Pasteurella haemolytica Leukotoxin and Its Interaction With Target Cells

A number of Pasteurella haemolytica isolates from cattle and sheep, including representatives of all serotypes and untypables, was examined for leukotoxin (LktA) production at the end of the log phase of growth in brain-heart infusion broth. The bacteria produced most toxin with very high and high aeration, at 3

The nanobiosensor of rfbE geneof detection

زمینه و هدف: علائم بالینی بیماران آلوده به باکتری اشریشیاکولی O157:H7 دامنه وسیعی دارد و تشخیص بیماری از روی علایم آن دشوار است. روش‌های تشخیص متعارف عوامل بیولوژیک دارای معایب بسیاری می باشند. هدف از انجام این تحقیق، تشخیص توالی ژن rfbE در نمونه واقعی باکتری اشریشیاکولی O157:H7 به وسیله نانوبیوسنسور الکتروشیمیایی بود. روش بررسی: در این مطالعه تجربی، برای آماده سازی نمونه قبل از سنجش با نانوبیوسنسور، ژنوم باکتری اشریشیاکولی O157 به روش CTAB-NaCl و با استفاده از کیت تخلیص DynaBioTM DNA Extraction Mini Kit استخراج گردید. هضم آنزیمی DNA ژنومی باکتری اشریشیاکولی O157 در دمای 37 درجه سانتی گراد و به مدت 4 دقیقه و دناتوراسیون حرارتی صورت گرفت. به منظور اطمینان، ژن rfbE باکتری اشریشیاکولی O157: H7 توسط واکنش PCR تشخیص داده شد. توالی ژن rfbE در نمونه واقعی باکتری اشریشیاکولی O157: H7 به وسیله نانوبیوسنسور مبتنی بر هیبریداسیون DNA مورد سنجش قرار گرفت. یافته ها: نتایج به دست آمده از مقاومت انتقال بار بیانگر بر هم کنش مناسب بین ssDNA تثبیت شده روی الکترود با DNA ژنوم هضم شده باکتری اشریشیاکولی O157:H7 و محصول PCR بوده و نشان دهنده تشخیص موفقیت آمیز قطعه ‌ای از ژن rfbE باکتری اشریشیاکولی O157:H7 می ‌باشد. این نانوبیوسنسور قادر به تشخیص ژنوم هضم شده باکتری تا رقت 6-10 بود که این رقت متناظر با غلظت 102 باکتری اشریشیاکولی در میلی لیتر است. نتیجه گیری: در این تحقیق، نانوبیوسنسور مبتنی بر هیبریداسیون DNA برای تشخیص توالی ژن rfbE در نمونه واقعی باکتری اشریشیاکولی O157:H7 با دقت و حساسیت مطلوب به کارگیری شد که می تواند زمینه ای برای ساخت ابزار تشخیص DNA باشد

Optimization of expression, extraction & purification of the N-terminal region of ipaD gene in Shigella dysenteriae by proteomics analysis

