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Jayol A.,University of Fribourg | Poirel L.,University of Fribourg | Villegas M.-V.,International Center for Medical Research and Training | Nordmann P.,University of Fribourg
International Journal of Antimicrobial Agents | Year: 2015

Gene modifications in the PmrAB and PhoPQ two-component regulatory systems, as well as inactivation of the mgrB gene, are known to be causes of colistin resistance in Klebsiella pneumoniae. The objective of this study was to characterise the mechanism involved in colistin resistance in a Klebsiella oxytoca isolate. A K oxytoca clinical isolate showing resistance to colistin was recovered in Cali, Colombia. The pmrA, pmrB, phoP, phoQ and mgrB genes were amplified and sequenced. Wild-type mgrB genes from K pneumoniae and K. oxytoca were cloned, and corresponding recombinant plasmids were used for complementation assays. By analysing the mgrB gene of the K. oxytoca isolate and its flanking sequences, an insertion sequence (IS) of 1196bp was identified in its promoter region. The insertion was located between nucleotides-39 and -38 when referring to the start codon of the mgrB gene, thus negatively interfering with expression of the mgrB gene by modifying its promoter structure. This IS was very similar to ISKpn26 (99% nucleotide identity) belonging to the IS5 family. Complementation assays with mgrB genes from wild-type K pneumoniae or K oxytoca restored full susceptibility to colistin. In conclusion, here we identified the mechanism involved in colistin resistance in a K oxytoca isolate. Modulation of mgrB gene expression was the key factor for this acquired resistance to colistin. © 2015 Published by Elsevier B.V.


Cuzon G.,French Institute of Health and Medical Research | Naas T.,French Institute of Health and Medical Research | Truong H.,French Institute of Health and Medical Research | Villegas M.-V.,International Center for Medical Research and Training | And 7 more authors.
Emerging Infectious Diseases | Year: 2010

Klebsiella pneumoniae isolates that produce carbapenemases (KPCs) are rapidly disseminating worldwide. To determine their genetic background, we investigated 16 blaKPC-2-harboring K. pneumoniae isolates from 5 countries. The isolates were multidrug resistant, possessed the blaKPC-2 gene, and differed by additional β-lactamase content. They harbored a naturally chromosome-encoded bla gene (blaSHV-1 [12.5%], blaSHV-11 [68.7%], or blaOKP-A/B [18.8%]) and several acquired and plasmid-encoded genes (blaTEM-1 [81.3%], blaCTX-M-2 [31.3%], blaCTX-M-12 [12.5%], blaCTX-M-15 [18.7%], and blaOXA-9 [37.5%]). The blaKPC-2 gene was always associated with 1 of the Tn4401 isoforms (a, b, or c). Tn4401 was inserted on different-sized plasmids that belonged to different incompatibility groups. Several blaKPC-containing K. pneumoniae clones were found: 9 different pulsotypes with 1 major (sequence type 258) and 7 minor distinct allelic profiles. Different clones harboring different plasmids but having identical genetic structure, Tn4401, could be at the origin of the worldwide spread of this emerging resistance gene.


Poirel L.,University of Fribourg | Poirel L.,French Institute of Health and Medical Research | Jayol A.,French Institute of Health and Medical Research | Bontron S.,University of Fribourg | And 5 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2015

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. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.


Naas T.,University Paris - Sud | Bonnin R.A.,University Paris - Sud | Cuzon G.,University Paris - Sud | Villegas M.,International Center for Medical Research and Training | Nordmann P.,University Paris - Sud
Journal of Antimicrobial Chemotherapy | Year: 2013

Objectives: KPC-producing Pseudomonas aeruginosa are increasingly isolated in the Americas and in the Caribbean islands. Here, we determined the whole-plasmid sequence of two plasmids carrying the blaKPC-2 gene from multidrug-resistant P. aeruginosa clinical isolates from Colombia. Methods: The two plasmids, pCOL-1 and pPA-2, were transferred to Escherichia coli recipient strain TOP10 and completely sequenced using high-throughput pyrosequencing for pCOL-1 and classical Sanger sequencing for pPA-2. Results: Both plasmids could be transferred to E. coli by transformation and displayed no other resistance marker besides KPC. Plasmid pCOL-1 was 31529 bp in size, contained 31 open reading frames (ORFs) and belonged to the IncP-6 replicon group. It exhibited genes involved in replication, mobilization and partitioning, but none involved in conjugation. Plasmid pPA-2 was 7995 bp in size and contained seven ORFs. It exhibited a replicase gene of IncU, but was lacking genes involved in mobilization, partitioning and conjugation. Only 2072 bp matched Tn4402, including the blaKPC-2 gene, part of ISKpnó and a 73 bp segment located upstream of the blaKPC-2 gene, containing the P1 promoter. Sequence identity was interrupted by a Tn3 transposon, itself interrupted by an IS26 element inserted within the (3-lactamase b/aTEM-1 gene. Conclusions: Here we present the genetic features of the very first plasmids carrying the blaKPC-2 gene from P. aeruginosa. The emergence of the blaKPC-2 gene on unrelated plasmids, differing in size and in incompatibility group, and harbouring different genetic structures containing the blaKPC-2 genes in P. aeruginosa isolates suggests that this resistance trait may follow a dissemination scheme in P. aeruginosa similar to that seen in Enterobacteriaceae. © The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.


Jayol A.,French Institute of Health and Medical Research | Poirel L.,French Institute of Health and Medical Research | Poirel L.,University of Fribourg | Brink A.,Ampath National Laboratory Services | And 4 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2014

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. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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