Journal of Microbiology

Journal Articles

Non-ureolytic calcium carbonate precipitation by Lysinibacillus sp. YS11 isolated from the rhizosphere of Miscanthus sacchariflorus: Yun Suk Lee , Hyun Jung Kim , Woojun Park; J. Microbiol. 2017;55(6):440-447. Published online May 28, 2017; DOI: https://doi.org/10.1007/s12275-017-7086-z

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Although microbially induced calcium carbonate precipita-tion (MICP) through ureolysis has been widely studied in en-vironmental engineering fields, urea utilization might cause environmental problems as a result of ammonia and nitrate production. In this study, many non-ureolytic calcium car-bonate-precipitating bacteria that induced an alkaline envi-ronment were isolated from the rhizosphere of Miscanthus sacchariflorus near an artificial stream and their ability to pre-cipitate calcium carbonate minerals with the absence of urea was investigated. MICP was observed using a phase-contrast microscope and ion-selective electrode. Only Lysinibacillus sp. YS11 showed MICP in aerobic conditions. Energy disper-sive X-ray spectrometry and X-ray diffraction confirmed the presence of calcium carbonate. Field emission scanning elec-tron microscopy analysis indicated the formation of morpho-logically distinct minerals around cells under these condi-tions. Monitoring of bacterial growth, pH changes, and Ca2+ concentrations under aerobic, hypoxia, and anaerobic con-ditions suggested that strain YS11 could induce alkaline con-ditions up to a pH of 8.9 and utilize 95% of free Ca2+ only under aerobic conditions. Unusual Ca2+ binding and its re-lease from cells were observed under hypoxia conditions. Bio-film and extracellular polymeric substances (EPS) formation were enhanced during MICP. Strain YS11 has resistance at high pH and in high salt concentrations, as well as its spore- forming ability, which supports its potential application for self-healing concrete.

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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.

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From simple to complex: Protein‐based biomarker discovery in tuberculosis
Zaynab Mousavian, Gunilla Källenius, Christopher Sundling
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Research Support, Non-U.S. Gov't

Morphological changes in human gastric epithelial cells induced by nuclear targeting of Helicobacter pylori urease subunit A: Jung Hwa Lee , So Hyun Jun , Jung-Min Kim , Seung Chul Baik , Je Chul Lee; J. Microbiol. 2015;53(6):406-414. Published online May 30, 2015; DOI: https://doi.org/10.1007/s12275-015-5085-5

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Abstract

Nuclear targeting of bacterial proteins and their pathological effects on host cells are an emerging pathogenic mechanism in bacteria. We have previously reported that urease subunit A (UreA) of Helicobacter pylori targets the nuclei of COS-7 cells through nuclear localization signals (NLSs). This study further investigated whether UreA of H. pylori targets the nuclei of gastric epithelial cells and then induces molecular and cellular changes in the host cells. H. pylori 26695 strain produced and secreted outer membrane vesicles (OMVs). UreA was translocated into gastric epithelial AGS cells through outer membrane vesicles (OMVs) and then targeted the nuclei of AGS cells. Nuclear targeting of rUreA did not induce host cell death, but resulted in morphological changes, such as cellular elongation, in AGS cells. In contrast, AGS cells treated with rUreAΔNLS proteins did not show this morphological change. Next generation sequencing revealed that nuclear targeting of UreA differentially regulated 102 morphogenesis- related genes, of which 67 and 35 were up-regulated and down-regulated, respectively. Our results suggest that nuclear targeting of H. pylori UreA induces both molecular and cellular changes in gastric epithelial cells.

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Journal Article

Use of Selected Lactic Acid Bacteria in the Eradication of Helicobacter pylori Infection: Jin-Eung Kim , Min-Soo Kim , Yeo-Sang Yoon , Myung-Jun Chung , Do-Young Yum; J. Microbiol. 2014;52(11):955-962. Published online October 3, 2014; DOI: https://doi.org/10.1007/s12275-014-4355-y

