Expanding the Use of a Fluorogenic Method to Determine Activity and Mode of Action ofBacillus thuringiensisBacteriocins Against Gram-Positive and Gram-Negative Bacteria

Abstract

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 × 108 cell/mL and ~7 × 108 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.

Antimicrobial activity of broccoli (Brassica oleracea var. italica ) cultivar Avenger against pathogenic bacteria, phytopathogenic filamentous fungi and yeast

“…aeruginosa ATCC 27853) maintained in our bacterial culture collection (de la Fuente‐Salcido et al . ). Inhibitory effects were also tested against fungi ( Colletotrichum gloeosporioides , Aspergillus niger ) (de la Fuente‐Salcido et al .…”

“…Supernatant was collected and heated for 3 min at 65°C, centrifuged at 51 200 g for 30 min and passed through a filter of pore size 0Á45 lm (Millipore, Bedford, MA). Filter-sterilized supernatants (hereafter called as crude extracts) were used to evaluate the inhibitory activity against bacteria and filamentous fungi and yeasts (Barboza-Corona et al 2007;de Beer and Vivier 2011;de la Fuente-Salcido et al 2016). Protein concentration in crude extracts was determined using the Quick Start Bradford 1x Dye reagent (BioRad, Hercules, CA).…”

Comparative study of composition, antioxidant and antimicrobial activity of two adult edible insects from Tenebrionidae family

“…Samples were assayed by the well diffusion method according to de la Fuente Salcido et al [26,27] with some modifications. Briefly: twenty-five mililiters of freshly prepared sterile Tryptic Soy Agar (cooled to 40-50 °C) was poured into sterile Petri dishes containing 115 µL (1 × 10 9 cells/mL) of the following bacteria: Micrococcus luteus, Bacillus aerius, Samonella spp., Bacillus wiedmannii, Streptococcus canis, Enterococcus casseliflavus, Bacillus cereus strain JCM 2152, Bacillus cereus strain 183, Listeria monocytogenes and Listeria innocua and Vibrio parahaemolyticus.…”