Analyses of the response of carbapenem-resistant Pseudomonas aeruginosa against monotherapy and combined therapy using quantum dots and proteomics.

Carbapenem-resistant P. aeruginosa (CRPA) has become a serious public health problem and the biofilm formation aggravates this problem. The study aimed to evaluate the occurrence of ß-lactamases and quorum sensing (QS) genes in CRPA isolates, analyze production of biofilm, evaluate the response against meropenem (MPM) and∕or polymyxin B (POL B) and its association with azythromicin (AZT) using quantum dots (QDs) and proteomic analysis. Six CRPA isolates were analyzed. ß-lactamases and QS genes were search using specific PCRs and were tested for biofilm production by quantitative technique. A CRPA isolate, containing blaKPC gene and biofilm-producing, was selected to assess its response to therapy using QDs and the MALDI-TOF. The ß-lactamase detected was blaKPC in 66.7% of the isolates. All isolates were biofilm producers and carriers of the QS genes. QDs-MPM conjugates triggered the formation of biofilm and the association with AZT inhibited this effect. Proteomics analysis showed that treatments with MPM or POL B suppressed the expression of the transglycosylase protein, while combined therapy with AZT induced expression of the RpoN protein. Thus, this study shows that the use of fluorescence combined with the proteomics analysis was promising to understand how a CRPA strain reacts to antimicrobial treatment.

A Novel Strategy Based on Zn(II) Porphyrins and Silver Nanoparticles to Photoinactivate Candida albicans.

Background: Photodynamic inactivation (PDI) is an attractive alternative to treat Candida albicans infections, especially considering the spread of resistant strains. The combination of the photophysical advantages of Zn(II) porphyrins (ZnPs) and the plasmonic effect of silver nanoparticles (AgNPs) has the potential to further improve PDI. Here, we propose the novel association of polyvinylpyrrolidone (PVP) coated AgNPs with the cationic ZnPs Zn(II) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin or Zn(II) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin to photoinactivate C. albicans. Methods: AgNPs stabilized with PVP were chosen to allow for (i) overlap between the NP extinction and absorption spectra of ZnPs and (ii) favor AgNPs-ZnPs interaction; prerequisites for exploring the plasmonic effect. Optical and zeta potential (ζ) characterizations were performed, and reactive oxygen species (ROS) generation was also evaluated. Yeasts were incubated with individual ZnPs or their respective AgNPs-ZnPs systems, at various ZnP concentrations and two proportions of AgNPs, then irradiated with a blue LED. Interactions between yeasts and the systems (ZnP alone or AgNPs-ZnPs) were evaluated by fluorescence microscopy. Results: Subtle spectroscopic changes were observed for ZnPs after association with AgNPs, and the ζ analyses confirmed AgNPs-ZnPs interaction. PDI using ZnP-hexyl (0.8 µM) and ZnP-ethyl (5.0 µM) promoted a 3 and 2 log10 reduction of yeasts, respectively. On the other hand, AgNPs-ZnP-hexyl (0.2 µM) and AgNPs-ZnP-ethyl (0.6 µM) systems led to complete fungal eradication under the same PDI parameters and lower porphyrin concentrations. Increased ROS levels and enhanced interaction of yeasts with AgNPs-ZnPs were observed, when compared with ZnPs alone. Conclusion: We applied a facile synthesis of AgNPs which boosted ZnP efficiency. We hypothesize that the plasmonic effect combined with the greater interaction between cells and AgNPs-ZnPs systems resulted in an efficient and improved fungal inactivation. This study provides insight into the application of AgNPs in PDI and helps diversify our antifungal arsenal, encouraging further developments toward inactivation of resistant Candida spp.

Photoinactivation of Yeast and Biofilm Communities of Candida albicans Mediated by ZnTnHex-2-PyP4+ Porphyrin.

