IDENTIFICATION OF SULFATE-REDUCING BACTERIA STRAINS OF HUMAN LARGE INTESTINE

The sulfate-reducing bacteria have been isolated from human intestine. These bacteria are identified by their morphological, physiological and biochemical characteristics as the Desulfovibrio sp. and Desulfomicrobium sp. according to Bergey’s Manual of Determinative Bacteriology. Growth of the isolated strains of bacteria has been studied under various conditions including types of media, effect of oxygen, pH, and tempe­ rature. Bacterial growth in the modified Kravtsov­Sorokin’s medium, and process of dissimilatory sulfate reduction by the selected strains of bacteria have been investiga­ ted. All obtained bacterial cultures used sulfate and lactate intensively as well as produced hydrogen sulfide and acetate in the medium. The Desulfovibrio sp. strain Vib­7 produced the highest concentration (up to 3.23 mM) of hydrogen sulfide, while the bacteria used about 99% of sulfate ions presented in the medium. This strain also produced the highest concentration of acetate ions (up to 15.87 mM) on the fifth day of cultivation. Among the studied Desulfomicrobium sp. strains, the highest concentration of hydrogen sulfide was produced by the Desulfomicrobium sp. strain Rod­9, and the highest concentration of acetate ions was produced by the Desulfomicrobium sp. strain Rod­4.


INTRODUCTION
Sulfatereducing bacteria Desulfovibrio, Desulfomicrobium, Desulfobulbus, Desulfobacter, Desulfomonas and Desulfotomaculum genera make an important component of human intestinal microbiocenosis [2,15,19,21,23]. An increased number of these microorganisms can be found in people with periodontitis, inflammatory bowel diseases, ulcerative colitis, rheumatic diseases and many other diseases [6,8,9,16,18,20,24]. Some scientists also suggest that the sulfatereducing bacteria (SRB) might cause of some forms of colon cancer, taking into account that these microorganisms produce hydrogen sulfide affecting the intestinal cells metabolism causing various diseases [6,15,16,22,24,28]. This is the main reason why it is necessary to study SRB in more detail. ISSN 1996-4536 • Біологічні Студії / Studia Biologica • 2013 • Том 7/№3 • С. 115-132 on the concentration of hydrogen sulfide, produced by the isolates is supposed to help in establishing and assessing a toxicity effect of hydrogen sulfide on the epithelial cells of the human intestine. Such studies might help in predicting the development of diseases in the gastrointestinal tract, by providing further details on the etiology of bowel diseases which are very important for the clinical diagnosis of these disease types.
The aim of this study was to obtain the sulfatereducing bacteria from the colon feces of healthy people, and identify some strains of the bacteria by their morphological, physiological and biochemical characteristics.

MATERIAL AND METHODS
Object of the study. Strains of sulfate-reducing bacteria which were obtained from human large intestine at the Microbiology, Virology and Immunology Department of the Danylo Halytsky Lviv National Medical University. As control, sulfate-reducing bacteria Desulfovibrio desulfuricans strain ІМV K6, isolated from Yavoriv lake water and identified at the Microbiology Department of Ivan Franko National University of Lviv were used.
Obtaining of the bacterial cultures. One gram of feces was suspended in 9 ml of the modified Kravtsov-Sorokin's liquid medium [13]. Before, the medium was heated in boiling water for 30 min in order to obtain an oxygenfree medium and cooled to +30°C temperature. After cooling, decimal dilutions were made to 10 -12 in a series of tubes containing Kravtsov-Sorokin's liquid medium of such composition (g/l): Na 2 SO 4 -0.5; Before bacterial seeding in the medium, 10 ml/l of sterile Mohr's salt solution [(NH 4 ) 2 Fe(SO 4 ) 2 ×6H 2 O] (10%), and 0.05 ml/l of sterile solution of Na 2 S×9H 2 O (1%) were added. Sterile 10N solution of NaOH (0.9 ml/l) provided a medium of pH 7.2. In each tube approximately 2 ml of low temperature melting point paraffin (+70°C) was poured over the surface of the medium and incubated at +37°C for 10 days under anaerobic conditions. Cultures were deemed positive when a black FeS precipitate occurred in the bottom of the tube.
Kravtsov-Sorokin's liquid medium, to which 12 g of agar was added per litre, was used for surface cultures. Each positive culture was streaked on the poured Petri plates of Kravtsov-Sorokin's agar for obtaining isolated colonies. Mohr's salt in the agar medium for detecting colonies of the sulfatereducing bacteria was added. As a result, FeS was formed by the bacterial cells that caused black coloured colonies. Plates were introduced into an anaerobic box with oxygen uptake generators (GENbox anaer, France) for anaerobiosis.
The black colonies obtained, both large and small, from each Petri plates were selected and suspended in sterile saline (0.9% solution of NaCl). They were seeded in the standard media: one with sulfate (concentration 3.5 mM), one without sulfate ions and another medium containing sulfur without sulfate ions, to be sure that the selected microorganisms belong to the sulfate-reducing bacteria.
Microscopy and morphological characteristics. Morphology of cells of the sulfatereducing bacteria has been studied by using light microscope "MBR1" (×1350). Bacteria were observed by Gram staining from Kravtsov-Sorokin's liquid culture and also from the surface Kravtsov-Sorokin's agar culture. Flagella were examined using flagella stain reagent (Difco) from a 24hr Kravtsov-Sorokin's agar slant in 160×16 mm tubes, which were incubated anaerobically. The bacteria were suspended in 0.2 ml of sterile saline.
Tests on bacterial growth in the non-selective media. Additional tests on the growth of the obtained strains in different non-selective media (Meat Peptone Agar, Wort-Agar, Starch-and-Ammonia Agar, Giltay's, Baalsrud's, Kravtsov-Sorokin's) were performed [13].
Determination of bacterial sporulation. To determine the ability of the bacteria to sporulate, two types of these colonies were selected and suspended in the Kravtsov-Sorokin's liquid medium. Thermoresistance was determined by heating 1 ml of an 8day culture in the liquid medium for 10 min at +80°C, then subculturing it in a similar medium incubated in anaerobiosis. To detect the bacterial spores the cells were additionally stained by Peshkov method.
Bacterial growth at various pH. The influence of acidity (pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0) on the growth of the sulfate-reducing bacteria in the KravtsovSorokin's liquid medium was established.
Bacterial biomass accumulation. Biomass accumulation of the various strains of sulfate-reducing bacteria in liquid medium by photometric method (nutritional medium was without Mohr's salt) has been examined [26].
Measuring of sulfate concentration. The sulfate ions concentration in the medium was determined by turbidymetric method after it had been precipitated by barium chloride. To stabilize the suspension glycerol was used [14].
Measuring of hydrogen sulfide concentration. Hydrogen sulfide concentration in the culture medium was determined by photometric method based on reaction of sulfide and n-aminodimethylaniline with methylene blue formation [25]. The concentration of hydrogen sulfide by calibration curve was established.
Measuring of lactate concentration. Determination of lactate concentration was carried out through dehydrogenation of lactate reaction by lactate dehydrogenase in the presence of NAD, with formation of pyruvate and NADH [27]. For the determination of lactate content, the following reagents were used: hydrazineglycine buffer pH 9.0 (glycine -0.1 M solution containing 0.1 M hydrazine); NAD + -0.03 M solution, pH 6.0; lactate dehydrogenase solution (protein content was about 2 mg/ml). The samples of glycine and hydrazine were dissolved in a small amount of distilled water, the pH of the mixture was stabilized by 2 N NaOH solution and it became 9.0 then diluted in the distilled water to 100 ml. Solutions of NAD + and lactate dehydrogenase were kept on ice. The tubes content was thoroughly mixed and then it was placed inside a thermostat at +25°C for 60 min. After the incubation, the samples were cooled and then the optical density of the samples was measured at 340 nm. Taking into account all the dilutions, the quantity of the lactic acid was calculated.
Measuring of acetate ions concentration. The accumulation of acetate ions by the bacteria cultures during their growth in the medium was determined by titration [5].
Determination of other characteristics [3]. Indole production. This was tested using a 24hr Kravtsov-Sorokin's liquid culture with nitric acid and isoamylic alcohol reagents (Salkowski's reaction).
Nitrate reduction. Sodium nitrate (5%) was added to Kravtsov-Sorokin's liquid medium. Nitrites were tested with a napthylaminesulfanilic acid reagent on a 24hr culture.
Carbohydrate fermentation. Lactate, formate, propionate, fumarate, malate, me tha nol, citrate, ethanol, acetate, glycerol, oleate, stearate and benzoate were aseptically added to Kravtsov-Sorokin's liquid medium without electron donor and carbone source to obtain a final concentration of 1%. Glucose and pyruvate fermentation was also checked in the Kravtsov-Sorokin's liquid medium. Acidity was analyzed by pH reaction and pH indicators.
Gas production. This was observed in Kravtsov-Sorokin's deep culture agar in 9×180 mm tubes, inoculated with a Kravtsov-Sorokin's liquid culture.
Desulfoviridin production. This was examined using UV light on Kravtsov-Sorokin's agar surface cultures after treatment with a 1 N NaOH solution.
