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An Optimized Method for Reconstruction of Transcriptional Regulatory Networks in Bacteria Using ChIP-exo and RNA-seq Datasets: Minchang Jang, Joon Young Park, Gayeon Lee, Donghyuk Kim; J. Microbiol. 2024;62(12):1075-1088. Published online November 11, 2024; DOI: https://doi.org/10.1007/s12275-024-00181-6

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Transcriptional regulatory networks (TRNs) in bacteria are crucial for elucidating the mechanisms that regulate gene expression and cellular responses to environmental stimuli. These networks delineate the interactions between transcription factors (TFs) and their target genes, thereby uncovering the regulatory processes that modulate gene expression under varying environmental conditions. Analyzing TRNs offers valuable insights into bacterial adaptation, stress responses, and metabolic optimization from an evolutionary standpoint. Additionally, understanding TRNs can drive the development of novel antimicrobial therapies and the engineering of microbial strains for biofuel and bioproduct production. This protocol integrates advanced data analysis pipelines, including ChEAP, DEOCSU, and DESeq2, to analyze omics datasets that encompass genome-wide TF binding sites and transcriptome profiles derived from ChIP-exo and RNA-seq experiments. This approach minimizes both the time required and the risk of bias, making it accessible to non-expert users. Key steps in the protocol include preprocessing and peak calling from ChIP-exo data, differential expression analysis of RNA-seq data, and motif and regulon analysis. This method offers a comprehensive and efficient framework for TRN reconstruction across various bacterial strains, enhancing both the accuracy and reliability of the analysis while providing valuable insights for basic and applied research.

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ChIP-mini: a low-input ChIP-exo protocol for elucidating DNA-binding protein dynamics in intracellular pathogens
Joon Young Park, Minchang Jang, Eunna Choi, Sang-Mok Lee, Ina Bang, Jihoon Woo, Seonggyu Kim, Eun-Jin Lee, Donghyuk Kim
Nucleic Acids Research.2025;[Epub] CrossRef

Unexpected Requirement of Small Amino Acids at Position 183 for DNA Binding in the Escherichia coli cAMP Receptor Protein: Marcus Carranza, Amanda Rea, Daisy Pacheco, Christian Montiel, Jin Park, Hwan Youn; J. Microbiol. 2024;62(10):871-882. Published online September 6, 2024; DOI: https://doi.org/10.1007/s12275-024-00169-2

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The Escherichia coli cAMP receptor protein (CRP) relies on the F-helix, the recognition helix of the helix-turn-helix motif, for DNA binding. The importance of the CRP F-helix in DNA binding is well-established, yet there is little information on the roles of its non-base-contacting residues. Here, we show that a CRP F-helix position occupied by a non-base-contacting residue Val183 bears an unexpected importance in DNA binding. Codon randomization and successive in vivo screening selected six amino acids (alanine, cysteine, glycine, serine, threonine, and valine) at CRP position 183 to be compatible with DNA binding. These amino acids are quite different in their amino acid properties (polar, non-polar, hydrophobicity), but one commonality is that they are all relatively small. Larger amino acid substitutions such as histidine, methionine, and tyrosine were made site-directedly and showed to have no detectable DNA binding, further supporting the requirement of small amino acids at CRP position 183. Bioinformatics analysis revealed that small amino acids (92.15% valine and 7.75% alanine) exclusively occupy the position analogous to CRP Val183 in 1,007 core CRP homologs, consistent with our mutant data. However, in extended CRP homologs comprising 3700 proteins, larger amino acids could also occupy the position analogous to CRP Val183 albeit with low occurrence. Another bioinformatics analysis suggested that large amino acids could be tolerated by compensatory small-sized amino acids at their neighboring positions. A full understanding of the unexpected requirement of small amino acids at CRP position 183 for DNA binding entails the verification of the hypothesized compensatory change(s) in CRP.

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SPD_0410 negatively regulates capsule polysaccharide synthesis and virulence in Streptococcus pneumoniae D39
Ye Tao, Li Lei, Shuhui Wang, Xuemei Zhang, Yibing Yin, Yuqiang Zheng
Frontiers in Microbiology.2025;[Epub] CrossRef

Mycobacterium tuberculosis PE_PGRS45 (Rv2615c) Promotes Recombinant Mycobacteria Intracellular Survival via Regulation of Innate Immunity, and Inhibition of Cell Apoptosis: Tao Xu , Chutong Wang , Minying Li , Jing Wei , Zixuan He , Zhongqing Qian , Xiaojing Wang , Hongtao Wang; J. Microbiol. 2024;62(1):49-62. Published online February 9, 2024; DOI: https://doi.org/10.1007/s12275-023-00101-0

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Tuberculosis (TB), a bacterial infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), is a significant global public health problem. Mycobacterium tuberculosis expresses a unique family of PE_PGRS proteins that have been implicated in pathogenesis. Despite numerous studies, the functions of most PE_PGRS proteins in the pathogenesis of mycobacterium infections remain unclear. PE_PGRS45 (Rv2615c) is only found in pathogenic mycobacteria. In this study, we successfully constructed a recombinant Mycobacterium smegmatis (M. smegmatis) strain which heterologously expresses the PE_PGRS45 protein. We found that overexpression of this cell wall-associated protein enhanced bacterial viability under stress in vitro and cell survival in macrophages. MS_PE_PGRS45 decreased the secretion of pro-inflammatory cytokines such as IL-1β, IL-6, IL-12p40, and TNF-α. We also found that MS_PE_PGRS45 increased the expression of the anti-inflammatory cytokine IL-10 and altered macrophage-mediated immune responses. Furthermore, PE_PGRS45 enhanced the survival rate of M. smegmatis in macrophages by inhibiting cell apoptosis. Collectively, our findings show that PE_PGRS45 is a virulent factor actively involved in the interaction with the host macrophage.

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Evolution of the PE_PGRS Proteins of Mycobacteria: Are All Equal or Are Some More Equal than Others?
Bei Chen, Belmin Bajramović, Bastienne Vriesendorp, Herman Pieter Spaink
Biology.2025; 14(3): 247. CrossRef
Recent advances in research on Mycobacterium tuberculosis virulence factors and their role in pathogenesis
Ming-Rui Sun, Jia-Yin Xing, Xiao-Tian Li, Ren Fang, Yang Zhang, Zhao-Li Li, Ning-Ning Song
Journal of Microbiology, Immunology and Infection.2025; 58(5): 497. CrossRef
Rv2741 Promotes Mycobacterium Survival by Modulating Macrophage Function via the IL-1α-MAPK Axis
Xintong He, Yonglin He, Xichuan Deng, Nan Lu, Anlong Li, Sijia Gao, Shiyan He, Yuran Wang, Nanzhe Fu, Zijie Wang, Yuxin Nie, Lei Xu
ACS Infectious Diseases.2025; 11(3): 676. CrossRef
The PE/PPE family proteins of Mycobacterium tuberculosis: evolution, function, and prospects for tuberculosis control
Zhijing Zhang, Le Dong, Xin Li, Taibing Deng, Qinglan Wang
Frontiers in Immunology.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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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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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
Particuology.2024; 90: 140. CrossRef
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

Review

Bacterial Crosstalk via Antimicrobial Peptides on the Human Skin: Therapeutics from a Sustainable Perspective: Seon Mi Lee , Hye Lim Keum , Woo Jun Sul; J. Microbiol. 2023;61(1):1-11. Published online January 31, 2023; DOI: https://doi.org/10.1007/s12275-022-00002-8

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The skin’s epidermis is an essential barrier as the first guard against invading pathogens, and physical protector from external injury. The skin microbiome, which consists of numerous bacteria, fungi, viruses, and archaea on the epidermis, play a key role in skin homeostasis. Antibiotics are a fast-acting and effective treatment method, however, antibiotic use is a nuisance that can disrupt skin homeostasis by eradicating beneficial bacteria along with the intended pathogens and cause antibioticresistant bacteria spread. Increased numbers of antimicrobial peptides (AMPs) derived from humans and bacteria have been reported, and their roles have been well defined. Recently, modulation of the skin microbiome with AMPs rather than artificially synthesized antibiotics has attracted the attention of researchers as many antibiotic-resistant strains make treatment mediation difficult in the context of ecological problems. Herein, we discuss the overall insights into the skin microbiome, including its regulation by different AMPs, as well as their composition and role in health and disease.

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The epidermal lipid-microbiome loop and immunity: Important players in atopic dermatitis
Junchao Wu, Lisha Li, Tingrui Zhang, Jiaye Lu, Zongguang Tai, Quangang Zhu, Zhongjian Chen
Journal of Advanced Research.2025; 68: 359. CrossRef
Marine algal polysaccharides: Multifunctional bioactive ingredients for cosmetic formulations
Si-Yuan Lu, Tao Zhou, Iqra Shabbir, Jaehwan Choi, Young Heui Kim, Myeongsam Park, Jude Juventus Aweya, Karsoon Tan, Saiyi Zhong, Kit-Leong Cheong
Carbohydrate Polymers.2025; 353: 123276. CrossRef
The interaction between the skin microbiome and antimicrobial peptides within the epidermal immune microenvironment: Bridging insights into atopic dermatitis
Shan Wang, Ge Peng, Alafate Abudouwanli, Mengyao Yang, Quan Sun, Wanchen Zhao, Arisa Ikeda, Yi Tan, Lin Ma, Hideoki Ogawa, Ko Okumura, François Niyonsaba
Allergology International.2025;[Epub] CrossRef
The dual role of skin microbiome modulation in precision care for atopic dermatitis: A review
Xue Chen
Health Sciences Review.2025; 17: 100245. CrossRef
A review on pathogenicity of Aeromonas hydrophila and their mitigation through medicinal herbs in aquaculture
Anurag Semwal, Avdhesh Kumar, Neelesh Kumar
Heliyon.2023; 9(3): e14088. CrossRef
Fırtına Deresindeki Gökkuşağı Alabalık Çiftliklerinde İzole Edilen Aeromonas spp. İzolatlarının Antimikrobiyel Hassasiyetin Belirlenmesi
Fikri BALTA
Journal of Anatolian Environmental and Animal Sciences.2020; 5(3): 397. CrossRef
Monitoring microbial community structure and succession of an A/O SBR during start-up period using PCR-DGGE
Xiuheng WANG, Kun ZHANG, Nanqi REN, Nan LI, Lijiao REN
Journal of Environmental Sciences.2009; 21(2): 223. CrossRef

Journal Articles

Assessing the microcystins concentration through optimized protein phosphatase inhibition assay in environmental samples: Kyoung-Hee Oh , Kung-Min Beak , Yuna Shin , Young-Cheol Cho; J. Microbiol. 2022;60(6):602-609. Published online April 30, 2022; DOI: https://doi.org/10.1007/s12275-022-2020-4

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Protein phosphatase (PPase) inhibition assay (PPIA) is widely used to analyze the concentration of microcystins (MCs) because it is comparatively less expensive and faster than other assays. This study aimed to optimize the PPIA by determining a suitable reaction terminator and an optimal methanol concentration in the sample. The most suitable reaction time was 90 min, with the corresponding methanol concentration in the sample being 15% or less. When p-nitrophenyl phosphate (pNPP) was used as a substrate, copper chloride solution was suitably used as a reaction terminator, and when 4- methylumbelliferyl phosphate (MUP) was used, a glycine buffer not only increased the measurement sensitivity of the reaction product but also terminated the enzymatic reaction. When PPase 1 and MUP were used as an enzyme and a substrate, respectively, the limit of quantitation for MC-leucine/ arginine (LR) was 0.02 μg/L, whereas it was 0.1 μg/L when pNPP was used as a substrate. The proposed method facilitated the measurement of MC-LR concentration without additional pretreatments, such as concentration or purification; therefore, this method was suitable and feasible for the continuous monitoring of MCs in drinking water.

