Journal of Microbiology

Volume 54(11); November 2016

Review

MINIREVIEW] High-resolution imaging of the microbial cell surface: Ki Woo Kim; J. Microbiol. 2016;54(11):703-708. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6348-5

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Abstract: Microorganisms, or microbes, can function as threatening pathogens that cause disease in humans, animals, and plants; however, they also act as litter decomposers in natural ecosystems. As the outermost barrier and interface with the environment, the microbial cell surface is crucial for cell-to-cell communication and is a potential target of chemotherapeutic agents. Surface ultrastructures of microbial cells have typically been observed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Owing to its characteristics of low-temperature specimen preparation and superb resolution (down to 1 nm), cryo-field emission SEM has revealed paired rodlets, referred to as hydrophobins, on the cell walls of bacteria and fungi. Recent technological advances in AFM have enabled high-speed live cell imaging in liquid at the nanoscale level, leading to clear visualization of celldrug interactions. Platinum-carbon replicas from freeze-fractured fungal spores have been observed using transmission electron microscopy, revealing hydrophobins with varying dimensions. In addition, AFM has been used to resolve bacteriophages in their free state and during infection of bacterial cells. Various microscopy techniques with enhanced spatial resolution, imaging speed, and versatile specimen preparation are being used to document cellular structures and events, thus addressing unanswered biological questions.

Journal Articles

Siphonobacter intestinalis sp. nov., a bacterium isolated from the feces of Pseudorhynchus japonicus: Shin Ae Lee , Jeong Myeong Kim , Jae-Hyung Ahn , Jae-Ho Joa , Soo-Jin Kim , Mee-Kyung Sang , Jaekyeong Song , Soon-Wo Kwon , Hang-Yeon Weon; J. Microbiol. 2016;54(11):709-712. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6451-7

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Abstract: Strain 63MJ-2T was isolated from the feces of broad-winged katydid (Pseudorhynchus japonicus) collected in Korea. The 16S rRNA gene sequence of this strain showed the highest sequence similarity with that of Siphonobacter aquaeclarae P2T (96.1%) and had low similarities (below 86.3%) with those of other members of family ‘Flexibacteraceae’. The strain 63MJ-2T is a strictly aerobic, Gram-stain-negative, non-motile, rod-shaped bacterium. The strain grew at 4–35°C (optimum, 25–30°C), pH of 5.0–9.0 (optimum, 6.0–7.0), and 0–2.0% (optimum, 1.0–2.0) (w/v) NaCl. The DNA G+C content of strain 63MJ-2T was 43.5 mol%. The major fatty acids were C16:1 ω5c (42.5%), iso-C17:0 3-OH (18.7%), and summed feature 3 (iso-C15:0 2-OH and/or C16:1 ω7c, 18.0%). The major menaquinone was MK-7 and polar lipids were phosphatidylethanolamine, six unknown aminolipids, and five unknown lipids. Based on the evidence from our polyphasic taxonomic study, we conclude that strain 63MJ-2T should be classified as a novel species of the genus Siphonobacter, and propose the name Siphonobacter intestinalis sp. nov. The type strain is 63MJ-2T (=KACC 18663T =NBRC 111883T).

Vertical distribution of bacterial community is associated with the degree of soil organic matter decomposition in the active layer of moist acidic tundra: Hye Min Kim , Min Jin Lee , Ji Young Jung , Chung Yeon Hwang , Mincheol Kim , Hee-Myong Ro , Jongsik Chun , Yoo Kyung Lee; J. Microbiol. 2016;54(11):713-723. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6294-2

