEN
Aeromonas caviae CIP 74.32 was resistant to amoxicillin, ticarcillin and cephalothin, and susceptible to cefoxitin, cefotaxime, ceftazidime, aztreonam and imipenem. This strain produced a cephalosporinase (pI 7.2) and an oxacillinase (pI 8.5). The cephalosporinase gene cav-1 was cloned and sequenced. Unlike A. caviae donor, Escherichia coli pNCE50 transformant producing CAV-1 beta-lactamase was resistant to cefoxitin. The deduced protein sequence CAV-1 contained 382 amino acids, and shared >96% homology with FOX-1 to FOX-5 cephalosporinase. CAV-1 presented only two amino acid substitutions (Thr270Ser and Arg271Ala) with FOX-1. CAV-1 is the chromosomal putative ancestor of the FOX family, a cluster of class C/group 1 plasmidic cephalosporinases spreading in Klebsiella and E. coli clinical isolates via conjugative plasmids.
Asunto(s)
Aeromonas/enzimología , Aeromonas/genética , Proteínas Bacterianas , Cefalosporinasa/química , Cefalosporinasa/genética , Resistencia betalactámica , Aeromonas/efectos de los fármacos , Secuencia de Aminoácidos , Antibacterianos/farmacología , Cefoxitina/farmacología , Cromosomas Bacterianos , Datos de Secuencia Molecular , Plásmidos , beta-Lactamasas/química , beta-Lactamasas/genéticaRESUMEN
In some inhibitor-resistant TEM-derived beta-lactamases, Met-69 is substituted by Leu, Ile or Val. Residue 69 is located in a region of strong structural constraints, at the beginning of H2 alpha-helix, and in the vicinity of B3 and B4 beta-strands. Analysis of the three-dimensional structure of TEM-1 beta-lactamase suggests that alteration of the substrate-binding site can be produced by changes of the size of residue 69 side chain. Met-69 was substituted by alanine or glycine in TEM-Bs beta-lactamase (a TEM-1-related enzyme) using site-directed mutagenesis. The minimum inhibitory concentrations of the mutants compared with the wild-type revealed an increased susceptibility to beta-lactamase inhibitor-beta-lactam combinations and to first-generation cephalosporins. Comparing the Met69Ala and Met69Gly beta-lactamases with TEM-Bs, K(m) constants of the mutants showed an increased affinity for most beta-lactams but the kcat for most substrates did not change substantially. Mutants also demonstrated lower IC50 for the three inhibitors (clavulanic acid, tazobactam and sulbactam). The two substitutions of the residue 69 by alanine and glycine had a noticeable effect on K(m) values of TEM-Bs beta-lactamase, and on affinity for beta-lactamase inhibitors.
Asunto(s)
Sustitución de Aminoácidos/genética , Antibacterianos/farmacología , Ácido Clavulánico/farmacología , Farmacorresistencia Microbiana/genética , Escherichia coli/efectos de los fármacos , Inhibidores de beta-Lactamasas , beta-Lactamasas/genética , Alanina/genética , Alanina/metabolismo , Antibacterianos/metabolismo , Ácido Clavulánico/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Glicina/genética , Glicina/metabolismo , Hidrólisis , Concentración 50 Inhibidora , Cinética , Metionina/genética , Metionina/metabolismo , Pruebas de Sensibilidad Microbiana , Mutagénesis Sitio-Dirigida/genética , Mutación , Especificidad por Sustrato , beta-Lactamasas/metabolismoRESUMEN
An atypical Enterobacteriaceae strain with a beta-lactam susceptibility pattern of inducible cephalosporinase was isolated in Tenon Hospital (Paris, France) from a patient's skull wound infection. Identifications by the API-50CHE biochemical system and 16S rRNA gene sequencing concluded that it was a member of the Buttiauxella genus. The bla gene was cloned and sequenced. The deduced translated product was a 383-amino acid protein (BUT-1) with 75-78% identity with the chromosomal AmpC beta-lactamases of Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae and Escherichia coli. The isoelectric point of 9.0 and the kinetic constants of BUT-1 were comparable with results described for other Ambler class C enzymes. bla(BUT-1) and the associated ampR transcriptional regulator gene were divergently transcribed from a common intercistronic region, a genetic organization already described for other inducible class C beta-lactamases. The deduced amino acid sequence of AmpR shared 85% and 81% identity with AmpR from E. cloacae and C. freundii respectively.
