Characterization of Lytic Bacteriophages Infecting Multidrug-Resistant Shiga Toxigenic Atypical Escherichia coli O177 Strains Isolated From Cattle Feces

Abstract

The increasing incidence of antibiotic resistance and emergence of virulent bacterial pathogens, coupled with a lack of new effective antibiotics, has reignited interest in the use of lytic bacteriophage therapy. The aim of this study was to characterize lytic Escherichia coli O177-specific bacteriophages isolated from cattle feces to determine their potential application as biocontrol agents. A total of 31 lytic E. coli O177-specific bacteriophages were isolated. A large proportion (71%) of these phage isolates produced large plaques while 29% produced small plaques on 0.3% soft agar. Based on different plaque morphologies and clarity and size of plaques, eight phages were selected for further analyses. Spot test and efficiency of plating (EOP) analyses were performed to determine the host range for selected phages. Phage morphotype and growth were analyzed using transmission electron microscopy and the one-step growth curve method. Phages were also assessed for thermal and pH stability. The spot test revealed that all selected phages were capable of infecting different environmental E. coli strains. However, none of the phages infected American Type Culture Collection (ATCC) and environmental Salmonella strains. Furthermore, EOP analysis (range: 0.1-1.0) showed that phages were capable of infecting a wide range of E. coli isolates. Selected phage isolates had a similar morphotype (an icosahedral head and a contractile tail) and were classified under the order Caudovirales, Myoviridae family. The icosahedral heads ranged from 81.2 to 110.77 nm, while the contractile tails ranged from 115.55 to 132.57 nm in size. The phages were found to be still active after 60 min of incubation at 37 and 40 • C. Incremental levels of pH induced a quadratic response on stability of all phages. The pH optima for all eight phages ranged between 7.6 and 8.0, while at pH 3.0 all phages were inactive. Phage latent period ranged between 15 and 25 min while burst size ranged from 91 to 522 virion particles [plaque-forming unit (PFU)] per infected cell. These results demonstrate that lytic E. coli O177-specific bacteriophages isolated from cattle feces are highly stable and have the capacity to infect different E. coli strains, traits that make them potential biocontrol agents.

Analysis of selection methods to develop novel phage therapy cocktails against antimicrobial resistant clinical isolates of bacteria

“…These three tests form the basis for the initial screening of a phage library to identify phages with the broadest host range. The data generated also allowed direct comparison of DST versus EOP, as these two assays are commonly used to characterise phages (Montso et al, 2019; Rivera et al, 2019; Sybesma et al, 2016) [Supplementary Data Table S5 & S6].…”

“…DST is commonly used in the literature to assess the host range of phage (Hyman, 2019; Montso et al, 2019; Sybesma et al, 2016). The data demonstrated that the DST overestimates the host range or clinical isolate coverage of the individual phage by approximately 50% compared with the EOP and the planktonic killing assay (Figure 2).…”

An Overview of the Elusive Passenger in the Gastrointestinal Tract of Cattle: The Shiga Toxin Producing Escherichia coli

“…STEC control using bacteriophages against STEC contaminated surfaces in cattle farms (e.g., cow hides) and meat products has demonstrated a significant decrease in STEC [ 114 , 139 ]. However, even though in vitro assays have shown promising results [ 137 , 138 , 139 ], in vivo trials have not always been successful. Administration of bacteriophages can result in STEC reduction, although in some studies, the reduction between treatment groups was not significant and in some other studies, there was no reduction at all [ 114 ].…”

“…T4-like Myoviridae phages have been found in higher titers in low shedding animals, suggesting that their presence is linked to reduced O157:H7 titers [ 114 ]. Recent studies have further suggested that cattle intestinal endemic phages target specific STEC strains: Myoviridae and Siphoviridae phages target various non-O157 groups [ 137 ]; Myoviridae phages (isolated from cattle feces) target O177, O157, O26 STEC serogroups [ 138 ]; a T4-like phage of the Myoviridae family isolated from the cattle intestine (not from feces) targeted O157:H7 and non-O157 serotypes but not Salmonella strains [ 139 ]. Ciliate protozoa have been reported to predate on STEC, but predation does not appear to be STEC specific [ 47 ].…”

An exegesis of bacteriophage therapy: An emerging player in the fight against anti-microbial resistance

“…These represent the first set of indigenous bacteriophages to be isolated from tropical Africa. Another study from South Africa [125] reported the characterization of lytic bacteriophages active against multi-drug resistant Escherichia coli 0177 strains isolated from cattle fecal matter. While a study reported the effective use of phage therapy against multi-drug resistant Staphylococcus aureus pneumonia in mice models [126] , no study reporting the successful use of bacteriophage therapy on humans in a clinical setting in Africa was found.…”