Microvirga jeongseonensis sp. nov., isolated from soil in South Korea.

A novel Gram-stain-negative, aerobic, rod-shaped, convex, and light pink-colored strain BT688T was isolated from a soil sample collected in Jeongseon City, South Korea. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain BT688T belongs to a distinct lineage within the genus Microvirga (family Methylobacteriaceae, order Rhizobiales, class Alphaproteobacteria, phylum Proteobacteria). The 16S rRNA gene sequence similarity between strain BT688T and Microvirga aerilata 5420S-16T was 98.5%. Strain BT688T had Q-10 as a major respiratory quinone and the major polar lipids were diphosphatidilglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). The major cellular fatty acids of strain BT688T were C18:1 ω7c (76.0%) and summed feature 3 (9.6%). Based on the polyphasic characteristics, strain BT688T represents a novel bacterial species within the genus Microvirga and the proposed name is Microvirga jeongseonensis. The type strain of Microvirga jeongseonensis is BT688T (= KCTC 82701T = NBRC 114857T).

Microvirga terrestris sp. nov., and Microvirga arvi sp. nov., isolated from soil in South Korea.

Two novel Gram-stain-negative, aerobic, rod shaped bacterial strains BT290T and BT689T were isolated from soil collected in South Korea. Colony morphologies of both strains were circular and convex while the colors of BT290T and BT689T were light-pink and white, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that BT290T and BT689T belong to a distinct lineage within the genus Microvirga (family Methylobacteriaceae, order Rhizobiales, class Alphaproteobacteria, phylum Proteobacteria, kingdom Bacteria). The 16S rR NA gene sequence similarity between two strains was 97.9%. Both strains had the similar quinone system, with ubiquinone 10 (Q-10) as the major respiratory quinone. The major polar lipids of strains BT290T and BT689T were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), and phosphatidylglycerol (PG). The major cellular fatty acids of strain BT290T were C18:1 ω7c (58.2%) and C16:0 (17.7%), while those of strain BT689T were C18:1 ω7c (61.8%) and C16:0 (10.8%). On the bases of polyphasic analysis (phylogenetic, chemotaxonomic, and biochemical), strains BT290T and BT689T can be suggested as novel bacterial species within the genus Microvirga and the proposed names are Microvirga terrestris and Microvirga arvi, respectively. The type strain of Microvirga terrestris is BT290T (= KCTC 72367T = NBRC 114844T) and the type strain of Microvirga arvi is BT689T (= KACC 22016T = NBRC 114858T), respectively.

Hymenobacter telluris sp. nov., isolated from soil in South Korea.

Two novel bacterial strains, designated as BT186T and BT505, were isolated from a soil sample collected in South Korea and characterized. Both strains were Gram-stain-negative, rod-shaped, aerobic, circular, convex, and had red-colored colonies. The level of 16S rRNA gene sequence similarity between the strains BT186T and BT505 was 100%, indicating that they represent an identical species. 16S rRNA sequence analysis indicated that strains BT186T and BT505 belong to a distinct lineage within the genus Hymenobacter (family Hymenobacteraceae, order Cytophagales, class Cytophagia, phylum Bacteroidetes, Kingdom Bacteria). Both strains were closely related to Hymenobacter norwichensis DSM 15439T (98.3% 16S rRNA gene similarity), Hymenobacter aquaticus JCM 31653T (96.8%), and Hymenobacter perfusus LMG26000T (96.5%). Strain BT186T was found to have the MK-7 as the major respiratory quinone. The major polar lipid of strain BT186T was identified to be phosphatidylethanolamine (PE). The major cellular fatty acid profiles of strain BT186T were C16:1 ω5c (24.3%), iso-C15:0 (20.3%) and summed feature 3 (C16:1 ω6c/C16:1 ω7c) (19.9%). Characterization based on polyphasic analysis indicated that strains BT186T and BT505 represent novel species of the genus Hymenobacter and the name Hymenobacter telluris sp. nov. is proposed. The type strain of Hymenobacter telluris is BT186T (= KCTC 72338T = NBRC 114968T).

