Identification and molecular characterization of plasmid-mediated verotoxin gene in non-o157 escherichia coli / Md. Fazlul Karim Khan

Faculty of Industrial Sciences and Technology

Thesis (Doctor of Philosophy) -- Universiti Malaysia Pahang – 2021

Includes bibliographical references

Diarrheagenic non-O157 verotoxin-producing Escherichia coli (VTEC) associates with endemic infantile diarrhoea, causing morbidity and mortality across the world. Thus, the existence and prevalence of non-O157 VTEC have emerged as a particular public health concern. This study investigated the biological activities of the plasmid-mediated Vtx gene in non-O157 VTEC isolates. Besides, the extracted Vtx gene sequence information was used in tertiary structure modelling followed by thermostability analysis as a computational approach of potential vaccine candidates. Standard microbiological techniques identified non-repeated, clinically significant E. coli isolates. Serological assays discerned non-O157 E. coli serogroups while all these isolates were subjected to a polymerase chain reaction for investigating virulent genes, Vtx1 and Vtx2. Subsequently, the Close and Rodriguez method and Perfectprep plasmid mini preparation kit were used to detect plasmid harbored isolates. These plasmid harbored isolates were further investigated using disk diffusion assay, phage induction, and plasmid curing followed by polymerase chain reaction. Meanwhile, the sequence of the Vtx gene was analyzed using several bioinformatics tools, including BioEdit, NCBI-BLAST, ClustalW-X, and MEGA 5.0, etc. Furthermore, most potential Vtx gene sequences were modelled using MODELLER and refined via molecular dynamics simulations using GROMACS. Finally, the Vtx gene as a potential vaccine candidate was evaluated by incorporating scales of beta turns, hydropathicity, surface accessibility, and antigenicity in MATLAB. In this cross-sectional study, 137 clinical isolates of E. coli were collected from Hospital Tengku Ampuan Afzan, Kuantan, Malaysia. Different PCR sets characterized 49 clinical isolates of sorbitol positive non-O157 E. coli while twenty-nine isolates harbored Vtx genes (Vtx1 and Vtx2). Among 29 (59.18 %) strains of Vtx producing non-O157 E. coli¸ Vtx1 in 21 isolates (42.85 %), Vtx2 in 5 isolates (10.20 %) and both Vtx1 and Vtx2 was observed in 3 (6.12 %) isolates. Besides that, 26 multidrugresistant isolates possessed single-sized and multiple plasmids with different molecular weight varied from 1.8 kb to 7.6 kb. Furthermore, eight isolates changed their susceptibility patterns, while three isolates were found to lose plasmid after post plasmid curing treatment, and the rest of the 15 isolates remained unchanged. Finally, different PCR sets characterized 3 plasmid-mediated Vtx gene producing non-O157 E. coli while the absence of lysogenic phages indicated that Vtx genes were not phage mediated. On the other hand, these three plasmids mediated Vtx gene in non-O157 VTEC isolates were used as a template DNA for sequences of interest. The sequencing results confirmed the occurrence of Vtx genes and demonstrated higher similarity in nucleotides level. Moreover, Verify 3D, ERRAT, and PROCHECK showed that the generated Vtx1-1 protein model has a high stereochemical quality. In addition, structural analysis of the thermal profile demonstrated the stability of Vtx1-1 models structure at 40 °C. As the temperature rises, the stability of the Vtx1-1 protein model was disrupted due to flexible secondary loops as well as the presence of non-charged and hydrophilic amino acid residues. However, DSGTGDNLF (47-55) and MIDSGTGDN (45-53), two 9-mer peptides with a score of 1.242 and 1.229 respectively, could be the most effective B-cell epitopes and a possible vaccine candidate against VTEC infections. The information obtained from this study is expected to improve the molecular synthesis applications in the future.