Whole genome sequencing of multidrug-resistant clinical Klebsiella Isolates from community acquired pneumonia patients at Adama Hospital Medical College, Ethiopia

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Klebsiella pneumoniae is a leading Gram-negative pathogen causing severe community-acquired pneumonia (CAP), classically characterized by rapid lung necrosis, cavitary lesions, and production of thick, blood-tinged sputum. CAP is an acute lung infection that develops in a person outside healthcare facilities or within 48 h of hospital admission for another reason. Its clinical significance is heightened by the emergence of multidrug-resistant (MDR) high-risk clones carrying specific fitness-associated virulence factors, which can complicate empirical treatment. Although K. pneumoniae is widely recognized as a major cause of healthcare-associated infections, it accounts for 3–15% of CAP cases globally, with a disproportionately higher incidence in low- and middle-income countries. Therefore, this study aimed to investigate antibiotic resistance genes, virulence genes, and clonal relationships of MDR Klebsiella species isolated from patients with CAP at Adama Hospital Medical College (AHMC). Sixteen Klebsiella isolates were collected from the sputum samples of patients with community-acquired pneumonia at AHMC. Bacterial identification employed conventional culture methods, a comprehensive biochemical panel, MALDI-TOF MS and phenotypic antimicrobial susceptibility testing by Kirby Buaer disk diffusion. Whole-genome sequencing (WGS) was conducted using both Illumina NovaSeq and Oxford Nanopore sequencing technologies. The resulting genomes were then processed using Bactopia pipeline for quality control, polishing, and annotation, followed by plasmid reconstruction and genomic mapping of key resistance and evolutionary markers via MOB-suite and BRIG. MALDI-TOF MS and WGS confirmed 12 Klebsiella pneumoniae and 4 Klebsiella variicola isolates from sputum samples. MLST revealed high genetic diversity, identifying 10 sequence types (STs) alongside high resistance rates to β-lactams, sulfonamides, and aminoglycosides. A total of 47 antimicrobial resistance (AMR) genes were detected with bla CTX−M−15 , tet(A) and fosA6 present in all isolates. Notably, the ST1805 lineage was known to harbor bla CTX−M−15 and bla NDM−1 , making it a carbapenem-resistant strain. Furthermore, the potential virulence of this isolate was indicated by presence of Type VI Secretion System (T6SS) and yersiniabactin. Overall, virulence profiling identified 92 genes, including fimbrial clusters (fimA , mrkA and mrkC ), immune evasion genes (gndA and wcaJ) and the most prevalent T6SS and yersiniabactin which are very important for competition and iron acquisition, in order were the most predominant virulent factors. These virulence factors are primarily associated with the predominant IncF-family plasmid replicon found across the isolates. In this study, whole-genome sequencing of the isolates identified high risk sequence types, including ST14, ST39, and ST101, as well as the presence of ESBL gene ( bla CTX−M−15 ), the carbapenemase gene ( bla NDM−1 ) and narrow-spectrum beta-lactamases ( bla TEM−1 and bla OXA−1 ) alongside various virulence genes. This finding underscores the need for robust genomic surveillance and strengthened antimicrobial stewardship programs to curb the emergence of multidrug-resistant, Klebsiella species harboring mobile, acquired virulome determinants in the region.

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