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Acid phosphatase detection using a colorimetric probe based on azo compound toward forensic applications for seminal fluid identification
Jéssica Raimundo da Rocha, Marcone Gomes dos Santos Alcântara, Verônica Diniz da Silva, Dimas José da Paz Lima, Josué Carinhanha Caldas Santos
Dyes and Pigments.2025; 239: 112806. CrossRef
Analyzing MC-LR distribution characteristics in natural lakes by a novel fluorescence technology
Xiangyu Hu, Zhaomin Wang, Xiao Ye, Ping Xie, Yong Liu
Environmental Pollution.2024; 342: 123123. CrossRef
Magnetic solid phase extraction coupled with high-performance liquid chromatography-diode array detection based on assembled magnetic covalent organic frameworks for selective extraction and detection of microcystins in aquatic foods
Tianliang Wang, Hongzhen Xie, Yuting Cao, Qing Xu, Ning Gan
Journal of Chromatography A.2022; 1685: 463614. CrossRef

[PROTOCOL] Flow cytometric monitoring of the bacterial phenotypic diversity in aquatic ecosystems: Jin-Kyung Hong , Soo Bin Kim , Seok Hyun Ahn , Yongjoo Choi , Tae Kwon Lee; J. Microbiol. 2021;59(10):879-885. Published online September 23, 2021; DOI: https://doi.org/10.1007/s12275-021-1443-7

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Flow cytometry is a promising tool used to identify the phenotypic features of bacterial communities in aquatic ecosystems by measuring the physical and chemical properties of cells based on their light scattering behavior and fluorescence. Compared to molecular or culture-based approaches, flow cytometry is suitable for the online monitoring of microbial water quality because of its relatively simple sample preparation process, rapid analysis time, and high-resolution phenotypic data. Advanced statistical techniques (e.g., denoising and binning) can be utilized to successfully calculate phenotypic diversity by processing the scatter data obtained from flow cytometry. These phenotypic diversities were well correlated with taxonomic-based diversity computed using nextgeneration 16S RNA gene sequencing. The protocol provided in this paper should be a useful guide for a fast and reliable flow cytometric monitoring of bacterial phenotypic diversity in aquatic ecosystems.

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Enhancing Bacterial Phenotype Classification Through the Integration of Autogating and Automated Machine Learning in Flow Cytometric Analysis
In Jae Jeong, Jin‐Kyung Hong, Young Jun Bae, Tea Kwon Lee
Cytometry Part A.2025; 107(3): 203. CrossRef
Assessing long-term ecological impacts of PCE contamination in groundwater using a flow cytometric fingerprint approach
Jin-Kyung Hong, Soo Bin Kim, Gui Nam Wee, Bo Ram Kang, Jee Hyun No, Susmita Das Nishu, Joonhong Park, Tae Kwon Lee
Science of The Total Environment.2024; 931: 172698. CrossRef
Phenotypic shifts induced by environmental pre-stressors modify antibiotic resistance in Staphylococcus aureus
Gui Nam Wee, Eun Sun Lyou, Susmita Das Nishu, Tae Kwon Lee
Frontiers in Microbiology.2023;[Epub] CrossRef

Raman spectroscopy reveals alteration of spore compositions under different nutritional conditions in Lysinibacillus boronitolerans YS11: Youngung Ryu , Minyoung Hong , Soo Bin Kim , Tae Kwon Lee , Woojun Park; J. Microbiol. 2021;59(5):491-499. Published online March 29, 2021; DOI: https://doi.org/10.1007/s12275-021-0679-6

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Little is known about final spores components when bacteria undergo sporulation under different nutrient conditions. Different degrees of resistance and germination rates were observed in the three types of spores of Lysinibacillus boronitolerans YS11 (SD, Spores formed in Difco sporulation mediumTM; SC and SF, Spores formed in an agricultural byproduct medium with 10 mM CaCl2 and with 10 mM FeSO4, respectively). Stronger UV resistance was recorded for SF with 1.8–2.3-fold greater survival than SC and SD under UV treatment. The three spore types showed similar heat resistances at 80°C, but survival rates of SC and SD were much higher (~1,000 times) than those of SF at 90°C. However, germination capacity of SF was 20% higher than those of SD and SC on Luria-Bertani agar plates for 24 h. SF germinated more rapidly in a liquid medium with high NaCl concentrations than SC and SD, but became slower under alkaline conditions. Raman spectroscopy was used to analyze the heterogeneities in the three types of vegetative cells and their spores under different nutritional conditions. Exponentially grown-each vegetative cells had different overall Raman peak values. Raman peaks of SC, SD, and SF also showed differences in adenine and amide III compositions and nucleic acid contents. Our data along with Raman spectroscopy provided the evidence that spores formed under under different growth conditions possess very different cellular components, which affected their survival and germination rates.

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Characterization of the Bacillus cereus spore killed by plasma-activated water (PAW)
Xiao Hu, Pengfei Ge, Xiaomeng Wang, Xinyu Liao, Jinsong Feng, Ruiling Lv, Tian Ding
Food Research International.2024; 196: 115058. CrossRef
Alleviation of H2O2 toxicity by extracellular catalases in the phycosphere of Microcystis aeruginosa
Yerim Park, Wonjae Kim, Yeji Cha, Minkyung Kim, Woojun Park
Harmful Algae.2024; 137: 102680. CrossRef
Effects of sporulation conditions on the growth, germination, and resistance of Clostridium perfringens spores
Dong Liang, Xiaoshuang Cui, Miaoyun Li, Yaodi Zhu, Lijun Zhao, Shijie Liu, Gaiming Zhao, Na Wang, Yangyang Ma, Lina Xu
International Journal of Food Microbiology.2023; 396: 110200. CrossRef
Lysinibacilli: A Biological Factories Intended for Bio-Insecticidal, Bio-Control, and Bioremediation Activities
Qazi Mohammad Sajid Jamal, Varish Ahmad
Journal of Fungi.2022; 8(12): 1288. CrossRef
Discrimination of Stressed and Non-Stressed Food-Related Bacteria Using Raman-Microspectroscopy
Daniel Klein, René Breuch, Jessica Reinmüller, Carsten Engelhard, Peter Kaul
Foods.2022; 11(10): 1506. CrossRef
Detection of low numbers of bacterial cells in a pharmaceutical drug product using Raman spectroscopy and PLS-DA multivariate analysis
R. A. Grosso, A. R. Walther, E. Brunbech, A. Sørensen, B. Schebye, K. E. Olsen, H. Qu, M. A. B. Hedegaard, E. C. Arnspang
The Analyst.2022; 147(15): 3593. CrossRef
Linkage between bacterial community-mediated hydrogen peroxide detoxification and the growth of Microcystis aeruginosa
Minkyung Kim, Wonjae Kim, Yunho Lee, Woojun Park
Water Research.2021; 207: 117784. CrossRef

Patterns and drivers of Vibrio isolates phylogenetic diversity in the Beibu Gulf, China: Xing Chen , Hong Du , Si Chen , Xiaoli Li , Huaxian Zhao , Qiangsheng Xu , Jinli Tang , Gonglingxia Jiang , Shuqi Zou , Ke Dong , Jonathan M. Adams , Nan Li , Chengjian Jiang; J. Microbiol. 2020;58(12):998-1009. Published online October 23, 2020; DOI: https://doi.org/10.1007/s12275-020-0293-z

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Members of the genus Vibrio are ubiquitous in aquatic environments and can be found either in a culturable or a viable but nonculturable (VBNC) state. Despite widespread concerns as to how to define the occurrence and dynamics of Vibrio populations by culture-independent approaches, further physiological research and relevant biotechnological developments will require the isolation and cultivation of the microbes from various environments. The present work provides data and perspectives on our understanding of culturable Vibrio community structure and diversity in the Beibu Gulf. Finally, we isolated 1,037 strains of Vibrio from 45 samples and identified 18 different species. Vibrio alginolyticus, V. cyclitrophicus, V. tasmaniensis, V. brasiliensis, and V. splendidus were the dominant species that had regional distribution characteristics. The correlation between the quantitative distribution and community structure of culturable Vibrio and environmental factors varied with the Vibrio species and geographical locations. Among them, salinity, nitrogen, and phosphorus were the main factors affecting the diversity of culturable Vibrio. These results help to fill a knowledge gap on Vibrio diversity and provide data for predicting and controlling pathogenic Vibrio outbreaks in the Beibu Gulf.

