Rapid Detection of New Delhi Metallo-β-Lactamase Gene Using Recombinase-Aided Amplification Directly on Clinical Samples From Children

Abstract

New Delhi metallo-β-lactamase, a metallo-β-lactamase carbapenemase type, mediates resistance to most β-lactam antibiotics including penicillins, cephalosporins, and carbapenems. Therefore, it is important to detect bla _NDM genes in children's clinical samples as quickly as possible and analyze their characteristics. Here, a recombinase-aided amplification (RAA) assay, which operates in a single one-step reaction tube at 39°C in 5-15 min, was established to target bla _NDM genes in children's clinical samples. The analytical sensitivity of the RAA assay was 20 copies, and the various bacterial types without bla _NDM genes did not amplify. This method was used to detect bla _NDM genes in 112 children's stool samples, 10 of which were tested positive by both RAA and standard PCR. To further investigate the characteristics of carbapenem-resistant bacteria carrying bla _NDM in children, 15 carbapenem-resistant bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Citrobacter freundii, Klebsiella oxytoca, Acinetobacter junii, and Proteus mirabilis) were isolated from the 10 samples. Notably, more than one bacterial type was isolated from three samples. Most of these isolates were resistant to cephalosporins, cefoperazone-sulbactam, piperacillin-tazobactam, ticarcillin-clavulanic acid, aztreonam, co-trimoxazole, and carbapenems. bla _{NDM - 1} and bla _{NDM - 5} were the two main types in these samples. These data show that the RAA assay has potential to be a sensitive and rapid bla _NDM gene screening test for clinical samples. The common existence of bla _NDM and multi-drug resistance genes presents major challenges for pediatric treatment.

Keywords: blaNDM; carbapenemase; character; pediatrics; recombinase-aided amplification.

FIGURE 1

Primer and probe regions in the 31 bla_NDM variants.

FIGURE 2

Specificity of the RAA assay. Klebsiella pneumoniae 2146, Acinetobacter baumannii 1 and Pseudomonas aeruginosa produced amplification signals, while the other bla_NDM-lacking bacterial types were negative (A–C). All the bacterial types produced 16S rRNA gene amplification signals (D–F). 1: K. oxytoca, 2: A. baumannii 2, 3: K. pneumoniae 700603, 4: P. aeruginosa ATCC27853, 5: S. sonnei, 6: E. coli 25922, 7: C. jejuni, 8: E. aerogenes, 9: P. mirabilis, 10: S. enteritidis, 11: E. cloacae, 12: C. freundii, 13: P. mirabilis.

FIGURE 3

Sensitivity of the RAA assay. An increase in the fluorescence signal was observed from 1 × 10⁷ to 1 × 10¹ copies/reaction.

FIGURE 4

Comparison of the RAA detection results with standard PCR on 112 clinical samples. Ten positive samples were identified. (A) The RAA detection results from the 10 positive samples. (B) The PCR results from the same 10 positive samples. M: Marker, 1–10: The 10 positive samples for patients 1–10; 11: negative control.