Journal of Microbiology

Reviews

Emerging synthetic biology-assisted technologies for overcoming antibiotic resistance: CRISPR-Cas, bacteriophage, microbiome, and metabolic engineering-based solutions: Yujeong Oh, Hyunjin Lee, Sungho Jang; J. Microbiol. 2026;64(3):e2512002. Published online March 31, 2026; DOI: https://doi.org/10.71150/jm.2512002

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Antibiotic resistance has become a critical global health challenge due to the decreased efficacy of existing antibiotics and the emergence of multidrug-resistant pathogens. In particular, the rapid horizontal transfer of resistance genes and the diverse mechanisms by which bacteria acquire resistance have significantly undermined the effectiveness of conventional therapeutic strategies, revealing fundamental limitations in current infectious disease management. In this context, synthetic biology provides a promising framework to overcome the limitations of conventional antibiotics by integrating engineering principles with bioengineering approaches, thereby enabling precise and programmable control of biological processes. These synthetic biology-based approaches offer substantial potential for developing sustainable and highly specific antimicrobial strategies. This review comprehensively examines recent advances in synthetic biology-assisted antimicrobial strategies, including CRISPR-Cas systems, bacteriophage engineering, microbiome engineering, and metabolic engineering-driven antibiotic discovery. Collectively, these approaches represent a precision antimicrobial paradigm that enables selective targeting of resistant bacteria while preserving microbiome homeostasis. These strategies also provide new directions for limiting resistance dissemination and guiding the development of next-generation therapeutics.

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Pioneering strategies for overcoming bacterial drug resistance
Byoung Sik Kim
Journal of Microbiology.2026; 64(3): e2603100. CrossRef

Armored RNA technology as a clinical diagnostics tool for future pandemic preparedness: Jin Hao Tan, Prashant Mainali, Wei Zhang, Dave Siak-Wei Ow; J. Microbiol. 2026;64(2):e2510016. Published online February 28, 2026; DOI: https://doi.org/10.71150/jm.2510016

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Abstract PDF: The COVID-19 pandemic highlighted the critical role of reliable molecular diagnostics in outbreak response and the vulnerabilities of existing systems to delays and reagent instability. Armored RNA technology, which packages RNA within bacteriophage-derived capsids, offers a robust solution by combining nuclease resistance, safety, and versatility into a single platform. Armored RNA has become a trusted internal and external control for RT-qPCR and RT-LAMP, enabling accurate detection across a wide range of viral pathogens. Also, recent advances in alternative expression systems, such as plant-based and cell-free platforms, as well as the use of more stable scaffolds from bacteriophage Qβ, are enhancing yield, stability, and accessibility of armored RNA. Engineering innovations, including capsid polymorphism and optimized downstream purification, further improve efficiency and broaden possible applications. Looking ahead, armored RNA holds promise not only as a diagnostic standard but also as a delivery vehicle for vaccines and therapeutics. Encapsulation of self-amplifying RNA, small interfering RNA, or microRNA could open new pathways for rapid-response vaccines and targeted therapies, aligning this technology with the future of precision medicine. By uniting stability, scalability, and adaptability, armored RNA represents a critical component of global health preparedness, with the potential to strengthen diagnostic resilience and accelerate biomedical countermeasures in future pandemics.

Articles

Characterization of novel bacteriophages for effective phage therapy against Vibrio infections in aquaculture: Kira Moon, Sangdon Ryu, Seung Hui Song, Se Won Chun, Nakyeong Lee, Aslan Hwanhwi Lee; J. Microbiol. 2025;63(5):e2502009. Published online May 27, 2025; DOI: https://doi.org/10.71150/jm.2502009

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Abstract PDF Supplementary Material

The widespread use of antibiotics in aquaculture has led to the emergence of multidrug-resistant pathogens and environmental concerns, highlighting the need for sustainable, eco-friendly alternatives. In this study, we isolated and characterized three novel bacteriophages from aquaculture effluents in Korean shrimp farms that target the key Vibrio pathogens, Vibrio harveyi, and Vibrio parahaemolyticus. Bacteriophages were isolated through environmental enrichment and serial purification using double-layer agar assays. Transmission electron microscopy revealed that the phages infecting V. harveyi, designated as vB_VhaS-MS01 and vB_VhaS-MS03, exhibited typical Siphoviridae morphology with long contractile tails and icosahedral heads, whereas the phage isolated from V. parahaemolyticus (vB_VpaP-MS02) displayed Podoviridae characteristics with an icosahedral head and short tail.

