Journal of Microbiology

Volume 40(2); June 2002

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: 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.

Characterization of Quinolone-Resistant Clinical Isolates of Escherichia coli in Korea: Yoojung Oh , Seohyung Park , Misun Ha , Yeonhee Lee; J. Microbiol. 2002;40(2):98-103.

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Abstract: Twenty-eight clinical isolates of Escherichia coli, composed of thirteen norfloxacin resistant isolates (MIC of >16 ug/ml), one intermediately resistant isolate (MIC of 8 ug/ml), and fourteen susceptible isolates (MIC of <4 ug/ml), were randomly selected to study the norfloxacin resistance mechanism and phylogeny in clinical isolates in Korea. Eleven norfloxacin resistant isolates and one susceptible isolate were multi-drug resistant (MDR). Every norfloxacin resistant isolate with MIC higher than 32 ug/ml had the same three mutations: Ser83->Leu and Asp87->Asn or Tyr in GyrA and Ser80->Ile in ParC. Whereas a resistant isolate with MIC of 16 ug/ml had three mutations but Asp87 in GyrA was replaced with Gly instead of Asn. The intermediately resistant isolate had the same two mutations in GyrA but a different mutation in ParC, Glu84->Lys. Among the susceptible isolates, two isolates with MIC of 4 ug/ml had one mutation: Ser83->Leu in GyrA, and no mutation was found in the susceptible isolates. Resistant isolates showed higher efflux activity than the susceptible ones, with random amplification of polymorphic DNA (RAPD), six susceptible isolates form a separate group from the rest of the isolates.

The First Study on Bacterial Flora and Biological Control Agent of Anoplus roboris (Sufr., Coleoptera): Ismail Demir , Kazim Sezen , Zihni Demirbag; J. Microbiol. 2002;40(2):104-108.

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Abstract: The hazelnut leaf holer (Anoplus roboris Sufr., Coleoptera: Curculionidae) is a devastating pest of hazelnut and oak trees. It causes approximately 20-30% economic damage to hazelnut production per year in Turkey. In the present study, in order to find a more effective and safe biological control agent against A. roboris, we investigated the bacterial flora of the hazelnut leave holer, and tested them for insecticidal effects on it. According to morphological, physiological and biochemical tests, bacterial flora were identified as Bacillus circulans (Ar1), Bacillus polymyxa (Ar2), Enterobacter sp. (Ar3) and Bacillus sphaericus (Ar4). Insecticidal effects of bacterial isolates were performed on adult A. roboris. The highest insecticidal effect determined was 67% by B. sphaericus within eight days. The insecticidal effects of the other isolates (Ar1, Ar2 and Ar3) were determined as 33%, 47% and 47% within the same period, respectively.

Isolation, Characterization and Numerical Taxonomy of Novel Oxalate-oxidizing Bacteria: Nurettin Sahin , Isa Gokler , Abdurrahman U. Tamer; J. Microbiol. 2002;40(2):109-118.

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Abstract: The present work is aimed at providing additional new pure cultures of oxalate utilizing bacteria and its preliminary characterization for further work in the field of oxalate-metabolism and taxonomic studies. The taxonomy of 14 mesophilic, aerobic oxalotrophic bacteria isolated by an enrichment culture technique from soil, rhizospheres, and the juice of the petiole/stem tissue of plants was investigated. Isolates were characterized with 95 morphological, biochemical and physiological tests. Cellular lipid components and carotenoids of isolates were also studied as an aid to taxonomic characterization. All isolates were Gram-negative, oxidase and catalase positive and no growth factors were required. In addition to oxalates, some of the strains grow on methanol and/or formate. The taxonomic similarities among isolates, reference strains or previously reported oxalotrophic bacteria were analysed by using the Simple Matching (S_SM ) and Jaccard (S_J ) Coefficients. Clustering was performed by using the unweighted pair group method with arithmetic averages (UPGMA) algorithm. The oxalotrophic strains formed five major and two single-member clusters at the 70-86% similarity level. Based on the numerical taxonomy, isolates were separated into three phenotypic groups. Pink-pigmented strains belonged to Methylobacterium extorquens, yellow-pigmented strains were most similar to Pseudomonas sp. YOx and Xanthobacter autotrophicus, and heterogeneous non-pigmented strains were closely related to genera Azospirillum, Ancylobacter, Burkholderia and Pseudomonas. New strains belonged to the genera Pseudomonas, Azospirillum and Ancylobacter that differ taxonomically from other known oxalate oxidizers were obtained. Numerical analysis indicated that some strains of the yellow-pigmented and non-pigmented clusters might represent new species.

