KPC-39-Mediated Resistance to Ceftazidime-Avibactam in a Klebsiella pneumoniae ST307 Clinical Isolate.
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ABSTRACT:
SUBMITTER: Jousset AB
PROVIDER: S-EPMC8597731 | biostudies-literature | 2021 Nov
REPOSITORIES: biostudies-literature
Publications
KPC-39-Mediated Resistance to Ceftazidime-Avibactam in a Klebsiella pneumoniae ST307 Clinical Isolate.
Jousset Agnès B AB Oueslati Saoussen S Emeraud Cécile C Bonnin Rémy A RA Dortet Laurent L Iorga Bogdan I BI Naas Thierry T
Antimicrobial agents and chemotherapy 20211004 12
Resistance to the ceftazidime (CAZ)-avibactam (AVI) combination is increasingly being reported. Here, we report a CAZ-AVI-resistant Klebsiella pneumoniae strain belonging to the high-risk sequence type 307 (ST307) clone and producing Klebsiella pneumoniae carbapenemase 39 (KPC-39), a single-amino-acid variant of KPC-3 (A172T). Cloning experiments, steady-state kinetic parameters, and molecular dynamics simulations revealed a loss of carbapenemase activity and increased affinity for CAZ. KPC-39 w ...[more]
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Project description:Carbapenem-resistant Enterobacterales, such as Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae, represent a major threat to public health due to their rapid spread. Novel drug combinations such as ceftazidime-avibactam (CZA), combining a broad-spectrum cephalosporin along with a broad-spectrum β-lactamase inhibitor, have recently been introduced and have been shown to exhibit excellent activity toward multidrug-resistant KPC-producing Enterobacterales strains. However, CZA-resistant K. pneumoniae isolates are now being increasingly reported, mostly corresponding to producers of KPC variants. In this study, we evaluated in vitro the nature of the mutations in the KPC-2 and KPC-3 β-lactamase sequences (the most frequent KPC-type enzymes) that lead to CZA resistance and the subsequent effects of these mutations on susceptibility to other β-lactam antibiotics. Single-step in vitro selection assays were conducted, resulting in the identification of a series of mutations in the KPC sequence which conferred the ability of those mutated enzymes to confer resistance to CZA. Hence, 16 KPC-2 variants and 10 KPC-3 variants were obtained. Production of the KPC variants in an Escherichia coli recombinant strain resulted in a concomitant increased susceptibility to broad-spectrum cephalosporins and carbapenems, with the exceptions of ceftazidime and piperacillin-tazobactam, compared to wild-type KPC enzymes. Enzymatic assays showed that all of the KPC variants identified exhibited an increased affinity toward ceftazidime and a slightly decreased sensitivity to avibactam, sustaining their impact on CZA resistance. However, their respective carbapenemase activities were concurrently negatively impacted.
| S-EPMC8370223 | 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:We identified a novel ceftazidime/avibactam resistance mechanism in sequence type 11 Klebsiella pneumoniae carbapenemase 2-producing K. pneumoniae. Plasmid recombination and chromosomal integration formed a novel virulence plasmid and provided an additional promoter for blaSHV-12, leading to blaSHV-12 overexpression and ceftazidime/avibactam resistance. Genetic rearrangement contributed to convergence of hypervirulence and ceftazidime/avibactam resistance.
| S-EPMC10617341 | biostudies-literature
Project description:Ceftazidime-avibactam (CAZ-AVI) shows promising activity against carbapenem-resistant Klebsiella pneumoniae (CRKP), however, CAZ-AVI resistance have emerged recently. Mutations in KPCs, porins OmpK35 and/or OmpK36, and PBPs are known to contribute to the resistance to CAZ-AVI in CRKP. To identify novel CAZ-AVI resistance mechanism, we generated 10 CAZ-AVI-resistant strains from 14 CAZ-AVI susceptible KPC-producing K. pneumoniae (KPC-Kp) strains through in vitro multipassage resistance selection using low concentrations of CAZ-AVI. Comparative genomic analysis for the original and derived mutants identified CAZ-AVI resistance-associated mutations in KPCs, PBP3 (encoded by ftsI), and LamB, an outer membrane maltoporin. CAZ-AVI susceptible KPC-Kp strains became resistant when complemented with mutated blaKPC genes. Complementation experiments also showed that a plasmid borne copy of wild-type lamB or ftsI gene reduced the MIC value of CAZ-AVI in the induced resistant strains. In addition, blaKPC expression level increased in four of the six CAZ-AVI-resistant strains without KPC mutations, indicating a probable association between increased blaKPC expression and increased resistance in these strains. In conclusion, we here identified a novel mechanism of CAZ-AVI resistance associated with mutations in porin LamB in KPC-Kp.
| S-EPMC8567916 | biostudies-literature
Project description:We describe in vivo evolution of carbapenem and ceftazidime-avibactam resistance by analyzing four longitudinal Klebsiella pneumoniae 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 Klebsiella pneumoniae. The infection started with a KPC-2-producing K. pneumoniae (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 in vivo was not stable and the subsequent use of imipenem against KPC-33-producing K. pneumoniae infection resulted in a reversion of KPC-2 producers (ZRKP03 and ZRKP04, ceftazidime-avibactam-S/carbapenem-R). Genetic analysis demonstrated that all four K. pneumoniae isolates belonged to sequence type 11and had identical capsular polysaccharide (KL47), identical porin genes, and same plasmid replicon types. Phylogenetic analysis indicated that four K. pneumoniae 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 K. pneumoniae harboring bla KPC-2 gene. This is the first report to observe the in vivo 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 bla KPC genes, which leading to the dynamic change of KPC enzymes and the dynamic emergence of resistance to ceftazidime-avibactam and carbapenems. Statement: 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 bla KPC 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 in vivo 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.