Whole-genome sequencing produced complete, circularized genomes of 81,710 bp for vB_VhaS-MS01, 81,874 bp for vB_VhaS-MS03, and 76,865 bp for vB_VpaP-MS02, each showing a modular genome organization typical of Caudoviricetes. Genomic and phylogenetic analyses based on the terminase large subunit gene revealed that although vB_VhaS-MS01 and vB_VhaS-MS03 were closely related, vB_VpaP-MS02 exhibited a distinct genomic architecture that reflects its unique morphology and host specificity. Collectively, these comparative analyses demonstrated that all three phages possess genetic sequences markedly different from those of previously reported bacteriophages, thereby establishing their novelty. One-step growth and multiplicity of infection (MOI) experiments demonstrated significant differences in replication kinetics, such as burst size and lytic efficiency, among the phages, with vB_VhaS-MS03 maintaining the most effective bacterial control, even at an MOI of 0.01. Additionally, host range assays showed that vB_VhaS-MS03 possessed a broader spectrum of activity, supporting its potential use as a stand-alone agent or key component of phage cocktails. These findings highlight the potential of region-specific phage therapy as a targeted and sustainable alternative to antibiotics for controlling Vibrio infections in aquaculture.

Citations

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Revolutionizing seafood safety with bacteriophages: emerging technologies and applications
Nigar Sultana Meghla, Soo-Jin Jung, Md Furkanur Rahaman Mizan, Syeda Roufun Nesa, IkSoon Kang, Sang-Do Ha
Food Microbiology.2026; 137: 105021. CrossRef
Genomic characterization of APEC phages and evaluation of the efficacy in reducing the loads of APEC O78 infections in chickens
Qin Lu, Xinxin Jin, Zui Wang, Rongrong Zhang, Yunqing Guo, Qiao Hu, Wenting Zhang, Tengfei Zhang, Qingping Luo
Frontiers in Microbiology.2026;[Epub] CrossRef
Feed Additives in Aquaculture: Benefits, Risks, and the Need for Robust Regulatory Frameworks
Ekemini Okon, Matthew Iyobhebhe, Paul Olatunji, Mary Adeleke, Nelson Matekwe, Reuben Okocha
Fishes.2025; 10(9): 471. CrossRef

Single nucleotide genome recognition and selective bacterial lysis using synthetic phages loaded with CRISPR-Cas12f1-truncated sgRNA: Ho Joung Lee, Song Hee Jeong, Sang Jun Lee; J. Microbiol. 2025;63(2):e2501012. Published online February 27, 2025; DOI: https://doi.org/10.71150/jm.2501012

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Abstract PDF Supplementary Material: Phage specificity primarily relies on host cell-surface receptors. However, integrating cas genes and guide RNAs into phage genomes could enhance their target specificity and regulatory effects. In this study, we developed a CRISPR-Cas12f1 system-equipped bacteriophage λ model capable of detecting Escherichia coli target genes. We demonstrated that synthetic λ phages carrying Cas12f1-sgRNA can effectively prevent lysogen formation. Furthermore, we showcased that truncating the 3'-end of sgRNA enables precise identification of single-nucleotide variations in the host genome. Moreover, infecting E. coli strains carrying various stx2 gene subtypes encoding Shiga toxin with bacteriophages harboring Cas12f1 and truncated sgRNAs resulted in the targeted elimination of strains with matching subtype genes. These findings underscore the ability of phages equipped with the CRISPR-Cas12f1 system to precisely control microbial hosts by recognizing genomic sequences with high resolution.

Characterization of Newly Isolated Bacteriophages Targeting Carbapenem-Resistant Klebsiella pneumoniae: Bokyung Kim, Shukho Kim, Yoon-Jung Choi, Minsang Shin, Jungmin Kim; J. Microbiol. 2024;62(12):1133-1153. Published online December 10, 2024; DOI: https://doi.org/10.1007/s12275-024-00180-7

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Abstract PDF

Klebsiella pneumoniae, a Gram-negative opportunistic pathogen, is increasingly resistant to carbapenems in clinical settings. This growing problem necessitates the development of alternative antibiotics, with phage therapy being one promising option. In this study, we investigated novel phages targeting carbapenem-resistant Klebsiella pneumoniae (CRKP) and evaluated their lytic capacity against clinical isolates of CRKP. First, 23 CRKP clinical isolates were characterized using Multi-Locus Sequence Typing (MLST), carbapenemase test, string test, and capsule typing. MLST classified the 23 K. pneumoniae isolates into 10 sequence types (STs), with the capsule types divided into nine known and one unknown type. From sewage samples collected from a tertiary hospital, 38 phages were isolated. Phenotypic and genotypic characterization of these phages was performed using Random Amplification of Polymorphic DNA-PCR (RAPD-PCR), transmission electron microscopy (TEM), and whole genome sequencing (WGS) analysis. Host spectrum analysis revealed that each phage selectively lysed strains sharing the same STs as their hosts, indicating ST-specific activity. These phages were subtyped based on their host spectrum and RAPD-PCR, identifying nine and five groups, respectively. Fourteen phages were selected for further analysis using TEM and WGS, revealing 13 Myoviruses and one Podovirus. Genomic analysis grouped the phages into three clusters: one closely related to Alcyoneusvirus, one to Autographiviridae, and others to Straboviridae. Our results showed that the host spectrum of K. pneumoniae-specific phages corresponds to the STs of the host strain. These 14 novel phages also hold promise as valuable resources for phage therapy against CRKP.

