Determining the Efficacy of CRISPR-Cas9 Mechanisms on the uppP Gene of Klebsiella pneumonia using Lysozyme Assay

@#Introduction: Multidrug resistance bacteria is alarming worldwide. A lot of research were done and are ongoing to search for the best, convenient and economically affordable ways to fight them. With the latest genome editing tool; Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, this research was performed to develop a novel strategy to genetically modify the genome and inhibit the growth of Klebsiella pneumoniae (UPM ESBLKP1), an Extended Spectrum Beta Lactamases (ESBL) organism. Methods: A CRISPR-Cas9 vector was constructed together with guide RNAs designed specifically for the targeted uppP gene, a gene responsible for bacterial cell growth and protection. Results: The growth and cell wall integrity of the modified Klebsiella pneumoniae (ΔUPM ESBLKP1) were significantly inhibited and reduced, respectively. Interestingly, wild type Klebsiella pneumoniae showed a normal growth curve while modified strains showed a faster doubling rate when supplemented with Luria-Bertani media. In contrast, slower growth rate of modified strain was observed in the M9 minimal media. This explained the higher doubling rate of mutants on nutrient rich medium earlier is being related to gene recovery. They grew slowly in the minimal media as they were adapting to a new environment while recovering the uppP gene and surviving, proving the success of its gene modification. Conclusion: The developed CRISPR-gRNA system was able to modify the targeted Klebsiella pneumoniae gene hence providing an opportunity to develop a new drug for Klebsiella pneumoniae infection as an alternative to antibiotics.

In vitro treatment of lipopolysaccharide increases invasion of Pasteurella multocida serotype B:2 into bovine aortic endothelial cells

Pasteurella multocida serotype B:2 causes hemorrhagic septicemia in cattle and buffalo. The invasion mechanism of the bacterium when invading the bloodstream is unclear. This study aimed to characterize the effects of immunomodulatory molecules, namely dexamethasone and lipopolysaccharide, on the invasion efficiency of P. multocida serotype B:2 toward bovine aortic endothelial cells (BAECs) and the involvement of actin microfilaments in the invasion mechanism. The results imply that treatment of BAECs with lipopolysaccharide at 100 ng/mL for 24 h significantly increases the intracellular bacteria number per cell (p < 0.01) compared with those in untreated and dexamethasone-treated cells. The lipopolysaccharide-treated cells showed a significant decrease in F-actin expression and an increase in G-actin expression (p < 0.001), indicating actin depolymerization of BAECs. However, no significant differences were detected in the invasion efficiency and actin filament reorganization between the dexamethasone-treated and untreated cells. Transmission electron microscopy showed that P. multocida B:2 resided in a vacuolar compartment of dexamethasone-treated and untreated cells, whereas the bacteria resided in cellular membrane of lipopolysaccharide-treated cells. The results suggest that lipopolysaccharide destabilizes the actin filaments of BAECs, which could facilitate the invasion of P. multocida B:2 into BAECs.

Occurrence of Escherichia coliand Escherichia ColiO157:H7 in cattle, farm environment, milk and beef

@#Aims:The aim of this study was to determine the occurrence of Escherichia coliand E. coliO157:H7 in the cattle, farm environment beef andmilk samples.Methodology and results:A total of 192 samples were collected from cattle (rectal faeces) (96) and their farm environment (96) including feed, floorof stall swabs, pooled flies and water samples and 30 milk samples from dairy cattle. A total of seven markets were selected to collect 60 beef samples. The presence of E. coli and E. coli O157:H7 was determined using culture method and confirmed using PCR assay. The overall occurrence rate of E. coli in the farm was 49%. In milk, the occurrence of E. coliwas 33.3% and in beef was 10%. The isolated E. coli were then screened for E. coli O157 using latex agglutination test (Oxoid) and 34.2% were found positive to the test. The identified E. coliO157 isolates were then subjected to multiplex PCR with five primer pairs. They were all negative except for 3.6% of the E. coliO157 (10.5%)isolates which were positive for the presence of fliCH7 genes. Conclusion, significance and impact of study: This study showed the absence of E. coli O157:H7 in the cattle, farm environment, milk and beef. This study may provide a base for conducting a broader spectrum study by having more risk factors included in the study of E. coliin cattle in Malaysia. Further detailed studies may provide a platform to control emergence and distribution of pathogenic bacteria and ensure good control and surveillance strategies and policies.