Isolation and Characterization of Bacteriophages Targeting Uropathogenic E. coli

Magwai, Thandi

Isolation and Characterization of Bacteriophages Targeting Uropathogenic E. coli

Master Thesis

2019

Abstract

Urinary tract infections (UTI)are one of the most common human bacterial infections, caused by uropathogenic E. coli (UPEC). Conventionally, UTI’s are treated with antibiotics, such as sulpha-mathoxazole/trimethoprim and ampicillin. However, a significant proportion of UPEC strains have become resistant to these antibiotics, resulting in a significant burden on the health care system worldwide. Bacteriophages (phages) that target E. coli strains could provide an alternative treatment for UTIs, particularly those resistant to antibiotics. This study aimed to screen a variety of environmental samples for phages that target a UPEC strains and characterize these phenotypically and genotypically, towards the development of a candidate phage preparation for treatment for antibiotic-resistant UPEC. Environmental samples (including faeces cattle, sheep, horse, goat faeces, river water, raw cow milk and mud) were collected from various sources around the City of Cape Town and screened for phages using an E. coli laboratory strain; K-12 MG1655. Lytic phages against E. coli MG1655 were purified and host-range testing was conducted against a panel of 11 UPEC strains form the E. coli reference collection (ECOR). These lytic phages were characterized phenotypically using transmission electron microscopy (TEM) using negative staining and genotypically using restriction enzyme HaeIII. In total, 41 phages that were lytic against MG1655 were isolated from six different environmental samples. Of these, 31/41 (76%) were lytic against 7/11 (64%) UPEC strains tested. Four phages (16-3, 16-4, 16-5 and 16-7; all isolated from raw milk) had the broadest host range of all the phages screened, being lyticagainst4/11(36%) UPEC strains. UPEC strain ECOR-40 was the most susceptible of the E. coli strains tested, being susceptible to 18/31 (58%) of the lytic phages isolated. In contrast, four UPEC strains (including ECOR-14, -60, -62 and -64) were not susceptible to any of the phages isolated. Ninety percent (28/31) of the isolated phages were structurally similar to four known phage families; including Myoviridae, Siphoviridae, Inoviridae and Rudiviridae. The remaining 10% (3/31) had structures that did not resemble any ofthe known phage families. DNA was x isolated from all 31 phage isolates to screen for similarities in restriction patterns of isolates. Of these, RFLP banding was clear for 5/31 samples; which showed that 5/5 phages were unique based on their banding patterns. In conclusion, this study demonstrated the existence of several unique E. coli phages in nature and their ability to target several of the UPEC strains known to cause UTI. Although time did not allow for sequencing of the full genomes of those isolates with unique characteristics in this study, this should be a priority for this research going forward.

Keywords

Medicine

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