Journal of Microbiology

Volume 44(4); August 2006

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

Protein Expression Analysis of Halobacillus dabanensis D-8T Subjected to Salt Shock: De Qin Feng , Bo Zhang , Wei Dong Lu , Su Sheng Yang; J. Microbiol. 2006;44(4):369-374.; DOI: https://doi.org/2418 [pii]

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Abstract: To investigate the mechanism of salt tolerance of gram-positive moderately halophilic bacteria, two-dimensional gel electrophoresis (2-D PAGE) was employed to achieve high resolution maps of proteins of Halobacillus dabanensis D-8T. Approximately 700 spots of proteins were identified from these 2-D PAGE maps. The majority of these proteins had molecular weights between 17.5 and 66 kDa, and most of them were distributed between the isoelectric points (pI) 4.0 and 5.9. Some protein spots were distributed in the more acidic region of the 2-D gel (pI <4.0). This pattern indicated that a number of proteins in the strain D-8T are acidic. To understand the adaptation mechanisms of moderately halophilic bacteria in response to sudden environmental changes, differential protein profiles of this strain were investigated by 2-D PAGE and ImagemasterTM 2D Platinum software after the cells were subjected to salt shock of 1 to 25% salinity for 5 and 50 min. Analysis showed 59 proteins with an altered level of expression as the result of the exposure to salt shock. Eighteen proteins had increased expression, 8 proteins were induced, and the expression of 33 proteins was down-regulated. Eight of the up-regulated proteins were identified using MALDI-TOF/MS and MASCOT, and were similar to proteins involved in signal transduction, proteins participating in energy metabolism pathways and proteins involved in stress.

Journal Article

Proteomic Analysis of Protein Expression in Streptococcus pneumoniae in Response to Temperature Shift: Myoung-Ro Lee , Song-Mee Bae , Tong-Soo Kim , Kwang-Jun Lee; J. Microbiol. 2006;44(4):375-382.; DOI: https://doi.org/2417 [pii]

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Abstract: From its initial colonization to causation of disease, Streptococcus pneumoniae has evolved strategies to cope with a number of stressful in vivo environmental conditions. In order to analyze a global view of this organism’s response to heat shock, we established a 2-D electrophoresis proteome map of the S. pneumoniae D39 soluble proteins under in vitro culture conditions and performed the comparative proteome analysis to a 37 to 42°C temperature up-shift in S. pneumoniae. When the temperature of an exponentially growing S. pneumoniae D39 culture was raised to 42°C, the expression level of 25 proteins showed changes when compared to the control. Among these 25 proteins, 12 were identified by MALDI-TOF and LC-coupled ESI MS/MS. The identified proteins were shown to be involved in the general stress response, energy metabolism, nucleotide biosynthesis pathways, and purine metabolism. These results provide clues for understanding the mechanism of adaptation to heat shock by S. pneumoniae and may facilitate the assessment <br>of a possible role for these proteins in the physiology and pathogenesis of this pathogen.

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

A Comparison of the Phenotypic and Genetic Stability of Recombinant Trichoderma spp. Generated by Protoplast- and Agrobacterium-Mediated Transformation: Rosa Elena Cardoza , Juan Antonio Vizcaino , Maria Rosa Hermosa , Enrique Monte , Santiago Gutierrez; J. Microbiol. 2006;44(4):383-395.; DOI: https://doi.org/2416 [pii]

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Abstract: Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 transformants/μg DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 transformants/107 spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transformants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.