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Environmental factors that regulate Vibrio spp. abundance and community structure in tropical waters
Yi You Wong, Choon Weng Lee, Chui Wei Bong, Joon Hai Lim, Ching Ching Ng, Kumaran Narayanan, Edmund Ui Hang Sim, Ai-jun Wang
Anthropocene Coasts.2024;[Epub] CrossRef
Co-occurrence of chromophytic phytoplankton and the Vibrio community during Phaeocystis globosa blooms in the Beibu Gulf
Qiangsheng Xu, Pengbin Wang, Jinghua Huangleng, Huiqi Su, Panyan Chen, Xing Chen, Huaxian Zhao, Zhenjun Kang, Jinli Tang, Gonglingxia Jiang, Zhuoting Li, Shuqi Zou, Ke Dong, Yuqing Huang, Nan Li
Science of The Total Environment.2022; 805: 150303. CrossRef
Virulence mechanisms of vibrios belonging to the Splendidus clade as aquaculture pathogens, from case studies and genome data
Weiwei Zhang, Chenghua Li
Reviews in Aquaculture.2021; 13(4): 2004. CrossRef

Phenotypic characterization of a conserved inner membrane protein YhcB in Escherichia coli: Chul Gi Sung , Umji Choi , Chang-Ro Lee; J. Microbiol. 2020;58(7):598-605. Published online April 22, 2020; DOI: https://doi.org/10.1007/s12275-020-0078-4

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Although bacteria have diverse membrane proteins, the function of many of them remains unknown or uncertain even in Escherichia coli. In this study, to investigate the function of hypothetical membrane proteins, genome-wide analysis of phenotypes of hypothetical membrane proteins was performed under various envelope stresses. Several genes responsible for adaptation to envelope stresses were identified. Among them, deletion of YhcB, a conserved inner membrane protein of unknown function, caused high sensitivities to various envelope stresses and increased membrane permeability, and caused growth defect under normal growth conditions. Furthermore, yhcB deletion resulted in morphological aberration, such as branched shape, and cell division defects, such as filamentous growth and the generation of chromosome- less cells. The analysis of antibiotic susceptibility showed that the yhcB mutant was highly susceptible to various anti-folate antibiotics. Notably, all phenotypes of the yhcB mutant were completely or significantly restored by YhcB without the transmembrane domain, indicating that the localization of YhcB on the inner membrane is dispensable for its function. Taken together, our results demonstrate that YhcB is involved in cell morphology and cell division in a membrane localization-independent manner.

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Inhibition of cardiolipin biosynthesis partially suppresses the sensitivity of an Escherichia coli mutant lacking OmpC to envelope stress
Dae-Beom Ryu, Umji Choi, Gyubin Han, Chang-Ro Lee
Journal of Microbiology.2025; 63(11): e2507004. CrossRef
Co-ordinated assembly of the multilayered cell envelope of Gram-negative bacteria
Elayne M Fivenson, Laurent Dubois, Thomas G Bernhardt
Current Opinion in Microbiology.2024; 79: 102479. CrossRef
Loss of YhcB results in overactive fatty acid biosynthesis
Hannah M. Stanley, M. Stephen Trent, K. Heran Darwin
mBio.2024;[Epub] CrossRef
A New Factor LapD Is Required for the Regulation of LpxC Amounts and Lipopolysaccharide Trafficking
Alicja Wieczorek, Anna Sendobra, Akshey Maniyeri, Magdalena Sugalska, Gracjana Klein, Satish Raina
International Journal of Molecular Sciences.2022; 23(17): 9706. CrossRef
Loss of YhcB results in dysregulation of coordinated peptidoglycan, LPS and phospholipid synthesis during Escherichia coli cell growth
Emily C. A. Goodall, Georgia L. Isom, Jessica L. Rooke, Karthik Pullela, Christopher Icke, Zihao Yang, Gabriela Boelter, Alun Jones, Isabel Warner, Rochelle Da Costa, Bing Zhang, James Rae, Wee Boon Tan, Matthias Winkle, Antoine Delhaye, Eva Heinz, Jean-F
PLOS Genetics.2021; 17(12): e1009586. CrossRef
The inner membrane protein LapB is required for adaptation to cold stress in an LpxC-independent manner
Han Byeol Lee, Si Hyoung Park, Chang-Ro Lee
Journal of Microbiology.2021; 59(7): 666. CrossRef

Soft sweep development of resistance in Escherichia coli under fluoroquinolone stress: Xianxing Xie , Ruichen Lv , Chao Yang , Yajun Song , Yanfeng Yan , Yujun Cui , Ruifu Yang; J. Microbiol. 2019;57(12):1056-1064. Published online September 25, 2019; DOI: https://doi.org/10.1007/s12275-019-9177-5

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We employed a stepwise selection model for investigating the dynamics of antibiotic-resistant variants in Escherichia coli K-12 treated with increasing concentrations of ciprofloxacin (CIP). Firstly, we used Sanger sequencing to screen the variations in the fluoquinolone target genes, then, employed Illumina NGS sequencing for amplicons targeted regions with variations. The results demonstrated that variations G81C in gyrA and K276N and K277L in parC are standing resistance variations (SRVs), while S83A and S83L in gyrA and G78C in parC were emerging resistance variations (ERVs). The variants containing SRVs and/or ERVs were selected successively based on their sensitivities to CIP. Variant strain 1, containing substitution G81C in gyrA, was immediately selected following ciprofloxacin exposure, with obvious increases in the parC SRV, and parC and gyrA ERV allele frequencies. Variant strain 2, containing the SRVs, then dominated the population following a 20× increase in ciprofloxacin concentration, with other associated allele frequencies also elevated. Variant strains 3 and 4, containing ERVs in gyrA and parC, respectively, were then selected at 40× and 160× antibiotic concentrations. Two variants, strains 5 and 6, generated in the selection procedure, were lost because of higher fitness costs or a lower level of resistance compared with variants 3 and 4. For the second induction, all variations/indels were already present as SRVs and selected out step by step at different passages. Whatever the first induction or second induction, our results confirmed the soft selective sweep hypothesis and provided critical information for guiding clinical treatment of pathogens containing SRVs.

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Could traces of fluoroquinolones in food induce ciprofloxacin resistance in Escherichia coli and Klebsiella pneumoniae ? An in vivo study in Galleria mellonel
Zina Gestels, Yuliia Baranchyk, Dorien Van den Bossche, Jolein Laumen, Said Abdellati, Basil Britto Xavier, Sheeba Santhini Manoharan-Basil, Chris Kenyon, Sadjia Bekal, Mustafa Sadek
Microbiology Spectrum.2024;[Epub] CrossRef

Transcriptome analysis to understand the effects of the toxoflavin and tropolone produced by phytopathogenic Burkholderia on Escherichia coli: Jungwook Park , Hyun-Hee Lee , Hyejung Jung , Young-Su Seo; J. Microbiol. 2019;57(9):781-794. Published online August 27, 2019; DOI: https://doi.org/10.1007/s12275-019-9330-1

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The phytopathogenic Burkholderia species B. glumae and B. plantarii are the causal agents of bacterial wilt, grain rot, and seedling blight, which threaten the rice industry globally. Toxoflavin and tropolone are produced by these phytopathogens and are considered the most hostile biohazards with a broad spectrum of target organisms. However, despite their nonspecific toxicity, the effects of toxoflavin and tropolone on bacteria remain unknown. RNA-seq based transcriptome analysis was employed to determine the genome-wide expression patterns under phytotoxin treatment. Expression of 2327 and 830 genes was differentially changed by toxoflavin and tropolone, respectively. Enriched biological pathways reflected the down-regulation of oxidative phosphorylation and ribosome function, beginning with the inhibition of membrane biosynthesis and nitrogen metabolism under oxidative stress or iron starvation. Conversely, several systems such as bacterial chemotaxis, flagellar assembly, biofilm formation, and sulfur/taurine transporters were highly expressed as countermeasures against the phytotoxins. In addition, our findings revealed that three hub genes commonly induced by both phytotoxins function as the siderophore enterobactin, an ironchelator. Our study provides new insights into the effects of phytotoxins on bacteria for better understanding of the interactions between phytopathogens and other microorganisms. These data will also be applied as a valuable source in subsequent applications against phytotoxins, the major virulence factor.

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AHL-Based Quorum Sensing Regulates the Biosynthesis of a Variety of Bioactive Molecules in Bacteria
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Reviews

MINIREVIEW] EAST1 toxin: An enigmatic molecule associated with sporadic episodes of diarrhea in humans and animals: J. Daniel Dubreuil; J. Microbiol. 2019;57(7):541-549. Published online June 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8651-4

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EAST1 is produced by a subset of enteroaggregative Escherichia coli strains. This toxin is a 38-amino acid peptide of 4100 Da. It shares 50% homology with the enterotoxic domain of STa and interacts with the same receptor. The mechanism of action of EAST1is proposed to be identical to that of STa eliciting a cGMP increase. EAST1 is associated with diarrheal disease in Man and various animal species including cattle and swine. Nevertheless, as EAST1-positive strains as well as culture supernatants did not provoke unequivocally diarrhea either in animal models or in human volunteers, the role of this toxin in disease is today still debated. This review intent is to examine the role of EAST1 toxin in diarrheal illnesses.

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REVIEW] Antibiotic-resistant clones in Gram-negative pathogens: presence of global clones in Korea: Kwan Soo Ko; J. Microbiol. 2019;57(3):195-202. Published online October 2, 2018; DOI: https://doi.org/10.1007/s12275-019-8491-2

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Antibiotic resistance is a global concern in public health. Antibiotic-resistant clones can spread nationally, internationally, and globally. This review considers representative antibiotic-resistant Gram-negative bacterial clones–CTX-M- 15-producing ST131 in Escherichia coli, extended-spectrum β-lactamase-producing ST11 and KPC-producing ST258 in Klebsiella pneumoniae, IMP-6-producing, carbapenem-resistant ST235 in Pseudomonas aeruginosa, and OXA-23- producing global clone 2 in Acinetobacter baumannii–that have disseminated worldwide, including in Korea. The findings highlight the urgency for systematic monitoring and international cooperation to suppress the emergence and propagation of antibiotic resistance.

Citations

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Journal Articles

Hydrogen sulfide inhibits the growth of Escherichia coli through oxidative damage: Liu-Hui Fu , Zeng-Zheng Wei , Kang-Di Hu , Lan-Ying Hu , Yan-Hong Li , Xiao-Yan Chen , Zhuo Han , Gai-Fang Yao , Hua Zhang; J. Microbiol. 2018;56(4):238-245. Published online February 28, 2018; DOI: https://doi.org/10.1007/s12275-018-7537-1

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Abstract PDF

Many studies have shown that hydrogen sulfide (H2S) is both detrimental and beneficial to animals and plants, whereas its effect on bacteria is not fully understood. Here, we report that H2S, released by sodium hydrosulfide (NaHS), significantly inhibits the growth of Escherichia coli in a dose-dependent manner. Further studies have shown that H2S treatment stimulates the production of reactive oxygen species (ROS) and decreases glutathione (GSH) levels in E. coli, resulting in lipid peroxidation and DNA damage. H2S also inhibits the antioxidative enzyme activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) and induces the response of the SoxRS and OxyR regulons in E. coli. Moreover, pretreatment with the antioxidant ascorbic acid (AsA) could effectively prevent H2S-induced toxicity in E. coli. Taken together, our results indicate that H2S exhibits an antibacterial effect on E. coli through oxidative damage and suggest a possible application for H2S in water and food processing.

