Development of a new spectrophotometric assay for rapid detection and differentiation of KPC, MBL and OXA-48 carbapenemase-producing Klebsiella pneumoniae clinical isolates.

The increased prevalence of carbapenemase-producing Enterobacteriaceae (CPE) has made essential the design of quicker tests for CPE detection. In the present study, a simple and rapid assay was developed based on measurement of the hydrolytic activity of imipenem at a final concentration of 65 µg/mL (100 µM) through ultraviolet-visible (UV-Vis) spectrophotometry. All measurements were conducted at 297 nm. A total of 83 carbapenem-non-susceptible CPE, consisting of Klebsiella pneumoniae clinical isolates and genotypically characterised as KPC-, VIM-, NDM- or OXA-48-producers, were tested. For comparison, 30 carbapenem-non-susceptible clinical isolates, consisting of Escherichia coli and K. pneumoniae and genotypically confirmed as non-CPE, were also examined. The spectrophotometric assay enabled efficient discrimination of CPE from non-CPE isolates even in 45 min (P < 0.0001). Moreover, the presence of phenylboronic acid (PBA) or ethylene diamine tetra-acetic acid (EDTA) in the reaction mixture was able to inhibit the hydrolytic capacity of KPC- or metallo-ß-lactamase (MBL)-producers, respectively, while the hydrolytic activity of OXA-48-producing strains was not affected by the presence of these inhibitors (P < 0.001). The newly developed assay presented 100% sensitivity and specificity to detect and differentiate KPC-, MBL- and OXA-48-producers compared with genotypic characterisation. Thus, the proposed spectrophotometric method can be considered as an easy, fast, accurate and cost-effective diagnostic tool for screening carbapenem-non-susceptible K. pneumoniae isolates in the clinical laboratory.

The olive constituent oleuropein exhibits proteasome stimulatory properties in vitro and confers life span extension of human embryonic fibroblasts.

Normal human fibroblasts undergo replicative senescence due to both genetic and environmental factors. Senescence and aging can be further accelerated by exposure of cells to a variety of oxidative agents that contribute among other effects to the accumulation of damaged proteins. The proteasome, a multicatalytic nonlysosomal protease, has impaired function during aging, while its increased expression delays senescence in human fibroblasts. The aim of this study was to identify natural compounds that enhance proteasome activity and exhibit antiaging properties. We demonstrate that oleuropein, the major constituent of Olea europea leaf extract, olive oil and olives, enhances the proteasome activities in vitro stronger than other known chemical activators, possibly through conformational changes of the proteasome. Moreover, continuous treatment of early passage human embryonic fibroblasts with oleuropein decreases the intracellular levels of reactive oxygen species (ROS), reduces the amount of oxidized proteins through increased proteasome-mediated degradation rates and retains proteasome function during replicative senescence. Importantly, oleuropein-treated cultures exhibit a delay in the appearance of senescence morphology and their life span is extended by approximately 15%. In summary, these data demonstrate the beneficial effect of oleuropein on human fibroblasts undergoing replicative senescence and provide new insights towards enhancement of cellular antioxidant mechanisms by natural compounds that can be easily up-taken through normal diet.

Alterations of senescence biomarkers in human cells by exposure to CrVI in vivo and in vitro.

Heavy metals like CrVI, CdII, PbII and SnII have many applications in industry. They also represent a group of labour pollutants, as they are involved in several physiological disorders, such as carcinogenesis and various tissue dysfunctions. However, limited knowledge exists regarding their effects on ageing. In the current work we provide evidence that workers chronically exposed to CrVI have considerably reduced serum levels of the biomarker of senescence and cell survival, Apolipoprotein J/Clusterin (ApoJ/CLU). Moreover, we have found that both the degree and the time of exposure to CrVI associate negatively with ApoJ/CLU serum levels. To further examine whether CrVI directly affects cellular senescence we treated for 10 weeks two adult skin fibroblasts cultures as well as embryonic fibroblasts with a range of CrVI concentrations that approximate the values recorded in the blood circulation of exposed workers. Cellular treatment with a CrVI concentration that approximates the highest concentration in the blood was extremely toxic and nearly all cells died immediately after the first treatment. Interestingly, continuous treatment with a 10-fold lower CrVI concentration resulted in the induction of premature senescence. More specifically, treated cells were growth arrested, acquired an irregular shape, were positive to beta-galactosidase staining, accumulated oxidized proteins and over-expressed the cyclin-dependent kinase inhibitor p21 and ApoJ/CLU. Similar treatments with three additional labour pollutants resulted in the induction of premature senescence by CdII, but not by SnII or PbII. In summary, our results indicate that exposure to CrVI induces alterations of senescence biomarkers both in vivo and in vitro. They also provide new valuable tools for monitoring CrVI cytotoxic effects in vivo as well as for re-evaluating the maximum permissive values of some labour pollutants, like CrVI and CdII.