Identification of sulfate-reducing bacteria. Identification of the sulfatereducing bacteria by morphological, physiological and biochemical characteristics was conduc ted according to Bergey's Manual of Determinative Bacteriology [11].
Statistical analysis. Using the experimental data, the basic statistical parameters (M -mean, m -standard error, M±m) have been calculated. For the estimation of the reliability between the statistical characteristics, Student's tcoefficient test was used. The difference was reliable when P>0.95 [1]. Statistical processing of the results has been performed using packet Excel, Origin [12].

RESULTS AND DISCUSSION
Description of the obtained bacterial colonies and cell morphology. The results of the study showed that the isolated colonies on the selective solid nutrition medium with Mohr's salt were of identical shape (round), color (black), profile and structure; they differ only in size (large and small colonies). The size of the large colonies measured 2 mm in diameter with the small colonies measuring up to 1 mm in diameter. The bacterial colonies were black in colour, indicating the intense sulfate reduction. In Kravtsov-Sorokin's liquid medium all the strains showed a homogenous turbidity and a precipitation of iron from the sulfide.
Colonies were black and smooth when young and displayed a crater when older. After exposure to air, colonies decolorized quickly becoming yellow; at this stage the cells were dead. In Kravtsov-Sorokin's deep agar all the colonies were surrounded with a black halo of iron precipitation. Gas production was positive for all obtained strains. Desulfoviridin was produced by strains with large colonies but not by those with small colonies. All the strains were catalase positive and reduced nitrate. In addition, indol production was negative for all studied strains.
The ability of the strains to metabolize lactate, formate, propionate, pyruvate, fumarate, malate, methanol, citrate, ethanol, acetate, glycerol, glucose, oleate, stearate and benzoate was studied. The level of bacterial biomass accumulation in the medium with each of these substances was different. From this wide range of organic compounds studied bacteria grew only in the media with lactate, pyruvate, fumarate, malate, citrate, ethanol and glucose. Moreover, the bacteria grow well in medium with molecular hydrogen in the presence of carbon dioxide and acetate. Thus, studied strains are capable of hemolitoheterotrophic growth. In addition to organic acids with different carbon chain lengths and alcohol, the strains assimilated some amino acids.
Total number of the obtained SRB in the feces equaled 10 5 colonyforming units per gram of feces. 157 obtained colonies of microorganisms were examined under the light microscope. The microscopic study showed that live bacterial preparations, made from 124 large bacterial colonies, contained active motile cells. The live bacterial preparations, taken from 33 small colonies, also contained motile cells. The cells of the large colonies were single vibrioshaped and the cells of the small colonies displayed single short rods on fixed preparations. The bacterial cells of both these types of colonies were Gram-negative.
In previous research, the author has studied the ultrastructure of cells of the isolated bacteria under the electron microscope [17]. In the photos it was visible that the cells were rodshaped or vibrioshaped, surrounded by a cell wall and cytoplasmic membrane. In the cytoplasm the presence of nucleoid, ribosomes, and reserve substances was also observed. The results of electronic microscopic study helped to confirm the fact that two types of isolated colonies of SRB contain morphologically different cells. The large isolated colonies contain vibriolike cells and the other small colonies contain short rods cells. The vibrio cells size is 0.6-0.8×2.7-3.5 μm and appear slightly curved with crescent forms. The short rods cells size is 0.5×0.8-1.0 mm and is similar to the coccobacilli forms. Thus, this is the morphology of the cells typical for sulfatereducing bacteria.
The effect of oxygen on growth of the isolated bacterial strains. The cultivation of these microorganisms in anaerobic, microaerobic and aerobic conditions showed that aeration leads to cells death. However, the short contact of the SRB cells with oxygen does not cause their instant death. This may be due to the high activity of antioxidant enzymes, catalase and superoxide dismutase [2,23]. The obtained strains are obligate (strict) anaerobes.
The effect of pH on growth of the selected bacterial strains. It was established that the most intensive growth of all the bacterial strains and hydrogen sulfide accumulation were observed at pH 7.0-8.0. The decreasing and increasing acidity of the medium was accompanied by the decrease of the bacteria growth rate and hydrogen sulfide production. Similar results were obtained by Furutani et al which established that at low pH the process of dissimilatory sulfate reduction was significantly slowed [7]. Increasing of the pH medium to 9.0-10.0 also caused growth inhibition of the studied bacteria.
Growth of the bacterial strains in various media. To determine which subgroups the identified bacteria belong to, the selected suspended bacterial colonies (large and small) were seeded in media with and without sulfates and in the medium with elemental sulfur without sulfates. All of these media contained sodium lactate as a carbon source.