Microvirga splendida sp. nov., bacteria isolated from soil.

Two bacterial strains, BT325T and BT690, were isolated from soil samples collected in Korea. Both strains were Gram stain-negative, short rod-shaped, and formed light-pink colored colonies. The 16S rRNA sequence similarity of strains BT325T and BT690 shared a sequence similarity of 99.7%. Both strains shared the highest 16S rRNA gene similarity of 98.6% with Microvirga arabica SV2184PT, followed by Microvirga ossetica V5/3 M T (98.5% and 98.2%, respectively), Microvirga soli R491T (98.3% and 98.2%, respectively), Microvirga aerilata (98.2% and 98.08%, respectively), Microvirga makkahensis (98.08% and 97.8%, respectively). Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain BT325T and BT690 were positioned in a distinct lineage within the family Methylobacteriaceae (order Rhizobiales, class Alphaproteobacteria). The genome size of strain BT325T was 5,200,315 bp and the genomic DNA G + C content was 64.3 mol%. The sole respiratory quinone of strain BT325T was Q-10 and the predominant cellular fatty acids were summed feature 3 (C16:1 ω7c/C16:1 ω6c) and summed feature 8 (C18:1 ω7c/C18:1 ω6c). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. Polyphasic taxonomic analysis of biochemical, chemotaxonomic, and phylogenetic analyses suggested that strains BT325T represents a novel bacterial species within the genus Microvirga, for which the name Microvirga splendida is proposed. The type strain of Microvirga splendida is BT325T (= KCTC 72406 T = NBRC 114847 T).

Synergistic inactivation of Listeria and E. coli using a combination of erythorbyl laurate and mild heating and its application in decontamination of peas as a model fresh produce.

We investigated the synergistic antimicrobial activity of erythorbyl laurate (EL) and mild heating co-treatment on the Gram-positive Listeria innocua and Gram-negative Escherichia coli O157:H7 bacteria. EL (2 mM) and mild heating (55 °C for 3 min) resulted in 3.1 and 0.5 log colony forming units (CFU)/mL reductions in the number of L. innocua, respectively, compared to a 6.4 log CFU/mL reduction induced by the combined treatment of EL and mild heating in saline. EL (10 mM) and mild heating (55 °C for 3 min) resulted in 1.3 and 0.7 log CFU/mL reductions in the number of E. coli O157:H7, respectively, compared to a 6.2 log CFU/mL reduction with the combined treatment in saline. EL, a membrane-active compound, showed a strong synergistic effect with mild heating, possibly due to enhanced disruption of the bacterial cell membrane. The synergistic antibacterial effect was evaluated using inoculated English peas (Pisum sativum) and this combined treatment (2 mM EL and mild heating against L. innocua and 10 mM EL and mild heating against E. coli O157:H7) resulted in more than 7 log reductions in the numbers of L. innocua and E. coli O157:H7, inoculated on the surface of fresh peas. The treatments did not show significant difference in the color or texture of treated peas compared to the non-treated controls. This is the first report illustrating synergistic activity of EL and mild heating for both the gram positive (L. innocua) and the gram negative (E. coli O157:H7) bacteria on food. Overall, this research will illustrate the development of more effective and rapid antibacterial surface disinfection method for application in the processing of minimally processed foods.

Flavisolibacter longurius sp. nov., isolated from soil.

A novel Gram-negative bacterial strain BT320T was isolated from soil collected in Uijeongbu city, Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BT320T belong to a distinct lineage within the genus Flavisolibacter (family Chitinophagaceae, order Chitinophagales, class Chitinophagia). The strain BT320T was closely related to Flavisolibacter galbus 17J28-26T (97.6% 16S rRNA gene similarity), Flavisolibacter nicotianae X7XT (96.7%), Flavisolibacter ginsengiterrae Gsoil 492T (96.2%), and Flavisolibacter ginsengisoli Gsoil 643 T (96.1%). The genome size of strain BT320T was 5,664,094 bp. Bacterial growth was observed at 10-37 °C (optimum 25 °C) and pH 6.0-8.0 (optimum pH 7.0) on R2A agar. The major cellular fatty acids of strain BT320T were iso-C15:0, summed feature 3 (C16:1 ω6c/C16:1 ω7c), and summed feature 1 (iso-C15:1 H/C13:0 3OH). Its predominant respiratory quinone was MK-7. The major polar lipid of strain BT320T was identified to be phosphatidylethanolamine (PE). Based on the biochemical, chemotaxonomic, and phylogenetic analysis, strain BT320T can be suggested as a novel bacterial species within the genus Flavisolibacter and the proposed name is Flavisolibacter longurius. The type strain of Flavisolibacter longurius is BT320T (= KCTC 72422T = NBRC 114375T).

