Identification of a Novel Ceftazidime-Avibactam-Resistant KPC-2 Variant, KPC-123, in Citrobacter koseri Following Ceftazidime-Avibactam Treatment.
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SUBMITTER: Wang L
PROVIDER: S-EPMC9251512 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:KPC-82 is a KPC-2 variant identified in a carbapenem-nonsusceptible Citrobacter koseri that confers high-level resistance to ceftazidime-avibactam. Genomic analysis revealed that blaKPC-82 is carried by a chromosomally integrated Tn4401 transposon (disrupting porin gene phoE) and evolved by a 6-nucleotide tandem repeat duplication causing a two-amino-acid insertion (Ser-Asp) within the Ala267-Ser275 loop. Similar to related KPC variants, KPC-82 showed decreased carbapenemase activity when expressed in a heterologous background and remained susceptible to carbapenem/β-lactamase inhibitor combinations.
| S-EPMC8218621 | biostudies-literature
Project description:A novel Klebsiella pneumoniae carbapenemase (KPC) variant, KPC-93, was identified in two Klebsiella pneumoniae clinical isolates from a patient from China treated with ceftazidime-avibactam. KPC-93 possessed a five-amino-acids insertion (Pro-Asn-Asn-Arg-Ala) between Ambler positions 267 and 268 in KPC-2. Cloning and expression of the blaKPC-93 gene in Escherichia coli, followed by determination of minimum inhibitory concentration (MIC) values and kinetic parameters, showed that KPC-93 exhibited increased resistance to ceftazidime-avibactam, but a drastic decrease in carbapenemase activity. Our data highlight that a KPC variant conferring resistance to ceftazidime-avibactam could be easily induced by ceftazidime-avibactam treatment and that actions are required to control dissemination of these determinants. IMPORTANCE Ceftazidime-avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor combination with activity against serine β-lactamases, including the Ambler class A enzyme KPC. However, during recent years, there have been increasing reports of emergence of new KPC variants that could confer resistance to CZA. This has limited its clinical application. Here, we reported a new KPC variant, KPC-93, that could confer CZA resistance. KPC-93 possessed a five-amino-acids insertion (Pro-Asn-Asn-Arg-Ala) between Ambler positions 267 and 268 in KPC-2. Our findings have revealed the potential risk of blaKPC gene mutations associated with CZA exposure over a short period of time.
| S-EPMC9045388 | biostudies-literature
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.
| S-EPMC6158515 | biostudies-literature
Project description:This study reports on the characterization of a Klebsiella pneumoniae clinical isolate showing high-level resistance to ceftazidime-avibactam associated with the production of KPC-53, a KPC-3 variant exhibiting a Leu167Glu168 duplication in the Ω-loop and a loss of carbapenemase activity. Whole-genome sequencing (WGS) revealed the presence of two copies of bla KPC-53, located on a pKpQIL-like plasmid and on a plasmid prophage of the Siphoviridae family, respectively. The present findings provide new insights into the mechanisms of resistance to ceftazidime-avibactam.
