Characterisation of moxifloxacin-resistant Clostridium difficile mutants selected in vitro
Abstract number: P1781
Spigaglia P., Barbanti F., Mastrantonio P.
Objectives: To perform an analysis of point mutations in the quinolone resistance-determining region (QRDR) of the DNA gyrase of Clostridium difficile strains after "in vitro" exposure to increasing concentrations of moxifloxacin (MX).
Methods: Five MX susceptible C. difficile strains were selected: strain C253 and 630 (toxinotype 0), strain CD5 (toxinotype V), strain A422 and BI1 (toxinotype III). MIC values for MX were determined using the Agar Dilution method. The breakpoint used was 8 mg/L. Selection of MX-resistant mutants was performed by growing bacteria on Mueller-Hinton (MH) plates with twofold increases in concentrations of MX (from 2 to 32 mg/L) for 48 h. Resistant mutants were grown for three repeated passages on MH plates without MX. The QRDRs of gyrA and gyrB were amplified and sequenced.
Results: Before induction, all C. difficile strains had MICs for MX between 1 and 2 mg/L. No strain showed mutations in GyrA or GyrB, except CD5 that had a mutation in GyrB already described in susceptible strains. BI1 resistant mutants, isolated from the first step of selection (2 mg/L of MX), showed the substitution Arg447 to Lys in GyrB. At the fourth step (16 mg/L of MX) these mutants acquired another mutation (Asp426 to Asn) in GyrB. MICs of BI1 mutants were comprised between 16 and 32 mg/L. C253, 630 and A422 mutants were isolated from the third step of selection (8 mg/L of MX) and showed the substitution Ala118 to Ser in GyrA, Asp426 to Val in GyrB and Asp81 to Asn in GyrA, respectively. The third mutation resulted new. MICs of C253 mutants ranged 816 mg/L, those of 630 mutants were 16 mg/L, whereas MICs of A422 mutants ranged 1624 mg/L. Two different CD5 mutants were isolated: those from the second step (4 mg/L of MX) showed the new mutation Ala92 to Glu in GyrA, whereas those from the third step (8 mg/L of MX) showed both mutations Ala118 to Ser and Thr82 to Ile in GyrA. The MICs of the mutants with Ala92 to Glu ranged 48 mg/L, whereas those of the mutants with Ala118 to Ser and Thr82 to Ile were comprised between 16 and 32 mg/L.
Conclusion: These results demonstrate that MX is able in vitro to generate different mutations in both GyrA and B of C. difficile strains. One or two mutations can be present in the same protein and two new mutations were identified in GyrA. The apparent easiness in developing resistant mutants may explain the circulation of highly resistant strains, such as C. difficile ribotype 027, in the hospital environment.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
|Back to top|