Methylobacterium radiodurans sp. nov., a novel radiation-resistant Methylobacterium.

A Gram-negative, aerobic, flagellated, rod-shaped, and pink-pigmented bacterium, strain 17Sr1-43 T, was isolated from a soil sample collected in Nowongu, Seoul, Korea. The isolate could grow at 18-37 °C (optimum, 28-30 °C), pH 6.0-8.0 (optimum, pH 7.0) and in the presence of 0-1.0% (w/v) NaCl (optimum, 0%) with aeration. The major cellular fatty acids were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) and summed feature 2 (iso-C16:1 I and/or C14:0 3-OH). The predominant respiratory quinone was Q-10 and the major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phospholipid, and diphosphatidylglycerol. The G + C content of genomic DNA was 69.1 mol%. Strain 17Sr1-43 T was closely related to Methylobacterium gregans KACC 14808 T (98.4% 16S rRNA gene sequence similarity), Methylobacterium hispanicum KACC 11432 T (97.9%), and Methylobacterium phyllosphaerae CBMB27T (96.1%). The complete genome of strain 17Sr1-43 T contains essential genes related to DNA repair processes including bacterial RecBCD dependent pathway and UmuCD system. Based on the phenotypic, genotypic, and chemotaxonomic characteristics, strain 17Sr1-43 T represents a novel species in the genus Methylobacterium, for which the name Methylobacterium radiodurans sp. nov. is proposed. The type strain is strain 17Sr1-43 T (= KCTC 52906 T = NBRC 112875 T).

Sphingomonas panacisoli sp. nov., bacterium isolated from soil in South Korea.

A newly isolated bacterial strain designated as HKS19 was isolated from a ginseng cultivation soil sample collected in South Korea. Cells of the strain HKS19 were Gram-stain-negative, rod, oval-shaped and they formed yellow colonies when grown on R2A agar at 30 °C. HKS19 showed the highest 16S rRNA gene sequence similarity (98.6%) with Sphingomonas asaccharolytica NBRC 15499T. Its growth was observed at 10-37 °C (optimum 30 °C), pH 6-9 (optimum pH 7), and in the presence of 0-1% NaCl (optimum 0%). The genome size of HKS19 was 3.4 Mb and the G+C content was 65.1 mol%. The main polar lipid of strain HKS19 was diphosphatidylglycerol (DPG), the predominant respiratory quinone was Q-10 and the major fatty acids were a summed feature 8 (C18 : 1 ω6c / C18 : 1 ω7c) and C16 : 0. Based on the phylogenetic, genotypic, phenotypic and chemotaxonomic analysis, strain HKS19 represents a newly isolated species of the genus Sphingomonas, for which the name Sphingomonas panacisoli is proposed. The type strain is HKS19T (=KACC 18881T=LMG 29564 T).

Effective inhibition of Salmonella Typhimurium in fresh produce by a phage cocktail targeting multiple host receptors.