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Gly184 of the Escherichia coli cAMP receptor protein provides optimal context for both DNA binding and RNA polymerase interaction: Matt N. Hicks , Sanjiva Gunasekara , Jose Serate , Jin Park , Pegah Mosharaf , Yue Zhou , Jin-Won Lee , Hwan Youn; J. Microbiol. 2017;55(10):816-822. Published online September 28, 2017; DOI: https://doi.org/10.1007/s12275-017-7266-x

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The Escherichia coli cAMP receptor protein (CRP) utilizes the helix-turn-helix motif for DNA binding. The CRP’s recognition helix, termed F-helix, includes a stretch of six amino acids (Arg180, Glu181, Thr182, Val183, Gly184, and Arg185) for direct DNA contacts. Arg180, Glu181 and Arg185 are known as important residues for DNA binding and specificity, but little has been studied for the other residues. Here we show that Gly184 is another F-helix residue critical for the transcriptional activation function of CRP. First, glycine was repeatedly selected at CRP position 184 for its unique ability to provide wild type-level transcriptional activation activity. To dissect the glycine requirement, wild type CRP and mutants G184A, G184F, G184S, and G184Y were purified and their in vitro DNA-binding activity was measured. G184A and G184F displayed reduced DNA binding, which may explain their low transcriptional activation activity. However, G184S and G184Y displayed apparently normal DNA affinity. Therefore, an additional factor is needed to account for the diminished transcriptional activation function in G184S and G184Y, and the best explanation is perturbations in their interaction with RNA polymerase. The fact that glycine is the smallest amino acid could not fully warrant its suitability, as shown in this study. We hypothesize that Gly184 fulfills the dual functions of DNA binding and RNA polymerase interaction by conferring conformational flexibility to the F-helix.

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cAMP-independent DNA binding of the CRP family protein DdrI from Deinococcus radiodurans
Yudong Wang, Jing Hu, Xufan Gao, Yuchen Cao, Shumai Ye, Cheng Chen, Liangyan Wang, Hong Xu, Miao Guo, Dong Zhang, Ruhong Zhou, Yuejin Hua, Ye Zhao, Paul Babitzke
mBio.2024;[Epub] CrossRef
Unexpected Requirement of Small Amino Acids at Position 183 for DNA Binding in the Escherichia coli cAMP Receptor Protein
Marcus Carranza, Amanda Rea, Daisy Pacheco, Christian Montiel, Jin Park, Hwan Youn
Journal of Microbiology.2024; 62(10): 871. CrossRef
cAMP Activation of the cAMP Receptor Protein, a Model Bacterial Transcription Factor
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Candida krusei isolated from fruit juices ultrafiltration membranes promotes colonization of Escherichia coli O157:H7 and Salmonella enterica on stainless steel surfaces: María Clara Tarifa , Jorge Enrique Lozano , Lorena Inés Brugnoni; J. Microbiol. 2017;55(2):96-103. Published online January 26, 2017; DOI: https://doi.org/10.1007/s12275-017-6300-3

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To clarify the interactions between a common food spoilage yeast and two pathogenic bacteria involved in outbreaks associated with fruit juices, the present paper studies the effect of the interplay of Candida krusei, collected from UF membranes, with Escherichia coli O157:H7 and Salmonella enterica in the overall process of adhesion and colonization of abiotic surfaces. Two different cases were tested: a) co-adhesion by pathogenic bacteria and yeasts, and b) incorporation of bacteria to pre-adhered C. krusei cells. Cultures were made on stainless steel at 25°C using apple juice as culture medium. After 24 h of co-adhesion with C. krusei, both E. coli O157:H7 and S. enterica increased their counts 1.05 and 1.11 log CFU cm2, respectively. Similar increases were obtained when incorporating bacteria to pre-adhered cells of Candida. Nevertheless C. krusei counts decreased in both experimental conditions, in a) 0.40 log CFU cm2 and 0.55 log CFU cm2 when exposed to E. coli O157:H7 and S. enterica and in b) 0.18 and 0.68 log CFU cm2, respectively. This suggests that C. krusei, E. coli O157:H7, and S. enterica have a complex relationship involving physical and chemical interactions on food contact surfaces. This study supports the possibility that pathogen interactions with members of spoilage microbiota, such as C. krusei, might play an important role for the survival and dissemination of E. coli O157:H7 and Salmonella enterica in food-processing environments. Based on the data obtained from the present study, much more attention should be given to prevent the contamination of these pathogens in acidic drinks.

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What We Still Don’t Know About Biofilms—Current Overview and Key Research Information
Tsvetozara Damyanova, Tsvetelina Paunova-Krasteva
Microbiology Research.2025; 16(2): 46. CrossRef
Efficacy of natamycin to reduce adhesion and biofilm formation of multispecies yeast biofilms on variable flow conditions
María del Rosario Agustín, Diego Bautista Genovese, Manuel Alejandro Palencia Díaz, Lorena Inés Brugnoni
Biofouling.2025; 41(6): 573. CrossRef
Effectiveness of sodium hypochlorite and benzalkonium chloride in reducing spoilage yeast biofilms on food contact surfaces
Manuel Alejandro Palencia Díaz, María Clara Tarifa, Patricia Liliana Marucci, Diego Bautista Genovese, Lorena Inés Brugnoni
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Application of natamycin and farnesol as bioprotection agents to inhibit biofilm formation of yeasts and foodborne bacterial pathogens in apple juice processing lines
María del Rosario Agustín, María Clara Tarifa, María Soledad Vela-Gurovic, Lorena Inés Brugnoni
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Candida krusei is the major contaminant of ultrafiltration and reverse osmosis membranes used for cranberry juice production
Sherazade Fikri, Marie-Hélène Lessard, Véronique Perreault, Alain Doyen, Steve Labrie
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Application of Natamycin and Farnesol as Biocontrol Agents of Multi-Species Biofilms on Industrial Surfaces in Apple Juice
María del Rosario Agustín, Maria Clara Tarifa, Maria Soledad Vela-Gurovic, Lorena Ines Brugnoni
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Ratka Hoferick, Angelos Ntovas, Qasim Alhusaini, Mareike Müller, Stéphan Barbe, Holger Schönherr
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Yeast biofilm in food realms: occurrence and control
Giacomo Zara, Marilena Budroni, Ilaria Mannazzu, Francesco Fancello, Severino Zara
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Disinfection efficacy over yeast biofilms of juice processing industries
María C. Tarifa, Jorge E. Lozano, Lorena I. Brugnoni
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C.G. Kouadio-Yapo, G.S.P. Dou, N.A.D. Aka, K.D. Zika, K.D. Adoubryn, M. Dosso
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Multispecies biofilms between Listeria monocytogenes and Listeria innocua with resident microbiota isolated from apple juice processing equipment
María del Rosario Agustín, Lorena Brugnoni
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Potential for colonization of O111:H25 atypical enteropathogenic E. coli: Marta O. Domingos , Keyde C.M. Melo , Irys Viana Neves , Cristiane M. Mota , Rita C. Ruiz , Bruna S. Melo , Raphael C. Lima , Denise S.P.Q. Horton , Monamaris M. Borges , Marcia R. Franzolin; J. Microbiol. 2016;54(11):745-752. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6015-x

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Using clonal phylogenetic methods, it has been demonstrated that O111:H25 atypical enteropathogenic E. coli (aEPEC) strains belong to distinct clones, suggesting the possibility that their ability to interact with different hosts and abiotic surfaces can vary from one clone to another. Accordingly, the ability of O111:H25 aEPEC strains derived from human, cat and dogs to adhere to epithelial cells has been investigated, along with their ability to interact with macrophages and to form biofilms on polystyrene, a polymer used to make biomedical devices. The results demonstrated that all the strains analyzed were able to adhere to, and to form pedestals on, epithelial cells, mechanisms used by E. coli to become strongly attached to the host. The strains also show a Localized-Adherence- Like (LAL) pattern of adhesion on HEp-2 cells, a behavior associated with acute infantile diarrhea. In addition, the O111:H25 aEPEC strains derived either from human or domestic animals were able to form long filaments, a phenomenon used by some bacteria to avoid phagocytosis. O111:H25 aEPEC strains were also encountered inside vacuoles, a characteristic described for several bacterial strains as a way of protecting themselves against the environment. They were also able to induce TNF-α release via two routes, one dependent on TLR-4 and the other dependent on binding of Type I fimbriae. These O111:H25 strains were also able to form biofilms on polystyrene. In summary the results suggest that, regardless of their source (i.e. linked to human origin or otherwise), O111:H25 aEPEC strains carry the potential to cause human disease.

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Surface Display Expression of Bacillus licheniformis Lipase in Escherichia coli Using Lpp’OmpA Chimera: Jae-Hyung Jo , Chan-Wook Han , Seung-Hwan Kim , Hyuk-Jin Kwon , Hyune-Hwan Lee; J. Microbiol. 2014;52(10):856-862. Published online August 27, 2014; DOI: https://doi.org/10.1007/s12275-014-4217-7

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The lipase from Bacillus licheniformis ATCC14580 was displayed on the cell surface of Escherichia coli using Lpp’OmpA as the anchoring protein. The expressed Lpp’OmpA-lipase fusion protein has a molecular weight of approximately 35 kDa, which was confirmed by SDS-PAGE and western blot analysis. The Lpp’OmpA-lipase fusion protein was located on the cell surface, as determined by immunofluorescence confocal microscopy and flow cytometry. The enzyme activity of the surface-displayed lipase showed clear halo around the colony. The cell surface-displayed lipase showed the highest activity of 248.12 ± 9.42 U/g (lyophilized cell) at the optimal temperature of 37°C and pH 8.0. The enzyme exhibited the highest activity toward the substrate p-nitrophenyl caprylate (C8). These results suggest that E. coli, which displayed the lipase on its surface, could be used as a whole cell biocatalyst.

