Emergence of Ceftazidime-Avibactam Resistance Due to Plasmid-Borne blaKPC-3 Mutations during Treatment of Carbapenem-Resistant Klebsiella pneumoniae Infections.
ABSTRACT: Ceftazidime-avibactam is a novel ?-lactam/?-lactamase inhibitor with activity against carbapenem-resistant Enterobacteriaceae (CRE) that produce Klebsiella pneumoniae carbapenemase (KPC). We report the first cases of ceftazidime-avibactam resistance to develop during treatment of CRE infections and identify resistance mechanisms. Ceftazidime-avibactam-resistant K. pneumoniae emerged in three patients after ceftazidime-avibactam treatment for 10 to 19 days. Whole-genome sequencing (WGS) of longitudinal ceftazidime-avibactam-susceptible and -resistant K. pneumoniae isolates was used to identify potential resistance mechanisms. WGS identified mutations in plasmid-borne blaKPC-3, which were not present in baseline isolates. blaKPC-3 mutations emerged independently in isolates of a novel sequence type 258 sublineage and resulted in variant KPC-3 enzymes. The mutations were validated as resistance determinants by measuring MICs of ceftazidime-avibactam and other agents following targeted gene disruption in K. pneumoniae, plasmid transfer, and blaKPC cloning into competent Escherichia coli In rank order, the impact of KPC-3 variants on ceftazidime-avibactam MICs was as follows: D179Y/T243M double substitution > D179Y > V240G. Remarkably, mutations reduced meropenem MICs ?4-fold from baseline, restoring susceptibility in K. pneumoniae from two patients. Cefepime and ceftriaxone MICs were also reduced ?4-fold against D179Y/T243M and D179Y variant isolates, but susceptibility was not restored. Reverse transcription-PCR revealed that expression of blaKPC-3 encoding D179Y/T243M and D179Y variants was diminished compared to blaKPC-3 expression in baseline isolates. In conclusion, the development of resistance-conferring blaKPC-3 mutations in K. pneumoniae within 10 to 19 days of ceftazidime-avibactam exposure is troubling, but clinical impact may be ameliorated if carbapenem susceptibility is restored in certain isolates.
Project description:We identified four <i>bla</i><sub>KPC-3</sub> mutations in ceftazidime-avibactam-resistant clinical <i>Klebsiella pneumoniae</i> isolates, corresponding to D179Y, T243M, D179Y/T243M, and EL165-166 KPC-3 variants. Using site-directed mutagenesis and transforming vectors into <i>Escherichia coli</i>, we conclusively demonstrated that mutant <i>bla</i><sub>KPC-3</sub> encoded enzymes that functioned as extended-spectrum ?-lactamases; mutations directly conferred higher MICs of ceftazidime-avibactam and decreased the MICs of carbapenems and other ?-lactams. Impact was strongest for the D179Y mutant, highlighting the importance of the KPC ?-loop.
Project description:A bla KPC-2-carrying Citrobacter freundii isolate developed ceftazidime-avibactam resistance during treatment with this agent. The initial and follow-up isolates exhibited ceftazidime-avibactam MICs of 4 and 64?µg/ml, respectively. Overexpression of AcrAB-TolC and porin alterations were detected in both isolates, but no other resistance mechanism was observed. After passaging the initial clinical isolate in ceftazidime-avibactam at a fixed concentration of 4?µg/ml and a 4:1 ratio, resistance to all ?-lactams was noted, and a percentage of the bla KPC-2 sequencing reads had mutations leading to the alterations D176Y (bla KPC-2-D176Y [78%]) or R164S plus P147L (bla KPC-2-R164S + P147L [82%]). Further investigation of the follow-up isolate showed that 11% of the bla KPC-2 reads had mutations leading to D179Y substitution (bla KPC-2-D179Y). In the absence of selective pressure, ceftazidime-avibactam MICs of the passaged and follow-up isolates revealed that 7 or 8 out of 20 screened colonies reverted to susceptible and possessed bla KPC-2 wild-type sequences. Recombinant plasmids carrying the bla KPC-2 alterations observed were transformed in Escherichia coli, and MIC values for ceftazidime ± avibactam were elevated. Lower MICs for ceftriaxone, cefepime, aztreonam, meropenem, and imipenem for the mutated KPC-2-producing isolates were observed compared to those of the isolates producing a wild-type KPC-2. Avibactam at a fixed concentration of 4?