Rapid and accurate etiologic diagnosis accelerates targeted antimicrobial therapy. Metagenomic analysis has played a critical role in pathogen identification. In this study, we leveraged the advantages of both the MinION and BGISEQ-500 platforms to make a bacteriologic diagnosis from a culture-negative lung tissue sample from an immunocompromised patient with severe pneumonia. Real-time nanopore sequencing rapidly identified Klebsiella pneumoniae by an 823 bp specific sequence within 1 min. Genomic analysis further identified bla SHV-12 , bla KPC-2 , bla TEM-1 , bla CTX-M-65 , and other resistance genes. The same sample was further sequenced on the BGISEQ-500 platform, which presented consistent results regarding the most top dominant pathogens and provided additional information of resistance genes. Revised antibiotic treatment was followed by the patient's clinical recovery. Though sample preparation and the interpretation of final results still need to be improved further, metagenomic sequencing contributes to the accurate diagnosis of culture-negative infections and facilitates the rational antibiotic therapy.
Background: Enterobacter cloacae is an opportunistic pathogen which is responsible for serious nosocomial infections. A gene which plays an important role in resistance to carbapenems is the New Delhi metallo-β-lactamase 1 (NDM-1). Currently, the spread of NDM-1-producing E. cloacae strains is a serious public threat. Methods: A multidrug-resistant E. cloacae ssp. dissolvens strain CBG15936 was recovered in 2017 in Guangzhou, China. PCR, S1-pulsed-field gel electrophoresis, and Southern blotting were performed to locate the bla NDM−1 gene. Susceptibility testing and conjugation experiments were also performed. Illumina HiSeq and Nanopore sequencers were used to perform whole-genome sequencing. Results: Strain CBG15936 belongs to ST932 and is resistant to carbapenems. The bla NDM−1 gene was found on a ∼62-kb plasmid, which has a conjugation frequency of 1.68 × 10 −3 events per donor cell. Genome sequencing and analysis revealed that the NDM-1-carrying IncN1 plasmid contained a new transposon Tn6696, which consists of an intact qnrS1-carrying Tn6292 element, an inverted 8.3-kb Tn3000 remnant, ISkpn19, tnpA, and IS26. Conclusion: A new transposon, Tn6696, has been detected on a bla NDM−1-carrying plasmid recovered from multidrug-resistant E. cloacae ssp. dissolvens CBG15936 from China. This finding provides a new perspective regarding the potential for bla NDM−1 to undergo horizontal transfer among drug-resistant bacteria.
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