Isolation and distribution of mosquito-larvicidal cry genes in Bacillus thuringiensis strains native to Saudi Arabia.

A total of 157 environmental samples were collected from 11 ecological regions across Saudi Arabia to isolate native Bacillus thuringiensis (Bt) strains. Bt isolates (n=103) were recovered by the 50% (v/v) ethanol treatment method with Bt index range of 0.01 to 0.4. Most of Bt isolates showed spherical crystals (54%), while, irregular, bi-pyramidal, and spore-attached crystal constituted 27, 16 and 3% respectively. PCR analysis with eight general and specific dipteran primers of Cry and Cyt genes, revealed positive amplification for cry4 & cyt1, and cry4A, cry4B and cyt2, and cry 10 and cry 11 genes in 28%, 26%, 22%, and 25% of tested strains respectively; whereas cry2 gene was not detected except with the reference Bt kurstaki HD-1 strain. Bioassays against Aedes caspuis and Culex pipiens larvae indicated that 11 strains displayed better larvicidal activity compared with Bacillus thuringiensis H14 (Bti) reference (LC50 0.6 µg/ml) strain against Ae. caspuis, but only two strains (620A & 633R1, LC50 of 0.09 µg/ml & 0.064 µg/ml) that gave significant enhancement. Additionally, one strain (633R1) showed LC50 similar to that of Bti H14 (LC50 0.064 µg/ml) against Cx. pipiens. With the exception of cyt primers, sequenced DNA of all positive primers amplicons revealed 95 to 99% identity in GenBank with Bacillus thuringiensis subsp. israelensis plasmid pBtoxis and also correlated with its SDS-PAGE expressed protein profiles analysis. It is hoped that our wild bio-insecticide Bt strains can be explored in future in the control of mosquito-vector borne diseases in Saudi Arabia.

Isolation and distribution of mosquito-larvicidal cry genes in Bacillus thuringiensis strains native to Saudi Arabia

A total of 157 environmental samples were collected from 11 ecological regions across Saudi Arabia to isolate native Bacillus thuringiensis (Bt) strains. Bt isolates (n=103) were recovered by the 50% (v/v) ethanol treatment method with Bt index range of 0.01 to 0.4. Most of Bt isolates showed spherical crystals (54%), while, irregular, bi-pyramidal, and sporeattached crystal constituted 27, 16 and 3% respectively. PCR analysis with eight general and specific dipteran primers of Cry and Cyt genes, revealed positive amplification for cry4 & cyt1, and cry4A, cry4B and cyt2, and cry 10 and cry 11 genes in 28%, 26%, 22%, and 25% of tested strains respectively; whereas cry2 gene was not detected except with the reference Bt kurstaki HD-1 strain. Bioassays against Aedes caspuis and Culex pipiens larvae indicated that 11 strains displayed better larvicidal activity compared with Bacillus thuringiensis H14 (Bti) reference (LC50 0.6 μg/ml) strain against Ae. caspuis, but only two strains (620A & 633R1, LC50 of 0.09 μg/ml & 0.064 μg/ml) that gave significant enhancement. Additionally, one strain (633R1) showed LC50 similar to that of Bti H14 (LC50 0.064 μg/ml) against Cx. pipiens. With the exception of cyt primers, sequenced DNA of all positive primers amplicons revealed 95 to 99% identity in GenBank with Bacillus thuringiensis subsp. israelensis plasmid pBtoxis and also correlated with its SDS-PAGE expressed protein profiles analysis. It is hoped that our wild bio-insecticide Bt strains can be explored in future in the control of mosquitovector borne diseases in Saudi Arabia.

In vitro activity of subinhibitory concentrations of quinolones on urea-splitting bacteria: effect on urease activity and on cell surface hydrophobicity.

