ABSTRACT:
Background: Klebsiella pneumoniae is one of the leading causes of death a worldwide due to hypervirulent strains developing in hospitals. Also, antibiotic-resistant K. pneumoniae is progressively being involved in invasive infections with high morbidity and mortality. The current study aimed to determine antimicrobial susceptibility patterns, identify the resistance genes (genomic and plasmid) among clinical isolates of K. pneumoniae, and also to carry out the assay of beta-lactamase activity. Isolation of Genomic DNA and Plasmid DNA is thus carried out to know which gene is responsible for resistance. Molecular and Docking studies were carried out to find the presence of a mutation in the beta-lactamase gene and their docking sites with the standard inhibitors and antibiotics. Methods: Fifty clinical sputum samples of Respiratory tract infected patients were collected from different hospitals and Labs (Government Medical College, Indira Gandhi Medical College, Vishakha Lab etc) in Nagpur (Maharashtra). Out of 50, only 21 isolates were confirmed as Klebsiella pneumoniae by performing morphological and biochemical testing. An antibiotic Sensitivity test was performed. Microiodometric assay and Nitrocefin method were carried out to find out the beta-lactamase producing bacteria from clinical isolates. These isolates were also checked for different ßL genes by PCR using specific primers for ESBL genes and showed the presence SHV-1, TEM, and CTX-M genes in the results. These genes were sent for gene sequencing for confirmation, and mutational analysis was done. After sequencing and mutational analysis were done for these isolated ßL genes, Phylogenetic analysis was done to find out the closest standard protein. Automated molecular docking with all inhibitors to beta- lactamase protein was performed by using the advanced docking program Autodock 4.0. Results: The present study concluded that out of six, the five gene sequences i.e kp 13(g) CTX-M, kp 16(g) CTX-M, kp 14(p) CTX-M, kp 17(p) SHV, and kp 18(p) CTX-M contained a less number of mutations which do not show the actual change at their protein level, whereas kp 18 (g) TEM showed a maximum number of mutations and showed actual change at their protein level. The binding affinities of various inhibitors and antibiotics against TEM, SHV, and CTX-M enzymes were calculated. Durlobactam and Relebactam ligands demonstrated strong binding affinities with both wild-type and mutant TEM enzymes, indicating potent inhibitory activity Durlobactam and Ciprofloxacin exhibited the highest binding energies against the SHV enzyme, while Norfloxacin and Aztreonam showed the highest binding energies against the CTX-M enzyme. These combinations demonstrate potential as therapeutic strategies for treating resistant K. pneumoniae infections. Conclusions: Thus, this study concluded that the mutations occurring in the beta-lactamase genes may be due to variations in the environmental conditions or due to the frequent use of antibiotics makes them resistant to antibiotics. Docking helps us to know which part of the beta-lactamase protein is responsible for resistance to antibiotics. The findings of this study conclude that if we design (drug) the combination of high-binding energy Antibiotics and Inhibitors against beta-lactamase enzymes, can be useful to treat MDR K. pneumoniae.