زمینه و هدف: باکتری شیگلا دیسانتری یکی از عوامل پاتوژن مهم است که علی‌رغم تلاش‌های چندین ساله برای تهیه واکسن علیه آن هنوز مطالعات گسترده پیرامون آن ادامه دارد. محصولات پلاسمید تهاجمی شیگلا (Ipa) نقش مهمی در تهاجم باکتری ایفا می‌کنند. پروتئین IpaD یکی از اعضای این خانواده است که به عنوان کاندید واکسن شیگلا مطرح می‌باشد. مطالعات متعدد بر روی این پروتئین نشان داده که ناحیه N- ترمینال آن نقش مهمی در فرآیند تهاجمی باکتری دارد. این مطالعه با هدف بهینه سازی بیان N- ترمینال ژن IpaD به منظور افزایش تولید پروتئین نو ترکیب انجام شد. روش بررسی: در این مطالعه تجربی-آزمایشگاهی باکتری E. coli BL21(DE3) حامل پلاسمید pET-28a که ژن ناحیه N- ترمینال IpaD در آن همسانه سازی شده بود جهت مطالعات مورد استفاده قرار گرفت. پس از کشت باکتری، تاثیر سه فاکتور زمان القا، دما و غلظت ماده القا کننده ایزو-پروپیل-تایوبتا دی گالاکتوپیرانوزید (IPTG) بر میزان بیان، با استفاده از ژل سدیم دو دسیل سولفات-پلی آکریل آمید (SDS-PAGE) به صورت کیفی بررسی گردید. با استفاده از تصاویر دو بعدی تهیه شده از ژل‌ها با کمک نرم افزار آنالیز ژل‌های دو بعدی بررسی کمی بیان پروتئین صورت پذیرفت. مراحل استخراج و تخلیص پروتئین نوترکیب با کمک روش شیب اوره آغاز و با عبور نمونه‌ها از ستون کروماتوگرافی پایان یافت. یافته‌ها: نتایج بر روی ژل‌های SDS-PAGE نشان داد که میزان تقریبا مشابهی از تولید پروتئین نوترکیب در زمان‌ القا، دما و غلظت های مختلف IPTG بیان وجود دارد، اما یافته‌های نرم افزاری نشان داد بهترین شرایط بیان ناحیه N- ترمینال پروتئین IpaD در وکتور pET-28a دمای 37 درجه سانتی‌گراد، غلظت 7/0 میلی مولار IPTG و زمان 3 ساعت بعد از القا می‌باشد. نتیجه‌گیری: بر اساس نتایج این مطالعـــه هر پروتئین بعد از فرآیند همسانه سازی شرایط بیان مخصوص به خود را دارا می‌باشد که شرایط دمایی و طول زمان القا سلول‌ها در مقدار تولید پروتئین موثرتر می‌باشند

Nano and microparticle drug delivery systems for the treatment of Brucella infections

Nano-based drug delivery systems are increasingly used for diagnosis, prevention and treatment of several diseases, thanks to several beneficial properties, including the ability to target specific cells or organs, allowing to reduce treatment costs and side effects frequently associated with chemotherapeutic medications, thereby improving treatment compliance of patients. In the field of communicable diseases, especially those caused by intracellular bacteria, the delivery of antibiotics targeting specific cells is of critical importance to maximize their treatment efficacy. Brucella melitensis, an intracellular obligate bacterium surviving and replicating inside macrophages is hard to be eradicated, mainly because of the low ability of antibiotics to enter these phagocityc cells . Although different antibiotics regimens including gentamicin, doxycycline and rifampicin are in fact used against the Brucellosis, no efficient treatment has been attained yet, due to the intracellular life of the respective pathogen. Nano-medicines responding to environmental stimuli allow to maximize drug delivery targeting macropages, thereby boosting treatment efficacy. Several drug delivery nano-technologies, including solid lipid nanoparticles, liposomes, chitosan, niosomes, and their combinations with chitosan sodium alginate can be employed in combination of antibiotics to successfully eradicate Brucellosis infection from patients

Detection of Sea, Seb, Sec, Seq genes in staphylococcus aureus isolated from nasal carriers in Tehran province, Iran; by multiplex PCR

Staphylococcus(S.) aureus  produces different extra-cellular protein toxins and virulence factors. One of the most important extra-cellular proteins is an enterotoxin which causes staphylococcal food poisoning (SFP) due to their enterotoxins. Different methods have been used to detect this toxin, each of which has advantages and disadvantages. DNA amplification methods, however, can show the presence of enterotoxigenic strains of S. aureus before the expression of enterotoxins on the basis of specific gene sequences. In this study, 150 S. aureus strains isolated from nasal carriers were confirmed by biochemical testing. PCR was used to amplify the staphylococcal enterotoxin A, B, C and Q genes, as well as the staphylococcal nuclease gene.  Among the 150 healthy human isolates from the nasal carrier, 95 were confirmed as S. aureus.  Only 58.9% of the isolates were diagnosed as sea, b, c, q positive. There were 24 (25.3%) isolates associated with the sea gene, 15.8% isolates associated with the seb gene, 9.5% of the isolates were associated with the sec gene, and 8.4% of the isolates associated with the seq gene. Of these isolates, 41% might be possessing additional se genes but they were not see (178 bp) and sed (319 bp) genes.  The nuc gene, which encodes thermo nuclease, was used as a target DNA to identify S. aureus. Additionally, one of these enterotoxigenic isolates carried more than one toxin gene

