Specific Cytotoxic Effects of Parasporal Crystal Proteins Isolated from Native Saudi Arabian Bacillus thuringiensis Strains against Cervical Cancer Cells.

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.

Larvicidal Activities of Indigenous Bacillus thuringiensis Isolates and Nematode Symbiotic Bacterial Toxins against the Mosquito Vector, Culex pipiens (Diptera: Culicidae).

BACKGROUND: The incidence of mosquito-borne diseases and the resistance of mosquitoes to conventional pesticides have recently caused a panic to the authorities in the endemic countries. This study was conducted to identify native larvicidal biopesticides against Culex pipiens for utilization in the battle against mosquito-borne diseases. METHODS: Larvicidal activities of new indigenous Bacillus thuringiensis isolates and crude toxin complexes (TCs) of two nematode bacterial-symbionts, Photorhabdus luminescens akhurstii (HRM1) and Ph. luminescens akhurstii (HS1) that tested against Cx. pipiens. B. thuringiensis isolates were recovered from different environmental samples in Saudi Arabia, and the entomopathogenic nematodes, Heterorhabditis indica (HRM1) and He. sp (HS1) were isolated from Egypt. Larvicidal activities (LC50 and LC95) of the potentially active B. thuringiensis strains or TCs were then evaluated at 24 and 48h post-treatment. RESULTS: Three B. thuringiensis isolates were almost as active as the reference B. thuringiensis israelensis (Bti-H14), and seven isolates were 1.6-5.4 times more toxic than Bti-H14. On the other hand, the TCs of the bacterial symbionts, HRM1 and HS1, showed promising larvicidal activities. HS1 showed LC50 of 2.54 folds that of HRM1 at 24h post-treatment. Moreover, histopathological examinations of the HS1-treated larvae showed deformations in midgut epithelial cells at 24h post-treatment. CONCLUSION: Synergistic activity and molecular characterization of these potentially active biocontrol agents are currently being investigated. These results may lead to the identification of eco-friend mosquito larvicidal product(s) that could contribute to the battle against mosquito-borne diseases.

Prevalence and risk factors of early fecal carriage of Enterococcus faecalis and Staphylococcus spp and their antimicrobial resistant patterns among healthy neonates born in a hospital setting in central Saudi Arabia.

OBJECTIVES: To investigate the prevalence, antibiotic resistant profiles, and risk factors of early fecal carriage of Enterococcus faecalis (E. faecalis) and staphylococci among 150 healthy Saudi neonates born in a hospital setting in central Saudi Arabia. METHODS: This prospective study was conducted in Al-Bukayriyah General Hospital, Qassim, Saudi Arabia, between June 2012 and January 2013. The E. faecalis and Staphylococcus spp. isolates were identified manually, and Vitek2 system was used for identity confirmation at the species level and minimum inhibitory concentration-susceptibility testing. RESULTS: Enterococcus faecalis (n=73) and Staphylococcus spp. (n=18) were recovered. Unlike staphylococci, E. faecalis colonization did not significantly vary from day one up to 7 days of life, regardless of the type of feeding, but it was relatively higher among vaginally versus cesarean delivery. Both Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus) carriage increase as the body weight increases, and this difference was significant (p=0.025) for S. epidermidis. High-level resistance in Gentamycin among E. faecalis isolates was 25% and 11% to Streptomycin. Thirty percent of S. epidermidis were resistant to oxacillin and exhibited multidrug-resistant (MDR) patterns of 5 resistant markers, which were also observed among 2/5 (40%) of Methicillin-resistant Staphylococcus aureus isolates. CONCLUSION: Enterococcus faecalis did not significantly vary in relation to type of delivery, age up to 7 days, and type of feeding. The neonatal fecal carriage of MDR isolates should be considered as a crucial reservoir to the further spread of antimicrobial resistance genes among hospitals, cross infections, and the community.

Risk factors of nasal carriage of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus among health care staff in a teaching hospital in central Saudi Arabia.

OBJECTIVES: To investigate possible risk factors of Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) nasal carriage associated with various health troubles among healthcare workers (HCWs) at King Khalid University Hospital (KKUH). METHOD: This prospective study was conducted between May 2012 and January 2013 in KKUH, Riyadh, Saudi Arabia. A total of 200 nasal swabs were collected from HCWs. Identification was carried out based on morphology, Gram stain, catalase and coagulase test, Staphaurex PlusH test, chromogenic medium, oxacillin, and cefoxitin test using disc diffusion method. Characterization was carried out using disk diffusion method and E-test. Polymerase chain reaction was carried out to confirm using GeneXpert® Dx System (Cepheid) to detect mecA gene. RESULTS: Among the 200 isolates, 80 (40%) were S. aureus carriers, and 36 (18%) of all HCWs were identified as MRSA carriers. There was a significant difference of S. aureus according to gender with male carriers (p=0.012), occupation particularly among nurses (p=0.006), and duration of working years in the hospital among 4-6 years group (p=0.002). Moreover, none of the risk factors assessed were significantly associated with the carriage rate of MRSA (p greater than 0.05). CONCLUSION: The current study revealed that nursing staff was the potential colonizers of S. aureus and MRSA compared with other HCWs. Regular screening of carriers is required for prevention of nosocomial infections.