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Recent advances in bioinspired multienzyme engineering for food applications
Xianhan Chen, Yujin Chen, Dandan Tang, Mengyu Li, Yuting Lu, Yi Cao, Quanyu Zhao, Shuai Jiang, Wei Liu, Ling Jiang
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Patricia L. A. Muñoz-Muñoz, Celina Terán-Ramírez, Rosa E. Mares-Alejandre, Ariana B. Márquez-González, Pablo A. Madero-Ayala, Samuel G. Meléndez-López, Marco A. Ramos-Ibarra
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Characterization of a novel subfamily 1.4 lipase from Bacillus licheniformis IBRL-CHS2: Cloning and expression optimization
Ammar Khazaal Kadhim Almansoori, Nidyaletchmy Subba Reddy, Mustafa Abdulfattah, Sarah Solehah Ismail, Rashidah Abdul Rahim, Estibaliz Sansinenea
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Liangyan Wang, Yudong Wang, Shang Dai, Binqiang Wang
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Jingjing Sun, Xuansheng Lin, Yige He, Baozhong Zhang, Nan Zhou, Jian-dong Huang
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Dan Wang, Linwei Duan, Min Wei, Baizhu Chen, Zhipeng Li, Qingyou Liu
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A Modular System for the Rapid Comparison of Different Membrane Anchors for Surface Display on Escherichia coli
Sabrina Gallus, Esther Mittmann, Kersten S. Rabe
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Sonia Nicchi, Maria Giuliani, Fabiola Giusti, Laura Pancotto, Domenico Maione, Isabel Delany, Cesira L. Galeotti, Cecilia Brettoni
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Shurong Chen, Li Pan, Siying Liu, Lijie Pan, Xuejie Li, Bin Wang
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Eszter Csibra, Marleen Renders, Vitor B. Pinheiro
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Sabrina Gallus, Theo Peschke, Malte Paulsen, Teresa Burgahn, Christof M. Niemeyer, Kersten S. Rabe
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Chen-Fu Chung, Shih-Che Lin, Tzong-Yuan Juang, Yung-Chuan Liu
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Ricardo Torres-Bañaga, Rosa E. Mares-Alejandre, Celina Terán-Ramírez, Ana L. Estrada-González, Patricia L.A. Muñoz-Muñoz, Samuel G. Meléndez-López, Ignacio A. Rivero, Marco A. Ramos-Ibarra
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Antimicrobial Resistance, Virulence Genes and PFGE-profiling of Escherichia coli Isolates from South Korean Cattle Farms: Seung Won Shin , Jae-Won Byun , Myounghwan Jung , Min-Kyoung Shin , Han Sang Yoo; J. Microbiol. 2014;52(9):785-793. Published online July 30, 2014; DOI: https://doi.org/10.1007/s12275-014-4166-1

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To estimate the prevalence of Escherichia coli with potential pathogenicity in cattle farm in South Korea, a total of 290 E. coli isolates were isolated from cattle farms over a period of 2 years in South Korea. These were examined for phenotypic and genotypic characteristics including antimicrobial susceptibility, serotype, and gene profiles of virulence and antimicrobial resistance. The most dominant virulence gene was f17 (26.2%), followed by stx2 (15.9%), ehxA (11.0%), stx1 (8.3%), eae (5.2%), and sta (4.1%). Some shiga-toxin producing E. coli isolates possessed eae (15.9%). All isolates except for one showed resistance to one or more antimicrobials, with 152 isolates exhibiting multidrug-resistance. The most prevalent resistance phenotype detected was streptomycin (63.1%), followed by tetracycline (54.5%), neomycin (40.3%), cephalothin (32.8%), amoxicillin (30.0%), ampicillin (29.7%), and sulphamethoxazole/trimethoprim (16.6%). The associated resistance determinants detected were strAstrB (39.0%), tet(E) (80.0%), tet(A) (27.6%), aac(3)-IV (33.1%), aphA1 (21.4%), blaTEM (23.8%), and sul2 (22.1%). When investigated by O serotyping and PFGE molecular subtyping, the high degree of diversity was exhibited in E. coli isolates. These results suggest that E. coli isolates from South Korean cattle farms are significantly diverse in terms of virulence and antimicrobial resistance. In conclusion, the gastroinstestinal flora of cattle could be a significant reservoir of diverse virulence and antimicrobial resistance determinants, which is potentially hazardous to public health.

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Underrepresented high diversity of class 1 integrons in the environment uncovered by PacBio sequencing using a new primer
Yu Yang, An-Ni Zhang, You Che, Lei Liu, Yu Deng, Tong Zhang
Science of The Total Environment.2021; 787: 147611. CrossRef
The prevalence of causative agents of calf diarrhea in Korean native calves
Jeong-Byoung Chae, Hyeon-Cheol Kim, Jun-Gu Kang, Kyoung-Seong Choi, Joon-Seok Chae, Do-Hyeon Yu, Bae-Keun Park, Yeon-su Oh, Hak-Jong Choi, Jinho Park
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O-serogroups, virulence genes, antimicrobial susceptibility, and MLST genotypes of Shiga toxin-producing Escherichia coli from swine and cattle in Central China
Zhong Peng, Wan Liang, Zizhe Hu, Xiaosong Li, Rui Guo, Lin Hua, Xibiao Tang, Chen Tan, Huanchun Chen, Xiangru Wang, Bin Wu
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Shivasharanappa Nayakvadi, Charlotte Alison Alemao, H.B. Chethan Kumar, R.S. Rajkumar, Susitha Rajkumar, Eaknath B. Chakurkar, Shivaramu Keelara
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Antimicrobial Resistance inEscherichia coli
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Wioletta Adamus-Białek, Anna Baraniak, Monika Wawszczak, Stanisław Głuszek, Beata Gad, Klaudia Wróbel, Paulina Bator, Marta Majchrzak, Paweł Parniewski
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Interrelationship between tetracycline resistance determinants, phylogenetic group affiliation and carriage of class 1 integrons in commensal Escherichia coli isolates from cattle farms
Kuastros Mekonnen Belaynehe, Seung Won Shin, Han Sang Yoo
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Occurrence of aminoglycoside-modifying enzymes among isolates of Escherichia coli exhibiting high levels of aminoglycoside resistance isolated from Korean cattle farms
Kuastros Mekonnen Belaynehe, Seung Won Shin, Park Hong-Tae, Han Sang Yoo
FEMS Microbiology Letters.2017;[Epub] CrossRef
Prevalence of Antimicrobial Resistance and Transfer of Tetracycline Resistance Genes in Escherichia coli Isolates from Beef Cattle
Seung Won Shin, Min Kyoung Shin, Myunghwan Jung, Kuastros Mekonnen Belaynehe, Han Sang Yoo, M. W. Griffiths
Applied and Environmental Microbiology.2015; 81(16): 5560. CrossRef
Profiling of antimicrobial resistance and plasmid replicon types in β-lactamase producingEscherichia coliisolated from Korean beef cattle
Seung Won Shin, Myunghwan Jung, Min-Kyung Shin, Han Sang Yoo
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Expression and Purification of Lacticin Q by Small Ubiquitin-Related Modifier Fusion in Escherichia coli: Qingshan Ma , Zhanqiao Yu , Bing Han , Qing Wang , Rijun Zhang; J. Microbiol. 2012;50(2):326-331. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1425-x

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Abstract PDF: Lacticin Q is a broad-spectrum class II bacteriocin with potential as an alternative to conventional antibiotics. The objective of this study was to produce recombinant lacticin Q using a small ubiquitin-related modifier (SUMO) fusion protein expression system. The 168-bp lacticin Q gene was cloned into the expression vector pET SUMO and transformed into Escherichia coli BL21(DE3). The soluble fusion protein was recovered with a Ni-NTA Sepharose column (95% purity); 130 mg protein was obtained per liter of fermentation culture. The SUMO tag was then proteolytically cleaved from the protein, which was re-applied to the column. Finally, about 32 mg lacticin Q (≥96% purity) was obtained. The recombinant protein exhibited antimicrobial properties similar to that of the native protein, demonstrating that lacticin Q had been successfully expressed by the SUMO fusion system.

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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Abstract PDF: 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.

Genetic Diversity and Population Structure of Escherichia coli from Neighboring Small-Scale Dairy Farms: Jesús Andrei Rosales-Castillo , Ma. Soledad Vázquez-Garcidueñas , Hugo Álvarez-Hernández , Omar Chassin-Noria , Alba Irene Varela-Murillo , María Guadalupe Zavala-Páramo , Horacio Cano-Camacho , Gerardo Vázquez-Marrufo; J. Microbiol. 2011;49(5):693-702. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-0461-2

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Abstract PDF: The genetic diversity and population structure of Escherichia coli isolates from small-scale dairy farms were used to assess the ability of E. coli to spread within the farm environment and between neighboring farms. A total of 164 E. coli isolates were obtained from bovine feces, bedding, cow teats and milk from 6 small-scale dairy farms. Ward’s clustering grouped the isolates into 54 different random amplified polymorphic DNA (RAPD) types at 95% similarity, regardless of either the sample type or the farm of isolation. This suggests that RAPD types are shared between bovine feces, bedding, cow teats, and milk. In addition, transmission of RAPD types between the studied farms was suggested by the Ward grouping pattern of the isolates, Nei’s and AMOVA population analyses, and genetic landscape shape analysis. For the first time, the latter analytical tool was used to assess the ability of E. coli to disseminate between small-scale dairy farms within the same producing region. Although a number of dispersal mechanisms could exist between farms, the genetic landscape shape analysis associated the flow of E. coli RAPD types with the movement of forage and milking staff between farms. This study will aid in planning disease prevention strategies and optimizing husbandry practices.

Journal Article

NOTE] Identification of PhoB Binding Sites of the yibD and ytfK Promoter Regions in Escherichia coli: Yusuke Yoshida , Shinichiro Sugiyama , Tomoya Oyamada , Katsushi Yokoyama , Soo-Ki Kim , Kozo Makino; J. Microbiol. 2011;49(2):285-289. Published online May 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0360-6

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Abstract PDF: By using a lacZ operon fusion genomic library of the Escherichia coli O157:H7 Sakai, we identified phosphatestarvation-inducible (psi) promoters located upstream of the yibD and ytfK genes. They have been previously proposed to belong to the phosphate regulon (pho regulon) by Beak and Lee (2006), based on the DNA array and in vivo transcriptional experiments. However, the direct interaction of these promoters with the activator protein of the pho regulon, PhoB, has not been determined. We determined the binding regions of PhoB in these promoter regions by DNase I footprinting. Both regions contained two pho boxes similar to the consensus sequence for PhoB binding.