| S-EPMC7927837 | biostudies-literature
Project description:Carbapenem-resistant Pseudomonas aeruginosa (CRPA) has become a serious challenge in the clinic. Recently, the prevalence of CRPA isolates carrying the blaKPC-2 gene has been increasing in China. Ceftazidime-avibactam (CZA) has shown good efficacy against large portions of KPC-2-producing CRPA strains. However, with the increasing usage of this drug, CZA resistance in CRPA strains has been reported. Here, we reported for the first time that resistance of the ST463 CRPA strain to CZA was caused by a novel variant in the KPC gene that arose after CZA exposure. The CRPA strain PA2207 is a carbapenem- and CZA-resistant strain that harbors a mutated blaKPC gene, named blaKPC-90. Cloning and expression of blaKPC-90 in Escherichia coli DH5α revealed that KPC-90 led to a 64-fold increase in the MIC value of CZA. Conjugation experiments further confirmed that blaKPC-90 was located on a conjugative plasmid. Whole-genome sequencing analysis showed that this plasmid had high sequence similarity to a previously reported novel blaKPC-2-harboring plasmid in a clinical P. aeruginosa strain isolated in China. In addition, overexpression of an efflux pump (MexXY-OprM) might be associated with the CZA resistance phenotype, as determined by reverse transcription-quantitative PCR and efflux pump inhibition experiments. For the first time, we reported a KPC variant, KPC-90, in a clinical ST463 CRPA strain with CZA resistance that was mediated by a 2 amino acid insertion outside the KPC omega-loop region. Our study further highlights that diverse KPC variants that mediate CZA resistance have emerged in the CRPA strain. Furthermore, KPC-90 mutation combined with efflux pump overexpression resulted in a high level of resistance to CZA in the PA2207 isolate. Effective surveillance should be conducted to prevent CZA resistance from spreading in the CRPA strain. IMPORTANCE For the first time, we reported a KPC variant, KPC-90, in a clinical ST463 CRPA strain with CZA resistance. CZA resistance was mediated by a 2 amino acid insertion outside the KPC omega-loop region in CRPA. Our study further emphasized that CZA resistance caused by blaKPC gene mutation could be selected in CRPA after CZA therapy. Considering the widespread presence of the ST463 CRPA strain in China, clinicians should pay attention to the risk of the development of CZA resistance in CRPA strains under treatment pressure.
| S-EPMC8754116 | biostudies-literature
Project description:The novel β-lactam/β-lactamase inhibitor combinations (βL-βLICs) are one of the last-line resources available against multidrug-resistant (MDR) Gram-negative bacteria. Among βL-βLICs, ceftazidime/avibactam (CAZ-AVI) demonstrated strong activity against carbapenem-resistant Enterobacterales (CRE). Avibactam was proven to restore bactericidal activity of ceftazidime, inhibiting both KPC and OXA-48-like β-lactamases. Despite this, emergence of CAZ-AVI-resistant strains in Enterobacterales has been reported. Herein, we evaluated the in vitro ceftazidime activity in the presence of increasing concentrations of avibactam by the broth microdilution method against CAZ-AVI-susceptible and resistant genome-characterized KPC-producing K. pneumoniae (KPC-Kp) clinical isolates. Strains expressing KPC and co-expressing KPC/OXA-181 carbapenemase were selected on the basis of the different phenotypic traits for novel βL-βLICs and cefiderocol. Notably, avibactam at 8 mg/L maintained the MIC of ceftazidime above the clinical breakpoint in 14 out of 15 (93%) KPC-Kp resistant to CAZ-AVI. A high concentration of avibactam (i.e., 64 mg/L) is required to observe a bactericidal activity of ceftazidime against 9 out of 15 (60%) CAZ-AVI-resistant isolates. In vitro evaluation showed that with the increase in the concentration of avibactam, ceftazidime showed high activity against CAZ-AVI-susceptible strains. High concentrations of avibactam in vivo are required for ceftazidime to be active against CAZ-AVI-resistant KPC-Kp.
