Hymenobacter armeniacus sp. nov. and Hymenobacter montanus sp. nov., two radiation-resistant bacteria from soil.

Two bacterial strains, designated BT189T and BT664T, were isolated from soil sampled in the Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strains BT189T and BT664T belonged to the genus Hymenobacter, family Hymenobacteraceae (order Cytophagales). The 16S rRNA genes of the two strains shared a sequence similarity of 93.7 %. The closely related species of strain BT189T were Hymenobacter rubidus DG7BT (97.1 % 16S rRNA similarity) and Hymenobacter terrae DG7AT (96.7 %). The closest related species to strain BT664T were Hymenobacter sedentarius DG5BT (95.3 %) and Hymenobacter terrenus MIMtkLc17T (95.2 %). The genome sizes of strains BT189T and BT664T were 5 285 287 and 5 475 357 bp, respectively. The genomic DNA G+C contents of strains BT189T and BT664T were 63.2 and 59.3 mol%, respectively. The main fatty acids of strain BT189T were iso-C15 : 0, anteiso-C15 : 0 and summed feature 3 (C16 : 1 ω6c/C16 : 1 ω7c), and those of strain BT664T were iso-C15 : 0, C16 : 1 ω5c and summed feature 3 (C16 : 1 ω6c/C16 : 1 ω7c). The main polar lipid in both strains was phosphatidylethanolamine and the predominant respiratory quinone was MK-7, supporting the affiliation of these strains with the genus Hymenobacter. Based on the results of biochemical, chemotaxonomic and phylogenetic analyses, two novel species, Hymenobacter armeniacus BT189T (=KCTC 72341T=NBRC 114843T) and Hymenobacter montanus BT664T (KACC 21967T=NBRC 114856T), are proposed.

Nocardioides panacis sp. nov., isolated from soil of a ginseng field.

A bacterial strain designated as G188T was isolated from ginseng field soil in the Republic of Korea. Phylogenetic analysis of 16S rRNA gene sequences showed that strain G188T formed a distinct lineage within the genus Nocardioides, family Nocardioidaceae, order Propionibacteriales. Sequence similarity revealed that strain G188T was most closely related to Nocardioides iriomotensis IR27-S3T (97.7 % 16S rRNA similarity). The genome size of strain G188T was 4 901 775 bp, and the genomic DNA G+C content was 72.3 mol%. The average nucleotide identity and DNA-DNA hybridization values with other Nocardioides species were less than 75.6 and 20.1 %, respectively. The main fatty acids of strain G188T were C17 : 0, C17 : 1 ω8c and iso-C16 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylinositol, and the major respiratory quinone was menaquinone 8, supporting that strain G188T was affiliated with the genus Nocardioides. Based on biochemical, chemotaxonomic and phylogenetic analyses, the novel species Nocardioides panacis G188T (KACC 21695T=LMG 31733T) is proposed.

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.

Isolation, screening and identification of key components having intense insect repellent activity against Plodia interpunctella from four different medicinal plant materials.

BACKGROUND: Global warming and the indiscriminate use of pesticides have increased the propagation of the stored-product insect pests, leading to enormous losses in the agriculture and food industries. The most used insect repellents are synthetic derivatives; however, these have an adverse effect on human health as well as on the environment. Therefore, we attempted to find materials with insect repellent activity in natural products. The present study aimed to identify the single chemical component with intense insect repellent activity in extracts from four different Oriental medicinal plant materials: (i) Anethum graveolens L. (dill) seeds; (ii) Artemisia capillaris Thunb. (capillary wormwood) leaves; (iii) smoked Prunus mume Siebold & Zucc. (mume) fruits; and (iv) Rhus javanica L. (galls). RESULTS: As a result of the bioassay-guided fractionation of each extract against the Plodia interpunctella, stored-product insect, the n-hexane fraction of dill seeds extract was confirmed as the optimal fraction between all of the fractions. In total, 32 chemical components were identified from the n-hexane fraction of dill seeds by gas chromatography-mass spectrometry analysis, and the two main components were dillapiole (47.51%) and carvone (26.76%). Of the two components, dillapiole was confirmed as the key component playing an essential role in insect repellent activity. CONCLUSION: Our study suggests that dillapiole has the potential to be used as a natural insect repellent for the control of P. interpunctella infestation in agricultural and food products during distribution and storage. © 2021 Society of Chemical Industry.

