O262 The mode of action of daptomycin against Staphylococcus aureus

Novel resistance mechanisms and quinolone resistance of aminoacidic identity in the NCBI database. A PCR-based strategy was used to clone and express the genes coding for Qnr-like pentapeptidic proteins of E. faecalis ATCC 29212, E. faecalis V583, E. faecium BM4147, Listeria monocytogenes 868 (clinical isolate), Clostridium perfringens 455 (clinical isolate) and Bacillus cereus ATCC 11778 in pPCR-Script vector (Stratagene) in Escherichia coli DH10B. According to CLSI guidelines, MICs of nalidixic acid, ofloxacin, norfloxacin, ciprofloxacin, levofloxacin and moxifloxacin were determined for clinical strains, reference strains and Escherichia coli DH10B harbouring recombinant plasmids containing genes coding for the pentapeptidic proteins. Results: The aminoacidic identity of Qnr-like pentapeptidic proteins of the Gram-positive strains compared to those of plasmid-mediated QnrA1, QnrB1 or QnrS1 quinolone resistance proteins ranged from 20% to 27%. When compared to the homologue of QnrA in E. faecalis V583, the aminoacidic identity ranged from 30% to 35%. All the clones obtained in E. coli DH10B expressing a pentapentidic protein from the Gram positives strains conferred reduced susceptibility to quinolones. The recombinant plasmids expressing pentapentidic proteins in E. coli DH10B increased the MICs from 4 to 12 folds for nalidixic acid, from 6 to 24 folds for ofloxacin, from 2 to 47 folds for norfloxacin, from 12 to 32 folds for ciprofloxacin, from 4 to 21 folds for levofloxacin, and from 4 to 32 folds for moxifloxacin. Conclusions: The pentapeptidic proteins analysed confer reduced susceptibility phenotype in E. coli. These data could provide further evidence about the possible role of pentapeptidic proteins of different Gram-positive species in their natural resistance to quinolones. These Gram positives species could constitute a reservoir of Qnr-like quinolone resistance proteins. O262 The mode of action of daptomycin against Staphylococcus aureus J. Hobbs, K. Miller, N. Read, I. Chopra (Leeds, UK) Objectives: The Silverman model for the mode of action of daptomycin (DAP) is based on the leakage of potassium ions (K+ ), membrane deenergisation and subsequent cell death without lysis. However, it is unknown whether loss of K+ is sufficient to cause cell death. In this study we determined the kinetics of both cell death and lysis of S. aureus treated with DAP to establish any relationships between the two. Methods: Time-kill experiments were performed at various concentrations of DAP to determine the kinetics of cell death and samples were taken and prepared for scanning and transmission electron microscopy (EM). Culture samples were taken at intervals and the extracellular and intracellular amounts of ATP determined. The extracellular and intracellular levels of b-galactosidase (b-gal) in a S. aureus strain expressing b-gal in its cytoplasm were also measured. Results: The time-kill experiments show DAP to be rapidly bactericidal at 32 mg/L. DAP at 4 mg/L shows a bacteriostatic effect for the first 60 min and then becomes slowly bactericidal. There is evidence for dosedependent leakage of ATP and b-gal from cells treated with DAP, but this leakage occurs at very different rates. After 30 min of treatment with DAP at 32 mg/L, 90% of the total ATP has been released but only a negligible amount of b-gal. The percentage of b-gal released increases steadily over a period of hours, reaching 40% after 6 h and 90% after 24 h. After 30 min of treatment with DAP at 4 mg/L, 50% of the total ATP has been released and this amount continues to increase steadily. The leakage of b-gal is less apparent and after 24 h the cells have released less than 30%. The EM images show some cellular disruption in the first hour after DAP treatment, with this damage becoming more apparent and widespread over the following hours. The images clearly show that the rate and extent of cellular lysis is dose-dependent. Conclusion: Our findings support the Silverman model for the mode of action of DAP and the hypothesis that it can cause rapid cell death without whole cell lysis. We have, however, shown that cell lysis does occur over time, through both EM images and the leakage of large amounts of b-gal. Our findings, while upholding the Silverman model, suggest that it is too simple to conclude that cell death is due only to

