Maria-Virginia Villegas

Genetic Structures at the Origin of Acquisition of the β-Lactamase blaKPC Gene

ABSTRACT Genetic structures surrounding the carbapenem-hydrolyzing Ambler class A blaKPC gene were characterized in several KPC-positive Klebsiella pneumoniae and Pseudomonas aeruginosa strains isolated from the United States, Colombia, and Greece. The blaKPC genes were associated in all cases with transposon-related structures. In the K. pneumoniae YC isolate from the United States, the β-lactamase blaKPC-2 gene was located on a novel Tn3-based transposon, Tn4401. Tn4401 was 10 kb in size, was delimited by two 39-bp imperfect inverted repeat sequences, and harbored, in addition to the β-lactamase blaKPC-2 gene, a transposase gene, a resolvase gene, and two novel insertion sequences, ISKpn6 and ISKpn7. Tn4401 has been identified in all isolates. However, two isoforms of this transposon were found: Tn4401a was found in K. pneumoniae YC and K. pneumoniae GR from the United States and Greece, respectively, and differed by a 100-bp deletion, located just upstream of the blaKPC-2 gene, compared to the sequence of Tn4401b, which was found in the Colombian isolates. In all isolates tested, Tn4401 was flanked by a 5-bp target site duplication, the signature of a recent transposition event, and was inserted in different open reading frames located on plasmids that varied in size and nature. Tn4401 is likely at the origin of carbapenem-hydrolyzing β-lactamase KPC mobilization to plasmids and its further insertion into various-sized plasmids identified in nonclonally related K. pneumoniae and P. aeruginosa isolates.

Worldwide diversity of Klebsiella pneumoniae that produce β-lactamase blaKPC-2 gene.

TOC summary: Clones harboring different plasmids with identical genetic structure could be the origin of worldwide spread.

The mgrB gene as a key target for acquired resistance to colistin in Klebsiella pneumoniae

OBJECTIVESnAlterations 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.nnnMETHODSnColistin-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.nnnRESULTSnOf 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.nnnCONCLUSIONnThe 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.

Resistance to Colistin Associated with a Single Amino Acid Change in Protein PmrB among Klebsiella pneumoniae Isolates of Worldwide Origin

ABSTRACT 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.

Wide Dissemination of Pseudomonas aeruginosa Producing β-Lactamase blaKPC-2 Gene in Colombia

ABSTRACT Ten blaKPC-2-harboring Pseudomonas aeruginosa isolates from hospitals located in five different Colombian cities have been characterized. Isolates were multidrug resistant, belonged to five different pulsotypes, and possessed naturally chromosome-encoded blaAmpC and blaOXA-50 genes and the acquired blaKPC-2 gene. In most cases, the blaKPC-2 genes were carried by plasmids of different sizes and were associated with Tn4401b or a new structure containing only part of the Tn4401 sequence. This study revealed that several clones of P. aeruginosa producing blaKPC-2 are disseminating in Colombia.

Complete sequence of two KPC-harbouring plasmids from Pseudomonas aeruginosa

OBJECTIVESnKPC-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.nnnMETHODSnThe 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.nnnRESULTSnBoth plasmids could be transferred to E. coli by transformation and displayed no other resistance marker besides KPC. Plasmid pCOL-1 was 31u200a529 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 Tn4401, including the blaKPC-2 gene, part of ISKpn6 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 β-lactamase blaTEM-1 gene.nnnCONCLUSIONSnHere 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 schemexa0in P. aeruginosa similar to that seen in Enterobacteriaceae.

Modulation of mgrB gene expression as a source of colistin resistance in Klebsiella oxytoca

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.

Development and validation of a multiplex PCR assay for identification of the epidemic ST-258/512 KPC-producing Klebsiella pneumoniae clone

The Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-KP) sequence type (ST)-258/512 clone is the dominant clone by which KPC has disseminated worldwide. Standard typing methods are time-consuming and are therefore impractical for identification of this clone in the course of an outbreak. Through comparative genomic study, we have previously identified several presumably unique genes of this clone: 1) PILV-like protein (pilv-l), 2) transposase, IS66-family (is-66), and a 3) phage-related protein (prp). Our aims were to 1) test for the presence of these genes using a multiplex PCR in a large, multinational collection of KPC-KP isolates and to 2) validate this assay as a typing method for the identification of the ST-258/512 clone. KPC-KP isolates (n=160) that included both ST-258/512 (group A, n=114) and non-ST-258 (group B, n=46) strains were collected from the following countries: Greece, 20; Israel, 93; Italy, 19; USA, 25; and Colombia, 3. Group B included 30 different STs from various lineages. The pilv-l gene was present in 111/114 of ST-258 isolates, including all of the KPC-negative isolates resulting in a sensitivity of 97%. Using primers for a unique ST-258 pilv-l allele resulted in a specificity of 100%. The sensitivity values of is-66 and prp genes for detecting KPC-KP ST-258 were 83 and 89%, respectively, and the specificity values were 67 and 93%, respectively. PCR for the unique pilv-l ST-258 allele provides a reliable tool for rapid detection of the ST-258 clone. This method can be helpful both in the setting of an outbreak and in a large-scale survey of KPC-KP strains.

Dissemination of the KPC-2 carbapenemase in non-Klebsiella pneumoniae enterobacterial isolates from Colombia ☆

Klebsiella pneumoniae carbapenemase (KPC)-type enzymes have largely disseminated worldwide among K. pneumoniae isolates. In this study, 11 non-K. pneumoniae KPC-producing enterobacterial isolates from four hospitals located in different Colombian cities were genetically investigated. All isolates were multidrug-resistant and harboured the bla(KPC-2) gene along with several other acquired β-lactamase genes. The bla(KPC-2) gene was associated with transposon Tn4401b inserted in different loci of plasmids varying in size and replicon type. The presence of KPC-2 in different enterobacterial species from different cities within Colombia underlines the spread of KPC beyond K. pneumoniae.

High-Level Resistance to Colistin Mediated by Various Mutations in the crrB Gene among Carbapenemase-Producing Klebsiella pneumoniae

ABSTRACT Mutations in crrAB genes encoding a two-component regulator involved in modifications of lipopolysaccharide were searched for among a collection of colistin-resistant Klebsiella pneumoniae isolates. Four isolates, respectively, producing carbapenemases NDM-1, OXA-181, or KPC-2 showed mutated CrrB proteins compared with those in wild-type strains. Complementation assays with a wild-type CrrB protein restored the susceptibility to colistin in all cases, confirming the involvement of the identified substitutions in the resistance phenotype.