Photodynamic inactivation of Leishmania braziliensis doubly sensitized with uroporphyrin and diamino-phthalocyanine activates effector functions of macrophages in vitro.

Photodynamic inactivation of Leishmania has been shown to render them non-viable, but retain their immunological activities. Installation of dual photodynamic mechanisms ensures complete inactivation of species in the Leishmania subgenus, raising the prospect of their safe and effective application as whole-cell vaccines against leishmaniasis. Here, we report the successful extension of this approach to L. braziliensis in the Viannia subgenus, viz. genetic engineering of promastigotes for cytosolic accumulation of UV-sensitive uroporphyrin (URO) and their loading with red light excitable phthalocyanines (PC) that was cationized by chemical engineering. The transgenic strategy used previously produced L. braziliensis transfectants, which gave the same phenotype of aminolevulinate (ALA)-inducible uroporphyria as found in Leishmania subgenus, indicative of pre-subgenus evolutionary origin for similar genetic deficiencies in porphyrin/heme biosynthesis. In the present study, 12 independent clones were obtained and were invariably ALA-responsive, albeit to different extent for uroporphyrinogenesis and UV-inactivation. In a separate study, L. braziliensis was also found, like other Leishmania spp., to take up diamino-PC (PC2) for red light inactivation. In vitro interactions of a highly uroporphyrinogenic clone with primary macrophages were examined with the intervention of URO/PC2-medated double-photodynamic inactivation to ascertain its complete loss of viability. Doubly sensitized L. braziliensis transfectants were photo-inactivated before (Strategy #1) or after (Strategy #2) loading of macrophages. In both cases, macrophages were found to take up L. braziliensis and degrade them rapidly in contrast to live Leishmania infection. The effector functions of macrophages became upregulated following their loading with L. braziliensis photodynamically inactivated by both strategies, including CD86 expression, and IL6 and NO production. This was in contrast to the immunosuppressive infection of macrophages with live parasites, marked by IL10 production. The results provide evidence that photodynamically inactivated L. braziliensis are susceptible to the degradative pathway of macrophages with upregulation of immunity relevant cytokine and co-stimulatory markers. The relative merits of the two loading strategies with reference to previous experimental vaccination were discussed in light of the present findings with L. braziliensis.

Agave: a natural renewable resource with multiple applications.

Agaves are a group of succulent plants that thrive in arid or semiarid environments. Indeed, genes associated with their resilience are a potential resource for genetic engineering of other agronomically important crops grown in adverse climates. Agave is mainly used for the production of distilled (spirits) and non-distilled alcoholic beverages, including tequila, mezcal, bacanora, raicilla, and pulque, all of which have special connections to Mexican history and culture, and contribute to the Mexican economy. In recent years, there has been growing interest to maximize the use of agave plant materials for other purposes, as the bulk of their biomass pre- and post-production is wasted. In traditional practice, during the passage from fields to factories, only agave cores are used, and the leaves and bagasse are not always harnessed. To place this in perspective, during the period from 2010 to 2019, 2674.7 × 106 L of tequila was produced in Mexico, which required 9 607 400 tons of agave cores. This generated approximately the same amount of leaves and 3 842 960 tons of bagasse. The economic base of agave plants can be expanded if expended biomass could be transformed into products that are useful for applications in food, forage, ensilage, agriculture, medicine, energy, environment, textiles, cosmetics, and esthetics. This review focuses on the current utility of agave plants, as well as our perspective for future studies and uses of this formidable plant. © 2020 Society of Chemical Industry.

Class I defensins (BraDef) from broccoli (Brassica oleracea var. italica) seeds and their antimicrobial activity.

The objective of this study was to determine whether seeds of Brassica oleracea var. italica (i.e. broccoli, an edible plant) produce defensins that inhibit phytopathogenic fungi and pathogenic bacteria of clinical significance. Crude extracts obtained from broccoli seeds were fractioned by molecular exclusion techniques and analyzed by liquid chromatography-high-resolution mass spectrometry. Two peptides were identified, BraDef1 (10.68 kDa) and BraDef2 (9.9 kDa), which were categorized as Class I defensins based on (a) their primary structure, (b) the presence of four putative cysteine disulfide bridges, and (c) molecular modeling predictions. BraDef1 and BraDef2 show identities of, respectively, 98 and 71%, and 67 and 85%, with defensins from Brassica napus and Arabidopsis thaliana. BraDef (BraDef1 + BraDef2) disrupted membranes of Colletotrichum gloeosporioides and Alternaria alternata and also reduced hyphal growth of C. gloeosporioides by ~ 56% after 120 h of incubation. Pathogenic bacteria (Bacillus cereus 183, Listeria monocytogenes, Salmonella typhimurium, Pseudomonas aeruginosa, and Vibrio parahaemolitycus) were susceptible to BraDef, but probiotic bacteria such as Bifidobacterium animalis, Lactobacillus acidophilus, and Lactobacillus casei were not inhibited. To our knowledge, this is the first report of defensins present in seeds of B. oleracea var. italica (i.e. edible broccoli). Our findings suggest an applied value for BraDef1/BraDef2 in controlling phytopathogenic fungi and pathogenic bacteria of clinical significance.