Candida albicans is the main cause of superficial candidiasis. While the antifungals available are defied by biofilm formation and resistance emergence, antimicrobial photodynamic inactivation (aPDI) arises as an alternative antifungal therapy. The tetracationic metalloporphyrin Zn(II) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (ZnTnHex-2-PyP4+) has high photoefficiency and improved cellular interactions. We investigated the ZnTnHex-2-PyP4+ as a photosensitizer (PS) to photoinactivate yeasts and biofilms of C. albicans strains (ATCC 10231 and ATCC 90028) using a blue light-emitting diode. The photoinactivation of yeasts was evaluated by quantifying the colony forming units. The aPDI of ATCC 90028 biofilms was assessed by the MTT assay, propidium iodide (PI) labeling, and scanning electron microscopy. Mammalian cytotoxicity was investigated in Vero cells using MTT assay. The aPDI (4.3 J/cm2) promoted eradication of yeasts at 0.8 and 1.5 µM of PS for ATCC 10231 and ATCC 90028, respectively. At 0.8 µM and same light dose, aPDI-treated biofilms showed intense PI labeling, about 89% decrease in the cell viability, and structural alterations with reduced hyphae. No considerable toxicity was observed in mammalian cells. Our results introduce the ZnTnHex-2-PyP4+ as a promising PS to photoinactivate both yeasts and biofilms of C. albicans, stimulating studies with other Candida species and resistant isolates.

Activity of carbonyl cyanide-3-chlorophenylhydrazone on biofilm formation and antimicrobial resistance in Pseudomonas aeruginosa using quantum dots-meropenem conjugates as nanotools.

Hospital infections associated with multidrug-resistant (MDR) Pseudomonas aeruginosa are a worldwide public health problem. Efflux systems and biofilm formation are mechanisms related to resistance to carbapenemics. In this study, quantum dots (QDs) were used to evaluate the effect of carbonyl cyanide-3-chlorophenylhydrazone (CCCP), an efflux pump system inhibitor, on biofilm formation and antimicrobial resistance profile of P. aeruginosa strains. For this, QDs were covalently conjugated to meropenem (MPM) and incubated with a P. aeruginosa resistant isolate (P118) or a control sensitive strain (ATCC Pa27853). P118 was also analyzed with conjugates after previous CCCP efflux inhibitor incubation. Fluorescence microscopy images showed that both sensitive and resistant bacteria were efficiently labeled. Nevertheless, P118 isolates presented fluorescent cell agglomerates, suggesting biofilm formation. The addition of the CCCP changed the labeling profile of the resistant isolate, and the absence of agglomerates was observed, indicating no biofilm formation. Genetic assays revealed the presence of MexA and MexE genes encoding channel proteins from efflux pump systems in both resistant and sensitive strains. Disk-diffusion and broth microdilution tests determined drug susceptibility profiles in the presence and absence of CCCP for P118 isolates. We verified that the CCCP efflux system inhibitor may contribute to P. aeruginosa resistant phenotype reduction for some antimicrobials. This study verified the efficiency of QD-MPM conjugates to trigger and study biofilm formation, or its inhibition, before and after CCCP addition. QDs conjugated to antimicrobials can be used as nanotools to investigate multidrug-resistant bacterial strains on biofilm formation.

Bauhinia monandra leaf lectin (BmoLL) conjugated with quantum dots as fluorescent nanoprobes for biological studies: application to red blood cells.

Carbohydrates perform important physiological functions in eukaryotic and prokaryotic cells. Indeed, alterations in glycan patterns may be associated with disorders. The analysis of these sugars can be reached using nanoprobes composed by lectins associated with fluorescent nanoparticles. This study reports the conjugation of a galactose-binding lectin (BmoLL) isolated from Bauhinia monandra leaves with quantum dots (QDs) by adsorption. QDs-BmoLL conjugates showed bright fluorescence and the hemagglutination assay revealed that the lectin preserved its carbohydrate-binding ability after the conjugation. To evaluate the efficiency/specificity of the bioconjugate, ABO human red blood cells (RBCs) were used as biological models and the labeling was analyzed by flow cytometry. Among ABO blood groups, higher labeling (71.7 ± 5.9%) was detected for B-type RBCs, whose antigens have galactose in their structure. The specificity of labeling was confirmed since A- and O-types RBCs incubated with QDs-BmoLL, as well as B-type cells incubated with previously galactose-inhibited conjugates, were labeled below 6%. In AB-type RBCs, which simultaneously have B and A (N-acetylgalactosamine) antigens on their membrane, the labeling was ca. 14.1 ± 4.8%. Therefore, a successful conjugation was reached and QDs-BmoLL conjugates can be considered promising fluorescent nanoprobes for biological investigations.