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

Two novel Gram-negative bacterial strains, BT507T and BT506, were isolated from soil collected in Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both strains BT507T and BT506 belong to a distinct lineage within the genus Hymenobacter (family Hymenobacteraceae; order Cytophagales; class Cytophagia). The 16S rRNA genes of the two strains shared a sequence similarity of 100.0%. Strains BT507T and BT506 are closely related to Hymenobacter profundi M2T (97.2% 16S rRNA gene similarity), Hymenobacter defluvii POA9T (97.1%), and Hymenobacter tenuis POB6T (95.3%). The genome size of strain BT507T is 5,078,289 base pairs. Bacterial growth was observed at 10-37 °C (optimum 25 °C) and pH 6.0-8.0 (optimum pH 6.0). The primary cellular fatty acids of strain BT507T are iso-C15:0, C16:1ω5c, and summed feature 3 (C16:1ω6c/C16:1ω7c). Its predominant respiratory quinone is MK-7. The primary polar lipids of strain BT507T are lipid, aminophospholipid, and phosphatidylethanolamine. Based on the biochemical, chemotaxonomic, and phylogenetic analysis, strains BT507T and BT506 can be described a novel bacterial species within the genus Hymenobacter, and the proposed name is Hymenobacter citatus. The type strain of H. citatus is BT507T is KCTC 82115T and NBRC 114850T.

Phyllobacterium pellucidum sp. nov., isolated from soil.

A bacterial strain, BT25T, was isolated from soil in Korea. The bacterial cells were Gram-negative and rod-shaped. Phylogenetic analysis using 16S rRNA gene sequences showed that the BT25T strain was related to the genus Phyllobacterium. BT25T was 96.6 and 96.5% similar to Phyllobacterium brassicacearum STM 196T and Phyllobacterium myrsinacearum DSM 5892T, respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between BT25T and the two closest phylogenetic neighbors were calculated to be 78.5 and 77.7, 21.1 and 21.2%, respectively. The major cellular fatty acids were summed feature 8 (C18:1 ω7c/C18:1 ω6c) (29.3%), cyclo-C19:0 ω8c (27.5%), and C16:0 (16.5%). The BT25T strain had menaquinone Q-10 as the predominant quinone, as well as phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and phosphatidylcholine as the major polar lipids. Based on the phenotypic, phylogenetic, and chemotaxonomic data, the BT25T strain was classified as a novel Phyllobacterium species. The name Phyllobacterium pellucidum sp. nov. was proposed. The type strain is BT25T (= KCTC 62765T = NBRC 114381T).

Pontibacter fetidus sp. nov. and Pontibacter burrus sp. nov., isolated from the soil.

Two novel strains, BT213T and BT327T, were isolated from the soil collected in Uijeongbu city, Korea. Cells of strains were Gram negative, aerobic, and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains BT213T and BT327T formed two distinct lineages within the family Hymenobacteraceae (order Cytophagales, class Cytophagia). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BT213T and BT327T belonged to the genus Pontibacter. Strain BT213T showed the highest similarities of 97.8% with Pontibacter deserti JC215T. Strain BT327T showed the highest sequence similarity of 96.5% with Pontibacter mucosus PB3T. Optimal growth occurred at 25 °C, pH 7, and in the absence of NaCl. The major cellular fatty acid of strains BT213T and BT327T were iso-C15:0 and summed feature 4 (iso-C17:1 I/anteiso-C17:1 B). Strains BT213T and BT327T had MK-7 as major respiratory quinone and phosphatidylethanolamine as major polar lipids. The genome size of strains BT213T and BT327T were 4,072,018 bp and 4,314,171 bp, respectively. The genomic G + C mol% of strains BT213T and BT327T are 45.6% and 46.1%, respectively. Based on biochemical, chemotaxonomic, and phylogenetic analysis, two novel species Pontibacter fetidus BT213T (KCTC 72345T = NBRC 114379T) and Pontibacter burrus BT327T (KCTC 72412T = NBRC 114376T) are proposed as type strains.

