Heteroresistance to colistin in the Enterobacter cloacae complex: Clinical characteristics, molecular epidemiology and mechanisms study

Chunli Wei ¹ Jiming Wu ¹ Jisheng Zhang ¹ Youtao Liang ¹ Kaixin Yu ^1,2 Mingjing Liao ¹ Xushan Liang ¹ Jianmin Wang ¹ Wenzhang Long ¹ Jin Wang ¹ Shijian Chen ¹ Yang Yang ¹ Xue Gong ¹ Jie Li ¹ Xiaoli Zhang ^1*

• ¹ Department of Microbiology, Yongchuan Hospital of Chongqing Medical University, Chongqing, China
• ² Department of Pathogenic Biology, Basic Medicine of Jiamusi University, Jiamusi, China

Colistin has emerged as the last resort for treating multidrug-resistant Enterobacter cloacae complex (ECC) infections. The primary purposes of this study were to demonstrate the presence of colistin heteroresistance in ECC and to further investigate their clinical characteristics, molecular epidemiology and mechanisms. Population analysis profiles (PAP) confirmed that a high proportion (24.4%) of the non-resistant strains were colistin-heteroresistant isolates. Among the several ECC species, Enterobacter kobei had the largest percentage (29.4%) of colistin-heteroresistant isolates, followed by Enterobacter hormaechei (20.5%) and Enterobacter bugandensis (20.0%). Notably, only one strain (0.8%; 1/132) of Enterobacter hormaechei was fully resistant to colistin. Different ECC species showed varying heteroresistance levels: Enterobacter roggenkampii, Enterobacter kobei, Enterobacter asburiae and Enterobacter bugandensis displayed high heteroresistance levels  (MIC ≥ 128 mg/L). 75% of all ST116 and ST56 strains were heteroresistant to colistin. A retrospective case-control study demonstrated the infection of ST116 and ST56 strains as well as exposure to cephalosporin antibiotics were independent risk factors for colistin-heteroresistant ECC infections. Mechanistic analysis revealed that heteroresistance strongly correlated with the overexpression of arnA, regulated by the PhoPQ two-component system (TCS). Notably, mgrB had minimal impact. AcrAB-TolC efflux pump genes showed unsynchronized expression; High acrB expression was strongly associated with colistin heteroresistance, while acrA and tolC were not. Colistin heteroresistance showed species-dependent variations in levels and prevalence rates. The colistin-heteroresistant mechanisms were complex, involving coordinated regulation of multiple genes. These results highlighted the need for tailored antimicrobial stewardship. In addition, the development of direct, reliable and rapid clinical methods for detecting heteroresistance is essential for improving infection management and prevention.