Salmonella contamination of fresh produce is the primary bacterial cause of a significant number of foodborne outbreaks and infections. Bacteriophages can be used as natural antibacterial agents to control foodborne pathogens. However, the rapid development of bacterial resistance to phage infection is a significant barrier to practical phage application. To overcome this problem, we developed a novel phage cocktail consisting of the three phages (BSPM4, BSP101 and BSP22A) that target different host receptors, including flagella, O-antigen and BtuB, respectively. Whole genome sequence analysis of the phages revealed that three phages do not harbor genes involved in lysogen formation or toxin production, suggesting they are safe for use as biocontrol agents in foods. In vitro treatment of the phage cocktail resulted in a significant reduction in the development of bacterial resistance. Phage cocktail treatments achieved 4.7-5.5 log CFU/cm2 reduction of viable cell number in iceberg lettuce and 4.8-5.8 log CFU/cm2 reduction in cucumber after 12 h at room temperature (25 °C). The phage cocktail exhibited good antimicrobial efficiency, suggesting that it could reduce S. Typhimurium contamination of fresh produce. The strategy of developing cocktails of phages that target multiple host receptors can be used to develop novel biocontrol agents of S. Typhimurium.

Mutation of a Staphylococcus aureus temperate bacteriophage to a virulent one and evaluation of its application.

Bacteriophages have been suggested as alternative antimicrobial agents based on their host specificity and lytic activity. Therefore, it is necessary to obtain a virulent phage from a temperate one using molecular techniques to control Staphylococcus aureus efficiently. SA13, a novel temperate phage infecting S. aureus, was isolated and characterized. From this phage, mutant phages were generated by random deletion mutations, and a virulent mutant phage SA13m was selected. Comparative genome analysis revealed that the SA13m genome contains various nucleotide deletions in six genes encoding three hypothetical proteins and three lysogeny-associated proteins, including putative integrase, putative CI, and putative anti-repressor proteins. Mitomycin C induction of SA13m-resistant strains revealed that this mutant phage does not form lysogen, suggesting that SA13m is a virulent phage. In addition, SA13m showed rapid and long-lasting host cell growth inhibition activity. Furthermore, application of SA13m in sterilized milk showed that S. aureus was reduced to non-detectable levels both at refrigerator temperature (4 °C) and room temperature (25 °C), suggesting that SA13m can efficiently control the growth of S. aureus in foods. The virulent mutant phage SA13m could be used as a promising biocontrol agent against S. aureus without lysogen formation.

A Novel Bacteriophage Targeting Cronobacter sakazakii Is a Potential Biocontrol Agent in Foods.

Cronobacter sakazakii is an important pathogen that causes high mortality in infants. Due to its occasional antibiotic resistance, a bacteriophage approach might be an alternative effective method for the control of this pathogen. To develop a novel biocontrol agent using bacteriophages, the C. sakazakii-infecting phage CR5 was newly isolated and characterized. Interestingly, this phage exhibited efficient and relatively durable host lysis activity. In addition, a specific gene knockout study and subsequent complementation experiment revealed that this phage infected the host strain using the bacterial flagella. The complete genome sequence analysis of phage CR5 showed that its genome contains 223,989 bp of DNA, including 231 predicted open reading frames (ORFs), and it has a G+C content of 50.06%. The annotated ORFs were classified into six functional groups (structure, packaging, host lysis, DNA manipulation, transcription, and additional functions); no gene was found to be related to virulence or toxin or lysogen formation, but >80% of the predicted ORFs are unknown. In addition, a phage proteomic analysis using SDS-PAGE and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) revealed that seven phage structural proteins are indeed present, supporting the ORF predictions. To verify the potential of this phage as a biocontrol agent against C. sakazakii, it was added to infant formula milk contaminated with a C. sakazakii clinical isolate or food isolate, revealing complete growth inhibition of the isolates by the addition of phage CR5 when the multiplicity of infection (MOI) was 10(5).

Identification and characterization of outer membrane vesicle-associated proteins in Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium is a primary cause of enteric diseases and has acquired a variety of virulence factors during its evolution into a pathogen. Secreted virulence factors interact with commensal flora and host cells and enable Salmonella to survive and thrive in hostile environments. Outer membrane vesicles (OMVs) released from many Gram-negative bacteria function as a mechanism for the secretion of complex mixtures, including virulence factors. We performed a proteomic analysis of OMVs that were isolated under standard laboratory and acidic minimal medium conditions and identified 14 OMV-associated proteins that were observed in the OMV fraction isolated only under the acidic minimal medium conditions, which reproduced the nutrient-deficient intracellular milieu. The inferred roles of these 14 proteins were diverse, including transporter, enzyme, and transcriptional regulator. The absence of these proteins influenced Salmonella survival inside murine macrophages. Eleven of these proteins were predicted to possess secretion signal sequences at their N termini, and three (HupA, GlnH, and PhoN) of the proteins were found to be translocated into the cytoplasm of host cells. The comparative proteomic profiling of OMVs performed in this study revealed different protein compositions in the OMVs isolated under the two different conditions, which indicates that the OMV cargo depends on the growth conditions and provides a deeper insight into how Salmonella utilizes OMVs to adapt to environmental changes.