Cloning and Expression of N-terminal Region of IpaD from Shigella dysenteriae in E. coli

Genus Shigella is one of the important members of the family enterobacteriacae. There are numerous antigens in Shigella carrying by a 220 kb plasmid. Among them, IpaD is the key virulence factor of S. flexneri. Apart from having effectors function that is essential for host cell invasion and intracellular survival, this protein also controls the secretion and translocation of other effector proteins into eukaryotic host cells. In the present study, we have cloned and expressed the ipaD in E. coli. The ipaD gene was amplified by PCR. Prokaryote expression vector pET-28a(+)- ipaD was constructed, and used to transform E. coli BL21DE3 plySs. The expression of recombinant protein induced by IPTG was examined by SDS-PAGE. Western blot were used to determine immunoreactivity of IpaD-His by a rabbit monoclonal antibodies against his-tag. SDS-PAGE demonstrated that the constructed prokaryotic expression efficiently produced IpaD at the 1 mmol/L of IPTG. IpaD protein was able to react with the rabbit monoclonal antibody against His-tag.  IpaD is essential for Shigella spp invasion. N-terminal region is most significant functional fragment of IpaD. Purification of IpaD from the wild type of Shigella is difficult furthermore profound study on a specific domain on the N-terminal of IpaD by using the wild type of purified IpaD is not feasible.

Engineered and construction of pDS132::∆virG as suicide vector for targeted gene deletion of virG from Shigella flexneri 2a in order to generation a live attenuated Shigella vaccine

Background & Objective: Shigella are Gram negative bacteria capable of inducing their entry into non-phagocytic cells via secretion of various effector proteins called invasion plasmid antigens (Ipas). The most important of them is VirG protein. Live attenuated Shigella vaccines have indicated promise in inducing protective immune responses in human clinical trials. In current situation, constructions of Shigella vaccine candidate strains based on classical allelic exchange systems are considered. The aim of this research was engineered and constructed pDS132::∆virG as a suicide plasmid for targeted deletion regions of virG gene by using allele exchange method in Shigella flexneri 2a. Method & Materials: In this applied study, species and serotype of shigella was confirmed by using serological and Polymerase Chain Reaction (PCR) tests. Detection primers of virG gene were designed and cloned to pGEM-5zf vector and finally, sequencing was done. According to virG restriction enzyme map, 1751 bp of virG gene was removed by using of HincП restriction enzyme and the ∆virG was successfully constructed. The pGEM∆virG vector was digested by use of SphI and SalI enzymes and then cloned to pSD132 as suicide vector. Precision of process were verified through phenotype and genotype experiment. Results: The Shigella flexneri type 2a strain was verified by serological and PCR tests. Sequence of the virG gene in native strain was sequentially identical with the strains submitted in the Gene-Bank database. Since the pDS132::∆virG contains 1484 bp which derived from virG gene, therefore, it can be utilized for interference in virG gene as specific suicide vector in shigella flexneri 2a. Conclusion: Application of suicide systems facilitated mutant construction in more specific and effective method in comparison with the other early techniques such as serial passage

Engineered and construction of pDS132::∆virG as suicide vector for targeted gene deletion of virG from Shigella flexneri 2a in order to generation a live attenuated Shigella vaccine