Asunto(s)
Enterobacteriaceae/genética , beta-Lactamasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , Enterobacteriaceae/clasificación , Enterobacteriaceae/enzimología , Punto Isoeléctrico , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/análisis , ARN Ribosómico 16S/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , beta-Lactamasas/biosíntesis , beta-Lactamasas/metabolismoRESUMEN
The statistical methods and parameters commonly used to define bacterial susceptibility to antibiotics in vitro such as MIC(50), linear regression or others, usually lead to a considerable loss of information: they do not take into account the heterogeneity of the bacterial population. In contrast, multivariate data analyses are more adapted to the description of biological systems. In this way, a population of a given bacterial species can be separated into homogenous classes corresponding to the different sensitivity and resistance phenotypes. The applications of this mathematical approach include: (i) a new model for more relevant interpretation of antimicrobial susceptibility test results; (ii) numerical estimation of breakpoints having a known risk; (iii) calibration of a technique relative to a reference technique; (iv) detection of strains with new phenotypes; (v) in vitro evaluation of the activity of new compounds.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Pruebas de Sensibilidad Microbiana/métodos , Modelos Teóricos , Automatización , Bacterias/clasificación , Bacterias/efectos de los fármacos , Bacterias/aislamiento & purificación , Infecciones Bacterianas/tratamiento farmacológico , Calibración , Heterogeneidad Genética/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/aislamiento & purificación , Fenotipo , Factores de RiesgoRESUMEN
Inhibitor-resistant TEM beta-lactamases (IRT) have been identified in Enterobacteriaceae which, however, remained susceptible to cephalosporins. We evaluated the combined inhibitory activity of clavulanic acid and imipenem at ratios of 1:1 and 1:3 against IRT-4, using the median effect principle of Chou and Talalay. The combination of the two drugs, which produced a nearly additive effect, meant their concentrations could be lowered 1.3-4.9-fold, while maintaining a 50% inhibitory effect against the IRT-4 in comparison with each drug alone. From a therapeutic point of view, such a combination is not efficient but this method of Chou and Talalay, used for the first time to assay combined inhibitory activity of beta-lactamase inhibitors, could be used to evaluate new molecules and/or strategies to inactivate beta-lactamase.
Asunto(s)
Antibacterianos/farmacología , Ácidos Clavulánicos/farmacología , Imipenem/farmacología , Modelos Teóricos , Inhibidores de beta-Lactamasas , Farmacorresistencia Microbiana , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , beta-LactamasasRESUMEN
In the search for novel biodegradable antifouling agents, a series of imides (N-substituted maleimides and succinimides) have been synthesized. A large majority of N-substituted maleimides and succinimides showed an antimicrobial activity toward gram-positive and gram-negative bacteria and fungi of marine origin with minimal inhibition concentrations in the range of 6 to 24 microg/ml. The imides with an alkyl substituent showed higher activities than aromatic analogues, but structure-activity relationships were not clearly established. Some compounds were powerful inhibitors of Mytilus edulis phenoloxidase. This inhibition was noncompetitive as demonstrated with 2 maleimides, a 3-acetoxy succinimide and a succinimide. Thus, this family of products has potential interest as antifouling agents.
