Determination of Whole Genome Sequence and Resistance Genes in Hypermucoviscous Klebsiella pneumoniae with Long-Read Sequencing Technology

Abstract

Numerous virulence factors are present in the hypervirulent/hypermucoviscous Klebsiella pneumoniae, which primarily causes community-acquired infections. In recent years, hypervirulent/hypermucoviscous K.pneumoniae has acquired resistance genes and has been linked to healthcare-associated infections. The aims of this study were to determine whole genome sequencing of hypermucoviscous K.pneumoniae that induces healthcare-associated bloodstream infection utilizing the Oxford Nanopore (MinION) platform, to identify resistance genes using various databases, and to compare the database results. K.pneumoniae isolates that were sent to the Dokuz Eylül University Research and Training Hospital Center Laboratory and were isolated from blood cultures were included in this study between January 2018 and December 2020. K.pneumoniae isolates were identified using the automated VITEK-2 system. The disc diffusion method was used to characterize the antimicrobial resistance profile, and the string test was used to assess the hypermucoviscous phenotype. By using specific primers blaOXA-48, blaNDM, blaKPC, blaIMP, and blaVIM, carbapenem resistance genes were investigated using the PCR method. To ascertain clonal relatedness among hypermucoviscous K.pneumoniae isolates, PFGE was used. The whole genome sequencing of five K.pneumoniae strains with different origins was determined by Oxford Nanopore (MinION) technology. Using the ResFinder, CARD and BacWGSTdb databases, resistance genes were examined. Capsule regulation genes were analyzed with the BacWGSTdb database. Resistance genes on the plasmid were discovered using the ResFinder database after plasmid analyses were carried out using the PLSDB and PlasmidFinder databases. A total of 244 K.pneumoniae isolates were included in the study. Ten isolates were found to be hypermucoviscous. A carbapenem-resistant hypermucoviscous K.pneumoniae isolate was discovered to carry the blaOXA-48. Five hypermucoviscous isolates of which whole genome sequences were determined had blaSHV types, oqxB, oqxA, and fosA genes on the chromosome. Capsule regulation genes rcsA, rcsB were detected in all five isolates, and rmpA/rmpA2 genes caused hypermucoviscous phenotype was detected in two of the five isolates. In the plasmid analysis, IncFIB (K) pCAV1099- 14, Col (pHAD28), IncFIA (HI1)-IncR, Col (pHAD28), Col440, IncFIB (pNDM_Mar)-IncHI1 (pNDM_MAR), IncL, IncFIB (K)-IncHI1 (K), IncFIB (K), IncR, IncFIB (pNDM_Mar)-IncHI1 (pNDM_MAR)-IncR, repB plasmids were identified. Resistance genes; aac(3)-IId, aph(6)-Id, aph(3")-Ib qnrB1, sul2, dfrA14, blaTEM-1A, blaCTX-M-15, armA, msr(E), mph(E), catB3, blaOXA-1, aac(6')-Ib-cr, catA1 and blaOXA-48 were detected on the plasmid. In the bioinformatic analyzes, it was determined that two study isolates with hypermucoviscous phenotype had various plasmids and carried many resistance genes on these plasmids. Various resistance and virulence genes are spread through plasmids and the number of resistant hypermucoviscous K.pneumoniae is increasing day by day. Appropriate infection control strategies should be implemented to prevent healthcare-associated infections and outbreaks caused by antimicrobial-resistant hypermucoviscous K.pneumoniae. For this purpose, resistance monitoring of antimicrobials used in the treatment of K.pneumoniae infections should be performed. There were some differences in the results of the three databases used to detect resistance genes in this study. For this reason, it is important to analyze different databases and compare the database results in studies while performing whole genome sequencing. Further studies and updates using different databases will increase the compatibility between the databases.