Hymenobacter negativus sp. nov., bacteria isolated from mountain soil collected in South Korea.

Two novel Gram-negative bacterial strains BT442T and BT584 were isolated from dry soil collected in mountains Busan and Guri, Korea during wintertime. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains BT442T and BT584 both belong to a distinct lineage within the genus Hymenobacter (family Hymenobacteraceae, order Cytophagales, class Cytophagia). Strain BT442T was closely related to Hymenobacter soli PB17T (98.0% 16S rRNA gene similarity) and Hymenobacter terrae POA9T (97.6%). No other recognized bacterial species showed more than 97% 16S rRNA gene sequence similarity to strains BT442T. The genome size of strain BT442T was 5,143,362 bp. Bacterial growth was observed at 10-30 °C (optimum 25 °C), pH 6.0-8.0 (optimum pH 6.0) in R2A agar and in the presence up to 1% NaCl. The major cellular fatty acids of strains BT442T and BT584 were iso-C15:0, anteiso-C15:0 and summed feature 3 (C16:1 ω6c / C16:1 ω7c). In addition, their predominant respiratory quinone was MK-7. The major polar lipids of strains BT442T and BT584 were identified to be phosphatidylethanolamine, aminophospholipid, and aminolipid. Based on the biochemical, chemotaxonomic, and phylogenetic analyses, strains BT442T and BT584 are novel bacterial species within the genus Hymenobacter, and the proposed name is Hymenobacter negativus. The strain type of Hymenobacter negativus is BT442T (= KCTC 72902T = NBRC XXXXT).

Nocardioides convexus sp. nov. and Nocardioides anomalus sp. nov., isolated from soil and mineral water.

Two bacterial strains designated as W3-2-3T and HKS04T were isolated from mineral water and a soil sample, respectively, in the Republic of Korea. The 16S rRNA genes of the two strains shared a sequence similarity of 93.5 %. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains W3-2-3T and HKS04T formed a distinct lineage within the genus Nocardioides of the family Nocardioidaceae (order Propionibacteriales). The closely related species of strain W3-2-3T were Nocardioides albidus (98.9 %), Nocardioides caeni (98.8 %), Nocardioides kongjuensis (98.6 %), Nocardioides aromaticivorans (98.5 %), Nocardioides nitrophenolicus (98.4 %), Nocardioides flava (98.2 %) and Nocardioides ginsengisoli (98.1 %). The closest species of strain HKS04T was Nocardioides halotolerans (98.7 %). The genome sizes of strains W3-2-3T and HKS04T were 4741198 and 5 120341 bp, respectively. The genomic DNA G+C contents of strains W3-2-3T and HKS04T were 73.3 and 72.1 mol%, respectively. The main fatty acids of strain W3-2-3T were C17:1 ω6c and iso-C16:0 and those of strain HKS04T were iso-C16:0 and iso-C16:0 H. The main polar lipids of both strains were diphosphatidylglycerol and phosphatidylglycerol and the predominant respiratory quinone was MK-8(H4), supporting the affiliation of these strains with the genus Nocardioides. Based on the results of biochemical, chemotaxonomic and phylogenetic analyses, two novel species, Nocardioides convexus W3-2-3T (KACC 21211T=LMG 31251T) and Nocardioides anomalus HKS04T (KACC 18879T=LMG 31249T), are proposed.

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.

Characterization of a novel cell wall binding domain-containing Staphylococcus aureus endolysin LysSA97.

