Identification of antarctic soil bacteria exhibiting antiproliferative activity against a colon cancer cell line / Identificación de bacterias de suelo antártico que presentan actividad antiproliferativa sobre una línea celular de cáncer de colon

SUMMARY: Cancer is the second leading cause of death in the world and colorectal cancer is the only cancer that has shown a sustained increase in mortality in the last decade. In the search for new chemotherapeutic agents against cancer, extremophilic microorganisms have shown to be a potential source to obtain molecules of natural origin and with selective cytotoxic action towards cancer cells. In this work we analyzed the ability of a collection of Antarctic soil bacteria, isolated on Collins Glacier from the rhizosphere of Deschampsia antarctica Desv plant, to secrete molecules capable of inhibiting cell proliferation of a colorectal cancer tumor line. Our results demonstrated that culture supernatants from the Antarctic bacteria K2I17 and MI12 decreased the viability of LoVo cells, a colorectal adenocarcinoma cell line. Phenotypic and genotypic characterization of the Antarctic bacteria showed that they were taxonomically related and nucleotide identity analysis based on the 16S rRNA gene sequence identified the bacterium K2I17 as a species belonging to the genus Bacillus.

El cáncer es la segunda causa de muerte en el mundo y el cáncer colorrectal es el único que presenta un aumento sostenido de la mortalidad en la última década. En la búsqueda de nuevos agentes quimioterapeúticos contra el cáncer, se ha propuesto a los microorganismos extremófilos como una fuente potencial para obtener moléculas de origen natural y con acción citotóxica selectiva hacia las células cancerígenas. En este trabajo analizamos la capacidad de una colección de bacterias de suelo antártico, aisladas en el glaciar Collins desde rizosfera de la planta de Deschampsia antarctica Desv, de secretar moléculas capaces de inhibir la proliferación celular de una línea tumoral de cáncer colorrectal. Nuestros resultados demostraron que los sobrenadantes de cultivo de las bacterias antárticas K2I17 y MI12 disminuyeron la viabilidad de la línea celular de adenocarcinoma colorrectal LoVo, en un ensayo de reducción metabólica de MTT. La caracterización fenotípica y genotípica de las bacterias antárticas, demostró que estaban relacionadas taxonómicamente y el análisis de la identidad nucleotídica en base a la secuencia del gen ARNr 16S identificó a la bacteria K2I17 como una especie perteneciente al género Bacillus.

Control potential of Meloidogyne javanica and Ditylenchus spp. using fluorescent Pseudomonas and Bacillus spp

ABSTRACT Plant Growth Promoting Rhizobacteria (PGPR) have different mechanisms of action in the development of plants, such as growth promotion, production of phytohormones and antibiotic substances and changes in root exudates. These help to control plant diseases. In order to evaluate the potential of microorganisms in the control of Meloidogyne javanica and Ditylenchus spp., five rhizobacteria isolated from rhizosphere of garlic cultivated in the Curitibanos (SC) region were tested. Hatching chambers were set on Petri dishes, in which were added 10 mL of bacterial suspension and 1 mL of M. javanica eggs suspension, at the rate of 4500, on the filter paper of each chamber. The same procedure was performed with 300 juvenile Ditylenchus spp. The experimental design was completely randomized, with four replications. The evaluations were performed every 72 h for nine days. The antagonized population of nematodes was determined in Peters counting chamber, determining the percentage hatching (for M. javanica) and motility (for Ditylenchus spp). Isolates CBSAL02 and CBSAL05 significantly reduced the hatching of M. javanica eggs (74% and 54.77%, respectively) and the motility of Ditylenchus spp. (55.19% and 53.53%, respectively) in vitro. Isolates were identified as belonging to the genera Pseudomonas (CBSAL05) and Bacillus (CBSAL02).