Research Support, Non-U.S. Gov't

Stable Expression and Secretion of Polyhydroxybutyrate Depolymerase of Paucimonas lemoignei in Escherichia coli: Se Whan Park , Moon Gyu Chung , Hwa Young Lee , Jeong Yoon Kim , Young Ha Rhee; J. Microbiol. 2008;46(6):662-669. Published online December 24, 2008; DOI: https://doi.org/10.1007/s12275-008-0283-z

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Abstract PDF: An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase (PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly synthesized mature form of PhaZ1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing PhaZ1 and its own N-terminal signal peptide (PrePhaZ1) enabled the secretion of active PhaZ1 into the extracellular medium. However, the PrePhaZ1 construct was harmful to the host cell and resulted in atypical growth and instability of the plasmid during the cultivation. In contrast, INPNC-PhaZ1 and INPNCPrePhaZ1 fusion constructs did not affect growth of host cells. INPNC-PhaZ1 was successfully displayed on the cell surface with its fusion form, but did not retain PhaZ1 activity. In the case of INPNC-PrePhaZ1, the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and active PhaZ1 was consequently released into the culture medium. The amount of PhaZ1 derived from E. coli (INPNC-PrePhaZ1) was almost twice as great as that directly expressed from E. coli (PrePhaZ1), and was predominantly (approximately 85%) located in the periplasm when cultivated at 22°C but was efficiently secreted into the extracellular medium when cultivated at 37°C.

Journal Article

Biologically Active and C-Amidated HinnavinII-38-Asn Produced from a Trx Fusion Construct in Escherichia coli: Chang Soo Kang , Seung-Yeol Son , In Seok Bang; J. Microbiol. 2008;46(6):656-661. Published online December 24, 2008; DOI: https://doi.org/10.1007/s12275-008-0214-z

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Abstract PDF: The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn (HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15~20% of the total cell proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography, and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus.

Research Support, Non-U.S. Gov't

Inactivation of Barotolerant Strains of Listeria monocytogenes and Escherichia coli O157:H7 by Ultra High Pressure and tert-Butylhydroquinone Combination: Yoon-Kyung Chung , Ahmed E. Yousef; J. Microbiol. 2008;46(3):289-294. Published online July 5, 2008; DOI: https://doi.org/10.1007/s12275-008-0090-6

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Abstract PDF

Antimicrobial efficacy of ultra-high-pressure (UHP) can be enhanced by application of additional hurdles. The objective of this study was to systematically assess the enhancement in pressure lethality by TBHQ treatment, against barotolerant strains of Escherichia coli O157:H7 and Listeria monocytogenes. Two L. monocytogenes Scott A and the barotolerant OSY-328 strain, and two E. coli O157:H7 strains, EDL-933 and its barotolerant mutant, OSY-ASM, were tested. Cell suspensions containing TBHQ (50 ppm, dissolved in dimethyl sulfoxide) were pressurized at 200 to 500 MPa (23±2°C) for 1 min, plated on tryptose agar and enumerated the survivors. The TBHQ-UHP combination resulted in synergistic inactivation of both pathogens, with different degrees of lethality among strains. The pressure lethality threshold, for the combination treatment, was lower for E. coli O157:H7 (≥ 200 MPa) than for L. monocytogenes (> 300 MPa). E. coli O157:H7 strains were extremely sensitive to the TBHQ-UHP treatment, compared to Listeria strains. Interestingly, a control treatment involving DMSO-UHP combination consistently resulted in higher inactivation than that achieved by UHP alone, against all strains tested. However, sensitization of the pathogens to UHP by the additives (TBHQ in DMSO) was prominently greater for UHP than DMSO. Differences in sensitivities to the treatment between these two pathogens may be attributed to discrepancies in cellular structure or physiological functions.

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Recent Progress in the Synergistic Bactericidal Effect of High Pressure and Temperature Processing in Fruits and Vegetables and Related Kinetics
Sinan Zhang, Maninder Meenu, Lihui Hu, Junde Ren, Hosahalli S. Ramaswamy, Yong Yu
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Journal Article

Characterization of the Bacillus subtilis WL-3 Mannanase from a Recombinant Escherichia coli: Ki-Hong Yoon , Seesub Chung , Byung-Lak Lim; J. Microbiol. 2008;46(3):344-349. Published online July 5, 2008; DOI: https://doi.org/10.1007/s12275-008-0045-y

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Abstract PDF

A mannanase was purified from a cell-free extract of the recombinant Escherichia coli carrying a Bacillus subtilis WL-3 mannanase gene. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. Optimal conditions for the purified enzyme occurred at pH 6.0 and 60°C. The specific activity of the purified mannanase was 5,900 U/mg on locust bean gum (LBG) galactomannan at pH 6.0 and 50°C. The activity of the enzyme was slightly inhibited by Mg2+, Ca2+, EDTA and SDS, and noticeably enhanced by Fe2+. When the enzyme was incubated at 4°C for one day in the presence of 3 mM Fe2+, no residual activity of the mannanase was observed. The enzyme showed higher activity on LBG and konjac glucomannan than on guar gum galactomannan. Furthermore, it could hydrolyze xylans such as arabinoxylan, birchwood xylan and oat spelt xylan, while it did not exhibit any activities towards carboxymethylcellulose and para-nitrophenyl-β-mannopyranoside. The predominant products resulting from the mannanase hydrolysis were mannose, mannobiose and mannotriose for LBG or mannooligosaccharides including mannotriose, mannotetraose, mannopentaose and mannohexaose. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose.

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Dong-Gwan Kim, Chang-Muk Lee, Young-Seok Lee, Sang-Hong Yoon, Su-Yeon Kim
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Research Support, Non-U.S. Gov'ts

Molecular Characterization of Antibiotic Resistant Escherichia coli Strains Isolated from Tap and Spring Waters in a Coastal Region in Turkey: Osman Birol Ozgumus , Elif Celik-Sevim , Sengul Alpay-Karaoglu , Cemal Sandalli , Ali Sevim; J. Microbiol. 2007;45(5):379-387.; DOI: https://doi.org/2600 [pii]

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Abstract PDF: A hundred and seventeen antibiotic-resistant Escherichia coli strains were isolated from public tap and spring waters which were polluted by fecal coliforms. There were no significant differences between two water sources as to the coliform pollution level (p> 0.05). All E. coli isolates were detected to be resistant to one or more antibiotics tested. Nearly 42% of the isolates showed multiresistant phenotype. Three (2.5%) of these isolates contained class 1 integron. Sequencing analysis of variable regions of the class 1 integrons showed two gene cassette arrays, dfr1-aadA1 and dhfrA17-aadA5. Resistance to ampicillin, tetracycline or trimethoprim-sulfamethoxazole was transferable according to the results of conjugation experiments. The rate of tetracycline resistance was 15%. tet(A)-mediated tetracycline resistance was widespread among tetracycline-resistant E. coli isolates. Genotyping by BOX-polymerase chain reaction (BOX-PCR) showed that some of the strains were epidemiologically related. This is the first report on the prevalence and characterization of class 1 integron-containing E. coli isolates of environmental origin in Turkey.

Genetic Characterization of the Escherichia coli O66 Antigen and Functional Identification of its wzy Gene: Jiansong Cheng , Bin Liu , David A. Bastin , Weiqing Han , Lei Wang , Lu Feng; J. Microbiol. 2007;45(1):69-74.; DOI: https://doi.org/2488 [pii]

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Abstract PDF: Escherichia coli is a clonal species, and occurs as both commensal and pathogenic strains, which are normally classified on the basis of their O, H, and K antigens. The O-antigen (O-specific polysaccharide), which consists of a series of oligosaccharide (O-unit) repeats, contributes major antigenic variability to the cell surface. The O-antigen gene cluster of E. coli O66 was sequenced in this study. The genes putatively responsible for the biosynthesis of dTDP-6-deoxy-L-talose and GDP-mannose, as well as those responsible for the transfer of sugars and for O-unit processing were identified based on their homology. The function of the wzy gene was confirmed by the results of a mutation test. Genes specific for E. coli O66 were identified via PCR screening against representatives of 186 E. coli and Shigella O type strains. The comparison of intergenic sequences located between galF and the O-antigen gene cluster in a range of E. coli and Shigella showed that this region may perform an important function in the homologous recombination of the O-antigen gene clusters.

Isolation of Quinolone-Resistant Escherichia coli Found in Major Rivers in Korea: Dahye Jung , Min Young Lee , Jung Min Kim , Je Chul Lee , Dong Taek Cho , Yeonhee Lee; J. Microbiol. 2006;44(6):680-684.; DOI: https://doi.org/2456 [pii]

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Abstract PDF: Twenty isolates resistant to seven quinolones were isolated from major rivers in Korea. All isolates had three mutations, Ser83→Leu and Asp87→Asn in GyrA and Ser80→Ile or Ser80→Arg in ParC and three isolates had an additional mutation Glu84→Gly or Glu84→Val in ParC. In addition, a clonal spread was not found in these isolates.

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.

Journal Article

Protective Immune Response of Bacterially-Derived Recombinant FaeG in Piglets: Huang Yahong , Wanqi Liang , Aihu Pan , Zhiai Zhou , Qiang Wang , Cheng Huang , Jianxiu Chen , Dabing Zhang; J. Microbiol. 2006;44(5):548-555.; DOI: https://doi.org/2442 [pii]

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Abstract PDF: FaeG is the key factor in the infection process of K88ad enterotoxigenic Escherichia coli(ETEC) fimbrial adhesin. In an attempt to determine the possibility of expressing recombinant FaeG with immunogenicity for a new safe and high-production vaccine in E. coli, we constructed the recombinant strain, BL21 (DE3+K88), which harbors an expression vector with a DNA fragment of faeG, without a signal peptide. Results of 15% SDS-polyacrylamide slab gel analysis showed that FaeG can be stably over-expressed in BL21 (DE3+K88) as inclusion bodies without FaeE. Immunoglobulin G (IgG) and M (IgM) responses in pregnant pigs, with boost injections of the purified recombinant FaeG, were detected 4 weeks later in the sera and colostrum. An in vitro villius-adhesion assay verified that the elicited antibodies in the sera of vaccinated pigs were capable of preventing the adhesion of K88ad ETEC to porcine intestinal receptors. The protective effect on the mortality rates of suckling piglets born to vaccinated mothers was also observed one week after oral challenge with the virulent ETEC strain, C83907 (K88ad, CT+, ST+). The results of this study proved that the adhesin of proteinaceous bacterial fimbriae or pili could be overexpressed in engineered E. coli strains, with protective immune responses to the pathogen.

