Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP) are challenging multidrug-resistant pathogens due to their extensively drug-resistant phenotypes and potential for epidemic dissemination in health care settings. Colistin is a key component of the combination antimicrobial regimens used for treatment of severe KPC-KP infections. We previously reported that insertional inactivation of the mgrB gene, encoding a negative-feedback regulator of the PhoQ-PhoP signaling system, can be responsible for colistin resistance in KPC-KP, due to the resulting upregulation of the Pmr lipopolysaccharide modification system. In this work we investigated the status of the mgrB gene in a collection of 66 colistin-resistant nonreplicate clinical strains of KPC-KP isolated from different hospitals in Italy and Greece. Overall, 35 strains (53%) exhibited alterations of the mgrB gene, including insertions of different types of mobile elements (IS 5 -like, IS 1F -like, or IS Kpn14 ), nonsilent point mutations, and small intragenic deletions. Four additional strains had a larger deletion of the mgrB locus, while the remaining 27 strains (41%) did not show mgrB alterations. Transcriptional upregulation of the phoQ and pmrK genes (part of the phoPQ and pmrHFIJKLM operon, respectively) was observed in all strains with mgrB alterations. Complementation experiments with a wild-type mgrB gene restored colistin susceptibility and basal expression levels of phoQ and pmrK genes in strains carrying different types of mgrB alterations. The present results suggest that mgrB alteration can be a common mechanism of colistin resistance among KPC-KP in the clinical setting.

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.

Consecutive non-replicate clinical isolates (n=191) of carbapenem non-susceptible Enterobacteriaceae were collected from 21 hospital laboratories across Italy from November 2013 to April 2014 as part of the European Survey on Carbapenemase-producing Enterobacteriaceae (EuSCAPE) project. Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) represented 178 (93%) isolates with 76 (43%) respectively resistant to colistin, a key drug for treating carbapenamase-producing Enterobacteriaceae. KPC-KP colistin-resistant isolates were detected in all participating laboratories. This underscores a concerning evolution of colistin resistance in a setting of high KPC-KP endemicity.

A novel mcr variant, named mcr-1.2 , encoding a Gln ₃ -to-Leu functional variant of MCR-1, was detected in a KPC-3-producing ST512 Klebsiella pneumoniae isolate collected in Italy from a surveillance rectal swab from a leukemic child. The mcr-1.2 gene was carried on a transferable IncX4 plasmid whose structure was very similar to that of mcr-1 -bearing plasmids previously found in Escherichia coli and K. pneumoniae strains from geographically distant sites (Estonia, China, and South Africa).