Production of recombinant flagellin to develop ELISA-based detection of Salmonella Enteritidis

ABSTRACT Food-borne diseases, caused by the pathogenic bacteria, are highly prevalent in the world. Salmonella is one of the most important bacterial genera responsible for this. Salmonella Enteritidis (SE) is one of the non-typhoid Salmonellae that can be transmitted to human from poultry products, water, and contaminated food. In recent years, new and rapid detection methods such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) have been developed. In this study, recombinant FliC (rFliC) was produced to be used as an antigen. The immunization was conducted in mice with the purified recombinant FliC (rFliC). The mice were subcutaneously immunized with rFliC and elicited significant rFliC specific serum IgG antibodies. An indirect ELISA system was established for the detection of Salmonella Enteritidis. Our results confirmed that the recombinant flagellin can be one of the excellent indicators for the detection of Salmonella Enteritidis.

An epidemiological study of road traffic accidents in guilan province, northern iran in 2012

Objective: To determine the epidemiological characteristics of the road traffic injuries [RTIs] in Guilan province, northern Iran

Methods: This study was a cross-sectional study which included all of RTIs admitted to medical centers of Guilan province [northern Iran] during 2012. ICD-10 was used as diagnostic criteria. Demographic variables also injury circumstance and in hospital variables such as length of stay, time of admission, type of surgery, ICU admission, final outcome and mechanism of injury, anatomical part of injury according to Abbreviated Injury Scale [AIS] classification were derives from records by trained research team. Descriptive data is reported

The predictors of mortality were also determined

Results: The prevalence of road traffic injuries in Guilan province was 31 in 10,000 populations. Of total 7671 accidents, 5976 [77.9%] were men and 1695 [22.1%] were women. Mean age of these victims was 33.3+/-17.289 years [32.64+/-16.939 for men, 35.62+/-18.312 for women]

Most of them [32.5%] were 20-29 years old. Motorcyclecar accidents had the highest frequency followed by car-car crashes and car accidents involving pedestrians

Most of the patients [85.9%] were hospitalized and 280 injured died [3.7%]

Upper extremities were the most sites of injuries. Male sex, length of hospital stay, multiple injuries and increased age were associated with road traffic accident associated mortality

Conclusion: RTIs cause enormous death and disability in this area and more road traffic preventive programs should be enforcement in these areas to reduce incidences RTI

Detection of E. coli O157:H7 and Shigella dysenteriae toxins in clinical samples by PCR-ELISA

Shiga toxin producing bacteria are potential causes of serious human disease such as hemorrhagic colitis, severe inflammations of ileocolonic regions of gastrointestinal tract, thrombocytopenia, septicemia, malignant disorders in urinary ducts, hemolytic uremic syndrome (HUS) Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. A lot of methods have been developed so far to detect shiga toxins such as cell culture, ELISA, and RFPLA, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. In this study, PCR-ELISA method was used to detect genes encoding shiga toxins 1 and 2 (stx1 and stx2). To detect stx1 and stx2 genes, two primer pairs were designed for Multiplex-PCR then PCR-ELISA. PCR products (490 and 275, respectively) were subsequently verified by sequencing. Sensitivity and specificity of PCR-ELISA method were determined by using genome serial dilution and Enterobacteria strains. PCR-ELISA method used in this study proved to be a rapid and precise approach to detect different types of shiga toxins and can be used to detect bacterial genes encoding shiga toxins.

Comparison of cyp141 and IS6110 for detection of mycobacterium tuberculosis from clinical specimens by PCR

