Methods: Antibiotic susceptibility was tested by broth microdilution. Kp isolates underwent whole genome sequencing (Illumina MiSeq). blaKPC-3 expression was measured by RT-PCR. Mutant blaKPC-3 were implicated in C/A resistance by targeted gene disruption, and expression of plasmids and cloned genes in isogenic E. coli.
Results: C/A resistant (MIC >8µg/mL) Kp were recovered in 3 pts following 10, 15 and 19d tx courses. C/A resistance was associated with reversion to meropenem (MER) susceptibility. All isolates were sequence-type 258, clade II (cps-2) clones. Baseline isolates harbored blaKPC-3. In pt 1, subsequent resistant isolates carried blaKPC-3 mutations (D179Y, T243M double substitution). In pts 2 and 3, resistant isolates carried single V240G (pt 2) or D179Y (pts 2 and 3) mutations. blaKPC-3 expression was down-regulated in C/A resistant, MER susceptible isolates, and increased as MER MICs increased. Mutant blaKPC-3 were on a Tn4401d element, carried on an IncFIA pBK30683-like plasmid. Transfer of mutant blaKPC containing plasmids into E. coli resulted in significantly increased C/A MICs and decreased MER MICs compared to transfer of wild-type (WT) blaKPC. Cloning of mutant and WT blaKPC into E. coli corroborated results. In rank order, E. coli transformants with WT blaKPC, blaKPC single mutants, and blaKPC double mutants (D179Y, T243M) demonstrated increased C/A MICs. Deletion of blaKPC in Kp isolates restored susceptibility to C/A and MER.
Conclusion: C/A resistance may emerge rapidly due to blaKPC-3 mutations in CRKp during tx. The presence of mutated KPCs on mobile genetic elements is troubling, and suggests resistance may disseminate as C/A is used widely. The clinical significance of reversion to MER susceptibility is unclear. Carbapenems should be used cautiously until the mechanisms and stability of the phenotype are understood.
R. K. Shields,
Astellas: Grant Investigator , Research support
S. Cheng, None
K. Chava, None
B. N. Kreiswirth, None
E. G. Press, None
M. H. Nguyen, None
C. J. Clancy, None