Dataset Information

Carbapenem antibiotic stress increases bla_KPC _-2 gene relative copy number and bacterial resistance levels of Klebsiella pneumoniae.

ABSTRACT:

Background

The clinical isolation rates of carbapenem-resistant Klebsiella pneumoniae (CR-KP) continue to increase. In China, clinical CR-KP isolates are mainly attributed to the bla_KPC-2 gene carried on plasmids, and the bla_KPC-2 copy number correlates with the expression of KPC enzymes, which can cause elevated carbapenem MICs.

Methods

Thirty-seven CR-KP isolates were collected at the Second People's Hospital of Lianyungang City between January 2020 and March 2021, with no duplicate isolates, and were screened for the bla_KPC-2 gene with PCR. Analysis of current CRKP resistance to clinically relevant antimicrobials using the bioMérieux VITEK^® 2 bacterial identification card. The multilocus sequence types of the strains were confirmed with PCR and DNA sequencing. Recombinant plasmids pET20b-bla_KPC-2 and pET20b-CpsG were constructed, and the copy numbers of the recombinant plasmids per unit volume was calculated based on the molecular weight of the plasmids. After the genomes DNA of clinical isolates of K. pneumoniae carrying the bla_KPC-2 gene were purified, the bla_KPC-2 gene relative copy number in individual K. pneumoniae strains was indicated by the double standard curve method. Detection of MIC values changes of K. pneumoniae under imipenem selection pressure by broth microdilution method.

Results

Among the 37 CR-KP strains isolated, only the bla_KPC-2 gene was detected in 30 strains, three strains were positive for the bla_NDM-1 gene, two strains carried both the bla_KPC-2 and bla_NDM-1 genes, and two strains without detectable carbapenem resistance genes. The ST11 clone was predominant among the 37 carbapenem-resistant K. pneumoniae isolates. Drug sensitivity testing showed that except for polymyxins (100% susceptible) and tigecycline (75.7% intermediate), the 37 CR-KP strains were resistant to almost all antimicrobial drugs. The bla_KPC-2 relative copy number in nine ST11 clinical isolates of K. pneumoniae was 7.64 ± 2.51 when grown on LB plates but 27.67 ± 13.04 when grown on LB plates containing imipenem. Among these nine isolates, five CRKP strains exhibited elevated MICs to imipenem, while the remaining four strains showed unchanged MIC values to imipenem.

Conclusion

Carbapenem-resistant Klebsiella pneumoniae isolates may have multiple pathways to achieve high levels of carbapenem resistance, and moderate carbapenem pressure can increase the copy number of KPC enzyme genes in CRKP strains and enhance the degree of carbapenem resistance in the strains.

SUBMITTER: Chen H

PROVIDER: S-EPMC9280016 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Dataset Information

Carbapenem antibiotic stress increases bla_KPC _-2 gene relative copy number and bacterial resistance levels of Klebsiella pneumoniae.

Background

Methods

Results

Conclusion

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Dataset Information

Carbapenem antibiotic stress increases blaKPC -2 gene relative copy number and bacterial resistance levels of Klebsiella pneumoniae.

Background

Methods

Results

Conclusion

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Carbapenem antibiotic stress increases bla_KPC _-2 gene relative copy number and bacterial resistance levels of Klebsiella pneumoniae.