Tuberculosis is a major public health problem throughout the world. TB's worldwide patterns of prevalence coupled with the increase in incidence of HIV infection threaten the health and lives of humans worldwide. Rapid detection of TB and the rapidly initiation of the administration of medication are important strategies for stopping the transmission of this disease transmission and its resistance to anti-TB drugs. Molecular methods are advantageous relative to conventional techniques due to their greater speed and sensitivity in the detection of TB. In this study, we targeted the cyp141 gene for the detection of Mycobacterium tuberculosis from clinical specimens [n = 123] by PCR and compared the sensitivity and specificity of this new target with those of IS6110 gene. Targeting of the cyp141 gene is more sensitive [97.1% for cultured isolates and 85.7% for direct specimens] than the targeting of the commonly used IS6110 gene [95.1% for cultured isolates and 42.9% for direct specimens], and the specificities of these two target genes were equal [100%]. The cyp141 gene can be used as a new target for the direct detection of Mycobacterium tuberculosis that seems to be superior to IS6110

in silico chimeric vaccine targeting breast cancer containing inherent adjuvant

Today, Lack of efficient therapeutic strategy for breast cancer [the most common cause of death in women] is one of the momentous problematic topics for all health care committees. Designing new specific vaccine, based on antigens located on the surface of cancer cells can be useful. Over expression of ROR1, lacked of HER2/neu, and hormone receptors on cell surface in the breast cancer, introduce this protein as an appropriate candidate for designing cancer vaccine. We hypothesized the extracellular domain of receptor tyrosine kinase like orphan receptor 1 [ROR-1] along with a super antigen such as staphylococcal enterotoxin B could be a potent vaccine for drug resistant breast cancer. Here, we assessed the findings of bioinformatics analysis to identify the antitumor immune properties of this chimeric construct. In addition, the stability, physic-chemical properties and allergic potency of designed fusion protein were investigated by valid bioinformatics software. Our result suggested that chimeric model is capable to be a stimulant of both T-cell and B- cell mediated immune responses with an acceptable accessibility and solubility but without any allergenicity. The ROR-1 with an enterotoxin B could be a potent vaccine for breast cancer

In silico analysis for identifying potential vaccine candidates against Staphylococcus aureus

PURPOSE: Staphylococcus aureus is one of the most important causes of nosocomial and community-acquired infections. The increasing incidence of multiple antibiotic-resistant S. aureus strains and the emergence of vancomycin resistant S. aureus strains have placed renewed interest on alternative means of prevention and control of infection. S. aureus produces a variety of virulence factors, so a multi-subunit vaccine will be more successful for preventing S. aureus infections than a mono-subunit vaccine. MATERIALS AND METHODS: We selected three important virulence factors of S. aureus, clumping factor A (ClfA), iron-regulated surface determinant (IsdB), and gamma hemolysin (Hlg) that are potential candidates for vaccine development. We designed synthetic genes encoding the clfA, isdB, and hlg and used bioinformatics tools to predict structure of the synthetic construct and its stabilities. VaxiJen analysis of the protein showed a high antigenicity. Linear and conformational B-cell epitopes were identified. RESULTS: The proteins encoded by these genes were useful as vaccine candidates against S. aureus infections. CONCLUSION: In silico tools are highly suited to study, design, and evaluate vaccine strategies.

Genetics study and transmission electron microscopy of pili in susceptible and resistant clinical isolates of Mycobacterium tuberculosis

Objective: To study genetic bases and morphology of pili in Mycobacterium tuberculosis (M. tuberculosis). Methods: PCR and sequencing were used to investigate two related pili, Mtp and Flp genes in clinical isolates of M. tuberculosis. The primers were designed and PCR program were set for whole genes amplification. PCR products of the two genes from all isolates were sequenced by an applied biosystems apparatus and the results were analysed by online software. In the other hands, harvested cells from fresh cultures of isolates were undergoing specific sample preparation for sectional and negative staining for transmission electron microscopy. Results: Electrophoresis revealed two specific bonds of 361 bp for Mtp and 150 bp for Flp genes and confirmed primer and PCR conditions designing. There were not any mutations in sequencing results of Mtp and Flp in comparison with reference sequence. Transmission electron microscopy examination revealed two distinct types of pili in the isolates as a bundle-forming pilus and rope-like pilus. From total investigated cells, 10% harbored pili in their structure. Conclusions: Two genes of pili in all clinical isolates of M. tuberculosis were conserved and two morphological types of pili were detected. We proposed that by targeting pili proteins by a suitable inhibitor, it could affect the pathogenesis especially in resistant forms.

