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Sequence Analysis of the 16S Rrna Gene of Sulfate-Reducing Bacteria Isolated from Human Intestine

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Sequence Analysis of the 16S Rrna Gene of Sulfate-Reducing Bacteria Isolated from Human Intestine Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 239-248 ISSN: 2319-7706 Volume 3 Number 2 (2014) pp. 239-248 http://www.ijcmas.com Original Research Article Sequence analysis of the 16s rRNA gene of sulfate-reducing bacteria isolated from human intestine Ivan Kushkevych*, Milan Barto and Ladislava Barto ová Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno Palackeho 1/3, CZ-61242 Brno, Czech Republic *Corresponding author A B S T R A C T K e y w o r d s The sulfate-reducing bacteria from the human intestine have been isolated. These Sulfate-reducing bacteria have been identified as the Desulfovibrio piger by sequence analysis of the bacteria; 16S rRNA gene. Comparative sequence analysis has shown the identity of DNA micro- sequences which encode for the 16S rRNA gene of the studied sulfate-reducing biocenosis; bacteria strain with similar sequences as those from the GenBank database. The inflammatory nucleotide sequence of isolated sulfate-reducing bacteria has 99% homology with bowel diseases. sequences Desulfovibrio piger ATCC 29098 deposited in the GenBank. Introduction Sulfate-reducing bacteria (SRB) belong to brain and abdomen etc.) in the presence of the intestine microbiocenosis of both other infections (Cummings et al., 2003; humans and animals (Barton et al., 2007; Loubinoux et al., 2000; 2002). This genus Brenner et al., 2005; Dzierzewicz et al., of bacteria has the most pathogenic role 2003). The species Desulfovibrio genus compared to other SRB genera and their are dominant SRB in the intestine (Gibson species [Loubinoux et al., 2000; Rowan et et al., 1991; 1993). They constitute 67 al., 2009]. Similar species, bacterium 91% of all SRB genera. There are bacteria Desulfovibrio fairfieldensis was isolated in of the genera Desulfobacter (9 16%), mono- and polymicrobial infections of the Desulfobulbus (5 8%) and gastrointestinal tract (Barton et al., 2007; Desulfotomaculum (2%) found in diseased Macfarlane et al., 2007). SRB are also intestines (Gibson et al., 1993; Zinkevich found in the human oral cavity et al., 2000). (Langendijk et al., 2001). Dissimilatory sulfate reduction by SRB and the Various species of the Desulfovibrio genus formation of hydrogen sulfide in the are often isolated during different intestinal lumen can cause a variety of illnesses. These species can cause a variety inflammatory processes (Kushkevych of diseases (cholecystitis, abscesses of the 2013; Pitcher et al., 1996). SRB, which 239 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 239-248 produces the largest amount of hydrogen because they are difficult to isolate from sulfide, were isolated from the feces of a among a large number of human intestinal distal human colon. It is probably due to a SRB. reaction medium where the proximal part of the colon is acidic (pH<5.5), and distal In previous studies, the sulfate-reducing part is neutral (Pitcher et al., 1996; Rowan bacteria were isolated from human et al., 2009). intestine and identified by their morphological, physiological and Loubinoux . et al. have isolated bacteria biochemical characteristics as Desulfovibrio genera from the human Desulfovibrio sp. according to Bergey s intestine (Loubinoux et al., 2000; 2002). It Manual of Determinative Bacteriology was established that 12 of 100 samples of (Holt et al., 1994). The physiological and purulent peritoneal and pleural cavities biochemical properties of obtained contained Desulfovibrio piger, D. bacteria were studied (Kushkevych, 2013). fairfieldensis or D. desulfuricans The isolated strain Desulfovibrio sp. Vib- (Loubinoux et al., 2002). Bacteria D. 7, which produces the largest amount of desulfuricans were also isolated from the hydrogen sulfide and acetate, was selected colon during bleeding microvilli, causing for sequence analysis because it is the bacteremia (Loubinoux et al., 2000). most interesting and promising for future These studies confirm that the main way study. of SRB penetration in the blood is going through the damaged intestinal microvilli The aim of our work was to carry out then bacteria can cause infection. sequence analysis of the 16S rRNA genes from the Desulfovibrio sp. Vib-7 which It is of vital importance to obtain new was isolated from human intestine in strains of the SRB from different people, preparation for future studies. identify them by molecular methods and then study their physiological, biochemical The aim was accomplished using the and genetic properties. Aside from that, methods of PCR, gel electrophoresis and process of dissimilatory sulfate reduction sequence analysis of the 16S rRNA gene by SRB and the production of hydrogen of the sulfate-reducing bacteria of the sulfide should be investigated in order to human intestine, and after statistical clarify the etiological role of these bacteria processing of the results, the obtained data in the development of various diseases. were compared with those from literature. Such studies might help in predicting the development of diseases in the gastrointestinal tract, by providing further Materials and Methods details on the etiology of bowel diseases which are very important for the clinical Object of the study diagnosis of these disease types. Strain Desulfovibrio sp. Vib-7 was Currently the D. desulfuricans strains are obtained from the human large intestine the most studied among bowel SRB. (Kushkevych, 2013). The strains are kept However, the physiological and in the collection of microorganisms at the biochemical characteristics of bacteria Biotechnology laboratory of Pharmacy Desulfovibrio piger are unexplored Faculty at the University of Veterinary and 240 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 239-248 Pharmaceutical Sciences Brno (Czech universal primers (according to Weisburg Republic). et al. 1991 (Weisburg et al., 1991), and Pershing book [Persing, 2011]): 8FPL 5´- Cultivating of the bacterial cultures AGT - TTG- ATC- CTG- GCT- CAG-3´ The bacteria were grown in nutrition position 8 27, 1492RPL 5´-GGT-TAC- modified Kravtsov-Sorokin's liquid CTT-GTT-ACG-ACT-T-3´ position medium of the following composition 1510 1492 (amplicon length approximate 1500 bp) and 8FPL 5´-AGT-TTG-ATC- (g/l): Na2SO4 0.5; KH2PO4 0.3; CTG-GCT-CAG-3´ position 8 27, 806R K2HPO4 0.5; (NH4)2SO4 0.2; NH4Cl 5´-GGA-CTA-CCA-GGG-TAT-CTA-AT- 1.0; CaCl2×6H2O 0.06; MgSO4×7H2O 3´ position 806 787 (amplicon length 0.1; C3H5O3Na 2.0; yeast extract 1.0; approximate 800 bp). FeSO4×7H2O 0.004; sodium citrate×2H2O 0.3. Before bacteria PCR procedure seeding in the medium, 0.05 ml/l of sterile solution of Na2S×9H2O (1%) was added. PCR was carried out on DNA isolated The sterile 10N solution of NaOH (0.9 from sulfate-reducing bacteria cells in a ml/l) in the medium was used to provide final volume of 20 µl consisting of 10.0 µl the final pH 7.2. The medium was heated Taq PCR Master Mix Kit (Cat. No in boiling water for 30 min in order to 201445), 0.1 µl of each primer, 0.1 µl obtain an oxygen-free medium, and cooled uracil D-glycosylase (Cat. No. M0280L), to +30°C temperature. The bacteria were 2.0 µl of DNA supernatant, and 7.7 µl grown for 72 hours at +37°C under deionised water. anaerobic conditions. The tubes were brim-filled with medium and closed to The amplicons were amplified by a provide anaerobic conditions. preliminary incubation at 94ºC for 5 min (initial denaturation) then 34 cycles of DNA isolation 94ºC for 1 min (denaturation), 55ºC for 1 min (annealing of primers), and 72ºC for 2 Isolation and purification of DNA was min (polymerization), using a carried out with three day old culture of Thermocycler (model MJ Research PTC- sulfate-reducing bacteria using a QIAmp 200, USA). After the last amplification DNA Mini Kit (QIAGEN), Cat. No cycle, the samples were incubated further 51304 . One single bacterial colony was at 72ºC for 2 min for complete elongation taken from the Kravtsov-Sorokin's of the final PCR products and cooled at medium and suspended in 50 µl of 10ºC. deionised water in a screw cap micro- centrifuge tube. The samples were boiled Analysis of PCR products at 98°C for 5 min prior to being centrifuged for 5 min/14000g to settle cell Analysis of PCR products was carried out debris. Two microliters of supernatant, by electrophoresis in 1.5% agarose gel, containing the genomic DNA, was used with a field strength of 5V/cm. for PCR amplification. Electrophoresis time was 40 min. The 100 bp ladder (Malamité, Czech Republic) was Amplification of gene fragments used as a size standard and molecular weight marker. Isolation and purification Amplification of 16S rRNA gene of fragments from agarose was performed fragments was carried out using the 241 Int.J.Curr.Microbiol.App.Sci (2014) 3(2): 239-248 by centrifugation of gel strips containing also compared by BLASTN analysis with DNA through aerosol filters. For the nucleotide sequences of 16S rRNA purification Desulfovibrio amplicons the gene of other strains found in table 2. commercial kit from QIAGEN MinElute Gel Extraction Kit was used. Thus, the nucleotide sequence of the 16S rRNA gene of studied sulfate-reducing Sequence was carried out using Genetic bacteria has the highest 99% homology Analyzer and reagents BigDye compared to deposited nucleotide Terminator v3.1 Cycle Sequencing Kit . sequence Desulfovibrio piger ATCC Search for homologous deposited in 29098 (AF192152) in the GenBank GenBank nucleotide sequence encoding database. the 16S rRNA gene was performed using BLASTN and Blast2 programs. The obtained Desulfovibrio piger strain belongs to sulfate-reducing bacteria which Results and Discussion are usually considered a commensal bacteria in humans [Holt et al., 1994; The isolated strain Desulfovibrio sp. Vib-7 Moore et al., 1976]. More recently, producing the largest amount of hydrogen Desulfovibrio piger has attracted more sulfide and acetate for sequence analysis interest as it was found to be the most was selected.
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