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Antibacterial activity of single Klebsiella phage and phage cocktail against clinical carbapenem-resistant Klebsiella pneumoniae in vitro
Min Li, Ruixuan Guo, Zitai Qi, Yongjie Liu, Jinfeng Xu, Fei Mao, Wei Zhang, XiaoXiang Zhou
Virology.2026; 618: 110840. CrossRef
Molecular Insights into Carbapenem Resistance in Klebsiella pneumoniae: From Mobile Genetic Elements to Precision Diagnostics and Infection Control
Ayman Elbehiry, Eman Marzouk, Adil Abalkhail
International Journal of Molecular Sciences.2026; 27(3): 1229. CrossRef
Evaluation of Bacteriophage and Antibiotic Synergy Against Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates
Bokyung Kim, Shukho Kim, Yoon-Jung Choi, Minsang Shin, Jungmin Kim
Journal of Bacteriology and Virology.2025; 55(2): 131. CrossRef
Possible regulatory network and associated pathways governing the expression of ADH2 in Saccharomyces cerevisiae
Pratima Sarkar, Rohan Nath, Prity Adhikary, Arindam Bhattacharjee
Current Genetics.2025;[Epub] CrossRef

Enterococcus Phage vB_EfaS_HEf13 as an Anti-Biofilm Agent against Enterococcus faecalis: Dongwook Lee, Jintaek Im, A Reum Kim, Woohyung Jun, Cheol-Heui Yun, Seung Hyun Han; J. Microbiol. 2024;62(8):683-693. Published online June 27, 2024; DOI: https://doi.org/10.1007/s12275-024-00150-z

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Abstract PDF

Enterococcus faecalis is a Gram-positive bacterium that is frequently found in the periapical lesion of patients with apical periodontitis. Its biofilm formation in root canal is closely related to the development of refractory apical periodontitis by providing increased resistance to endodontic treatments. Phage therapy has recently been considered as an efficient therapeutic strategy in controlling various periodontal pathogens. We previously demonstrated the bactericidal capacities of Enterococcus phage vB_EfaS_HEf13 (phage HEf13) against clinically-isolated E. faecalis strains. Here, we investigated whether phage HEf13 affects biofilm formation and pre-formed biofilm of clinically-isolated E. faecalis, and its combinatory effect with endodontic treatments, including chlorhexidine (CHX) and penicillin. The phage HEf13 inhibited biofilm formation and disrupted pre-formed biofilms of E. faecalis in a dose- and time-dependent manner. Interestingly, phage HEf13 destroyed E. faecalis biofilm exopolysaccharide (EPS), which is known to be a major component of bacterial biofilm. Furthermore, combined treatment of phage HEf13 with CHX or penicillin more potently inhibited biofilm formation and disrupted pre-formed biofilm than either treatment alone. Confocal laser scanning microscopic examination demonstrated that these additive effects of the combination treatments on disruption of pre-formed biofilm are mediated by relatively enhanced reduction in thickness distribution and biomass of biofilm. Collectively, our results suggest that the effect of phage HEf13 on E. faecalis biofilm is mediated by its EPS-degrading property, and its combination with endodontic treatments more potently suppresses E. faecalis biofilm, implying that phage HEf13 has potential to be used as a combination therapy against E. faecalis infections.

