Natural variation within Staphylococcus aureus populations determines concentration and time-dependent phenotypic daptomycin resistance

Abstract number: 1732_166

Morosini M., García-Castillo M., Cantón R., Levin B., Baquero F.

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 DAP-resistance 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 the ATCC 25923 strain using the Luria-Delbrück fluctuation test (in duplicate) at 2×, 4×, and 8× DAP concentration (using both 3 ml- and 10 ml-tubes) along 6 days of incubation. Non-degraded DAP was determined by a microbioassay.

Results: In time kill studies, DAP displayed bactericidal activity against all ATCC strains at 3 hours. However, an after-killing increase (re-growth) of DAP-susceptible viable cells was observed along prolonged incubation despite of no DAP degradation over time. Fluctuation test indicates that natural phenotypic variation in propensity to DAP killing is generated at random before DAP exposure. The frequency of DAP-phenotypic resistant variants is inversely proportional to DAP concentration and time of exposure. Bacterial densities of 3×105 contain variants able to re-grow in 4 out of 6 tubes at 72 h after exposure to 2×MIC, only in 1/6 tubes at 4×MIC, and none (0/6 tubes) at 8×MIC. On the contrary, 1×106 bacterial density contains variants able to grow in all tubes at 2×, 4× and 8×MIC at 24 h. However, after 6 days of exposure, bacteria from 4 out of 6 tubes were killed.

Conclusions: A stochastic phenotypic variability in propensity to being killed by DAP occurs in S. aureus. The number of phenotypically resistant variants able to persist and re-grow in the presence of DAP is inversely proportional to drug concentration and time of exposure. Studies on putative implication of physiological, reversible heterogeneity in cytoplasmic membrane in this behaviour are awaited to interpret these findings.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Location: ICC, Munich, Germany
Presentation type:
Back to top