Cefiderocol Resistance Conferred by Plasmid-Located Ferric Citrate Transport System in KPC-Producing Klebsiella pneumoniae

Abstract

Cefiderocol (FDC), a siderophore-cephalosporin conjugate, is the newest option for treating infection with carbapenem-resistant gram-negative bacteria. We identified a novel mechanism contributing to decreased FDC susceptibility in Klebsiella pneumoniae clinical isolates. The mechanism involves 2 coresident plasmids: pKpQIL, carrying variants of bla_KPC carbapenemase gene, and pKPN, carrying the ferric citrate transport (FEC) system. We observed increasing FDC MICs in an Escherichia coli model system carrying different natural pKpQIL plasmids, encoding different K. pneumoniae carbapenemase (KPC) variants, in combination with a conjugative low copy number vector carrying the fec gene cluster from pKPN. We observed transcriptional repression of fiu, cirA, fepA, and fhuA siderophore receptor genes in bla_KPC-fec-E. coli cells treated with ferric citrate. Screening of 27,793 K. pneumoniae whole-genome sequences revealed that the fec cluster occurs frequently in some globally distributed different KPC-producing K. pneumoniae clones (sequence types 258, 14, 45, and 512), contributing to reduced FDC susceptibility.

Keywords: Italy; KPC; Klebsiella pneumoniae; antimicrobial resistance; bacteria; carbapenemase-producing Klebsiella pneumoniae; cefiderocol; ferric citrate-dependent iron transport system; siderophore.

Figure 1

Schematic representation of R69c and R69c-FEC conjugation in Escherichia coli DH5-α recipient carrying the pKpQIL plasmids R69c and R69c-FEC and pKpQIL major features in study of cefiderocol resistance conferred by plasmid-located ferric citrate transport system in KPC-producing Klebsiella pneumoniae. The left panel (blue) represents construction of the R69c and R69c-FEC donor vectors, both introduced by transformation in E. coli DH5-α chemically competent cells. The central panel (yellow) shows the pKpQIL transformation of E. coli DH5-α chemically competent cells with different pKpQIL natural plasmids extracted from K. pneumoniae strains. The right panel (green) represents the exconjugant pairs obtained by conjugation of the R69c vectors into the recipients carrying the different pKpQIL plasmids. FEC, ferric citrate transport system. KPC, Klebsiella pneumoniae carbapenemase.

Figure 2

FDC MICs of KPC-producing Escherichia coli in a study of cefiderocol resistance conferred by plasmid-located ferric citrate transport system in KPC-producing Klebsiella pneumoniae. FDC susceptibility tests were performed according to manufacturer directives, with concentrations of 0 µM, 0.5 µM, and 5 µM ammonium ferric citrate on Escherichia coli DH5-α cells carrying different combinations of pKpQIL, R69c, and R69c-FEC plasmids. FDC, cefiderocol; KPC, Klebsiella pneumoniae carbapenemase.

Figure 3

Expression analysis of siderophore receptor genes in the presence and absence of plasmidic fec gene cluster and ferric citric inducer in an Escherichia coli model used in a study of cefiderocol resistance conferred by plasmid-located ferric citrate transport system KPC-producing Klebsiella pneumoniae. A) Transcription of the fecA genes in the DH5-α strain carrying R69c or R69c- FEC, determined by using primer pairs able to discern the chromosomal fecA allele from the K. pneumoniae fecA gene in the fecABCDE operon or a primer pair recognizing both chromosomal and plasmidic fecA alleles (Appendix 1 Table 2). The relative quantitative analysis of the transcripts was based on the 2^−ΔΔCT method (31). In both bar graphs, the relative values were calculated with respect to the transcript level observed in the R69c carrying strains and set to 1. B) Transcription of the siderophore receptor genes fiu, cirA, fepA, and fhuA in the R69c-FEC and R69c strains grown in the absence of ferric citrate or in the presence of 0.5 μM or 5.0 μM ferric citrate, relative to the R69c strain grown without ferric citrate, which is set to 1. The relative quantitative analysis of the transcripts was based on the 2^−ΔΔCT method (31). Error bars represent SDs. Statistical significance was determined by using a paired 2-tailed Student t-test comparing the dataset obtained from the 2 strains grown under the same conditions. FEC, ferric citrate transport system; KPC, Klebsiella pneumoniae carbapenemase.

Figure 4

Distribution of the fec gene cluster among prevalent STs of Klebsiella pneumoniae. Distribution of the fec gene cluster, represented by a black bar (number of positive genomes) or a white bar (number of negative genomes), across the total analyzed genomes for prevalent STs in the K. pneumoniae database (Appendix 3, Table). ST, sequence type.

Figure 5

Phylogenetic analysis of Klebsiella pneumoniae ST512 based on core-genome alignment of 510 K. pneumoniae ST512 isolates. The tree is midpoint rooted, and the scale bar represents the number of substitutions per site. The presence of the fec operon is indicated in red; bla_KPC, bla_VIM, and bla_NDM genes in black; and the FIB(K) replicon in orange. Yellow shading indicates genomes sequenced in this study or our previous studies (Appendix 1, Table 1). The best-fit model was selected by ModelFinder (http://www.iqtree.org/ModelFinder), The tree was visualized with Microreact (https://microreact.org) and adjusted by using the InkScape software (https://www.inkscape.org). ST, sequence type.

Figure 6

Phylogenetic analysis based on core-genome alignments of 468 Klebsiella pneumoniae ST101 isolates in study of cefiderocol resistance conferred by plasmid-located ferric citrate transport system in K. pneumoniae carbapenemase–producing K. pneumoniae. The trees are midpoint rooted, and the scale bar represents the number of substitutions per site. The presence of the fec operon is indicated in red; bla_KPC, bla_VIM, and bla_NDM genes in black; and the FIB(K) replicon in orange. Yellow shading indicates genomes sequenced in this study or our previous studies (Appendix 1, Table 1). The best-fit model was selected by ModelFinder (34). The trees were visualized with Microreact (https://microreact.org) and adjusted by using the InkScape software (https://www.inkscape.org). ST, sequence type.

Figure 7

Phylogenetic analysis based on core-genome alignments of 1,516 Klebsiella pneumoniae ST307 isolates in a study of cefiderocol resistance conferred by plasmid-located ferric citrate transport system in K. pneumoniae carbapenemase–producing K. pneumoniae. The trees are midpoint rooted, and the scale bar represents the number of substitutions per site. The presence of the fec operon is indicated in red; bla_KPC, bla_VIM, and bla_NDM genes in black; and the FIB(K) replicon in orange. Yellow shading indicates genomes sequenced in this study or our previous studies (Appendix 1, Table 1). The best-fit model was selected by ModelFinder (34). The trees were visualized with Microreact (https://microreact.org) and adjusted by using the InkScape software (https://www.inkscape.org). ST, sequence type.