• The Plant Pathology Journal
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• v.37 no.6
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• pp.555-565
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• 2021

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King's B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

• Microbiology and Biotechnology Letters
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• v.49 no.4
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• pp.510-518
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• 2021

Bacteriophages are considered excellent sensing elements for platforms detecting bacteria. However, their lytic cycle has restricted their efficacy. Here, we used the minor coat protein pIII domain (N1N2) of phage CTXφ to construct a novel surface plasmon resonance (SPR) biosensor that could detect Vibrio cholerae. N1N2 harboring the domains required for phage adsorption and entry was obtained from Escherichia coli using recombinant protein expression and purification. SDS-PAGE revealed an approximate size of 30 kDa for N1N2. Dot blot and transmission electron microscopy analyses revealed that the protein bound to the host V. cholerae but not to non-host E. coli K-12 cells. Next, we used amine-coupling to develop a novel recombinant N1N2 (rN1N2)-functionalized SPR biosensor by immobilizing rN1N2 proteins on gold substrates and using SPR to monitor the binding kinetics of the proteins with target bacteria. We observed rapid detection of V. cholerae in the range of approximately 10³ to 10⁹ CFU/ml but not of E. coli at any tested concentration, thereby confirming that the biosensor exhibited differential recognition and binding. The results indicate that the novel biosensor can rapidly monitor a target pathogenic microorganism in the environment and is very useful for monitoring food safety and facilitating early disease prevention.

• Journal of Microbiology and Biotechnology
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• v.32 no.6
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• pp.816-823
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• 2022

The rapid spread of superbugs leads to the escalation of infectious diseases, which threatens public health. Endolysins derived from bacteriophages are spotlighted as promising alternative antibiotics against multi-drug resistant bacteria. In this study, we isolated and characterized the novel Salmonella typhimurium phage PBST08. Bioinformatics analysis of the PBST08 genome revealed putative endolysin ST01 with a lysozyme-like domain. Since the lytic activity of the purified ST01 was minor, probably owing to the outer membrane, which blocks accessibility to peptidoglycan, antimicrobial peptide cecropin A (CecA) was fused to the N-terminus of ST01 to disrupt the outer membrane. The resulting CecA::ST01 has been shown to have increased bactericidal activity against gram-negative pathogens including Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Enterobacter cloacae and the most affected target was A. baumannii. In the presence of 0.25 µM CecA::ST01, A. baumannii ATCC 17978 strain was completely killed and CCARM 12026 strain was wiped out by 0.5 µM CecA::ST01, which is a clinical isolate of A. baumannii and resistant to multiple drugs including carbapenem. Moreover, the larvae of Galleria mellonella could be rescued up to 58% or 49% by the administration of CecA::ST01 upon infection by A. baumannii 17978 or CCARM 12026 strain. Finally, the antibacterial activity of CecA::ST01 was verified using 31 strains of five gram-negative pathogens by evaluation of minimal inhibitory concentration. Thus, the results indicate that a fusion of antimicrobial peptide to endolysin can enhance antibacterial activity and the spectrum of endolysin where multi-drug resistant gram-negative pathogens can be efficiently controlled.

• Animal Bioscience
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• v.36 no.8
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• pp.1285-1292
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• 2023

Objective: The objective of this study was to develop a novel endolysin (PanLys.1) for the specific killing of the ruminal hyper-ammonia-producing bacterium Peptostreptococcus anaerobius (P. anaerobius). Methods: Whole genome sequences of P. anaerobius strains and related bacteriophages were collected from the National Center for Biotechnology Information database, and the candidate gene for PanLys.1 was isolated based on amino acid sequences and conserved domain database (CDD) analysis. The gene was overexpressed using a pET system in Escherichia coli BL21 (DE3). The lytic activity of PanLys.1 was evaluated under various conditions (dosage, pH, temperature, NaCl, and metal ions) to determine the optimal lytic activity conditions. Finally, the killing activity of PanLys.1 against P. anaerobius was confirmed using an in vitro rumen fermentation system. Results: CDD analysis showed that PanLys.1 has a modular design with a catalytic domain, amidase-2, at the N-terminal, and a cell wall binding domain, from the CW-7 superfamily, at the C-terminal. The lytic activity of PanLys.1 against P. anaerobius was the highest at pH 8.0 (p<0.05) and was maintained at 37℃ to 45℃, and 0 to 250 mM NaCl. The activity of PanLys.1 significantly decreased (p<0.05) after Mn²⁺ or Zn²⁺ treatment. The relative abundance of P. anaerobius did not decrease after administration PanLys.1 under in vitro rumen conditions. Conclusion: The application of PanLys.1 to modulate P. anaerobius in the rumen might not be feasible because its lytic activity was not observed in in vitro rumen system.

