Bacteriophage and endolysin engineering for biocontrol of food pathogens/pathogens in the food: recent advances and future trends

Figures & data

Figure 1. Mechanisms of action of (A) bacteriophage and (B) endolysin. The figure was created with Biorender (http://biorender.com).

Table 1. Phage applications in the foods and biofilm.

Types of Food	Phage	Target pathogens	Main outcomes	Reference
Dairy products	PhageGuard Listex (Intralytix)	L. monocytogenes	Application of PhageGuard Listex, eradication of Listeria viable counts in surface-ripened red-smear soft cheese	Carlton et al. (2005)
	ListShield (Intralytix)	L.monocytogenes	Treatment of ListShield™, a commercially available phage cocktail Prevention of Listeria contamination by 0.7 log on cheese	Perera et al. (2015)
	JN01	E. coli O157:H7	Significant reduction of the viable E. coli O157:H7 in UHT milk at 4 °C	Li et al. (2021)
	vB_SauM_ME18, and vB_SauM_ME126	S. aureus	Elimination of MDR S. aureus artificially inoculated in UHT milk.	Gharieb et al. (2020)
Meats	SalmoFresh (Intralytix)	Salmonella spp.	Reduction of artificially inoculated Salmonella on Chicken breast fillets and inhibited re-growth of Salmonella up to 7 days at 4 °C	Sukumaran et al. (2016)
	PhageGuard S (PhageGuard)	Salmonella spp.	PhageGuard S, phage cocktail consisting of 2 lytic phages (S16 and FO1a) Significant reduction of Salmonella population (1 log) on experimentally contaminated ground beef trim.	Yeh et al. (2018)
	ShigaShield (Intralytix)	Shigella sonnei	Prevention of Shigella contamination on deli meat, smoked salmon, pre-cooked chicken, lettuce, melon, and yogurt.	Soffer et al. (2017)
Vegetables	SalmoFresh (Intralytix)	Salmonella spp.	Application of SalmoFresh immersion to Salmonella spp. on Romain lettuce and sprouts Reduction of Salmonella on lettuce and sprouts by 2–3 log CFU/g More effective control of Salmonella at 2 °C and 10 °C than at 25 °C	Zhang et al. (2019)
	Ecoshield (Intralytix)	E. coli O157:H7	Reduction of artificially contaminated E. coli O157:H7 in tomatoes, broccoli, spinach, and lettuce	Abuladze et al. (2008), Sharma et al. (2009)
	ListShield (Intralytix)	Listeria spp.	Reduction of Listeria population on melon and apple slices	Leverentz et al. (2004)
	PhageGuard (Intralytix)	Listeria spp.	Efficient biocontrol of artificially infected Listeria in melon and pear juice	Oliveira et al. (2014b)
Biofilms	Listex P100	L. monocytogenes	Removal of biofilms formed by 13 different serotypes of L. monocytogenes in 3.5–5.4 log/cm^2	Soni and Nannapaneni (2010)
	SalmoFresh, SalmoLyse (Intralytix)	Salmonella spp.	Protection of protected stainless and glass surface from Salmonella contamination by using SalmoFresh and SalmoLyse	Woolston et al. (2013)

Table 2. Published strategies for phage engineering.

