Bacterial flora of marine fish doma (Sciaenoids) (Small spp.) from Bombay coast

Marine fish doma (Sciaenoids) (Small spp.) from Bombay coast was studied for total bacterial counts on the surface and gut. Large number of Micrococcus species (77.4%) was found whereas few species from Achromobacter, Bacillus, Bacterium, Flavobacter, Pseudomonas and Sarcina were noted.

Use of skin graft punch graft type for the healing of leg ulcers in treated Hansen's disease patients

Hansen's disease is an infectious illness caused by Mycobacterium leprae. It affects preferentially the skin and the peripheral nervous system leading to incapacities, such as leg ulcers, which happens due to the direct action of the bacillus on the organs or its indirect action on the peripheral nervous system. Leg ulcers can occur by two physiopathologic processes. There are many treatments for general leg ulcers, which include the ones caused by Hansen's disease sequels. Among them, surgical treatment shows to be effective when using skin graft, which can be performed by several techniques. Considering the low number of techniques known for treating leg ulcers in Hansen's disease sequels, the aims of this work were to standardize alternative techniques, to detect the main bacteria found in ulcer secretion cultures, to analyze the patients profile and the ulcers, to describe the histophatologies found, and to correlate these data with those of literature from all over the world. Skin graft punch type was carried out and analyzed; males had a mean age of 59.4 years old and females, 54.2 years old. Patients were 73.6% male and 26.3% female. Lepromatous type was present in 89.4% patients and tuberculoid type was seen in 10.5% of them. Associated systemic diseases were observed in 26.3% patients. Mean time of ulcers evolution was 11.6 years in male and 12.8 years in women. The average diameter of ulcers in the pre-treatment period was 8.5 X 9.5 cm in male and 10.2 X 6.8 cm in women. After the graft, their average diameters were 3.2 X 2.7 cm in male and 5.1 X 5.6 cm in women. Statistical analysis showed that there was no significant correlation between the ulcer diameter and its reduction or not in the post-surgery period (p=0.269732). The mean age of patients whose ulcers diameter did not change or reduced by only 20% was 63.5 years. Using the Spearman's coefficient, it was possible to observe that there was no significant correlation between the patients' age and the ulcers diameter reduction after the skin graft (p=0.222531). Evolution time of ulcers that did not present any satisfactory result in the post-surgery period was 12.1 years. The Spearman's coefficient showed that there was no significant correlation between the ulcers evolution time and the ulcers diameter reduction in the post-surgery period (p=0.191655). Cultures presented 50% of cases with Pseudomonas aeruginosa. Statistical analysis showed there is no correlation between the bacterial types found and the ulcer evolution in the post-surgery period (p=0.697531). The average of the ulcers diameter reduction was 42.4%, and in 26.3% of the patients the lesions disappeared after the skin graft.

First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): New perspectives for an insect-bacteria association

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.

Bacillus solisalsi sp nov., a halotolerant, alkaliphilic bacterium isolated from soil around a salt lake

A novel Gram-positive, motile, rod-shaped bacterium isolated from a saline soil in China was characterized by a polyphasic taxonomic approach. The strain, designated YC1(T), was halotolerant [tolerating up to 15 % (w/v) NaCl] and alkaliphilic (growing at

Lysis of Aphanizomenon flos-aquae (Cyanobacterium) by a bacterium Bacillus cereus

A phytoplankton-lytic (PL) bacterium, Bacillus cereus, capable of lysing the bloom-forming cyanobacterium. Aphanizomenon flos-aquae was isolated from Lake Dianchi of Yunnan province, China. This bacterium showed lytic activities against a wide range of cyanobacteria/algae, including A. flos-aquae, Microcystis viridis, Microcystis wesenbergi, Microcystis aeruginosa, Chlorella ellipsoidea, Oscillatoria tenuis, Nostoc punctiforme, Anabaena flos-aquae, Spirulina maxima, and Selenastrum capricornutum. Chlorophyll a contents, phycocyanin contents, and photosynthetic activities of the A. flos-aquae decreased evidently in an infected culture for a period. Bacterium B. cereus attacked rapidly A. flos-aquae cells by cell-to-cell contact mechanism. It was shown that the lysis of A. flos-aquae began with the breach of the cyanobacterial cell wall, and the cyanobacterial cell appeared abnormal in the presence of the PL bacterium. Moreover, transmission electron microscope examinations revealed that a close contact between the bacterium and the cyanobacterium was necessary for lysis. Some slime extrusions produced from B. cereus assisted the bacterial cells to be in close association with and lyse the cyanobacterial cells. These findings suggested that this bacterium could play an important role in controlling the Aphanizomenon blooms in freshwaters. (c) 2006 Elsevier Inc. All rights reserved.