Plant growth promoting rhizobacteria reduce aphid population and enhance the productivity of bread wheat

Abstract Plant growth promoting rhizobacteria increase plant growth and give protection against insect pests and pathogens. Due to the negative impact of chemical pesticides on environment, alternatives to these chemicals are needed. In this scenario, the biological methods of pest control offer an eco-friendly and an attractive option. In this study, the effect of two plant growth promoting rhizobacterial strains (Bacillus sp. strain 6 and Pseudomonas sp. strain 6K) on aphid population and wheat productivity was evaluated in an aphid susceptible (Pasban-90) and resistant (Inqlab-91) wheat cultivar. The seeds were inoculated with each PGPR strain, separately or the combination of both. The lowest aphid population (2.1 tiller−1), and highest plant height (85.8 cm), number of spikelets per spike (18), grains per spike (44), productive tillers (320 m−2), straw yield (8.6 Mg ha−1), and grain yield (4.8 Mg ha−1) were achieved when seeds were inoculated with Bacillus sp. strain 6 + Pseudomonas sp. strain 6K. The grain yield of both varieties was enhanced by 35.5-38.9% with seed inoculation with both bacterial strains. Thus, the combine use of both PGPR strains viz. Bacillus sp. strain 6 + Pseudomonas sp. strain 6K offers an attractive option to reduce aphid population tied with better wheat productivity.

Squamocin, an annonaceous acetogenin, enhances naphthalene degradation mediated by Bacillus atrophaeus CN4 / Squamocin, una acetogenina annonácea, estimula la degradación de naftaleno mediada por Bacillus atrophaeus CN4

Squamocin belongs to a group of compounds called annonaceous acetogenins. They are secondary products of Annonaceae metabolism and can be isolated from Annona cherimolia seeds. This paper deals with the stimulation of biofilm formation of Bacillus atrophaeus CN4 by employing low squamocin concentrations to increase naphthalene degradation. Bacillus atrophaeus CN4, isolated from contaminated soil, has the ability to degrade naphthalene as the only source of carbon and energy. In the absence of additional carbon sources, the strain removed 69% of the initial concentration of naphthalene (approx. 0.2 mmol/l) in the first 12 h of incubation. The addition of squamocin in LB medium stimulated Bacillus atrophaeus CN4 biofilm formation and enhanced naphthalene removal. Squamocin (2.5 pg/ml) does not affect planktonic growth and therefore, the observed increases are solely due to the stimulation of biofilm formation.

Squamocin pertenece a un grupo de compuestos llamados acetogeninas annonáceas (ACG). Las ACG son productos secundarios del metabolismo de plantas de la familia Annonaceae y se pueden aislar a partir de semillas de Annona cherimola. Este artículo trata de la estimulación de la formación de biofilm de Bacillus atrophaeus CN4 mediante el empleo de bajas concentraciones de squamocin para optimizar la degradación de naftaleno. B. atrophaeus CN4, aislado de suelo contaminado, tiene la capacidad de emplear naftaleno como única fuente de carbono y energía. En ausencia de fuentes de carbono adicionales, la cepa degradó el 69% de la concentración inicial de naftaleno (aprox. 0,2 mmol/l) en las primeras 12h de incubación. La adición de squamocin en medio LB estimula la formación de biofilm y la remoción naftaleno de B. atrophaeus CN4. Squamocin (2,5 µg/ml) no afecta al crecimiento planctónico y, por lo tanto, los incrementos observados se deben únicamente a la estimulación de la formación de biofilm.

Survival strategies of Bacillus spores in food.

Control of bacterial spores is one of the major problem in the food preservation. Spores of Bacillus genus are commonly present in different environments, including soil and the gut of insects and animals and, as a result, they can be spread to all kind of foods. Due to their high resistance properties, their complete inactivation in food is often impossible without changing the product characteristics. Surviving spores can germinate and grow out to vegetative cells, with the consequent great risk of food spoilage and food poisoning after consumption. Spores have evolved various mechanisms, including phenotypic variability, to protect themselves from a wide range of damage resulting from food preservation treatments. Even if the phenotypic heterogeneity contributes to increase the chances of survival of Bacillus spore to conventional preservation treatments, in some specific instances, an homogeneous response could be the result of a strategy adopted by the spores to increase resistance to those treatments.

In vivo Evaluation of Hypolipidemic Potentials of Bacillus Species Isolated from Fermented Locust Bean (Parkia fillicoides Welw) Seeds (Iru).

