Molecular Identification of Co-Existence of Carbapenemase and Extended-Spectrum iβ/i-Lactamase Genes in iKlebsiella pneumoniae/iClinical Isolates, and Their Phylogenetic Patterns in Kenya

摘要

The increasing incidence of multidrug-resistant Klebsiella pneumoniae strains has become a serious global healthcare problem. Additionally, the carriage of both extended-spectrum ß-lactamase and carbapenemase genes on plasmid and genomic DNA in K. pneumoniae clinical isolates has not been documented in Kenya. This study aimed to assess the presence of extended spectrum β-lactamase (ESBL) and carbapenemase genes on genomic and plasmid DNA in K. pneumoniae, and classify these super-bug clinical isolates based on their phylogenetic patterns. The identification of Klebsiella-like clinical isolates (n = 20) collected from Kenyatta National Hospital in Nairobi was performed using API 20E Kit. Screening and confirmation for ESBL and carbapenemase phenotypes were conducted using Kirby-Bauer disk diffusion susceptibility test protocol. Conventional PCR technique was used to characterize ESBL and carbapenemase resistant genes on both genomic and plasmid DNA. Subsequently, 16S rRNA gene amplification and sequencing were performed. The 16S rRNA gene contiguous sequences of the bacterial isolates were analyzed using the ChromasPro. The gene sequence was compared with the sequences in GenBank database, using the BLAST program of NCBI to obtain the nearest phylogenetic neighbours from the databases. Then, the sequences of MDR K. pneumoniae and its relatives were aligned using ClustalW. The evolutionary history was inferred by using the maximum likelihood algorithm in MEGA MX. The phenotypic data of antibiotic susceptibility testing revealed that 2/20 (10%) clinical isolates were resistant both to imipenem and meropenem and producers of carbapenemase. These isolates were carbapenemase producers but not extended β-lactamases. However, 3/20 (15%) isolates that co-harboured blaNDM-1, blaIMP, blaTEM, and bla-OXA were identified during genotypic analysis. The positive control used separately yielded the expected band sizes for blaIMP (275 bp), blaOXA-48 (438 bp), and BlaKPC (798). The phylogenetic analysis showed the dual ESBL and carbapenemase producing Klebsiella pneumoniae could be classified as K. pneumoniae strain DSM 30104 and K. pneumonia subsp. pneumoniae strain GMH1080. This study confirmed the co-existence of ESBL and carbapenemase genes in Klebsiella pneumoniae on both bacterial genomic and Plasmid DNA, and demonstrated that the isolates are evolutionarily distinct. These findings raise a concern about the genotypic diversity of antibiotic resistance genes in bacterial isolates and their location. We, therefore, recommend an alternative management approach to combat these MDR bacterial isolates as well as frequent molecular surveillance programs to support antimicrobial stewardship.