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Antibiotic susceptibility testing of Mycobacterium avium subspecies under iron starvation

Abstract number: P2028

Kopinc R., Ocepek M., Lapanje A.

Objectives:Mycobacterium avium ssp. are environmental opportunistic pathogens, that infect a wide range of hosts, including humans. Upon entry into macrophages they dwell and replicate inside phagosomes, eventually killing the macrophage. Since macrophage environment is known to be hostile to most of bacteria, especially by limiting available iron, we wanted to test if susceptibility of Mycobacterium avium ssp. to antibiotics is affected by iron concentrations. Previous extensive genomic analysis of Mycobacterium avium ssp. paratuberculosis K-10 genome pointed to existence of potential antibiotic resistance genes under regulation of iron responsive repressor IdeR.

Methods: MICs for different first and second line anti-tuberculous antibiotics under iron replete or deplete growth conditions were measured for Mycobacterium avium strain 104 and several field isolates of Mycobacterium avium ssp. paratuberculosis with resazurin viability assay. Strains were first grown in Sauton's medium with 100 uM iron to late exponential phase. Cells were then washed and dilluted in Sauton's medium without iron (leqslant R: less-than-or-eq, slant1 uM) to an OD600 of 0,010 and transferred to 96-well plates. Antibiotic was added at different concentrations in triplicates along with controls and plates were incubated for 6 days at 37°C. Resazurin redox dye was then added to each well and the plates incubated for 24 hours. Fluorescence was measured at 590 nm for each well. MICs and growth inhibition were determined for each antibiotic in iron deplete/replete medium.

Results: MICs for antibiotics tested were different between subspecies and compared to MICs, determined for Mycobacterium tuberculosis. However MICs were not significantly different in iron replete compared to iron deplete medium. Extent of growth inhibition showed different patterns in presence of iron compared to growth without iron. Subinhibitory concentrations of antibiotics have shown to increase growth of bacteria in iron deplete compared to iron replete medium.

Conclusion: Growth conditions can significantly affect antibiotic susceptibility, therefore it is very important to closely mimic the natural environment of pathogen. Current in vitro antibiotic susceptibility testing for mycobacteria is carried out predominantely in nutrient rich media, optimal for bacterial growth, which by no means resembles host environments. This should be taken into consideration in future development of antibiotics.

Session Details

Date: 07/05/2011
Time: 00:00-00:00
Session name: Abstracts of 21st ECCMID / 27th ICC
Subject:
Location: Milan, Italy, 7 - 10 May 2011
Presentation type:
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