Dataset Information

Ceftazidime-Avibactam Resistance Associated with Increased bla KPC-3 Gene Copy Number Mediated by pKpQIL Plasmid Derivatives in Sequence Type 258 Klebsiella pneumoniae.

ABSTRACT: This study reports on the characterization of two ceftazidime-avibactam (CZA)-resistant KPC-producing Klebsiella pneumoniae strains (KP-14159 and KP-8788) sequentially isolated from infections occurred in a patient never treated with CZA. Whole-genome sequencing characterization using a combined short- and long-read sequencing approach showed that both isolates belonged to the same ST258 strain, had altered outer membrane porins (a truncated OmpK35 and an Asp137Thr138 duplication in the L3 loop of OmpK36), and carried novel pKpQIL plasmid derivatives (pIT-14159 and pIT-8788, respectively) harboring two copies of the Tn4401a KPC-3-encoding transposon. Plasmid pIT-8788 was a cointegrate of pIT-14159 with a ColE replicon (that was also present in KP-14159) apparently evolved in vivo during infection. pIT-8788 was maintained at a higher copy number than pIT-14159 and, upon transfer to Escherichia coli DH10B, was able to increase the CZA MIC by 32-fold. The present findings provide novel insights about the mechanisms of acquired resistance to CZA, underscoring the role that the evolution of broadly disseminated pKpQIL plasmid derivatives may have in increasing the bla KPC gene copy number and KPC-3 expression in bacterial hosts. Although not self-transferable, similar elements, with multiple copies of Tn4401 and maintained at a high copy number, could mediate transferable CZA resistance upon mobilization.

SUBMITTER: Coppi M

PROVIDER: S-EPMC7179273 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Ceftazidime-Avibactam Resistance Associated with Increased blaKPC-3 Gene Copy Number Mediated by pKpQIL Plasmid Derivatives in Sequence Type 258 Klebsiella pneumoniae.

Coppi Marco M Di Pilato Vincenzo V Monaco Francesco F Giani Tommaso T Conaldi Pier Giulio PG Rossolini Gian Maria GM

Antimicrobial agents and chemotherapy 20200324 4

This study reports on the characterization of two ceftazidime-avibactam (CZA)-resistant KPC-producing Klebsiella pneumoniae strains (KP-14159 and KP-8788) sequentially isolated from infections occurred in a patient never treated with CZA. Whole-genome sequencing characterization using a combined short- and long-read sequencing approach showed that both isolates belonged to the same ST258 strain, had altered outer membrane porins (a truncated OmpK35 and an Asp137Thr138 duplication in the L ...[more]

PMID: 31964792

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Project description:As the first-line antimicrobial agent for the infection caused by carbapenem-resistant Enterobacterales, ceftazidime-avibactam develops drug resistance during its ever-growing clinical use. In this study, we report multiple novel variants in blaKPC-2-positive Klebsiella pneumoniae from two separate patients during their exposure to ceftazidime-avibactam. For one patient, the blaKPC-2 gene carried by K. pneumoniae mutated into blaKPC-35, blaKPC-78, and blaKPC-33 over the same period, while that for the other patient mutated into blaKPC-79 and further evolved into blaKPC-76 to enhance resistance level, among which blaKPC-76 and blaKPC-79 were reported for the first time. In contrast with blaKPC-2, the emergent mutations within the Ω-loop conferred high-level resistance to ceftazidime-avibactam with a sharp reduction of carbapenemase activity. These blaKPC-positive K. pneumoniae isolated from sputum (both patients) and cerebrospinal fluid (patient 2) belonged to ST11 and ST859, respectively. All strains located blaKPC alleles on IncFII/IncR plasmids, except one on an IncFII plasmid. Such blaKPC-2 variants first appeared after 9 to 18 days of ceftazidime-avibactam usage, but the lack of its feasible detection method often led to the assumption of ceftazidime-avibactam sensitivity resulting in clinical incorrect usage. Subsequent substitution of ceftazidime-avibactam with carbapenems also failed, because the blaKPC-2-containing K. pneumoniae dominated again. Ultimately, treatment failed even with the therapeutic regimen of ceftazidime-avibactam combined with carbapenems, because of the inadequate concentration of avibactam in infection sites and decreased drug sensitivity of strains caused by increased expression of blaKPC and point mutation of ompK35 and ompK36. As novel KPC variants conferring resistance to ceftazidime-avibactam are constantly emerging worldwide, quick and efficient laboratory detection and surveillance are urgently needed for infection control. IMPORTANCE Carbapenem-resistant K. pneumoniae which was classified as the most urgent threat by World Health Organization, is the most critical public health concern due to its high mortality rate. Recently, the rapid mutation of blaKPC has occurred during anti-infective therapy, which posed an unexpected challenge for both the diagnostic laboratory and clinical practice.