New bacteriocin-like substances produced by Streptomyces species with activity against pathogens.

Streptomyces spp. are Gram-positive bacteria well-known for their ability to produce antibiotics and other metabolites, but few studies on bacteriocins produced by these bacteria have been reported. We tested eight Streptomyces strains against different pathogenic bacteria, and selected S. griseus, S. nigrescens, S. bottroprensis, and S. violaceoruber for further study based on their inhibitory effects against bacteria, including human pathogens. S. bottropensis reached its highest activity at 312 h and was higher than the activities of S. violaceoruber and S. nigrescens. The best condition for bacteriocin precipitation was using diammonium sulfate at 50% saturation. Bacteriocins were susceptible to proteinase treatments and stable at high temperature (up to 100 °C). The highest inhibitory activities were observed between pH 5 and 6. Cross-activity assays indicated that each Streptomyces strain produced different bacteriocins. When preparations of S. griseus and S. nigrescens were subjected to SDS-PAGE, bands of inhibition were observed in the gel overlay assay at a position corresponding to ~ 2 and 3 kDa, respectively, suggesting that both strains are potential sources for novel bacteriocins.

Chitinases of Bacillus thuringiensis: Phylogeny, Modular Structure, and Applied Potentials.

The most important bioinsecticide used worldwide is Bacillus thuringiensis and its hallmark is a rich variety of insecticidal Cry protein, many of which have been genetically engineered for expression in transgenic crops. Over the past 20 years, the discovery of other insecticidal proteins and metabolites synthesized by B. thuringiensis, including chitinases, antimicrobial peptides, vegetative insecticidal proteins (VIP), and siderophores, has expanded the applied value of this bacterium for use as an antibacterial, fungicidal, and nematicidal resource. These properties allow us to view B. thuringiensis not only as an entity for the production of a particular metabolite, but also as a multifaceted microbial factory. In particular, chitinases of B. thuringiensis are secreted enzymes that hydrolyze chitin, an abundant molecule in the biosphere, second only to cellulose. The observation that chitinases increase the insecticidal activity of Cry proteins has stimulated further study of these enzymes produced by B. thuringiensis. Here, we provide a review of a subset of our knowledge of B. thuringiensis chitinases as it relates to their phylogenetic relationships, regulation of expression, biotechnological potential for controlling entomopathogens, fungi, and nematodes, and their use in generating chitin-derived oligosaccharides (ChOGs) that possess antibacterial activities against a number of clinically significant bacterial pathogens. Recent advances in the structural organization of these enzymes are also discussed, as are our perspective for future studies.

Expression of ChiA74∆sp and its truncated versions in Bacillus thuringiensis HD1 using a vegetative promoter maintains the integrity and toxicity of native Cry1A toxins.

Our objective was to determine whether a recombinant chitinase ChiA74∆sp of Bacillus thuringiensis and its truncated versions (ChiA74∆sp-60, ChiA74∆sp-50) could be produced in B. thuringiensis HD1 with no detrimental effect on the size and insecticidal activity of the native bipyramidal Cry crystal. chiA-p, the promoter used to drive chitinase gene expression, was active during vegetative growth of Cry-B. HD1 recombinants showed increases from ~7- to 12-fold in chitinase activity when compared with parental HD1 and negligible or no effect on the volume of bipyramidal crystals was observed. HD1/ChiA74∆sp-60 showed increases from 20% to 40% in the yield of Cry1A per unit of culture medium when compared with parental HD1 and HD1/ChiA74∆sp-50, HD1/ChiA74∆sp. Inclusion bodies presumably composed of the enzyme attached to native Cry1A crystals of recombinant strains were observed; these inclusions were likely responsible for the enhancements in chitinase activity. Western blot analysis using polyclonal anti-ChiA74∆sp showed a weak signal with proteins of ~50 kDa in sporulated and lysed cells of recombinant strains. Bioassays against Spodoptera frugiperda using sporulated/lysed samples of the recombinant strains did not show statistically significant differences in LC50s when compared with HD1.

Co-synthesis of kenyacin 404 and heterologous thurincin H enhances the antibacterial activity of Bacillus thuringiensis.

OBJECTIVES: To develop a recombinant strain of Bacillus thuringiensis that synthesizes two bacteriocins that enhance the antibacterial potency of the strain and that could have applied clinical and industrial value. RESULTS: We cloned the thurincin H cluster into the pHT3101 vector by assembling two genetic cassettes harboring genes for the synthesis, modification, immunity and transport of thurincin H. This construct was used to transform a thurincin H-sensitive strain of B. thuringiensis that synthesizes the kenyacin 404 to generate the recombinant Btk 404/pThurH which was immune to thurincin H and produces bacteriocins of approximately 3 kDa. A significant increase in the inhibitory activity, respectively, ~ 40 and 300%, was observed when compared with parental Btm 269 and Btk 404. Btk 404/pThurH showed increased activity against ten Gram-positive bacteria, including B. cereus, Listeria monocytogenes and B. pseudomycoides, and the Gram-negative bacterium, Sphingobacterium cabi. However, an antagonistic effect against Vibrio parahaemolyticus, relative to native strains, was observed. CONCLUSIONS: We have generated a recombinant strain of B. thuringiensis that co-synthesizes two bacteriocins (kenyacin 404, thurincin H) with improved inhibitory activity, when compared with parental strains. To our knowledge, this is the first study that shows that B. thuringiensis could be manipulated to produce two bacteriocins, one being of heterologous origin, that enhance the antibacterial activity of the recombinant strain.

Regulator ThnR and the ThnDE ABC transporter proteins confer autoimmunity to thurincin H in Bacillus thuringiensis.

The structural gene that encodes thurincin H, a bacteriocin produced by Bacillus thuringiensis, is harboured in a genetic cluster (thnP, E, D, R, A1, A2, A3, B, T, I) that controls its synthesis, modification, secretion and autoimmunity. The specific genes in the cassette that confer immunity in B. thuringiensis to thurincin H are unknown. To identify these immunity determinants, we generated constructs that were used to transform a natural thurincin H-sensitive B. thuringiensis strain (i.e. Btk 404), and resistance or susceptibility to the bacteriocin in resultant recombinants was evaluated. When Btk 404/pHT3101-ThnARDEP and Btk 404/pHT3101-ThnABTI were exposed to thurincin H, immunity was demonstrated by the former only, indicating that ThnI does not play a role in resistance to the bacteriocin as previously proposed. Furthermore, we generated different sub-cassettes under the control of divergent promoters pThnR and pThur of the thurincin H locus, and pChi, and using the green fluorescent protein gene as reporter, which demonstrated that all promoters were recognised by ThnR, except pChi. We show for the first time that the small operon composed of thnR, thnD and thnE is required for immunity of B. thuringiensis to thurincin H, and thnI is not necessary for this response.

Photodynamic Vaccination of BALB/c Mice for Prophylaxis of Cutaneous Leishmaniasis Caused by Leishmania amazonensis.

Background: Photosensitizers (PS), like porphyrins and phthalocyanines (PC) are excitable by light to generate cytotoxic singlet oxygen and other reactive oxygen species in the presence of atmospheric O2. Photodynamic inactivation of Leishmania by this means renders them non-viable, but preserves their effective use as vaccines. Leishmania can be photo-inactivated after PS-sensitization by loading via their endocytic uptake of PC or endogenous induction of transgenic mutants with delta-aminolevulinate (ALA) to accumulate cytosolic uroporphyrin I (URO). Here, PS-sensitization and photo-inactivation of Leishmaniaamazonensis was further examined in vitro and in vivo for vaccination against cutaneous leishmaniasis (CL). Methods and Results:Leishmania amazonensis promastigotes were photodynamically inactivated in vitro by PC-loading followed by exposure to red light (1-2 J/cm2) or ALA-induction of uroporphyrinogenic transfectants to accumulate cytosolic URO followed by longwave UV exposure. When applied individually, both strategies of photodynamic inactivation were found to significantly, albeit incompletely abolish the MTT reduction activities of the promastigotes, their uptake by mouse bone marrow-derived macrophages in vitro and their infectivity to mouse ear dermis in vivo. Inactivation of Leishmania to completion by using a combination of both strategies was thus used for the sake of safety as whole-cell vaccines for immunization of BALB/c mice. Different cutaneous sites were assessed for the efficacy of such photodynamic vaccination in vivo. Each site was inoculated first with in vitro doubly PS-sensitized promastigotes and then spot-illuminated with white light (50 J/cm2) for their photo-inactivation in situ. Only in ear dermis parasites were photo-inactivated beyond detection. Mice were thus immunized once in the ear and challenged 3 weeks later at the tail base with virulent L. amazonensis. Prophylaxis was noted in mice photodynamically vaccinated with doubly photo-inactivated parasites, as indicated by a significant delay in the onset of lesion development and a substantial decrease in the parasite loads. Conclusion: Leishmania doubly PS-sensitized and in situ photo-inactivated as described proved to be safe and effective when used for one-time immunization of ear dermis, as indicated by its significant protection of the inherently very susceptible BALB/c mice against CL.