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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:Antibiotic combinations, including ceftazidime/avibactam (CAZ/AVI), are frequently employed to combat KPC-producing Klebsiella pneumoniae (KPC-Kp), though such combinations have not been rationally optimized. Clinical KPC-Kp isolates with common genes encoding aminoglycoside-modifying enzymes (AMEs), aac(6')-Ib' or aac(6')-Ib, were used in static time-kill assays (n = 4 isolates) and the hollow-fiber infection model (HFIM; n = 2 isolates) to evaluate the activity of gentamicin, amikacin, and CAZ/AVI alone and in combinations. A short course, one-time aminoglycoside dose was also evaluated. Gentamicin plus CAZ/AVI was then tested in a mouse pneumonia model. Synergy with CAZ/AVI was more common with amikacin for aac(6')-Ib'-containing KPC-Kp but more common with gentamicin for aac(6')-Ib-containing isolates in time-kill assays. In the HFIM, although the isolates were aminoglycoside-susceptible at baseline, aminoglycoside monotherapies displayed variable initial killing, followed by regrowth and resistance emergence. CAZ/AVI combined with amikacin or gentamicin resulted in undetectable counts 50 h sooner than CAZ/AVI monotherapy against KPC-Kp with aac(6')-Ib'. CAZ/AVI monotherapy failed to eradicate KPC-Kp with aac(6')-Ib and a combination with gentamicin led to undetectable counts 70 h sooner than with amikacin. A one-time aminoglycoside dose with CAZ/AVI provided similar killing to aminoglycosides dosed for 7 days. In the mouse pneumonia model (n = 1 isolate), gentamicin and CAZ/AVI achieved a 6.0-log10 CFU/lung reduction at 24 h, which was significantly greater than either monotherapy (P < 0.005). Aminoglycosides in combination with CAZ/AVI were promising for KPC-Kp infections; this was true even for a one-time aminoglycoside dose. Selecting aminoglycosides based on AME genes or susceptibilities can improve the pharmacodynamic activity of the combination.
| S-EPMC8370209 | biostudies-literature
Project description:Klebsiella pneumoniae carbapenemase 2 (KPC-2) is an important source of drug resistance as it can hydrolyze and inactivate virtually all β-lactam antibiotics. KPC-2 is potently inhibited by avibactam via formation of a reversible carbamyl linkage of the inhibitor with the catalytic serine of the enzyme. However, the use of avibactam in combination with ceftazidime (CAZ-AVI) has led to the emergence of CAZ-AVI resistant variants of KPC-2 in clinical settings. One such variant, KPC-44, bears a 15 amino-acid duplication in one of the active site loops (270-loop). Here, we show that the KPC-44 variant exhibits higher catalytic efficiency in hydrolyzing ceftazidime, lower efficiency towards imipenem and meropenem, and a similar efficiency in hydrolyzing ampicillin, when compared to the wild-type KPC-2 enzyme. In addition, the KPC-44 variant enzyme exhibits 12-fold lower avibactam carbamylation efficiency than the KPC-2 enzyme. An X-ray crystal structure of KPC-44 showed that the 15 amino acid duplication results in an extended and partially disordered 270-loop and also changes the conformation of the adjacent 240-loop, which in turn has altered interactions with the active-site omega loop. Furthermore, a structure of KPC-44 with avibactam revealed that formation of the covalent complex results in further disorder in the 270-loop, suggesting that rearrangement of the 270-loop of KPC-44 facilitates avibactam carbamylation. These results suggest that the duplication of 15 amino acids in the KPC-44 enzyme leads to resistance to CAZ-AVI by modulating the stability and conformation of the 270-, 240-, and omega-loops.
| S-EPMC10716778 | 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:From January 2019 to April 2020, 32 KPC-producing, ceftazidime-avibactam (CZA)-resistant Klebsiella pneumoniae strains were isolated in a university hospital in Rome, Italy. These strains belonged to the sequence type 512 (ST512), ST101, and ST307 high-risk clones. Nine different CZA-resistant KPC-3 protein variants were identified, five of them never previously reported (KPC-66 to KPC-70). Among the nine, KPC-31, KPC-39, KPC-49, KPC-66, KP-68, KPC-69, and KPC-70 showed amino acid substitutions, insertions, and deletions in the Ω loop of the protein. KPC-29 has a duplication, while the novel KPC-67 has a triplication, of the KDD triplet in the 270-loop, a secondary loop of the KPC-3 protein. Genomics performed on contemporary resistant and susceptible clones underlined that these novel mutations emerged in blaKPC-3 genes located on conserved plasmids: in ST512, all blaKPC-3 mutant genes were located in pKpQIL plasmids, while the three novel blaKPC-3 mutants identified in ST101 were on FIIk-FIA(HI1)-R plasmids. Selection also promoted multiplication of the carbapenemase gene copy number by transposition, recombination, and fusion of resident plasmids. When expressed in Escherichia coli recipient cells cloned in the high-copy-number pTOPO vector, the Ω loop mutated variants showed the CZA-resistant phenotype associated with susceptibility to carbapenems, while KPC variants with insertions in the 270-loop showed residual activity on carbapenems. The investigation of CZA resistance mechanisms offered the unique opportunity to study vertical, horizontal, and oblique evolutionary trajectories of K. pneumoniae high-risk clones.
| S-EPMC8448114 | biostudies-literature