Klebsiella pneumoniae (K. pneumoniae) is an opportunistic bacterium that has drawn attention due to its resistance to carbapenem antibiotics. The treatment of patients with severe infections has been challenging. Thus, silver nanoparticles (AgNPs) have been applied for their antimicrobial effects. This work aims to analyze the synergistic effect of the carbapenem antibiotic Imipenem with AgNPs against different susceptibility clinical profiles of K. pneumoniae. The silver nanoparticles were synthesized by bottom-up methodology and capped with alpha-lipoic acid. Susceptibility tests were performed using four K. pneumoniae strains with different susceptibility profiles to Imipenem. The strains were induced to form a biofilm for 48 h. Crystal violet and Resazurin assays were performed to determine biofilm formation and minimal inhibitory concentration, respectively. The reduction in Imipenem concentration with the association of nanoparticles was found in all strains studied in planktonic form, and the synergism between silver nanoparticles and Imipenem was demonstrated through the analysis of the fractional inhibitory concentration index. The viability percentage was reduced at rates ≥80% in the biofilm analysis, characterized by the minimal biofilm inhibitory concentration. The study’s proposed association resulted in inhibitory effects on different K. pneumoniae profiles, both in planktonic forms and biofilm, with peculiar behavior in the Imipenem-resistant profile.

In recent years FT-IR microspectroscopy has been developed for microbiology analysis and applied successfully in pure cultures of microorganisms to rapidly identify strains of bacteria, yeasts and fungi. The investigation and characterization of microorganism mixed cultures is also of growing importance, especially in hospitals where it is common to poly-microbial infections. In this work, the rapid identification of bacteria in pure and mixed cultures was studied. The bacteria were obtained from the Institute Oswaldo Cruz culture collection at Brazil. Escherichia coli ATCC 10799 and Staphylococcus aureus ATCC 14456 were analyzed, 3 inoculations were examined in triplicate: Escherichia coli, Staphylococcus aureus and a mixed culture of them. The inoculations were prepared according to McFarland 0.5, incubated at 37 ° C for 6 hours, diluted in saline, placed in the CaF2 window and store for one hour at 50°C to obtain thin film. The measurement was performed by Spectrum Spotlight 400 (Perkin-Elmer) equipment in the range of 4000-900 cm^-1, with 32 scans using a transmittance technique with point and image modes. The data were processed (baseline, normalization, calculation of first derivate followed by smoothing with 9 point using a Savitzky-Golay algorithm) and a cluster analysis were done by Ward's algorithm and an excellent discrimination between pure and mixed culture was obtained. Our preliminary results indicate that the FT-IR microspectroscopy associated with cluster analysis can be used to discriminate between pure and mixed culture.

Introduction: Nanotechnology has been studied for several years for its applications in medicine, particularly silver nanoparticles (AgNPs), which have demonstrated excellent antimicrobial results and have been proposed as an alternative to combat bacterial resistance to antibiotic therapy. However, the development of bacterial resistance to AgNPs has been minimally explored, and bacteria of medical and dental interest have already been reported as resistant, raising concerns about their efficacy. This resistance appears to stems from the production of bacterial appendage proteins, which trigger the aggregation of nanoparticles, rendering them ineffective. Objective: This study aims to evaluate the stability of AgNPs at different concentrations when administered to a standard strain of Klebsiella pneumoniae, a Gram-negative bacterium particularly in that produces bacterial appendage. Methodology: AgNPs were synthesized using the bottom-up chemical method. Silver nitrate was reduced with sodium borohydride to produce a colloidal solution, which was stabilized by cappeing with alpha lipoic acid. An aliquot of 30 mL of the sodium borohydride solution was cooled and stirred for 1 hour at 0 ºC an ultra-thermostatic bath. Subsequently, 10 mL of silver nitrate was added to the solution and, after seven minutes in the dark, a yellow-colored colloidal silver was observed. The silver nanoparticles were characterized using the following techniques: Uv-Vis spectroscopy, dynamic light scattering, infrared spectroscopy and X-ray diffraction. Antimicrobial susceptibility tests, such as minimum inhibitory concentration and minimum bactericidal concentration, were carried out on nanoparticles with and without cappeing at different concentration and performed in triplicate. The minimum inhibitory concentration test followed CLSI (Clinical and Laboratory Standards Institute) guidelines M07A11 and M100S22, while the minimum bactericidal concentration was determined using the microdrop technique on brain-heart-infusion agar. Results: Agglomeration of the uncapped AgNPs was observed at all concentrations tested, with resulted in bacterial growth during both the minimum inhibitory concentration and minimum bactericidal concentration assays. In contrast, the nanoparticles capped with alpha lipoic acid exhibited both inhibitory and bactericidal effect against planktonic cells. In the analysis of variance with Tukey's post-hoc test of the group without cappeing, there was no significance between the control group and the other concentrations studied, however, in the group with cappeing, all groups presented significance ˂ 0.0001. Conclusion: The mechanism of bacterial resistance to AgNPs can be overcome by stabilization with alpha lipoic acid, enabling an inhibitory and bactericidal effect. The capping provides stability in biological systems, where various biomacromolecules can bind to the surface of the nanoparticles and hinder their interaction. Furthermore, the stabilized nanoparticle retain the antimicrobial action of the AgNPs and hold potential for applications in human and animal health, particularly in diagnostics and targeted drug delivery.