Bacillus lehensis-an alkali-tolerant bacterium isolated from cassava starch wastewater: optimization of parameters for cyclodextrin glycosyltransferase production

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Biomineralization of manganese by Bacillus spp isolated from a marine biofilm

Biomineralization of manganese on titanium condenser material exposed to seawater has been illustrated. Biomineralization occurs when the fouling components, namely, the microbes, are able to oxidize minerals present in water and deposit them as insoluble oxides on biofilm surfaces. Extensive biofilm characterization studies Showed that an alarmingly large number of bacteria in these biofilms are capable of oxidizing manganese and are, thereby, capable of causing biomineralization on the condenser material exposed to seawater. This paper addresses studies on understanding the exact role of the microbes in bringing about oxidation of manganese. The kinetics of manganese oxidation by marine Gram-positive manganese oxidizing bacterium Bacillus spp. that was isolated front the titanium surface was studied in detail. Manganese oxidation in the presence of Bacillus cells, by cell free extract (CFE) and heat-treated cell free extract was also studied. The study confirmed that bacteria mediate manganese oxidation and lead to the formation of biogenic oxides of MnO2 eventually leading to biomineralization on titanium surface exposed to seawater.

Bacillus cereus spores and cereulide in food-borne illness

B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.

Assessment and control of Bacillus cereus emetic toxin in food

Despite of improving levels of hygiene, the incidence of registered food borne disease has been at the same level for many years: there were 40 to 90 epidemics in which 1000-9000 persons contracted food poisoning through food or drinking water in Finland. Until the year 2004 salmonella and campylobacter were the most common bacterial causes of food borne diseases, but in years 2005-2006 Bacillus cereus was the most common. Similar developement has been published i.e. in Germany already in the 1990´s. One reason for this can be Bacillus cereus and its emetic toxin, cereulide. Bacillus cereus is a common environmental bacterium that contaminates raw materials of food. Otherwise than salmonella and campylobacter, Bacillus cereus is a heat resistant bacterium, capable of surviving most cooking procedures due to the production of highly thermo resistant spores. The food involved has usually been heat treated and surviving spores are the source of the food poisoning. The heat treatment induces germination of the spore and the vegetative cells then produce toxins. This doctoral thesis research focuses on developing methods for assessing and eliminating risks to food safety by cereulide producing Bacillus cereus. The biochemistry and physiology of cereulide production was investigated and the results were targeted to offer tools for minimizing toxin risk in food during the production. I developed methods for the extraction and quantitative analysis of cereulide directly from food. A prerequisite for that is knowledge of the chemical and physical properties of the toxin. Because cereulide is practically insoluble in water, I used organic solvents; methanol, ethanol and pentane for the extraction. For extraction of bakery products I used high temperature (100C) and pressure (103.4 bars). Alternaties for effective extraction is to flood the plain food with ethanol, followed by stationary equilibration at room temperature. I used this protocol for extracting cereulide from potato puree and penne. Using this extraction method it is also possible also extract cereulide from liquid food, like milk. These extraction methods are important improvement steps for studying of Bacillus cereus emetic food poisonings. Prior my work, cereulide extraction was done using water. As the result, the yield was poor and variable. To investigate suspected food poisonings, it is important to show actual toxicity of the incriminated food. Many toxins, but not cereulide, inactivate during food processing like heating. The next step is to identify toxin by chemical methods. I developed with my colleague Maria Andesson a rapid assay for the detection of cereulide toxicity, within 5 to 15 minutes. By applying this test it is possible to rapidly detect which food was causing the food poisoning. The chemical identification of cereulide was achieved using mass spectrometry. I used cereulide specific molecular ions, m/z (+/-0.3) 1153.8 (M+H+), 1171.0 (M+NH4+), 1176.0 (M+Na+) and 1191.7 (M+K+) for reliable identification. I investigated foods to find out their amenability to accumulate cereulide. Cereulide was formed high amounts (0.3 to 5.5 microg/g wet wt) when of cereulide producing B. cereus strains were present in beans, rice, rice-pastry and meat-pastry, if stored at non refrigerated temperatures (21-23C). Rice and meat pastries are frequently consumed under conditions where no cooled storage is available e.g. picnics and outdoor events. Bacillus cereus is a ubiquitous spore former and is therefore difficult to eliminate from foods. It is therefore important to know which conditions will affect the formation of cereulide in foods. My research showed that the cereulide content was strongly (10 to 1000 fold differences in toxin content) affected by the growth environment of the bacterium. Storage of foods under nitrogen atmosphere (> 99.5 %) prevented the production of cereulide. But when also carbon dioxide was present, minimizing the oxygen contant (< 1%) did not protect the food from formation of cereulide in preliminary experiments. Also food supplements affected cereulide production at least in the laboratory. Adding free amino acids, leucine and valine, stimulated cereulide production 10 to 20 fold. In peptide bonded form these amino acids are natural constituents in all proteins. Interestingly, adding peptide bonded leucine and valine had no significant effect on cereulide production. Free amino acids leucine and valine are approved food supplements and widely used as flawour modifiers in food technology. My research showed that these food supplements may increase food poisoning risk even though they are not toxic themselves.