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Characteristics, Whole Genome Analysis of a Virulent Phage from Avian-Derived Enterococcus faecalis and Its Application in Poultry Product Processing Safety
Xiaoming Li, Mengli Zhao, Lei Zhang, Guobo Sun, Xiujun Duan, Guoshun Chen
Microorganisms.2026; 14(2): 490. CrossRef
Size-dependent ecotoxicological impacts of tire wear particles on zebrafish physiology and gut microbiota: Implications for aquatic ecosystem health
Yun Zhang, Qianqian Song, Qingxuan Meng, Tianyu Zhao, Xiaolong Wang, Xinrui Meng, Jing Cong
Journal of Hazardous Materials.2025; 487: 137215. CrossRef
Phage therapy as a revitalized weapon for treating clinical diseases
Yingjie Wang, Yamei Yu
Microbiome Research Reports.2025;[Epub] CrossRef

Protocol

Use of Cas9 Targeting and Red Recombination for Designer Phage Engineering: Shin-Yae Choi , Danitza Xiomara Romero-Calle , Han-Gyu Cho , Hee-Won Bae , You-Hee Cho; J. Microbiol. 2024;62(1):1-10. Published online February 1, 2024; DOI: https://doi.org/10.1007/s12275-024-00107-2

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Abstract PDF

Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa. This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35 gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA. The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules. This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.

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Climate change and the immune system
Ger T. Rijkers, Zhibek Timraliyeva, Emma Mackie, Matei Lupuşoara
Expert Review of Clinical Immunology.2026; 22(1): 61. CrossRef
CRISPR/Cas9-engineered Salmonella phage displaying antimicrobial peptide LL37 for enhanced antibacterial activity
Su Jin Jo, Se Chang Park, Sang Guen Kim
International Journal of Antimicrobial Agents.2026; 67(4): 107734. CrossRef
Pilin regions that select for the small RNA phages in Pseudomonas aeruginosa type IV pilus
Hee-Won Bae, Hyeong-Jun Ki, Shin-Yae Choi, You-Hee Cho, Kristin N. Parent
Journal of Virology.2025;[Epub] CrossRef
Synthetic and Functional Engineering of Bacteriophages: Approaches for Tailored Bactericidal, Diagnostic, and Delivery Platforms
Ola Alessa, Yoshifumi Aiba, Mahmoud Arbaah, Yuya Hidaka, Shinya Watanabe, Kazuhiko Miyanaga, Dhammika Leshan Wannigama, Longzhu Cui
Molecules.2025; 30(15): 3132. CrossRef
Characteristics of bioaerosols under high-ozone periods, haze episodes, dust storms, and normal days in Xi’an, China
Yiming Yang, Liu Yang, Xiaoyan Hu, Zhenxing Shen
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Airborne desert dust and aeromicrobiology over the Turkish Mediterranean coastline
Dale W. Griffin, Nilgün Kubilay, Mustafa Koçak, Mike A. Gray, Timothy C. Borden, Eugene A. Shinn
Atmospheric Environment.2007; 41(19): 4050. CrossRef

Article

Molecular mechanism of Escherichia coli H10407 induced diarrhoea and its control through immunomodulatory action of bioactives from Simarouba amara (Aubl.): Hegde Veena , Sandesh K. Gowda , Rajeshwara N. Achur , Nayaka Boramuthi Thippeswamy; J. Microbiol. 2021;59(4):435-447. Published online February 25, 2021; DOI: https://doi.org/10.1007/s12275-021-0423-2

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Abstract PDF

Enterotoxigenic Escherichia coli (ETEC) infection is a major cause of death in children under the age of five in developing countries. ETEC (O78:H11:CFA/I:LT+:ST+) mechanism has been studied in detail with either heat labile (LT) or heat stable (ST) toxins using in vitro and in vivo models. However, there is no adequate information on ETEC pathogenesis producing both the toxins (LT, ST) in BALB/c mice model. In this study, female mice have been employed to understand ETEC H10407 infection induced changes in physiology, biochemical and immunological patterns up to seven days post-infection and the antidiarrhoeal effect of Simarouba amara (Aubl.) bark aqueous extract (SAAE) has also been looked into. The results indicate that BALB/c is sensitive to ETEC infection resulting in altered jejunum and ileum histomorphology. Withal, ETEC influenced cAMP, PGE2, and NO production resulting in fluid accumulation with varied Na+, K+, Cl-, and Ca2+ levels. Meanwhile, ETEC subverted expression of IL-1β, intestine alkaline phosphatase (IAP), and myeloperoxidase (MPO) in jejunum and ileum. Our data also indicate the severity of pathogenesis reduction which might be due to attainment of equilibrium after reaching optimum rate of infection. Nevertheless, degree of pathogenesis was highly significant (p < 0.01) in all the studied parameters. Besides that, SAAE was successful in reducing the infectious diarrhoea by inhibiting ETEC H10407 in intestine (jejunum and ileum), and shedding in feces. SAAE decreased cAMP, PGE2, and fluid accumulation effectively and boosted the functional activity of immune system in jejunum and ileum IAP, MPO, IL-1β, and nitric oxide.