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Abstract

Helicobacter pylori is among the major pathogenic bacteria that cause chronic gastritis and peptic ulcer disease and is related to the development of gastric cancer. Several chemicals, including antibiotics, have been used to eradicate H. pylori; however, they do not always curb the infection. Ten representative type strains of lactic acid bacteria (LAB) were screened for antagonism toward H. pylori via inhibition of urease activity. Strains inhibiting the binding of H. pylori to human gastric cell line cells and suppressing H. pylori-induced interleukin-8 (IL-8) production were also screened. Of these, Pediococcus pentosaseus (SL4), which inhibited the adhesion of H. pylori to MKN-45 gastric cancer cells, Bifidobacterium longum (BG7), with urease inhibiting activity, and Lactococcus lactis (SL3), and Enterococcus faecalis (SL5), which suppressed H. pylori-induced IL-8 production within MKN-45 and AGS cells, were selected. In mouse model, these LAB stains in combination significantly suppressed IL-8 levels in serum. Gastric pH also recovered to normal values after the administration of these LAB. These stains effectively suppressed H. pylori viability, although not to the extent of antibiotic treatment. When used as probiotics, LAB may help decrease the occurrence of gastritis and reduce the risk of H. pylori infection without, inducing side effects.

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

Screening of Mutant Strain Streptomyces mediolani sp. AC37 for (-)-8-O-Methyltetrangomycin Production Enhancement: Jakeline Trejos Jiménez , Maria Sturdíková , Vlasta Brezová , Emil Svajdlenka , Marta Novotová; J. Microbiol. 2012;50(6):1014-1023. Published online December 30, 2012; DOI: https://doi.org/10.1007/s12275-012-2025-5

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Abstract: Streptomyces mediolani sp. AC37 was isolated from the root system of higher plant Taxus baccata and produced metabolite identified as (-)-8-O-methyltetrangomycin according to LC/MS/MS analysis. In our screening program for improvements of bioactive secondary metabolites from plant associate streptomycetes, mutation was used as a tool for the induction of genetic variations for selection of higher (-)-8-O-methyltetrangomycin producers of isolates. S. mediolani sp. AC37 was treated with UV irradiation and chemical mutagenic treatment (N-nitroso-N-methyl-urea). The radical scavenging and antioxidant capacity of (-)-8-O-methyltetrangomycin and extracts isolated from mutants were tested using EPR spin trapping technique and ABTS􀁹+ assay. Comparison of electron microscopic images of Streptomyces sp. AC37 and mutant strains of Streptomyces sp. AC37 revealed substantial differences in morphology and ultrastructure.

Protection Against Helicobacter pylori Infection by a Trivalent Fusion Vaccine Based on a Fragment of Urease B-UreB414: Li Wang Wang , Xiao-Fei Liu , Shi Yun , Xiao-Peng Yuan , Xu-Hu Mao , Chao Wu , Wei-Jun Zhang , Kai-Yun Liu , Gang Guo , Dong-Shui Lu , Wen-De Tong , Ai-Dong Wen , Quan-Ming Zou; J. Microbiol. 2010;48(2):223-228. Published online May 1, 2010; DOI: https://doi.org/10.1007/s12275-009-0233-4

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Abstract: A multivalent fusion vaccine is a promising option for protection against Helicobacter pylori infection. In this study, UreB414 was identified as an antigenic fragment of urease B subunit (UreB) and it induced an antibody inhibiting urease activity. Immunization with UreB414 partially protected mice from H. pylori infection. Furthermore, a trivalent fusion vaccine was constructed by genetically linking heat shock protein A (HspA), H. pylori adhesin A (HpaA), and UreB414, resulting in recombinant HspA-HpaA-UreB414 (rHHU). Its protective effect against H. pylori infection was tested in BALB/c mice. Oral administration of rHHU significantly protected mice from H. pylori infection, which was associated with H. pylori-specific antibody production and Th1/Th2-type immune responses. The results show that a trivalent fusion vaccine efficiently combats H. pylori infection, and that an antigenic fragment of the protein can be used instead of the whole protein to construct a multivalent vaccine.

