Searching for a Bacteriophage Lysin to Treat Corynebacterium bovis in Immunocompromised Mice

Abstract

Corynebacterium bovis is the causative agent of Corynebacterium-associated hyperkeratosis in immunocompromised mice. The resulting skin pathology can be profound and can be associated with severe wasting, making the animals unsuitable for research. Although the administration of antibiotics is effective in resolving clinical symptoms, antibiotics do not eradicate the offending bacterium. Furthermore, antibiotic use may be contraindicated as it can affect tumor growth and is associated with Clostridioides difficile enterotoxemia in highly immunocompromised murine strains. Lysins, which are lytic enzymes obtained from bacteriophages, are novel antimicrobial agents for treating bacterial diseases. The advantage of lysins are its target specificity, with minimal off-target complications that could affect the host or the biology of the engrafted tumor. The aim of this study was to identify lysins active against C. bovis. Chemical activation of latent prophages by using mitomycin C in 3 C. bovis isolates did not cause bacteriophage induction as determined through plaque assays and transmission electron microscopy. As an alternative approach, 8 lysins associated with other bacterial species, including those from the closely related species C. falsenii, were tested for their lytic action against C. bovis but were unsuccessful. These findings were congruentwith the previously reported genomic analysis of 21 C. bovis isolates, which failed to reveal bacteriophage sequences by usingthe PHAST and PHASTER web server tools. From these results, we suggest C. bovis is among those rare bacterial species devoid of lysogenic bacteriophages, thus making the identification of C. bovis-specific lysins more challenging. However, C. bovis may be a useful model organism for studying the effects of antiphage systems.

Establishing the Median Infectious Dose (ID50) and Characterizing the Clinical Manifestations of Mouse, Rat, Cow, and HumanCorynebacterium bovisIsolates in Select Immunocompromised Mouse Strains

“…All Cb isolates grew slowly, which was consistent with our previous findings for this species and is the reason why cultures should be held for 5 to 7 days before ascertaining that the organism is absent in samples. 10 The majority of isolates tended to cluster together, with most mice and human isolates having similar mid-log growth times (13 to 20 and 30 to 35 hours, respectively). Although collectively the isolates growth was slow, isolates grew at different rates with only the mouse isolates, CUAMC1 ,13-1426, and 17-0240, and the rat isolate 16-2004 having similar mid-log growth times of 13 and 20 h, respectively.…”

“…5,10 To determine the growth kinetics of each Cb isolate, 3 growth curves were established for each isolate, except isolate 7894 because its' growth curve was previously established. 10 Frozen stocks (Microbank TM, Pro-lab Diagnostics, Rounds Rock, TX) of each Cb isolate were grown as described above. Three colonies of each isolate were transferred to a 15-mL conical tube (Corning™ Falcon™, Fischer Scientific, Hampton, NH) containing 1.5 ml of brain-heart infusion broth ([BHI]; Becton Dickinson, Franklin Lakes, NJ) supplemented with 0.1% Tween 80 (VWR Chemicals, Solon, OH) and were incubated at 37°C with 5% CO 2, lying flat on a titer plate shaker (4625 Titer Shaker, Thermo Scientific, Waltham, MA) at 200 RPM.…”

“…Cb isolates from skin and buccal swabs or frozen stocks were grown on trypticase soy agar supplemented with 5% sheep blood (BBL™ TSA II 5% SB, Becton, Dickinson and Company, Sparks, MD, USA) at 37°C with 5% CO 2 for 48 hours as previously described. 5,10 To determine the growth kinetics of each Cb isolate, 3 growth curves were established for each isolate, except isolate 7894 because its’ growth curve was previously established. 10 Frozen stocks (Microbank TM, Pro-lab Diagnostics, Rounds Rock, TX) of each Cb isolate were grown as described above.…”

“…Time to reach mid-log growth for 9 Corynebacterium bovis isolates from various species 10. Experimental design: Eight groups of 6 nude mice were inoculated with increasing doses of Cb.…”

Phage Therapy as a Focused Management Strategy in Aquaculture

“…These proteins with enzymatic activity have also been used to destroy preformed biofilms by Gram-positive or Gram-negative bacteria, although most studies have been carried out in vitro [ 24 , 25 , 26 ]. Moreover, while some naturally occurring lysins have potent in vitro activity, they can be engineered to be more effective in binding or traversing the bacterial wall [ 27 , 28 , 29 , 30 , 31 , 32 ]. Although not well known as lysins, phage enzymes with depolymerase activity—or depolymerases—can be of great interest for fighting pathogens and the biofilms that they form since a critical component of the biofilm matrix is usually polymers of a different nature [ 33 , 34 , 35 ].…”