Whole-genome sequence-based analysis of Klebsiella pneumoniae JM45 / 中华临床感染病杂志

ABSTRACT

Objective To investigate the distribution of β-lactamase genes in a pan-drug resistant Klebsiella pneumoniae isolate JM45.Methods Klebsiella pneumoniae JM45 was isolated from the blood sample of a patient admitted in the intensive care unit,the Second Affiliated Hospital,Zhejiang University School of Medicine on April 7,2010.The susceptibilities to 26 antibiotics were tested using E-test method.Cica-β-Test was performed to detect β-lactams,and modified Hodge test was performed to detect carbapenemase.Resistant genotypes were detected using PCR,DNA sequencing and BLAST algorithm.Whole genome sequencing (complete graph) was performed by high throughput Roche 454 sequencing approach to analyze the distribution of β-lactamase genes.Results Except polymyxin B and tigecycline,JM45 was resistant to other 24 kinds of antibiotics including cephalosporins and carbapenems.Several β-lactamases were positive in Cica-β-Test,and modified Hodge test was positive.Based on PCR typing,TEM-1,SHV-11,CTX-M-24 and VEB-3 were positive,but carbapenemase genes and metallo-β-lactamase genes were negative.A complete genome (chromosome) sequence (GenBank accession numberCP006656) and 2 plasmids sequences (GenBank accession numberCP006657,CP006658) were obtained by wholegenome sequencing.CTX-M-24 (Locus tagN559_5233),TEM-1 (Locus tagN559_5242) and VEB-3 (Locus tagN559_5248) were positive in plasmid 1.CTX-M-24 located in insertion sequence (IS903-CTXM-24-ISEcp1),while TEM-1 and VEB-3 located in transposons (tnpA-TEM-1-rmtB and VEB-3-tnpA).SHV-11 (Locus tagN559_2715) was positive in genome (chromosome),and 4 putative β-lactamase genes or β-lactamase domains were obtained(1) metallo-β-lactamase domain protein (Locus tagN559_0119,780 bp) ; (2) putative β-lactamase (Locus tagN559_1633,1308 bp) ; (3) β-lactamase domain protein (Locus tagN559_2279,813 bp); (4) β-lactamase domain protein (Locus tagN559_3769,1101 bp).No insertion sequence or transposase gene was observed near SHV-11.Conclusion The resistance to antibiotics including cephalosporins and carbapenems is correlated with TEM-1,SHV-11,CTX-M-24,VEB-3 and 4 kinds of putative β-lactamase genes or β-lactamase domains.