Emergence of Tigecycline and Carbapenem-Resistant Citrobacter freundii Co-Carrying tmexCD1-toprJ1, bla(KPC-2), and bla(NDM-1) from a Sepsis Patient

PURPOSE: This research aims to profile ten novel strains of carbapenem-resistant Enterobacteriaceae (CRE) co-carrying bla(KPC) and bla(NDM). METHODS: Clinical CRE strains, along with corresponding medical records, were gathered. To ascertain the susceptibility of the strains to antibiotics, antimicrobial susceptibility tests were conducted. To validate the transferability and cost of fitness of plasmids, conjugation experiments and growth curves were employed. For determining the similarity between different strains, ERIC-PCR was utilised. Meanwhile, whole genome sequencing (WGS) was performed to characterise the features of plasmids and their evolutionary characteristics. RESULTS: During the course of this research, ten clinical CRE strains co-carrying bla(KPC) and bla(NDM) were gathered. It was discovered that five out of these ten strains exhibited resistance to tigecycline. A closer examination of the mechanisms underlying tigecycline resistance revealed that tmexCD1-toprJ1, bla(KPC-2), and bla(NDM-1) existed concurrently within a single Citrobacter freundii strain (CF10). This strain, with a minimum inhibitory concentration (MIC) of 32 mg/L to tigecycline, was obtained from a sepsis patient. Furthermore, an investigation of genome evolution implied that CF10 belonged to a novel ST type 696, which lacked analogous strains. Aligning plasmids exposed that similar plasmids all had less than 70% coverage when compared to pCF10-tmexCD1, pCF10-KPC, and pCF10-NDM. It was also found that tmexCD1-toprJ1, bla(KPC-2), and bla(NDM-1) were transferred by Tn5393, IS5, and Tn6296, respectively. CONCLUSION: This research presents the first report of coexistence of tmexCD1-toprJ1, bla(KPC-2), and bla(NDM-1) in a carbapenem and tigecycline-resistant C. freundii strain, CF10. IMPORTANCE: Tigecycline is considered a “last resort” antibiotic for treating CRE infections. The ongoing evolution of resistance mechanisms to both carbapenem and tigecycline presents an alarming situation. Moreover, the repeated reporting of both these resistance mechanisms within a single strain poses a significant risk to public health. The research revealed that the genes tmexCD1-toprJ1, bla(KPC-2), and bla(NDM-1), which cause carbapenem and tigecycline-resistance in the same strain, were located on mobile elements, suggesting a potential for horizontal transmission to other Gram-negative bacteria. The emergence of such a multi-resistant strain within hospitals should raise significant concern due to the scarcity of effective antimicrobial treatments for these “superbugs”.