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Relaxed Cleavage Specificity of Hyperactive Variants of Escherichia coli RNase E on RNA I
Dayeong Bae, Hana Hyeon, Eunkyoung Shin, Ji-Hyun Yeom, Kangseok Lee
Journal of Microbiology.2023; 61(2): 211. CrossRef
A systematic antidiarrhoeal evaluation of a vegetable root Begonia roxburghii and its marker flavonoids against nonpathogenic and pathogenic diarrhoea
Rupali S. Prasad, Nikhil Y. Yenorkar, Suhas R. Dhaswadikar, Saurabh K. Sinha, Nitish Rai, Pravesh Sharma, Onkar Kulkarni, Neeraj Kumar, Mahaveer Dhobi, Damiki Laloo, Shailendra S. Gurav, Prakash R. Itankar, Satyendra K. Prasad
Food Bioscience.2023; 53: 102672. CrossRef

Review

Dissection of plant microbiota and plant-microbiome interactions: Kihyuck Choi , Raees Khan , Seon-Woo Lee; J. Microbiol. 2021;59(3):281-291. Published online February 23, 2021; DOI: https://doi.org/10.1007/s12275-021-0619-5

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Abstract PDF

Plants rooted in soil have intimate associations with a diverse array of soil microorganisms. While the microbial diversity of soil is enormous, the predominant bacterial phyla associated with plants include Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. Plants supply nutrient niches for microbes, and microbes support plant functions such as plant growth, development, and stress tolerance. The interdependent interaction between the host plant and its microbes sculpts the plant microbiota. Plant and microbiome interactions are a good model system for understanding the traits in eukaryotic organisms from a holobiont perspective. The holobiont concept of plants, as a consequence of co-evolution of plant host and microbiota, treats plants as a discrete ecological unit assembled with their microbiota. Dissection of plant-microbiome interactions is highly complicated; however, some reductionist approaches are useful, such as the synthetic community method in a gnotobiotic system. Deciphering the interactions between plant and microbiome by this reductionist approach could lead to better elucidation of the functions of microbiota in plants. In addition, analysis of microbial communities’ interactions would further enhance our understanding of coordinated plant microbiota functions. Ultimately, better understanding of plantmicrobiome interactions could be translated to improvements in plant productivity.

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Articles

Leucobacter coleopterorum sp. nov., Leucobacter insecticola sp. nov., and Leucobacter viscericola sp. nov., isolated from the intestine of the diving beetles, Cybister brevis and Cybister lewisianus, and emended description of the genus Leucobacter: Dong-Wook Hyun , Hojun Sung , Pil Soo Kim , Ji-Hyun Yun , Jin-Woo Bae; J. Microbiol. 2021;59(4):360-368. Published online January 26, 2021; DOI: https://doi.org/10.1007/s12275-021-0472-6

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Three novel bacterial strains, HDW9AT, HDW9BT, and HDW9CT, isolated from the intestine of the diving beetles Cybister lewisianus and Cybister brevis, were characterized as three novel species using a polyphasic approach. The isolates were Gram-staining-positive, strictly aerobic, non-motile, and rod-shaped. They grew optimally at 30°C (pH 7) in the presence of 0.5% (wt/vol) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that they belong to the genus Leucobacter and are closely related to L. denitrificans M1T8B10T (98.4–98.7% sequence similarity). Average nucleotide identity (ANI) values among the isolates were 76.4–84.1%. ANI values for the isolates and the closest taxonomic species, L. denitrificans KACC 14055T, were 72.3–73.1%. The isolates showed ANI values of < 76.5% with all analyzable Leucobacter strains in the EzBioCloud database. The genomic DNA G + C content of the isolates was 60.3–62.5%. The polar lipid components were phosphatidylglycerol, diphosphatidylglycerol, and other unidentified glycolipids, phospholipids, and lipids. The major cellular fatty acids were anteiso- C15:0, iso-C16:0, and anteiso-C17:0. MK-10 was the major respiratory quinone, and MK-7 and MK-11 were the minor respiratory quinones. The whole-cell sugar components of the isolates were ribose, glucose, galactose, and mannose. The isolates harbored L-2,4-diaminobutyric acid, L-serine, L-lysine, L-aspartic acid, glycine, and D-glutamic acid within the cell wall peptidoglycan. Based on phylogenetic, phenotypic, chemotaxonomic, and genotypic analyses, strains HDW9AT, HDW9BT, and HDW9CT represent three novel species within the genus Leucobacter. We propose the name Leucobacter coleopterorum sp. nov. for strain HDW9AT (= KACC 21331T = KCTC 49317T = JCM 33667T), the name Leucobacter insecticola sp. nov. for strain HDW9BT (= KACC 21332T = KCTC 49318T = JCM 33668T), and the name Leucobacter viscericola sp. nov. for strain HDW9CT (= KACC 21333T = KCTC 49319T = JCM 33669T).

