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Fungi are the primary infectious agents in plant crops and many post-harvest fungal diseases of fruit and vegetables causing significant economic losses worldwide. Here, the antifungal effect of Prosopis glandulosa extract (PgE) against phytopathogenic fungi was evaluated. The effect with PgE (5, 4, 2, 1, 0%) as AI (%) and radial growth rate reduction (Kr %) were determined in vitro in Colletotrichum gloeosporoides, Fusarium oxysporum, Rhizopus oryzae and R. stolonifer (1 × 105 spores/mL). The phytopathogenicity of fungal strains was performed under in vivo conditions (room temperature, 25-30 °C and refrigeration, ~ 4 °C) by fruit surface inoculation method on strawberries, tomatoes and carrots by recording the development of mycelial growth, necrosis, soft rot and dehydration symptoms showed on each fruit at 14 days. The extract (5%) showed the highest AI against C. gloesporioides (~ 96%), and F. oxysporum (~ 79%) and growth rate reduction of 74.92% and 64.82% respectively. Likewise, the extract controls the development of phytopathogenicity symptoms against C. gloesporioides and F. oxysporum in vivo conditions, nevertheless, was less efficiency against both Rhizopus species. The P. glandulosa extract represents an efficient, economical, and eco-friendly alternative to preserve the quality of the agricultural products and to increase their shelf life.
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Nowadays, consumers are demanding nutrient-rich products for health optimal benefits. In this regard, Brassicaceae family plants, previously named cruciferous, group a large number of widely consumed species around the world. The popularity of Brassica is increasing due to their nutritional value and pharmacological effects. The group includes a large number of vegetable foods such as cabbages, broccoli, cauliflower, mustards as well as, oilseed rapeseed, canola, among others. In recent years, the phytochemical composition of Brassicaceae has been studied deeply because they contain many valuable metabolites, which are directly linked to different recognized biological activities. The scientific evidence confirms diverse medical properties for the treatment of chronic diseases such as obesity, type-2 diabetes, cardiovascular diseases (hypertension, stroke), cancer, and osteoporosis. The unique features of Brassicaceae family plants conferred by their phytochemicals, have extended future prospects about their use for beneficial effects on human nutrition and health worldwide. PRACTICAL APPLICATIONS: For years, the Brassicaceae plants have been a fascinating research topic, due to their chemical composition characterized by rich in bioactive compounds. The implementation of extracts of these vegetables, causes various beneficial effects of high biological value in the treatment of diseases, owing to their bioactive properties (anti-obesity, anticancer, antimicrobial, antioxidant, hepatoprotective, cardioprotective, gastroprotective, anti-inflammatory, antianemic, and immunomodulator). Therefore, this review summarizes the chemical composition, describes the bioactive compounds isolated in the plant extracts, and highlights diverse biological activities, mainly the antimicrobial and antioxidant capacity. Brassica plants, as source of natural bioactive agents, have a great potential application to improve the human nutrition and health.
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Currently, there are few reports on the isolation of microorganisms from goat milk and goat cheese that have antibacterial activity. In particular, there are no reports on the isolation of microorganisms with antibacterial activity from these products in central Mexico. Our objective was to isolate bacteria, from goat products, that synthesized antimicrobial peptides with activity against a variety of clinically significant bacteria. We isolated and identified Lactobacillus rhamnosus, L. plantarum, L. pentosus, L. helveticus and Enterococcus faecium from goat cheese, and Aquabacterium fontiphilum, Methylibium petroleiphilum, Piscinobacter aquaticus and Staphylococcus xylosus from goat milk. These bacteria isolated from goat cheese were able to inhibit Staphylococcus aureus, Bacillus cereus, Escherichia coli, Listeria monocytogenes, L. inoccua, Pseudomona aeruginosa, Shigella flexneri, Serratia marcescens, Enterobacter cloacae and Klebsiella pneumoniae. In addition, bacteria from goat milk showed inhibitory activity against B. cereus, L. lactis, E. coli, S. flexneri, E. cloacae and K. pneumonia; S. aureus, L. innocua, S. agalactiae and S. marcescens. The bacteriocins produced by these isolates were shown to be acid stable (pH 2-6) and thermotolerant (up to 100 °C), but were susceptible to proteinases. When screened by PCR for the presence of nisin, pediocin and enterocin A genes, none was found in isolates recovered from goat milk, and only the enterocin A gene was found in isolates from goat cheese.
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Bacillus thuringiensis subsp. tenebrionis DSM-2803 has been studied extensively and spore/crystal mixtures of this strain are used widely in commercial products to control coleopteran pests. The endochitinase chiA Btt gene of B. thuringiensis subsp. tenebrionis DSM-2803 was cloned and expressed in Escherichia coli. The recombinant 6x-histidine tagged protein (rChiA Btt, ~74 kDa), was purified by a HiTrap Ni affinity column. The Km of rChiA Btt was 0.847 µmol L-1 and its optimal activity occurred at pH 7 and ~40°C. Most divalent cations reduced endochitinase activity but only Hg+2 abolished activity of the enzyme. We report for the first time the characterization of a chitinase synthesized by B. thuringiensis subsp. tenebrionis DSM-2803, and show that the purified rChiA74 Btt reduced the radial growth and increased the hyphal density of Colletotrichium gloeosporioides, the etiological agent of "anthracnose" in plants.
Asunto(s)
Antifúngicos/farmacología , Bacillus thuringiensis/enzimología , Quitinasas/genética , Colletotrichum/efectos de los fármacos , Enfermedades de las Plantas/microbiología , Proteínas Recombinantes/farmacología , Antifúngicos/metabolismo , Agentes de Control Biológico , Quitinasas/metabolismo , Quitinasas/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Mexican Tuba (M-Tuba) and Tepache are Mexican fermented beverages prepared mainly with pineapple pulp and coconut palm, respectively. At present, reports on the microbiota and nutritional effects of both beverages are lacking. The purpose of this study was to determine whether M-Tuba and Tepache contain cultivable lactic acid bacteria (LAB) capable of producing bacteriocins. Tepache and M-Tuba contain mesophilic aerobic bacteria, LAB, and yeast. Bacillus subtilis, Listeria monocytogenes, Listeria innocua, Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, and Salmonella spp, were the microorganisms most susceptible to metabolites produced by bacterial isolates. M-Tuba and Tepache contain bacteria that harbor genes coding for nisin and enterocin, but not pediocin. The presence of Lactococcus lactis and E. faecium in M-Tuba and Tepache, was identified by 16S rDNA. These bacteria produced bacteriocins of â¼3.5 kDa and 4.0-4.5 kDa, respectively. Partial purified bacteriocins showed inhibitory effect against Micrococcus luteus, L. monocytogenes, L. innocua, Str. agalactiae, S. aureus, Bacillus cereus, B. subtilis, E. faecalis, and K. pneumoniae. We characterized, for the first time, cultivable microbiota of M-Tuba and Tepache, and specifically, identified candidate lactic bacteria (LAB) present in these beverages that were capable of synthesizing antimicrobial peptides, which collectively could provide food preservative functions.
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Bacteriocins synthesized by entomopathogenic Bacillus thuringiensis are gaining attention owing to their inhibitory effects against a wide variety of pathogenic bacteria. In the present study, we purified and characterized Tolworthcin 524, a bacteriocin synthesized by B. thuringiensis subsp. tolworthi, and compared it with other bacteriocins synthesized by B. thuringiensis. Tolworthcin 524 was separated and purified from the secretome of B. thuringiensis by fast protein liquid chromatography with a gel filtration column to obtain yields of 17% and a specific activity of â¼3600U/mgprotein. The purified product showed two peptides of â¼9 and 6kDa with antimicrobial activity in a gel-screening assay. The purified product was analyzed by two-dimensional electrophoresis and the resolved peptides of â¼9 and 6kDa with isoelectric points of â¼8 were sequenced. Partial sequences (METPVVQPR and DWTCWSCLVCAACS) were obtained suggesting that the â¼9 and 6kDa correspond to the prebacteriocin and mature Tolworthcin 524, respectively. Sequences showed high identity with Thurincin H and Thuricin 17 and had a conserved motif with other bacteriocins of B. thuringiensis. Based on sequence data, Tolworthcin 524 was classified in subclass II.2 (Thuricin-like peptides) of the Bacillus bacteriocin classification scheme. The larger peptide did not harbor a sequence suggestive of a signal peptide neither did it contain the double-glycine (GG) motif characteristic of the secretion leader recognized by the ABC transport system. Implications of these properties in Tolworthcin 524 secretion are discussed.