Genetics study and transmission electron microscopy of pili in susceptible and resistant clinical isolates of Mycobacterium tuberculosis

OBJECTIVE@#To study genetic bases and morphology of pili in Mycobacterium tuberculosis (M. tuberculosis).@*METHODS@#PCR and sequencing were used to investigate two related pili, Mtp and Flp genes in clinical isolates of M. tuberculosis. The primers were designed and PCR program were set for whole genes amplification. PCR products of the two genes from all isolates were sequenced by an applied biosystems apparatus and the results were analysed by online software. In the other hands, harvested cells from fresh cultures of isolates were undergoing specific sample preparation for sectional and negative staining for transmission electron microscopy.@*RESULTS@#Electrophoresis revealed two specific bonds of 361 bp for Mtp and 150 bp for Flp genes and confirmed primer and PCR conditions designing. There were not any mutations in sequencing results of Mtp and Flp in comparison with reference sequence. Transmission electron microscopy examination revealed two distinct types of pili in the isolates as a bundle-forming pilus and rope-like pilus. From total investigated cells, 10% harbored pili in their structure.@*CONCLUSIONS@#Two genes of pili in all clinical isolates of M. tuberculosis were conserved and two morphological types of pili were detected. We proposed that by targeting pili proteins by a suitable inhibitor, it could affect the pathogenesis especially in resistant forms.

Drug resistance pattern of Mycobacterium tuberculosis isolates from patients of five provinces of Iran

OBJECTIVE@#To determine the patterns of resistance to first line anti-tuberculosis (TB) drugs among a collection of Mycobacterium tuberculosis (MTB) isolates from 5 provinces of Iran.@*METHODS@#A total of the 6 426 clinical specimens from patients suspected of active TB were collected from March 2010 to June 2012. All specimens were subjected for microscopy and culture tests in the TB centers of studies provinces. Drug susceptibility testing to the first line anti-TB drugs for culture positive MTB was performed on Löwenstein-Jensen (LJ) medium using proportion method.@*RESULTS@#Of 6 426 clinical specimens, 261 were culture positive for mycobacteria, of which 252 were MTB and 9 were MOTT (mycobacteria other than tuberculosis). Of 252 MTB isolates, 211 (83.7%) were pan-susceptible and 41 (16.3%) were resistant to at least one drug. Resistance was most common to streptomycin, 30 isolates (12.0%), followed by isoniazid, 20 isolates (8.0%), rifampin, 15 isolates (6.0%) and ethambutol, 14 isolates (5.5%). Sixteen (6.3%) MTB isolates were MDR. A clear evidence of heterogeneity amongst the 5 provinces in the proportions with resistance to one or more drugs was observed [χ(2); = 12.209 (4 degrees of freedom), P values = 0.015 9].@*CONCLUSIONS@#The prevalence of drug resistance in this study area underscoring the need for further enforcement of TB control strategies in the Iran. Drug susceptibility testing for all TB cases to provide optimal treatment, establishing advanced diagnostic facilities for rapid detection of MDR-TB and continuous monitoring of drug resistance are recommended for prevention and control of drug-resistant TB.

Prevalence of exfoliative toxin A and B genes in Staphylococcus aureus isolated from clinical specimens