Aim: Iru is a popular West Africa fermented soup condiment which is also consumed without cooking as snack. This product is mainly fermented by Bacillus species. The hypolipidemic activities of Bacillus spp. isolated from iru have not been documented hence the aim of this study. Place and Duration of Study: Iru sample was bought in an open market in Iworoko-Ekiti, Nigeria and transferred to the Laboratory of the Department of Microbiology, Ekiti State University, Nigeria where other studies were carried out. The study was conducted between January and June, 2012. Methodology: The properties and in vivo hypolipidemic potential of Bacillus species from iru were investigated using standard microbiological and haematological methods. Results: The cell free extracts of the Bacillus spp. did not produce significant inhibition on the selected Gram positive and Gram negative pathogens. Qualitative enzyme screening of the isolates showed all were haemolysin negative. Only B. subtilis was positive to gelatinase while all the isolates produced catalase and lipase. The average weight of the animals after inducement of hyper-cholesterolemia ranged between 60.5g - 95.3g. The amount of serum total cholesterol (TC) in the animals ranged between 124.9 mg/dl – 127.4 mg/dl while that of serum triglycerides (TG), high density protein (HDL) and low density protein (LDL) were 122.5 – 155.3 mg/dl, 10.0 – 15.3 mg/dl and 76.6 – 81.0 mg/dl respectively. The weights of hyper-cholesterolemia induced rats challenged with different species of Bacillus were relatively lower than those in the control group and also differ significantly from the control, at p˂ 0.05. The values of TC, TG, and LDL were highest in the control (saline) group while the values in the treatment group ranged between 121.3 ± 1.5 and 102.3 ± 6.8 mg/dl for TC. The treatment groups recorded lower values of values for TG (104.7 ± 1.6 - 117.4 ± 9.1 mg/dl) and LDL (42.6 ±7.4 - 59.0 ± 10.2 mg/dl) compared to the control. B. subtilis had the highest values of TC but least amount of LDL. TG in all the groups was higher than TC, HDL and LDL. The TC/HDL and the LDL/HDL of the animals in the iru group was higher than the other treatment groups but lower than the control. Conclusion: Compared to the control, hypolipidemic activities of B. lichenliformis was the best followed by B. subtilis. Iru had the least hypo-cholesterolemic effect.

Facultative alkalophilic bacteria from mangrove soil with varying buffering capacity and H+ conductance.

Facultative alkalophilic bacteria Planococcus sp. (EMGA-26), Bacillus sp. (EMGA-29) and Corynebacterium spp. (EMGA-33 and 130) were isolated from mangrove soil samples. Neutrophiles were predominant than alkalophiles. Buffering capacity and membrane H+ conductance were investigated for the strains grown in PPYG medium at pH 10.5 using acid pulse technique. Bacillus sp. showed higher buffering capacity than Planococcus sp. and Corynebacterium spp. Buffering capacity was two-fold higher in Corynebacterium sp. EMGA-33 than in EMGA-130. The membrane H+ conductance was high in Bacillus sp. and was directly proportional to the buffering capacity values. The Bacillus sp. (EMGA-29) had higher cell membrane adaptability in high pH environment than the Planococcus sp. and Corynebacterium spp.

Inhibition of the growth of Ascosphaera apis by Bacillus and Paenibacillus strains isolated from honey / Inhibición del crecimiento de Ascosphaera apis mediante cepas de Bacillus y Paenibacillus aisladas de miel

The fungus Ascosphaera apis, the causative agent of chalkbrood disease in honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of 249 aerobic spore-forming bacterial antagonists isolated from honey samples was evaluated. Each isolate was screened against A. apis by a central disk test assay. Ten bacterial strains that showed the best antagonistic effect to A. apis were selected for further study and identified as Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), and Paenibacillus alvei (m321). For testing the efficiency of the selected strains, a paired culture test was used with 5 replicates of each combination bacterial antagonist / A. apis strain, and 5 replications for each control on 4 different culture media. The analysis of variance and posterior comparison of means according to LSD method showed that the best antagonists when using YGPSA medium were B. subtilis (m329) and B. megaterium (m435), and in the case of MYPGP medium the most efficient were B. circulans strains Fr 231 and m448b.

La cría yesificada es una micosis invasiva ocasionada por el hongo heterotálico Ascosphaera apis que afecta exclusivamente a las larvas de las abejas. La enfermedad tiene difusión mundial y en la Argentina se halla diseminada en todas las áreas donde se realiza apicultura. Se estudió la potencialidad de 249 cepas de bacterias esporuladas aeróbicas aisladas de miel como agentes biocontroladores del hongo mediante un ensayo en disco central en condiciones de laboratorio. Se seleccionaron como mejores antagonistas 10 cepas bacterianas identificadas como Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), y Paenibacillus alvei (m321). Para probar la eficiencia de las cepas seleccionadas, se empleó la técnica de cultivo dual con 5 repeticiones de cada combinación antagonista bacteriano / cepa de A. apis, 5 para cada control y 4 medios de cultivo diferentes empleando 10 cepas del hongo de distintos orígenes geográficos. El análisis de la variancia y posterior comparación de medias LSD (Least Square Dfferences) mostró que los mejores antagonistas fueron B. subtilis (m329) y B. megaterium (m435) para el caso del medio YGPSA, mientras que para MYPGP las más eficientes fueron las cepas de B. circulans Fr 231 y m448b.

