Modulation of peptidases by 2,4-diamine-quinazoline derivative induces cell death in the amitochondriate parasite Trichomonas vaginalis.

Trichomonas vaginalis is an amitochondriate protozoan and the agent of human trichomoniasis, the most prevalent non-viral sexually transmitted infection (STI) in the world. In this study we showed that 2,4-diamine-quinazoline derivative compound (PH100) kills T. vaginalis. PH100 showed activity against fresh clinical and American Type Culture Collection (ATCC) T. vaginalis isolates with no cytotoxicity against cells (HMVI, 3T3-C1 and VERO) and erythrocytes. In addition, PH100 showed synergistic action with metronidazole, indicating that these compounds act by different mechanisms. When investigating the mechanism of action of PH100 to ATCC 30236, apoptosis-like characteristics were observed, such as phosphatidylserine exposure, membrane alterations, and modulation of gene expression and activity of peptidases related to apoptosis. The apoptosis-like cell death features were not observed for the fresh clinical isolate treated with PH100 revealing distinct profiles. Our data revealed the heterogeneity among T. vaginalis isolates and contribute with the understanding of mechanisms of cell death in pathogenic eukaryotic organisms without mitochondria.

N4 -benzyl-N2 -phenylquinazoline-2,4-diamine compound presents antibacterial and antibiofilm effect against Staphylococcus aureus and Staphylococcus epidermidis.

Staphylococcus aureus and Staphylococcus epidermidis are the main agents involved with implant-related infections. Their ability to adhere to medical devices with subsequent biofilm formation is crucial to the development of these infections. Herein, we described the antibacterial and antibiofilm activities of a quinazoline-based compound, N4 -benzyl-N2 -phenylquinazoline-2,4-diamine, against both biofilm-forming pathogens. The minimum inhibitory concentrations (MIC) were determined as 25 µM for S. aureus and 15 µM for S. epidermidis. At sub-MIC concentrations (20 µM for S. aureus and 10 µM for S. epidermidis), the compound was able to inhibit biofilm formation without interfere with bacterial growth, confirmed by scanning electron microscopy. Moreover, surfaces coated with the quinazoline-based compound were able to prevent bacterial adherence. In addition, this compound presented no toxicity to human red blood cells at highest MIC 25 µM and in vivo toxicity assay using Galleria mellonella larvae resulted in 82% survival with a high dose of 500 mg/kg body weight. These features evidence quinazoline-based compound as interesting entities to promising applications in biomedical fields, such as antimicrobial and in anti-infective approaches.

Rapid tools to gain insights into the interaction dynamics of new 8-hydroxyquinolines with few fungal lines.

The combination of tools such as time-kill assay with subsequent application of mathematical modeling can clarify the potential of new antimicrobial compounds, since minimal inhibitory concentration (MIC) value does not provide a very detailed characterization of antimicrobial activity. Recently, our group has reported that the 8-hydroxy-5-quinolinesulfonic acid presents relevant antifungal activity. However, its intrinsic acidity could lead to an ionization process, decreasing fungal cell permeability. To overcome this potential problem and enhance activity, the purpose of this study was to synthesize and evaluate a novel series of hybrids between the 8-hydroxyquinoline core and sulfonamide and to prove their potential using broth microdilution method, obtaining the pharmacodynamic parameters of the most active derivatives combining time-kill studies and mathematical modeling and evaluating their toxicity. Compound 5a was the most potent, being active against all the fungal species tested, with low toxicity in normal cells. 5a and 5b have presented important antibacterial activity against Staphylococcus aureus strain. The EC50 values obtained by combination of time-kill studies with mathematical model were similar to those of MIC, which confirms the potential of compounds. In addition, these derivatives are non-irritant molecules with the absence of topical toxicity. Finally, 5a and 5b are promising candidates for treatment of dermatomycosis and candidiasis.

Prevalence of ColV Plasmid-Linked Genes and In Vivo Pathogenicity of Avian Strains of Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes extraintestinal infections in birds, leading to an increase in the cost of poultry production. The ColV plasmid-linked genes iroN, ompT, hlyF, iss, and iutA have previously been suggested to be predictors of the virulence of APEC. In this research, we analyzed the frequencies of these genes in a Brazilian collection of E. coli isolated from birds with colibacillosis (APEC) and from apparently healthy birds (avian fecal [A(fecal)]), as well as from the litter of poultry houses of apparently healthy flocks (avian litter [A(litter)]). All the isolates that harbored ompT also harbored hlyF, so they were considered as one trait for statistical analysis. The relationship between in vivo virulence in 1-day-old chicks, expressed as a pathogenicity score, and the number of genes in each isolate showed that isolates with less than two of the four genes were rarely pathogenic, while most pathogenic isolates contained two or more genes. Nevertheless, about half of the nonpathogenic isolates also harbored two or more genes, in agreement with previous observations that commensal E. coli isolates from the birds' microbiota can serve as a reservoir of virulence genes. Thus, the pentaplex polymerase chain reaction can be used to indicate that a strain carrying none or only one gene would be nonpathogenic, but it cannot be used to indicate that a strain with two to four genes would be an APEC. Isolates allocated to phylogenetic group B2, which is frequently associated with extraintestinal infections, had the highest pathogenicity scores, while isolates allocated to group B1 had the lowest.

Genotypes and pathogenicity of cellulitis isolates reveal traits that modulate APEC virulence.

We characterized 144 Escherichia coli isolates from severe cellulitis lesions in broiler chickens from South Brazil. Analysis of susceptibility to 15 antimicrobials revealed frequencies of resistance of less than 30% for most antimicrobials except tetracycline (70%) and sulphonamides (60%). The genotyping of 34 virulence-associated genes revealed that all the isolates harbored virulence factors related to adhesion, iron acquisition and serum resistance, which are characteristic of the avian pathogenic E. coli (APEC) pathotype. ColV plasmid-associated genes (cvi/cva, iroN, iss, iucD, sitD, traT, tsh) were especially frequent among the isolates (from 66.6% to 89.6%). According to the Clermont method of ECOR phylogenetic typing, isolates belonged to group D (47.2%), to group A (27.8%), to group B2 (17.4%) and to group B1 (7.6%); the group B2 isolates contained the highest number of virulence-associated genes. Clonal relationship analysis using the ARDRA method revealed a similarity level of 57% or higher among isolates, but no endemic clone. The virulence of the isolates was confirmed in vivo in one-day-old chicks. Most isolates (72.9%) killed all infected chicks within 7 days, and 65 isolates (38.1%) killed most of them within 24 hours. In order to analyze differences in virulence among the APEC isolates, we created a pathogenicity score by combining the times of death with the clinical symptoms noted. By looking for significant associations between the presence of virulence-associated genes and the pathogenicity score, we found that the presence of genes for invasins ibeA and gimB and for group II capsule KpsMTII increased virulence, while the presence of pic decreased virulence. The fact that ibeA, gimB and KpsMTII are characteristic of neonatal meningitis E. coli (NMEC) suggests that genes of NMEC in APEC increase virulence of strains.