Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 13 de 13
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Molecules ; 26(18)2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-34576958

RESUMO

Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some ß-lactams. Significant reduction of kcat values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of TmB and TmD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some ß-lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2-OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues.

2.
Nutrients ; 13(8)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445055

RESUMO

BACKGROUND: We previously reported that severe COVID-19 patients had higher chances of survival and a reduced risk of developing respiratory failure when administered with the probiotic formulation SLAB51. This study aimed to investigate further bacteriotherapy mechanisms and how early they are activated. METHODS: We performed an analysis on the blood oxygenation parameters collected in sixty-nine severe COVID-19 patients requiring non-invasive oxygen therapy and presenting a CT lung involvement ≥50%. Twenty-nine patients received low-molecular-weight heparin, azithromycin and Remdesivir. In addition, forty subjects received SLAB51. Blood gas analyses were performed before the beginning of treatments and at 24 h. RESULTS: The patients receiving only standard therapy needed significantly increased oxygen amounts during the 24 h observation period. Furthermore, they presented lower blood levels of pO2, O2Hb and SaO2 than the group also supplemented with oral bacteriotherapy. In vitro data suggest that SLAB51 can reduce nitric oxide synthesis in intestinal cells. CONCLUSIONS: SARS-CoV-2 infected patients may present lesions in the lungs compromising their gas exchange capability. The functionality of the organs essential for these patients' survival depends mainly on the levels of pO2, O2Hb and SaO2. SLAB51 contains enzymes that could reduce oxygen consumption in the intestine, making it available for the other organs.


Assuntos
COVID-19/terapia , Oxigênio/uso terapêutico , Probióticos/uso terapêutico , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/uso terapêutico , Idoso , Alanina/análogos & derivados , Alanina/uso terapêutico , Antivirais/uso terapêutico , Azitromicina/uso terapêutico , Gasometria , Linhagem Celular , Feminino , Heparina , Humanos , Hipóxia , Itália , Pulmão , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos
3.
Artigo em Inglês | MEDLINE | ID: mdl-33722888

RESUMO

The Guiana extended-spectrum (GES) ß-lactamase GESG170H, GESG170L, and GESG170K mutants showed k cat, Km , and k cat/Km values very dissimilar to those of GES-1 and GES-5. The enhancement of the hydrolytic activity against carbapenems is potentially due to a shift of the substrate in the active site that provides better positioning of the deacylating water molecule caused by the presence of the imidazole ring of H170 and of the long side chain of K170 and L170.


Assuntos
Carbapenêmicos , Laboratórios , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Ácido Clavulânico/farmacologia , Hidrólise , beta-Lactamases/genética
4.
Int J Antimicrob Agents ; 57(1): 106228, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33246038

RESUMO

OBJECTIVE: This study aimed to investigate the in vitro activity of taniborbactam (VNRX-5133), a novel broad-spectrum bicyclic boronate, against NDM-1 and Q119E, Q119K, Q119C, Q119F, Q119V, and Q119Y NDM-1 variants, which showed an increased activity towards some ß-lactams, including cefepime. METHODS: Inhibition kinetic assays were spectrophotometrically performed using cefepime (50 µM) as the reporter substrate and 80 nM of each enzyme. Taniborbactam behaves as a competitive inhibitor towards NDM-1 and NDM-1 Q119 variants with lower Ki values (range 3-16 nM). The phenotypic profile was assessed in both Enterobacterales clinical isolates and engineered Escherichia coli BL21(DE3) strains by conventional broth microdilution procedures according to the Clinical and Laboratory Standards Institute (CLSI). RESULTS: Taniborbactam at a fixed concentration of 4 mg/L was able to restore activity of cefepime in 24 of 26 Enterobacterales clinical isolates harbouring metallo-ß-lactamases with MIC50/MIC90 values of 14 mg/L. Cefepime MICs were drastically reduced in all clinical isolates and in NDM-1 and Q119X producing Escherichia coli BL21(DE3). Taniborbactam was unable to restore susceptibility to cefepime in two IMP variants producing clinical isolates. CONCLUSION: The inhibition level of NDM enzymes provided by taniborbactam protects the antibacterial activity of cefepime from this important metallo-ß-lactamase.