Field evaluation of Spicbiomoss, a biolarvicidal formulation of Bacillus sphaericus against immatures of Culex quinquefasciatus & Anopheles species.

Spicbiomoss, an aqueous suspension formulation of Bacillus sphaericus was evaluated for its efficacy against immatures of Culex quinquefasciatus at the application rate of 120 l/ha in cement tanks, cesspits and drains in Pondicherry and in drains in Kochi, Kerala. The formulation was also tested against anophelines (Anopheles fluviatilis and An. culicifacies) breeding in stream pools in Malkangiri district, Orissa. In cement tanks and cesspits more than 80 per cent reduction in immature density was observed for a period of 6-13 days (mean 9.8 +/- 1.25 days) and 3-8 days (mean 5.2 +/- 0.7 days) respectively. The same level of reduction was found to last for 1-4 days (mean 2.2 +/- 0.52 days) in drains in Pondicherry and 2-9 days (mean 4.8 +/- 1.17 days) in Kochi. In bunded stream pools 40-79 per cent reduction in immature density of Anopheles was obtained for an average period of 1-7 (mean 3.83 +/- 0.98) days. There was no improvement in the efficacy of the formulation against anophelines even at the higher application rate (240 l/ha). Thus, Spicbiomoss can be used against Cx. quinquefasciatus in an integrated vector management programme.

Efficacy of a Bacillus sphaericus formulation as influenced by the quality of Culex quinquefasciatus breeding waters.

To understand the physico-chemical factors that influence the efficacy of B. sphaericus formulation in the breeding sites of Culex quinquefasciatus, a study was carried out in Mayiladuturai area of Tamil Nadu (India). The factors studied were hydrogen ion concentration (pH), acidity, alkalinity, chlorides, phosphates, total hardness, sulphates, total solids, dissolved solids, suspended solids, nitrate nitrogen, ammoniacal nitrogen, dissolved oxygen, biological oxygen demand (BOD) and chemical oxygen demand (COD). Efficacy of the formulation was assessed in terms of reduction in larval population in the treated habitats. pH of water in the treated sites was around neutral range (mean +/- SD 7.65 +/- 0.23). Phosphate content was low (2.27 +/- 1.34 ppm) whereas chlorides (326.1 +/- 55.8 ppm) and sulphates (38.9 +/- 23.8 ppm) were high. Total hardness ranged from 206 to 462.5 ppm with a mean of 312.1 +/- 80.5 ppm. The chlorides and sulphates, though present in considerable quantity, did not have any influence on the efficacy of B. sphaericus formulation. However, the proportion of insoluble chlorides and sulphates which contribute to total hardness seemed to influence the formulation adversely.

Optimization of different cultural conditions for m-cresol degradation by free and immobilized bacillus laterosporus cells

Optimization of the cultural conditions that facilitate the degradation of m-cresol by a locally isolated bacterial strain Bacillus laterosporus free and entrapped cells was attempted. Bacterial cells entrapped in 2% Ca-allginate beads were more active than free cells and showed a 20% higher rate of m-cresol degradation. Medium No. 1 was most favorable for m-cresol utilization. Supplementing the medium with yeast extract stimulated degradation especially at concentration 0.2 g/l. Optimum concentration of ammonium salts, phosphate salts and magnesium sulfate were 3 g/l and 1.5 g/l, respectively. Optimum concentration of trace elements was 4 ml/l and its omission reduced the rate of degradation. Initial pH of the medium that gave the highest rate of degradation was pH 7 and incubation temperature of 35C. Addition of some amino acids to the mineral medium did not improve degradation rate. The best alginate concentration was 2% and the optimum quantity of beads was 20 ml/ 100 ml medium. The rate of degradation increased by increasing the m- cresol concentration up to 500 mg/l, while higher concentrations [1 g/l] decreased the degradation ability of the entrapped cells

Degradation of meta-cresol in waste waters by entrapped bacillus laterosporus cells