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54 Citations

Abstract: The increasing temperature in Arctic tundra deepens the active layer, which is the upper layer of permafrost soil that experiences repeated thawing and freezing. The increasing of soil temperature and the deepening of active layer seem to affect soil microbial communities. Therefore, information on soil microbial communities at various soil depths is essential to understand their potential responses to climate change in the active layer soil. We investigated the community structure of soil bacteria in the active layer from moist acidic tundra in Council, Alaska. We also interpreted their relationship with some relevant soil physicochemical characteristics along soil depth with a fine scale (5 cm depth interval). The bacterial community structure was found to change along soil depth. The relative abundances of Acidobacteria, Gammaproteobacteria, Planctomycetes, and candidate phylum WPS-2 rapidly decreased with soil depth, while those of Bacteroidetes, Chloroflexi, Gemmatimonadetes, and candidate AD3 rapidly increased. A structural shift was also found in the soil bacterial communities around 20 cm depth, where two organic (upper Oi and lower Oa) horizons are subdivided. The quality and the decomposition degree of organic matter might have influenced the bacterial community structure. Besides the organic matter quality, the vertical distribution of bacterial communities was also found to be related to soil pH and total phosphorus content. This study showed the vertical change of bacterial community in the active layer with a fine scale resolution and the possible influence of the quality of soil organic matter on shaping bacterial community structure.

Dynamics of bacterial communities in rice field soils as affected by different long-term fertilization practices: Jae-Hyung Ahn , Shin Ae Lee , Jeong Myeong Kim , Myung-Sook Kim , Jaekyeong Song , Hang-Yeon Weon; J. Microbiol. 2016;54(11):724-731. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6463-3

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28 Citations

Abstract: Fertilization and the response of the soil microbial community to the process significantly affect crop yield and the environment. In this study, the seasonal variation in the bacterial communities in rice field soil subjected to different fertilization treatments for more than 50 years was investigated using 16S rRNA sequencing. The simultaneous application of inorganic fertilizers and rice straw compost (CAPK) maintained the species richness of the bacterial communities at levels higher than that in the case of non-fertilization (NF) and application of inorganic fertilizers only (APK) in the initial period of rice growth. The seasonal variation in the bacterial community structure in the NF and APK plots showed cyclic behavior, suggesting that the effect of season was important; however, no such trend was observed in the CAPK plot. In the CAPK plot, the relative abundances of putative copiotrophs such as Bacteroidetes, Firmicutes, and Proteobacteria were higher and those of putative oligotrophs such as Acidobacteria and Plactomycetes were lower than those in the other plots. The relative abundances of organotrophs with respiratory metabolism, such as Actinobacteria, were lower and those of chemoautotrophs that oxidize reduced iron and sulfur compounds were higher in the CAPK plot, suggesting greater carbon storage in this plot. Increased methane emission and nitrogen deficiency, which were inferred from the higher abundances of Methylocystis and Bradyrhizobium in the CAPK plot, may be a negative effect of rice straw application; thus, a solution for these should be considered to increase the use of renewable resources in agricultural lands.

An in vitro study of the antifungal activity of Trichoderma virens 7b and a profile of its non-polar antifungal components released against Ganoderma boninense: Lee Pei Lee Angel , Mohd Termizi Yusof , Intan Safinar Ismail , Bonnie Tay Yen Ping , Intan Nur Ainni Mohamed Azni , Norman Hj Kamarudin , Shamala Sundram; J. Microbiol. 2016;54(11):732-744. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6304-4

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47 Citations

Abstract: Ganoderma boninense is the causal agent of a devastating disease affecting oil palm in Southeast Asian countries. Basal stem rot (BSR) disease slowly rots the base of palms, which radically reduces productive lifespan of this lucrative crop. Previous reports have indicated the successful use of Trichoderma as biological control agent (BCA) against G. boninense and isolate T. virens 7b was selected based on its initial screening. This study attempts to decipher the mechanisms responsible for the inhibition of G. boninense by identifying and characterizing the chemical compounds as well as the physical mechanisms by T. virens 7b. Hexane extract of the isolate gave 62.60% ± 6.41 inhibition against G. boninense and observation under scanning electron microscope (SEM) detected severe mycelial deformation of the pathogen at the region of inhibition. Similar mycelia deformation of G. boninense was observed with a fungicide treatment, Benlate® indicating comparable fungicidal effect by T. virens 7b. Fraction 4 and 5 of hexane active fractions through preparative thin layer chromatography (P-TLC) was identified giving the best inhibition of the pathogen. These fractions comprised of ketones, alcohols, aldehydes, lactones, sesquiterpenes, monoterpenes, sulphides, and free fatty acids profiled through gas chromatography mass spectrometry detector (GC/MSD). A novel antifungal compound discovery of phenylethyl alcohol (PEA) by T. virens 7b is reported through this study. T. virens 7b also proved to be an active siderophore producer through chrome azurol S (CAS) agar assay. The study demonstrated the possible mechanisms involved and responsible in the successful inhibition of G. boninense.

