Dataset Information

Increased Expression and Amplification of bla_KPC-2 Contributes to Resistance to Ceftazidime/Avibactam in a Sequence Type 11 Carbapenem-Resistant Klebsiella pneumoniae Strain.

ABSTRACT: Ceftazidime/avibactam (CAZ/AVI) is regarded as an effective alternative antibiotic for the clinical treatment of Klebsiella pneumoniae carbapenemase (KPC)-producing isolates. As resistance has been reported in some strains, it is critical to understand the key mechanisms contributing to the acquired resistance to CAZ/AVI. From January 2018 to April 2020, 127 KPC-producing carbapenem-resistant Klebsiella pneumoniae strains (CRKPs) were isolated at a university hospital in Chongqing, China, and 25 strains showed reduced susceptibility to CAZ/AVI. All reduced-susceptibility CRKPs were deficient in Ompk35 and Ompk36 porins, and 24 strains had a premature termination at amino acid position 63 in Ompk35 and 134 to 135 glycine and aspartic acid (GD) insertion in OmpK36, while the bla_KPC-2 expression level showed no significant difference compared to that of strain BAA-1705. Four reduced-susceptibility strains evolved resistance under selective pressure of CAZ/AVI with the bla_KPC-2 expression level increased, and two of these strains had mutations in the Ω-loop. The study found a strain of CRKP55 with changes in the resistance phenotype during conjugation, evolving from reduced sensitivity to high-level resistance to CAZ/AVI. Through plasmid sequencing and reverse transcription-quantitative PCR, it was speculated that insertion sequence (IS)26-mediated bla_KPC-2 gene amplification caused the MIC value change in the conjugant JKP55. Our findings illustrated the potential of CAZ/AVI resistance under antibiotic stress and demonstrated that IS26 may mediate bla_KPC-2 replication transposition, leading to high-level resistance during horizontal gene transfer. Investigation of CAZ/AVI resistance mechanisms may offer a unique opportunity to study the horizontal evolutionary trajectories of K. pneumoniae high-risk clones. IMPORTANCE Klebsiella pneumoniae carbapenemase (KPC) production is the most common mechanism of K. pneumoniae resistance to carbapenems in China. Currently, CAZ/AVI is considered a potential alternative therapeutic option for infections caused by these isolates. However, there have been increasing reports of resistant or reduced-sensitivity strains since the approval of this agent. In this study, resistance to CAZ/AVI was induced under drug-selective pressure and was caused by bla_KPC-2 overexpression and/or substitutions in the Ω-loop of KPC. Additionally, it was demonstrated that a conjugative plasmid carrying bla_KPC-2 could transfer horizontally between species, and perhaps, IS26-derived tandem amplification of bla_KPC-2 during this period led to high-level resistance to CAZ/AVI. Our research suggests that IS26-mediated resistance evolution may have important implications in guiding clinical antibiotic use.

SUBMITTER: Li X

PROVIDER: S-EPMC9430841 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Increased Expression and Amplification of <i>bla</i><sub>KPC-2</sub> Contributes to Resistance to Ceftazidime/Avibactam in a Sequence Type 11 Carbapenem-Resistant Klebsiella pneumoniae Strain.

Li Xinhui X Zhang Jisheng J Yang Chengru C Li Jie J Wang Jianmin J Huang Wan W Zeng Lingyi L Liang Xushan X Long Wenzhang W Zhang Xiaoli X

Microbiology spectrum 20220728 4

Ceftazidime/avibactam (CAZ/AVI) is regarded as an effective alternative antibiotic for the clinical treatment of Klebsiella pneumoniae carbapenemase (KPC)-producing isolates. As resistance has been reported in some strains, it is critical to understand the key mechanisms contributing to the acquired resistance to CAZ/AVI. From January 2018 to April 2020, 127 KPC-producing carbapenem-resistant Klebsiella pneumoniae strains (CRKPs) were isolated at a university hospital in Chongqing, China, and 25 ...[more]

PMID: 35900090

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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:To illustrate the genomic and drug resistance traits of the Klebsiella pneumoniae Kpn_XM9, which harbors a transposon (Tn) As1 and was barely susceptible to ceftazidime-avibactam (CZA). Whole-genome sequencing, gene deletion, antimicrobial susceptibility, and conjugation tests were carried out to illustrate the traits of Kpn_XM9. As confirmed by whole-genome sequencing, the Kpn_XM9 harbored a 5,523,536 bp chromosome and five plasmids with lengths being 128,129, 196,512, 84,812, 43,695, and 5,596 bp, respectively. Plasmid p1_Kpn_XM9 (128,219 bp) contained four resistance genes, blaCTX-M-65, blaTEM-1B, rmtB, and two copies of blaKPC-2. Genes blaKPC-2 were bracketed by ISKpn17 and ISKpn16 within a new composite Tn3-like TnAs1. The two tandem repeats, positioned opposite each other, were spaced 93,447 bp apart in p1_Kpn_XM9. Kpn_XM9 belonged to K64 and sequence type (ST) 11. The Kpn_XM9 was resistant to amikacin, aztreonam, ticarcillin/clavulanic acid, piperacillin/tazobactam, ceftazidime, cefepime, imipenem, meropenem, tobramycin, ciprofloxacin, levofloxacin, doxycycline, minocycline, tigecycline, colistin, and trimethoprim/sulfamethoxazole; it was barely susceptible to CZA with a minimum inhibitory concentration of 8/4 µg/mL, which declined to 2/4 µg/mL after a 18,555 bp nucleotide was knocked out and one copy of blaKPC-2 was sustained on p1_Kpn_XM9. Kpn_XM9 had virulence genes encoding Types 1 and 3 fimbriae, four siderophores, and capsular polysaccharide anchoring protein but no genes upregulating capsular polysaccharide synthesis. The Kpn_XM9 presented a classical phenotype with extreme drug resistance. The emergence of double copies of blaKPC-2 in a single plasmid from the predominant ST11 K. pneumoniae represents a new therapeutic challenge.IMPORTANCEWith the wide use of ceftazidime-avibactam against carbapenem-resistant organisms, its resistance is increasingly documented; among the corresponding resistance mechanisms, mutations of blaKPC-2 or blaKPC-3 into other subtypes are dominant to date. However, more copies of blaKPC-2 may also greatly increase the minimum inhibitory concentration of ceftazidime-avibactam, which could be conferred by transposon As1 and insertion sequence 26 and should be of concern.

Dataset Information

Increased Expression and Amplification of bla_KPC-2 Contributes to Resistance to Ceftazidime/Avibactam in a Sequence Type 11 Carbapenem-Resistant Klebsiella pneumoniae Strain.

Publications

Increased Expression and Amplification of <i>bla</i><sub>KPC-2</sub> Contributes to Resistance to Ceftazidime/Avibactam in a Sequence Type 11 Carbapenem-Resistant Klebsiella pneumoniae Strain.

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Dataset Information

Increased Expression and Amplification of blaKPC-2 Contributes to Resistance to Ceftazidime/Avibactam in a Sequence Type 11 Carbapenem-Resistant Klebsiella pneumoniae Strain.

Publications

Increased Expression and Amplification of <i>bla</i><sub>KPC-2</sub> Contributes to Resistance to Ceftazidime/Avibactam in a Sequence Type 11 Carbapenem-Resistant Klebsiella pneumoniae Strain.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Increased Expression and Amplification of bla_KPC-2 Contributes to Resistance to Ceftazidime/Avibactam in a Sequence Type 11 Carbapenem-Resistant Klebsiella pneumoniae Strain.