RESUMEN
We have synthesized a series of 3-substituted succinimides and their in vitro antibacterial activities have been tested towards Gram-positive and Gram-negative bacteria from the ATCC collection. Some of them possess significant antibacterial activity against Gram-positive organisms (Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212) but all are poorly active or inactive against Gram-negative organisms (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853). The compounds with the lowest minimal inhibitory concentrations (esters of 3-hydroxy succinimides) are also the most cytotoxic against green monkey Vero cell line (ATCC CCL-81) and could explain that perhaps apoptosis should be implicated in eukaryotic cell cytotoxicity of succinimides.
Asunto(s)
Antibacterianos/farmacología , Succinimidas/farmacología , Animales , Antibacterianos/síntesis química , Chlorocebus aethiops , Enterococcus faecalis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Succinimidas/síntesis química , Células VeroRESUMEN
In the field of our research programs concerning novel antimicrobial agents, a series of N-substituted imides was synthesized. These compounds were obtained by cyclization of amido-acids in acetic anhydride/sodium acetate or hexamethyldisilazane/zinc bromide for the hydroxy-aromatic derivatives. The hydroxy-alkyl maleimides were directly prepared by condensation of the corresponding amino-alcohol with maleic anhydride in boiling toluene. Most of N-substituted maleimides showed an interesting antimicrobial activity towards bacteria from the ATCC collection (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) but the MIC values for P. aeruginosa were always high (128 microg/ml). The imides with alkyl substituents showed higher activities than aromatic analogues with MIC values in the range of 8-32 microg/ml. Comparatively, succinimides were practically inactive.
Asunto(s)
Antibacterianos/farmacología , Maleimidas/síntesis química , Maleimidas/farmacología , Succinimidas/síntesis química , Succinimidas/farmacología , Antibacterianos/síntesis química , Enterococcus faecalis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Técnicas In Vitro , Maleimidas/química , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , Succinimidas/químicaRESUMEN
Eighty-five extended-spectrum beta-lactamase-producing Enterobacteriaceae from a Slovak hospital have been studied. SHV-2a was predominant, but other variants have been detected, namely, SHV-5, SHV-12, TEM-12, TEM-15, and TEM-132, which differed from TEM-1 by amino acid substitutions R164H, E240K, and I173V and had kinetic properties similar to those of TEM-28.
Asunto(s)
Infecciones por Enterobacteriaceae/epidemiología , Enterobacteriaceae/efectos de los fármacos , Hospitales , beta-Lactamasas/metabolismo , Antibacterianos/farmacología , Enterobacteriaceae/enzimología , Infecciones por Enterobacteriaceae/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Eslovaquia/epidemiología , Resistencia betalactámica , beta-Lactamasas/genética , beta-Lactamas/farmacologíaRESUMEN
OBJECTIVES: The aim of this study was to characterize the ampC beta-lactamase gene of a clinical isolate of Serratia marcescens resistant to ceftazidime. METHODS: S. marcescens SMSA was isolated from an intra-abdominal wound of a patient previously treated with ceftazidime. A susceptible strain, SLS73, was used as a control. Susceptibility testing, PCR, DNA sequencing, molecular cloning, site-directed mutagenesis and determination of kinetic parameters were carried out to investigate the mechanism of resistance to ceftazidime. RESULTS: MICs of ceftazidime were 64 and 0.2 mg/L for SMSA and SLS73, respectively. Sequencing of the ampC gene of SMSA was carried out. When compared with the closest AmpC enzyme, the S. marcescens S3 beta-lactamase, the novel protein showed E57Q, Q129K and S220Y substitutions. The S220Y substitution is located in the omega loop. Introduced by mutagenesis in the ampC gene of SLS73, this substitution conferred the same level of resistance to ceftazidime. The catalytic efficiency (k(cat)/K(m)) of the mutated enzyme toward ceftazidime was increased by about 100-fold. CONCLUSIONS: We present another example of in vivo selection of broad-spectrum resistance by amino acid substitution in the omega loop of chromosomal AmpC beta-lactamase in S. marcescens.