S53 loss of K+ and membrane de-energisation. The rapid loss of ATP would be an equally likely cause of cell death. O263 The correlation between resistance phenotype and expression levels of efflux pumps of Pseudomonas aeruginosa X.J. Lu (Chengdu, CN) Objective: To study the effects of efflux pump inhibitors (CCCP and PAbN) on carbapenems in P. aeruginosa (Pa); to investigate the correlation between the resistance phenotypes and expression levels of efflux pumps of Pa and discuss the mechanism of different phenotypes in resistant Pa. Methods: MICs of imipenem (IMP) or meropenem (MEP) combined with efflux pump inhibitors against IMP resistant strains were determined by agar dilution method. For 32 strains with different resistant phenotypes to IMP and MEP, the mRNA expression levels of three efflux pump genes (mexA, mexD, mexF) were quantified by real time fluorescent quantitative PCR. Results: The resistance rate of IMP and MEP descended gently after combined with efflux pump inhibitors. The strains with sustained MICs reached over 50%. MICs of only 8 strains descended to 1/4 original MIC values. The amount of mRNA of efflux pump genes in ATCC27853 was defined as 1. The amounts of mexA in susceptible strains were from 0.0003 to 0.99, those of mexD were from 0.0001 to 1.11, and those of mexF were from 0.013 to 0.31. The amounts of mexA in IMP resistant but MEP strains were from 0.28 to 4.48, mexD were from 0.37 to 20.06 and mexF were from 0.44 to 5.56. The amounts of mexA in both resistant strains were from 1.42 to 138.25, mexD were from 0.1 to 16.66 and mexF were from 0.22 to 12.61. That appeared overexpressions of efflux pumps accounted for 88.89% in 27 strains resistant to carbapenems. The statistic result showed the expression levels of three efflux pumps of strains resistant to carbapenems were much higher than susceptible strains. Conclusions: When CCCP was incorporated in the Mueller-Hinton agar at a concentration of 5 mg/mL and PAbN was 20 mg/mL in vitro they could not obviously decrease the carbapenems resistance of Pa in this study, and there were rare relativity between the amount of mRNA of efflux pump genes and effects on MICs of carbapenems with efflux pump inhibitors. Overexpression of efflux pumps were the main reason for the resistance to carbapenems of Pa in our hospital. A phenomenon that manifold efflux pumps co-existed in a Pa isolate was common. It demonstrated that overexpression of MexAB-OprM plays an important role in resistance differences between IMP and MEP. Overexpressions of MexCD-OprJ and MexEF-OprN played important roles for carbapenems resistance in Pa, but had fewer effects on resistance difference between IMP and MEP. O264 Natural variation within Staphylococcus aureus populations determines concentration and time-dependent phenotypic daptomycin resistance M. Morosini, M. Garc´ıa-Castillo, R. Cant´on, B. Levin, F. Baquero (Madrid, ES; Atlanta, US) Daptomycin (DAP) is a rapid-killing lipopeptide antibiotic acting on MSSA, MRSA, and GISA strains. However, we have seen that, after killing, extended incubation with 8×MIC DAP concentrations results in a slow but sustained increase (from 24 h to 5 days) in viable staphylococcal cells. Such increase occurs in the presence of non-degraded DAP, and sub-cultivation of viable cells renders only DAP-susceptible organisms, suggesting heterogeneous phenotypic DAP-resistance. Objective: To determine whether heterogeneous phenotypic DAPresistance occurs as a result of natural variation in S. aureus populations before DAP exposure. Methods: Time kill studies (in triplicate) with DAP 8×MIC were performed in 10 ml-tubes inoculated with 5×105 CFU/mL of 4 S. aureus ATCC strains (29213, 25923, 43300, and 700789) and incubated along 5 days. Natural variation in propensity to DAP-killing was studied with