The results shown that the studied bacteria grow well only in the medium with sulfate ions and sodium lactate (Table 1). Due to the results obtained, the isolated strains belong to the group of sulfatereducing bacteria.
The studied bacterial strains do not grow in the medium without sulfates and also do not grow in the medium with molecular sulfur and lactate without sulfates. Therefore they are unable to use sulfur as an electron acceptor. This was helped by confirming the absence among isolated bacteria of the Desulfurella and Desulfuromonas genera. Thus, the obtained bacterial strains do not belong to the fourth subgroup of sulfurredu cing bacteria, since the bacteria do not use the sulfur.
This research has helped to check the purity of isolated cultures and further confirm that the selected strains are the sulfatereducing bacteria since bacterial growth was only in the Kravtsov-Sorokin's medium. Table 2.
Staining bacterial cells of isolated colonies by the Peshkov method has helped to confirm that none of the colonies contain sporeforming bacteria. This has helped to reach the conclusion that the isolated bacteria can not form spores. Therefore, among the isolated cultures there are no representatives of the Desulfotomaculum genus (the first subgroup of the sulfate-reducing bacteria according to Bergey's Manual of Determinative Bacteriology). Table 3.
Compared to the other rodshaped strains, the highest biomass was accumulated at +35°C temperature by SRB Rod3, SRB Rod4, SRB Rod9 and SRB Rod10 strains (Fig. 1B). At +20°C and +45°C temperature less intensive growth of bacterial strains was observed. The least biomass was accumulated by the isolates at +14°C temperature. Bacterial growth at +4°C temperature was not observed. Thus, +35°C temperature was the optimum temperature for growth of the selected strains of bacteria.
Bacterial growth and dissimilatory sulfate reduction by the selected bacterial strains. Bacterial growth and sulfate reduction processes performed by different strains of SRB were studied. The results of the study showed that all the isolates were actively growing, using sulfate as an electron acceptor and accumulating hydrogen sulfide in the medium. These bacterial strains use lactate as the electron donor which is incompletely oxidized by the bacteria, to acetate (Figs. 2-3).
Different growth rates of various strains of SRB in the modified Kravtsov-Sorokin's medium have been studied. The biomass accumulated by the studied vibrioshaped bacterial strains was highest when compare with the rodshaped strains (Fig. 2).
The studied SRB use sulfate and lactate with varying intensity of hydrogen sulfide and acetate producing. The intense growth of these bacteria depended on the use of sulfate, lactate, and the accumulation of hydrogen sulphide and acetate. A strong reverse negative correlation between these processes was established.
Having used all the sulfate and lactate in the medium, the bacteria stopped growing and the stationary growth phase began. The limiting factors of the growth of these bacteria were hydrogen sulfide and acetate, produced by the bacteria in the processes of dissimilatory sulfate reduction and lactate oxidation respectively.
In comparison to the vibrioshaped strains, the rodshaped strains of the SRB reduced sulfate, oxidized lactate, and produced hydrogen sulfide in the medium less intensively (Fig. 3). Such usage of acceptor and donor electrons by the rodshaped bacteria had influence on their growth in the medium. This growth was less intense in comparison to the vibrioshaped strains.
Among all the isolated bacteria the highest biomass (up to 3.89 gram per liter) was accumulated by the SRB Vib7 strain on the sixth day of cultivation. The lowest biomass (up to 3.41 gram per liter) among vibrios strains was accumulated by the SRB Vib10   The highest concentration of hydrogen sulfide (up to 3.23 mM) among the vibrioshaped strains was produced by the bacterial SRB Vib7 strain on the eighth day of cultivation; while the bacteria used about 99% of the sulfate in the medium, compared to the initial concentration of the sulfate. The SRB Vib8 strain produced hydrogen sulfide in the lowest concentration (up to 2.67 mM) on the sixth day of cultivation, compared to the vibrioshaped strains; under these conditions, about 98% of sulfate ions were used in the medium compared to its initial concentration.
Among the rodshaped strains, the highest concentration of hydrogen sulfide (up to 3.14 mM) was produced by the SRB Rod9 strain on the eighth day of cultivation. At this time, the strain used about 98% of the sulfate in the medium. The bacterial SRB Rod2 strains produced the lowest concentration of hydrogen sulfide (up to 1.91 mM) on the sixth day of cultivation; they used about 97% of the sulfate in the medium, compared to its initial concentration.
