Determination of efflux activity in Enterococci by Hoechst accumulation assay and the role of zinc oxide nanoparticles in inhibition of this activity.

BACKGROUND: Contribution of efflux pumps in development of antimicrobial resistance has been largely addressed in Gram negative and to a much lesser extent in Gram positive bacteria. Measuring accumulation of Hoechst (H) dye is known as a safe and rapid method for monitoring efflux activity in bacteria. Antimicrobial effects of metal nanoparticles have been attributed in part to inhibition of efflux pumps. This study aimed to first determine efflux activity in enterococci by Hoechst accumulation assay, and to second characterize the role of zinc oxide nanoparticles (ZnONPs) in inhibition of these pumps. RESULTS: Increased accumulation of Hoechst dye showed more potential of ZnONPs in efflux inhibition compared with CCCP. H33258 represented more suitability for accumulation studies in enterococci. Two to six-fold reduction in minimum inhibitory concentration (MIC) values of antimicrobial agents in the presence of ZnONPs was observed. CONCLUSIONS: Efflux activity in enterococcal strains can be measured by H33258 accumulation assay. Application of ZnONPs as an efflux inhibitor, may rejuvenate the use of conventional antimicrobial agents against these bacteria.

Antiproliferative properties of Turmerone on Leishmania major: Modes of action confirmed by antioxidative and immunomodulatory roles.

Treatment of leishmaniasis by conventional synthetic compounds has faced a serious challenge worldwide. This study was performed to evaluate the effect and modes of action of aromatic Turmerone on the Leishmania major intra-macrophage amastigotes, the causative agent of zoonotic cutaneous leishmaniasis in the Old World. In the findings, the mean numbers of L. major amastigotes in macrophages were significantly decreased in exposure to Turmerone plus meglumine antimoniate (Glucantime®; MA) than MA alone, especially at 50 µg/mL. In addition, Turmerone demonstrated no cytotoxicity as the selectivity index (SI) was 21.1; while it induced significant apoptosis in a dose-dependent manner on L. major promastigotes. In silico molecular docking analyses indicated an affinity of Turmerone to IL-12, with the MolDock score of - 96.8 kcal/mol; which may explain the increased levels of Th1 cytokines and decreased level of IL-10. The main mechanism of action is more likely associated with stimulating a powerful antioxidant and promoting the immunomodulatory roles in the killing of the target organism.

Reduced Susceptibility to Biocides among Enterococci from Clinical and Non-Clinical Sources.

BACKGROUND: Wide use of biocidal agents such as benzalkonium chloride (BCC) and chlorhexidine digluconate (CHX) in hospitals and non-hospital environments, has raised concerns over the emergence of non-susceptible strains. Efflux pumps are of known main mechanisms in biocide tolerance which have been rarely addressed in enterococci - members of gut microbiota which can cause serious problems particularly in hospitalized patients. The purpose of this study was to investigate the susceptibility of enterococci from different sources (clinical and fecal isolates) toward BCC and CHX, and its correlation with efflux associated genes. Also, possible link between biocide tolerance and antibiotic resistance was examined. MATERIALS AND METHODS: One hundred and four enterococcus isolates including clinical (n = 54) and fecal isolates (n = 50) were studied for susceptibility toward BCC, CHX, ciprofloxacin, gentamicin and vancomycin. Twelve efflux associated genes were investigated by polymerase chain reaction assay. RESULTS: In clinical isolates, reduced susceptibility to CHX and resistance to gentamicin and ciprofloxacin were significantly higher than fecal isolates. Vancomycin resistance was associated with increasing minimum inhibitory concentration of CHX. Among all investigated genes, only three ones, efrA, efrB and emeA were detected which were significantly associated with reduced susceptibility to CHX and were more frequent among clinical isolates. Also, high level resistance to gentamicin was significantly associated with the presence of efrA/B as well as with reduced susceptibility to CHX. CONCLUSION: As expected, reduced susceptibility to CHX, was significantly higher in clinical isolates. However, the presence of a vancomycin-resistant enterococci among fecal isolates of healthy people which showed resistance/tolerance to studied antimicrobial agents, was unexpected and highlights the need to investigate other non-hospital environments to avoid dissemination of antimicrobial resistance. Correlation between reduced susceptibility to CHX and high level resistance to gentamicin, substantiates monitoring of biocide tolerance particularly in the healthcare settings to control the establishment of antimicrobial resistant strains.

Genotypic Diversity of Multidrug Resistant Shigella species from Iran.

BACKGROUND: In many developing countries, shigellosis is endemic and also occurs in epidemics and treatment of multidrug-resistant (MDR) isolates are important. The aims of this study were to determine the antimicrobial susceptibility, prevalence of class 1 and 2 integrons and the clonal relatedness of isolates. MATERIALS AND METHODS: Antimicrobial susceptibility tests were performed by disc diffusion method. Polymerase chain reaction (PCR)-sequencing technique was employed for detection and characterization of integrons. The genetic relatedness was evaluated by using enterobacterial repetitive intergenic consensus (ERIC) PCR. RESULTS: There was a high percentage of resistance to trimethoprim-sulfamethoxazole (TMP/SMX) (93.7%), ampicillin (AMP) (87.3%), streptomycin (STR) (84.5%) and tetracycline (TET) (78.9%). Multidrug resistant phenotype was seen in 95.1% of total isolates. Most common MDR profile was TMP/SMX/STR/AMP resistant pattern. Among the 142 Shigella spp. analyzed in this study, 28 isolates were positive for class 1 integron with two types of gene cassette arrays (dfrA17/aadA5 = 31.7% and dfrA7 = 3.8%). The class 2 integron was more frequently detected among the isolates (94.7%) with dfrA1/sat1/aadA1 (69.4%) and dfrA1/sat1 (30.6%) gene cassettes. ERIC-PCR results showed 6, 5, 4 and 3 main genotypes among S. flexneri, S. sonnei, S. boydii and S. dysenteriae isolates, respectively. CONCLUSIONS: Our findings revealed that multidrug resistant Shigella species with high prevalence of class 2 integron were very common in Iran. In addition, ERIC-PCR patterns showed limited variety of clones are responsible for shigellosis in the region of the study.