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Bacillus thuringiensis/metabolismo , Bacteriocinas/química , Bacteriocinas/clasificación , Secuencia de Aminoácidos , Bacillus thuringiensis/química , Bacteriocinas/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Electroforesis en Gel Bidimensional , Datos de Secuencia Molecular , Péptidos/químicaRESUMEN
Various strains of Bacillus thuringiensis are among the most successful entomopathogenic bacteria used commercially as biopesticides owing to their ability to synthesize insecticidal crystal (Cry) and cytolytic (Cyt) protein toxins during sporulation, and vegetative insecticidal (VIPs) proteins during the vegetative phase of growth. Whereas much is known about the molecular biology of Cry, Cyt, and VIPs, comparatively little is known about other proteins and metabolites synthesized by B. thuringiensis that could also have applied value. Here, we review recent reports on bacteriocins synthesized by this bacterium as they relate to antibacterial activity, molecular genetics, biophysical and biochemical properties, and methods used to separate and purify these antimicrobial peptides. We highlight the potential of bacteriocins for use as food preservatives, antibiotics, plant protection, and plant growth promoters. We suggest that B. thuringiensis could be used not only in biological control of insects but also in other agronomical and industrial areas of public interest.
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Bacillus thuringiensis/fisiología , Bacteriocinas/metabolismo , Animales , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Bacteriocinas/biosíntesis , Bacteriocinas/genética , Bacteriocinas/aislamiento & purificación , Agentes de Control Biológico , Humanos , InsecticidasRESUMEN
In the present study, we expressed the chiA74 gene of Bacillus thuringiensis in Escherichia coli K12 and demonstrated that the active ChiA74 enzyme was produced at a high level in this strain. The ChiA74 enzymatic activity (in units per milliliter) was approximately 500 % greater in E. coli K12 when compared to that produced in E. coli DH5α. Moreover, we showed that, when using our protocol, ChiA74 preparations obtained from recombinant E. coli K12 did not contain live bacteria, although transformable DNA (erm, bla genes) was detected. Nucleic acids were subsequently easily eliminated when samples were treated with magnesium. Importantly, ChiA74 was secreted by E. coli K12 and the active enzyme was shown to generate chitin-derived oligosaccharides (C-OGS) with degrees of polymerization of 2, 3, 4, 5, and 6. From an applied perspective, the C-OGS showed activity against various pathogenic bacteria. In addition, we demonstrated that ChiA74 was not toxic to Hek 293 and 3T3 L1 cells, i.e., the enzyme did not induce apoptosis or affect normal cellular cycle and also did not produce abnormal changes in cell morphology. The potential biotechnological use of producing endochitinase of B. thuringiensis in a microorganism recognized as safe (i.e., E. coli K12) is discussed.
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Bacillus thuringiensis/enzimología , Biotecnología/métodos , Quitinasas/aislamiento & purificación , Quitinasas/metabolismo , Animales , Bacillus thuringiensis/genética , Línea Celular , Supervivencia Celular/efectos de los fármacos , Quitinasas/genética , Escherichia coli K12/enzimología , Escherichia coli K12/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Expresión Génica , Humanos , Ratones , Oligosacáridos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Previously we described a rapid fluorogenic method to measure the activity of five bacteriocins produced by Mexican strains of Bacillus thuringiensis against B. cereus 183. Here we standardize this method to efficiently determine the activity of bacteriocins against both Gram-positive and Gram-negative bacteria. It was determined that the crucial parameter required to obtain reproducible results was the number of cells used in the assay, that is, ~4 × 10(8) cell/mL and ~7 × 10(8) cell/mL, respectively, for target Gram-positive and Gram-negative bacteria. Comparative analyses of the fluorogenic and traditional well-diffusion assays showed correlation coefficients of 0.88 to 0.99 and 0.83 to 0.99, respectively, for Gram-positive and Gram-negative bacteria. The fluorogenic method demonstrated that the five bacteriocins of B. thuringiensis have bacteriolytic and bacteriostatic activities against all microorganisms tested, including clinically significant bacteria such as Listeria monocytogenes, Proteus vulgaris, and Shigella flexneri reported previously to be resistant to the antimicrobials as determined using the well-diffusion protocol. These results demonstrate that the fluorogenic assay is a more sensitive, reliable, and rapid method when compared with the well-diffusion method and can easily be adapted in screening protocols for bacteriocin production by other microorganisms.
