WITHDRAWN: Characterization of the bla VEB-3 bearing IncC plasmid and bla KPC-2 bearing IncFII plasmid in a type I-E CRISPR-Cas system harboring Klebsiella oxytoca stain
Authors: Zhong Yu-Xia , Liu Wan-Ting , Yuan Pei-Bo , Yang Ling , Xu Zhenbo , Chen Ding-Qiang 
Published: February 2023

Background: Klebsiella oxytoca has become an important pathogen for opportunistic infections. Plasmid-mediated multidrug-resistant strains pose a significant risk to public health, especially when the strains produce carbapenemase. Here, we report the first detection of a blaKPC-2-IncFII plasmid and a blaVEB-3-IncC plasmid in a clinical multidrug-resistant K. oxytoca strain YL6, which has an entire cluster regularly spaced short palindromic repeat (CRISPR)-Cas immune system.
Case presentation and methods: A 46-year-old man was hospitalized with chronic fibrous pneumonia. The K. oxytoca strain YL6 was isolated from his sputum sample. It was resistant to all tested antimicrobials, including almost all β-lactams, cephalosporins, quinolones, and aminoglycosides, except for colistin and tigecycline. To investigate this strain further, we conducted whole-genome sequencing and sequence alignment. Furthermore, the emerging plasmids were compared with similar ones available in the gene bank better to understand the genetic composition and evolution of the plasmids.
Results: K. oxytoca strain YL6 belongs to ST27. This stain possesses a chromosome with 6,111,288 bp and two plasmids (An IncFII plasmid pYL6-1 and An IncC plasmid pYL6-2) with 101,530 bp and 145,051 bp, respectively. Genomic comparative analysis revealed that pYL-1 shared 70%~85% query cover and 98.65%-99.85% nucleotide identity with nineteen similar IncFII plasmids, and pYL-2 shared 79%~93% query cover and 99.74%-99.99% nucleotide identity with twenty-one similar IncC plasmids. Both groups of plasmids retained the complete coding region of the tra gene during their evolution. We also observed that the blaKPC-2 gene was located on an ISKpn19-orf1-orf2-Tn3-ISKpn27-blaKPC-2-ISKpn6-orf2-orf1-ISKpn19 structure in the multidrug resistance (MDR) region of pYL-1. Meanwhile, the blaVEB-3 gene in pYL6-2 was situated in an IS26-IS6100-blaVEB-3-transpone-ISAs1-qacEΔ1-sul1-ISCR1 casset. Based on additional data, the YL6 strain appears to possess an intact type I-E CRISPR -Cas immune system, which had been confirmed to inhibit the invasion and existence of the blaKPC-IncF plasmid effectively. The proto-spacers gene in pYL6-1, which are essential for the function of the CRISPR-Cas immune system, was no match to the CRISPR system sequence of K. oxytoca.
Conclusions: We first reported a multidrug-resistant K. oxytoca with an intact CRISPR-Cas immune system harbors a blaVEB-3-IncC plasmid and a blaKPC-2-IncFII plasmid. There are no proto-spacer sequences matching the CRISPR system of K. oxytoca on the IncFII plasmid pYL6-1, which is crucial for identifying the foreign gene. It is inferred that the blaKPC-2-IncFII plasmid might escape the host's immune recognition by avoiding its proto-spacer DNA Base sequences complementing the gene arrangements of the strain's CRISPR system. The mutual adaptation between the invasive plasmid and host bacteria is bound to increase bacterial drug resistance further and lead to an antimicrobial public crisis, which calls for considerable attention.