Dataset Information

Design of Rapid Detection System for Five Major Carbapenemase Families (bla _KPC, bla _NDM, bla _VIM, bla _IMP and bla _OXA-48-Like) by Colorimetric Loop-Mediated Isothermal Amplification.

ABSTRACT:

Purpose

Carbapenemase-producing Enterobacteriaceae (CPE) infection constitutes a public health threat. Timely and efficient diagnosis is of paramount importance for prompt and effective therapy. In order to quickly and comprehensively detect the five major families of carbapenemases (bla _KPC, bla _NDM, bla _VIM, bla _IMP, and bla _OXA-48-like), colorimetric loop-mediated isothermal amplification (LAMP) was employed.

Materials and methods

Five sets of LAMP primers were designed, each of which can, respectively, amplify all the carbapenemase subtypes described in this work. Twenty whole genome sequencing-verified-"standard strains", including 1 bla _NDM-1, 1 bla _NDM-5, 1 bla _NDM-6, 1 bla _NDM-7, 2 bla _IMP-4, 1 bla _IMP-8, 2 bla _KPC-2, 1 bla _KPC-3, 1 bla _KPC-4, 1 bla _KPC-5, 1 bla _KPC-6, 1 bla _KPC-7, 1 bla _OXA-48 and 1 bla _OXA-181 carrier, and 1 bla _VIM and bla _OXA-244, 1 bla _KPC-2 and bla _IMP-4, 1 bla _KPC-2 and bla _VIM-1 and 1 bla _KPC-2 and bla _NDM-1-co-carriers, were used to establish a 25-microliter visual LAMP reaction system (kept at 65°C for 30 minutes in water bath). Color change from bright pink to yellow indicated positive amplification. In addition, 126 pre-verified clinical carbapenem-resistant Enterobacteriaceae (CRE) isolates, including 65 CPE (23 bla _NDM, 2 bla _OXA-48-like, 1 bla _KPC and bla _VIM, 2 bla _IMP, and 37 bla _KPC carriers) and 61 non-CPE, were also detected.

Results

With the lowest detection limit of 10 colony forming units (CFU) per reaction for LAMP and 10³ CFU per reaction for PCR, the LAMP system demonstrated dramatically higher sensitivity while retaining the same specificity. Furthermore, we demonstrated concordant results between the two methods for the 126 clinical isolates.

Conclusion

Therefore, LAMP could be used for rapid identification of the five major carbapenemase gene families in routine clinical laboratories.

SUBMITTER: Feng W

PROVIDER: S-EPMC8164214 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The emergence of carbapenemase-producing Enterobacterales (CPE) infections is a major global public health threat. Rapid and accurate detection of pathogenic bacteria is essential to optimize treatment and timely avoid further transmission of these bacteria. Here, we aimed to develop a rapid on site visualization detection method for CPE using improved recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) method, based on four most popular carbapenemase genes: bla KPC, bla NDM, bla OXA-48-like, and bla IMP. All available allelic variants of the above carbapenemases were downloaded from the β-lactamase database, and the conserved regions were used as targets for RPA assay. Five primer sets were designed targeting to each carbapenemase gene and the RPA amplification products were analyzed by agarose gel electrophoresis. FITC-labeled specific probes were selected, combined with the best performance primer set (Biotin-labeled on the reverse primer), and detected by RPA-LFS. Mismatches were made to exclude the false positive signals interference. This assay was evaluated in 207 clinically validated carbapenem-resistant Enterobacterales (CRE) isolates and made a comparison with conventional PCR. Results showed that the established RPA-LFS assay for CPE could be realized within 30 min at a constant temperature of 37°C and visually detected amplification products without the need for special equipment. This assay could specifically differentiate the four classes of carbapenemases without cross-reactivity and shared a minimum detection limit of 100 fg/reaction (for bla KPC, bla NDM, and bla OXA-48-like) or 1000 fg/reaction (for bla IMP), which is ten times more sensitive than PCR. Furthermore, the detection of 207 pre-validated clinically CRE strains using the RPA-LFS method resulted in 134 bla KPC, 69 bla NDM, 3 bla OXA-48-like, and 1 bla IMP. The results of the RPA-LFS assay were in consistent with PCR, indicating that this method shared high sensitivity and specificity. Therefore, the RPA-LFS method for CPE may be a simple, specific, and sensitive method for the rapid diagnosis of carbapenemase Enterobacterales.

Project description:PurposeTo explore the genetic characteristics of the IMP-4, NDM-1, OXA-1, and KPC-2 co-producing multidrug-resistant (MDR) clinical isolate, Citrobacter freundii wang9.MethodsMALDI-TOF MS was used for species identification. PCR and Sanger sequencing analysis were used to identify resistance genes. In addition to agar dilution, broth microdilution was used for antimicrobial susceptibility testing (AST). We performed whole genome sequencing (WGS) of the strains and analyzed the resulting data for drug resistance genes and plasmids. Phylogenetic trees were constructed with maximum likelihood, plotted using MAGA X, and decorated by iTOL.ResultsCitrobacter freundii carrying blaKPC-2, blaIMP-4, blaOXA-1, and blaNDM-1 are resistant to most antibiotics, intermediate to tigecycline, and only sensitive to polymyxin B, amikacin, and fosfomycin. The blaIMP-4 coexists with the blaNDM-1 and the blaOXA-1 on a novel transferable plasmid variant pwang9-1, located on the integron In1337, transposon TnAS3, and integron In2054, respectively. The gene cassette sequence of integron In1337 is IntI1-blaIMP-4-qacG2-aacA4'-catB3Δ, while the gene cassette sequence of In2054 is IntI1-aacA4cr-blaOXA-1-catB3-arr3-qacEΔ1-sul1. The blaNDM-1 is located on the transposon TnAS3, and its sequence is IS91-sul-ISAba14-aph (3')-VI-IS30-blaNDM-1-ble-trpF-dsbD-IS91. The blaKPC-2 is located on the transposon Tn2 of plasmid pwang9-1, and its sequence is klcA-korC-ISkpn6-blaKPC-2-ISkpn27-tnpR-tnpA. Phylogenetic analysis showed that most of the 34\u00B0C. freundii isolates from China were divided into three clusters. Among them, wang1 and wang9 belong to the same cluster as two strains of C. freundii from environmental samples from Zhejiang.ConclusionWe found C. freundii carrying blaIMP-4, blaNDM-1, blaOXA-1, and blaKPC-2 for the first time, and conducted in-depth research on its drug resistance mechanism, molecular transfer mechanism and epidemiology. In particular, we found that blaIMP-4, blaOXA-1, and blaNDM-1 coexisted on a new transferable hybrid plasmid that carried many drug resistance genes and insertion sequences. The plasmid may capture more resistance genes, raising our concern about the emergence of new resistance strains.

Dataset Information

Design of Rapid Detection System for Five Major Carbapenemase Families (bla _KPC, bla _NDM, bla _VIM, bla _IMP and bla _OXA-48-Like) by Colorimetric Loop-Mediated Isothermal Amplification.

Purpose

Materials and methods

Results

Conclusion

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Design of Rapid Detection System for Five Major Carbapenemase Families (bla KPC, bla NDM, bla VIM, bla IMP and bla OXA-48-Like) by Colorimetric Loop-Mediated Isothermal Amplification.

Purpose

Materials and methods

Results

Conclusion

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Design of Rapid Detection System for Five Major Carbapenemase Families (bla _KPC, bla _NDM, bla _VIM, bla _IMP and bla _OXA-48-Like) by Colorimetric Loop-Mediated Isothermal Amplification.