Bacillus subtilis, growth inhibition

In 1998, two bioassay methods were considered by the Chilean Regulation Institute (INN) as the first attempts for the introduction of microbioassays for routine testing in Chilean regulations (1) the Bacillus subtilis growth inhibition test for toxicity evaluation of industrial effluents discharged into sewers, to detect interference with the BOD, is near endorsement and (2) the assessment of acute toxicity in receiving waters using D. pulex is presently under discussion. 

Fig. 4. a) Inhibition of microbial growth by GA, from Bacillus subtilis. Micrococcus luteus and Pseudomonas aeruginosa, b) Growth curve of Bacillus subtilis at 32 ° C ( ) Milk, (O) milk with addedGA47pM. 

Several methods for performing the test have been described, the guideline describes the diffusion and suspension test. Bacteria should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The response is expressed in the preferential inhibition of growth or the preferential killing of the DNA repair deficient strain. Escherichia coli polA (W3110/p3478) or Bacillus subtilis rec (H17/M45) pairs are recommended. The test should initially be performed over a broad range of concentrations. 

Kalihinols G (277) and H (278) were trace components of a species of Acanthella from Guam and kalihinol X (279) was isolated from a Fijian species of Acanthella. All inhibited growth of Bacillus subtilis, Staphylococcus aureus and Candida albicans [278]. 10-Epi-isokalihinol H (280) and 15-isothiocyanato-l-epi-kalihinene (281) were isolated from Acanthella cavernosa from the Seychelles [279]. A Japanese specimen of A. cavernosa contained a sesquiterpene isothiocyanate (282) and 10 3-formamido-5p-isothiocyanatokalihinol A (283). Structures were assigned by spectral data interpretation [280]. Phakellia pulcherrima from the Philippines contained the minor diterpene isothiocyanates kalihinol L (284), 10-isothiocyanatokalihinol G (285), 10-epi-kalihinol H (286) and 10-isothiocyanatokalihinol C (287) [281]. 10-Epi-kalihinol I (288) and 5,10-bisisothiocyanatokalihinol G (289) were isolated from an Acanthella sp. from Okinawa [282]. 

There have been three reports of the same dimeric disulfide. It was first isolated from an unidentified sponge from Guam and the structure elucidated by analysis of spectral data. The (E,E) stereochemistry of the disulfide (500) was defined by comparing the I3C NMR spectroscopic data with those of the (E,Z)-isomer (501) that was obtained as an unstable minor product [425]. Compound 500 was isolated from a species of Psammaplysilla and was called psammaplin A [426]. It was also isolated from Thorectopsamma xana, collected from the same location in Guam, together with a minor dimeric metabolite bisaprasin (502). Both compounds inhibited growth of Staphylococcus aureus and Bacillus subtilis [427]. Psammaplin A (bisprasin) (500) was later isolated from a Dysidea species of sponge and shown to act on Ca2+-induced Ca2+ release channels of skeletal muscle [428]. 

The Aria test is a routine assay in which a heat-treated milk sample is added to a well of a microtiter plate containing a freeze-dried tablet containing Bacillus subtilis ATCC 6633, nutrients, and triphenyltetrazoliumchloride as redox indicator (33). Following incubation, the normal growth of the organism is inhibited if antibacterials are present, and the uncolored indicator is not reduced into its red form. Detection of sulfonamides requires prior addition of trimethoprim in the milk samples analyzed. 

The CAST uses Bacillus megatherium and a 16-24 h incubation at 44-45 C, whereas STOP employs Bacillus subtilis and a 16-24 h incubation at 27-29 C. A zone of inhibition around the swab suggests the presence of a microbial inhibitor in the sample. In the US, bob veal calf carcasses are condemned on the basis of a positive CAST without further confirmation. For FAST, the organism and temperature are the same as those for the CAST, but the CAST medium is supplemented with dextrose and bromocresol purple. The faster growth rate of bacteria with FAST, allows reduction of minimum incubation from 16 to 6h. 

Besides physicochemical methods, the use of microbiological growth-inhibition assays to test meat and milk for the presence of antibiotics residues is popular over a long period of time. These tests use antibiotic-sensitive bacterial reporter strains, such as Bacillus subtilis and Bacillus stearothermophilus var. calidolactis. These bacteria are inoculated under optimal conditions with and without sample. After culturing, results are read from visible inhibition zones or from the color change of the bacterial suspension in agar gels [6]. 

DNA repair. Polymerase-deficient, and thus DNA repair-deficient, E. coli has provided the basis for a test that depends on the fact that the growth of a deficient strain is inhibited more by a DNA-damaging agent than is that of a repair-competent strain. The recombinant assay using Bacillus subtilis is conducted in much the same way because recombinant deficient strains are more sensitive to DNA-damaging agents. 

Figure 2. Inhibition of growth of Bacillus subtilis by triazolopyrimidine reversal by valine, leucine and isoleucine.

The aqueous/methanol fraction of the dichloromethane extract of Carthamus lanatus L. exhibited a high rate of antibacterial activity against Staphylococcus aureus and Escherichia coli [35], while crude dichloromethane extracts of the aerial parts and roots of Leontopodium alpinum Cass, exhibited significant growth inhibition of Bacillus subtilis, Escherichia coli. Pseudomonas aeruginosa. Staphylococcus aureus and Streptococcus pyogenes [36]. 

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