Asunto(s)
Proteínas Bacterianas/genética , Ceftazidima/farmacología , Resistencia a las Cefalosporinas/genética , Serratia marcescens/efectos de los fármacos , Serratia marcescens/genética , beta-Lactamasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , beta-Lactamasas/químicaRESUMEN
Escherichia hermannii showed a low level of resistance to amoxicillin and ticarcillin, reversed by clavulanate, and a moderate susceptibility to piperacillin but was susceptible to all cephalosporins. A bla gene was cloned and encoded a typical class A beta-lactamase (HER-1, pI 7.5), which shares 45, 44, 41, and 40% amino acid identity with other beta-lactamases, AER-1 from Aeromonas hydrophila, MAL-1/Cko-1 from Citrobacter koseri, and TEM-1 and LEN-1, respectively. No ampR gene was detected. Only penicillins were efficiently hydrolyzed, and no hydrolysis was observed for cefuroxime and broad-spectrum cephalosporins. Sequencing of the bla gene in 12 other strains showed 98 to 100% identity with bla(HER-1).
Asunto(s)
Escherichia/enzimología , Escherichia/genética , beta-Lactamasas/genética , Secuencia de Aminoácidos , Antibacterianos/farmacología , ADN Bacteriano/genética , ADN Recombinante/genética , Escherichia/efectos de los fármacos , Focalización Isoeléctrica , Cinética , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Plásmidos/genética , beta-Lactamasas/metabolismoRESUMEN
Enterobacter cloacae Ecl261 was isolated with Escherichia coli Ec257 from the urine of a patient living in a nursing home. Both isolates were resistant to ticarcillin (MICs, 1,024 microg/ml), without significant potentiation of its activity by 2 microg of clavulanate per ml (MICs, 512 microg/ml), and susceptible to naturally active cephalosporins. This inhibitor-resistant phenotype was conferred in both strains by similar conjugative plasmids of 40 kb (Ecl261) and 30 kb (Ec257), which also conveyed resistance to sulfonamides and trimethoprim. Clinical and transconjugant strains produced a beta-lactamase with a pI of 5.2 which belonged to the TEM family, as indicated by specific PCR amplification. Compared with TEM-1, this enzyme exhibited lower catalytic efficiencies (14- and 120-fold less for amoxicillin and ticarcillin, respectively), and higher concentrations of beta-lactamase inhibitors were required to yield a 50% reduction in benzylpenicillin hydrolysis (750-, 82-, and 50-fold higher concentrations for clavulanate, sulbactam, and tazobactam, respectively). Gene sequencing revealed four nucleotide differences with the nucleotide sequence of bla(TEM-1A). The first replacement (T32C), located in the promoter region, was described as being responsible for the increase in the level of beta-lactamase production. The three other changes led to amino acid substitutions that define a new inhibitor-resistant TEM (IRT) beta-lactamase, TEM-80 (alternate name, IRT-24). Two of them, Met69Leu and Asn276Asp, have previously been related to inhibitor resistance. The additional mutation, Ile127Val, was demonstrated by site-directed mutagenesis to have a very weak effect, at least alone, on the IRT phenotype. This is the first description of an IRT beta-lactamase in E. cloacae. The horizontal transfer of bla(TEM-80) may have occurred either from Ec257 to Ecl261 or in the reverse order.
