In vivo emergence of colistin resistance in Klebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrB regulator

Abstract

Colistin is one of the few agents that retain activity against extensively drug-resistant strains of Klebsiella pneumoniae producing KPC-type carbapenemases (KPC-KP). However, resistance to colistin is increasingly reported among KPC-KP. Comparative genomic analysis of a pair of sequential KPC-KP isolates from the same patient including a colistin-susceptible isolate (KKBO-1) and a colistin-resistant isolate (KKBO-4) selected after colistin exposure revealed that insertional inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, is a genetic mechanism for acquired colistin resistance. The role of mgrB inactivation in acquired colistin resistance was confirmed by complementation experiments with wild-type mgrB, which restored colistin susceptibility in KKBO-4, and by construction of an mgrB deletion mutant from KKBO-1, which exhibited a colistin-resistant phenotype. Insertional mgrB inactivation was also detected in 60% of colistin-resistant mutants selected from KKBO-1 in vitro, following plating on colistin-containing medium, confirming the role (although not unique) of this mechanism in the emergence of acquired colistin resistance. In colistin-resistant mutants carrying insertional inactivation or deletion of the mgrB gene, upregulated transcription of phoP, phoQ, and pmrK (which is part of the pmrHFIJKLM operon) was detected. These findings confirmed the MgrB regulatory role in K. pneumoniae and were in agreement with the known association between upregulation of the PhoQ/PhoP system and activation of the pmrHFIJKLM operon, which eventually leads to resistance to polymyxins by modification of the lipopolysaccharide target.

Fig 1

Construction of the mgrB deletion mutant of K. pneumoniae KKBO-1 by using plasmid pKOV via homologous recombination. (a) Outline of construction of plasmid pKOVΔmgrB. The positions of primers used for amplification of the flanking regions (KOmgrB1_XbaI_F and KOmgrB1_XhoI_R; KOmgrB2_XhoI_F and KOmgrB2_BglII_R) are indicated by thin black arrows. (b) Chromosomal integration of pKOVΔmgrB in K. pneumoniae KKBO-1 followed by excision of plasmid and generation of K. pneumoniae ΔKKBO-1, carrying a deletion of mgrB. Open reading frames are indicated by arrows. Black backbone lines represent chromosomal regions; dashed backbone lines represent plasmid regions. Gene identifications (IDs) refer to the K. pneumoniae HS11286 chromosome (31).

Fig 2

PFGE profiles of XbaI-digested genomic DNAs showing genomic relatedness among KKBO-1 (left) and KKBO-4 (right) K. pneumoniae isolates. DNA size standards are indicated (in kb) on the left.

Fig 3

Schematic representation of the mgrB loci of K. pneumoniae KKBO-1 (colistin susceptible) and KKBO-4 (colistin resistant). Open reading frames are indicated by arrows. The IS5-like element is shown as a striped rectangle, and its inverted repeats (IR-L and IR-R) are shown as black vertical lines. The interrupted mgrB fragments are shown with dotted borders. Nucleotide sequence details at the insertion junctions of the IS5-like element are also shown: mgrB sequences flanking the insertion point are shown in italics, and the 4-bp direct repeats generated by transposition are boldfaced; the external portions of the IR-L and IR-R of the IS5-like element are in uppercase letters. The 11-bp segment of mgrB representing the putative recombination hot spot is boxed, and the highly similar sequences within the IRs of the IS5-like element are underlined. Gene IDs refer to the K. pneumoniae HS11286 chromosome (31).