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    Resistance to colistin associated with a single amino acid change in protein PmrB among Klebsiella pneumoniae isolates of worldwide origin
    (American Society for Microbiology, 2014) Jayol, Aurelie; Poirel, Laurent; Brink, Adrian; Villegas, Maria-Virginia; Yılmaz, Mesut; Nordmann, Patrice
    A series of colistin-resistant Klebsiella pneumoniae isolates recovered from different countries was investigated in order to evaluate the involvement of the PmrA/PmrB two-component system in this resistance. Six isolates possessed a mutated PmrB protein, which is encoded by the pmrB gene, part of the pmrCAB operon involved in lipopolysaccharide modification. The same amino acid substitution (Thr157Pro) in PmrB was identified in the six isolates. The six isolates belonged to four distinct clonal groups, recovered in South Africa (sequence type 14 [ST14]), Turkey (ST101), and Colombia (ST258 and ST15). Three out of the four clones produced a carbapenemase, OXA-181, OXA-48, or KPC-3, while a single isolate did not produce any carbapenemase. Expression assays revealed an overexpression of the pmrA (70-fold), pmrB (70-fold), pmrC (170-fold), and pmrK (40-fold) genes in the pmrB-mutated isolate compared to expression of the pmrB wild-type isogenic K. pneumoniae isolate, confirming that the PmrB substitution was responsible for increased expression levels of those genes. Complementation assays leading to the expression of a wild-type PmrB protein restored the susceptibility to colistin in all isolates, confirming that the substitution in PmrB was responsible for the resistance phenotype. This study identified a key amino acid located in the PmrB protein as being responsible for the overexpression of pmrCAB and pmrHFIJKLM operons, leading to resistance to colistin.
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    The mgrB gene as a key target for acquired resistance to colistin in Klebsiella pneumoniae
    (Oxford University Press, 2015) Poirel, Laurent; Jayol, Aurelie; Bontron, Severine; Villegas, Maria-Virginia; Özdamar, Melda; Türkoğlu, Salih; Nordmann, Patrice
    Objectives: Alterations in the PhoPQ two-component regulatory system may be associated with colistin resistance in Klebsiella pneumoniae. MgrB is a small transmembrane protein produced upon activation of the PhoPQ signalling system, and acts as a negative regulator on this system. We investigated the role of the MgrB protein as a source of colistin resistance in a series of K. pneumoniae. Methods: Colistin-resistant K. pneumoniae isolates were recovered from hospitalized patients worldwide (France, Turkey, Colombia and South Africa). The mgrB gene was amplified and sequenced. A wild-type mgrB gene was cloned and the corresponding recombinant plasmid was used for complementation assays. Clonal diversity was evaluated by MLST and Diversilab analysis. Results: Of 47 colistin-resistant isolates, 12 were identified as having a mutated mgrB gene. Five clonally unrelated isolates had an mgrB gene truncated by an IS5-like IS, while one clone also harboured an insertional inactivation at the exact same position of the mgrB gene, but with ISKpn13. Another clone harboured an insertional inactivation due to ISKpn14 at another location of the mgrB gene. Two clonally related isolates harboured an IS (IS10R) in the promoter region of mgrB. Finally, three clonally unrelated isolates harboured substitutions leading to anticipated stop codon in the MgrB protein. Complementation assays with a wild-type MgrB protein restored full susceptibility to colistin for all colistin-resistant isolates identified with qualitative or quantitative MgrB modifications. Conclusion: The inactivation or down-regulation of the mgrB gene was shown to be a source of colistin resistance in K. pneumoniae. Interestingly, identical genetic events were identified among clonally unrelated isolates.