目的：阐明摩根摩根菌的临床和分子流行特征，分析其种群结构及进化趋势，探究产碳青霉烯酶摩根摩根菌的全球流行分布，明确溶血素（HlyA）的编码基因hlyCABD操纵子在摩根摩根菌中的分布特征及其致病功能。

方法：本研究依托科技部基础资源调查专项（2019FY101200），纳入2020年和2021年在全国26个省份和自治区45家医院收集的206株临床分离的摩根摩根菌。采用微量肉汤稀释法检测19种抗菌药物对摩根摩根菌的最低抑菌浓度。对206株摩根摩根菌进行全基因组测序，并整合国家生物技术信息中心（National Center for Biotechnology Information，NCBI）基因组数据库中225株摩根摩根菌的参考基因组数据，开展分子流行病学分析。通过平均核苷酸一致性（Average nucleotide identity，ANI）和主成分分析（Principal component analysis，PCA）进行摩根摩根菌的种群分析，结合比较基因组学和海藻糖发酵实验分析不同种群特征。构建系统发育树对摩根摩根菌进行分型，并分析每个序列分簇的分子特征。统计分析产碳青霉烯酶摩根摩根菌在全球的流行分布特征，并通过探究碳青霉烯酶基因定位及其基因环境来阐述产碳青霉烯酶摩根摩根菌的传播机制。此外，基于生物信息学分析hlyCABD操纵子阳性摩根摩根菌的分子特征，并通过比较hlyCABD操纵子在不同物种中的基因环境阐述其可能来源。采用基因敲除和毒力表型实验等方法，明确HlyA在摩根摩根菌中的致病作用。

结果：在431株摩根摩根菌中，尿道是其最常见的标本来源。药物敏感性试验结果显示，摩根摩根菌对碳青霉烯类（除亚胺培南）、β内酰胺类和β内酰胺酶抑制剂复合制剂（除氨苄西林-舒巴坦）、头孢菌素类（除头孢他啶）、头孢西丁和阿米卡星的耐药率较低（低于10%），而对喹诺酮类、磷霉素、复方新诺明、庆大霉素和氯霉素呈现高水平耐药（超过30%）。全基因组分析结果显示，431株摩根摩根菌共携带了110种耐药基因（Antimicrobial resistance genes，ARGs），可介导15种不同种类的抗菌药物耐药，包括碳青霉烯类（bla_KPC、bla_NDM和bla_IMP）、多黏菌素类（mcr）和替加环素[tet(X4)和tmexCD-toprJ]。摩根摩根菌具有高度保守的毒力基因（Virulence-related genes，VRGs）谱，除了III型分泌系统相关基因（yscC、ipaD和sctC）、部分毒素基因以及umoB、sodC和btuB，其余VRGs在所有菌株中均存在。此外，摩根摩根菌含有丰富的可移动遗传元件（Mobile genetic elements，MGEs），平均每株菌携带17.19个。种群分析结果显示，除了摩根亚种和塞诺亚种，摩根摩根菌还存在其他三个新的亚种，分别为陈氏亚种、变异亚种1和变异亚种2。陈氏亚种通过MGEs介导的水平转移获得丰富的鞭毛等细菌运动相关基因，增强其环境适应能力。系统发育分析进一步将菌株划分为12个序列分簇（Sequence clusters，SCs），其中SC9簇在ARGs、VRGs和MGEs数量上具有显著优势。