Reflection on Kinetic Models to the Chlorine Disinfection for Drinking Water Production: Yoon-jin Lee , Sang-ho Nam; J. Microbiol. 2002;40(2):119-124.

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Abstract: Experiments for the characterization of inactivation were performed in a series of batch processes with the total coliform used as a general indicator organism based on the chlorine residuals as a disinfectant. The water samples were taken from the outlet of a settling basin in a conventional surface water treatment system that is provided with the raw water drawn from the mid-stream of the Han River. The inactivation of total coliform was experimentally analyzed for the dose of disinfectant, contact time, filtration and mixing intensity. The curves obtained from a series of batch processes were shaped with a general tailing-off and biphasic mode of inactivation, i.e. a sharp loss of bacterial viability within 15 min followed by an extended phase. In order to observe the effect of carry-over suspended solids on chlorine consumption and disinfection efficiency, the water samples were filtered, prior to inoculation with coliforms, with membranes of both 2.5 um and 11.0 um pore size, and with a sand filter of 1.0 mm in effective size and of 1.4 in uniformity coefficient. As far as the disinfection efficiency is concerned, there were no significant differences. The parameters estimated by the models of Chick-Watson, Hom and Selleck from our experimental data obtained within 120 min are: log(N/N_0 )=-0.16CT with n=1, log(N/N_0 )=-0.71C^0.87 T with n=/1 for the Chick-Watson model, log (N/N_0 )=-1.87C^0.47 T^ 0.36 for the Hom model, log (N/N_0 )=-2.13log (1+CT/0.11) for the Selleck model. It is notable that among the models reviewed with regard to the experimental data obtained, the Selleck model appeared to most closely resemble the total coliform survival curve.

Respiratory Chain-Linked Components of the Marine Bacterium Vibrio alginolyticus Affect Each Other: Young Jae Kim; J. Microbiol. 2002;40(2):125-128.

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Abstract: The aerobic respiratory chain of Vibrio alginolyticus possesses two different kinds of NADH oxidase systems, i.e., an Na^+ -dependent NADH oxidase system and an Na^+ -independent NADH oxidase system. When deamino-NADH, which is the only substrate for the Na^+ -dependent NADH oxidase system, was used as a substrate, the maximum activities of Na^+ -dependent NADH:quinone oxidoreductase and Na^+ -dependent NADH oxidase were obtained at about 0.06 M and 0.2 M NaCl, respectively. When NADH, which is a substrate for both Na^+ -dependent and Na^+ -independent NADH oxidase systems was used as a substrate, the NADH oxidase activity had a pH optimum at about 8.0. In contrast, when deamino-NADH was used as a substrate, the NADH oxidase activity had a pH optimum at about 9.0. On the other hand, inside-out membrane vesicles prepared from the wild-type bacterium generated only a very small [delta]pH by the NADH oxidase system, whereas inside-out membrane vesicles prepared from Nap1, which is a mutant defective in the Na^+ pump, generated [delta]pH to a considerable extent by the NADH oxidase system. On the basis of the results, it was concluded that the respiratory chain-linked components of V. alginolyticus affect each other.

Purification and Characterization of Poly(3-hydroxybutyrate) Depolymerase from a Fungal Isolate, Emericellopsis minima W2: Do Young Kim , Ji Hye Yun , Hyung Woo Kim , Kyung Sook Bae , Young Ha Rhee; J. Microbiol. 2002;40(2):129-133.