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Characterization of a Salmonella Enteritidis bacteriophage showing broad lytic activity against Gram-negative enteric bacteria: Shukho Kim , Sung-Hun Kim , Marzia Rahman , Jungmin Kim; J. Microbiol. 2018;56(12):917-925. Published online October 25, 2018; DOI: https://doi.org/10.1007/s12275-018-8310-1

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In this study, we sought to isolate Salmonella Enteritidis-specific lytic bacteriophages (phages), and we found a lytic phage that could lyse not only S. Enteritidis but also other Gramnegative foodborne pathogens. This lytic phage, SS3e, could lyse almost all tested Salmonella enterica serovars as well as other enteric pathogenic bacteria including Escherichia coli, Shigella sonnei, Enterobacter cloacae, and Serratia marcescens. This SS3e phage has an icosahedral head and a long tail, indicating belong to the Siphoviridae. The genome was 40,793 base pairs, containing 58 theoretically determined open reading frames (ORFs). Among the 58 ORFs, ORF49, and ORF25 showed high sequence similarity with tail spike protein and lysozyme-like protein of Salmonella phage SE2, respectively, which are critical proteins recognizing and lysing host bacteria. Unlike SE2 phage whose host restricted to Salmonella enterica serovars Enteritidis and Gallinarum, SS3e showed broader host specificity against Gram-negative enteric bacteria; thus, it could be a promising candidate for the phage utilization against various Gram-negative bacterial infection including foodborne pathogens.

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Comparative Genomic Analysis of Bacteriophage EP23 Infecting Shigella sonnei and Escherichia coli: Ho-Won Chang , Kyoung-Ho Kim; J. Microbiol. 2011;49(6):927-934. Published online December 28, 2011; DOI: https://doi.org/10.1007/s12275-011-1577-0

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Bacteriophage EP23 that infects Escherichia coli and Shigella sonnei was isolated and characterized. The bacteriophage morphology was similar to members of the family Siphoviridae. The 44,077 bp genome was fully sequenced using 454 pyrosequencing. Comparative genomic and phylogenetic analyses showed that EP23 was most closely related to phage SO-1, which infects Sodalis glossinidius and phage SSL-2009a, which infects engineered E. coli. Genomic comparison indicated that EP23 and SO-1 were very similar with each other in terms of gene order and amino acid similarity, even though their hosts were separated in the level of genus. EP23 and SSL-2009a displayed high amino acid similarity between their genes, but there was evidence of several recombination events in SSL-2009a. The results of the comparative genomic analyses further the understanding of the evolution and relationship between EP23 and its bacteriophage relatives.

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Antibacterial Efficacy of Lytic Pseudomonas Bacteriophage in Normal and Neutropenic Mice Models: Birendra R. Tiwari , Shukho Kim , Marzia Rahman , Jungmin Kim; J. Microbiol. 2011;49(6):994-999. Published online December 28, 2011; DOI: https://doi.org/10.1007/s12275-011-1512-4

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Abstract PDF

Recently, lytic bacteriophages (phages) have been focused on treating bacterial infectious diseases. We investigated the protective efficacy of a novel Pseudomonas aeruginosa phage, PA1Ø, in normal and neutropenic mice. A lethal dose of P. aeruginosa PAO1 was administered via the intraperitoneal route and a single dose of PA1Ø with different multiplicities of infection (MOI) was treated into infected mice. Immunocompetent mice infected with P. aeruginosa PAO1 were successfully protected by PA1Ø of 1 MOI, 10 MOI or 100 MOI with 80% to 100% survival rate. No viable bacteria were found in organ samples after 48 h of the phage treatment. Phage clearing patterns were different in the presence or absence of host bacteria but PA1Ø disappeared from all organs after 72 h except spleen in the presence of host bacteria. On the contrary, PA1Ø treatment could not protect neutropenic mice infected with P. aeruginosa PAO1 even though could extend their lives for a short time. In in vitro phage-neutrophil bactericidal test, a stronger bactericidal effect was observed in phage-neutrophil co-treatment than in phage single treatment without neutrophils, suggesting phage-neutrophil co-work is essential for the efficient killing of bacteria in the mouse model. In conclusion, PA1Ø can be possibly utilized in future phage therapy endeavors since it exhibited strong protective effects against virulent P. aeruginosa infection.

