Molecular characterisation of Escherichia coli O157:H7 specific bacteriophages from cattle faeces
Akindolire, Muyiwa Ajoke
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Escherichia coli (E. coli) O157:H7 is the main cause of food-borne diseases including haemorrhagic colitis, which can progress to a complication referred to as haemolytic uremic syndrome (HUS) especially in children. The bacteria colonize the gastrointestinal tract of ruminants, particularly cattle, and contamination of carcasses and meat can arise at abattoirs if hygienic procedures are compromised and the bacteria in the intestine or cattle hide is transferred to sterile meat surfaces. The current methods of meat decontamination have various problems and limitations, especially regarding the organoleptic impact and the environmental effect of these methods. A novel, safe and environmentally friendly approach to mitigate E. coli O157:H7 involves the use of bacteriophages. The success of such strategy requires an in-depth understanding of the bacteriophages that infect E. coli O157:H7. This thesis therefore describes the isolation and detailed characterisation of E. coli O157:H7 bacteriophages as a basis to investigate their biocontrol potential. Prior to the isolation and characterisation of virulent phages, the host bacteria − E. coli O157:H7 was isolated from cattle faecal samples obtained from some commercial farms in the North-West province. The isolates were characterised by determinining their virulence profiles and genetic relationships using pulsed-field gel electrophoresis (PFGE). The characterised bacteria were used as host strains for the isolation of phages and phages were further characterised by lytic profile determination, TEM, PFGE analysis, growth characteristics, stability under different physico-chemical conditions and finally by genome sequencing and analysis. A total of 69 virulent E. coli O157:H7 isolates that was obtained in the study were genetically similar by PFGE grouping even though they originated from different farms. Using the characterised E. coli O157:H7 as host strains, 15 virulent phages were isolated from cattle faeces that were obtained from different farms. The spot test assay facilitated the identification and selection of three lytic bacteriophages designated V3, V7 and V8 as potential candidates for the control of E. coli O157:H7. Further characterisation indicated that the three phages displayed a high degree of similar characteristics despite isolation from different farms. Transmission Electron microscopy (TEM) of the phages revealed that they all had isometric heads of about 73 − 77 nm in diameter and short tails of about 20-25 nm in diameter. Accordingly, phages V3, V7 & V8 were assigned to the family Podoviridae based on their morphology. Pulsed-field gel electrophoresis (PFGE) genome estimation of the 3 phages demonstrated identical genome sizes of − 69 nm. The latent periods and the burst sizes of these phages were 20 min, 15 min, and 20 min for V3, V7 and V8 respectively while the burst sizes were 374, 349 and 419 PFU/ infected cell respectively. While all the phages were relatively stable over a wide range of salinity, temperatures and pH values, their range of infectivity was rather narrow on environmental E. coli O157:H7 strains isolated from cattle feaces. However, a novel Escherichia coli phage G17 (originally designated V3) that showed promising features was submitted for genomic sequencing. The obtained DNA sequence of phage G17 has allowed for genomic characterisation with bio-informatics analysis identifying the functions of a number of coding sequences. These molecular tools revealed high homology to the newly assigned G7cvirus genus making the novel phage G17 a possible member of the genus. Moreover, the genome did not show any homology to genes encoding virulence such as Shiga toxin genes, antibiotic resistance genes and other genes that are involved in lysogeny. Thus, phage phi-G17 possessed favourable characteristics and could be utilized as an alternative in the control of E. coli O157:H7.