Escherichia coli O157:H7 has been global concern that caused serious diseases such as hemolytic uremic syndrome (HUS) and bloody diarrhea. Chronic environmental resistance of E. coli O157:H7 caused by biofilm formation has also been considered major cause of food poisoning. Due to the limitation and...
Escherichia coli O157:H7 has been global concern that caused serious diseases such as hemolytic uremic syndrome (HUS) and bloody diarrhea. Chronic environmental resistance of E. coli O157:H7 caused by biofilm formation has also been considered major cause of food poisoning. Due to the limitation and low efficacy of classical sterilization methods, bacteriophages (phage) have been proposed as newly attractive strategies to control the foodborne.
To develop a novel biocontrol agent against E. coli O157:H7, 25 new E. coli infecting phages were isolated and two phages of ECP26 and BECP10 were selected based on the host range and lysis properties. Unlike E. coli infecting phages, ECP26 and BECP10 have very unique host spectrum and lytic activity agianst pathogenic E. coli. Especially, BECP10 was able to infect E. coli O157 strains only and it did not infect other tested strains. Based on further morphological analysis, ECP26 and BECP10 were classified as Myoviridae and Siphoviridae respectively. The genome of ECP26 and BECP10 did not contain lysogeny-related modules and toxin-associated genes, suggesting that both phages were strictly lytic and then suitable for biocontrol. Interestingly, tailspike protein (TSP) sequence of BECP10 has very low homology with the T1-like phages, implying that TSP is related to the unique host spectrum of BECP10. Periodate-based adsorption assay result suggested that lipopolysaccharide (LPS) of E. coli O157:H7 may be the host receptor of BECP10, and it was verified by Tn5 random mutant library. Both phages remained viability in wide range of temperature (25°C–55°C) and pH (3–10). Bacterial challenge assays revealed that BECP10 exhibited more strong antibacterial activity than ECP26 against planktonic E. coli O157:H7 and could minimize the development of bacteriophage insensitive mutants (BIMs). In food application studies, both phages sustained the viability and were able to reduce the E. coli O157:H7 to non-detectable level in romaine and burger patty. Therefore, ECP26 and BECP10 would be useful agents for the control of E. coli O157:H7.
Endolysin, a phage-derived lysis protein, is able to lyse the target bacteria without any special resistance, and thus has been suggested as a powerful antimicrobial agent. Therefore, rV5-like phage endolysin targeting E. coli O157:H7, named as LysECP26, was identified and purified. This endolysin had a lysozyme-like catalytic domain, but differed markedly from the sequence of lambda phage endolysin. Purified LysECP26 exhibited strong activity with a broad lytic spectrum against various gram-negative strains (29/29) and was relatively stable at a broad temperature range (4°C–55°C). The optimum temperature and pH ranges of LysECP26 were identified at 37°C–42°C and pH 7–8, respectively. NaCl supplementation did not affect the lytic activity. Although action of LysECP26 was limited in that it could not pass the outer membrane, E. coli O157:H7 could be effectively controlled by adding EDTA and citric acid. However, practical food application will have limitations because it is difficult to implement buffer conditions. In order to enhance the lytic activity, synergistic effects of LysECP26 and essential oils were investigated. As a result, more strong synergistic effects with eugenol and thymol against E. coli O157:H7 were confirmed. Therefore, LysECP26 might serve as an effective biocontrol agent for E. coli O157:H7 and gram-negative pathogens.
The process of recognizing and attaching the surface of host bacteria is one of the very important steps in phage infection. Phage depolymerase, derived from the TSP, is not only functions as receptor binding protein (RBP) but also can specifically degrade the sugar residues in bacterial surface decorating components in the initial step. Hence, the depolymreases have been considered potential anti-biofilm agents. In this respect, TSP of BECP10 designated Dpo10 was identified and characterized. Bioinformatics analysis revealed that Dpo10 possesses O-specific polysaccharide lyase activity, but no homologous to the previously reported siphophage. Purified Dpo10 consisted of the putative homotrimer form (294 kDa) in native state and showed specific glycosidase activity against O-antigen of E. coli O157:H7 LPS, suggesting that Dpo10 is responsible for the unique host spectrum of phage BECP10. Dpo10 did not inhibit the cell growth but significantly increased complement-mediated serum lytic activity against E. coli O157:H7 by 4.34 log CFU/mL reduction for 2 h treatment. In addition, Dpo10 could inhibit the biofilm formation of E. coli O157:H7 on abiotic surfaces such as polystyrene (8-fold) and stainless steel (2.56 log CFU/coupon). Dpo10 is expected to become a promising anti-biofilm agent against E. coli O157:H7.
In this study, I suggested various approaches to control E. coli O157:H7 by using phages and phage-derived lytic enzymes as biocontrol agents. These results would be helpful for the development of phage-based biocontrol strategies for preventing E. coli O157:H7 associated foodborne disease in the future.