µg/ml restored the activity of all ?-lactams tested for the recombinant strains. The heterogenous population of wild-type and mutated bla KPC-2 and the reversibility of the genotypes observed suggest a significant challenge for managing KPC-producing isolates that develop ceftazidime-avibactam resistance during therapy.IMPORTANCE The development of ceftazidime-avibactam resistance among KPC-producing isolates during treatment with this agent has been reported. Usually isolates that become resistant have a mutated bla KPC gene that confers resistance to ceftazidime-avibactam and susceptibility to meropenem. We report a Citrobacter freundii isolate that developed ceftazidime-avibactam resistance due to mutations within the coding region of the bla KPC-2 ?-loop previously reported; however, in this case, only 11% of the whole-genome sequencing reads had mutations, making this alteration difficult to detect and the treatment of these isolates more challenging. In addition to bla KPC, the initial and the follow-up patient isolates displayed hyperexpression of the AcrAB-TolC efflux system and disruption of the outer membrane protein (OMP) OmpF, which contribute to carbapenem resistance. Experiments performed to confirm our findings included generating mutant isolates from the initial patient isolate, passaging the isolates for purity in drug-free medium, resulting in a reversible phenotype, and cloning the mutations to demonstrate the resistance conferred.
Project description:Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae isolates have been increasingly reported worldwide, and therapeutic options to treat infections caused by these organisms are limited. We evaluated the activity of ceftazidime-avibactam and comparators against 456 Enterobacteriaceae isolates carrying blaKPC collected from 79 U.S. hospitals during 2012 to 2015. Overall, ceftazidime-avibactam (MIC50/90, 0.5/2 μg/ml; 99.3% susceptible) and tigecycline (MIC50/90, 0.5/1 μg/ml; 98.9% susceptible at ≤2 μg/ml) were the most active agents. Only 80.5% and 59.0% of isolates were susceptible to colistin and amikacin, respectively. All three isolates (0.7%) displaying resistance to ceftazidime-avibactam (K. pneumoniae; MICs, ≥16 μg/ml) were evaluated using whole-genome sequencing analysis and relative quantification of expression levels of porins and efflux pump. Two isolates carried metallo-β-lactamase genes, blaNDM-1 or blaVIM-4, among other β-lactam resistance mechanisms, and one displayed a premature stop codon in ompK35 and decreased expression of ompK36 Ceftazidime-avibactam was active against 100.0 and 99.3% of isolates carrying blaKPC-3 (n = 221) and blaKPC-2 (n = 145), respectively. Isolates carrying blaKPC were more commonly recovered from pneumonia (n = 155), urinary tract (n = 93), and skin/soft tissue (n = 74) infections. Ceftazidime-avibactam (97.8 to 100.0% susceptible) was consistently active against isolates from all infection sites. K. pneumoniae (83.3% of the collection) susceptibility rates were 99.2% for ceftazidime-avibactam, 98.9% for tigecycline, and 80.1% for colistin. Ceftazidime-avibactam susceptibility did not vary substantially when comparing isolates from intensive care unit (ICU) patients to those from non-ICU patients. Ceftazidime-avibactam was active against this large collection of isolates carrying blaKPC and represents a valuable addition to the armamentarium currently available for the treatment of infections caused by KPC-producing Enterobacteriaceae.