The utility of Bacillus subtilis as a bioflocculant for fine coal

The application of Bacillus subtilis as a flocculant for fine coal has been reported here. Zeta-potential measurements showed that both the coal and bacteria had similar surface charge as a function of pH. Surface free energy calculations showed that the coal was hydrophobic while the bacterium was hydrophilic. The adhesion of the bacteria to coal and subsequent settling was studied in detail. Adhesion of bacteria to coal surface and subsequent settling of coal was found to be quick. Both adhesion and settling were found to be independent of pH, which makes the process very attractive for field applications. The presence of an electrolyte along with the bacterium was found to not only enhance adhesion of bacteria, but also produce a clear supernatant. Further, the settled fraction was more compact than with bacteria alone. Interaction energy calculations using the extended DLVO theory showed that the electrical forces along with the acid-base interaction energy play a dominant role in the lower pH range. Above pH 7, the acid-base interaction energy is the predominant attractive force and is sufficient enough to overcome the repulsive forces due to electrical charges to brine about adhesion and thus settling of fine coal. With increase in electrolyte concentration, the change in total interaction energy with pH is minimal which probably leads to better adhesion and hence settling. (C) 2003 Elsevier Science B.V. All rights reserved.

Selective biodissolution of calcium and iron from bauxite in the presence of Bacillus polymyxa

A bacterium Bacillus polymyxa was found to be capable of selective removal of calcium and iron from bauxite. The bioleached residue was found to be enriched in its alumina content with insignificant amounts of iron and calcium as impurities. The developed bio- process was found to be capable of producing a bauxite product which meets the specifica- tions as a raw material for the manufacture of alumina based ceramics and refractories. The role of bacterial cells and metabolic products in the selective dissolution of calcium (present as calcite) and iron (present as hematite and goethite) from bauxite was assessed and possi- ble mechanisms illustrated. The effect of different parameters such as sucrose concentra- tion, pH, pulp density and time on selective biodissolution was studied. It was observed that periodic decantation and replenishment of the leach medium was beneficial in improving the dissolution kinetics. Calcium removal involves chelation with bacterial exopolysaccha- tides and acidolysis by organic acid generation. Hematite could be solubilized through a reductive dissolution mechanism.

Isolation and characteristics of a bacteriophage for bacillus stearothermophilus

A bacteriophage (TØ3) which infects the thermophilic bacterium Bacillus stearothermophilus ATCC 8005 was isolated and characterized. Infection of the bacterium by the bacteriophage was carried out at 60°C, the optimum growth temperature of the host. At 60°C the phage has a latent period of 18 minutes and a burst size of about 200. The phage is comparatively thermostable in broth. The half life of the phage is 400 minutes at 60°C, 120 minutes at 65°C, 40 minutes at 70°C and 12 minutes at 75°C. The activation energy for the heat inactivation of TØ3 is 56,000 cal. The buoyant density of TØ3 in a cesium chloride density gradient is 1.526.

Electron micrographs of TØ3 indicate that the phage has a regular hexagonal shaped head 57 mμ long. The morphology of the head is compatible with icosahedral symmetry. Each edge of the head is 29 mμ long, and there are 6 or 7 subunits along each edge. The tail of TØ3 is 125 mμ long and 10 mμ wide. There are about 30 cross striations that are spaced at 3.9 mμ intervals along the tail.