Among all the vibrioshaped strains, the bacterial SRB Vib7 strain produced the highest concentration of acetate ions (up to 15.87 mM) on the fifth day of cultivation; while the bacteria used about 97% of lactate in the medium, compared to its initial concentration. The SRB Vib5 strain produced the lowest concentration (up to 14.36 mM) of acetate ions on the eighth day of cultivation, compared to the vibrioshaped strains; under these conditions, the strain used about 98% of lactate in the medium, compared to its initial concentration. Among the rodshaped strains the highest concentration of acetate ions (up to 16.24 mM) produced by the SRB Rod4 strain on the eighth day of cultivation; at this time the strain used about 99% of lactate in the medium. The bacterial SRB Rod5 strains produced the lowest concentration of acetate ions (up to 15.64 mM) on the tenth day of cultivation; they used about 98% of lactate in the medium compared to its initial concentration.
Therefore, the studied bacteria actively reduced sulfates and produced hydrogen sulfide due to incomplete lactate oxidation by the strains accumulating acetate ions in the medium. This data has helped to establish that the obtained bacterial strains do not belong to the third subgroup, containing asporogenous sulfate-reducing bacteria that completely oxidize the organic substrates to CO 2 .
All isolated strains of bacteria actively produced acetate and lactate oxidation was incomplete. Thus, the isolated strains belong to the second subgroup that contains asporogenous sulfatereducing bacteria; they oxidize organic compounds incompletely to acetate.
The next task of the study was to establish to which genera the selected bacteria strains belong. The subgroup includes Desulfobulbus, Desulfomicrobium, Desulfomonas, Desulfovibrio and Thermodesulfobacterium genera. For the identification the table of Bergey's Manual of Determinative Bacteriology was used (Table 4) [11].
Growth of all the isolated strains in the modified KravtsovSorokin's medium with sulfates and propionate (without lactate) was investigated. The results of this study showed that none of the bacterial cultures grow in this medium with propionate, which eliminates the possibility of belonging to the Desulfobulbus genus.
Notes: "+" -the presence of the feature; "-" -the absence of the feature Примітки: "+" -наявність ознаки; "-" -ознаки нема It was established that +35°C is the optimum temperature for growth of the studied bacterial strains. Increase of temperature to +45°C caused a decrease in the intensity of the bacterial growth. It has helped to establish that none of the isolated strains belong to the Thermodesulfobacterium genus which grow at high temperatures (+65...+70°C).
The isolated strains also can not belong to the Desulfomonas genus because the studies have shown that all the isolated bacteria are motile due to a single polar flagellum.
The growth of the obtained strains in the KravtsovSorokin's medium (without lactate) with different carbon sources was studied. The studies have shown that bacteria grew in the medium with acetate, pyruvate, ethanol, malate, and fumarate.
Taking into consideration all the obtained results, investigation of bacterial growth in different media; ability to form spores; temperature and acidic optima; the ability to reduce sulfate to hydrogen sulfide; production of acetate as a result of utilization of lactate; morphology; growth in the medium with lactate; acetate and other carbon sources, the isolated bacteria may therefore be identified as Desulfovibrio sp. and Desulfomicrobium sp.

CONCLUSIONS
Twenty pure cultures of the sulfatereducing bacteria of human intestine were obtained. The results of studies of their morphological, physiological and biochemical characteristics showed that the obtained bacterial strains belong to the Desulfovibrio and Desulfomicrobium genera according to the Bergey's Manual of Determinative Bacteriology. All the obtained bacterial cultures extensively used sulfate, lactate and produced hydrogen sulfide as well as acetate during growth in the modified Kravtsov Sorokin's medium. The Desulfovibrio sp. strain Vib7 produced the highest concentration (up to 3.23 mM) of hydrogen sulfide on the eighth day of cultivation among the studied Desulfovibrio sp. strains; while the bacteria used about 99% of the sulfate in the medium, compared to the initial concentration of the sulfate. Among all the studied Desul fovibrio sp. strains, the Desulfovibrio sp. strain Vib7 produced the highest concentration of acetate ions (up to 15.87 mM) on the fifth day of cultivation. Among the studied Desulfomicrobium sp. strains the highest concentration of hydrogen sulfide (up to 3.14 mM) produced by the Desulfomicrobium sp. Rod9 strain on the eighth day of cultivation; the strain used about 98% of sulfate in the medium. The Desulfomicrobium sp. strain Rod4 produced the highest concentration of acetate ions (up to 16.24 mM) among all the studied Desulfomicrobium sp. strains.
The optimum growth of the studied bacteria at +35°C temperature suggests that the isolated cultures can actively develop in the human intestine and extensively produce hydrogen sulfide and acetate in its lumen. The presence and activity of these micro organisms in the human intestine may be considerably responsible for the development of very serious pathological conditions of the human intestine, causing disease.