In vitro susceptibility of methicillin-resistant Staphylococcus aureus isolates from skin and soft tissue infections to vancomycin, daptomycin, linezolid and tedizolid

Abstract Introduction Treatment of multidrug-resistant Gram-positive infections caused by Staphylococcus aureus remains as a clinical challenge due to emergence of new resistance mechanisms. Tedizolid is a next-generation oxazolidinone, recently approved for skin and soft tissues infections. We conducted a study to determine in vitro susceptibility to vancomycin, daptomycin, linezolid and tedizolid in MRSA clinical isolates from adult patients with skin and soft tissue infections. Material and methods Methicillin-resistant S. aureus isolates were collected in three tertiary-care hospitals of Medellin, Colombia, from February 2008 to June 2010 as part of a previous study. Clinical characteristics were assessed by medical records and MIC values were determined by Epsilometer test. Genotypic analysis included spa typing, MLST, and SCCmec typing. Results A total of 150 MRSA isolates were evaluated and tedizolid MIC values obtained showed higher in vitro activity than other antimicrobials, with MIC values ranging from 0.13 µg/mL to 0.75 µg/mL and lower values of MIC50 and MIC90 (0.38 µg/mL and 0.5 µg/mL). In contrast, vancomycin and linezolid had higher MIC values, which ranged from 0.5 µg/mL to 2.0 µg/mL and from 0.38 µg/mL to 4.0 µg/mL, respectively. Tedizolid MICs were 2- to 5-fold lower than those of linezolid. Clinical characteristics showed high previous antimicrobial use and hospitalization history. The majority of the strains belong to the CC8 harboring the SCCmec IVc and were associated with the spa t1610 (29.33%, n = 44). Conclusion In vitro effectiveness of tedizolid was superior for isolates from skin and soft tissue infections in comparison with the other antibiotics evaluated. The above added to its less toxicity, good bioavailability, daily dose and unnecessity of dosage adjustment, make tedizolid in a promising alternative for the treatment of infections caused by MRSA.

Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

A quantitative structure-activity relationship (QSAR) study on a few series of potent, highly selective inhibitors of nitric oxide synthase.

QSAR study was performed on a series of 1,2-dihydro-4-quinazolinamines, 4,5-dialkylsubstituted-2-imino-1,3-thiazolidine derivatives and 4,5-disubstituted-1,3-oxazolidin-2-imine derivatives studied by Tinker et al. [J Med Chem (2003), 46, 913-916], Ueda et al. [Bioorg Med Chem (2004) 12, 4101-4116] and Ueda et al. [Bioorg Med Chem Lett (2004) 14, 313-316], respectively, as potent, highly selective inhibitors of inducible nitric oxide synthase (iNOS). The iNOS inhibition activity of the whole series of compounds was analyzed in relation to the physicochemical and molecular properties of the compounds. The QSAR analysis revealed that the inhibition potency of the compounds was controlled by a topological parameter 1v (Kier’s first order valence molecular connectivity index), density (D), surface tension (St) and length (steric parameter) of a substituent. This suggested that the drug-receptor interaction predominantly involved the dispersion interaction, but the bulky molecule would face steric problem because of which the molecule may not completely fit in active sites of the receptor and thus may not have the optimum interaction.

Inhibition of azoxymethane-induced colon carcinogenesis in rats due to JTE-522, a selective cyclooxygenase-2 inhibitor.

Prostaglandin E2, which is produced by cyclooxygenase (COX) during arachidonic acid metabolism, is considered to be related to colon carcinogenesis and selective COX-2 inhibitors may be effective for chemoprevention without the adverse side effects of non-selective, nonsteroid anti-inflammatory drugs. Therefore, the influence of JTE-522 (4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzensulfonamide), a selective COX-2 inhibitor, was examined in azoxymethane(AOM)-induced rat colon carcinogenesis. A total of 40 male F344 rats were randomly divided into two groups. Group 1 received diet containing 0.015% JTE-522 and group 2 the normal diet without supplement as a control group; one week later, all rats were administered axozymethane (AOM) s.c. at a dose of 15 mg/kg body weight once a week for 3 successive weeks. At the termination of the experiment (30 weeks after the start), the multiplicity of colon cancer in group 1 was significantly less than that of group 2. The proliferating cell nuclear antigen (PCNA) indices for non-neoplastic cells of the colon mucosa in group 1 were also lower. These data thus suggest that JTE-522 has chemopreventive potential against colon carcinogenesis with decrease of mucosal cell proliferation in rats.

Calyculin A modulates activation of the NADPH-oxidase in Me2SO-differentiated HL-60 cells

Human promyelocytic leukemia cells (HL-60) have been used as a model system in which to study the effects of protein phosphatase inhibitors on NADPH-oxidase activation. Since O2- is generated by NADPH-oxidase, we examined the effect of calyculin A pretreatment on oxidase activation in response to various agonists. When Me2SO-differentiated HL-60 cells were treated with calyculin A prior to the addition of phorbol 12-myristate 13-acetate (PMA), O2- production was inhibited; however, calyculin A enhanced O2- production by N-formyl-methionyl-leucyl-phenylalanine (FMLP). The decreased O2- production seen with calyculin A pretreatment followed by PMA may be due to diminished translocation of the p47-phox and p67-phox, cytosolic components of the oxidase, and inhibition of arachidonic acid release. Interestingly calyculin A pretreatment followed by either agonist significantly enhanced mitogen-activated-protein kinase (MAPK) activity. The differential effects of pretreatment with calyculin A on subsequent oxidase stimulation elicited by FMLP or PMA provide further evidence for substantial heterogeneity in the activation of the respiratory burst.

A culture medium for cultivation of mycobacteria, probably Mycobacterium leprae from Mycobacterium leprae infected tissues.

Mycobacterium leprae suspensions were prepared from infected armadillos. The M. leprae cells were inoculated into culture media containing KH2PO4 4.7. g. Na2HPO4 2 g, sodium thioglycolate 1 g, (NH4)2SO4 2 g, MgSO4 0.1 g, ferric ammonium citrate 0.05 g, and lipoic acid (thioctic acid) 0.1 g in one liter distilled water. The solution was enriched with heat killed, sonicated leprosy derived Mycobacterium X or crude mycobactin extract from M. phlei to contain + 0.2 micrograms mycobactin per 1 ml in the final medium. Twenty ml media was distributed into each of 25 ml screw cap tubes and autoclaved for 30 minutes. Positive growth was obtained from seven out of ten specimens when incubated at 34 degrees C. The cultures developed as a sediment in the liquid media, suggesting preference for microaerophylic conditions. No growth was seen on the surface of the semi-solid agar media containing the same ingredients. Latency period of growth was estimated as 10-16 days and time of division as 6 days. Subcultures were obtained. Cells were long, acid fast, arranged side by side or end to end, with a tendency to form long spiral cords or clumps when sedimented on siliconized slides. Pyridine extraction eliminated acid fastness, but not gram positivity. Cultures did not grow on Dubos, Lowenstein or 7H10 media. They produce the disease in the foot pads of mice characteristic of M. leprae. Subcultures remain dependent on the heat killed sonicated mycobacteria, or crude mycobactin extract, and reduced oxygen tension in the media. Results suggest that cultures might be identical to M. leprae.