Enhancement of antibiotics antimicrobial activity due to the silver nanoparticles impact on the cell membrane.

The ability of microorganisms to generate resistance outcompetes with the generation of new and efficient antibiotics; therefore, it is critical to develop novel antibiotic agents and treatments to control bacterial infections. An alternative to this worldwide problem is the use of nanomaterials with antimicrobial properties. Silver nanoparticles (AgNPs) have been extensively studied due to their antimicrobial effect in different organisms. In this work, the synergistic antimicrobial effect of AgNPs and conventional antibiotics was assessed in Gram-positive and Gram-negative bacteria. AgNPs minimal inhibitory concentration was 10-12 µg mL-1 in all bacterial strains tested, regardless of their different susceptibility against antibiotics. Interestingly, a synergistic antimicrobial effect was observed when combining AgNPs and kanamycin according to the fractional inhibitory concentration index, FICI: <0.5), an additive effect by combining AgNPs and chloramphenicol (FICI: 0.5 to 1), whereas no effect was found with AgNPs and ß-lactam antibiotics combinations. Flow cytometry and TEM analysis showed that sublethal concentrations of AgNPs (6-7 µg mL-1) altered the bacterial membrane potential and caused ultrastructural damage, increasing the cell membrane permeability. No chemical interactions between AgNPs and antibiotics were detected. We propose an experimental supported mechanism of action by which combinatorial effect of antimicrobials drives synergy depending on their specific target, facilitated by membrane alterations generated by AgNPs. Our results provide a deeper understanding about the synergistic mechanism of AgNPs and antibiotics, aiming to combat antimicrobial infections efficiently, especially those by multi-drug resistant microorganisms, in order to mitigate the current crisis due to antibiotic resistance.

Synthesis and Complete Antimicrobial Characterization of CEOBACTER, an Ag-Based Nanocomposite.

The antimicrobial activity of silver nanoparticles (AgNPs) is currently used as an alternative disinfectant with diverse applications, ranging from decontamination of aquatic environments to disinfection of medical devices and instrumentation. However, incorporation of AgNPs to the environment causes collateral damage that should be avoided. In this work, a novel Ag-based nanocomposite (CEOBACTER) was successfully synthetized. It showed excellent antimicrobial properties without the spread of AgNPs into the environment. The complete CEOBACTER antimicrobial characterization protocol is presented herein. It is straightforward and reproducible and could be considered for the systematic characterization of antimicrobial nanomaterials. CEOBACTER showed minimal bactericidal concentration of 3 µg/ml, bactericidal action time of 2 hours and re-use capacity of at least five times against E. coli cultures. The bactericidal mechanism is the release of Ag ions. CEOBACTER displays potent bactericidal properties, long lifetime, high stability and re-use capacity, and it does not dissolve in the solution. These characteristics point to its potential use as a bactericidal agent for decontamination of aqueous environments.

Overexpression and purification of Rhizobium etli glutaminase A by recombinant and conventional procedures.

Rhizobium etli glutaminase A was purified to homogeneity by conventional procedures that included ammonium sulfate differential precipitation, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration, and dye-ligand chromatography. Alternatively, the structural glsA gene that codifies for glutaminase A was amplified by PCR and cloned in the expression vector pTrcHis. The recombinant protein was purified to homogeneity by affinity chromatography. This protein showed the same kinetic properties as native glutaminase A (K(m) for glutamine of 1.5 mM and V(max) of 80 micromol ammonium min(-1) mg protein(-1)). Physicochemical and biochemical properties of native and recombinant glutaminase were identical. The molecular mass of recombinant glutaminase A (M(r) 106.8 kDa) and the molecular mass of the subunits (M(r) 26.9 kDa) were estimated by mass spectrometry. These results suggest that R. etli glutaminase A is composed of four identical subunits. The high-level production of recombinant glutaminase A elevates the possibilities for determination of its three-dimensional structure through X-ray crystallography.

Sequence and molecular analysis of the Rhizobium etli glsA gene, encoding a thermolabile glutaminase.

We sequenced a 2.1 kb fragment of DNA carrying the structural glsA gene, which codes for the Rhizobium etli thermolabile glutaminase (A). The glsA gene complements the R. etli LM16 mutant that lacks glutaminase A activity, and is expressed in the heterologous host Sinorhizobium meliloti. The deduced amino acid sequence consists of 309 residues, with a calculated molecular mass of 33 kDa. The amino acid sequence shares 53% and 43% identity with two hypothetical glutaminases of E. coli; 42% identity with liver-type; 38% identity with kidney-type glutaminase; 41% and 40% identity hypothetical glutaminases of Bacillus subtilis; and 41% and 37% identity with two putative glutaminases of Caenorhabditis elegans. The glsA gene represents the first glutaminase gene cloned and sequenced in prokaryotes.

Identification of two glutaminases in Rhizobium etli.

We present evidence that Rhizobium etli has two glutaminases differentiated by their thermostability and electrophoretic mobility. The thermostable glutaminase (B) is constitutive, in contrast with the thermolabile glutaminase (A), which is positively regulated by glutamine and negatively regulated by ammonium and by the carbon source. In distinction to glutaminase A, glutaminase B plays a minor role in the utilization of glutamine as a carbon source, but it may play a role in maintaining the balance of glutamine and glutamate. By complementation of the Rhizobium etli LM16 mutant that lacks glutaminase A, we have cloned the gene that codes for this enzyme.