RESUMO
Carbapenems are "last resort" ß-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-ß-lactamases (MßLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all ß-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MßLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MßLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different ß-lactams in all MßLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MßLs.
Assuntos
Antibacterianos/química , Ceftazidima/química , Imipenem/química , Meropeném/química , Zinco/química , beta-Lactamases/química , Sequência de Aminoácidos , Antibacterianos/metabolismo , Domínio Catalítico , Cefepima/química , Cefepima/metabolismo , Cefotaxima/química , Cefotaxima/metabolismo , Ceftazidima/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Imipenem/metabolismo , Cinética , Meropeném/metabolismo , Modelos Moleculares , Piperacilina/química , Piperacilina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Zinco/metabolismo , Resistência beta-Lactâmica , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
INTRODUCTION: Expanded-spectrum betalactamases (ESBLs) are the main source of resistance to oxyimino cephalosporins and monobactams in Enterobacteriaceae. Most of them derive from TEM or SHV, however the incidence of other families like CTX-M, OXA and PER has increased. In Argentina, the most frequent ESBL in Enterobacteriaceae is CTX-M-2. This specific circumstance, which differs from the situation in the Northern Hemisphere, motivated us to study new diagnostic strategies for the detection of ESBLs in our region. METHOD: Microbiological ESBL detection was performed by double-disk synergy tests, cefotaxime and ceftazidime disks with and without clavulanic acid (NCCLS), and cefotaxime and ceftazidime disks in Müeller-Hinton agar supplemented with lithium clavulanate (MH-cla). Betalactamases were characterized by isoelectric focusing, hydrolysis profile and PCR amplification. RESULTS: Among 575 clinical isolates of Enterobacteriaceae, 14% were oxyimino cephalosporin-resistant. Two different ESBLs were detected in 31 resistant strains: CTX-M-2 (28) and PER-2 groups (3). The double-disk synergy test was the least sensitive method for ESBL detection. ESBLs were detected by the other two methods in all isolates with the use of cefotaxime disks, but not with ceftazidime disks. CONCLUSION: The microbiological method employing MH-cla with cefotaxime disks had a sensitivity and specificity comparable to the referral test using the same antibiotic proposed by the NCCLS for the detection of ESBLs.
Assuntos
Proteínas de Bactérias/análise , Cefalosporinas/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/enzimologia , Testes de Sensibilidade Microbiana/métodos , Monobactamas/metabolismo , Resistência beta-Lactâmica , beta-Lactamases/análise , Argentina/epidemiologia , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Cefotaxima/metabolismo , Cefotaxima/farmacologia , Ceftazidima/metabolismo , Ceftazidima/farmacologia , Cefalosporinas/classificação , Cefalosporinas/farmacologia , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/epidemiologia , Humanos , Hidrólise , Focalização Isoelétrica , Monobactamas/classificação , Monobactamas/farmacologia , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Especificidade por Substrato , Resistência beta-Lactâmica/genética , beta-Lactamases/classificação , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
INTRODUCTION: The aim of this study was to evaluate betalactam resistance within the genus Proteus and characterize the betalactamases responsible for this resistance. METHODS: We analyzed 99 strains (87, P. mirabilis; 10 P. vulgaris, and 2, P. penneri) isolated from patients at one University Hospital. Antibiotic susceptibility tests were performed according to NCCLS recommendations. Presence of extended spectrum betalactamases (ESBL) was inferred by both double disk diffusion tests and minimum inhibitory concentration (MIC) of third and fourth generation cephalosporins alone and in the presence of clavulanic acid. Isoelectric points (pI) of the enzymes were estimated by isoelectrofocusing and the presence of the encoding genes was confirmed by polymerase chain reaction (PCR). RESULTS: A broad spectrum betalactamase could be detected in those isolates (28%) resistant to penicillin and first generation cephalosporins while CTX-M-2 enzyme could be detected in P. mirabilis isolates resistant to third and fourth generation cephalosporins (18%). One of the P. vulgaris displayed reduced susceptibility to cefotaxime due to an enzyme of pI 7.4, while resistance to cefotaxime in one P. penneri was related to an enzyme of pI 6.8. Both enzymes were active on cefotaxime (1,000 mg/l) in the iodometric assay. CONCLUSION: The broad extended spectrum betalactamase within genus Proteus was TEM-1, while CTX-M-2 was the ESBL responsible for the third and fourth generation cephalosporins in P. mirabilis. In P. vulgaris and P. penneri this resistance was associated with the hyperproduction of the chromosomal encoded betalactamase.
Assuntos
Proteínas de Bactérias/genética , Cefalosporinas/farmacologia , Proteus/efeitos dos fármacos , Resistência beta-Lactâmica , beta-Lactamases/genética , Argentina/epidemiologia , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cefotaxima/metabolismo , Cefotaxima/farmacologia , Cefalosporinas/classificação , Cefalosporinas/metabolismo , Cromossomos Bacterianos/genética , Genes Bacterianos , Genótipo , Humanos , Ponto Isoelétrico , Testes de Sensibilidade Microbiana , Fenótipo , Proteus/enzimologia , Proteus/genética , Infecções por Proteus/epidemiologia , Infecções por Proteus/microbiologia , Proteus mirabilis/efeitos dos fármacos , Proteus mirabilis/enzimologia , Proteus mirabilis/genética , Proteus penneri/efeitos dos fármacos , Proteus penneri/enzimologia , Proteus penneri/genética , Proteus vulgaris/efeitos dos fármacos , Proteus vulgaris/enzimologia , Proteus vulgaris/genética , Especificidade da Espécie , Resistência beta-Lactâmica/genética , beta-Lactamases/isolamento & purificação , beta-Lactamases/metabolismoRESUMO
From a collection of cefotaxime-resistant Klebsiella pneumoniae isolated from neonatal blood culture specimens in a maternity hospital in Aracaju, Brazil, two isolates (strains KPBRZ-842 and -843, indistinguishable by pulsed-field gel electrophoresis) were found to produce beta-lactamases with isoelectric points (pI) of 5.4 and 8.2, respectively. Using a gel overlay method, cefotaxime hydrolysis was shown to be associated with the pI 8.2 protein. Nucleotide sequencing of the gene encoding the pI 8.2 beta-lactamase revealed a bla(SHV-ESBL)-type gene differing from the gene encoding SHV-1 by three silent point mutations, and a fourth that resulted in an amino acid substitution, aspartate for glycine, at position 156. This novel SHV-type extended-spectrum beta-lactamase is designated SHV-27.
Assuntos
Antibacterianos/metabolismo , Cefotaxima/metabolismo , Resistência Microbiana a Medicamentos/genética , Klebsiella pneumoniae/enzimologia , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Aztreonam/farmacologia , Brasil , Cefotaxima/farmacologia , Ceftazidima/farmacologia , Análise Mutacional de DNA , Eletroforese em Gel de Campo Pulsado , Hospitais , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação Puntual/genética , Polimorfismo de Fragmento de Restrição , beta-Lactamases/isolamento & purificaçãoRESUMO
Escherichia coli R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin, and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem. This particular strain contained three different plasmids that encoded two beta-lactamases with pIs of 7.0 and 9.0. Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and sulfamethoxazole was transferred by conjugation from E. coli R170 to E. coli J53-2. The transferred plasmid, RZA92, which encoded a single beta-lactamase, was 150 kb in length. The cefotaxime resistance gene that encodes the TLA-1 beta-lactamase (pI 9.0) was cloned from the transconjugant by transformation to E. coli DH5alpha. Sequencing of the bla(TLA-1) gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to class A beta-lactamases: (70)SXXK, (130)SDN, and (234)KTG. The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A beta-lactamase from Chryseobacterium (Flavobacterium) meningosepticum; 48.8% identity with the VEB-1 class A beta-lactamase from E. coli; 40 to 42% identity with CblA of Bacteroides uniformis, PER-1 of Pseudomonas aeruginosa, and PER-2 of Salmonella typhimurium; and 39% identity with CepA of Bacteroides fragilis. The partially purified TLA-1 beta-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime. The enzyme was markedly inhibited by sulbactam, tazobactam, and clavulanic acid. TLA-1 is a new extended-spectrum beta-lactamase of Ambler class A.
Assuntos
Escherichia coli/enzimologia , Escherichia coli/genética , Plasmídeos/genética , beta-Lactamases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cefotaxima/metabolismo , Cefalosporinas/farmacologia , Clonagem Molecular , Conjugação Genética/genética , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/microbiologia , Humanos , Focalização Isoelétrica , Cinética , México , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , beta-Lactamases/genética , beta-Lactamases/isolamento & purificaçãoRESUMO
The frequency of enterobacterial isolates with high resistance to expanded-spectrum beta-lactam antibiotics (mainly cefotaxime or ceftriaxone) has increased notoriously in Argentina, mainly because of the spread of extended-spectrum beta-lactamases. The aim of this work was the study of extended-spectrum beta-lactamases in several Morganella morganii isolates with unusually high resistance to ceftriaxone. These strains produced at least two beta-lactamases, of apparent pIs of 5.4 and 8.2, molecular weight 23 000, well inhibited by clavulanate, compatible with a broad-spectrum beta-lactamase - perhaps TEM-1 - and an extended-spectrum beta-lactamase, respectively. The extended-spectrum beta-lactamase was identified as a CTX-M-type beta-lactamase - probably CTX-M-2 - by polymerase chain reaction, restriction profile analysis and DNA-DNA hybridisation. The remaining isolates studied produced either the broad-spectrum beta-lactamase plus the ubiquitous AmpC beta-lactamase (13 strains), or the AmpC beta-lactamase only (10 strains).
Assuntos
Cefotaxima/metabolismo , Cefalosporinas/metabolismo , Morganella morganii/enzimologia , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Hidrólise , Testes de Sensibilidade Microbiana , Morganella morganii/efeitos dos fármacosRESUMO
With the simultaneous use of an isoelectric focusing gel (IEF) and a nitrocefin/cefotaxime bioassay, it is possible to identify the Extended-Spectrum beta-lactamases (ESBL) with precision. A mixture of soft agar and susceptible bacterial cells are layered over the gel following overnight incubation and areas of cell growth are detected where the antibiotic has been hydrolyzed by specific enzymes. This innovative method improves sensitivity and specificity for the identification of ESBLs in those enterobacteria strains producing more than one beta-lactamase and are resistant to third generation cephalosporines.
Assuntos
Proteínas de Bactérias/análise , Bioensaio , Enterobacteriaceae/enzimologia , Focalização Isoelétrica , Testes de Sensibilidade Microbiana , Resistência beta-Lactâmica , beta-Lactamases/análise , Proteínas de Bactérias/metabolismo , Cefotaxima/metabolismo , Cefalosporinas/metabolismo , Escherichia coli/enzimologia , Klebsiella pneumoniae/enzimologia , Sensibilidade e Especificidade , beta-Lactamases/metabolismoRESUMO
We evaluated the concentration of cefotaxime in the exudate from ischemic leg ulcers in 9 subjects with severe obstructive arterial disease. The administration of the drug was either systemic (1 g in 250 ml of saline given in 30 min) or regional at a vein of the foot while a tourniquet was applied at the level of the thigh during 30 min. Hygroscopic discs were used to collect samples of ulcer exudate at hourly intervals for 4 hr. Cefotaxime concentration was determined by HPL chromatography. A significantly greater concentration of antibiotic was obtained with regional as compared to systemic administration (46 +/- 16 vs 25 +/- 14, p less than 0.01) and a greater percentage of patients attained MIC 90. A stable concentration of the drug was observed during the 4 hr period indicating a decreased rate of elimination of the antibiotic from the ulcer tissue. Thus, regional administration of antibiotics affords greater concentration than systemic administration, for treatment of ischemic leg ulcers.
Assuntos
Cefotaxima/metabolismo , Complicações do Diabetes , Exsudatos e Transudatos/metabolismo , Úlcera da Perna/metabolismo , Adulto , Idoso , Cefotaxima/administração & dosagem , Cefalosporinas/administração & dosagem , Cefalosporinas/metabolismo , Diabetes Mellitus/metabolismo , Feminino , Humanos , Infusões Intravenosas , Úlcera da Perna/tratamento farmacológico , Masculino , Pessoa de Meia-IdadeRESUMO
The pharmacokinetics of ceftriaxone was studied in the plasma, urine, and cerebrospinal fluid of seven neonates and seven infants with meningitis. In addition, plasma and urine data were obtained in five neonates and one infant receiving ceftriaxone for other serious infections. All neonates younger than 14 days received daily doses of 50 mg/kg ceftriaxone; all other patients but two received 100 mg/kg. The average weight-corrected values for total body clearance (ClT), volume of distribution (Vdss), and biologic half-life (t 1/2) were 0.37 ml/min/kg, 0.45 L/kg, and 16.2 hours in neonates younger than 1 week; 0.77 ml/min/kg, 0.48 L/kg, and 9.2 hours in neonates older than 1 week; and 1.03 ml/min/kg, 0.39 L/kg, and 7.1 hours in older infants, respectively. There was a significant difference in ClT and t 1/2 between the neonates younger and both neonates older than 1 week, and infants. The Vdss was not significantly different among the three age groups. The average renal clearance in neonates younger than 1 week (0.28 ml/min/kg was 70%, in neonates older than 1 week (0.54 ml/min/kg) was 77%, and in older infants (0.49 ml/min/kg) was 47% of ClT, indicating that nonrenal elimination was less developed in neonates. The quantitation of CSF diffusion of ceftriaxone was assessed by comparison of the areas under the CSF and plasma concentration-time curve. The mean ceftriaxone penetration into the CSF in neonates and infants with bacterial meningitis was 17%. On the other hand, penetration in patients with aseptic meningitis amounted to only 4%. Mean ceftriaxone concentrations in the CSF in patients with bacterial meningitis were 2.8 mg/L after 24 hours, exceeding by many times the minimum inhibitory concentration of the common meningitis pathogens at this time.
Assuntos
Cefotaxima/análogos & derivados , Meningite/tratamento farmacológico , Cefotaxima/sangue , Cefotaxima/líquido cefalorraquidiano , Cefotaxima/metabolismo , Cefotaxima/uso terapêutico , Cefotaxima/urina , Ceftriaxona , Humanos , Lactente , Recém-Nascido , Cinética , Taxa de Depuração MetabólicaAssuntos
Cefotaxima/análogos & derivados , Ventrículos Cerebrais , Encefalite/metabolismo , Cefotaxima/metabolismo , Cefotaxima/uso terapêutico , Ceftriaxona , Criança , Pré-Escolar , Encefalite/tratamento farmacológico , Humanos , Lactente , Recém-Nascido , Cinética , Meningite/tratamento farmacológico , Meningite/metabolismoRESUMO
Ceftriaxone has greater in vitro and in vivo efficacy against many common bacteria than other third-generation cephalosporins. Single-dose ceftriaxone pharmacokinetics were studied in 17 patients, aged 0.6 to 52 months, with infections of the central nervous system. Patients received a randomized dose of 50 or 75 mg/kg ceftriaxone intravenously over 5 minutes on the second to fifth day of illness. Serial blood samples were collected over 24 hours in all patients, and cerebrospinal fluid (CSF) was obtained 1 to 4.5 hours after injection. Ceftriaxone mean peak plasma concentrations, determined by high-power liquid chromatography, were 267 and 184 microgram/ml for the 75 and 50 mg/kg dosage groups, respectively. The harmonic mean elimination half-life was 4.2 hours, and the mean percent drug penetrance into CSF was 4.8 +/- 3.5%. Of CSF studies evaluated, the glucose concentration was correlated most closely (inversely) with CSF penetration of ceftriaxone. Individual CSF concentrations of ceftriaxone exceeded the minimal inhibitory concentrations of the respective bacteria causing infection by 480 to 5,600 times. Ceftriaxone may be useful in the treatment of serious pediatric infections, including meningitis.