Project description:Ceftazidime-avibactam (CZA) therapy has significantly improved survival rates for patients infected by carbapenem-resistant bacteria, including KPC producers. However, resistance to CZA is a growing concern, attributed to multiple mechanisms. In this study, we characterized four clinical CZA-resistant Klebsiella pneumoniae isolates obtained between July 2019 and December 2020. These isolates expressed novel allelic variants of blaKPC-2 resulting from changes in hotspots of the mature protein, particularly in loops surrounding the active site of KPC. Notably, KPC-80 had an K269_D270insPNK mutation near the Lys270-loop, KPC-81 had a del_I173 mutation within the Ω-loop, KPC-96 showed a Y241N substitution within the Val240-loop and KPC-97 had an V277_I278insNSEAV mutation within the Lys270-loop. Three of the four isolates exhibited low-level resistance to imipenem (4 µg/mL), while all remained susceptible to meropenem. Avibactam and relebactam effectively restored carbapenem susceptibility in resistant isolates. Cloning mutant blaKPC genes into pMBLe increased imipenem MICs in recipient Escherichia coli TOP10 for blaKPC-80, blaKPC-96, and blaKPC-97 by two dilutions; again, these MICs were restored by avibactam and relebactam. Frameshift mutations disrupted ompK35 in three isolates. Additional resistance genes, including blaTEM-1, blaOXA-18 and blaOXA-1, were also identified. Interestingly, three isolates belonged to clonal complex 11 (ST258 and ST11) and one to ST629. This study highlights the emergence of CZA resistance including unique allelic variants of blaKPC-2 and impermeability. Comprehensive epidemiological surveillance and in-depth molecular studies are imperative for understanding and monitoring these complex resistance mechanisms, crucial for effective antimicrobial treatment strategies.ImportanceThe emergence of ceftazidime-avibactam (CZA) resistance poses a significant threat to the efficacy of this life-saving therapy against carbapenem-resistant bacteria, particularly Klebsiella pneumoniae-producing KPC enzymes. This study investigates four clinical isolates exhibiting resistance to CZA, revealing novel allelic variants of the key resistance gene, blaKPC-2. The mutations identified in hotspots surrounding the active site of KPC, such as K269_D270insPNK, del_I173, Y241N and V277_I278insNSEAV, prove the adaptability of these pathogens. Intriguingly, low-level resistance to imipenem and disruptions in porin genes were observed, emphasizing the complexity of the resistance mechanisms. Interestingly, three of four isolates belonged to clonal complex 11. This research not only sheds light on the clinical significance of CZA resistance but also shows the urgency for comprehensive surveillance and molecular studies to inform effective antimicrobial treatment strategies in the face of evolving bacterial resistance.

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.

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.

Dataset Information

Ceftazidime-Avibactam Resistance Associated with Increased bla KPC-3 Gene Copy Number Mediated by pKpQIL Plasmid Derivatives in Sequence Type 258 Klebsiella pneumoniae.

Publications

Ceftazidime-Avibactam Resistance Associated with Increased <i>bla</i><sub>KPC-3</sub> Gene Copy Number Mediated by pKpQIL Plasmid Derivatives in Sequence Type 258 <i>Klebsiella pneumoniae</i>.

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