Project description:Ceftazidime-avibactam is an antibiotic with activity against serine beta-lactamases, including Klebsiella pneumoniae carbapenemase (KPC). Recently, reports have emerged of KPC-producing isolates resistant to this antibiotic, including a report of a wild-type KPC-3 producing sequence type 258 Klebsiella pneumoniae that was resistant to ceftazidime-avibactam. We describe a detailed analysis of this isolate, in the context of two other closely related KPC-3 producing isolates, recovered from the same patient. Both isolates encoded a nonfunctional OmpK35, whereas we demonstrate that a novel T333N mutation in OmpK36, present in the ceftazidime-avibactam resistant isolate, reduced the activity of this porin and impacted ceftazidime-avibactam susceptibility. In addition, we demonstrate that the increased expression of blaKPC-3 and blaSHV-12 observed in the ceftazidime-avibactam-resistant isolate was due to transposition of the Tn4401 transposon harboring blaKPC-3 into a second plasmid, pIncX3, which also harbored blaSHV-12, ultimately resulting in a higher copy number of blaKPC-3 in the resistant isolate. pIncX3 plasmid from the ceftazidime-avibactam resistant isolate, conjugated into a OmpK35/36-deficient K. pneumoniae background that harbored a mutation to the ramR regulator of the acrAB efflux operon recreated the ceftazidime-avibactam-resistant MIC of 32 ?g/ml, confirming that this constellation of mutations is responsible for the resistance phenotype.
Project description:We determined imipenem, imipenem-relebactam, ceftazidime, and ceftazidime-avibactam MICs against 100 CRE isolates that underwent whole-genome sequencing. Klebsiella pneumoniae carbapenemases (KPCs) were the most common carbapenemases. Forty-six isolates carried extended-spectrum ?-lactamases (ESBLs). With the addition of relebactam, imipenem susceptibility increased from 8% to 88%. With the addition of avibactam, ceftazidime susceptibility increased from 0% to 85%. Neither imipenem-relebactam nor ceftazidime-avibactam was active against metallo-?-lactamase (MBL) producers. Ceftazidime-avibactam (but not imipenem-relebactam) was active against OXA-48-like producers, including a strain not harboring any ESBL. Major OmpK36 porin mutations were independently associated with higher imipenem-relebactam MICs (P < 0.0001) and showed a trend toward independent association with higher ceftazidime-avibactam MICs (P = 0.07). The presence of variant KPC-3 was associated with ceftazidime-avibactam resistance (P < 0.0001). In conclusion, imipenem-relebactam and ceftazidime-avibactam had overlapping spectra of activity and niches in which each was superior. Major OmpK36 mutations in KPC-K. pneumoniae may provide a foundation for stepwise emergence of imipenem-relebactam and ceftazidime-avibactam resistance.
Project description:Ceftazidime-avibactam (CAZ-AVI) is a promising novel treatment for infections caused by carbapenem-resistant Enterobacteriaceae (CRE). Despite improved treatment outcomes compared to those achieved with aminoglycoside- and colistin-based regimens, the rapid evolution of CAZ-AVI resistance during treatment has previously been reported in Klebsiella pneumoniae sequence type 258 (ST258) blaKPC-3-harboring isolates. Here, we report the stepwise evolution and isolation of two phenotypically distinct CAZ-AVI-resistant Klebsiella pneumoniae isolates from a patient with pancreatitis. All susceptible (n = 3) and resistant (n = 5) isolates were of the ST307 clonal background, a rapidly emerging clone. Taking advantage of short-read Illumina and long-read Oxford Nanopore sequencing and full-length assembly of the core chromosome and plasmids, we demonstrate that CAZ-AVI resistance first occurred through a 532G ? T blaKPC-2 point mutation in blaKPC-2 (D179Y protein substitution) following only 12 days of CAZ-AVI exposure. While subsequent isolates exhibited substantially decreased meropenem (MEM) MICs (?2 ?g/ml), later cultures demonstrated a second CAZ-AVI resistance phenotype with a lower CAZ-AVI MIC (12 ?g/ml) but also MEM resistance (MIC > 128 ?g/ml). These CAZ-AVI- and MEM-resistant isolates showed evidence of multiple genomic adaptations, mainly through insertions and deletions. This included amplification and transposition of wild-type blaKPC-2 into a novel plasmid, an IS1 insertion upstream of ompK36, and disruption of the rfb gene locus in these isolates. Our findings illustrate the potential of CAZ-AVI resistance to emerge in non-K. pneumoniae ST258 clonal backgrounds and alternative blaKPC variants. These results raise concerns about the strong selective pressures incurred by novel carbapenemase inhibitors, such as avibactam, on isolates previously considered invulnerable to CAZ-AVI resistance. There is an urgent need to further characterize non-KPC-mediated modes of carbapenem resistance and the intrinsic bacterial factors that facilitate the rapid emergence of resistance during treatment.