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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Abstract: 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.

The in vitro and in vivo efficacy of fluconazole in combination with farnesol against Candida albicans isolates using a murine vulvovaginitis model: Aliz Bozó , Marianna Domán , László Majoros , Gábor Kardos , István Varga , Renátó Kovács; J. Microbiol. 2016;54(11):753-760. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6298-y

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Abstract: Farnesol is a quorum-sensing molecule that inhibits biofilm formation in Candida albicans. Previous in vitro data suggest that, in combination with certain antifungals, farnesol may have an adjuvant anti-biofilm agent. However, the in vivo efficacy of farnesol is very questionable. Therefore, the in vitro and in vivo activity of fluconazole combined with farnesol was evaluated against C. albicans biofilms using fractional inhibitory concentration index (FICI) determination, time-kill experiments and a murine vulvovaginitis model. The median biofilm MICs of fluconazole-sensitive C. albicans isolates ranged between 4 -> 512 mg/L and 150–300 μM for fluconazole and farnesol, respectively. These values were 512 -> 512 mg/L and > 300 μM for fluconazole-resistant clinical isolates. Farnesol decreased the median MICs of fluconazole by 2-64-fold for biofilms. Based on FICI, synergistic interaction was observed only in the case of the sessile SC5314 reference strain (FICIs: 0.16–0.27). In time-kill studies, only the 512 mg/L fluconazole and 512 mg/L fluconazole + 75 μM farnesol reduced biofilm mass significantly at each time point in the case of all isolates. The combination reduced the metabolic activity of biofilms for all isolates in a concentration- and time-dependent manner. Our findings revealed that farnesol alone was not protective in a murine vulvovaginitis model. Farnesol was not beneficial in combination with fluconazole for fluconazole-susceptible isolates, but partially increased fluconazole activity against one fluconazole- resistant isolate, but not the other one.

The assessment of host and bacterial proteins in sputum from active pulmonary tuberculosis: Hsin-Chih Lai , Yu-Tze Horng , Pen-Fang Yeh , Jann-Yuan Wang , Chin-Chung Shu , Jang-Jih Lu , Jen-Jyh Lee , Po-Chi Soo; J. Microbiol. 2016;54(11):761-767. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6201-x

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Abstract: Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis. The protein composition of sputum may reflect the immune status of the lung. This study aimed to evaluate the protein profiles in spontaneous sputum samples from patients with active pulmonary TB. Sputum samples were collected from patients with pulmonary TB and healthy controls. Western blotting was used to analyze the amount of interleukin 10 (IL-10), interferon-gamma (IFN-γ), IL-25, IL- 17, perforin-1, urease, albumin, transferrin, lactoferrin, adenosine deaminase (also known as adenosine aminohydrolase, or ADA), ADA-2, granzyme B, granulysin, and caspase- 1 in sputum. Results of detection of IL-10, IFN-γ, perforin- 1, urease, ADA2, and caspase-1, showed relatively high specificity in distinguishing patients with TB from healthy controls, although sensitivities varied from 13.3% to 66.1%. By defining a positive result as the detection of any two proteins in sputum samples, combined use of transferrin and urease as markers increased sensitivity to 73.2% and specificity to 71.1%. Furthermore, we observed that the concentration of transferrin was proportional to the number of acidfast bacilli detected in sputum specimens. Detection of sputum transferrin and urease was highly associated with pulmonary TB infection. In addition, a high concentration of transferrin detected in sputum might correlate with active TB infection. This data on sputum proteins in patients with TB may aid in the development of biomarkers to assess the severity of pulmonary TB.

GPH1 is involved in glycerol accumulation in the three-dimensional networks of the nematode-trapping fungus Arthrobotrys oligospora: Qin-Yi Wu , Yue-Yan Zhu , Cheng-Gang Zou , Ying-Qian Kang , Lian-Ming Liang; J. Microbiol. 2016;54(11):768-773. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6272-8

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Abstract: Turgor is very important for the invasive growth of fungal pathogens. Glycerol, a highly osmotic solvent, is considered to play an important role in turgor generation. The nematophagous fungus Arthrobotrys oligospora mainly lives as a saprophyte. In the presence of nematodes, A. oligospora enters the parasitic stage by forming three-dimensional networks (traps) to capture nematodes. In A. oligospora, we found that glycerol accumulated during nematode-induced trap formation. We demonstrated that deleting gph1, which encodes glycogen phosphorylase, decreased the glycerol content, compared with that of a wild-type strain. Although the number of traps induced by nematodes was not affected in the Δgph1 mutant, the capture rate was lower. Meanwhile, deleting gph1 also affected the growth rate and conidiation capacity of the fungus. These results indicate that glycerol derived from GPH1 is essential for the full virulence of A. oligospora against nematodes.

A computationally simplistic poly-phasic approach to explore microbial communities from the Yucatan aquifer as a potential sources of novel natural products: Marfil-Santana Miguel David , O’Connor-Sánchez Aileen , Ramírez-Prado Jorge Humberto , De los Santos-Briones Cesar , López- Aguiar , Lluvia Korynthia , Rojas-Herrera Rafael , Lago-Lestón Asunción , Prieto-Davó Alejandra; J. Microbiol. 2016;54(11):774-781. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6092-x

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Abstract: The need for new antibiotics has sparked a search for the microbes that might potentially produce them. Current sequencing technologies allow us to explore the biotechnological potential of microbial communities in diverse environments without the need for cultivation, benefitting natural product discovery in diverse ways. A relatively recent method to search for the possible production of novel compounds includes studying the diverse genes belonging to polyketide synthase pathways (PKS), as these complex enzymes are an important source of novel therapeutics. In order to explore the biotechnological potential of the microbial community from the largest underground aquifer in the world located in the Yucatan, we used a polyphasic approach in which a simple, non-computationally intensive method was coupled with direct amplification of environmental DNA to assess the diversity and novelty of PKS type I ketosynthase (KS) domains. Our results suggest that the bioinformatic method proposed can indeed be used to assess the novelty of KS enzymes; nevertheless, this in silico study did not identify some of the KS diversity due to primer bias and stringency criteria outlined by the metagenomics pipeline. Therefore, additionally implementing a method involving the direct cloning of KS domains enhanced our results. Compared to other freshwater environments, the aquifer was characterized by considerably less diversity in relation to known ketosynthase domains; however, the metagenome included a family of KS type I domains phylogenetically related, but not identical, to those found in the curamycin pathway, as well as an outstanding number of thiolases. Over all, this first look into the microbial community found in this large Yucatan aquifer and other fresh water free living microbial communities highlights the potential of these previously overlooked environments as a source of novel natural products.

Latent Kaposi’s sarcoma-associated herpesvirus infection in bladder cancer cells promotes drug resistance by reducing reactive oxygen species: Suhyuk Lee , Jaehyuk Jang , Hyungtaek Jeon , Jisu Lee , Seung-Min Yoo , Jinsung Park , Myung-Shin Lee; J. Microbiol. 2016;54(11):782-788. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6388-x

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7 Citations

Abstract: Kaposi’s sarcoma-associated herpesvirus (KSHV) is the major etiologic agent of Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. Recent studies have indicated that KSHV can be detected at high frequency in patient-derived bladder cancer tissue and might be associated with the pathogenesis of bladder cancer. Bladder cancer is the second most common cancer of the genitourinary tract, and it has a high rate of recurrence. Because drug resistance is closely related to chemotherapy failure and cancer recurrence, we investigated whether KSHV infection is associated with drug resistance of bladder cancer cells. Some KSHV-infected bladder cancer cell lines showed resistance to an anti-cancer drug, cisplatin, possibly as a result of downregulation of reactive oxygen species. Additionally, drug resistance acquired from KSHV infection could partly be overcome by HDAC1 inhibitors. Taken together, the data suggest the possible role of KSHV in chemo-resistant bladder cancer, and indicate the therapeutic potential of HDAC1 inhibitors in drug-resistant bladder cancers associated with KSHV infection.

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