| S-EPMC10740727 | biostudies-literature
Project description:IntroductionCeftazidime/avibactam-resistance in Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) is a topic of great interest for epidemiological, diagnostic, and therapeutical reasons. However, data on its prevalence and burden on mortality in patients with bloodstream infection (BSI) are lacking. This study was aimed at identifying risk factors for mortality in patients suffering from ceftazidime/avibactam-resistant KPC-Kp BSI.MethodsAn observational retrospective study (January 2018-December 2022) was conducted at a tertiary hospital including all consecutive hospitalized adult patients with a ceftazidime/avibactam-resistant KPC-Kp BSI. Data on baseline clinical features, management, and admission outcomes were analyzed.ResultsOver the study period, among all the KPC-Kp BSI events recorded, 38 (10.5%) were caused by ceftazidime/avibactam-resistant KPC-Kp strains, 37 events being finally included. The ceftazidime/avibactam-resistant KPC-Kp strains revealed susceptibility restoration to at least one carbapenem in more than 60% of cases. In-hospital and 30-day all-cause mortality rates were 22% and 16.2%, respectively. Non-survivors suffered from more baseline comorbidities and experienced a more severe ceftazidime/avibactam-resistant KPC-Kp BSI presentation (i.e., both the Pitt Bacteremia and INCREMENT-CPE scores were significantly higher). Presenting with a higher Charlson Comorbidity Index, chronic kidney disease-KDIGO stage 3A or worse-having recently gone through renal replacement therapy, having suffered from an acute kidney injury following the ceftazidime/avibactam-resistant KPC-Kp BSI, and being admitted for cardiac surgery were the strongest predictors of mortality.ConclusionCeftazidime/avibactam resistance in KPC-Kp BSI easily emerged in our highly KPC-Kp endemic area with remarkable mortality rates. Our findings might provide physicians possibly actionable information when managing patients with a ceftazidime/avibactam-resistant KPC-Kp BSI.
| S-EPMC10774640 | biostudies-literature
Project description:BackgroundCeftazidime-avibactam was approved by the FDA to treat infections caused by Enterobacterales carrying blaKPC-2. However, variants of KPC-2 with amino acid substitutions at position 179 have emerged and confer resistance to ceftazidime-avibactam.MethodsThe activity of imipenem-relebactam was assessed against a panel of 19 KPC-2 D179 variants. KPC-2 and the D179N and D179Y variants were purified for biochemical analyses. Molecular models were constructed with imipenem to assess differences in kinetic profiles.ResultsAll strains were susceptible to imipenem-relebactam, but resistant to ceftazidime (19/19) and ceftazidime-avibactam (18/19). KPC-2 and the D179N variant hydrolyzed imipenem, but the D179N variant's rate was much slower. The D179Y variant was unable to turnover imipenem. All three β-lactamases hydrolyzed ceftazidime at varying rates. The acylation rate of relebactam for the D179N variant was ~2.5× lower than KPC-2. Poor catalytic turnover by the D179Y variant precluded the determination of inhibitory kinetic parameters. Acyl-complexes with imipenem and ceftazidime were less prevalent with the D179N variant compared to the D179Y variant, supporting the kinetic observations that the D179Y variant was not as active as the D179N variant. Relebactam was slower to form an acyl-complex with the D179Y variant compared to avibactam. The D179Y model with imipenem revealed that the catalytic water molecule was shifted, and the carbonyl of imipenem was not within the oxyanion hole. Conversely in the D179N model, imipenem was oriented favorably for deacylation.ConclusionsImipenem-relebactam overcame the resistance of the D179 variants, suggesting that this combination will be active against clinical isolates harboring these derivatives of KPC-2.