The pathogenicity of Staphylococcus aureus is based on the production of various virulence factors. The frequency of these factors can markedly differ according to the geographical region. In this study, we investigated the prevalence of two frequent isoforms of exfoliative toxins and mecA genes using PCR in 197 clinical isolates obtained from clinical samples during the years 2011 and 2012. The samples were obtained from an educational hospital in northern Tehran, Iran. In addition, the antibiotic susceptibility pattern was studied for each isolate using the disc diffusion method. In this study, 186 [94.4%], 15 [7.6%] and 172 [86.3%] of the 197 isolates expressed the eta, etb and mecA genes, respectively. In addition, 164 [88.2%] and 12 [80%] strains, which harbored the eta and etb genes, respectively, were MRSA [methicillin resistant S, aureus]. Furthermore, the prevalence of the mecA, eta and etb genes was higher among the wound samples [61.2%, 55.8% and 6.09%, respectively]. We observed high rates of MDR [multi drug resistance] among our isolates. A significant correlation was detected between the presence of the mecA gene and the resistance to oxacillin, gentamicin, kanamycin, erythromycin, tetracycline, cotrimoxazole, clindamycin, and cephazolin as well as the multi-drug resistant property [P<0.05]. In addition to penicillin, the MDR properties and resistances to the tested antibiotics in the etb-positive strains were significantly lower compared to the etb-negative strains [P< 0.05]. The prevalence of the eta, etb and mecA genes might be due to the specific geographic region

In silico design and analysis of TGF alpha L3-SEB fusion protein as "a new antitumor agent" candidate by ligand-targeted superantigens technique

Bacterial superantigen Staphylococcal Enterotoxins [SEs], has stimulated polyclonal T cells irrespective of their antigen specificity, resulted a massive release of cytokines, and suggested that they could be assigned as a candidate of new antitumor agents. Recent attempts have done to specifically target superantigens towards tumors, subsequently Monoclonal antibodies and tumor-related ligands have employed as targeting molecules of superantigen for the preclinical treatment of different tumors. Here, we have evaluated TGF alpha L3-SEB fusion protein as a new antitumor candidate by genetically fusing the third loop of transforming growth factor alpha [TGF alpha L3] to Staphylococcal Enterotoxin type B. An in silico techniques have launched to characterize the properties and structure of the protein, before initiating the experimental study, we have predicted physicochemical properties, structures, stability, MHC binding properties and ligand-receptor interaction of this chimeric protein by means of computational bioinformatics tools and servers. Our results have indicated codon adaptation index of tgf alpha l3-seb fusion gene has increased from 0.5 in the wild type sequences to 0.85 in the chimeric optimized gene. The mfold data has shown the tgf alpha l3-seb mRNA was stable enough for efficient translation in the new host. Based on Ramachandran plot TGF alpha L3-SEB has classified as a stable fusion protein. Our result has shown fusing of TGFalphaL3 in N-terminal of the TGF alpha L3-SEB construct, had no effects on MHC binding and subsequently superantigenic activity of SEB. Finally based on ligand-receptor docking the binding ability of TGFalphaL3 was strong enough to its receptor, so TGF alpha L3-SEB could be assigned as a new antitumor candidate in cancer immunotherapy. Our results have proposed that TGF alpha L3-SEB was a stable fusion protein with proper affinity to its receptor that overexpressed in various human carcinomas, so it could generate potent immune response towards tumors

Cellular immunity survey against urinary tract infection using pVAX/fimH cassette with mammalian and wild type codon usage as a DNA vaccine

PURPOSE: FimH (the adhesion fragment of type 1 fimbriae) is implicated in uropathogenic Escherichia coli (UPEC) attachment to epithelial cells through interaction with mannose. Recently, some studies have found that UPEC can thrive intracellularly causing recurrent urinary tract infection (UTI). Almost all vaccines have been designed to induce antibodies against UPEC. Yet, the humoral immune response is not potent enough to overcome neither the primary UTI nor recurrent infections. However, DNA vaccines offer the possibility of inducing cell mediated immune responses and may be a promising preventive tool. MATERIALS AND METHODS: In this study, we employed two different open reading frames within mammalian (mam) and wild type (wt) codons of fimH gene. Optimized fragments were cloned in pVAX-1. Expression of the protein in COS-7 was confirmed by western blot analysis after assessing pVAX/fimH(mam) and pVAX/fimH(wt). The constructs were injected to BALB/c mice at plantar surface of feet followed by electroporation. RESULTS: The mice immunized with both constructs following booster injection with recombinant FimH showed increased interferon-gamma and interleukin-12 responses significantly higher than non-immunized ones (p<0.05). The immunized mice were challenged with UPEC and then the number of bacteria recovered from the immunized mice was compared with the non-immunized ones. Decreased colony count in immunized mice along with cytokine responses confirmed the promising immune response by the DNA vaccines developed in this study. CONCLUSION: In conclusion, DNA vaccines of UPEC proteins may confer some levels of protection which can be improved by multiple constructs or boosters.

