Characterization of Bacteriophages Infecting Clinical Isolates of Clostridium difficile

Abstract

Clostridium difficile is recognized as a problematic pathogen, causing severe enteric diseases including antibiotic-associated diarrhea and pseudomembranous colitis. The emergence of antibiotic resistant C. difficile has driven a search for alternative anti-infection modalities. A promising strategy for controlling bacterial infection includes the use of bacteriophages and their gene products. Currently, knowledge of phages active against C. difficile is still relatively limited by the fact that the isolation of phages for this organism is a technically demanding method since bacterial host themselves are difficult to culture. To isolate and characterize phages specific to C. difficile, a genotoxic agent, mitomycin C, was used to induce temperate phages from 12 clinical isolates of C. difficile. Five temperate phages consisting of ΦHR24, ΦHN10, ΦHN16-1, ΦHN16-2, and ΦHN50 were successfully induced and isolated. Spotting assays were performed against a panel of 92 C. difficile isolates to screen for susceptible bacterial hosts. The results revealed that all the C. difficile phages obtained in this work displayed a relatively narrow host range of 0–6.5% of the tested isolates. Electron microscopic characterization revealed that all isolated phages contained an icosahedral head connected to a long contractile tail, suggesting that they belonged to the Myoviridae family. Restriction enzyme analysis indicated that these phages possess unique double-stranded DNA genome. Further electron microscopic characterization revealed that the ΦHN10 absorbed to the bacterial surface via attachment to cell wall, potentially interacting with S-layer protein. Bacteriophages isolated from this study could lead to development of novel therapeutic agents and detection strategies for C. difficile.

Isolation and Characterization of the Novel Phage JD032 and Global Transcriptomic Response during JD032 Infection of Clostridioides difficile Ribotype 078

“…1D) and decreased by only 0.57 and 0.24 log units at pH 3.0 and pH 4.0, respectively, compared with pH 7.5. Overall, phage JD032 virions were tolerant to wide pH ranges and high temperatures, which is similar to other C. difficile phages, such as HN10, HN16-1, HN16-2, and HN50 (25).…”

“…Biological characteristics and bacteriolytic activity in vitro. The thermostability of phage JD032 was tested using 10 8 PFU/ml purified phage lysate that was subjected to different temperatures (4,25,37,45,50,55,60, and 65°C) for 1 h. To test pH stability, the purified phage lysate was incubated at different pH ranges from pH 2.0 to pH 11.0 for 1-h intervals. After incubation in a temperature-controlled water bath or pH-controlled environment, the phage activity was determined by a double-layer plating experiment.…”

Phages in Anaerobic Systems

“…Attempts have also been made to isolate phages for control purposes, but the isolates obtained are of phages with lysogenic lifecycles ( Figure 1 ). These isolates were obtained from environmental and clinical samples, as well as by induction with mitomycin C or UV light [ 22 , 23 , 24 ]. Until 2018, 24 genomes of C. difficile phages were found in public databases, most of them belonging to the family Myoviridae [ 25 ].…”

The Novel Enterococcus Phage vB_EfaS_HEf13 Has Broad Lytic Activity Against Clinical Isolates of Enterococcus faecalis

“…Since little is known about the pH stability of E. faecalis phages, we compared the pH stability of phage HEf13 with those of other Gram-positive bacteria phages. Phage HEf13 titers were not affected by incubation at pH 11 and remained at around 30% at pH 12 ( Figure 4B), whereas reported titers of other phages were almost completely abolished at pH 11 (Li and Zhang, 2014;Chen et al, 2016;Phothichaisri et al, 2018). These comparisons demonstrated that phage HEf13 is relatively more stable to high temperatures and strongly basic conditions than other phages.…”