CRIPSR-Cas9 Mediated WaaL Gene Excision of the Avian Pathogenic E. Coli (APEC) for its Pathogenicity Evaluation

Abstract

Avian Pathogenic Escherichia coli (APEC) is considered amongst the leading causes of colibacillosis in poultry flocks and it is responsible for nearly 18% mortality in poultry which leads to significant economic loss to the poultry industry every year. The study focuses on employment of a new antibacterial strategy, in order to combat colibacillosis caused by multi drug resistant APEC strain. This study aims on the effective gene knockout of the lipopolysaccharide (LPS) synthesis gene “waaL” via CRISPR/CAS9 system, in order to assess its effect on pathogenicity of APEC. Sequence of suitable gRNA of 24bps was designed in the functional part of the gene based on GC content, minimal off-target sites and PAM sequence. The gRNA was ligated in the pCRISPR and along with pCas9 both were successfully transformed in the APEC wild type strain. The mutant strains M1-O1 and M12 -O1 along with WT-O1 were sent for sequencing for confirmation of gene knockout followed by several in vitro pathogenicity evaluation tests. Under different physiological stress conditions, mutants M1-O1 and M12-O1 were found to be more sensitive to heat shock, alkali, acid and osmolarity shock as compared to WT-O1. Furthermore, WT-O1 have the potential to survive and grow in urinary bladder and might have zoonotic potential as well whereas on the other hand M1-O1 and M12-O1 showed decreased growth in human urine as compared to WT-O1 thus indicating waaL gene might be involved in pathogenicity. Antibiotic susceptibility testing (AST) was performed, where strains were evaluated against 22 most commonly used antibiotics and the results showed that M1-O1 and M12-O1 showed resistance against more antibiotics when compared with WT-O1. Biofilm quantification and motility assays were performed which showed reduction in biofilm forming ability of M1-O1 and M12-O1 and reduction in swimming and swarming motility of M1-O1 and M12-O1 in comparison with WT-O1 thus proposing the role of waaL gene in bacterial virulence and pathogenicity of APEC.