Isolation, characterization, and application of a lytic bacteriophage SSP49 to control Staphylococcus aureus contamination on baby spinach leaves.

Staphylococcus aureus, a major foodborne pathogen, is frequently detected in fresh produce. It often causes food poisoning accompanied by abdominal pain, diarrhea, and vomiting. Additionally, the abuse of antibiotics to control S. aureus has resulted in the emergence of antibiotics-resistant bacteria, such as methicillin resistant S. aureus. Therefore, bacteriophage, a natural antimicrobial agent, has been suggested as an alternative to antibiotics. In this study, a lytic phage SSP49 that specifically infects S. aureus was isolated from a sewage sample, and its morphological, biological, and genetic characteristics were determined. We found that phage SSP49 belongs to the Straboviridae family (Caudoviricetes class) and maintained host growth inhibition for 30 h in vitro. In addition, it showed high host specificity and a broad host range against various S. aureus strains. Receptor analysis revealed that phage SSP49 utilized cell wall teichoic acid as a host receptor. Whole genome sequencing revealed that the genome size of SSP49 was 137,283 bp and it contained 191 open reading frames. The genome of phage SSP49 did not contain genes related to lysogen formation, bacterial toxicity, and antibiotic resistance, suggesting its safety in food application. The activity of phage SSP49 was considerably stable under various high temperature and pH conditions. Furthermore, phage SSP49 effectively inhibited S. aureus growth on baby spinach leaves both at 4 °C and 25 °C while maintaining the numbers of active phage during treatments (reductions of 1.2 and 2.1 log CFU/cm2, respectively). Thus, this study demonstrated the potential of phage SSP49 as an alternative natural biocontrol agent against S. aureus contamination in fresh produce.

Characterization of KMSP1, a newly isolated virulent bacteriophage infecting Staphylococcus aureus, and its application to dairy products.

Staphylococcus aureus is one of the major pathogens causing foodborne outbreaks and severe infections worldwide. Generally, various physical and chemical treatments have been applied to control S. aureus in the food industry. However, conventional treatments usually affected food quality and often produced toxic compounds. Therefore, bacteriophage (phage), a natural antimicrobial agent, has been suggested as an alternative strategy to control foodborne pathogens including S. aureus. In this study, KMSP1, a bacteriophage infecting S. aureus was isolated from a raw milk sample and characterized. Transmission electron microscopy (TEM) analysis revealed that phage KMSP1 belongs to the Myoviridae family. Phage KMSP1 efficiently inhibited bacterial growth for >28 h post-infection. In addition, phage KMSP1 could infect a broad spectrum of S. aureus strains, including methicillin-resistant S. aureus (MRSA) strains. Whole-genome sequence analysis showed that KMSP1 is a lytic phage with the absence of genes related to lysogen formation, toxin production, and antibiotics resistance, respectively. In the genome of KMSP1, the presence of putative tail lysin containing a cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain could be one of the reasons for the effective antimicrobial activity of KMSP1. Furthermore, high stability of phage KMSP1 at temperature ranging from 4 to 55 °C and pH ranging from 5 to 11, suggested its potential use in various food systems. Receptor analysis revealed that KMSP1 utilized cell wall teichoic acid (WTA), one of the major virulence factors of S. aureus, as a host receptor. Application of phage KMSP1 at an MOI of 104 achieved a significant reduction of log 8.8 CFU/mL of viable cell number in pasteurized milk and log 4.3 CFU/cm2 in sliced cheddar cheese after 24 h. Taken together, the strong antimicrobial activity of phage KMSP1 suggested that it could be developed as a biocontrol agent in dairy products to control S. aureus contamination.