Background & Objective: Shigella are Gram negative bacteria capable of inducing their entry into non-phagocytic cells via secretion of various effector proteins called invasion plasmid antigens (Ipas). The most important of them is VirG protein. Live attenuated Shigella vaccines have indicated promise in inducing protective immune responses in human clinical trials. In current situation, constructions of Shigella vaccine candidate strains based on classical allelic exchange systems are considered. The aim of this research was engineered and constructed pDS132::∆virG as a suicide plasmid for targeted deletion regions of virG gene by using allele exchange method in Shigella flexneri 2a. Method & Materials: In this applied study, species and serotype of shigella was confirmed by using serological and Polymerase Chain Reaction (PCR) tests. Detection primers of virG gene were designed and cloned to pGEM-5zf vector and finally, sequencing was done. According to virG restriction enzyme map, 1751 bp of virG gene was removed by using of HincП restriction enzyme and the ∆virG was successfully constructed. The pGEM∆virG vector was digested by use of SphI and SalI enzymes and then cloned to pSD132 as suicide vector. Precision of process were verified through phenotype and genotype experiment. Results: The Shigella flexneri type 2a strain was verified by serological and PCR tests. Sequence of the virG gene in native strain was sequentially identical with the strains submitted in the Gene-Bank database. Since the pDS132::∆virG contains 1484 bp which derived from virG gene, therefore, it can be utilized for interference in virG gene as specific suicide vector in shigella flexneri 2a. Conclusion: Application of suicide systems facilitated mutant construction in more specific and effective method in comparison with the other early techniques such as serial passage

Double Sandwich ELISA Modified Method for the Detection of Clostridium Botulinum Type E

Background & Objective: A very small amount of botulinum toxin can cause death and on the other hand, there is no cure for its poison other than antitoxin. Therefore, a diagnostic method that can detect very small amounts of botulinum toxin in a short time is very important. In this study, rapid and accurate detection of botulinum toxin type E has been performed with the double sandwich ELISA method. Materials & Methods: based on the double sandwich ELISA method two different antibodies that bind to epitopes without the interference of antigens is required. For this purpose, the rabbit antibody was poured at the bottom (Capture antibody) and mouse antibody at the top of the toxin (Detection antibody) of each well and the toxin was titrated from the first wells. Then, the modified sandwich ELISA system was set up and optimized for the detection of botulinum toxin type E.Results: The modified double sandwich ELISA method is able to detect Clostridium botulinum type E toxin to a value of 1 ng in less than 6 hours. Conclusion: The modified double sandwich ELISA method is very precise and fast in detecting Clostridium botulinum type E. This method is used for determining specific antigen in the unknown sample

Detection of sea, sec and seq genes in Staphylococcus aureus nasal sampling acquiring from healthy carrier

Background: Various assays have been used to identify of enterotoxins produced by Staphylococcus aureus and because of antigenic similarities among enterotoxins, serological assay may not always be practical. The aim of this study was to detect of S. aureus enterotoxins (SEA, SEC and SEQ) genes by multiplex PCR assay. Methods: Of 150 strains obtained from nasal carriers, 95 S. aureus were confirmed by biochemical test. Multiplex PCR assay for the detection of genes encoding staphylococcal enterotoxins A, C and Q genes (sea, c and q) S. aureus was used. The nuc gene, which encodes thermonuclease was used as a target DNA to identify S. aureus.Results: DNA amplification fragments for the staphylococcal nuclease gene (nuc) was 397 bp, 552 bp for staphylococcal enterotoxin A gene (sea), 271 bp for staphylococcal enterotoxin C gene (sec) and 122 bp for staphylococcal enterotoxin Q gene (seq). S. epidermidis used as negative control and did not yield a PCR product. Among the 95 healthy human isolates from nasal carriage, forty one isolates (43/1%) were diagnosed as sea, sec or seq-positive. Twenty four (25/3%) isolates were sea gene, nine (9/5%) isolates were the sec gene and eight (8/4%) isolates were the seq gene and 54 (56/8%) of them were other se genes. Conclusion: Because Staphylococcus aureus was isolated in nasal healthy carrier, so the PCR assay could be useful in the routine direct detection of staphylococcal enterotoxin A, C and Q genes