Isolation and Characterization of Cold-adapted Strains Producing β-Galactosidase: Jeong-Won Park , Yong-Sik Oh , Jai-Yun Lim , Dong-Hyun Roh; J. Microbiol. 2006;44(4):396-402.; DOI: https://doi.org/2414 [pii]

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Abstract: β-Galactosidase is extensively employed in the manufacture of dairy products, including lactose-reduced milk. Here, we have isolated two gram-negative and rod-shaped coldadapted bacteria, BS 1 and HS 39. These strains were able to break down lactose at low temperatures. Although two isolates were found to grow well at 10°C, the BS 1 strain was unable to grow at 37°C. Another strain, HS-39, evidenced retarded growth at 37°C. The biochemical characteristics and the results of 16S rDNA sequencing identified the BS 1 isolate as Rahnella aquatilis, and showed that the HS 39 strain belonged to genus Buttiauxella. Whereas the R. aquatilis BS 1 strain generated maximal quantities of β-galactosidase when incubated for 60 h at 10°C, Buttiauxella sp. HS-39 generated β-galactosidase earlier, and at slightly lower levels, than R. aquatilis BS 1. The optimum temperature for β-galactosidase was 30°C for R. aquatilis BS-1, and was 45°C for Buttiauxella sp. HS-39, thereby indicating that R. aquatilis BS-1 was able to generate a cold-adaptive enzyme. These two cold-adapted strains, and most notably the β-galactosidase from each isolate, might prove useful in some biotechnological applications.

Validation Study

Rapid Detection of Noroviruses in Fecal Samples and Shellfish by Nucleic Acid Sequence-based Amplification: Xiaoxia Kou , Qingping Wu , Jumei Zhang , Hongying Fan; J. Microbiol. 2006;44(4):403-408.; DOI: https://doi.org/2413 [pii]

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Abstract: The purpose of this study was to determine the efficacy of a nucleic acid sequence-based amplification (NASBA) method of detecting noroviruses in artificially and naturally contaminated shellfish. We used 58 fecal samples that tested positive for noroviruses with electron microscopy (EM) to develop an NASBA assay for these viruses. Oligonucleotide primers targeting the polymerase coding region were used to amplify the viral RNA in an isothermal process that resulted in the accumulation of RNA amplicons. These amplicons were detected by hybridization with digoxigenin-labeled oligonucleotide probes that were highly specific for genogroup I (GI) and genogroup II (GII) of noroviruses. The expected band of 327 bp appeared in denaturing agarose gel without any nonspecific band. The specific signal for each amplicon was obtained through Northern blotting in many repeats. All fecal samples of which 46 (79.3%) belonged to GII and 12 (20.6%) belonged to GI were positive for noroviruses by EM and by NASBA. Target RNA concentrations as low as 5 pg/ml were detected in fecal specimens using NASBA. When the assay was applied to artificially contaminated shellfish, the sensitivity to nucleic acid was 100 pg/1.5 g shellfish tissue. The potential use of this assay was also confirmed in naturally contaminated shellfish collected from different ponds in Guangzhou city of China, of which 24 (18.76%) out of 128 samples were positive for noroviruses; of these, 19 (79.6%) belonged to GII and 5 (20.4%) belonged to GI. The NASBA assay provided a more rapid and efficient way of detecting noroviruses in fecal samples and demonstrated its potential for detecting noroviruses in food and environmental samples with high specificity and sensitivity.

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

Clarithromycin Resistance Prevalence and Icea Gene Status in Helicobacter Pylori Clinical Isolates in Turkish Patients with Duodenal Ulcer and Functional Dyspepsia: Peren H. Baglan , Gulendam Bozdayi , Muhip Ozkan , Kamruddin Ahmed , A. Mithat Bozdayi , Ali Ozden; J. Microbiol. 2006;44(4):409-416.; DOI: https://doi.org/2412 [pii]