• Animal Bioscience
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• v.37 no.2_spc
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• pp.337-345
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• 2024

Ruminants possess a specialized four-compartment forestomach, consisting of the reticulum, rumen, omasum, and abomasum. The rumen, the primary fermentative chamber, harbours a dynamic ecosystem comprising bacteria, protozoa, fungi, archaea, and bacteriophages. These microorganisms engage in diverse ecological interactions within the rumen microbiome, primarily benefiting the host animal by deriving energy from plant material breakdown. These interactions encompass symbiosis, such as mutualism and commensalism, as well as parasitism, predation, and competition. These ecological interactions are dependent on many factors, including the production of diverse molecules, such as those involved in quorum sensing (QS). QS is a density-dependent signalling mechanism involving the release of autoinducer (AIs) compounds, when cell density increases AIs bind to receptors causing the altered expression of certain genes. These AIs are classified as mainly being N-acyl-homoserine lactones (AHL; commonly used by Gram-negative bacteria) or autoinducer-2 based systems (AI-2; used by Gram-positive and Gram-negative bacteria); although other less common AI systems exist. Most of our understanding of QS at a gene-level comes from pure culture in vitro studies using bacterial pathogens, with much being unknown on a commensal bacterial and ecosystem level, especially in the context of the rumen microbiome. A small number of studies have explored QS in the rumen using 'omic' technologies, revealing a prevalence of AI-2 QS systems among rumen bacteria. Nevertheless, the implications of these signalling systems on gene regulation, rumen ecology, and ruminant characteristics are largely uncharted territory. Metatranscriptome data tracking the colonization of perennial ryegrass by rumen microbes suggest that these chemicals may influence transitions in bacterial diversity during colonization. The likelihood of undiscovered chemicals within the rumen microbial arsenal is high, with the identified chemicals representing only the tip of the iceberg. A comprehensive grasp of rumen microbial chemical signalling is crucial for addressing the challenges of food security and climate targets.

• Journal of Food Hygiene and Safety
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• v.34 no.6
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• pp.591-594
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• 2019

Some strains of Escherichia coli are categorized as pathogenic bacteria and alternative antimicrobials including bacteriophages for controlling these bacteria have been studied. In this study we screened antimicrobial candidates that present synergistic inhibition of the growth of E. coli DH5α as a model when co-treated with the bacteriophage ECP27 to target the bacteria. As candidates, CaCl₂, lactic acid, and citric acid were tested. CaCl₂ showed a synergistic inhibition against the strain by dose-dependent manner at 6 h of incubation but the viable cell count was recovered at 12 h. However, lactic acid and citric acid at 30 mM concentration showed synergistic inhibitions at 6 h of incubation and cleared the viable cells of E. coli DH5α at 12 h when co-treated with the bacteriophage even though lactic acid or citric acid alone was effective. Therefore, co-treatment using the bacteriophage and organic acids such as lactic acid and citric acid can be a solution for synergistic inhibition of the growth of E. coli.

• Korean Journal of Poultry Science
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• v.37 no.3
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• pp.283-287
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• 2010

In the present study we report in vivo inhibitory potential of single strain of bacteriophage ($CJ{\phi}07$) in day-old SPF chicks experimentally infected with Salmonella enteritidis (SE). The bacteriophages prepared by feed additives and drinking water were given to chicks for 20 days starting prior 10 days before challenge with SE. Chicks were euthanized at 10 days after challenge for quantitative salmonella isolation from intestine and determination of environmental contamination level of salmonella. Bacteriophage therapy as additives in feed and drinking water resulted in significant inhibition of the SE replication in intestines of SPF chickens (P<0.05). In addition, environmental contamination by SE fecal shedding was decreased in bacteriophage-treated birds. Therefore, bacteriophage $CJ{\phi}07$ examined in this study may be a plausible alternative to antibiotics for the reduction of salmonella infection both in poultry.