Recombinant Bacillus thuringiensis subsp. kurstaki HD73 strain that synthesizes Cry1Ac and chimeric ChiA74∆sp chitinase inclusions.

In this study, the endochitinase chiA74 gene lacking its secretion signal peptide sequence (chiA74∆sp) was fused in frame with the sequence coding for the C-terminal crystallization domain and transcription terminator of cry1Ac. The chimeric gene was expressed under the strong pcytA-p/STAB-SD promoter system in an acrystalliferous Cry-B strain of Bacillus thuringiensis and B. thuringiensis subsp. kurstaki HD73. We showed that the chimeric ChiA74∆sp produced amorphous inclusions in both Cry-B and HD73. In addition to the amorphous inclusions putatively composed of the chimera, bipyramidal Cry1Ac crystals, smaller than the wild-type crystal, were observed in recombinant HD73, and chitinase activity was remarkably higher (75-fold) in this strain when compared with parental HD73. Moreover, we observed that lyophilized samples of a mixture containing Cry1Ac, amorphous inclusions, and spores maintained chitinase activity. Amorphous inclusions could not be separated from Cry1Ac crystals by sucrose gradient centrifugation. Interestingly, the chitinase activity of purified Cry1Ac/amorphous inclusions was 51-fold higher compared to purified Cry1Ac inclusions of parental HD73, indicating that the increased enzymatic activity was due primarily to the presence of the atypical amorphous component. The possibility that the chimera is occluded with the Cry1Ac crystal, thereby contributing to the increased endochitinolytic activity, cannot be excluded. Finally, bioassays against larvae of Spodoptera frugiperda with spore/crystals of HD73 or spore-crystal ChiA74∆sp chimeric inclusions of recombinant HD73 strain showed LC50s of 396.86 and 290.25 ng/cm2, respectively. Our study suggests a possible practical application of the chimera in formulations of B. thuringiensis-based lepidopteran larvicides.

The thnR gene is a negative transcription regulator of the thurincin H genetic cassette in Bacillus thuringiensis subsp. morrisoni.

Thurincin H is a bacteriocin synthesized by some strains of Bacillus thuringiensis. In this study, the thurincin H genetic cassette, which contains ten genes, from a Mexican strain of B. thuringiensis subsp. morrisoni (Btm) was cloned and sequenced. To study the function of the thnR gene component in the cassette, we generated various constructs with or without thnR for expression in Btm. All transformants harboring thnR in recombinant plasmids showed a decrease of ~15 to ~90 % in inhibitory activity against the target strain, Bacillus cereus 183. Importantly, a ~90 % reduction in inhibition occurred with Btm harboring a construct containing thnR alone, suggesting that ThnR, indeed, functions as a negative transcription regulator of the thurincin H cassette. Based on sequence homology, ThnR was classified as a member of the YtrA subfamily of the GntR superfamily of transcriptional regulators.

Bacteriocinogenic Bacteria Isolated from Raw Goat Milk and Goat Cheese Produced in the Center of México.

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.

Isolation and characterization of bacteriocinogenic lactic bacteria from M-Tuba and Tepache, two traditional fermented beverages in México.

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 ∼3.5 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.

Heterologous expression, purification and biochemical characterization of endochitinase ChiA74 from Bacillus thuringiensis.