Project description:We describe <i>in vivo</i> evolution of carbapenem and ceftazidime-avibactam resistance by analyzing four longitudinal <i>Klebsiella pneumoniae</i> clinical isolates from a patient with pneumonia following antimicrobial treatment. The patient had fever, cough associated with expectoration, and new infiltration was found on the chest CT. Antimicrobial susceptibility was determined, and whole genome sequencing (WGS) was performed to investigate its dynamic change of resistance phenotype. Population analysis profile was performed to investigate the population of <i>Klebsiella pneumoniae</i>. The infection started with a KPC-2-producing <i>K. pneumoniae</i> (ZRKP01, ceftazidime-avibactam-S/carbapenem-R). Then, after ceftazidime-avibactam treatment, the strain switched to D179Y mutant that is KPC-33 (ZRKP02, ceftazidime-avibactam-R/carbapenem-S), which restored carbapenem susceptibility. However, the restored carbapenem susceptibility <i>in vivo</i> was not stable and the subsequent use of imipenem against KPC-33-producing <i>K. pneumoniae</i> infection resulted in a reversion of KPC-2 producers (ZRKP03 and ZRKP04, ceftazidime-avibactam-S/carbapenem-R). Genetic analysis demonstrated that all four <i>K. pneumoniae</i> isolates belonged to sequence type 11and had identical capsular polysaccharide (KL47), identical porin genes, and same plasmid replicon types. Phylogenetic analysis indicated that four <i>K. pneumoniae</i> isolates showed a high degree of relatedness. Single nucleotide polymorphisms analysis indicated that the number of mutations observed in the KPC-33 isolate was more than in the wild-type KPC-2 isolates and the four KPC-Kp isolates evolved from a longitudinal evolution of <i>K. pneumoniae</i> harboring <i>bla</i> <sub>KPC-2</sub> gene. This is the first report to observe the <i>in vivo</i> evolution of wild-type KPC-2 to KPC-33 and then the reversion to its original wild-type KPC-2. Through WGS, we demonstrated the role of selective pressure of antibiotic in the mutation and reversion of <i>bla</i> <sub>KPC</sub> genes, which leading to the dynamic change of KPC enzymes and the dynamic emergence of resistance to ceftazidime-avibactam and carbapenems. <b>Statement:</b> Recently, studies reported the emergence of ceftazidime-avibactam-resistant strains. The KPC mutations mediating ceftazidime-avibactam resistance are generally associated with the restoration of carbapenem susceptibility. However, clinical significance of this observation is unclear. In this manuscript, we demonstrate the role of selective pressure of antibiotic in the mutation and reversion of <i>bla</i> <sub>KPC</sub> genes, which leading to the dynamic change of KPC enzymes and the dynamic emergence of resistance to ceftazidime-avibactam and carbapenems. To the best of our knowledge, this is the first report to observe the <i>in vivo</i> evolution of wild-type KPC-2 to KPC-33 and then the reversion to its original wild-type KPC-2. It should be noted that understanding the clinical significance of this observation is of critical importance, and reversion to carbapenem susceptibility would not imply a potential role for carbapenems monotherapy. We hope our study will draw attention to clinicians, so that this agent can be used most effectively for the longest period of time.
Project description:Resistance to ceftazidime-avibactam due to mutations in KPC genes has been reported both <i>in vitro</i> and in clinical settings. The most frequently reported mutation leads to the amino acid substitution D179Y in the ? loop of the enzyme. Bacterial cells that carry mutant KPC acquire a higher level of ceftazidime resistance, become more sensitive to other cephalosporins, and almost completely lose resistance to carbapenems. In this study, we demonstrated that two substitutions in KPC-2, D179Y and L169P, reduce the ability of avibactam to enhance the activity of ceftazidime, cefepime, or piperacillin against isogenic efflux-deficient strains of <i>Pseudomonas aeruginosa</i>, 8- to 32-fold and 4- to 16-fold for the D179Y and L169P variants, respectively, depending on the antibiotic. In contrast, the potency of vaborbactam, the structurally unrelated ?-lactamase inhibitor that was recently approved by the FDA in combination with meropenem, is reduced no more than 2-fold. Experiments with purified enzymes demonstrate that the D179Y substitution causes an ?20-fold increase in the 50% inhibitory concentration (IC<sub>50</sub>) for inhibition of ceftazidime hydrolysis by avibactam, versus 2-fold for vaborbactam, and that the L169P substitution has an ?4.5-fold-stronger effect on the affinity for avibactam than for vaborbactam. In addition, the D179Y and L169P variants hydrolyze ceftazidime with 10-fold and 4-fold-higher efficiencies, respectively, than that of wild-type KPC-2. Thus, microbiological and biochemical experiments implicate both decreased ability of avibactam to interact with KPC-2 variants and an increase in the efficiency of ceftazidime hydrolysis in resistance to ceftazidime-avibactam. These substitutions have a considerably lesser effect on interactions with vaborbactam, making the meropenem-vaborbactam combination a valuable agent in managing infections due to KPC-producing carbapenem-resistant <i>Enterobacteriaceae</i>.