Modification strategy	Phage	Target pathogens	Advantages	Reference
Homologous recombination	T2	E. coli O157:H7	Alteration of tail fiber protein gp38 of T2 phage to infect E. coli O157:H7 Site-specific recombination of T2 phage using IP008 long tail fiber genes for expanding host range	Yoichi et al. (2005),Mahichi et al. (2009)
	PaP1, JG004	Pseudomonas	Exchange each tail fiber gene to target phage for altering host specificity	Le et al. (2013)
	T3, T7	E. coli	Structure-based tail fiber engineering Construction of phagebody libraries to engineer host-range-determining regions Various phagebodies with broad host range and suppression of bacterial growth	Yehl et al. (2019)
	Y2	Erwinia amylovora	Insertion of depolymerase gene, dpoL1-C for efficient control	Born et al. (2017)
	T7	E. coli	Enhancing the biofilm-removal efficacy of T7 phage	Lu and Collins (2007)
	Che9c	Mycobacterium tuberculosis	Expression of RecE/RecT-like proteins to promote homologous recombination (Phage Recombineering of Electroporated DNA, BRED)	Marinelli et al. (2008)
	T7	E. coli	Using recombinase proteins derived from lambda phage to improve recombination efficiency	Jensen et al. (2020)
	SPN9CC	Salmonella	Conversion of phage life cycle by deletion of cI gene using a BRED method	Shin et al. (2014)
	T7	E. coli	First case of phage engineering using type I-E CRISPR-Cas system	Kiro, Shitrit, and Qimron (2014)
	T4	E. coli	Engineering T4 phage genome using type II or type V CRISPR-Cas system to enhance recombination Successive gene deletion and point mutation In vivo display of foreign peptides and proteins on capsid protein (Hoc and Soc)	Tao et al. (2017),Duong et al. (2020, 2021)
	T5	E. coli	Deletion of dmp gene by using CRISPR-Cas9	Ramirez-Chamorro et al. (2021)
	Phage 2972	S. thermophilus	First report of virulent phage engineering using type II-A CRISPR-Cas system (Cas9) to enhance recombination Successive gene insertion, deletion, and point mutation	Martel and Moineau (2014)
	Andhra, ISP	S. epidermidis	First report of phage engineering using type III CRISPR-Cas system (Cas10) to enhance recombination	Bari et al. (2017)

Table 2. (Continued)

Modification strategy	Phage	Target pathogens	Advantages	Reference
Genome assembly/ rebooting	P70, A511, B035, phage TP21, and phage K	L. monocytogenes, Bacillus cereus, and S. aureus	Synthesis of full-length phage genome by using enzymatic in vitro assembly (Gibson assembly) Employing Listeria L-form cells to transfect phage genome efficiently Rebooting genome of phage targeting Listeria, Bacillus, and Staphylococcus	Kilcher et al. (2018)
	T3	E. coli	Enhanced stability of two recombinant phages (T3p37 and T3p45) containing mutations within gene 37 and gene 45	Favor et al. (2020)
	phiX174	E. coli	The first study of yeast-based phage genome assembly Ligation of whole phage genome with linear yeast artificial chromosome (YAC) Reactivation of phage particles in E. coli cells	Jaschke et al. (2012)
	T3, T7, and K11	E. coli, Klebsiella, and Yersinia	Rebooting recombinant phages using a yeast-based platform Modulation of host range by exchange phage tail fiber	Ando et al. (2015)
	FelixO1	Salmonella	Synthesis of the larger phage genome through a yeast-based assembly	Timothy Kuan Ta Lu et al. (2011)
	KP32, KP34, KP36, and K11	Klebsiella	Random assemble of the domains of receptor binding proteins (RBPs) with depolymerase activity Switch of capsule serotype specificity	Latka et al. (2021)
Cell-Free genome synthesis	MS2	E. coli	The first application to produce MS2 phage coat protein virus-like particles (MS2 VLP) using TX-TL system	Bundy, Franciszkowicz, and Swartz (2008)
	T7, phiX174, and MS2	E. coli	Use the optimized TX-TL system to synthesize the E. coli phage T7, phiX174, and MS2 phage	Shin et al. (2012),Garamella et al. (2016),
	T4	E. coli	Synthesis of the larger phage genome by using TX-TL system	Rustad et al. (2018),
	AP205	Acinetobacter	de novo phage assembly using chemically synthesized oligonucleotides Low cost and low error rate compared to conventional engineering methods	Garenne et al. (2021)

Table 3. Endolysin applications in the foods and biofilm.