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Abstract: The fungus, Emericellopsis minima W2, capable of degrading poly(3-hydroxybutyrate) (PHB) was isolated from a waste water sample. Production of the PHB depolymerase from E. minima W2 (PhaZ_Emi ) was significantly repressed in the presence of glucose. PhaZ_Emi was purified by column chromatography on Octyl-Sepharose CL-4B and Sephadex G-100. The molecular mass of the PhaZ_Emi , which consisted of a single polypeptide chain, was estimated to be 48.0 kDa by SDS-PAGE and its pI value was 4.4. The maximum activity of the PhaZ_Emi was observed at pH 9.0 and 55 C. It was significantly inactivated by 1 mM dithiothreitol, 2 mM diisopropyl fluorophosphate, 0.1 mM Tween 80, and 0.1 mM Triton X-100, but insensitive to phenylmethylsulfonyl fluoride and N-ethylmaleimide. The PhaZ_Emi efficiently hydrolyzed PHB and its copolyester with 30 mol% 3-hydroxyvalerate, but did not act on poly(3-hydroxyoctanoate). It also hydrolyzed p-nitrophenylacetate and p-nitrophenylbutyrate but hardly affected the longer-chain forms. The main hydrolysis product of PHB was identified as a dimer of 3-hydroxybutyrate.

Effects of Thiamine Pyrophosphate on the Inhibition of Self-splicing of Primary Transcripts of T4 phage Thymidylate Synthase Gene in the Presence of GTP: Hyun Joo Lee , Sung Joon Ahn , Sook Shin , In Kook Park; J. Microbiol. 2002;40(2):134-139.

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Abstract: Effects of GTP on the inhibition of self-splicing of primary transcripts of the phage T4 thymidylate synthase gene (td) by thiamine pyrophosphate and its analogs have been investigated. The order of the inhibitory efficiency for compounds tested was as follows: thiamine pyrophosphate > thiamine mono-phosphate > thiamine. Of all compounds examined, thiamine pyrophosphate was the most potent inhibitor. Increasing GTP concentration in splicing reaction tended to overcome the suppressive effects of self-splicing by thiamine pyrophosphate and its analogs. The inhibition by thiamine pyrophosphate was most sensitized to a higher concentration of GTP. It has been speculated that the key structural features in thiamine pyrophosphate and its analogs responsible for the inhibition of splicing may be a thiamine moiety in which the phosphorylation of 2-hydroxylethyl group on 5-position of thiazolium ring rendered further stimulation of inhibition in self-splicing reaction.

Stable Secretion Vector Derived from the RCR (rolling-circle replication) Plasmid of Bacillus mesentericus: Seung-Soo Lee , Jeong-Sun Han , In Hyung Lee , Young- Yel l Yang , Soon-Kwang Hong , Joo-Won Suh; J. Microbiol. 2002;40(2):140-145.

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Abstract: The 5.8 kb pMMH1, rolling-circle replication (RCR) plasmid of the wild type soil Bacillus mesentericus was developed into a novel secretion vector system in Bacillus subtilis. The pMMH1 turned out to have a replication origin and two open reading frames (ORFs) of the putative [gamma]-GTP and type I signal peptidase (sipP). To characterize the regions necessary for plasmid stability and high copy number, five vectors (pPS, pPP, pEN, pMN, pME) were constructed by disruption or deletion of each region in pMMH1. Like pMMH1, all constructed vectors were stable over 100 generations in a non-selective medium. Since pPS was the smallest (2.3 kb)of all, it was selected for the construction of a novel secretion vector. Using the [alpha]-amylase promoter/signal sequence of B. subtilils, the novel plasmid pJSN was constructed. When [beta]-glucosidase was expressed using pJSN, we found [beta]-glucosidase activity in the medium. This result strongly suggested that plasmid pJSN can be used for the production of bioactive peptides in B. subtilis.

Multiplex Polymerase Chain Reaction Assay for Simultaneous Detection of Candida albicans and Candida dubliniensis: Young-Hee Lim , Do-Hyun Lee; J. Microbiol. 2002;40(2):146-150.

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Abstract: A multiplex polymerase chain reaction (PCR) assay was developed for the identification of two Candida species-albicans and dubliniensis. Three sets of primers were selected from different genomic sequences to specifically amplify a 516 bp fragment within the top2 gene, specific for several species of the genus Candida (CCL primers); a 239 bp fragment within the [alpha]INT1 gene, specific for Candida albicans (CAL primers); and a 175 bp fragment within the ALSD1 gene, specific for Candida dubliniensis (CDL primers). Using the primers in conjunction (multiplex PCR), we were able to detect both C. albicans and C. dubliniensis and to differentiate between them. The detection limit of the PCR assay was approximately 10 cells per milliliter of saline. Thus, this multiplex PCR assay can be applied for differentiation of C. albicans and C. dubliniensis from clinical specimens.

