Genomics of KPC-producing Klebsiella pneumoniae sequence type 512 clone highlights the role of RamR and ribosomal S10 protein mutations in conferring tigecycline resistance

Abstract

Full genome sequences were determined for five Klebsiella pneumoniae strains belonging to the sequence type 512 (ST512) clone, producing KPC-3. Three strains were resistant to tigecycline, one showed an intermediate phenotype, and one was susceptible. Comparative analysis performed using the genome of the susceptible strain as a reference sequence identified genetic differences possibly associated with resistance to tigecycline. Results demonstrated that mutations in the ramR gene occurred in two of the three sequenced strains. Mutations in RamR were previously demonstrated to cause overexpression of the AcrAB-TolC efflux system and were implicated in tigecycline resistance in K. pneumoniae. The third strain showed a mutation located at the vertex of a very well conserved loop in the S10 ribosomal protein, which is located in close proximity to the tigecycline target site in the 30S ribosomal subunit. This mutation was previously shown to be associated with tetracycline resistance in Neisseria gonorrhoeae. A PCR-based approach was devised to amplify the potential resistance mechanisms identified by genomics and applied to two additional ST512 strains showing resistance to tigecycline, allowing us to identify mutations in the ramR gene.

FIG 1

Mutations identified in proteins potentially implicated in tigecycline resistance. (A) The entire RamR deduced protein sequence of the susceptible KP3-S strain is shown in comparison with the RamR mutation identified in KP5-R (by premature termination of translation) and KP2-R, KP6-R, and KP7-R (by insertion of the ISKpn18 element, represented by arrows). (B) The entire S10 deduced protein sequence of the susceptible KP3-S strain is shown in comparison with the mutated S10 identified in the KP4-R strain. The positions of the Val57→Leu57 mutation are indicated by asterisks.