5.
Molecules ; 25(19)2020 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-32998307

RESUMO

New Delhi Metallo-ß-lactamase-1 (NDM-1) is the most prevalent type of metallo-ß-lactamase, able to hydrolyze almost all antibiotics of the ß-lactam group, leading to multidrug-resistant bacteria. To date, there are no clinically relevant inhibitors to fight NDM-1. The use of dromedary polyclonal antibody inhibitors against NDM-1 represents a promising new class of molecules with inhibitory activity. In the current study, immunoreactivities of dromedary Immunoglobulin G (IgG) isotypes containing heavy-chain and conventional antibodies were tested after successful immunization of dromedary using increasing amounts of the recombinant NDM-1 enzyme. Inhibition kinetic assays, performed using a spectrophotometric method with nitrocefin as a reporter substrate, demonstrated that IgG1, IgG2, and IgG3 were able to inhibit not only the hydrolytic activity of NDM-1 but also Verona integron-encoded metallo-ß-lactamase (VIM-1) (subclass B1) and L1 metallo-ß-lactamase (L1) (subclass B3) with inhibitory concentration (IC50) values ranging from 100 to 0.04 µM. Investigations on the ability of IgG subclasses to reduce the growth of recombinant Escherichia coli BL21(DE3)/codon plus cells containing the recombinant plasmid expressing NDM-1, L1, or VIM-1 showed that the addition of IgGs (4 and 8 mg/L) to the cell culture was unable to restore the susceptibility of carbapenems. Interestingly, IgGs were able to interact with NDM-1, L1, and VIM-1 when tested on the periplasm extract of each cultured strain. The inhibitory concentration was in the micromolar range for all ß-lactams tested. A visualization of the 3D structural basis using the three enzyme Protein Data Bank (PDB) files supports preliminarily the recorded inhibition of the three MBLs.


Assuntos
Anticorpos/farmacologia , beta-Lactamases/imunologia , Animais , Camelus , Ensaios Enzimáticos , Feminino , Soros Imunes , Imunidade Humoral/efeitos dos fármacos , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Concentração Inibidora 50 , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , beta-Lactamases/química
6.
Microb Drug Resist ; 26(8): 976-981, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32101080

RESUMO

Antibiotic-resistant bacteria (ARB) are widespread in nature and represent a serious public and environmental problem. In the present study, we report for the first time the presence of bacterial ß-lactamases in two macroinvertebrate species with different feeding traits. The class A ß-lactamases, SHV-1 and TEM-1, were found in Citrobacter freundii isolated from Gammarus elvirae and Escherichia coli from water samples, respectively. The metallo-ß-lactamase CphA was found in Aeromonas veronii and Aeromonas hydrophila strains isolated from the predator Dina lineata. The presence of a large plasmid was ascertained only in E. coli strains isolated from water. In all strains studied, an integrase I typical of class I integrin was found. In contaminated freshwater habitats, ARB and antibiotic resistance genes could be disseminated through trophic links with important ecological implications. Transmission through the food chain may contribute to spreading and transferring antibiotic resistance not only in freshwater ecosystems but also outside the aquatic habitat.


Assuntos
Aeromonas/isolamento & purificação , Antibacterianos/farmacologia , Citrobacter freundii/isolamento & purificação , Resistência Microbiana a Medicamentos/fisiologia , Escherichia coli/isolamento & purificação , Invertebrados/microbiologia , Aeromonas/efeitos dos fármacos , Aeromonas/genética , Animais , Proteínas de Bactérias/isolamento & purificação , Citrobacter freundii/efeitos dos fármacos , Citrobacter freundii/genética , Crustáceos/microbiologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Resistência Microbiana a Medicamentos/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Integrons/genética , Itália , Sanguessugas/microbiologia , Testes de Sensibilidade Microbiana , Plasmídeos , Rios , beta-Lactamases/isolamento & purificação
7.
Diagn Microbiol Infect Dis ; 96(3): 114968, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31924425

RESUMO

Klebsiella pneumoniae strain is an important opportunistic pathogen that causes severe nosocomial infections. In the present study a molecular characterization of carbapenem resistant K. pneumoniae, isolated from blood samples of hospitalized patients of Verona University Hospital, was performed. The simultaneous presence of SHV-1/CTX-M-15/KPC-3 and SHV-1/CTX-M-15/OXA-48 serin-ß-lactamases was ascertained in the 89% and 11% of K. pneumoniae ST512 and K. pneumoniae ST14, respectively. Molecular characterization of bla genes showed that blaKPC-3 was found in Tn4401a transposon with the tnpR, tnpA, ISKpn6, and ISKpn7 mobile elements whereas blaCTX-M-15 was detected downstream ISEcp1 genetic element. A class 1 integron with a gene cassette of 780 bp corresponding to aadA2 gene was identified in 33 K. pneumoniae ST512 isolates.


