Objective: The aim of this study was to use whole-genome sequencing to characterize Klebsiella pneumoniae  SKp2F and Klebsiella variicola  SKv2E, both carrying bla _KPC, co-isolated from the same sputum specimen.
Methods: Antimicrobial susceptibility testing was performed using microbroth dilution. Biofilm formation was determined by crystal violet staining and virulence was measured by a serum killing assay. Whole-genome sequencing of SKp2F and SKv2E was performed using an Illumina sequencer and the genetic characteristics were analyzed by computer.
Results: SKp2F and SKv2E were sensitive only to tigecycline and polymyxin among the tested antibiotics. The biofilm-forming ability of SKv2E is stronger than that of SKp2F. The grades of serum resistance of SKp2F and SKv2E are 4 and 3. MLST analysis of the 6,115,610 bp and 5,403,687 bp of SKv2E and SKp2F showed associations with ST1615 and ST631, respectively. SKv2E carried 13 resistance genes (bla _KPC-2, bla _TEM-1A, bla _LEN17, aadA16 , arr-3 , qnrB4 , oqxA/B , dfrA27 , sul1 , tetD , fosA , qacEΔ 1 ) and SKp2F carried 23 (bla _KPC-2, bla _CTX-M-3, bla _TEM-1B, bla _CTX-M-65, bla _SHV-27, aac(6ʹ)-IIa , rmtB , arr-3 , aph(3ʹ)-Ia , aadA16 , qnrS1 , aac(6ʹ)-Ib-cr , qnrB91 , oqxA/B , mph(A) , tet(A) , fosA , dfrA27 , and two copies of qacEΔ 1-sul1 ). Most of them were carried by various mobile genetic elements, such as IncFIB(K)/IncFII(K)/IncFII(Yp), IncFII(K) plasmid, Tn6338, and In469. Both SKv2E and SKp2F carried a large number of virulence factors, including type 1 and 3 fimbriae, capsule, aerobactin (iutA), ent siderophore (entABCDEFS , fepABCDGfes ), and salmochelin (iroE/iroEN ). SKv2E also carried type IV pili (pilW ), fimbrial adherence (steB , stfD ), and capsule biosynthesis gene (glf ).
Conclusion: bla _KPC-2-carrying K. variicola  and K. pneumoniae , which carried multiple resistance genes, virulence factors, and highly similar mobile genetic elements, were identified from the same specimen, indicating that clinical samples may carry multiple bacteria. We should avoid misidentification, and bear in mind that resistance genes carrying mobile genetic elements can be transmitted or integrated between bacteria in the same host.
Keywords: Klebsiella variicola , Klebsiella pneumoniae , carbapenem-resistant Enterobacteriaceae, CRE, bla _KPC