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General Orthopaedics

PROPIONIBACTERIUM ACNES CAN INCREASE ITS ARSENAL OF RESISTANCE: IN VITRO EMERGENCE OF FLUOROQUINOLONE RESISTANCE AND MOLECULAR CHARACTERISATION OF THE gyrA GENE MUTATIONS INVOLVED

European Bone and Joint Infection Society (EBJIS), Nantes, France, September 2017



Abstract

Aim

Although there are no treatment guidelines for Propionibacterium acnes (PA) bone and joint infections (Corvec et al Acta Orthopedica 2016), these infections can be treated with a combination of fluoroquinolones and rifampicin. Rifampicin resistance have already been reported either in in vitro selected mutants or clinical isolates (Furustrand et al JAC 2013, Anaerobe 2015). Minimal inhibitory concentrations of levofloxacin (LVX) ranging from 0,12 to 0.5mg/L are regularly observed but resistance has not yet been investigated. We investigated the in vitro emergence of LVX resistance and characterized the mutations involved in gyrA gene.

Method

The strain of PA ATCC11827 (MIC LVF = 0.25 mg/L) was used. The frequency of mutation was determined after inoculation of 108 PA on blood agar containing concentrations of 2 to 128 times the MIC incubated for 7 days in anaerobiosis at 35 ° C. The emergence of high-level of resistance was also studied from the low-level mutants after a second exposure. For the resistant mutants, the gyrA and parC genes were sequenced and compared to the PA reference sequences.

Results

The mutation frequency was 3.8 cfu × 10–8 (8×MIC) and 1.6 cfu × 10–7 (4×MIC), respectively. A low or high-level resistance to LVX was observed. MICs varied between 0.75 and> 32 mg/L and were stable after three subcultures. 87 mutants were studied including 40 with a mutation in gyrA gene. 10 different genotypes could be demonstrated with either high-level resistance: G99 (n = 4), G99 D (n = 3), D100N (n = 1), S101 L (n = 14), S101W N = 5) or low-level resistance D100H (n=1), D100G (n=1), A102P (n=5), D105 H (n = 4), D105 G (n = 2). Substitution 101 always leads to a high level of resistance. No mutation was found in parC gene.

Conclusions

To our knowledge, this is the first description of the emergence of LVX resistance in PA. The MIC increases from sensitivity to low or high-level resistance. This resistance is stable and associated exclusively with mutations in the gyrA gene. Six different positions give rise to ten different genotypes. The passage from a low to a high-level resistance is done mainly by the selection of the mutation at position 101. Finally, some mutants do not exhibit mutations in QRDRs, suggesting the existence of an efflux system.


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