Effective control of antibiotic resistance using a sonication-based combinational treatment and its application to fresh food.

Antibiotics have been widely used to treat several infectious diseases. However, the overuse of antibiotics has promoted the emergence and spread of antibiotic resistant bacteria (ARB) in various fields, including the food industry. In this study, the antimicrobial efficacies of two conventional sterilization methods, mild heat, and sonication, were evaluated and optimized to develop a new strategy against ARB. Simultaneous mild heat and sonication (HS) treatment led to a significant reduction in viable cell counts, achieving a 5.58-log reduction in 4 min. However, no remarkable decrease in viable cell counts was observed in individually treated groups. Interestingly, the release of antibiotic resistance genes (ARGs) increased in a time-dependent manner in the heat-treated and HS-treated groups. The inactivation levels of ARGs increased as the HS treatment time increased from 2 to 8 min, and most ARGs were degraded after 8 min. In contrast, no significant inactivation of ARGs was observed in the heat-treated and sonication-treated groups after 8 min. These results reveal the synergistic effect of the combination treatment in controlling not only ARB but also ARGs. Finally, on applying this newly developed combination treatment to fresh food (cherry tomato and carrot juice), 3.97- and 4.28-log microbial inactivation was achieved, respectively. In addition, combination treatment did not affect food quality during storage for 5 days. Moreover, HS treatment effectively inactivated ARGs in fresh food systems.

Inactivation of Indigenous Microorganisms and Salmonella in Korean Rice Cakes by In-Package Cold Plasma Treatment.

The antimicrobial effects of in-package cold plasma (CP) treatment on Korean rice cakes (KRC) were evaluated. The CP treatment (25 kV) inactivated indigenous mesophilic aerobic bacteria by 0.8-1.0 log CFU/g, irrespective of the position of KRC in the package. The addition of a shaking step during CP treatment increased the reduction in microbes by ~1 log CFU/g. The microbial inactivation efficiency increased significantly when the treatment time increased from 1 to 3 min. Microbial inactivation activity was highest for packages containing eight rice cakes. The optimized CP treatment achieved a 2.0 ± 0.1 log CFU/g reduction in indigenous bacteria. In addition, the optimum CP treatment inactivated indigenous yeast and molds and Salmonella in KRC by 1.7 ± 0.1 log CFU/g and 3.9 ± 0.3 log CFU/g, respectively. No significant changes in color and firmness were observed, and the surface temperature of KRC did not exceed 22 °C after CP treatment. Moreover, CP treatment damaged the cellular membrane of Salmonella, mainly by inducing lipid peroxidation. This study demonstrates the potential use of in-package CP treatment for the non-thermal microbial inactivation of KRC.

Synergistic Inactivation of Bacteria Using a Combination of Erythorbyl Laurate and UV Type-A Light Treatment.

This study evaluated the synergistic antimicrobial activity of erythorbyl laurate (EL) and UV type-A (UVA). To investigate the mode of synergism, changes in gene expression and bacterial inactivation activity were examined. Individual treatments with EL (10 mM) or UVA caused a 1.9- or 0.5-log CFU/ml reduction respectively, whereas EL/UVA co-treatment resulted in a 5.5-log CFU/ml reduction in Escherichia coli viable cell numbers. Similarly, treatment with either EL (2 mM) or UVA for 30 min resulted in a 2.8- or 0.1-log CFU/ml reduction in Listeria innocua, respectively, whereas combined treatment with both EL and UVA resulted in a 5.4-log CFU/ml reduction. Measurements of gene expression levels showed that EL and UVA treatment synergistically altered the gene expression of genes related to bacterial membrane synthesis/stress response. However, addition of 10-50-fold excess concentration of exogenous antioxidant compared to EL reduced the synergistic effect of EL and UVA by approximately 1 log. In summary, the results illustrate that synergistic combination of EL and UVA enhanced membrane damage independent of the oxidative stress damage induced by UVA and thus illustrate a novel photo-activated synergistic antimicrobial approach for the inactivation of both the Gram-positive and Gram-negative bacteria. Overall, this study illustrates mechanistic evaluation of a novel photochemical approach for food and environmental applications.

