| Background:As one of the most common gram-negative Enterobacteriaceae in the clinic,Klebsiella pneumoniae frequently causes infections in elderly patients,immunocompromised patients and intensive care patients in hospitals.In recent years,the detection rate of carbapenem-resistant Klebsiella pneumoniae（CRKP）has been on the rise due to the misuse of antibiotics.And ST15 CRKP is a high-risk clonal strain that has emerged in recent years and frequently outbreaks in hospitals,seriously endangering public health safety.In this paper,we performed phenotypic and genomic analyses of ST15 CRKP collected from a tertiary hospital in Anhui,China,to further explore the potential factors of ST15 CRKP outbreaks.Method:Non-replicated multi-resistant K.pneumoniae isolated in the clinical microbiology laboratory from July 2021 to December 2021 were collected for mass spectrometry identification,multilocus sequence typing（MLST）and podotyping,determination of their minimum inhibitory concentrations（MIC）,and phenotypic experiments such as growth curves,drawing assays,mucoadhesive assays,Galleria mellonella infection models and biofilm assays by using the hypervirulent K.pneumoniae isolate NTUH-K2044 as a control;virulence and resistance gene analysis,phylogenetic tree analysis and single nucleotide polymorphism（SNP）analysis.Genetic environment and homology analysis were performed by whole genome sequencing,assembly and annotation.Results:A total of 13 non-repeated multi-resistant isolates were collected during this period,with MLST of ST15 and capsule typing of KL19.13 ST15 CRKP isolates had high resistance to common antibiotics,including cephalosporins,quinolones,and carbapenems,but were sensitive to tigecycline and polymyxin B.In the G.mellonella infection model,23%of the isolates showed high lethality but negative pull-down assays,and another 31%of the isolates had strong biofilm formation ability.A total of 15 resistance genes were detected by genome sequencing analysis,six of which wereβ-lactamase genes,including bla SHV-106,bla KPC-2,bla LAP-2,bla TEM-1B,bla OXA-1,bla CTX-M-15;the phosphomycin resistance gene fos A,the quinolone efflux pump Oqx AB,the tetracycline resistance gene tet A,and the aminoglycoside resistance genes aac（3）-lid and aac（6’）-lb-cr.For virulence factors,ST15 CRKP carries siderophores genes such as aerobactin,yersin and enterobactin as well as the active efflux pump Acr AB.The upstream and downstream insertion elements and transposons of the 13ST15 CRKP isolates bla SHV-106,bla KPC-2,bla CTX-M-15,bla OXA-1 and bla TEM-1B genes were identical,and bla KPC-2was carried by the plasmid.single nucleotide polymorphism（SNP）analysis of ST15 CRKP revealed a maximum of 993 loci and a minimum of 21.The construction of a phylogenetic tree revealed that ST15 CRKP was divided into three subgroups with nosocomial transmission.The global phylogenetic analysis of these strains showed that they were highly homologous with isolates from Zhejiang and Jiangsu,and ST15 CRKP may have been spread in East China.Conclusion:The present study indicates that ST15 CRKP with co-producing SHV-106,KPC-2,CTX-M-15 and OXA-1 is spreading locally and may be evolving into hypervirulent isolates.Therefore,environmental surveillance and clinical infection control in hospitals should be strengthened to prevent further transmission of ST15 CRKP. |
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