BackgroundWorldwide, mosquito vectors are transmitting several etiological agents of important human diseases, including malaria, causing millions of deaths every year. In Saudi Arabia, as elsewhere, vector-control is based mostly on chemical insecticides which may be toxic and cause environmental deprivation. Here, to support the development of bio-pesticide alternatives, a study was conducted to identify native Bacillus thuringiensis (Bt) isolates with improved toxicity against the malaria vector, Anopheles gambiae (s.l.).MethodsSixty-eight Bt isolates were obtained from 300 soil and other samples collected from 16 sites across Saudi Arabia. Bt identification was based on morphological characteristics of colonies, shape of parasporal crystals and biochemical profiles. After characterization of their mosquitocidal activity, larvicidal strains were described through 16S ribosomal DNA gene sequencing, cry, cyt and chi genes PCR-amplification profiles, and SDS-PAGE protein analyses.ResultsSpherical Bt crystals were predominant amongst the 68 isolates (34%), while irregular, bi-pyramidal and spore-attached crystals were found in 32, 13 and 21% of strains, respectively. LC50 and LC90 bioassays showed that 23/68 isolates were larvicidal, with distinct biochemical activity profiles compared to non-larvicidal Bt strains. Eight larvicidal strains showed larvicidal activity up to 3.4-fold higher (LC50 range: 3.90–7.40 μg/ml) than the reference Bti-H14 strain (LC50 = 13.33 μg/ml). Of these, 6 strains had cry and cyt gene profiles similar to Bti-H14 (cry4Aa, cry4Ba, cry10, cry11, cyt1Aa, cyt1Ab, cyt2Aa). The seventh strain (Bt63) displaying the highest larvicidal activity (LC50 = 3.90 μg/ml) missed the cry4Aa and cyt1Ab genes and had SDS-PAGE protein profiles and spore/crystal sizes distinct from Bti-H14. The eight strain (Bt55) with LC50 of 4.11μg/ml had cry and cyt gene profiles similar to Bti-H14 but gave a chi gene PCR product size of 2027bp. No strains harbouring cry2, cry17 + 27, cry24 + 40, cry25, cry29, cry30, or cyt2Ba were detected.ConclusionThis study represents the first report of several Saudi indigenous Bt strains with significantly higher larvicidal efficacy against An. gambiae than the reference Bti-H14 strain. The very high toxicity of the Bt63 strain, combined with distinct cry and cyt genes and SDS-PAGE-protein profiles makes it a promising candidate for future applications in mosquito bio-control.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1922-6) contains supplementary material, which is available to authorized users.
Currently, global efforts are being intensified towards the discovery of local Bacillus thuringiensis (Bt) isolates with unique anticancer properties. Parasporins (PS) are a group of Bt non-insecticidal crystal proteins with potential and specific in vitro anticancer activity. However, despite the significant therapeutic potential of PS-producing Bt strains, our current knowledge on the effects of these proteins is limited. Hence, the main objective of this study was to screen Bt-derived parasporal toxins for cytotoxic activities against colon (HT-29) and cervical (HeLa) cancerous cell lines. Nine non-larvicidal and non-hemolytic Bt strains, native to Saudi Arabia, were employed for the isolation of their parasporal toxins. 16S rDNA sequencing revealed a 99.5% similarity with a reference Bt strain. While PCR screening results indicated the absence of selected Cry (Cry4A, Cry4B, Cry10 and Cry11), Cyt (Cyt1 and Cyt2) and PS (PS2, PS3 and PS4) genes, it concluded presence of the PS1 gene. SDS-PAGE analysis revealed that proteolytically-cleavaged PS protein profiles exhibit patterns resembling those observed with PS1Aa1, with major bands at 56 kDa and 17 kDa (Bt7), and 41 kDa and 16 kDa (Bt5). Solubilized and trypsinized PS proteins from all Bt strains exhibited a marked and dose-dependent cytotoxicity against HeLa cancerous cells but not against HT-29 cells. IC50 values ranged from 3.2 (Bt1) to 14.2 (Bt6) with an average of 6.8 µg/mL. The observed cytotoxicity of PS proteins against HeLa cells was specific as it was not evident against normal uterus smooth muscle cells. RT-qPCR analysis revealed the overexpression of caspase 3 and caspase 9 by 3.7, and 4.2 folds, respectively, indicative of the engagement of intrinsic pathway of apoptosis. To the best of our knowledge, this is the first report exploring and exploiting the versatile repertoire of Saudi Arabian environmental niches for the isolation of native and possibly novel Saudi Bt strains with unique and specific anticancer activity. In conclusion, native Saudi Bt-derived PS proteins might have a potential to join the arsenal of natural anticancer drugs.
Incubation of spores, washed mycelium or whole cultures of a Streptomyces sp.
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.
A total of 100 samples of raw milk, various cheeses, labnah, yogurt, and egett were collected from appropriate suppliers and markets in Riyadh region. Bacteriological analysis for typing of enterococci and other lactic Acid Bacteria (LAB) was carried out by plating appropriate dilutions of each sample on sheep blood agar and Edwards blood agar plates. After overnight aerobic incubation at 37°, the presumptive identification was done by colony morphology, cultural characteristics, Gram-stain and catalase production. Final identification to the genera and species level of the total 125 bacterial isolates was completed by API-20 strips as well as Lancefield-serogrouping. Results revealed that Enterococcus faecium (88 isolates) accounts of 70% of total bacterial isolates, while Enterococcus faecalis (26 isolates) accounts of up to 21% and other LAB constituted about 9% of total recovered isolates. The later isolates comprises 3, 3, and 5 isolates of Enterococcus gallinarum, Enterococcus durans, and Aerococcus viridans respectively.The results revealed that nature of sample, its pH, and salinity clearly affect the incidence and number of recovered bacterial isolates. Thus as pH rises towards neutrality, with no salt or low salinity, E. faecalis and other LAB were recovered more frequently, and vice versa. In contrast, E. faecium was routinely isolated from most of the examined samples regardless of their pH range and salinity-content, reflecting its ubiquitous nature and its tolerance to drastic environmental conditions, thereby facilitating person to person transmission. The dominance or persistence of enterococci in examined samples is most probably attributed to their wide range of growth temperatures, their tolerance to heat, salt and acid.In addition, the MIC of each of the tested 120 isolates was determined by serial dilution in Muller Hinton sheep blood agar against 9 antibiotics. All isolates were sensitive to ampicilin with the exception of one E. faecalis strain that showed an MIC of 4 ug/ml. While Erythromycin (EM) exhibited also a good activity with an MIC50/MIC90 of 1/1, 1/4, 2/8 and 4/4 ug/ml. for E. gallinarum, E. faecalis, E. faecium, and E. durans or Aerococcus viridans isolates respectively. Whereas all isolates were resistant to cefoxitin and about 50% were also resistant to Chloramphenicol (CM), Tetracycline (TC), or Trimethoprim / Sulfamethoxazole (SXT). Thus at the breakpoint of MIC (>16 ug/ml.) (TC) resistance rate for E. faecalis was 16% and for E. faecium and E. gallinarum was 35% and 100% respectively. Whereas that for vancomycin (VM) the figures were 44%, 19% and 100% respectively. It is concluded that the examined samples may constitute a potential source for the dissemination of antibiotic resistant determinants to human.
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