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Bacillus thuringiensis/química , Bacteriocinas/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacosRESUMEN
We have shown previously that in the presence of inducer Bacillus cereus 183, significant increases in bacteriocin production and bactericidal activity of B. thuringiensis occur when the latter is cultivated at pH 7.2, 28°C, and 180 rpm. Here we show that this activity can be further improved when B. thuringiensis is induced with B. cereus 183 and then cultivated with modification of pH, temperature, and agitation. Five native strains of B. thuringiensis, LBIT 269, LBIT 287, LBIT 404, LBIT 420, and LBIT 524 which synthesize, respectively, morricin 269, kurstacin 287, kenyacin 404, entomocin 420, and tolworthcin 524, were cultivated in four different fermentation media. Of these, fermentation in tryptic soy broth (TSB) yielded the highest level of bacteriocin activity (~100-133 FU). Bacteria grown in TSB were induced with B. cereus 183 and cultivated at different pH (6.0, 7.2, 8.0), temperature (26, 28, 30°C), and agitation (150, 180, 210 rpm). Full factorial design was performed and results were analyzed with analysis of variance (ANOVA) and Tukey multiple comparison tests at significant level of α ≤ 0.05 to study the influence of the three variables on bacterial growth and bacteriocin production. Our data show that the highest bacteriocin activity was found with LBIT 269 and LBIT 404 with an increase of ~95-100% compared with induced B. thuringiensis strains cultivated under fixed conditions (pH 7.2, 28°C, 180 rpm), for which the data were set at 0%. The optimal conditions for morricin 269 and kenyacin 404 production were, respectively, pH 8, 30°C, 210 rpm and pH 7.2, 26°C, 210 rpm.
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Antibacterianos/biosíntesis , Bacillus thuringiensis/metabolismo , Bacteriocinas/biosíntesis , Bacillus cereus/fisiología , Bacillus thuringiensis/crecimiento & desarrollo , Medios de Cultivo , Concentración de Iones de Hidrógeno , México , TemperaturaRESUMEN
In this study, 13% of fresh lettuce (Lactuca sativa) samples collected from markets and supermarkets in two cities of Mexico were contaminated with Salmonella spp. From those samples, amplicons of â¼300 base pairs (bp) were amplified, corresponding to the expected size of the invasion (invA) and internal transcribed spacer regions of the 16S and 23S rRNA genes of Salmonella spp. Additionally, Salmonella strains were isolated and harbored plasmids ranging from â¼9 to 16 kbp. From these strains, 91% were resistant to ampicillin and nitrofurantoin, whereas 55% were resistant to cephalothin and chloramphenicol. No resistance was detected to amikacin, carbenicillin, cefotaxime, gentamicin, netilmicin, norfloxacin, and sulfamethoxazole-trimethoprim. When Salmonella isolates were tested against novel bacteriocins (morricin 269, kurstacin 287, kenyacin 404, entomocin 420, and tolworthcin 524) produced by five Mexican strains of Bacillus thuringiensis, 50% were susceptible to these antimicrobial peptides. This is the first report showing that Salmonella strains isolated from lettuce are susceptible to bacteriocins produced by the most important bioinsecticide worldwide, suggesting the potential use of these antibacterial peptides as therapeutic agents or food preservatives to reduce or destroy populations of Salmonella spp.
