Peptidoglycan synthesis gene disruption using CRISPR/Cas9 on Extended Spectrum Beta Lactamase Klebsiella pneumoniae strain UPM ESBLKP1

The resistance ability of several bacteria towards drugs and antibiotics has been alarming worldwide. These bacteria are known as multiple drug resistance (MDR) organisms. A group of bacteria-harbouring beta-lactamase has become increasingly resistant to the most used antibiotics. Extended Spectrum Beta Lactamase (ESBLs) producing Enterobacteriaceae is an emerging public health concern where ESBL Klebsiella pneumoniae is a significant problem in major hospitals. In Europe and the United States, K. pneumoniae caused 8% of total nosocomial infections in hospitals, ranking them second after Escherichia coli. They also caused up to 17% of urinary tract infections in both countries. Research is done to find the best, most convenient, and most economically affordable ways to fight them. This research investigated the efficacy of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology as a novel strategy to genetically modify the genome and inhibit the growth of UPM ESBLKP1 K. pneumoniae. A CRISPR-Cas9 vector was constructed with the designed guide RNA (gRNA) targeted specifically to the uppP gene, a gene responsible for cell wall peptidoglycan synthesis, which is responsible for bacterial cell growth and protection. Three mutants, uppP1, uppP2, and uppP3, were constructed using high voltage transformation of pCasSA+CmR plasmid that carries both gRNA and Cas9 coding gene. The negative control and mutant strains were observed analysed using rich and limiting media for growth performance and lysozyme assay for cell wall integrity. Interestingly, the negative control, contrlKlebsiella pneumoniae, showed a standard growth curve, while the mutant strains showed a faster doubling rate. Meanwhile, the cell wall integrity of the modified K. pneumoniae was significantly reduced with mean value at alpha = 0.05 with 0.5130, 0.5160, 0.3700, and 0.2290 when compared to the negative control with Mutant uppP1, Mutant uppP2, and Mutant uppP3 analysed with lysozyme assay respectively. Cell wall appearance was observed using Transfer Electron Microscopy (TEM). Images show positive disruption of mutants’ strain cell walls. The cell wall of Mutant uppP1 appeared to be burst, and the Mutant uppP2 cell wall, on the other hand, appeared to have been snapped and clumped together; meanwhile, the Mutant cell wall of the Mutant uppP3 cell walls cannot be seen and identified. This indicates a successful modification of the uppP gene of all three mutants. The pathogenicity study shows that cells treated with Mutant uppP3 have a higher percentage of cell viability followed by both Mutant upp2 and Mutant uppP1 compared to wild type for all three-time frames (24h, 48h, and 72h). The developed CRISPR-Cas9 system has successfully modified the targeted K. pneumoniae uppP gene hence providing an opportunity to create an alternative treatment for K. pneumoniae infection.

Text 122728.pdf Download (1MB) Official URL or Download Paper: http://ethesis.upm.edu.my/id/eprint/18641

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