Endolysin from Staphylococcus aureus phage SA97 (LysSA97) was cloned and investigated. LysSA97 specifically lyse the staphylococcal strains and effectively disrupted staphylococcal biofilms. Bioinformatic analysis of LysSA97 revealed a novel putative cell wall binding domain (CBD) as well as two enzymatically active domains (EADs) containing cysteine, histidine-dependent amidohydrolases/peptidases (CHAP, PF05257) and N-acetylmuramoyl-L-alanine amidase (Amidase-3, PF01520) domains. Comparison of 98 endolysin genes of S. aureus phages deposited in GenBank showed that they can be classified into six groups based on their domain composition. Interestingly, approximately 80.61 % of the staphylococcal endolysins have a src-homology 3 (SH3, PF08460) domain as CBD, but the remaining 19.39 %, including LysSA97, has a putative C-terminal CBD with no homology to the known CBD. The fusion protein containing green fluorescent protein and the putative CBD of LysSA97 showed a specific binding spectrum against staphylococcal cells comparable to SH3 domain (PF08460), suggesting that the C-terminal domain of LysSA97 is a novel CBD of staphylococcal endolysins.

Characterization of a novel endolysin LysSA11 and its utility as a potent biocontrol agent against Staphylococcus aureus on food and utensils.

Here we show that the LysSA11 endolysin, derived from the virulent Staphylococcus aureus phage SA11, has lytic activity against staphylococcal strains. Bioinformatics analysis revealed an enzymatically active CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain at the N-terminus of LysSA11 that showed amidase activity. A novel cell wall binding domain (CBD) in the C-terminus could bind to a broad spectrum of staphylococcal cells. The bactericidal activity of LysSA11 was determined in food and utensils artificially contaminated with methicillin-resistant S. aureus (MRSA). The amounts of MRSA bacteria in milk and on ham were significantly reduced by 1.44-log CFU/mL and 3.12-log CFU/cm3, respectively, within 15 min at refrigeration temperature (4 °C) and by 2.02-log CFU/mL and 3.37-log CFU/cm2, respectively, within 15 min at room temperature (25 °C). Moreover, a polypropylene plastic cutting board and a stainless steel knife artificially contaminated with approximately 4-log CFU/cm2 of MRSA also showed complete bacterial elimination after a 30-min treatment with 1.35 µM of LysSA11. The data presented here strongly suggest that the novel CBD-containing staphylococcal endolysin LysSA11 can be used both as a food antimicrobial and as a practical sanitizer for utensils.

Oregano essential oil-based natural antimicrobial packaging film to inactivate Salmonella enterica and yeasts/molds in the atmosphere surrounding cherry tomatoes.

This study investigated the effectiveness of a polyvinyl alcohol (PVA) film containing the natural antimicrobial oregano essential oil (OEO) as an active packaging application for decreasing the microbial growth. The film exerted an antimicrobial effect via the atmosphere surrounding the food rather than direct contact, thereby preserving the quality of cherry tomatoes. A packaging film containing microencapsulated OEO was developed. The loading content increased gradually (104.29-234.29 µg OEO/mg film) with the amount of OEO incorporated (1%, 2%, and 3%), where the PVA films containing 2% OEO had the highest loading efficiency (91.64%), followed by 1% OEO (90.96%) and 3% OEO (88.38%). The antimicrobial activities of the films were evaluated by applying it to fresh cherry tomatoes at 4 °C and 22 °C for 7 days. The large 2% OEO film as well as both the small and large 3% OEO films had strong antimicrobial effects against Salmonella enterica, molds and yeasts, and mesophilic aerobic bacteria. The changes in the hardness, weight, and color of the cherry tomatoes during storage did not differ significantly. The films could be utilized as a packaging material for fresh produce with antimicrobial effects because of the controlled atmosphere surrounding the food rather than by direct contact.

Development of Biopolymer Composite Films Using a Microfluidization Technique for Carboxymethylcellulose and Apple Skin Particles.

Biopolymer films based on apple skin powder (ASP) and carboxymethylcellulose (CMC) were developed with the addition of apple skin extract (ASE) and tartaric acid (TA). ASP/CMC composite films were prepared by mixing CMC with ASP solution using a microfluidization technique to reduce particle size. Then, various concentrations of ASE and TA were incorporated into the film solution as an antioxidant and an antimicrobial agent, respectively. Fourier transform infrared (FTIR), optical, mechanical, water barrier, and solubility properties of the developed films were then evaluated to determine the effects of ASE and TA on physicochemical properties. The films were also analyzed for antioxidant effect on 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and antimicrobial activities against Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica, and Shigella flexneri. From the results, the ASP/CMC film containing ASE and TA was revealed to enhance the mechanical, water barrier, and solubility properties. Moreover, it showed the additional antioxidant and antimicrobial properties for application as an active packaging film.