Yeast Surface Display System for Facilitated Production and Application of Phage Endolysin.

Bacteriophage endolysin is one of the potential alternatives of conventional antibiotics, but the intrinsic limitations of the bacterial expression system may undermine the comprehensive application of this therapeutic protein. To circumvent such limitations, we adopted a yeast surface display system as a novel expression platform for endolysin. Endolysin LysSA11 from staphylococcal phage SA11 was expressed and surface-displayed in Saccharomyces cerevisiae to exhibit sufficient antimicrobial activity against Staphylococcus aureus. Without any protein isolation or purification procedures, we showed that direct treatment of LysSA11-displaying yeast cells could accomplish a 5-log reduction of viable Staphylococcus aureus within 3 h. Furthermore, the surface-displayed LysSA11 exhibited superior stability over the soluble form of purified LysSA11 during 14 days of storage in a refrigerated environment. We suggest that the yeast surface display system is an efficient, stable, and straightforward platform for the production and antibacterial applications of endolysin.

Identification and in vitro Characterization of a Novel Phage Endolysin that Targets Gram-Negative Bacteria.

Most double-stranded (ds) DNA phages utilize holin proteins to secrete endolysin for host peptidoglycan lysis. In contrast, several holin-independent endolysins with secretion sequences or signal-arrest-release (SAR) sequences are secreted via the Sec pathway. In this study, we characterized a novel lysis protein (M4Lys) encoded by the dsDNA phage BSPM4, whose lysis function is not dependent on either holin or the Sec pathway in vitro. In silico analysis of M4Lys revealed that it contains a putative virion protein domain and an unusual C-terminal transmembrane domain (TMD). Turbidity reduction assays and liquid chromatography-mass spectrometry using purified peptidoglycan showed that the virion protein domain of M4Lys has peptidoglycan lysis activity. In vitro overproduction of M4Lys in Escherichia coli revealed that M4Lys alone caused rapid cell lysis. Treatment of E. coli with a Sec inhibitor did not inhibit the lysis activity of M4Lys, indicating that the Sec pathway is not involved in M4Lys-mediated cell lysis. Truncation of the TMD eliminated the cell lysis phenomenon, while production of the TMD alone did not induce the cell lysis. All these findings demonstrate that M4Lys is a novel endolysin that has a unique mosaic structure distinct from other canonical endolysins and the TMD plays a critical role in M4Lys-mediated in vitro cell lysis.

Simultaneous Control of Staphylococcus aureus and Bacillus cereus Using a Hybrid Endolysin LysB4EAD-LysSA11.

Bacteriophage endolysins have attracted attention as promising alternatives to antibiotics, and their modular structure facilitates endolysin engineering to develop novel endolysins with enhanced versatility. Here, we constructed hybrid proteins consisting of two different endolysins for simultaneous control of two critical foodborne pathogens, Staphylococcus aureus and Bacillus cereus. The full-length or enzymatically active domain (EAD) of LysB4, an endolysin from the B. cereus-infecting phage B4, was fused to LysSA11, an endolysin of the S. aureus-infecting phage SA11, via a helical linker in both orientations. The hybrid proteins maintained the lytic activity of their parental endolysins against both S. aureus and B. cereus, but they showed an extended antimicrobial spectrum. Among them, the EAD of LysB4 fused with LysSA11 (LysB4EAD-LyaSA11) showed significantly increased thermal stability compared to its parental endolysins. LysB4EAD-LysSA11 exhibited high lytic activity at pH 8.0-9.0 against S. aureus and at pH 5.0-10.0 against B. cereus, but the lytic activity of the protein decreased in the presence of NaCl. In boiled rice, treatment with 3.0 µM of LysB4EAD-LysSA11 reduced the number of S. aureus and B. cereus to undetectable levels within 2 h and also showed superior antimicrobial activity to LyB4EAD and LysSA11 in combination. These results suggest that LysB4EAD-LysSA11 could be a potent antimicrobial agent for simultaneous control of S. aureus and B. cereus.