Types of Food	Endolysin	Target pathogens	Main outcomes	Reference
Dairy products	LysH5	S. aureus	8-log CFU/mL bacterial reduction in milk Wide host range of clinical staphylococcal strains, including S. aureus and S. epidermidis	Obeso et al. (2008)
			Synergistic sterilization effect with nisin against S. aureus in milk	García et al. (2010a)
	LysSA97	S. aureus	Synergistic antibacterial effect in whole milk and skimmed milk	Chang et al. (2017b)
	LysSA11	S. aureus	2-log CFU/mL bacterial reduction in milk	Chang, Kim, and Ryu (2017a)
	Ctl1L	C. sporogenes	Effective but weaker antibacterial activity in milk compared to the activity in broth condition	Mayer et al. (2010)
	LysZ5	L. monocytogenes	4-log CFU/mL bacterial inhibition in soya milk within 3 h at refrigerator temperature (4 °C)	Zhang et al. (2012)
	Ply511, Ply118	L. monocytogenes	Significant reduction of viable Listeria cells in whole cow milk Broad host spectrum of Listeria strains and high thermal resistance	Schmelcher, Waldherr, and Loessner (2012b)
	Ply100	L. monocytogenes	Stable in cheese for up to 4 weeks Synergistic anti-listerial effect with nisin in Queso Fresco cheese	Ibarra-Sánchez, Van Tassell, and Miller (2018)
Meats	LysSA11	Multidrug-resistant S. aureus	3-log CFU/mL bacterial reduction in ham artificially contaminated with MRSA within 15 min at refrigerator temperature (4 °C)	Chang, Kim, and Ryu (2017a)
	Trx-SA1	S. aureus	Reduction of somatic cells and S. aureus numbers after infusion of 20 mg of Trx-SA1 in udder quarters	Fan et al. (2016)
	LysRODI	S. aureus S. epidermidis	Protective efficacy against mammary infections in mice	Gutiérrez et al. (2020)
Vegetables	LysWL59, LysWL60	S. Typhimurium	Reduction of 93% of S. Typhimurium cells on lettuce in 1 h when treated with 2.5 μM of LysWL59 and 0.5 mM EDTA	Liu et al. (2019)
	Ply511, Ply118, Ply500	L. monocytogenes	Reduction of viable L. monocytogenes cells in a spiked iceberg (Ply511, Ply118) Broad lytic spectrum against Listeria (Ply511)	Schmelcher, Donovan, and Loessner (2012a)
Biofilms	LysH5	S. aureus	Notable staphylococcal biofilm removal activity against persister cells obtained after treatment with rifampicin and ciprofloxacin	Gutiérrez et al. (2014) Gutiérrez et al. (2017)
	LysCSA13	S. aureus	Reduction of staphylococcal biofilms mass up to 80–90% on various food utensil surfaces, including polystyrene, stainless steel, and glass	Cha et al. (2019)
	PlyC	S. pyogenes	Destruction of biofilm matrixes of S. pyogenes which showed rapid resistance to traditional antibiotics	Shen et al. (2013)
	Lys68	Salmonella	Synergistic biofilm-reducing effect in combination with malic or citric acid by 1-log CFU	Oliveira et al. (2014a)
	LysPA26	P. aeruginosa	2–3 log reduction of viable biofilms of P. aeruginosa 8327 on a polystyrene plate for 48 h	Guo et al. (2017)
	PlyLM	Listeria	Disrupting ability against Listeria biofilms, Synergistic effect with a protease	Simmons et al. (2012)

Table 4. Published strategies for endolysin engineering.