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Abstract: Clarithromycin resistance in Helicobacter pylori is a principal cause of failure of eradication therapies, and its prevalence varies geographically. The IceA gene is a virulence factor associated with clinical outcomes. The objective of this study was to determine the current state of clarithromycin resistance prevalence, and to investigate the role of iceA genotypes in 87 Turkish adult patients (65 with functional dyspepsia and 22 with duodenal ulcer). A2143G and A2144G point mutations were tested by PCR-RFLP for clarithromycin resistance. Among the patients in the study, 28 patients were tested by agar dilution as well. Allelic variants of the iceA gene were identified by PCR. A total of 24 (27.6%) strains evidenced one of the mutations, either A2143G or A2144G. IceA1 was found to be positive in 28 of the strains (32.2%), iceA2 was positive in 12 (13.8%) and, both iceA1 and iceA2 were positive in 22 (25.3%) strains. In conclusion, we discovered no relationships between iceA genotypes and functional dyspepsia or duodenal ulcer, nor between clarithromycin resistance and iceA genotypes. Clarithromycin resistance appears to be more prevalent in Turkish patients.

Journal Article

Antibacterial Effect of Electrolyzed Water on Oral Bacteria: Sung-Hoon Lee , Bong-Kyu Choi; J. Microbiol. 2006;44(4):417-422.; DOI: https://doi.org/2411 [pii]

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Abstract: This study investigated the antibacterial effect of electrolyzed water on oral bacteria both in vitro and in vivo. Tap water was electrolyzed in a water vessel using platinum cell technology. The electrolyzed tap water (called Puri-water) was put in contact with five major periodontopathogens or toothbrushes contaminated with these bacteria for 30 sec. In addition, Puri-water was used as a mouthwash for 30 sec in 16 subjects and the antibacterial effect on salivary bacteria was evaluated. Puri-water significantly reduced the growth of all periodontopathogens in culture and on toothbrushes, and that of aerobic and anaerobic bacteria in saliva, when compared to the effect of tap water. It also significantly reduced mutans streptococci growing on mitis salivarius-bacitracin agar. Our results demonstrate that the electrolyzed tap water is effective as a mouthwash and for toothbrush disinfection.

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

Outbreaks of Imipenem-Resistant Acinetobacter baumannii Producing Carbapenemases in Korea: Seok Hoon Jeong , Il Kwon Bae , Kwang Ok Park , Young Jun An , Seung Ghyu Sohn , Seon Ju Jang , Kwang Hoon Sung , Ki Suk Yang , Kyungwon Lee , Dongeun Young , Sang Hee Lee; J. Microbiol. 2006;44(4):423-431.; DOI: https://doi.org/2410 [pii]

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Abstract: Among 53 Acinetobacter baumannii isolates collected in 2004, nine imipenem-resistant isolates were obtained from clinical specimens taken from patients hospitalized in Busan, Korea. Nine carbapenemase-producing isolates were further investigated in order to determine the mechanisms underlying resistance. These isolates were then analyzed via antibiotic susceptibility testing, microbiological tests of carbapenemase activity, pI determination, transconjugation test, enterobacterial repetitive consensus (ERIC)-PCR, and DNA sequencing. One outbreak involved seven cases of infection by A. baumannii producing OXA-23 β-lactamase, and was found to have been caused by a single ERIC-PCR clone. During the study period, the other outbreak involved two cases of infection by A. baumannii producing IMP-1 β-lactamase. The two clones, one from each of the outbreaks, were characterized via a modified cloverleaf synergy test and an EDTA-disk synergy test. The isoelectric focusing of the crude bacterial extracts detected nitrocefin-positive bands with pI values of 6.65 (OXA-23) and 9.0 (IMP-1). The PCR amplification and characterization of the amplicons via direct sequencing showed that the clonal isolates harbored blaIMP-1 or blaOXA-23 determinants. The two clones were characterized by a multidrug resistance phenotype that remained unaltered throughout the outbreak. This resistance encompassed penicillins, extended-spectrum cephalosporins, carbapenems, monobactams, and aminoglycosides. These results appear to show that the imipenem resistance observed among nine Korean A. baumannii isolates could be attributed to the spread of an IMP-1- or OXA-23-producing clone. Our microbiological test of carbapenemase activity is a simple method for the screening of clinical isolates producing class D carbapenemase and/or class B metallo-β-lactamase, in order both to determine their clinical impact and to prevent further spread.