• Journal of Life Science
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• v.28 no.1
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• pp.78-82
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• 2018

Detection of pathogenic microorganisms takes several days by conventional methods. It is necessary to assess microorganisms in a timely manner to reduce the risk of spreading infection. For this purpose, bacteriophages are chosen for use as a biosensing tool due to their host specificity, wide abundance, and safety. However, their lytic cycle limits their efficacy as biosensors. Phage proteins involved in binding to bacteria could be a robust alternative in resolving this drawback. Here, a fragment of tail protein J (residues 784 to 1,132) of phage lambda fused with 6X His-tag (6HN-J) at its N-terminus was cloned, overexpressed, purified, and characterized for its binding with microorganisms. The purified protein demonstrated a size of about 38 kDa in sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and bound with anti-His monoclonal antibodies. It bound specifically to Escherichia coli K-12, and not Salmonella typhimurium, Bacillus subtilis, or Pseudomonas aeruginosa in dot blotting. Binding of the protein to E. coli K-12 inhibited about 50% of the in vivo adsorption of the phage lambda to host cells at a concentration of $1{\mu}g/ml$ 6HN-J protein and almost 100% at $25{\mu}g/ml$ 6HN-J. The results suggest that a fusion viral protein could be utilized as a biosensing element (e.g., protein chips) for detecting microorganisms in real time.

• Korean Journal of Microbiology
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• v.17 no.4
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• pp.165-178
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• 1979

Phages of Lactobaciilus casei (PLC) isolated from plant drainage were classified and characterized. The results are as follows : 1. On the basis of host range pattern, phages could be divided into 2 groups (PLC-B and PLC-C). PLC-B group phages could be further divided into 5 sub-groups $(B_1, \;B_2, \;B_3, \;B_4, \;and\;B_5)$. Although PLC-C group phages had the same host range, they could be also divided into 2 sub-groups $(C_1\;and\;C_2)$ by morphlogical type. 2. It was $B_3$ group phages that represented a major proportion (44.4%) of phages tested. However, $B_1$ group phages were shown to have the widest host range. 3. Electron micrographs revealed that the phages fell into three different morphological types. $(B_1, \;B_2, \;and\;B_3)$ group phages hd a hexagonal head (52nm in diameter) and a sheathless noncontractile (245 nm in length). $B_4\;and\;C_2$ group phages had a hexagonal head (56 nm) and a short flexible tail (169nm) having no sheath. $B_5\;and\;C_1$ group phages were shown to have a hexagonal head (81 nm) and a contractile tail (140 nm) having a sheath, a base plate and tail fibers. 4. The inactivation of the phages by antisera indicated that serological relationships correlated completely with morphological types. 5. $B_1, \;C_1\;and\;C_2$ group phages produced a large (1, 2 mm in diameter) plaque with a clear ring. The morphology of plaques of $B_3\;and\;B_5$ group phages was the same as those produced by the above, but the average plaque sizes for $B_3\;and\;B_5$ were 0.8 mm abd 0.5 mm, respectively. $B_2\;and\;B_4$ group phages produced a small (0.5 mm) turbid plaque with an irregular edge. 6. The latent period and the average burst size of $B_1\;and\;B_3$ group phages were 90 min and 100, respectively. These phages reuqired calcium ions for their miltiplication. 7. $B_3$ group phages could not be absrobed to R-variant $KC_1$. 8. The order of resistance of phages to heat was $B_2\;>\;B_1, B_4\;and\;B_5\;>\;B_3\;and\;C_2, \;B_5$ group phages were more stable than $B_3$ in various pH values. $C_2$ group phages were more sensitive to UV irradiation than $B_1\;and\;B_3$ group phages. 9. Strains YIT9018 and IAM 1043 were induced by mitomycin C treatment. Phage particles detected in the lysates had a hexagonal head (38 and 49 nm, respectively), but no tail. Any sensitive indicator strain could not be isolated in spite of repaeated trials.

• Journal of Korean Society of Environmental Engineers
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• v.32 no.11
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• pp.1063-1068
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• 2010

This study compared UV and UV/$H_2O_2$ inactivation of E.coli, a possible indicator microorganism for fecal contamination of water, and $Q{\ss}$ phage, an indicator for pathogenic viruses. UV inactivation of $Q{\ss}$, T4 and lambda phages in actual secondary effluent was investigated, too. As a result, similar inactivation efficiency between $Q{\ss}$ phage and E.coli was observed during UV treatment, while $Q{\ss}$ phage showed higher resistance to UV/$H_2O_2$ than E.coli. $Q{\ss}$ phage resistance to UV or UV/$H_2O_2$ does not reflect those of all pathogenic viruses. However, the result tells that the use of E.coli inactivation efficiency in evaluating microbiological safety of water could not always ensure the sufficient safety from pathogenic viruses. Meanwhile, $Q{\ss}$ phage showed less resistance to UV than T4 and lambda phages, indicating that the use of $Q{\ss}$ phage as an indicator virus may bring insufficient disinfection effectiveness by causing the introduction of lower UV dose than required. Consequently, it can be thought that T4 or lambda phages would be more desirable indicators in ensuring the sufficient disinfection effectiveness for various pathogenic viruses.