Modification strategy	Endolysin	Target pathogens	Key Advantages	Reference
Biocontrol of Gram-positive bacteria
Truncation or deletion of domains	PlyGBS90-1	Streptococcus	20-fold improved lytic activity compared to the full-length of PlyGBS	Cheng and Fischetti (2007)
	CHAPk	Staphylococcus	Truncation of amidase domain and CBD from LysK Better lytic activity against staphylococcal strains	Horgan et al. (2009)
Direct mutagenesis	L98WCD27L1-179	L. monocytogenes	Targeting conserved residues responsible for a hydrogen bond networking of amidase domain of CD27L Increased lytic activity against several L. monocytogenes serovars	Mayer et al. (2011)
	Cpl-7S	Pneumococcus	Substitution of 15 amino acids in the CBD Increased the net charge of CBD, inducing better lytic activity	Díez-Martínez et al. (2013)
	LysF1 Glu88Met & Glu88Leu variants	E. coli	Mutation non-conserved, hydrophobic core based on the understanding of mutable positions within the LysF1 fold Increased thermal stability	Love et al. (2021)
Domain swapping	CHAPSH3b	S. aureus	Fusion of CHAP domain of HydH5 and SH3b domain of lysostaphin Killing S. aureus to undetectable levels in pasteurized milk after 15 min Powerful anti-staphylococcal efficacy with a low concentration (1 µM)	Rodríguez-Rubio et al. (2012)
	Ply187AN-KSH3b	S. aureus	Domain shuffling of the Ply187 endopeptidase domain and SH3b domain of LysK Enhanced the lytic activity of Ply187 in milk	Mao et al. (2013)
	PlyGVE2CpCWB	Clostridium	Fusion of thermophilic phage ΦGVE2 and CBD of Clostridial endolysin Increased thermal stability up 50° C	Swift et al. (2015)
	ClyR	Streptococcus	Broader host spectrum, especially against Streptococci First establishment of lysis zone-based on-plate screening system for chimeric endolysins after domain shuffling	Yang et al. (2015)
	Lys109	S. aureus	Use of spanin for cell lysis during lysis zone-based screening step Improved killing ability against S. aureus in milk and stainless steel	Son et al. (2021)
	ClyC	S. aureus	Construction of chimeric endolysin library through shuffling domains from 12 natural staphylococcal endolysins Powerful antibacterial activity in buffer, milk, and animal blood	Lee, Kim, et al. (2021)

Table 4. (Continued)

Modification strategy	Endolysin	Target pathogens	Key Advantages	Reference
Biocontrol of Gram-negative bacteria
Fusion with OM-penetrating peptides	Art-175	A. baumannii P. aeruginosa, and E. coli	Fusion of a polycationic nonapeptide (SMAP-29) with an endolysin (KZ144) Bactericidal activity against various Gram-negative strains 8-log CFU/mL eradication of cells	Briers et al. (2014)
	N-terminus of T4 lysozyme - FyuA	Yersinia pestis	Fusion of binding domain of pesticin with the N-terminus of T4 lysozyme Advantage of targeting primarily disease-causing bacteria	Lukacik et al. (2012)
Fusion with OM-translocating protein or receptor-binding domain	Colicin-Lysep3	Multidrug-resistant E. coli	Fusion of the translocation and receptor binding domains of colicin A with an E. coli endolysin Lysis of E. coli in broad in vitro experiments Reduction of E. coli cells in an intestinal infection mouse model	Yan et al. (2017)
	PlyS2-GN4	Multidrug-resistant P. aeruginosa	Fusion of the P. aeruginosa bacteriocin pyocin S2 (PlyS2) with GN4 endolysin Log-fold anti-pseudomonal activity with minimal endotoxin release, disruption ability against biofilms, and protective efficacy in a bacteremia mice model	Heselpoth et al. (2019)
	Innolysin Ec21	E. coli	Fusion of phage T5 endolysin and receptor binding proteins (RBPs) PB5 in different configurations Potential bactericidal activity killing more than 3 logs reduction of E. coli cells which are resistant to third-generation cephalosporins	Zampara et al. (2020)
Encapsulation strategy	BSP16Lys	S. Typhimurium and E. coli	Encapsulation of BSPLys with cationic liposome Increased antibacterial activity up to 2.2 log CFU/mL reduction of S. Typhimurium cells and 1.6 logs of E. coli cells	Bai et al. (2019)
	HEW lysozyme T4 lysozyme	E. coli and P. mendocina	Conjugation of lysozyme with cellulose nanocrystals (CNC) to immobilize the enzyme Increased bactericidal activity against E. coli and P. mendocina compared to the free enzyme Enhanced storage stability at 4 °C and 22 °C	Abouhmad et al. (2017)
	Cpl-1	Streptococcus pneumoniae	Loading of Cpl-1 in chitosan nanoparticles Increased bioavailability of enzyme which can increase its in vivo half-life	Gondil et al. (2020)

OM: Outer Membrane

Figure 2. Future research needs to address the remaining concerns still seen in the application of phages and endolysins in the food industry. The figure was created with Biorender (http://biorender.com).

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