Characterization of a Vibrio parahaemolyticus-targeting lytic bacteriophage SSJ01 and its application in artificial seawater.

Vibrio parahaemolyticus is a major foodborne pathogen causing serious illnesses. In this study, a new lytic bacteriophage SSJ01 that infects V. parahaemolyticus was isolated and characterized. It had a short non-contractile tail and belonged to the Caudoviricetes class. It rapidly adsorbed onto host cells, exhibited a short latent period, and has a large burst size. It showed lytic activities under a broad range of temperature (- 18 to 60 °C), pH (5 to 11), and salinity (0 to 6%). It contained 35 open reading frames with a G + C content of 49.16% without toxic or lysogen-forming genes. The MOI of 105 phage-treated group in vitro reduced the target cells up to 3.49-log CFU/mL at 6 °C and 3.47-log CFU/mL at 25 °C, respectively. In aquatic environments (6 and 25 °C), bactericidal activities showed a significant decrease within 2 h. Therefore, the bacteriophage SSJ01 has potential as a biocontrol agent to control V. parahaemolyticus in marine culture.

Characterization and genomic analysis of two Staphylococcus aureus bacteriophages isolated from poultry/livestock farms.

Staphylococcus aureus is one of the most important pathogens, causing various diseases in humans and animals. As methicillin-resistant S. aureus (MRSA) has become increasingly prevalent, controlling this pathogen with standard antibiotic treatment has become challenging. Bacteriophages (phages) have attracted interest as alternative antibacterial agents to control MRSA. In this study, we isolated six S. aureus phages from soils of poultry/livestock farms. Based on the results of host range determination with 150 S. aureus strains and restriction enzyme treatment of phage DNA, two phages, designated SP5 and SP6, were selected for further characterization and genome sequencing. Both SP5 and SP6 were classified as members of the family Siphoviridae. The genome of SP5 comprises 43 305 bp and contains 63 ORFs, while the SP6 genome comprises 42 902 bp and contains 61 ORFs. Although they have different host spectra, the phage genomes exhibit high nucleotide similarity to each other. Adsorption assay results suggested that the host range determinants of the two phages are involved in both adsorption and infection. Comparative genomic analyses of the two phages provided evidence that the lysogenic/lytic control module and tail proteins may be important for host specificity.

Characterization and complete genome sequence analysis of Staphylococcus aureus bacteriophage SA12.

Staphylococcus aureus is a well-known pathogen that causes several serious diseases in humans and animals. As part of the efforts to control this pathogen, a newly isolated bacteriophage, SA12, which specifically targets S. aureus, was characterized, and its genome was completely sequenced. Host range and bacteriophage challenge tests demonstrated its specific and efficient host lysis of S. aureus. The genome of phage SA12 consists of 42,902 bp length double-stranded DNA with 58 predicted ORFs-encoding phage structure, DNA manipulation, packaging, host lysis, and regulation proteins. The characterization and genome study of phage SA12 in this report is useful for understanding S. aureus-targeting bacteriophages and provides basic information for the further development of phage-based biocontrol agents against S. aureus.

Complete Genome Sequence of the Bacillus cereus Temperate Bacteriophage BSG01.

The Bacillus cereus phage BSG01 has a siphovirus morphology that can belong to the order Caudovirales. It consists of 81,366 bp, with a GC content of 34.6%, and contains 70 predicted open reading frames. BSG01 includes lysogeny-related genes (tyrosine recombinase and antirepressor protein), indicating that it is a temperate phage.

Complete Genome Sequence of Bacteriophage EO1, Which Infects Both Escherichia coli O157:H7 and Shigella.

The lytic bacteriophage EO1 has been newly isolated. This phage infects Escherichia coli O157:H7 and has a broad antibacterial spectrum, including against Shigella. The complete genome sequence of phage EO1 was determined; its full length is 166,941 bp, and it has a G+C content of 35.46%.