Journal Articles

Meroparamycin Production by Newly Isolated Streptomyces sp. Strain MAR01: Taxonomy, Fermentation, Purification and Structural Elucidation: Moustafa Y. El-Naggar , Samy A. El-Assar , Sahar M. Abdul-Gawad; J. Microbiol. 2006;44(4):432-438.; DOI: https://doi.org/2409 [pii]

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Abstract: Twelve actinomycete strains were isolated from Egyptian soil. The isolated actinomycete strains were then screened with regard to their potential to generate antibiotics. The most potent of the producer strains was selected and identified. The cultural and physiological characteristics of the strain identified the strain as a member of the genus Streptomyces. The nucleotide sequence of the 16S rRNA gene (1.5 kb) of the most potent strain evidenced a 99% similarity with Streptomyces spp. and S. aureofaciens 16S rRNA genes, and the isolated strain was ultimately identified as Streptomyces sp. MAR01. The extraction of the fermentation broth of this strain resulted in the isolation of one major compound, which was active in vitro against gram-positive, gram-negative representatives and Candida albicans. The chemical structure of this bioactive compound was elucidated based on the spectroscopic data obtained from the application of MS, IR, UV, 1H NMR, 13C NMR, and elemental analysis techniques. Via comparison to the reference data in the relevant literature and in the database search, this antibiotic, which had a molecular formula of C19H29NO2 and a molecular weight of 303.44, was determined to differ from those produced by this genus as well as the available known antibiotics. Therefore, this antibiotic was designated Meroparamycin.

Improved Production of Live Cells of Lactobacillus rhamnosus by Continuous Cultivation using Glucose-yeast Extract Medium: Liew Siew Ling , Rosfarizan Mohamad , Raha Abdul Rahim , Ho Yin Wan , Arbakariya Bin Ariff; J. Microbiol. 2006;44(4):439-446.; DOI: https://doi.org/2408 [pii]

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Abstract: In this study, the growth kinetics of Lactobacillus rhamnosus and lactic acid production in continuous culture were assessed at a range of dilution rates (0.05 h?1 to 0.40 h?1) using a 2 L stirred tank fermenter with a working volume of 600 ml. Unstructured models, predicated on the Monod and Luedeking-Piret equations, were employed to simulate the growth of the bacterium, glucose consumption, and lactic acid production at different dilution rates in continuous cultures. The maximum specific growth rate of L. rhamnosus, ┢max, was estimated at 0.40 h?1, and the Monod cell growth saturation constant, Ks, at approximately 0.25 g/L. Maximum cell viability (1.3 ≠ 1010 CFU/ml) was achieved in the dilution rate range of D = 0.28 h?1 to 0.35 h?1. Both maximum viable cell yield and productivity were achieved at D = 0.35 h?1. The continuous cultivation of L. rhamnosus at D = 0.35 h?1 resulted in substantial improvements in cell productivity, of 267% (viable cell count) that achieved via batch cultivation.

Role of a Third Extracellular Domain of an Ecotropic Receptor in Moloney Murine Leukemia Virus Infection: Eun Hye Bae , Sung-Han Park , Yong-Tae Jung; J. Microbiol. 2006;44(4):447-452.; DOI: https://doi.org/2407 [pii]