antimicrobial activity of grape seed extract against two important oral pathogens

The antimicrobial properties of plant extracts have shown promise for development of new drugs. This study was conducted to measure the antibacterial activity of grape [Vitis vinifera] seed extract against Streptococcus mutans and Aggregatibacter actinomycetemcomitans. In this experimental study the grape seed extract have been prepared with maceration method. The antimicrobial activity of the extract was examined by determining Minimum Inhibitory Concentration [MIC] and Minimal Bactericidal Concentration [MBC] using the macro dilution broth technique. MIC and MBC for Aggregatibacter actinomycetemcomitans was 3.84 mg/mL and 7.68 mg/mL respectively. There were not any inhibitory effects against Streptococcus mutans. The Grape seed extract has inhibitory and bactericidal effects against Aggregatibacter actinomycetemcomitans. There were not any bactericidal or bacteriostatic effects against Streptococcus mutans

High level of gentamicin resistance [HLGR] among enterococcus strains isolated from clinical specimens

Enterococci are pathogens that can cause nosocomial infections and acquire resistance properties via several molecular mechanisms. The aac [6']Ie-aph[2"]Ia gene plays a significant role in the emergence of high-level gentamicin-resistant [HLGR] strains. The screening of resistant strains and the provision of appropriate antibiotic therapy can decide the outcome of serious nosocomial infections. In the present study, 142 enterococci were isolated from patients, and the species were identified using standard methods. An antimicrobial susceptibility test was performed using the disc diffusion method, and the minimum inhibition concentration [MIC] of gentamicin was determined according to the broth micro-dilution method. Additionally, PCR was utilized to detect the aac[6']Ie-aph[2"]Ia gene, the presence of which was confirmed by digestion with Sca1 and sequencing. Of the 142 isolates, 62 [43.7%] were found to exhibit the HLGR phenotype. All except one of the HLGR isolates contained the aac[6']Ie-aph[2"]Ia gene. The prevalence of resistance to other antibiotics and multi-drug resistance [MDR] was higher among the HLGR isolates compared to the non-HLGR isolates. Our results indicate that high prevalence rates of MDR and HLGR enterococci are an important problem associated with medical treatment. Furthermore, the presence of the aacaacaac[6']Ie-aph[2"]Ia gene was shown to correspond to the presence of the HLGR phenotype among enterococci

Prevalence and evaluation of toxin genes among uropathogenic escherichia coli clinical isolates by duplex PCR

One of the most common infections in human is urinary tract infection [UTI] and Uropathogenic Escherichia coli is one of its major causative agents. UTI is extremely common among young women. Children under age 5 are also highly at risk. Considering the prevalence of this disease, it is necessary to design an appropriate diagnostic method for its effective diagnosis. The aim of present study was to identify the prevalence of two virulence genes [sat and vat] among Uropathogenic E. coli isolates. Urine samples were taken from 350 patients with urinary tract infection. The samples were cultured on EMB agar and Blood agar. The suspected E. coli colonies were isolated and confirmed by biochemical tests. The genomic DNA was extracted from 297 isolated E. coli and target genes were amplified by PCR. The amplicons were sequenced and analyzed with ClustalW software. Moreover, data analysis was performed by using SPSS software. Subsequently, Duplex PCR was optimized for simultaneous detection of two genes. The prevalence of sat and vat genes were 75 [n: 225] and 36 [n: 106] percent, respectively. In addition, less than 4% [n: 11] of clinical isolates comprised two genes. According to the conducted research, molecular identification of Uropathogenic E .coli strains according to detection of sat gene is potentially an appropriate method and could be noted for diagnosis.