Research Support, Non-U.S. Gov'ts

Effect of Titanium Ion and Resistance Encoding Plasmid of Pseudomonas aeruginosa ATCC 10145: Sung Min Park , Hyun Soo Kim , Tae Shick Yu; J. Microbiol. 2006;44(3):255-262.; DOI: https://doi.org/2388 [pii]

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Abstract PDF: Titanium and its alloys are technically superior and cost-effective materials, with a wide variety of aerospace, industrial, marine, and commercial applications. In this study, the effects of titanium ions on bacterial growth were evaluated. Six strains of bacteria known to be resistant to both metal ions and antibiotics were used in this study. Two strains, Escherichia coli ATCC 15489, and Pseudomonas aeruginosa ATCC 10145, proved to be resistant to titanium ions. Plasmid-cured P. aeruginosa resulted in the loss of one or more resistance markers, indicating plasmid-encoded resistance. The plasmid profile of P. aeruginosa revealed the presence of a 23-kb plasmid. The plasmid was isolated and transformed into DH5α. Interestingly, the untransformed DH5α did not grow in 300 mg/l titanium ions, but the transformed DH5α grew quite well under such conditions. The survival rate of the transformed DH5α also increased more than 3-fold compared to that of untransformed DH5α.

Sterilization of Bacteria, Yeast, and Bacterial Endospores by Atmospheric-Pressure Cold Plasma using Helium and Oxygen: Kyenam Lee , Kwang-hyun Paek , Won-Tae Ju , Yoenhee Lee; J. Microbiol. 2006;44(3):269-275.; DOI: https://doi.org/2386 [pii]

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Abstract PDF: Atmospheric-pressure cold plasma (APCP) using helium/oxygen was developed and tested as a suitable sterilization method in a clinical environment. The sterilizing effect of this method is not due to UV light, which is known to be the major sterilization factor of APCP, but instead results from the action of reactive oxygen radicals. Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae deposited on a nitrocellulose filter membrane or Bacillus subtilis spores deposited on polypropylene plates were exposed to helium/oxygen plasma generated with AC input power at 10 kHz, 6 kV. After plasma treatment, nitrocellulose filter membranes were overlaid on fresh solid media and CFUs were counted after incubation overnight. D-values were 18 sec for E. coli, 19 sec for S. aureus, 1 min 55 sec for S. cerevisiae, and 14 min for B. subtilis spores. D-values of bacteria and yeast were dependent on the initial inoculation concentration, while the D-value of B. subtilis spores showed no correlation. When treated cells were observed with a scanning electron microscope, E. coli was more heavily damaged than S. aureus, S. cerevisiae exhibited peeling, and B. subtilis spores exhibited shrunken morphology. Results showed that APCP using helium/oxygen has many advantages as a sterilization method, especially in a clinical environment with conditions such as stable temperature, unlimited sample size, and no harmful gas production.

Review

Rho-dependent Transcription Termination: More Questions than Answers: Sharmistha Banerjee , Jisha Chalissery , Irfan Bandey , Ranjan Sen; J. Microbiol. 2006;44(1):11-22.; DOI: https://doi.org/2342 [pii]

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Abstract PDF: Escherichia coli protein Rho is required for the factor-dependent transcription termination by an RNA polymerase and is essential for the viability of the cell. It is a homohexameric protein that recognizes and binds preferably to C-rich sites in the transcribed RNA. Once bound to RNA, it utilizes RNA-dependent ATPase activity and subsequently ATPase-dependent helicase activity to unwind RNA-DNA hybrids and release RNA from a transcribing elongation complex. Studies over the past few decades have highlighted Rho as a molecule and have revealed much of its mechanistic properties. The recently solved crystal structure could explain many of its physiological functions in terms of its structure. Despite all these efforts, many of the fundamental questions pertaining to Rho recognition sites, differential ATPase activity in response to different RNAs, translocation of Rho along the nascent transcript, interactions with elongation complex and finally unwinding and release of RNA remain obscure. In the present review we have attempted to summarize ‘the knowns’ and ‘the unknowns’ of the Rho protein revealed by the recent developments in this field. An attempt has also been made to understand the physiology of Rho in the light of its phylogeny.

Journal Article

Expression of Escherichia coli Heat-labile Enterotoxin B Subunit (LTB) in Saccharomyces cerevisiae: Mohammad Ahangarzadeh Rezaee , Abbas Rezaee , Seyed Mohammad Moazzeni , Ali Hatef Salmanian , Yoko Yasuda , Kunio Tochikubo , Shahin Najar Pirayeh , Mohsen Arzanlou; J. Microbiol. 2005;43(4):354-360.; DOI: https://doi.org/2254 [pii]

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Abstract PDF: Heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli (ETEC) is both a strong mucosal adjuvant and immunogen. It is a subunit vaccine candidate to be used against ETEC-induced diarrhea. It has already been expressed in several bacterial and plant systems. In order to construct yeast expressing vector for the LTB protein, the eltB gene encoding LTB was amplified from a human origin enterotoxigenic E. coli DNA by PCR. The expression plasmid pLTB83 was constructed by inserting the eltB gene into the pYES2 shuttle vector immediately downstream of the GAL1 promoter. The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The LTB protein was detected in the total soluble protein of the yeast by SDS-PAGE analysis. Quantitative ELISA showed that the maximum amount of LTB protein expressed in the yeast was approximately 1.9% of the total soluble protein. Immunoblotting analysis showed the yeast-derived LTB protein was antigenically indistinguishable from bacterial LTB protein. Since the whole-recombinant yeast has been introduced as a new vaccine formulation the expression of LTB in S. cerevisiae can offer an inexpensive yet effective strategy to protect against ETEC, especially in developing countries where it is needed most.

Research Support, Non-U.S. Gov'ts

Comparative Evaluation of Three Purification Methods for the Nucleocapsid Protein of Newcastle Disease Virus from Escherichia coli Homogenates: Yan Peng Tan , Tau Chuan Ling , Khatijah Yusoff , Wen Siang Tan , Beng Ti Tey; J. Microbiol. 2005;43(3):295-300.; DOI: https://doi.org/2210 [pii]

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Abstract PDF: In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni^2^+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.

Purification and Characterization of NADPH-Dependent Cr(VI) Reductase from Escherichia coli ATCC 33456: Woo-Chul Bae , Han-Ki Lee , Young-Chool Choe , Deok-Jin Jahng , Sang-Hee Lee , Sang-Jin Kim , Jung-Hyun Lee , Byeong-Chul Jeong; J. Microbiol. 2005;43(1):21-27.; DOI: https://doi.org/2143 [pii]

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Abstract PDF: A soluble Cr(VI) reductase was purified from the cytoplasm of Escherichia coli ATCC 33456. The molecular mass was estimated to be 84 and 42 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, indicating a dimeric structure. The pI was 4.66, and optimal enzyme activity was obtained at pH 6.5 and 37^oC. The most stable condition existed at pH 7.0. The purified enzyme used both NADPH and NADH as electron donors for Cr(VI) reduction, while NADPH was the better, conferring 61% higher activity than NADH. The K_m values for NADPH and NADH were determined to be 47.5 and 17.2 umol, and the V_max values 322.2 and 130.7 umol Cr(VI) min^-1mg^-1 protein, respectively. The activity was strongly inhibited by N-ethylmalemide, Ag^2+, Cd^2+, Hg^2+, and Zn^2+. The antibody against the enzyme showed no immunological cross reaction with those of other Cr(VI) reducing strains.

A Proteomic Approach to Study msDNA Function in Escherichia coli: Mi-Ae Jeong , Dongbin Lim; J. Microbiol. 2004;42(3):200-204.; DOI: https://doi.org/2089 [pii]

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Abstract PDF: Retron is a prokaryotic genetic element that produces multicopy single-stranded DNA covalently linked to RNA (msDNA) by a reverse transcriptase. It was found that cells producing a large amount of msDNA, rather than those that did not, showed a higher rate of mutation. In order to understand the molecular mechanism connecting msDNA production to the high mutation rate the protein patterns were compared by two dimensional gel electrophoresis. Ten proteins were found to be differentially expressed at levels more than three fold greater in cells with than without msDNA, nine of which were identified by MALDI TOF MS. Eight of the nine identified proteins were repressed in msDNA-producing cells and, surprisingly, most were proteins functioning in the dissimilation of various carbon sources. One protein was induced four fold greater in the msDNA producing cells and was identified as a 30S ribosomal protein S2 involved in the regulation of translation. The molecular mechanism underlying the elevated mutation in msDNA-producing cell still remains elusive.

Regulation of fpr Gene encoding NADPH :Ferredoxin oxidoreductase by the soxRS locus in escherichia coli: Koh, Young Sang , Chouh, Jenny , Roe, Jung Hye; J. Microbiol. 1996;34(2):137-143.

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Abstract PDF: We isolated a promoter inducible by paraquat, a superoxide-generating agent, from Escherichia coli using a promoter-probing plasmid pRS415. From sequence analysis we found out the promoter is for fpr ENCODING nadph : ferredoxin oxidoreductase. We constructed on operon fusion of lacZ gene with fpr promoter to monitor the expression of the gene in the single-copy state. LacZ expression generators, menadione and plumbagin, also induced the expression of β-galactosidase in the fusion strain. On the other hand, no significant induction was observed by treatment with hydrogen peroxide, ethanol, and heat shock. Induction of β-galactosidase was significantly reduced by introducing a Δsox 8 :: cat of soxS3 :: Tn10 mutation into the fusion strain, indicating that fpr gene is a member of the soxRS regulon. The transcriptional start site was determined by primer extension analysis. Possible roles of fpr induction in superoxide stress were discussed.

Isolation and Characterization of Paraquat-inducible Promoters from Escherichia coli: Lee, Joon Hee , Roe, Jung Hye; J. Microbiol. 1997;35(4):277-283.

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Abstract PDF: Promoters inducible by paraquat, a superocide-generating agent, were isolated from Escherichia coli using a promoter-probing plasmid pRS415 with promoterless lacA gene. Twenty one promoters induced by paraquat were selected and further characterized. From sequence analysis, thirteen of the promoters were mapped to their specific loci on the Escherichia coli chromosome. Several promoters were mapped to the upstream of known genes such as usgl, katG, and mglB, whose relationships with superoxide response have not been previously reported. Other promoters were mapped to the upstream region of unknown open reading frames. Downstream of HC 96 promoter are uncharacterized ORFs whose sequences are homologous to ABC-transporter subunits. Downstream of HC84 promoter is an ORF encoding a transcriptional regulator-like protein, which contains a LysR family-specific HTH (helix-turn-helix) DNA bindign motif. We investigated whether these promoters belong to the soxRS regulon. All promoters except HC96 were found to belong to the soxRS regulon. The HC96 promoter was significantly induced by paraquat in the soxRS deletion mutant strain. The basal transcription level of three promoters (HE43, HC71, HD94) significantly increased at the stationary phase, implying that they are regulated by RpoS. However, paraquat inducibility of all promoters disappeared in the stationary phase, suggesting that SoxRS regulatory system is active only in rapidly growing cells.