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Antibacterianos/farmacología , Bacteriocinas/farmacología , Farmacorresistencia Bacteriana , Lactuca/microbiología , Salmonella/efectos de los fármacos , Bacillus thuringiensis/metabolismo , Bacteriocinas/biosíntesis , Recuento de Colonia Microbiana , Seguridad de Productos para el Consumidor , Relación Dosis-Respuesta a Droga , Contaminación de Alimentos/análisis , Contaminación de Alimentos/prevención & control , Microbiología de Alimentos , Conservantes de Alimentos/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , ARN Bacteriano/análisis , ARN Ribosómico 16S/análisis , ARN Ribosómico 23S/análisis , Salmonella/aislamiento & purificaciónRESUMEN
Bacillus thuringiensis HD-73 was transformed with the endochitinase gene chiA74 under the control of a strong promoter (pcytA) and a 5' mRNA stabilizing (STAB-SD) sequence (HD-73-pEBchiA74). Expression levels were compared with those observed from the wild type strain (HD-73) and the recombinant HD-73 strain expressing chiA74 under the control of its native promoter (HD-73-pEHchiA74). The chitinolytic activity of HD-73-pEBchiA74 was markedly elevated, being ~58- and 362-fold higher than, respectively, HD-73-pEHchiA74 and parental HD-73, representing the highest levels of chitinase expression in recombinant B. thuringiensis reported to date. Parasporal crystals measured under transmission electron microscopy showed that HD-73 produced crystals of 1.235 (+/-0.214) and 1.356 (+/-0.247) mum in length when the bacterium was grown in respectively, NBS and NBS with glucose. Otherwise, HD-73-pEBchiA74 synthesized crystals of 1.250 (+/-0.222) and 1.139 (+/-0.202) mum in length when cultivated in NBS and NBS with glucose, respectively, values that showed a diminution of ~10 and 20% compared with crystals produced by HD-73-pEHchiA74 grown under the same conditions. Comparison of viable spore counts per ml showed that HD-73-pEBchiA74 produced fewest viable spores (1.5 x 10(9), 1.3 x 10(9)), compared to HD-73-pEHchiA74 (4.9 x 10(9), 5.3 x 10(9)) and HD-73 (6.8 x 10(9), 8.8 x 10(9)) when grown in NBS and NBS supplemented with glucose, respectively. No change in cellular protease activity was observed despite the overproduction of the chitinase.
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Bacillus thuringiensis/enzimología , Bacillus thuringiensis/fisiología , Proteínas Bacterianas/biosíntesis , Quitinasas/biosíntesis , Endotoxinas/biosíntesis , Proteínas Hemolisinas/biosíntesis , Esporas Bacterianas/crecimiento & desarrollo , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/ultraestructura , Quitina/metabolismo , Quitinasas/genética , Recuento de Colonia Microbiana , Expresión Génica , Proteínas Hemolisinas/ultraestructura , Microscopía Electrónica de Transmisión , Transformación BacterianaRESUMEN
Antimicrobial therapy is a useful tool to control bovine mastitis caused by Staphylococcus aureus, as consequence an increase in staphylococci resistant cases has been registered. Alternative strategies are desirable and bacteriocins represent attractive control agents to prevent bovine mastitis. The aim of this work was to evaluate the activity of five bacteriocins synthesized by Bacillus thuringiensis against S. aureus isolates associated to bovine mastitis. Fifty S. aureus isolates were recovered from milk composite samples of 26 Holstein lactating cows from one herd during September 2007 to February 2008 in México and susceptibility of those isolates to 12 antibiotics and 5 bacteriocins from B. thuringiensis was evaluated. S. aureus isolates were mainly resistant to penicillin (92%), dicloxacillin (86%), ampicillin (74%) and erythromycin (74%); whereas susceptibility to gentamicin, trimethoprim and tetracycline was detected at, respectively, 92%, 88%, and 72%. All S. aureus isolates showed susceptibility to the five bacteriocins synthesized by B. thuringiensis, mainly to morricin 269 and kurstacin 287 followed by kenyacin 404, entomocin 420 and tolworthcin 524. Our results showed that S. aureus isolates had differences in the antimicrobial resistance patterns and were susceptible to bacteriocins produced by B. thuringiensis, which could be useful as an alternative method to control bovine mastitis.
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Bacillus thuringiensis/fisiología , Bacteriocinas/farmacología , Mastitis Bovina/microbiología , Staphylococcus aureus/efectos de los fármacos , Animales , Antibacterianos/farmacología , Bacteriocinas/biosíntesis , Bacteriocinas/aislamiento & purificación , Bovinos , Femenino , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
We describe a novel bacteriocin screening assay based on fluorescence emitted by berberine following its influx into compromised cells. This technique showed agreement with the conventional well-diffusion method, and results can be obtained within one hour. This assay could facilitate the rapid identification of bacteriocinogenic bacterial isolates.