在267株产碳青霉烯酶摩根摩根菌中，最常见的酶型分别为NDM-1（40.43%）、KPC-2（15.88%）、IMP-27（13.00%）和OXA-48（11.19%）。产碳青霉烯酶菌株携带的ARGs数量显著多于非产酶菌株，但两者的VRGs数量无统计学差异。近半数碳青霉烯酶基因（50.8%）位于质粒，30.2%位于染色体，其余基因尚无法定位。研究发现一个携带bla_KPC-2的新型质粒，该质粒由转座元件介导bla_KPC-2水平转移，整合进入摩根摩根菌的特有质粒中形成。基因环境分析表明，bla_IMP-27基因的遗传环境高度保守，几乎均位于染色体Tn7转座子的Ⅱ类整合子中。产碳青霉烯酶摩根摩根菌垂直传播现象广泛存在，包括欧洲流行的产NDM-1和KPC-2酶菌株及北美的产IMP-27酶菌株。在431株摩根摩根菌中，125株菌（29.00%）携带hlyCABD操纵子，阳性菌株多分离于血流标本及ICU病房，且携带的VRGs和MGEs数量均显著高于阴性菌株（P < 0.05）。系统发育分析显示，阳性菌株分为SC1和SC2两簇，其中SC1簇菌株携带更多与分泌系统等相关基因，提示其可能具有更强的致病潜力。对不同物种的hlyCABD操纵子及其基因环境进行比较分析，推测摩根摩根菌、大肠埃希菌、副溶血弧菌和恶魔弧菌中的hlyCAB操纵子可能起源于彭氏变形杆菌。研究证实，摩根摩根菌分泌的HlyA由hlyCABD操纵子编码，其在菌株的侵袭、粘附、生物膜形成及宿主免疫反应等致病过程中具有重要的作用。

结论：本研究揭示了摩根摩根菌携带丰富的ARGs，VRGs和MGEs。在环境选择压力和MGEs作用下，摩根摩根菌演化出新的亚种及具有高毒高耐潜力的序列分簇。尽管摩根摩根菌对碳青霉烯类抗菌药物的耐药率较低，但产碳青霉烯酶摩根摩根菌通过质粒介导的水平转移和垂直转移可能加剧碳青霉烯类抗菌药物耐药性的传播。此外，hlyCABD操纵子阳性摩根摩根菌具有更强的基因可塑性和毒力表型，对临床致病性具有重要意义，值得进一步关注和研究。本研究为摩根摩根菌的防控及精准治疗提供了重要的理论依据和实践参考。

Objective: To elucidate the clinical and molecular epidemiology of Morganella morganii, to analyze its population structure and evolutionary trends, to investigate the global prevalence of carbapenemase-producing M. morganii, and to characterize the distribution and pathogenic role of the α-hemolysin (HlyA)-encoding hlyCABD operon.

Methods: This study was conducted under the Special Foundation for National Science and Technology Basic Research Program of China (2019FY101200). A total of 206 clinical isolates of M. morganii were collected from 45 hospitals across 28 provinces and municipalities in China from 2020 to 2021. The minimum inhibitory concentrations of 19 antimicrobial agents against M. morganii were determined using the broth microdilution method. Whole-genome sequencing was performed on the 206 isolates, and reference genomic data of 225 additional M. morganii strains from the National Center for Biotechnology Information (NCBI) genome database was integrated for molecular epidemiological analysis. Population analysis was conducted using average nucleotide identity (ANI) and principal component analysis (PCA), combined with comparative genomics and trehalose fermentation experiments to investigate evolutionary features among different populations. A phylogenetic tree was constructed to classify M. morganii and molecular characteristics of each sequence cluster were analyzed. Statistical analysis of the global epidemiological distribution characteristics of carbapenemase-producing M. morganii was conducted, and molecular investigations through exploring the localization of carbapenemase genes and their genomic context were performed to elucidate the transmission mechanisms of carbapenemase-producing M. morganii strains. Additionally, bioinformatics analyses were conducted to examine the molecular features of hlyCABD operon-positive M. morganii, and comparisons of the genomic environments of hlyCABD operon across different species were used to infer the origin of the operon. Gene knockout and virulence phenotype experiments were performed to clarify the pathogenic role of HlyA in M. morganii.