| S-EPMC10215400 | biostudies-literature
Project description:Ceftazidime-avibactam is an effective antibiotic combination of a β-lactam and a β-lactamase inhibitor against Klebsiella pneumoniae-carbapenemase (KPC)-producing Enterobacterales. Despite a relatively low resistance rate, reports of resistance to ceftazidime-avibactam mainly caused by the mutations in KPC have increased in recent years. Here, we report a ceftazidime-avibactam-resistant and carbapenem-susceptible Klebsiella pneumoniae strain carrying a novel KPC variant, KPC-112, which differs from KPC-2 by 4-amino-acid deletions at Ambler positions 166L/167E and 242G/243T. The isolate was identified as K. pneumoniae by a Vitek mass spectrometer (bioMérieux, France). The MICs of antimicrobial agents were determined using broth microdilution susceptibility method. The result showed that the isolate was resistant to ceftazidime-avibactam (MIC = >128 mg/L) but susceptible to imipenem (MIC = 0.5 mg/L), meropenem (MIC = 1 mg/L), and tigecycline (MIC = 2 mg/L). The carbapenemase genes were confirmed by PCR-based sequencing. Plasmid transformation assay showed that the blaKPC-112-positive transformant increased MICs of ceftazidime-avibactam, ceftazidime, and cefepime by at least 256-fold, 128-fold, and 128-fold, respectively, compared with the recipient Escherichia coli DH5α. According to the whole-genome sequencing analysis, many common resistance genes were identified, including blaKPC-112, blaOXA-1, blaCTX-M-15, blaTEM-1B, blaSHV-28, aac(6')Ib-cr, aac(3)-IId, qnrS1, catA2, catB4, and fosA6, and mutations of GyrA (GyrA-83F and GyrA-87A) and ParC (ParC-80I) were also found. Overall, our study highlights the importance of monitoring susceptibility during ceftazidime-avibactam treatment and accurate detection of KPC variants. IMPORTANCE Carbapenem-resistant Enterobacterales (CRE) are one of the most serious antimicrobial resistance problems in the world, listed as an "urgent" threat by the U.S. Centers for Disease Control and Prevention. Among CRE, K. pneumoniae-carbapenemase-producing Klebsiella pneumoniae (KPC-KP) has become a significant health threat due to its rapid transmissibility and high mortality. With the wider clinical use of ceftazidime-avibactam, reports of resistance have increased in recent years even though the overall resistance rate remains relatively low. Among the reported resistance mechanisms are mainly mutations derived from the blaKPC-2 or blaKPC-3 gene. Here, we describe the characterization of a ceftazidime-avibactam-resistant blaKPC-112-positive K. pneumoniae clinical isolate for the first time. A number of Enterobacteriaceae isolates producing these kinds of KPC variants might be missed by conventional antimicrobial susceptibility testing (AST) methods and lead to irrational drug use. So, this study of KPC-112 will help to establish the diversity of KPCs and remind researchers of the challenge of drug resistance and detection brought by the KPC variants.
| S-EPMC9769832 | biostudies-literature
Project description:Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection poses a great threat to public health worldwide, and KPC-2-producing strains are the main factors responsible for resistance to carbapenems in China. Ceftazidime/avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor combination with good activity against KPC-2 carbapenemase and is becoming the most important option for treating KPC-producing CRKP infection. Here, we report the emergence of a novel KPC-2 variant, designated KPC-74, produced by K. pneumoniae strain KP55, that conferred CZA resistance in a patient after CZA exposure. The novel bla KPC-74 variant showed a deletion of 6 nucleotides at positions 712-717 compared with bla KPC-2, and this deletion resulted in the consequent deletion of glycine and valine at positions 239 and 240. Antimicrobial susceptibility testing showed that KP55 presents multidrug resistance, including resistance to CZA and ertapenem, but is susceptible to imipenem, meropenem, and colistin. The bla KPC-74 gene was located on a plasmid, as determined by S1-nuclease pulsed-field gel electrophoresis followed by southern blotting, and confirmed to be 133,766 bp in length by whole-genome sequencing on both the Illumina and MinION platforms. The CZA resistance phenotype of the novel KPC variant was confirmed by both transformation of the bla KPC-74-harboring plasmid and a bla KPC-74 gene cloning assay, showing a 64-fold higher CZA minimum inhibitory concentration (MIC) than the recipient strains. The G239_V240del observed in KPC-74 was outside the omega-loop region but was still close to the active site Ser70 and omega-loop in the protein tertiary structure. The enzyme kinetic parameters and IC50 values further indicated that the hydrolytic activity of the KPC-74 enzyme against ceftazidime was potentiated twofold and that the affinity between KPC-74 and avibactam was alleviated 17-fold compared with that of the KPC-2 allele. This CZA resistance mediated by KPC-74 could be selected after CZA therapy and evolved to be more diverse and heterogeneous. Surveillance of CZA resistance is urgently needed in clinical settings.
| S-EPMC8415713 | biostudies-literature