Project description:<h4>Background</h4>Resistance to ceftazidime-avibactam was reported, and it is important to investigate the mechanisms of ceftazidime/avibactam resistance in K. pneumoniae with mutations in bla<sub>KPC</sub>.<h4>Results</h4>We report the mutated bla<sub>KPC</sub> is not the only mechanism related to CZA resistance, and investigate the role of outer porin defects, efflux pump, and relative gene expression and copy number of bla<sub>KPC</sub> and ompk35/36. Four ceftazidime/avibactam-sensitive isolates detected wild type bla<sub>KPC-2</sub>, while 4 ceftazidime/avibactam-resistant isolates detected mutated bla<sub>KPC</sub> (bla<sub>KPC-51</sub>, bla<sub>KPC-52</sub>, and bla<sub>KPC-33</sub>). Compared with other ceftazidime/avibactam-resistant isolates with the minimal inhibitory concentration of ceftazidime/avibactam ranging 128-256 mg/L, the relative gene expression and copy number of bla<sub>KPC</sub> was increased in the isolate which carried bla<sub>KPC-51</sub> and also showed the highest minimal inhibitory concentration of ceftazidime/avibactam at 2048 mg/L. The truncated Ompk35 contributes rare to ceftazidime/avibactam resistance in our isolates. No significant difference in minimal inhibitory concentration of ceftazidime/avibactam was observed after the addition of PABN.<h4>Conclusions</h4>Increased gene expression and copy number of mutated bla<sub>KPC</sub> can cause high-level ceftazidime/avibactam resistance.
Project description:Ceftazidime/avibactam uniquely demonstrates activity against both KPC and OXA-48-like carbapenemase-expressing Enterobacterales. Clinical resistance to ceftazidime/avibactam in KPC-producers was foreseen in in-vitro resistance studies. Herein, we assessed the resistance selection propensity of ceftazidime/avibactam in <i>K. pneumoniae</i> expressing OXA-48-like β-lactamases (<i>n</i> = 10), employing serial transfer approach. Ceftazidime/avibactam MICs (0.25-4 mg/L) increased to 16-256 mg/L after 15 daily-sequential transfers. The whole genome sequence analysis of terminal mutants showed modifications in proteins linked to efflux (AcrB/AcrD/EmrA/Mdt), outer membrane permeability (OmpK36) and/or stress response pathways (CpxA/EnvZ/RpoE). In-vitro growth properties of all the ceftazidime/avibactam-selected mutants were comparable to their respective parents and they retained the ability to cause pulmonary infection in neutropenic mice. Against these mutants, we explored the activities of various combinations of β-lactams (ceftazidime or cefepime) with structurally diverse β-lactamase inhibitors or a β-lactam enhancer, zidebactam. Zidebactam, in combination with either cefepime or ceftazidime, overcame ceftazidime/avibactam resistance (MIC range 0.5-8 mg/L), while cefepime/avibactam was the second best (MIC: 0.5-16 mg/L) in yielding lower MICs. The present work revealed the possibility of ceftazidime/avibactam resistance in OXA-48-like <i>K. pneumoniae</i> through mutations in proteins involved in efflux and/or porins without concomitant fitness cost mandating astute monitoring of ceftazidime/avibactam resistance among OXA-48 genotypes.