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Phenotypic Characterization and Genomic Analysis of the Shigella sonnei Bacteriophage SP18: Kyoung-Ho Kim , Ho-Won Chang , Young-Do Nam , Seong Woon Roh , Jin-Woo Bae; J. Microbiol. 2010;48(2):213-222. Published online May 1, 2010; DOI: https://doi.org/10.1007/s12275-010-0055-4

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Abstract PDF

A novel bacteriophage that infects Shigella sonnei was isolated from the Gap River in Korea, and its phenotypic and genomic characteristics were investigated. The virus, called SP18, showed morphology characteristic of the family Myoviridae, and phylogenetic analysis of major capsid gene (gp23) sequences classified it as a T4-like phage. Based on host spectrum analysis, it is lytic to S. sonnei, but not to Shigella flexneri, Shigella boydii or members of the genera Escherichia and Salmonella. Pyrosequencing of the SP18 bacteriophage genome revealed a 170-kb length sequence. In total, 286 ORFs and 3 tRNA genes were identified, and 259 ORFs showed similarity (BLASTP e-value<0.001) to genes of other bacteriophages. The results from comparative genomic analysis indicated that the enterophage JS98, isolated from human stool, is the closest relative of SP18. Based on phylogenetic analysis of gp23 protein-coding sequences, dot plot comparison and BLASTP analysis of genomes, SP18 and JS98 appear to be closely related to T4-even phages. However, several insertions, deletions, and duplications indicate differences between SP18 and JS98. Comparison of duplicated gp24 genes and the soc gene showed that duplication events are responsible for the differentiation and evolution of T4-like bacteriophages.

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An Examination of the Bacteriophages and Bacteria of the Namib Desert: Eric Prestel , Sylvie Salamitou , Michael S. DuBow; J. Microbiol. 2008;46(4):364-372. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0007-4

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Abstract PDF

Bacteria and their viruses (called bacteriophages, or phages), have been found in virtually every ecological niche on Earth. Arid regions, including their most extreme form called deserts, represent the single largest ecosystem type on the Earth''s terrestrial surface. The Namib desert is believed to be the oldest (80 million years) desert. We report here an initial analysis of bacteriophages isolated from the Namib desert using a combination of electron microscopy and genomic approaches. The virus-like particles observed by electron microscopy revealed 20 seemingly different phage-like morphologies and sizes belonging to the Myoviridae and Siphoviridae families of tailed phages. Pulsed-field gel electrophoresis revealed a majority of phage genomes of 55~65 kb in length, with genomes of approximately 200, 300, and 350 kb also observable. Sample sequencing of cloned phage DNA fragments revealed that approximately 50% appeared to be of bacterial origin. Of the remaining DNA sequences, approximately 50% displayed no significant match to any sequence in the databases. The majority of the 16S rDNA sequences amplified from DNA extracted from the sand displayed considerable (94~98%) homology to members of the Firmicutes, and in particular to members of the genus Bacillus, though members of the Bacteroidetes, Planctomycetes, Chloroflexi, and delta-Proteobacteria groups were also observed.

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Development of a Simple Cell Lysis Method for Recombinant DNA Using Bacteriophage Lambda Lysis Genes: Boyun Jang , Yuna Jung , Dongbin Lim; J. Microbiol. 2007;45(6):593-596.; DOI: https://doi.org/2602 [pii]

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Abstract PDF: In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.

Isolation and Characterization of the Smallest Bacteriophage P4 Derivatives Packaged into P4-Size Head in Bacteriophage P2-P4 System: Kyoung-Jin Kim , Jaeho Song; J. Microbiol. 2006;44(5):530-536.; DOI: https://doi.org/2444 [pii]

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Abstract PDF: Bacteriophage P4, a satellite phage of coliphage P2, is a very useful experimental tool for the study of viral capsid assembly and cos-cleavage. For an in vitro cos-cleavage reaction study of the P2-P4 system, new shortened and selectable markers containing P4 derivative plasmids were designed as a substrate molecules. They were constructed by swapping the non-essential segment of P4 DNA for either the kanamycin resistance (kmr) gene or the ampicillin resistance (apr) gene. The size of the genomes of the resulting markers were 82% (P4 ash8 delRI:: kmr) and 79% (P4 ash8 delRI:: apr) of the wild type P4 genome. To determine the lower limit of genome size that could be packaged into the small P4-size head, these shortened P4 plasmids were converted to phage particles with infection of the helper phage P2. The conversion of plasmid P4 derivatives to bacteriophage particles was verified by the heat stability test and the burst size determination experiment. CsCl buoyant equilibrium density gradient experiments confirmed not only the genome size of the viable phage form of shortened P4 derivatives, but also their packaging into the small P4-size head. P4 ash8 delRI:: apr turned out to be the smallest P4 genome that can be packaged into P4-sized head.