The DNA of phage TØ3 has a melting temperature of 88.5°C. Heat denatured TØ3 DNA can be extensively annealed in a high ionic strength environment. The buoyant density of TØ3 DNA in a cesium chloride density gradient is 1.695. TØ3 DNA contains: 42.7% guanine plus cytosine, as determined from the melting temperature; 43% guanine plus cytosine, as determined from the buoyant density; and 40.2% guanine plus cytosine, as determined by chromatographic separation and spectrophotometric estimation of the bases. The molecular weight of TØ3 DNA is 16.7 X 10⁶ as determined from the band width of the TØ3 DNA concentration distribution in a cesium chloride density gradient. Electron microscopy of TØ3 DNA revealed a single linear molecule that is 11.7 μ long. This corresponds to a molecular weight of 22.5 X 10⁶.

Heat denatured TØ3 DNA forms two bands in a cesium chloride density gradient, one at a density of 1.707 and the other at a density of 1.715. After the separated bands are mixed and annealed in the centrifuge cell, the renatured TØ3 DNA forms a single band at a density of 1.699. These results indicate that the two complementary strands of TØ3 DNA have different buoyant densities in cesium chloride, presumably because they have different base compositions.

The characteristics of TØ3 are compared with those of other phages. A hypothesis is presented for a relationship between the base composition of one strand of TØ3 DNA and the amino acid composition of the proteins of TØ3.

Bacillus pallidus sp nov., isolated from forest soil

A Gram-positive bacterium, designated strain CW 7(T), was isolated from forest soil in Anhui Province, south-east China. Cells were strictly aerobic, motile with peritrichous flagella and rod-shaped. The strain grew optimally at 30-37 degrees C and pH 7.0-8.0. The major fatty acids of strain CW 7(T) were anteiso-C-15:0, iso-C-15:0 and anteiso-C-17:0. The predominant menaquinone was MK-7. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The G + C content of the genomic DNA was 42.3 mol%. Phylogenetic analysis indicated that strain CW 7(T) belonged to a monophyletic cluster within the genus Bacillus and showed 16S rRNA gene sequence similarities of less than 96.5% to recognized species of the genus Bacillus. The results of the polyphasic taxonomic study, including phenotypic, chemotaxonomic and phylogenetic analyses, showed that strain CW 7(T) represents a novel species of the genus Bacillus, for which the name Bacillus pallidus sp. nov. is proposed. The type strain is CW 7(T) (=KCTC 13200(T)=CCTCC AB 207188(T)=LMG 24451(T)).

Influence of a fish bacterium Lactobacillus sp. on the production of swordtail xiphophorus helleri (Heckel 1848)

The influence of a fish gut bacterium Lactobacillus sp on the production of swordtail Xiphophorus helleri was studied for a period of one year. The Lactobacillus sp P21 produced bacteriocin-like inhibitory substance and exhibited wide spectrum of action against Aeromonas hydrophila, Bacillus spp, Pseudomonas spp and Citrobacter freundi in vitro. The growth performance of X. helleri reared in the presence of Lactobacillus P21 at 106/ml rearing water was better than the control. The total plate counts, total MRS agar counts and the counts of motile aeromonads, presumptive pseudomonads, lactose fermenters and lactose non-fermenters in the gut of probiotic group were comparatively low than the control. On day 60 the count of Lactobacillus sp P21 was observed to be log 5.28/g in the gut of X. helleri indicating colonization of this bacterium in the gastrointestinal tract. The fecundity of X. helleri was in the range of 9-134. On average, it produced from 39.42±18.72 fry/female in control group to 53.00±23.57 fry/female in probiotic group. The increase in average fecundity in probiotic group over the control group was about 25%. There existed significant difference between probiotic group and control in respect of average fecundity/female (p<0.02), average number of fry survived /female (p<0.006) and average number of fry dead/female (p<0.029). The results of the present study demonstrated that the rearing of X. helleri in probiotic-enriched water have growth inducing ability and favourably influenced the reproductive performance in terms of high fecundity, high fry survival, reduced fry mortality and reduced fry deformity.

Pesquisa computacional de inibidores de fatores de virulência de bacillus anthracifs

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas