Molecular detection of resistance genes

DNA was extracted using the AccuPrep® genomic DNA extraction kit according to the manufacturer’s instructions. PCR was performed for antibiotic resistance-related genes described below.

The following carbapenemase-encoding genes were detected: class A β-lactamases genes: bla_KPC [26]; class B metallo-β-lactamases genes: bla_IMP [26], bla_VIM [26], bla_SPM [26], and bla_NDM [26]; and class D oxacillinases genes: bla_OXA-23-like [27], bla_{OXA-24/40-like} [27], bla_OXA-51-like [27], and bla_OXA-58-like [27]. The colistin-related resistance gene: mcr-1 [28], mcr-2 [28], mcr-3 [28], mcr-4 [28], and mcr-5 [28]. The protein-related gene carO [29] (A. baumannii outer membrane protein involved in carbapenem resistance) and the drug efflux pump component-related genes adeA [30], adeB [31], adeC [31], adeI [31], adeJ [31], and adeK [31] were also detected.

Database searches for DNA sequence similarities were performed using BLAST (https://blast.ncbi.nlm.nih.gov). The construction of a carO phylogenetic tree was obtained by MEGA using the neighbor-joining method. A model of the p-distance was used to estimate distances for nucleotide sequences. The significance of the groups observed in constructed trees was determined by bootstrap analysis with 1000 replicates.

Mobile genetic element-related resistance genes, including integrons (intI1 [32, 33], intI2 [32, 34], intI3 [32]), transposons (tnpU [35] and tnp513 [35]), the variable region of class 1 integron and class 2 integron, and insertion sequences (IS26 [36], ISAba1 [37], and ISAba125 [37]) were also analyzed. The PCR cycle consisted of denaturation at 94 °C for 5 min, followed by 35 cycles of 30 s at 94 °C, annealing for 40 s at optimal annealing temperature, and extension at 72 °C for 40 s for the genes and 4 min for the variable region of intI1 and intI2.

The primers used for PCR are shown in Table ​Table1.1. Primer synthesis and sequencing of PCR products were performed by Sangon Biotech Co., Ltd. (Shanghai, China).

Gene primers used for PCR amplification of genes associated with antimicrobial resistance

^aThe forward primer hybridizes to a region located 294 bp upstream of the 5′ end of the carO initiation codon, while the reverse primer anneals to a sequence located 131 bp downstream of the carO termination codon