Heparin-benzyl alcohol enhancement of biofilms formation and antifungal susceptibility of vaginal Candida species isolated from pregnant and nonpregnant Saudi women.

Biofilm formation by Candida species is a major contribute to their pathogenic potential.The aim of this study was to determine in vitro effects of EDTA, cycloheximide, and heparin-benzyl alcohol preservative on C. albicans (126) and non-albicans (31)vaginal yeast isolates biofilm formations and their susceptibility against three antifungal Etest strips. Results of the crystal violet-assay, indicated that biofilms formation were most commonly observed [100%] for C. kefyr, C. utilis, C. famata, and Rhodotorula mucilaginosa, followed by C. glabrata [70%], C. tropicalis [50%], C. albicans [29%], Saccharomyces cerevisiae [0.0%]. EDTA (0.3mg/ml) significantly inhibited biofilm formation in both C. albicans and non-albicans isolates (P=0.0001) presumably due to chelation of necessary metal cations for the process-completion. In contrast, heparin (-benzyl alcohol preservative) stimulated biofilm formation in all tested isolates, but not at significant level (P=0.567). Conversely, cycloheximide significantly (P=0.0001) inhibited biofilm formation in all C. albicans strains(126) and its effect was even 3 fold more pronounced than EDTA inhibition, probably due to its attenuation of proteins (enzymes) and/or complex molecules necessary for biofilm formation. Results also showed that all nonalbicans yeasts isolates were susceptible to 5-flucytosine (MIC50, 0.016 µg/ml; MIC90, 0.064 µg/ml), but 14% of C. albicans isolates were resistant (MIC50, 0.064 µg/ml; MIC90 >32 µg/ml). The MIC50 value of amphotricin B for all C. albicans and non-albicans isolates was at a narrow range of 0.023 µg /ml, and the MIC90 values were 0.047 µg/ml and 0.064 µg/ml respectively, thereby confirming its efficacy as a first line empiric- treatment of Candida spp infections.

Detection of genital colonization of group B streptococci during late pregnancy.

OBJECTIVE: To detect group B streptococcal carrier state of Saudi females during 3rd trimester of pregnancy and to assess type of specimens and the techniques used for the organism detection. METHODS: A total of 867 consecutive vaginal and rectal swabs were obtained from 217 pregnant women at > 28 weeks of gestation and their follow up testing from King Khalid University Hospital, Riyadh, Kingdom of Saudi Arabia. Swab-specimens were cultured comparatively on Islam and Edwards blood agar plates, and into selective Lim broth. Enrichment Lim broth cultures (>12 hours) with and without positive modified coagglutination test were then subcultured on Islam and Edwards sheep blood agar plates. Presumptive colonies were then tested for group B streptococcus identity by convential biochemical reactions, serogrouping and serotyping. Collected neonatal swab-specimens (184) were also treated similarly. RESULTS: In comparison to Lim broth enrichment culture, the direct swab specimen culture on Edwards blood agar or Islam agar plates technique revealed 84% sensitivity and 100% specificity, whereas modified coagglutination test after selective Lim broth enrichment revealed 100% sensitivity and 96% specificity. Group B streptococcus was isolated in at least one of the specimens from the 217 patients in 66 cases. Of these 66 cases, group B streptococcus was isolated from both vaginal and rectal swabs in 33 (50%) cases and only from vaginal swabs in 22 (33%) and rectal swabs in 11 (17%) cases. Of the group B streptococcus positive cases, 10 (15%) cases had spontaneously lost their carriage, upon follow up testing, whereas out of the 151 negative cases, 4 (2.6%) cases became positive for group B streptococcus colonization upon follow up testing with an overall carriage rate of (60/217) 27.6%. Certain demographic factors were found to alter such rate of carriage. Additionally, 50% of group B streptococcal colonized mothers vertically transmitted the homologous serotypes of the organism to their newborns, but clinical infection was not recorded during the study period. CONCLUSION: Group B streptococci colonization rate among term Saudi pregnant women is relatively high (27.6%); and thereby constitutes a group of women whose infants are at great risk of early-onset invasive disease. The modified coagglutination test after growth amplification seems rapid and cost-effective to detect lightly or heavily group B streptococcal colonized women. Vaginal and rectal swab specimens at late pregnancy appeared necessary to accurately identify group B streptococcus maternal colonization.