Characterization of the novel class A β-Lactamase gene bla_KOB-1 in an Enterobacter isolate

Class A serine β-lactamases are important enzymes that contribute to bacterial resistance to β-lactam antibiotics, especially in gram-negative bacilli (e.g., Enterobacteriaceae). These enzymes are highly diverse with many molecular variants, and some enzymes (such as SHV and TEM) can be used to extend the inactivation spectrum to the latest generation of cephalosporins by mutation. Consequently, the mechanisms of resistance to antimicrobial agents in bacteria and the clinical implications of antimicrobial resistance need to be more comprehensively studied.

Animal fecal samples were collected with sterile swabs, and individual colonies were isolated via the agar plate streaking method. The minimum inhibitory concentration (MIC) of the antimicrobial agent was tested by the standard agar dilution method. The complete bacterial genome sequence was obtained using the Illumina and PacBio sequencing platforms. Potential resistance genes were annotated according to the Comprehensive Antibiotic Resistance Database (CARD) and validated by molecular cloning. The β-lactamase KOB-1 was acquired by using the expression vector pCold I, after which the enzymatic kinetics were determined. The evolutionary relationships and genetic environments of the sequences associated with this resistance gene were analyzed via bioinformatics methods.

A novel chromosomal class A β-lactamase gene, bla_KOB-1, was identified in the Enterobacter kobei strain DW108, which was isolated from goose feces. bla_KOB-1 induced varying degrees of resistance to penicillins. Enzyme kinetic parameter assays revealed that KOB-1 could hydrolyze penicillins as well as some cephalosporins (such as cefazolin and cefotaxime), while its hydrolytic activities could be inhibited by tazobactam and avibactam. The bla_KOB-1(-like) genes were uniquely distributed in strains of the genus Enterobacter. Comparative analysis of the bla_KOB-1(-like) gene related sequences showed that, except for a fragment encoding two genes (bla-phoX) that was present only in the sequences from Enterobacter, the gene context of the sequences from the genus Leclercia were similar with that from Enterobacter in terms of gene content or gene order. No mobile genetic element was identified next to the bla_KOB-1 gene.

The identification of additional resistance genes aids in the elucidation of bacterial resistance mechanisms and application of effective clinical treatments for infections caused by pathogens carrying similar resistance genes.