[Effects of intra-oral intake of different concentrations of sucrose on biobehavioral pain response to immunizations in infants]

An uncontrolled pain in infants can lead to permanent damage of the components of cognitive growth and development and change in response to painful stimuli. The aim of this study was to compare the effect of oral sucrose%20 and%50 on biobehavioural pain response to hepatitis B virus [HBV] vaccine injection in term newborns. In this triple blind clinical trial study, which was performed in Semnan Amir-Al-Momenin Hospital, 91 term infants were selected and randomly divided into three groups. Two minutes prior to immunization with HBV vaccine by intramuscular injection, 2 ml of 20% or 50% sucrose solution was given to the infants in, experimental groups, while, the infants in the control group were orally received only 2 ml of sterile distilled water. The pain response was measured by using Neonatal Infant Pain Scale [NIPS] tools before the administration of sucrose, immediately after injection and 5 min after injection. The results showed that the administration of 50% sucrose significantly reduces pain response in newborns immediately and 5 minutes after HBV vaccination [P < 0.05]. But the administration of 20% sucrose was effective only 5 minutes after HBV vaccine injection. The administration of 50% sucrose can effectviley reduce the pain due to HBV vaccination in infants.

Smad molecules expression pattern in human bronchial airway induced by sulfur mustard

Airway remodelling is characterized by the thickening and reorganization of the airways seen in mustard lung patients. Mustard lung is the general description for the chronic obstructive pulmonary disease induced by sulfur mustard[SM]. Pulmonary disease was diagnosed as the most important disorder in individuals that had been exposed to sulfur mustard. Sulfur mustard is a chemical warfare agent developed during Wars. Iraqi forces frequently used it against Iranian during Iran -Iraq in the 1980-1988. Peribronchial fibrosis result from airway remodeling that include excess of collagen of extracellular matrix deposition in the airway wall. Some of Smads families in association with TGF-are involved in airway remodeling due to lung fibrosis. In the present study we compared the mRNA expression of Smad2, Smad3, and Smad4 and Smad7 genes in airway wall biopsies of chemical-injured patients with non-injured patients as control. We used airway wall biopsies of ten unexposed patients and fifteen SM-induced patients. Smads expression was evaluated by RT-PCR followed by bands densitometry. Expression levels of Smad3 and Smad4 in SM exposed patients were upregulated but Smad2 and Smad7 was not significantly altered. Our results revealed that Smad3, and 4 may be involved in airway remodeling process in SM induced patients by activation of TGF-. Smad pathway is the most represented signaling mechanism for airway remodeling and peribronchial fibrosis. The complex of Smads in the nucleus affects a series of genes that results in peribronchial fibrosis in SM-induced patients

[Morphologic changes of Mycobacterium tuberculosis after exposure with choloformic garlic extract]

Tuberculosis is one of the major problem facing of globle health. Drug resistance of mycobacterium tuberculosis to antimicrobial agent has strongly emerged the need for achiving the new drugs. Garlic as medical plants has long been taken under investigation. This study for antibacterial effect was done to determine the morphological alteration of Mycobacterium tuberculosis due to garlic choloformic extract. Garlic extract contains allicine [thio-2-propen-sulfonic acid-s-allil ester] is one of its effective antimicrobacterial substance. In a in-vitro study, the standard strain of Mycobacterium tuberculosis H37RV and clinical isolated strain was cultured in the middle broke 7H9 broth with different concentration of garlic extract in different 12, 24, 48, 72 hours. Morphological althertits of mycobacterium inspected with macroscopic and microscopic studies. The garlic exteract caused conversion of rough colonies to smooth and mucoid colonies and in microscopic studies morphologic change of mycobacterium from bacilli form to coccobacilli and cocci was observed. Also 0.67 mg/ml of garlic exteract on 48h period inhibited both of sensitive [standard strain of H37RV] and resistance [clinical strains] Mycobacterium tuberculosis. This study showed that garlic extract in addition to inhibiting growth, change the morphology of Mycobacterium tuberculosis from baccilli to cocoibaccill form and also alter the colony apearance from rough to smooth shap