Purification of Filamentous Bacteriophage M13 by Expanded Bed Anion Exchange Chromatography: Tau Chuan Ling , Chee Kin Loong , Wen Siang Tan , Beng Ti Tey , Wan Mohammad Wan Abdullah , Arbakariya Ariff; J. Microbiol. 2004;42(3):228-232.; DOI: https://doi.org/2084 [pii]

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Abstract PDF: In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock. M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography. In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed. In the single step expanded bed anion exchange adsorption, UpFront FastLine^TM 20 (20 mm i.d.) from UpFront Chromatography was used as the contactor, while 54 ml (H_o=15cm) of STREAMLINE DEAE (r=1.2 g/cm^3) from Amersham Pharmacia Biotechnology was used as the anion exchanger. The performance of the two methods were evaluated, analysed, and compared. It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86%. The conventional multiple step method yielded the lower recovery percentage, 36.07%. The generic application of this integrated technique has also been assessed.

An Analysis of the Arm-type Site Binding Domain of Bacteriophage γ Integrase: Cho , Eun Hee; J. Microbiol. 1995;33(2):165-170.

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Abstract PDF: The 356 amino acid long lambda integrase protein of bacteriophage λ constains two autonomous DNA binding domains with distinct sequence specificities. The amino terminal domain of integrase is implicated to bind to the arm-type sequences and the carboxyl domain interacts with the coretype sequencess. As a first step to understand the molecular mechanism of the integrase-DNA interaction at the arm-type site, the int(am)94 gene carrying an amber mutation at the 94th codon of the int was cloned under the control of the P_tac promoter and the lacI^q gene. The Int(am)94 mutant protein of amino terminal 93 amino acid residues can be produced at high level from a suppressor free strain harboring the plasmid pInt(am)94. The arm-type binding activity of Int(am)94 were measured in vivo and in vitro. A comparison of the arm-type binding properties of the wild-type integrase and the truncated Int(am)94 mutant indicated that the truncated fragment containing 93 amino acid residues carry all the determinants for DNA binding at the arm-type sites.

Characterization of a Phage Library Displaying Random 22mer Peptides: Lee, Seung Joo , Lee, Jeong Hwan , Brian K. Kay , Gideon Dreyfuss , Park, Yong Keun , Kim, Jeong Kook; J. Microbiol. 1997;35(4):347-353.

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Abstract PDF: We have characterized a phage library displaying random 22mer peptides which were produced as N-terminal fusions to the pIII coat protein of M13 filamentous phages. Among the sixty phages randomly picked from the library, 25 phages had the 22mer peptide inserts. The DNA sequence analysis of the 25 inserts showed the following results: first, each nucleotide was represented almost equally at each codon position except that there were some biases toward G bases at the first position of the codons. Secondly, the expected 47 sense codons were represented. The deduced amino acid sequences of the 25 inserts were analyzed to examine its diversity. Glycine and glutamate were the two most overrepresented residues above the expected value, whereas cysteine and threonine residues were underrepresented. The range of dicersity in dipeptide sequences showed that the amino acid residues were randomly distributed along the peptide insert. Acidic, basic, polar, and nonpolar amino acid residues were represented to the extent expected at most positions of the peptide inserts. The predicted isoelectric points and hydropathy indices of the 25 peptides showed that a variety of the peptide were represented in the library. These results indicate that this phage display library could be useful in fiuding ligands for a broad spectrum of receptors by affinity screening.

Characterization and Identification of the Bacteriophage P4 Mutant Suppressin sir Mutations of Bacteriophage P2: Kim, Kyoung Jin , Sunshine, Melvin G. , Six, Erich W.; J. Microbiol. 1998;36(4):262-265.

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Abstract PDF: Bacteriophage P4 ost1 was isolated as a suppressor mutant of P2 sir3 and identified by restriction enzyme site analysis. The mutant DNA turned out to be an imperfect P4 trimer containing deletions. It was suggested that the deletion resulted from int-mediated site-specific recombination. CsCl equilibrium density gradient experiment confirmed the genome size of P4 ostl.

Construction of a Hexapeptide Library using Phage Display for Bio-panning: Cho, Won Hee , Yoo, Seung Ku; J. Microbiol. 1999;37(2):97-101.

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Abstract PDF: Random hexapeptide library on the surface of filamentous bacteriophage was constructed using the SurfZAP vector. The size of the library was approximately 105. The peptide insert was flanked by two cysteines to constrain the peptide structure with a disulfide bond. This library was screened for the topoisomerase II binding peptide. Dramatic enrichment of the fusion phage over the VCS M13 helper phage was demonstrated by bio-panning affinity selection.

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