Degradation of m-cresol in different waste water samples instead of the mineral medium was carried out. Meta-cresol degradation in nonsterile waste water was slower than in sterile. However, the entrapped Bacillus laterosporus cells proved to actively degrade m- cresol in all the tested water samples except in the case of two samples collected from El-Max region near Alexandria Harbor. The growth and rate of degradation in both free and entrapped cell cultures were affected by Pb and Hg salts at concentrations higher than 10 mg/l. The rate of degradation was reduced by 75% when 100 mg/l and salts of these metals were present in the medium. Also, the rate of degradation was reduced in presence of 5 mg/l of zinc or cadmium salts. It was noticed that entrapped cells did not show higher resistance than the free cells in presence of the tested heavy metals. The presence of 4-chlorophenol, 4-bromophenol or p- nitrophenol in the medium delayed the degradation time and the rate of degradation was much lower in presence of these substituted phenols together with m-cresol. However, the degradation activity was not completely lost

A modified bioassay for microbial mosquito larvicides.

The proposed bioassay-the grid assay-eliminates the influence of cannibalism while allowing recycling to take place. The LC50 values of the grid assay were intermediate to those of the group and the individual assay. The grid assay thus reflects the true toxicity values of the microbial pesticides being tested. Viable count studies of the microbial pesticides employed, viz., Bacillus thuringiensis H-14 and Bacillus sphaericus 2362, performed along with the assay support the results of the bioassay.

Antagonistic activities of mosquito larvicidal Bacillus sphaericus (Neide) against some pathogenic bacteria.

Mosquitocidal strains of B. sphaericus serotype H-5a5b were shown for the first time to exhibit antagonistic activities against several human pathogens especially Salmonella. These strains of B. sphaericus also exhibited high larval toxicity against several mosquitoes.

Bioassays of Bacillus sphaericus (strain 1593) against mosquitoes of public health importance in Malaysia.

The pathogenicity of Bacillus sphaericus strain 1593 was tested against laboratory-reared larvae of four local species of mosquitoes of public health importance in Malaysia; Aedes aegypti, Anopheles balabacensis, Mansonia uniformis and Culex quinquefasciatus. The bacteria was shake-cultured at 28 +/- 1 degrees C for three days, using Glucose-Yeast Extract Salts medium. After which, the spores and vegetative cells were harvested and stored at 4 degrees C before use. Conditions for bioassays were mean temperature of 25 +/- 1 degrees C and relative humidity 65 +/- 5.0. Twenty third-instar larvae of each species were assayed in 90 ml of diluted spore solution. Each concentration and a control were replicated three times for each bioassay. Larval mortalities at 24 hours and 48 hours were taken and analyzed through Probit Analysis using a computer (IBM 370). LC50 values after 48 hours of exposure showed an increasing order of larval susceptibility as follows: Ae. aegypti (417.70 x 10(4)), An. balabacensis (45.84 x 10(4)), Ma. uniformis (18.23 x 10(4)) and Cx. quinquefasciatus (4.14 x 10(4) spores/ml). With the ability to kill 90% of the Cx. quinquefasciatus larvae tested with just a concentration of 10(5) spores/ml, B. sphaericus (strain 1593) has shown good potential as a biocontrol agent for this species of mosquito.

Taxonomía numérica del género Bacilus / Numerical taxonomy of the genus Bacillus

Se estudiaron por taxonomía numérica 70 cepas del género Bacillus de diferentes orígenes. Se emplearon 81 caracteres morfológicos y fisiológicos. se aplicaron un método de codificación y cuatro de agrupamiento (método de los pares no pesados usando promedios aritméticos, método de los apres pesados usando promedios aritméticos, método de ligamiento simple y método de ligamiento completo) obteniéndose cuatro dendrogramas. Utilziando los datos del primer método de agrupamiento, que presenta el coeficiente de correlación cofenética más alto (0,718), se clasifican las cepas en 11 grupos 14 subgrupos, estableciéndose como grupo al conjunto de cepas que se unen con niveles de similtud mayores de 0,700 y como subgrupo a las que se unen con niveles mayores de 0,800. Se observó un comportamiento muy similar al analizar el dendrograma obtenido utilizando como método de agrupamiento el de los pares pesados usando promedios aritméticos. La variabilidad morfológica y fisiológica de los miembros de este género también se manifiesta en la clasificación por taxonomia numérica