Assuntos
Proteínas de Bactérias/biossíntese , Infecções por Klebsiella/sangue , Klebsiella pneumoniae/genética , Centros de Atenção Terciária/estatística & dados numéricos , beta-Lactamases/biossíntese , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla , Variação Genética , Hospitais Universitários/estatística & dados numéricos , Humanos , Unidades de Terapia Intensiva/estatística & dados numéricos , Itália , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Testes de Sensibilidade Microbiana , Quartos de Pacientes/estatística & dados numéricos , beta-Lactamases/genética
8.
Microb Drug Resist ; 25(7): 1041-1049, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30994417

RESUMO

The main goal of this study was to identify Gram-negative bacteria resistant to antibiotics, in particular ß-lactams, in stream waters and effluents from urban wastewater treatment plants draining into Fino, Tavo, and Saline rivers of the Abruzzo region, Italy. Eight sampling sites were selected because they were the most contaminated by coliforms during previous sampling campaign. One sample for each site was collected for the detection of total and fecal coliforms, Escherichia coli and Enterococcus species by Colilert-18 and Enterolert-E Quanti-Tray/2000. Antibiotic-resistant bacteria, selected on ampicillin and cefotaxime-supplemented agar plates, were identified by EnteroPluri test systems and then confirmed by MALDI-TOF. The resistant determinants were identified and characterized by PCR and sequencing. The microbiological analysis allowed to detect E. coli, total coliforms, fecal coliforms, and enterococci with a coefficient of variation of 215.7%, 212.8%, 242.5%, and 188.5%, respectively. Several Gram-negative bacteria were identified: Serratia liquefaciens, E. coli, Enterobacter cloacae, Citrobacter freundii, Raoultella ornithinolytica, Acinetobacter johnsonii, Aeromonas veronii, Aeromonas hydrophila, and Pseudomonas koreensis. All strains possessed class 1 integrons, insertion sequences, and genes encoding for serin- and metallo-ß-lactamases. Extended-spectrum ß-lactamases, such as CTX-M-15 and CTX-M-27, were found in Enterobacteriaceae, whereas CphA metallo-ß-lactamase was found in A. veronii and A. hydrophila. The main resistance's mechanism to ß-lactams observed among the analyzed strains is represented by the production of serin ß-lactamases (CTX-M-15, CTX-M-27, and SHV-1) and metallo ß-lactamase (CphA).


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/imunologia , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Rios/microbiologia , beta-Lactamases/genética , Ampicilina/uso terapêutico , Antibacterianos/uso terapêutico , Cefotaxima/uso terapêutico , Fezes/microbiologia , Humanos , Integrons/genética , Itália , Testes de Sensibilidade Microbiana/métodos
9.
Artigo em Inglês | MEDLINE | ID: mdl-30917978

RESUMO

The New Delhi metallo-ß-lactamase-1 (NDM-1) enzyme is the most common metallo-ß-lactamase identified in many Gram-negative bacteria causing severe nosocomial infections. The aim of this study was to focus the attention on non-active-site residues L209 and Y229 of NDM-1 and to investigate their role in the catalytic mechanism. Specifically, the effect of the Y229W substitution in the L209F variant was evaluated by antimicrobial susceptibility testing, kinetic, and molecular dynamic (MD) studies. The Y229W single mutant and L209F-Y229W double mutant were generated by site-directed mutagenesis. The Km , k cat, and k cat/Km kinetic constants, calculated for the two mutants, were compared with those of (wild-type) NDM-1 and the L209F variant. Compared to the L209F single mutant, the L209F-Y229W double mutant showed a remarkable increase in k cat values of 100-, 240-, 250-, and 420-fold for imipenem, meropenem, benzylpenicillin, and cefepime, respectively. In the L209F-Y229W enzyme, we observed a remarkable increase in k cat/Km of 370-, 140-, and 80-fold for cefepime, meropenem, and cefazolin, respectively. The same behavior was noted using the antimicrobial susceptibility test. MD simulations were carried out on both L209F and L209F-Y229W enzymes complexed with benzylpenicillin, focusing attention on the overall mechanical features and on the differences between the two systems. With respect to the L209F variant, the L209F-Y229W double mutant showed mechanical stabilization of loop 10 and the N-terminal region. In addition, Y229W substitution destabilized both the C-terminal region and the region from residues 149 to 154. The epistatic effect of the Y229W mutation jointly with the stabilization of loop 10 led to a better catalytic efficiency of ß-lactams. NDM numbering is used in order to facilitate the comparison with other NDM-1 studies.


Assuntos
Substituição de Aminoácidos/genética , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Cefalosporinas/farmacologia , Mutação/genética , Penicilinas/farmacologia , beta-Lactamases/genética , Substituição de Aminoácidos/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/genética , Hidrólise/efeitos dos fármacos , Cinética , Testes de Sensibilidade Microbiana/métodos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida/métodos , Mutação/efeitos dos fármacos
10.
Artigo em Inglês | MEDLINE | ID: mdl-29941651

RESUMO

TEM-184, a novel TEM-derived extended-spectrum ß-lactamase (ESBL), was isolated from an Escherichia coli ST354 clinical strain. Compared to TEM-1, TEM-184 contains the mutations Q6K, E104K, I127V, R164S, and M182T. Kinetic analysis of this enzyme revealed extended-spectrum activity against aztreonam in particular. TEM-184 was also susceptible to inhibitors, including clavulanic acid, tazobactam, and avibactam.


Assuntos
Antibacterianos/farmacologia , Aztreonam/farmacologia , beta-Lactamases/genética , Sequência de Aminoácidos , Compostos Azabicíclicos/farmacologia , Ácido Clavulânico/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Humanos , Cinética , Testes de Sensibilidade Microbiana/métodos , Alinhamento de Sequência , Tazobactam/farmacologia , Inibidores de beta-Lactamases/farmacologia
11.
Artigo em Inglês | MEDLINE | ID: mdl-29507065

RESUMO

GES-type ß-lactamases are a group of enzymes that have evolved their hydrolytic activity against carbapenems. In this study, the role of residue 174 inside the Ω-loop of GES-1 and GES-5 was investigated. GES-1P174E and GES-5P174E mutants, selected by site saturation mutagenesis, were purified and kinetically characterized. In comparison with GES-1 and GES-5 wild-type enzymes, GES-1P174E and GES-5P174E mutants exhibited lower kcat and kcat/Km values for cephalosporins and penicillins. Concerning carbapenems, GES-1P174E shared higher kcat values but lower Km values than those calculated for GES-1. The GES-1P174E and GES-5P174E mutants showed high hydrolytic efficiency for imipenem, with kcat/Km values 100- and 660-fold higher, respectively, than those of GES-1. Clavulanic acid and tazobactam are good inhibitors for both GES-1P174E and GES-5P174E Molecular dynamic (MD) simulations carried out for GES-1, GES-5, GES-1P174E, and GES-5P174E complexed with imipenem and meropenem have shown that mutation at position 174 induces a drastic increase of enzyme flexibility, in particular in the Ω-loop. The circular dichroism (CD) spectroscopy spectra of the four enzymes indicate that the P174E substitution in GES-1 and GES-5 does not affect the secondary structural content of the enzymes.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Imipenem/farmacologia , Meropeném/farmacologia , beta-Lactamases/genética , Proteínas de Bactérias/antagonistas & inibidores , Cefalosporinas/farmacologia , Dicroísmo Circular , Ácido Clavulânico/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Penicilinas/farmacologia , Estrutura Secundária de Proteína/genética , Tazobactam/farmacologia , beta-Lactamases/metabolismo
12.
J Glob Antimicrob Resist ; 10: 95-100, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28711560

RESUMO

OBJECTIVES: The aim of this study was to evaluate the role of residue 238 in CTX-M-15 and CTX-M-15G238C mutant with respect to carbapenems and various ß-lactamase inhibitors. METHODS: A CTX-M-15G238C laboratory mutant was generated by site-directed mutagenesis from CTX-M-15 enzyme by replacing glycine 238 with cysteine. Thiol titration and p-chloromercuribenzoate (PCMB) inactivation assays were used to ascertain the presence of a disulfide bridge in the active site of CTX-M-15G238C. Kinetic parameters were determined both for CTX-M-15 and CTX-M-15G238C enzymes by analysing either the complete hydrolysis time courses or under initial rate conditions. RESULTS: In CTX-M-15G238C mutant, the two cysteines (C69 and C238) located in the enzyme active site were unable to form a disulfide bridge. CTX-M-15 and thermostable CTX-M-15G238C were used to study the kinetic interaction with carbapenems, which behaved as poor substrates for both enzymes. Meropenem and ertapenem acted as transient inactivators for CTX-M-15 and CTX-M-15G238C, and for these compounds the variation of kobs versus the inactivator concentration was linear. Imipenem behaved as a transient inactivator for CTX-M-15 and as an inactivator (with k+3=0) for CTX-M-15G238C. In any case, the k+2/K values for CTX-M-15G238C were higher than those for CTX-M-15. CONCLUSIONS: Compared with CTX-M-15, CTX-M-15G238C mutant appears to have a more favourable conformation for carbapenem acylation and higher activity against cefotaxime, which could be due to the presence of free -SH groups in the enzyme active site.


Assuntos
Carbapenêmicos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/efeitos dos fármacos , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/genética , Cefotaxima/farmacologia , Clonagem Molecular , Interações Medicamentosas , Ativação Enzimática/genética , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/genética , Escherichia coli/genética , Imipenem/farmacologia , Cinética , Mutagênese Sítio-Dirigida , Conformação Proteica , Análise de Sequência de Proteína , beta-Lactamases/genética
13.
Antimicrob Agents Chemother ; 60(5): 3123-6, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26883708

RESUMO

Site-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-ß-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.


Assuntos
Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , beta-Lactamases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Prolina/química , Prolina/metabolismo , Análise de Sequência de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato/genética , Especificidade por Substrato/fisiologia , Zinco/farmacologia , beta-Lactamases/química , beta-Lactamases/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...