Insertion sequences in mgrB and mutations in two-component system genes confer high polymyxin resistance to carbapenem-resistant Enterobacter cloacae complex strains

Due to the complexity of identifying the <i>Enterobacter cloacae</i> complex (ECC) at the species level, little is known about the distribution of carbapenem-resistant ECC (CRECC). Plasmid-mediated <i>mcr</i> family genes are significant contributors to polymyxin resistance. The emergence of the <i>mcr-9</i> gene has further complicated the landscape of polymyxin resistance in CRECC. Our study aimed to ascertain the prevalence of CRECC and the <i>mcr-9</i> gene, and to elucidate the mechanisms underlying high-level resistance to polymyxin B (PB). In this study, we collected 212 non-replicating ECC strains, identifying 38 CRECC strains (17.9%, 38/212) and <i>Enterobacter hormaechei</i> (71.1%, 27/38) as the predominant endemic strains. Among these, 10 CRECC strains (36.3%, 10/38) were found to harbor the <i>mcr-9</i> gene. Interestingly, the presence of <i>mcr-9</i> did not significantly impact PB resistance or impose a fitness cost. While overexpression of <i>mcr-9</i> can enhance PB resistance within a certain range and may incur fitness costs, it does not result in high-level PB resistance. The PB resistance of 17 CRECC strains was notably increased (from 16 to 128 mg/L), accompanied by mutations in the <i>phoP</i>/<i>Q</i> and <i>mgrB</i> genes. Notably, two novel insertion sequences, IS<i>5D</i> and IS<i>1X2</i>, were discovered within the <i>mgrB</i> gene. The inactivation of <i>mgrB</i> results in the loss of its negative regulatory effect on the two-component system. Protein structure predictions indicated that mutations in <i>phoQ</i> primarily affect the phosphatase (HAMP) and histidine kinase domains. This research significantly expands our comprehension of the complexities of PB resistance, highlighting the multifactorial nature of antibiotic resistance mechanisms.