Genomic determinants of resistance in extended-pectrum beta lactamases (ESBLs)-producing Klebsiella pneumoniae isolates from selected hospitals in Malaysia

The emergence of Klebsiella pneumoniae (Kp) producing extended-spectrum beta- lactamases (ESBLs) is critically undermining the efficacy of essential antibiotics, as demonstrated by recent global surveillance data. This escalating resistance not only compromises treatment options but also accelerates the spread of antimicrobial resistance, necessitating urgent and coordinated action. The purpose of this study was to assess the extended-spectrum beta-lactamases producing K. pneumoniae (ESBLs- Kp) archived isolates from Hospital Pakar Sultanah Fatimah Muar (HPSFM), Johor and recent isolates from Hospital Sultan Abdul Aziz Shah, Universiti Putra Malaysia (HSAAS UPM) in terms of their genetic resistance. The recent isolates were obtained from HSAAS between August 2021 to February 2022 and archived isolates were obtained from HPSFM in 2012. An analytical profile index (API) 20E kit was used to confirm 100 isolates of Klebsiella pneumoniae (36 archive clinical samples and 64 recent clinical samples). The disc diffusion method was used to determine antimicrobial susceptibility, which was followed by the phenotypic and genotypic detection of ESBLs. Six representative strains were selected from antibiogram clusters and were subjected to whole genome sequencing (WGS), multilocus sequence typing (MLST), and phylogenetic tree was constructed. The study showed that 46 % (n= 46) of the 100 isolates were positive for ESBL production and the presence of ESBL genes was high among the positive isolates (89%, n = 41). Antibiotic susceptibility testing against 19 antibiotics revealed resistance to aztreonam (49%), ampicillin/sulbactam (40%), cefuroxime (51%), cefotaxime (49%), and ceftriaxone (47%). All ESBL- positive isolates (89%, n=41) harboured the CTXM-15 gene, followed by TEM (85%, n=39) and SHV (74%, n=34). According to this study, 72% (n=33) of the isolates harboured all three ESBL genes; SHV, TEM, CXTM-15. However, none of the ESBL genes were detected in 11% (n=5) of the isolates. The selected representative ESBL- Kp from recent clinical samples of HSAAS were CIKP1, CIKP2, CIKP6, CIKP16 and CIKP12 and archived clinical sample was AIKPB13 from HPSFM. The WGS analysis of the representative strains detected genes encoding resistance to aminoglycoside, trimethoprim, sulphonamide, quinolone, tetracycline, fosfomycin, macrolide, rifampicin, β-lactam. Additionally, the MLST analysis revealed four heterogeneous STs; ST394, ST985, ST17, and ST11 for strains AIKPB13, CIKP1, CIKP6, and CIKP16, respectively. Two novel sequence types were discovered; CIKP2 with ST NF* and CIKP12 with ST NF. All of the clades had lineages with other reference strains, and the representative strains' phylogenetic tree likewise revealed closed clonal relationships. More importantly, CIKP12, shared the same lineage with some reference strains but does not have a clonal relationship with them. The results of this study indicated a high prevalence of ESBL-Kp in the study area as well as heterogeneous multi-locus sequence types with multiple antibiotic resistance determinants. The diverse resistance genes, identification of novel STs, clonal and non-clonal genetic relationships underscores the pathogen's genetic adaptability. These findings emphasize the urgent need for enhanced infection control measures, stronger antibiotic stewardship programs, and the development of novel therapeutic strategies. Molecular genomic tools, such as WGS and MLST, offer critical insights into resistance mechanisms and strain evolution, providing a foundation for more targeted and effective interventions to checkmate the spread of ESBL-Kp.

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