Synthesis and Requirement of Escherichia coli Heat Shock Proteins GroEL and DnaK for Survival under Phenol Stress Conditions: Jeon, Taeck Joong , Lee, Kil Jae; J. Microbiol. 1998;36(1):26-33.

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Abstract PDF: Exposure of Escherichia coli strain MC4100 to various concentrations of phenol at temperatures higher than 20℃ led to induction of stress proteins such as GroEL and DnaK, as analyzed by SDS-PAGE and Western blotting methods. The optimum range of phenol concentration for the induction of GroEL and DnaK was slightly different at each temperature of bacterial growth and phenol treatment. The level of GroEL increased as the temperatures of growth and phenol treatment were increased from 30℃ to 40℃. The level of induced FroEL was maximal in the wild type cells which had been grown and treated by 2000㎍/㎖ phenol at 40℃. In contrast to GroEL, the level of DnaK decreased as the temperatures of growth and phenol treatment were increased from 25℃ to 40℃. Dnak was maximally induced in the cells grown and exposed to 1000㎍/㎖ phenol at 25℃. In rpoH mutant cells KY1601, GroEL was not additionally induced by phenol treatment and DnaK was not even detectable under normal and phenol stress conditions. Viability of cells under the same conditions of phenol treatment showed that the phenol resistance in much more induced in wild type cells than rpoH mutant cells. These results suggest that the induction of GroEL and DnaK is required for the enhanced viability of cells under conditions of phenol stress.

Laboratory Developed fluoroquinolone Resistant Escherichia coli Has a new Missense Mutation in QRDR of PartC: Lee, Soon Deuk , Lee, Yeon Hee; J. Microbiol. 1998;36(2):106-110.

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Abstract PDF: The fluoroquinolone resistance mechanism of four laboratory developed fluorquinolone resistant strains of Escherichia coli was studied. Fluoroquinolone concentrations inside the resistant cells were similar to the concentrations in the susceptible cells. DNA sequencing of the quinolone resistance determining regions (QRDR) in gyrA and parC revealed the presence of Ser 83Leu and Asp87Gly mutations in GyrA, and Gly78Cys and Ser80Arh mutations in ParC of the ofloxacin, norfloxacin, and HK3140 resistant strains, while the ciprofloxacin resistant strain had Ser83Leu and Aasp87Tyr mutations in GyrA, and Gly78Cys and Ser80Ile mutations in ParC. A Gly78Cys substitution in ParC was newly detected in this work and seemed to be responsible for the extremely high MICs to fluroquinolones.

Cloning and Nucleotide Sequence Analysis of Verotoxin Gene from Escherichia coli O157 KNIH317 Isolated in Korea: Park, Yong-Chjun , Shin, Hee Jung , Kim, Young Chang; J. Microbiol. 1999;37(3):168-173.

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Abstract PDF: Escherichia coli O157 is an important pathogenic organism which causes diarrhea, haemorrhagic colitis, and haemolytic ureamic syndrome (HUS) in human. E. coli O157 KNIH317 was isolated form patients suffering with HUS in Korea. We designed a primer set for cloning shiga-like toxin (slt) gene. The amplified PCR product was used to Southern and colony hybridization as a probe. As a result, we cloned 4.5-kb KpnI fragment containing the slt gene encoding shiga-like toxin from chromosomal DNA of E. coli O157 KNIH317. This recombinant plasmid was named pOVT45. E. coli XL1-Blue harboring pOVT45 showed cytotoxicity in Vero cells. We sequenced the slt gene of this strain. The A-subunit gene of the slt was composed of 960 base pairs with ATG initiation codon and TAA terminationcodon. The B-subunit was composed of 270 base paris with ATG initiation codon and TGA termination codon. Nucleotide sequence comparison of the slt gene exhibited 100%, 98.4%, 93.7%, and 93.7% identity with that of shiga-like toxin type II (sltII) of E. coli bacteriophage 933W, variant slt of E. coli, slt of E. coli, and variant sltII of E. coli, respectively. From these results, it was concluded that the cloned slt gene belongs to SltII family and that the strain used in this study may be a lysogeny of E. coli bacteriphage 933W.

Reduction of Hexavalent Chromium by Escherichia coli ATCC 33456 in Batch and Continuous Cultures: Woo Chul Bae , Tae Gu Kang , In Kyong Kang , You Jung Won , Byeong Chul Jeong; J. Microbiol. 2000;38(1):36-39.

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Abstract PDF: Toxic hexavalent chromium, Cr(VI), was reduced to a less toxic trivalent chromium form by E. coli ATCC 33456. The suitable electron donor for Cr(VI) reduction was glucose. E. coli ATCC 33456 was more resistant to metal cations than other reported Cr(VI) reducing microorganisms. Cell growth was inhibited by the presence of Cr(VI) in a liquid medium and Cr(VI) reduction accompanied cell growth. With a hydraulic retention time of 20 h, Cr(VI) reducing efficiency was 100% to 84% when Cr(VI) concentration in the influent was in the range of 10 to 40 mg L^-1. Specific rate of Cr(VI) concentration in the influent was 2.41 mg Cr(VI) g DCW^-1 h^-1 when 40 mg :^-1 of Cr(VI) influent was used. This result suggested the potential application of E. coli ATCC 33456 for the detoxification of Cr(VI) in Cr(VI) contaminated wastewater.

Continuous Synthesis of Escherichia coli GroEL at a High Temperature: Young Hak Kwak , Kyong Sun Lee , Ji Yeon Kim , Dong Seok Lee , Han Bok Kim; J. Microbiol. 2000;38(3):145-149.

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Abstract PDF: GroEL is a typical molecular chaperone. GroEL synthesis patterns at various culture temperatures in Escherichia coli were investigated in this study. No significant differences in the amount of GroEL produced from the chromosome were found at 30 and 37 C. However, GroEL production increased 3.4-fold at 42 C. GroEL synthesis was not transient but continuous at 42 C, although most heat shock gene expression is known to be transient. To understand the role of the groEL structural gene, a groE promoter-lacZ fusion was constructed. Interestingly, while transcriptional fusion is not thermally inducible, it is inducible by ethanol, suggesting that the secondary structure of the groEL transcript is involved in thermal regulation of the groEL gene. Secondary structures of groE mRNA at 37 and 42 C were compared using the computer program RNAdraw. Distinct structures at the two temperatures were found, and these structures may be related to a high level of GroEL expression at 42 C.

Regulation of Glycogen Concentration by the Histidine-Containing Phosphocarrier Protein HPr in Escherichia coli: Byoung-Mo Koo , Yeong-Jae Seok; J. Microbiol. 2001;39(1):24-30.

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Abstract PDF: In addition to effecting the catalysis of sugar uptake, the bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes. In a previous paper [Seok et al., (1997) J. Biol. Chem. 272, 26511-26521], we reported the interaction with and allosteric regulation of Escherichia coli glycogen phosphorylase activity by the histidine-containing phosphocarrier protein HPr in vitro. Here, we show that the specific interaction between HPr and glycogen phosphorylase occurs in vivo. To address the physiological role of the HPr-glycogen phosphorylase complex, intracellular glycogen levels were measured in E. coli strains transformed with various plasmids. While glycogen accumulated during the transition between exponential and stationary growth phases in wildtype cells, it did not accumulate in cells overproducing HPr or its inactive mutant regardless of the growth stage. From these results, we conclude that HPr mediates crosstalk between sugar uptake through the phosphoenolpyruvate:sugar phosphotransferase system and glycogen breakdown. The evolutionary significance of the HPr-glycogen phosphorylase complex is suggested.

Isolation of Norfloxacin Resistant Escherichia coli from the Han River and Characterization of Resistance Mechanism: Yoosun Jung , Hyunjin Hong , Hyeran Nam , Yeonhee Lee; J. Microbiol. 2002;40(1):63-69.

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Abstract PDF: A total of twenty-five norfloxacin resistant Escherichia coli were isolated from Joongrang-chun stream, a branch of the Han River in Seoul, Korea from May to July in 2000 and their norfloxacin resistance mechanism was characterized for target site mutation, permeability, and efflux pump. Fourteen isolates contained the same three mutations, Ser83->Leu and Asp87->Asn in GyrA and Ser90->Ile in ParC. Six isolates had Ser83->Leu and Asp87->Tyr in GyrA and Ser80->Ile in ParC while one isolate had Ser83->Leu and Val103->Ala in GyrA and Ser80->Ile in ParC. Two isolates had mutation(s) in GyrA without any mutation in ParC. Two isolates had Ser80->Arg in ParC instead of the commonly found Ser80->Ile. Every norfloxacin resistant isolate had an efflux system but the correlation between the efflux activity and MIC was not observed. The amount of OmpF for norfloxacin permeability decreased in resistant isolates compared to the susceptible strains. When amplified polymorphic DNA (RAPD) and pulse field gel electrophoresis (PFGE) were performed, these isolates showed no similarity to each other or clinical isolates.

Histological Alterations and Immune Response Induced by Pet Toxin During Colonization with Enteroaggregative Escherichia coli (EAEC) in a Mouse Model: Teresita Sainz , Julia Perez , Ma. Cristina Fresan , Veronica Flores , Luis Jimenez , Ulises Hernandez , Ismael Herrera , Carlos Eslava; J. Microbiol. 2002;40(2):91-97.

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Abstract PDF: Enteroaggregative E. coli (EAEC) is an important aethiological causal agent of diarrhea in people of developed and undeveloped countries. Different in vitro and in vivo models have been proposed to study the pathogenic and immune mechanisms of EAEC infection. The aim of this study was to analyze whether BALB/c mice could be used as an animal model to study EAEC pathogenesis. Six-week-old BALB/c mice were inoculated with EAEC strain 042 (O44:H18) nalidixic acid resistant, and re-inoculated ten days after. Mice feces were monitored for the presence of the EAEC strain over a period of 20 days. Bacteria were enumerated on MacConkey agar containing 100 ug of nalidixic acid per ml. Results showed that 35% of the animals were colonized for 3 days, 15% for 5 and 10% for more than 7 days. After re-inoculation only 16% of the animals remained colonized for more than 3 days. During the necropsy, the intestinal fluid of some of the infected animals presented mucus and blood. Six of these fluids showed the presence of IgA antibodies against Pet toxin and IgG antibodies raised against the toxin were also detected in the animal serum. Histopathologic evidence confirms the stimulation of mucus hypersecretion, an increased amount of goblet cells and the presence of bacterial aggregates in the apical surfaces of intestinal epithelial cells. Edema was present in the submucosa. These results suggest that BALB/c mice could be used as an animal model for the in vivo study of EAEC infection.

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