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Abstract: The murine ecotropic retroviral receptor has been demonstrated to function as a mouse cationic amino acid transporter 1 (mCAT1), and is comprised of multiple membranespanning domains. Feral mouse (Mus dunni) cells are not susceptible to infection by the ecotropic Moloney murine leukemia virus (MoMLV), although they can be infected by other ecotropic murine leukemia viruses, including Friend MLV and Rauscher MLV. The relative inability of MoMLV to replicate in M. dunni cells has been attributed to two amino acids (V214 and G236) located within the third extracellular loop of the M. dunni CAT1 receptor (dCAT1). Via the exchange of the third extracellular loop of the mCAT1 cDNA encoding receptor from the permissive mouse and the corresponding portion of cDNA encoding for the nonpermissive M. dunni receptor, we have identified the most critical amino acid residue, which is a glycine located at position 236 within the third extracellular loop of dCAT1. We also attempted to determine the role of the third extracellular loop of the M. dunni CAT1 receptor with regard to the formation of the syncytium. The relationship between dCAT1 and virus-induced syncytia was suggested initially by our previous identification of two MLV isolates (S82F in Moloney and S84A in Friend MLV), both of which are uniquely cytopathic in M. dunni cells. In an attempt to determine the relationship existing between dCAT1 and the virally-induced syncytia, we infected 293-dCAT1 or chimeric dCAT1 cells with the S82F pseudotype virus. The S82F pseudotype virus did not induce the formation of syncytia, but did show increased susceptibility to 293 cells expressing dCAT1. The results of our study indicate that S82F-induced syncytium formation may be the result of cell-cell fusion, but not virus-cell fusion.

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

VanB-vanA Incongruent VRE Isolated from Animals and Humans in 1999: Enjoo Shin , Hyunjin Hong , Yasuyoshi Ike , Kyungwon Lee , Yong Ho Park , Dong Taek Cho , Yeonhee Lee; J. Microbiol. 2006;44(4):453-456.; DOI: https://doi.org/2406 [pii]

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Abstract: 16 chicken isolates and four clinical isolates of VanB-vanA incongruent vancomycinresistant Enterococcus faecium strains without vanS were isolated in 1999. Pulsed-field gel electrophoresis revealed only a peripheral relationship between the chicken isolates and clinical isolates, but suggested clonal spread in the chicken isolates.

Journal Article

Laboratory Confirmation of A Suspicious Meningococcal Meningitis Death Case: Tie-gang Zhang , Xiong He , Li-juan Chen , Jing-guo He , Ming Luo , Jie Yang , Zhu-jun Shao , Mei-ping Sun; J. Microbiol. 2006;44(4):457-460.; DOI: https://doi.org/2405 [pii]

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Abstract: A suspicious meningococcal meningitis death case was reported to the Beijing CDC. The blood specimen was analyzed via multi-PCR and MLST. 6 isolates from close contacts were analyzed via PFGE and MLST. According to the results of the above analyses, the cause of this case was identified as a serogroup A Neisseria meningitidis, which, in terms of sequence typing, belonged the ST7 group.

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

Occurrence of Thioredoxin Reductase in Deinococcus Species, the UV resistant Bacteria: Hee Jeong Seo , Young Nam Lee; J. Microbiol. 2006;44(4):461-465.; DOI: https://doi.org/2404 [pii]

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Abstract: The occurrence of thioredoxin reductase (NAD(P)H: oxidized-thioredoxin reductase, EC 1.6.4.5, TrxR) in five mesophilic species of Deinococcus was investigated by PAGE. Each species possessed a unique TrxR pattern, for example, a single TrxR characterized D. radiopugnans while multiple forms of TrxR occurred in other Deinococcal spp. Most of TrxRs occurring in Deinococcus showed dual cofactor specificity, active with either NADH or NADPH, although the NADPH specific-TrxR was observed in D. radiophilus and D.proteolyticus.

Decomposition of Biological Macromolecules by Plasma Generated with Helium and Oxygen: Seong-Mi Kim , Jong-Il Kim; J. Microbiol. 2006;44(4):466-471.; DOI: https://doi.org/2403 [pii]

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Abstract: In this study, we attempted to characterize the biomolecular effects of an atmospheric pressure cold plasma (APCP) system which utilizes helium/oxygen (He/O2). APCP using He/O2 generates a low level of UV while generating reactive oxygen radicals which probably serve as the primary factor in sterilization; these reactive oxygen radicals have the advantage of being capable to access the interiors of the structures of microbial cells. The damaging effects of plasma exposure on polypeptides, DNA, and enzyme proteins in the cell were assessed usiing biochemical methods.

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