ChiA74 is a secreted endochitinase produced by Bacillus thuringiensis. Previously we have partially characterized the physical parameters that affect enzymatic activity of ChiA74 in crude preparations of bacterial secretomes. In the present study, we cloned the chiA74 open reading frame (ORF) lacking the 5' sequence coding for its secretion signal peptide (chiA74Δsp) into a cold shock expression vector (pColdI) for production of the enzyme in Escherichia coli BL21-Rosetta 2. As a result, the N-terminal end of ChiA74Δsp ORF was fused to an artificial sequence of 28 amino acid, including a 6× histidine tag for purification of recombinant 6×His tagged-ChiA74Δsp (rChiA74, ∼74kDa). Along with a protein of ∼74kDa, we co-purified its ∼55kDa processed form which was confirmed by Western blot analysis. Optimal endochitinase activity of purified rChiA74 occurred at pH 7 and 40°C. Most divalent cations (e.g. Ba(+2), Ca(+2), Mn(+2), Mg(+2), Zn(+2) and Cu(+2)) at concentration of 10mM reduced chitinase activity by ∼30%, and Hg(+2) (10mM) drastically inhibited ChiA74 activity by ∼75-100%. The Vmax, Km and kcat for rChiA74 were 0.11±0.01nmol/min, 2.15µM±0.45 and 3.81s(-1), respectively, using 4-MU-GlcNAc3 as substrate. Using purified rChiA74 and colloidal chitin as substrate, chitin-derived oligosaccharides with degree of polymerization of 2 and 1 were detected.

Asthma in the elderly: what we know and what we have yet to know.

In the past, asthma was considered mainly as a childhood disease. However, asthma is an important cause of morbidity and mortality in the elderly nowadays. In addition, the burden of asthma is more significant in the elderly than in their younger counterparts, particularly with regard to mortality, hospitalization, medical costs or health-related quality of life. Nevertheless, asthma in the elderly is still been underdiagnosed and undertreated. Therefore, it is an imperative task to recognize our current challenges and to set future directions. This project aims to review the current literature and identify unmet needs in the fields of research and practice for asthma in the elderly. This will enable us to find new research directions, propose new therapeutic strategies, and ultimately improve outcomes for elderly people with asthma. There are data to suggest that asthma in older adults is phenotypically different from young patients, with potential impact on the diagnosis, assessment and management in this population. The diagnosis of AIE in older populations relies on the same clinical findings and diagnostic tests used in younger populations, but the interpretation of the clinical data is more difficult. The challenge today is to encourage new research in AIE but to use the existing knowledge we have to make the diagnosis of AIE, educate the patient, develop a therapeutic approach to control the disease, and ultimately provide a better quality of life to our elderly patients.

Loss of expression and function of SOCS3 is an early event in HNSCC: altered subcellular localization as a possible mechanism involved in proliferation, migration and invasion.

BACKGROUND: Suppressor of cytokine signaling 3 (SOCS3) is an inducible endogenous negative regulator of signal transduction and activator of transcription 3 (STAT3). Epigenetic silencing of SOCS3 has been shown in head and neck squamous cell carcinoma (HNSCC), which is associated with increased activation of STAT3. There is scarce information on the functional role of the reduction of SOCS3 expression and no information on altered subcellular localization of SOCS3 in HNSCC. METHODOLOGY/PRINCIPAL FINDINGS: We assessed endogenous SOCS3 expression in different HNSCC cell lines by RT-qPCR and western blot. Immunofluorescence and western blot were used to study the subcellular localization of endogenous SOCS3 induced by IL-6. Overexpression of SOCS3 by CMV-driven plasmids and siRNA-mediated inhibition of endogenous SOCS3 were used to verify the role of SOCS3 on tumor cell proliferation, viability, invasion and migration in vitro. In vivo relevance of SOCS3 expression in HNSCC was studied by quantitative immunohistochemistry of commercially-available tissue microarrays. Endogenous expression of SOCS3 was heterogeneous in four HNSCC cell lines and surprisingly preserved in most of these cell lines. Subcellular localization of endogenous SOCS3 in the HNSCC cell lines was predominantly nuclear as opposed to cytoplasmic in non-neoplasic epithelial cells. Overexpression of SOCS3 produced a relative increase of the protein in the cytoplasmic compartment and significantly inhibited proliferation, migration and invasion, whereas inhibition of endogenous nuclear SOCS3 did not affect these events. Analysis of tissue microarrays indicated that loss of SOCS3 is an early event in HNSCC and was correlated with tumor size and histological grade of dysplasia, but a considerable proportion of cases presented detectable expression of SOCS3. CONCLUSION: Our data support a role for SOCS3 as a tumor suppressor gene in HNSCC with relevance on proliferation and invasion processes and suggests that abnormal subcellular localization impairs SOCS3 function in HNSCC cells.

Expanding the use of a fluorogenic method to determine activity and mode of action of Bacillus thuringiensis bacteriocins against Gram-positive and Gram-negative bacteria.

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.

Effects of physical culture parameters on bacteriocin production by Mexican strains of Bacillus thuringiensis after cellular induction.

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.