Molecular Cloning of the Superoxide Dismutase Gene from Orientia tsutsugamushi, the Causative Agent of Scrub Typhus: Ji-Hyun Yun , Young-Sang Koh , Se-Jae Kim; J. Microbiol. 2002;40(2):151-155.

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Abstract: A superoxide dismutase (SOD) gene from the obligate intracellular bacterium Orientia tsutsugamushi has been cloned by using the polymerase chain reaction with degenerate oligonucleotide primers corresponding to conserved regions of known SODs. Nucleotide sequencing revealed that the predicted amino acid sequence was significantly more homologous to known iron-containing SODs (FeSOD) than to manganese-containing SODs (MnSOD). Conserved regions in bacterial FeSOD could also be seen. Isolation of the oriential SOD gene may provide an opportunity to examine its role in the intracellular survival of this bacterium.

Expression of the Galactokinase Gene (galK) from Lactococcus lactis ssp. lactis ATCC7962 in Escherichia coli: Jae Yeon Choi , Jong-Hoon Lee , Jung Min Lee , Jeong Hwan Kim , Hae Choon Chang , Dae Kyun Chung , Somi Kim Cho , Hyong Joo Lee; J. Microbiol. 2002;40(2):156-160.

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Abstract: The whole gal/lac operon genes of Lactococcus lactis ssp. lactis 7962 were reported as follows: galA-galM-galK-galT-lacA-lacZ-galE. The galK gene encoding a galactokinase involved in one of the Leloir pathways for galactose metabolism was found to be 1,197 bp in length and encodes a protein of 43,822 Da calculated molecular mass. The deduced amino acid sequence showed over 50% homology with GalK proteins from several other lactic acid bacteria. The galK gene was expressed in E. coli and the product was identified as a 43 kDa protein which corresponds to the estimated size from the DNA sequence. The galactokinase activity of recombinant E. coli was about 8 times greater against that of the host strain and more than 3 times higher than the induced L. lactis 7962.

Morphological Diversity of Marine Microorganisms on Different Isolation Media: Shin Hye Park , Kae Kyoung Kwon , Deuk-Soo Lee , Hong Kum Lee; J. Microbiol. 2002;40(2):161-165.

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Abstract: Isolation frequency of microorganisms from marine sources was examined with different media and samples collected from the coastal area of Cheju Island. From sea water samples, about 1% of microorganisms from the total number of bacteria were recovered. Microorganisms were cultured at the much lower frequency of 10^-4 ?0^-6 from other marine sources, such as sediment, sponges and corals. The frequency of duplicated isolation was examined with 140 morphologically different colonies isolated on different media. Fourteen percent of them exhibited the same morphology on two different media. The duplication frequency of the isolates among three different media was 33%.

Serotyping of Cryptococcus neoformans Strains Isolated in Korea: Soo-Myung Hwang; J. Microbiol. 2002;40(2):166-169.

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Abstract: Twenty strains of Cryptococcus neoformans isolated from environmental and clinical sources in Korea were examined for their serotypes. Two environmental isolates from pigeon excreta belonged to C. neoformans var. neoformans serotype A. Of the 18 isolates from clinical specimens, 17 belonged to C. neoformans var. neoformans (serotype A : 16, serotype D : 1) and one belonged to C. neoformans var. gattii serotype B, which was culturally unusual, producing mucous colonies. This is the first report of the identification of C. neoformans var. gattii serotype B from a patient in Korea.

Isolation, Identification and Characterization of Vancomycin-Resistant Enterococci from Raw Milk: Sung-Sook Choi , Bong-Su Kim , Nam-Joo Ha; J. Microbiol. 2002;40(2):170-172.

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Abstract: To determine the occurrence of vancomycin-resistant Enterococci in a raw milk sample, raw milk samples were examined for a period of 6 months. Enterococci were isolated directly from Enterococcal selective agar plates supplemented with 2 mg of vancomycin per liter. Nineteen strains were selected and identified by applying the Vitek system. To determine resistance patterns, 19 isolates were tested with vancomycin and teicoplanin. Vancomycin-resistant Enterococci were genotyped by using a PCR analysis and 5 out of 19 isolates were of the VanC type.

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