Phylogeny of a Novel “Helicobacter heilmannii” Organism from a Japanese Patient with Chronic Gastritis Based on DNA Sequence Analysis of 16S rRNA and Urease Genes: Takehisa Matsumoto , Masatomo Kawakubo , Mayumi Shiohara , Toshiko Kumagai , Eiko Hidaka , Kazuyoshi Yamauchi , Kozue Oana , Kenji Matsuzawa , Hiroyoshi Ota , Yoshiyuki Kawakami; J. Microbiol. 2009;47(2):201-207. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0313-x

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Abstract: “Helicobacter heilmannii” is an uncultivable spiral-shaped bacterium inhabiting the human gastric mucosa. It is larger and more tightly-coiled than H. pylori. We encountered a patient with chronic gastritis infected a “H. heilmannii”-like organism (HHLO), designated as SH6. Gastric mucosa derived from the patient was orally ingested by specific pathogen free mice. Colonization of the mice by SH6 was confirmed by electron microscopy of gastric tissue specimens. In an attempt to characterize SH6, 16S rRNA and urease genes were sequenced. The 16S rRNA gene sequence was most similar (99.4%; 1,437/1,445 bp) to HHLO C4E from a cheetah. However, the urease gene sequence displayed low similarity (81.7%; 1,240/1,516 bp) with HHLO C4E. Taxonomic analysis disclosed that SH6 represents a novel strain and should constitute a novel taxon in the phylogenetic trees, being discriminated from any other taxon, with the ability of infecting human gastric mucosa.

Rapid One Step Detection of Pathogenic Bacteria in Urine with Sexually Transmitted Disease (STD) and Prostatitis Patient by Multiplex PCR Assay (mPCR): Sang Rok Lee , Ji Min Chung , Young Gon Kim; J. Microbiol. 2007;45(5):453-459.; DOI: https://doi.org/2590 [pii]

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Abstract: We developed a multiplex PCR (mPCR) assay to simultaneously detect Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Corynebacterium spp. and seudomona aeruginosa. This method employs a single tube and multiple specific primers which yield 200, 281, 346, 423, 542, and 1,427 bp PCR products, respectively. All the PCR products were easily detected by agarose gel electrophoresis and were sequenced to confirm the specificity of the reactions. To test this method, DNA extracted from urine samples was collected from 96 sexually transmitted disease or prostatitis patients at a local hospital clinical center, and were subjected to the mPCR assay. The resulting amplicons were cloned and sequenced to exactly match the sequences of known pathogenic isolates. N. gonorrhoeae and Corynebacterium spp. were the most frequently observed pathogens found in the STDs and prostatitis patients, respectively. Unexpectedly, P. aeruginosa was also detected in some of the STD and prostatitis samples. More than one pathogen species was found in 10% and 80.7% of STD and prostatitis samples, respectively, indicating that STD and prostatitis patients may have other undiagnosed and associates. The sensitivity of the assay was determined by sing purified DNA from six pathogenic laboratory strains and revealed that this technique could detect pathogenic DNA at concentrations ranging from 0.018 to 1.899 pg/μl. Moreover, the specificities of this assay were found to be highly efficient. Thus, this mPCR assay may be useful for the rapid diagnosis of causative infectious STDs and prostatitis. useful for the infectious STDs and prostatitis.

Characteristics of Urease from Vibrio parahaemolyticus Possessing tdh and trh Genes Isolated in Korea: Young Hee Kim , Jong Sook Kim; J. Microbiol. 2001;39(4):279-285.

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Abstract: Vibrio parahaemolyticus is a halophilic bacterium associated with seafood gastroenteritis. An unusual strain of Kanagawa-positive urease producing Vibrio parahaemolyticus O1 : K1 was isolated from the environment and identified. A polymerase chain reaction assay revealed that this strain harbored both the tdh and trh genes. The urease from this strain was studied. Maximum urease production was induced in LB medium containing 0.2% urea, 0.5% glucose, 2% NaCl and pH 5.5 with 6 h of cultivation at 37 C under aeration. Purification of urease was achieved by the process of whole cell lysate, 65% ammonium sulphate precipitation, DEAE-cellulose ion exchange column chromatography, Sepharose CL-6B gel filtration and oxirane activated Sepharose 6B-urea affinity chromatography with 203 fold purification and 2.2% yield. Analysis of the purified enzyme by SDS-PAGE demonstrated the presence of the subunits with a molecular weight of 85 kDa, 59 kDa, 41 kDa and the molecular weight for the native enzyme by nondenaturing PAGE and gel filtration chromatography was 255 kDa. The purified urease was stable at pH 7.5 and the optimal pH in HEPES buffer was 8.0. The enzyme was stable at 60 C for 2 h with a residual activity of 32%. The addition of 10 uM of NiCl_2 maintained stability for 30 min. The Km value of the purified enzyme was 35.6 mM in urea substrate. The TD_50 (median toxic dose) of the purified urease was 2.5 ug/ml on human leukemia cells.

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