Escherichia coli O157:H7 has been global concern that caused serious diseases such as hemolytic uremic syndrome (HUS) and bloody diarrhea. Chronic environmental resistance of E. coli O157:H7 caused by biofilm formation has also been considered major cause of food poisoning. Due to the limitation and low efficacy of classical sterilization methods, bacteriophages (phage) have been proposed as newly attractive strategies to control the foodborne.
To develop a novel biocontrol agent against E. coli O157:H7, 25 new E. coli infecting phages were isolated and two phages of ECP26 and BECP10 were selected based on the host range and lysis properties. Unlike E. coli infecting phages, ECP26 and BECP10 have very unique host spectrum and lytic activity agianst pathogenic E. coli. Especially, BECP10 was able to infect E. coli O157 strains only and it did not infect other tested strains. Based on further morphological analysis, ECP26 and BECP10 were classified as Myoviridae and Siphoviridae respectively. The genome of ECP26 and BECP10 did not contain lysogeny-related modules and toxin-associated genes, suggesting that both phages were strictly lytic and then suitable for biocontrol. Interestingly, tailspike protein (TSP) sequence of BECP10 has very low homology with the T1-like phages, implying that TSP is related to the unique host spectrum of BECP10. Periodate-based adsorption assay result suggested that lipopolysaccharide (LPS) of E. coli O157:H7 may be the host receptor of BECP10, and it was verified by Tn5 random mutant library. Both phages remained viability in wide range of temperature (25°C–55°C) and pH (3–10). Bacterial challenge assays revealed that BECP10 exhibited more strong antibacterial activity than ECP26 against planktonic E. coli O157:H7 and could minimize the development of bacteriophage insensitive mutants (BIMs). In food application studies, both phages sustained the viability and were able to reduce the E. coli O157:H7 to non-detectable level in romaine and burger patty. Therefore, ECP26 and BECP10 would be useful agents for the control of E. coli O157:H7.
Endolysin, a phage-derived lysis protein, is able to lyse the target bacteria without any special resistance, and thus has been suggested as a powerful antimicrobial agent. Therefore, rV5-like phage endolysin targeting E. coli O157:H7, named as LysECP26, was identified and purified. This endolysin had a lysozyme-like catalytic domain, but differed markedly from the sequence of lambda phage endolysin. Purified LysECP26 exhibited strong activity with a broad lytic spectrum against various gram-negative strains (29/29) and was relatively stable at a broad temperature range (4°C–55°C). The optimum temperature and pH ranges of LysECP26 were identified at 37°C–42°C and pH 7–8, respectively. NaCl supplementation did not affect the lytic activity. Although action of LysECP26 was limited in that it could not pass the outer membrane, E. coli O157:H7 could be effectively controlled by adding EDTA and citric acid. However, practical food application will have limitations because it is difficult to implement buffer conditions. In order to enhance the lytic activity, synergistic effects of LysECP26 and essential oils were investigated. As a result, more strong synergistic effects with eugenol and thymol against E. coli O157:H7 were confirmed. Therefore, LysECP26 might serve as an effective biocontrol agent for E. coli O157:H7 and gram-negative pathogens.
The process of recognizing and attaching the surface of host bacteria is one of the very important steps in phage infection. Phage depolymerase, derived from the TSP, is not only functions as receptor binding protein (RBP) but also can specifically degrade the sugar residues in bacterial surface decorating components in the initial step. Hence, the depolymreases have been considered potential anti-biofilm agents. In this respect, TSP of BECP10 designated Dpo10 was identified and characterized. Bioinformatics analysis revealed that Dpo10 possesses O-specific polysaccharide lyase activity, but no homologous to the previously reported siphophage. Purified Dpo10 consisted of the putative homotrimer form (294 kDa) in native state and showed specific glycosidase activity against O-antigen of E. coli O157:H7 LPS, suggesting that Dpo10 is responsible for the unique host spectrum of phage BECP10. Dpo10 did not inhibit the cell growth but significantly increased complement-mediated serum lytic activity against E. coli O157:H7 by 4.34 log CFU/mL reduction for 2 h treatment. In addition, Dpo10 could inhibit the biofilm formation of E. coli O157:H7 on abiotic surfaces such as polystyrene (8-fold) and stainless steel (2.56 log CFU/coupon). Dpo10 is expected to become a promising anti-biofilm agent against E. coli O157:H7.
In this study, I suggested various approaches to control E. coli O157:H7 by using phages and phage-derived lytic enzymes as biocontrol agents. These results would be helpful for the development of phage-based biocontrol strategies for preventing E. coli O157:H7 associated foodborne disease in the future.
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#Escherichia coli O157:H7 bacteripohage endolysin depolymerase biofilm biocontrol
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