Development of a specific fluorescent phage endolysin for in situ detection of Clostridium species associated with cheese spoilage

Abstract

SummaryLate blowing defect (LBD) is a major cause of spoilage in cheeses, caused by the growth of Clostridium spp. in the cheese matrix. We investigated the application of CTP1L, a bacteriophage endolysin active against Clostridium tyrobutyricum, and its enzymatically active and cell wall‐binding domains (EAD and CBD) attached to green fluorescent protein (GFP) to detect dairy‐related Clostridium species by fluorescence microscopy. GFP‐CTP1L and GFP‐CBD demonstrated specificity for Clostridium spp. by labelling 15 and 17 of 20 Clostridium strains, respectively, but neither bound to other members of the cheese microbiota. However, GFP‐EAD did not label any Clostridium strain tested. Unexpectedly, GFP‐CTP1L and GFP‐CBD were also able to bind to clostridial spores. In addition, GFP‐CBD allowed us to visualize the vegetative cells of C. tyrobutyricum directly in the matrix of a LBD cheese. Site‐directed mutants of GFP‐CTP1L and GFP‐CBD were made to examine the amino acids involved in binding and oligomer formation. Oligomerization was not essential for binding, but specific mutations in the CBD which affected oligomer formation also affected binding and lytic activity. We conclude that GFP‐CTP1L and GFP‐CBD could be good biomarkers for rapid detection of Clostridium spores in milk, so measures can be taken for the prevention of LBD in cheese, and also provide effective tools to study the development of Clostridium populations during cheese ripening.

Engineered Bacteriophages for Practical Applications

“…As mentioned for tuberculosis, even though there are PCR-based assays faster than the standard method, they cannot differentiate among live and dead cells, which is critical for relevant results, and it is solved with this assay. 43) Similar techniques have been also reported for the detection of Salmonella, 44,45) E. coli, 46) and Clostridium 47,48) in different types of samples.…”

Molecular Basis of Bacterial Host Interactions by Gram-Positive Targeting Bacteriophages

“…The natural ability of phages to infect (and kill) a certain bacterial host range has led to their exploitation over the past decades for the development of diagnostic tools and antibacterials for use in medicine [ 25 , 26 ], food production [ 27 , 28 , 29 , 30 , 31 ], and biotechnology [ 32 , 33 , 34 ]. For example, phages can be used to treat Campylobacter [ 35 ] or Salmonella [ 27 ] infections of chicken flocks, or added to feed to kill Clostridium and intestinal coliforms in pigs [ 36 ]; added to ready-to-eat foods to prevent Salmonella [ 37 ] or Listeria [ 38 ] contaminations; or mixed with various food products to act as bio-preservatives [ 39 , 40 , 41 ].…”

LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain

“…Alternatively, the localization of LysPBC2 on the developing spores may be evolutionarily beneficial to both phage and host if the endolysin promotes spore germination under favorable environmental conditions. Recently, one study revealed that a Clostridium tyrobutyricum phage endolysin, CTP1L, can bind to clostridial spores (40). Although they did not find an SBD within CTP1L, it is intriguing to identify additional SBDs from phages infecting spore-forming bacteria.…”