The effect of subinhibitory concentrations of ciprofloxacin, lomefloxacin, norfloxacin, ofloxacin, and sparfloxacin on urease activity and on cell surface hydrophobicity of urea-splitting bacteria was examined. Quinolones at 0.5 MICs demonstrated variable effects on bacterial-urease activity. Norfloxacin inhibited enzyme activity in Proteus vulgaris and Proteus mirabilis, while other quinolones had no effects. In Morganella morganii, sparfloxacin and ciprofloxacin enhanced urease activity, particularly at the initial phase of growth. All quinolones tested showed no marked effect on urease activity by Providencia rettgeri. Quinolones at the same concentrations induced an increase in the cell surface hydrophobicity, which was strain-dependent. There was no correlation between urease inhibition and cell surface hydrophobicity. Inhibition of urease activity by quinolones, in addition to their antibacterial activities, may prevent the progression of urinary tissue damage and stone formation.

Biotransformation of antibiotics. II. Investigation of the chloramphenicol acetyltransferase in Streptomyces griseus.

Additional parameters for the chloramphenicol acetyltransferase (CAT) activity in spores of S. griseus are substantiated. A linear increase in activity was observed with increasing spore number up to a concentration of 5 x 10(10) spores/ml. Similarly an increase of the chloramphenicol concentration up to 500 mug/ml increased the activity. However, a drastic decrease in activity was noted above this level suggesting inhibition of the enzyme by the substrate. The CAT activity in the spores was highly influenced by the pH of the medium reaching a maximum at pH 6.5. This may suggest that CAT is apparently located to the outer surface of the spores and therefore very sensitive to variations in pH of the medium. The CAT showed a marked specificity for D-threo and D-erythrochloramphenicol, while no activity was observed with L-isomers. The enzyme acetylates D,L-erythrodechlor-chloramphenicol with a yield of 45% as compared to the D-threo parent antibiotic. While the tyrosinase characteristics (melanin formation) of S. griseus was eliminated by acriflavine or ethidium bromide treatment the CAT characteristic was persistent. The melanin negative variants retained all otherproperties of the parent strain including the production of antimicrobial agents; and revertants were not detected. The results suggest that the tyrosinase determinant gene is apparently located on an extrachromosomal element (plasmid). On the other hand, the location of the gene for CAT is not assigned yet. The nature of CAT in growing cells and the spores of S. griseus was investigated. The results show that CAT accumulated during the sporulation phase or the vegetative growth is inducible in nature; therefore the morphogenetic sequence in the strain bears no influence on CAT induction.

Biotransformation of antibiotics. I. Acylation of chloramphenicol by spores of Streptomyces griseus isolated from the Egyptian soil .

Incubation of spores, washed mycelium or whole cultures of a Streptomyces sp. with chloramphenicol (I) resulted in the loss of in vitro bioactivity of the antibiotic. Gas chromatographic estimation of an appropriate extract revealed that more than 95% of the antibiotic was inactivated under the specified conditions. The spores inactivated chloramphenicol in an inorganic buffer solution, or in distilled water, without the addition of carbohydrate or external co-factor. However, addition of certain carbon sources to the spores showed a pronounced effect on the chloramphenicol transformation process and on the relative concentration of the inactivated products. Time-course studies on the spore-catalyzed chloramphenicol transformation activity showed a maximum activity at 12-hour incubation. Addition of glucose or acetate at this point maintained maximum activity. The transformation products were identified as: chloramphenicol-1-acetate (IIa); chloramphenicol-3-acetate (IIb); chloramphenicol-3-propionate (III); CHLORAMPHENICOL-O-ISOBUTYRATE (IV); chloramphenicol-3-butyrate (V); and chloramphenicol-3-isovalerate (VI), by techniques of TLC, CPC, GC, UV, IR, MS and NMR. The microbial characteristics of the isolated strain include the formation of flexuous gray aerial mycelium with smooth to rough spores, irregular in size. It is an H2S and melanin former, non-chromogenic, and was inhibited by a streptomycin-producing strain of Streptomyces griseus (Krainsky 1914) Waksman and Henrici(1948).