Results: Among the 431 M. morganii strains, the urinary tract was the most common source of specimens. Antimicrobial susceptibility testing revealed that M. morganii exhibited low resistance rates (less than 10%) to carbapenems (except imipenem), β-lactam-β-lactamase inhibitors (except ampicillin-sulbactam), cephalosporins (except ceftazidime), cefoxitin, and amikacin. However, the strains showed high-level resistance (over 30%) to quinolones, fosfomycin, trimethoprim-sulfamethoxazole, gentamicin, and chloramphenicol. Whole-genome analysis identified 110 antimicrobial resistance genes (ARGs) in the 431 isolates, mediating resistance to 15 classes of antimicrobial agents, including carbapenems (bla_KPC, bla_NDM, and bla_IMP), polymyxins (mcr), and tigecycline [tet(X4) and tmexCD-toprJ]. M. morganii exhibited a highly conserved virulence-related genes (VRGs) profile. Except for genes related to the type III secretion system (yscC, ipaD, and sctC), some toxin genes, as well as umoB, sodC, and btuB, the remaining VRGs were present in almost all strains. Additionally, M. morganii harbored abundant mobile genetic elements (MGEs), with an average of 17.19 MGEs per isolate. Population analysis revealed that M. morganii consisted of not only M. morganii and M. sibonii genospecies but also three novel genospecies: M. chanii, M. variant1, and M. variant2. M. chanii acquired numerous flagellar-related genes through horizontal transfer mediated by MGEs, potentially enhancing its environmental adaptability. Phylogenetic analysis further grouped the isolates into 12 sequence clusters (SCs), with SC9 exhibiting significant advantages in terms of ARGs, VRGs, and MGEs.

Among 267 carbapenemase-producing M. morganii isolates, the most prevalent carbapenemases were NDM-1 (40.43%), KPC-2 (15.88%), IMP-27 (13.00%), and OXA-48 (11.19%). The carbapenemase-producing isolates carried significantly more non-carbapenem ARGs than non-carbapenemase-producing isolates, although no significant difference in VRGs numbers was observed. Nearly half of the carbapenemase genes (50.8%) were located on plasmids, 30.2% were on chromosomes, and the remaining could not be localized. A novel plasmid carrying bla_KPC-2 was identified, which was mediated by a transposition unit for horizontal transfer of bla_KPC-2, and was integrated into the unique plasmid of M. morganii. Genetic environment analysis demonstrated that bla_IMP-27 exhibited a conserved structure, primarily embedded within class II integrons of chromosomally located Tn7 transposons. Vertical transmission of carbapenemase-producing M. morganii was widespread, including NDM-1 and KPC-2-producing isolates in Europe and IMP-27-producing isolates in North America. Among the 431 isolates, 125 strains (29.00%) carried the hlyCABD operon. The positive strains were more frequently isolated from bloodstream specimens and ICU wards, with significantly higher numbers of VRGs and MGEs, compared to negative isolates (P < 0.05). Phylogenetic analysis revealed that positive strains were grouped into two clusters, SC1 and SC2, with SC1 carrying more secretion system-related genes, suggesting potentially enhanced pathogenicity. Comparative analysis of the hlyCABD operon and its genetic environment across species suggested that the hlyCABD operon in M. morganii, Escherichia coli, Vibrio parahaemolyticus, and Vibrio diabolicus may have originated from Proteus penneri. Experimental evidence confirmed that the HlyA secreted by M. morganii is encoded by the hlyCABD operon, and it plays a significant role in the pathogenic processes such as invasion, adhesion, biofilm formation, and host immune response.

Conclusions: This study reveals that M. morganii carries a rich variety of ARGs, VRGs, and MGEs. Under environmental selective pressure and the action of MGEs, M. morganii has evolved novel genetic genospecies and sequence clusters with high virulence and resistance potential. Although the resistance rate of M. morganii to carbapenems is relatively low, carbapenemase-producing M. morganii may exacerbate the spread of carbapenem resistance through plasmid-mediated horizontal transfer and vertical transmission. Furthermore, hlyCABD operon-positive strains exhibit greater genetic plasticity and enhanced virulence phenotypes, which has important clinical pathogenic significance and warrants further attention and research. This study provides important theoretical and practical references for the prevention, control, and precision treatment of M. morganii.