Asunto(s)
Enterobacter cloacae/enzimología , Inhibidores Enzimáticos/farmacología , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Anciano , Anciano de 80 o más Años , Farmacorresistencia Microbiana , Enterobacter cloacae/efectos de los fármacos , Infecciones por Enterobacteriaceae/microbiología , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , beta-Lactamas/farmacologíaRESUMEN
Desulfovibrio spp. are gram-negative anaerobes phylogenetically related to Bacteroides spp., which are rarely isolated and which are mostly isolated from intra-abdominal abscesses. Desulfovibrio desulfuricans clinical isolate D3 had a clavulanic acid-inhibited beta-lactam resistance profile and was resistant to some expanded-spectrum cephalosporins. A beta-lactamase gene, bla(DES-1), was cloned from whole-cell DNA of isolate D3 and expressed in Escherichia coli. Purified beta-lactamase DES-1, with a pI value of 9.1, had a relative molecular mass of ca. 31 kDa and a mature protein of 288 amino acids. DES-1 was distantly related to Ambler class A beta-lactamases and most closely related to PenA from Burkholderia pseudomallei (48% amino acid identity). It was weakly related to class A beta-lactamases CblA, CepA, CfxA, and CfxA2 from other anaerobic species, Bacteroides spp. and Prevotella intermedia. Its hydrolysis spectrum included amino- and ureidopenicillins, narrow-spectrum cephalosporins, ceftriaxone, and cefoperazone. bla(DES-1)-like genes were not identified in phylogenetically related Desulfovibrio fairfieldensis isolates. However, they were found in some but not all D. desulfuricans strains, thus suggesting that these genes may be present in a given D. desulfuricans subspecies.
Asunto(s)
Desulfovibrio/enzimología , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Desulfovibrio/efectos de los fármacos , Desulfovibrio/genética , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , beta-Lactamasas/química , beta-Lactamas/farmacologíaRESUMEN
Kluyvera ascorbata produces a beta-lactamase that results in an atypical susceptibility pattern, including low-level resistance to penicillins, cephalothin, and cefuroxime, but this resistance is reversed by clavulanate. Ten nucleotide sequences of the corresponding gene, bla(KLUA), were obtained and were found to have minor variations (96 to 100%). Otherwise, bla(KLUA) was found to be similar (95 to 100%) to some plasmid-encoded CTX-M-type beta-lactamases. Finally, mobilization of bla(KLUA) on a plasmid was found to be mediated probably by a genetic mobile element like ISEcp1.
Asunto(s)
Enterobacteriaceae/enzimología , Plásmidos/genética , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/genética , Genes Bacterianos/genética , Genotipo , Cinética , Pruebas de Sensibilidad Microbiana , Datos de Secuencia MolecularRESUMEN
OBJECTIVE: To understand the mechanisms and epidemiology of resistance to oxyiminocephalosporins in Escherichia coli over a 2-year period in a French hospital. METHODS: Forty-four strains, resistant or intermediately resistant to one of the oxyiminocephalosporins or aztreonam, were collected from 35 patients. MIC determinations were carried out for the 44 isolates using a panel of beta-lactam antibiotics, and characterization of the beta-lactamases they produced by isoelectric focusing and catalytic activity measurement. Extended-spectrum beta-lactamase production was studied by use of the double disk diffusion test. Conjugation experiments were used to search for plasmidic cephalosporinase. An epidemiologic study was then performed, by use of molecular typing of the strains with an ERIC-PCR method and a case-control analysis. RESULTS: Less than 1% of all the E. coli isolates at our hospital showed decreased susceptibility to oxyiminocephalosporins. Only three of the 44 isolates showed synergy between clavulanate and a third-generation cephalosporin and produced an extended-spectrum beta-lactamase. For the other strains, a beta-lactamase with a highly basic isoelectric point was detected. Spectrophotometric measures confirmed that most of these isolates were AmpC hyper-producers. No plasmidic cephalosporinase could be detected by conjugation experiments. Molecular typing showed all isolates to be different, except for two strains isolated in two patients of the same hospital unit, and for the repeated isolates of some patients. When 20 case patients were compared to 40 randomly selected control patients, prior receipt of an antimicrobial and more specifically of a beta-lactam agent was significantly associated with case patients. CONCLUSIONS: Although it appears to be very rare, the resistance to broad-spectrum cephalosporins needs our attention, because of the high frequency of E. coli infections and beta-lactam use in their treatment.