Table 1_Clinical characteristics, molecular epidemiology and mechanisms of colistin heteroresistance in Enterobacter cloacae complex.docx

IntroductionColistin 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.
MethodsPopulation analysis profiles (PAP) were performed to confirm the heteroresistance phenotype. Average nucleotide identity (ANI) was determined to classify ECC species. Phylogenetic analysis based on core genome single nucleotide polymorphisms (cg-SNPs), multilocus sequence typing (MLST) and core genome MLST (cg-MLST). Risk factors and clinical outcomes of infections were analyzed through a retrospective case-control study. Potential mechanisms of colistin heteroresistance were evaluated using polymerase chain reaction (PCR), efflux pump inhibition assays and reverse transcription quantitative PCR (RT-qPCR).
ResultsA 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. 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.
DiscussionColistin 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.

引言 多黏菌素（colistin）已成为治疗多重耐药阴沟肠杆菌复合群（Enterobacter cloacae complex, ECC）感染的最后一线治疗手段。本研究的核心目的在于明确ECC中多黏菌素异质性耐药（colistin heteroresistance）的存在情况，并进一步探究其临床特征、分子流行病学特征与耐药机制。
方法 本研究采用群体分析谱（population analysis profiles, PAP）验证异质性耐药表型；通过平均核苷酸同源性（average nucleotide identity, ANI）分析对ECC菌株进行物种分型；基于核心基因组单核苷酸多态性（core genome single nucleotide polymorphisms, cg-SNPs）、多位点序列分型（multilocus sequence typing, MLST）及核心基因组MLST（core genome MLST, cg-MLST）开展系统发育分析；通过回顾性病例对照研究（retrospective case-control study）分析感染相关危险因素与临床转归；采用聚合酶链式反应（polymerase chain reaction, PCR）、外排泵抑制试验及逆转录定量PCR（reverse transcription quantitative PCR, RT-qPCR）评估多黏菌素异质性耐药的潜在分子机制。
结果 本研究中，24.4%的非耐药菌株为多黏菌素异质性耐药分离株。在各类ECC物种中，科贝肠杆菌（Enterobacter kobei）的多黏菌素异质性耐药分离株占比最高（29.4%），其次为霍氏肠杆菌（Enterobacter hormaechei，20.5%）与布甘达肠杆菌（Enterobacter bugandensis，20.0%）。值得注意的是，仅1株霍氏肠杆菌（占比0.8%，1/132）对多黏菌素完全耐药。不同ECC物种的异质性耐药水平存在差异：罗根坎普肠杆菌（Enterobacter roggenkampii）、科贝肠杆菌、阿斯布尔肠杆菌（Enterobacter asburiae）及布甘达肠杆菌呈现最低抑菌浓度（minimum inhibitory concentration, MIC）≥128mg/L的高水平异质性耐药。所有ST116与ST56型菌株中，75%对多黏菌素呈现异质性耐药。感染ST116及ST56型菌株、暴露于头孢菌素类抗生素，均为多黏菌素异质性耐药ECC感染的独立危险因素。机制分析显示，异质性耐药与PhoPQ双组分系统（two-component system, TCS）调控的arnA基因过表达显著相关；值得注意的是，mgrB基因对该耐药表型影响极小。AcrAB-TolC外排泵基因的表达存在不同步性：acrB高表达与多黏菌素异质性耐药显著相关，而acrA与tolC基因表达则无此关联。
讨论 多黏菌素异质性耐药的水平与流行率存在物种依赖性差异。多黏菌素异质性耐药的分子机制较为复杂，涉及多基因的协同调控。本研究结果提示，需制定针对性的抗菌药物管理策略。此外，开发直接、可靠且快速的临床异质性耐药检测方法，对于优化感染防控与管理至关重要。