The incidence of hypervirulent (hv) carbapenem-resistant (CR) Klebsiella pneumoniae (Kp) is increasing globally among various clones and is also responsible for nosocomial infections. The CR-hvKp is formed by the uptake of a virulence plasmid by endemic high-risk clones or by the uptake of plasmids carrying antimicrobial resistance genes by the virulent clones. Here, we describe CR-hvKp from India belonging to high-risk clones that have acquired a virulence plasmid and are phenotypically unidentified due to lack of hypermucoviscosity.

Twenty-seven CRKp isolates were identified to possess rmpA2 by whole-genome sequencing; and resistance and virulence determinants were characterized. By in silico protein modeling (and validation), protein backbone stability analysis, and coarse dynamics study, the fitness of RmpA, RmpA2, and aerobactin-associated proteins-IucA and IutA, were determined to establish a reliable marker for clinical identification of CR-hvKp.

The CR-hvKp belonged to multidrug-resistant (MDR) high-risk clones such as CG11, CG43, ST15, and ST231 and carried OXA-232 as the predominant carbapenemase followed by NDM. The virulence plasmid belonged to IncHI1B replicon type and carried frameshifted and truncated rmpA and rmpA2. This resulted in a lack of hypermucoviscous phenotype. However, functional aerobactin was expressed in all high-risk clones. In silico analysis portrayed that IucA and IutA were more stable than classical RmpA. Furthermore, IucA and IutA had lower conformational fluctuations in the functional domains than the non-functional RmpA2, which increases the fitness cost of the latter for its maintenance and expression among CR-hvKp. Hence, RmpA and RmpA2 are likely to be lost among CR-hvKp owing to the increased fitness cost while coding for essential antimicrobial resistance and virulence factors.

Increasing incidence of convergence of AMR and virulence is observed among K. pneumoniae globally, which warrants the need for reliable markers for identifying CR-hvKp. The presence of non-functional RmpA2 among high-risk clones highlights the significance of molecular identification of CR-hvKp. The negative string test due to non-functional RmpA2 among CR-hvKp isolates challenges phenotypic screening and faster identification of this pathotype. This can potentially be counteracted by projecting aerobactin as a stable, constitutively expressed, and functional marker for rapidly evolving CR-hvKp.

高毒力 (hv) 碳青霉烯耐药 (CR) 的发生率 肺炎克雷伯菌(Kp) 在各种克隆中在全球范围内增加，并且也是医院感染的原因。CR-hvKp 是由地方性高危克隆吸收毒力质粒或毒力克隆吸收携带抗微生物基因的质粒形成的。在这里，我们描述了来自印度的 CR-hvKp，属于高风险克隆，这些克隆已获得毒力质粒，并且由于缺乏高黏膜粘度而在表型上未鉴定。

鉴定出 27 种 CRKp 分离株具有 rmpA2通过全基因组测序；并对耐药性和毒力决定因素进行了表征。经过电脑模拟 通过蛋白质建模（和验证）、蛋白质骨架稳定性分析和粗动力学研究，确定了 RmpA、RmpA2 和需氧菌素相关蛋白 IucA 和 IutA 的适合度，为临床鉴定 CR-hvKp 建立了可靠的标记。

CR-hvKp 属于多重耐药（MDR）高危克隆，如 CG11、CG43、ST15 和 ST231，携带 OXA-232 作为主要的碳青霉烯酶，其次是 NDM。毒力质粒属于IncHI1B复制子类型，携带移码和截短rmpA 和 rmpA2. 这导致缺乏高粘液表型。然而，功能性需氧菌素在所有高危克隆中均有表达。电脑模拟分析表明 IucA 和 IutA 比经典 RmpA 更稳定。此外，IucA 和 IutA 在功能域中的构象波动低于非功能性 RmpA2，这增加了后者在 CR-hvKp 中维持和表达的适应度成本。因此，RmpA 和 RmpA2 可能会在 CR-hvKp 中丢失，因为在编码基本的抗菌素耐药性和毒力因子时增加了适应度成本。

观察到 AMR 和毒力收敛的发生率增加 肺炎克雷伯菌全球范围内，需要可靠的标记来识别 CR-hvKp。高风险克隆中非功能性 RmpA2 的存在突出了 CR-hvKp 分子鉴定的重要性。由于 CR-hvKp 分离株中非功能性 RmpA2 导致的阴性字符串测试挑战了表型筛选和更快地识别这种病理类型。这可以通过将产气杆菌素投射为快速进化的 CR-hvKp 的稳定、组成型表达和功能性标记物来抵消。