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 blaNDM 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 blaNDM genes in children’s clinical samples. The analytical sensitivity of the RAA assay was 20 copies, and the various bacterial types without blaNDM genes did not amplify. This method was used to detect blaNDM 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 blaNDM 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. blaNDM–1 and blaNDM–5 were the two main types in these samples. These data show that the RAA assay has potential to be a sensitive and rapid blaNDM gene screening test for clinical samples. The common existence of blaNDM and multi-drug resistance genes presents major challenges for pediatric treatment.

Rapid Detection of Multi-Resistance Strains Carrying mcr-1 Gene Using Recombinase-Aided Amplification Directly on Clinical Samples

“…A commercial RAA kit (Jiangsu Qitian Bio-Tech Co., Ltd., China) was used for the RAA assays. The RAA assays were performed as described previously ( Feng et al, 2021 ). Briefly, a 50 μL reaction mixture was prepared first, which was made of reaction buffer (25 μL), DNase-free water (15.7 μL), 10 μM primer F (2.1 μL), 10 μM primer R (2.1 μL), DNA template (2 μL), 280 mM magnesium acetate (2.5 μL), and 10 μM probe (0.6 μL).…”

“…RAA assay is a highly efficient method for the rapid detection of specific target genes. Based on isothermal amplification technology, the RAA assay can be completed within 15–30 min at 39°C and has been widely used in clinical applications, such as for identifying New Delhi Metallo-β-Lactamase Gene ( Feng et al, 2021 ), bla KPC ( Zhang et al, 2021 ) and other applications ( Fan et al, 2019 ; Qi et al, 2019 ; Shen et al, 2019 ; Xue et al, 2020a , b ). Here, an RAA assay was developed to detect the mcr -1 gene in clinical samples, which was proven to have high specificity and sensitivity.…”

Emergence of uncommon KL38-OCL6-ST220 carbapenem-resistant Acinetobacter pittii strain, co-producing chromosomal NDM-1 and OXA-820 carbapenemases

“…Carbapenem resistance in A. pittii is mainly caused by class D carbapenemases such as OXA-23, OXA-40, and OXA-58 and, in some cases, by metallo-b-lactamases (MBLs), including the New Delhi metallo-b-lactamase (NDM) (Kaase et al, 2014). NDM could mediate resistance to most b-lactam antimicrobial agents, including penicillins, cephalosporins, and carbapenems (Feng et al, 2021). NDM in A. pittii strains was relevant to sporadic human infection with high mortality rates and hospital transmissions in various countries, and serves as a potential reservoir for the bla NDM-1 -carrying plasmids.…”

Emergence of a High-Risk Klebsiella michiganensis Clone Disseminating Carbapenemase Genes

“…Carbapanemases are classified into Ambler molecular classes A, B, and D, with New Delhi metallo-β-lactamase (NDM) as a class B enzyme ( Khan et al, 2017 ). NDM-1 was first discovered in a clinical setting in 2008, after which 31 unique variants have been described ( Feng et al, 2021 ). NDM-5 differs from NDM-1 by two amino substitutions and has enhanced carbapenemase activity ( Hornsey et al, 2011 ).…”