Molecular Identification of Co-Existence of Carbapenemase and Extended-Spectrum <i>β</i>-Lactamase Genes in <i>Klebsiella pneumoniae</i> Clinical Isolates, and Their Phylogenetic Patterns in Kenya

Abstract

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 like-

Formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing Klebsiella pneumoniae in vitro in Kenya

“…We previously reported the presence of extended-spectrum beta-lactamase (ESBL) genes (blaTEM and blaOXA) and carbapenemase genes (blaNDM-1, blaIMP). 13 The ESBL and carbapenemase genes produced by KP20 are indicated in Table 1 .…”

“… NA, not applicable. † , beta -lactamase gene, ‡ , Oxacillinase gene, § , New Delhi metallo-beta-lactamase-1 gene, ¶ , Imipenemasegene, †† , Oxacillinase-48 gene, ‡‡ , Data reported by Michodigni et al (2021) 13 . …”

“…The clinical isolate of CP K. pneumoniae (KP20) was obtained from the stock of the recently published data in Kenya. 13 Bacteriophages were isolated from waste water samples of Dandora Estate Sewage Treatment Works (Ruai) and Rongai effluent, Nairobi, Kenya. They were purified from waste water samples through spot assay and double-layer plaque method as described, previously.…”

“…A spot assay was conducted to determine the host range of precipitated bacteriophages and phage cocktails as described by Kutter. 19 Bacterial strains tested in our study included reference strains and MDR bacterial strains reported by Michodigni et al 13 and described in online Supplementary Methods 2.…”

Evaluation of the Genetic Diversity of Antibiotic-Resistant Klebsiella Pneumoniae Isolated from Diarrheal Humans and Poultry using Multilocus Sequence Typing

“…Many variables contribute to the irrational use of antibiotics, including policymakers' perceptions of their expertise, manufacturers' prescriptions, consumers' perceptions of their knowledge, and dispensers' perceptions of their knowledge (Wall, 2019). rpoB gene, which codes for the RNA polymerase ß-subunit, has emerged as a core gene candidate for phylogenetic analysis, allowing for the separation of closely related isolates (Michodigni et al, 2021). K. pneumoniae and other Klebsiella species can be correctly identified and differentiated using phylogenetic analysis of the rpoB gene.…”