Cloning, bioinformatics study and evaluation expression of gene of enterotoxigenic Escherichia coli CFA/I major subunit (CfaB)

زمینه و هدف: اشرشیاکولی انتروتوکسیژنیک (ETEC) عامل اصلی اسهال کودکان در کشورهای در حال توسعه و مسافران به این مناطق می‌باشد. از طریق ساختارهای سطحی به نام فاکتورهای کلونیزاسیون به سلول‌های میزبان متصل می شود. اشرشیاکولی انتروتوکسیژنیک در بسیاری از مناطق شیوع فاکتور کلونیزاسیون نوع یک (CFA/I) از سایر فاکتورهای کلونیزاسیون بیشتر می باشد و این ترکیب به عنوان ترکیب کلیدی در تولید واکسن محسوب می‌گردد. مولکول کد کننده زیر واحد اصلی آنتی ژن فاکتور کلونیزاسیون (CfaB) به عنوان زیر واحد اصلی این فیمبریه کاندیدای مناسبی برای واکسن می‌باشد. این مطالعه با هدف همسانه سازی ژن CfaB و بررسی تولید پروتئین نوترکیب انجام شد. روش بررسی: در این مطالعه توصیفی آزمایشگاهی اطلاعات مربوط به ژن فاکتور کلونیزاسیون CfaB از بانک ژن استخراج شد و پرایمرهای مناسب آن طراحی شدند. با استفاده از واکنش PCR، ژن مورد نظر تکثیر و سپس درون ناقل همسانه‌سازی (pTZ57R/T) و سپس در ناقل بیانی (pET28a(+)) همسانه سازی شد و بیان ژن CfaB مورد ارزیابی قرار گرفت. یافته‌ها: صحت همسانه سازی با استفاده از واکنش هضم آنزیمی و تعیین توالی تایید شد. بیان این توالی در شرایط مختلف مانند دما، میزبان و محیط کشت های مختلف بررسی گردید اما توالی طبیعی در pET 28a بیان نداشت. نتیجه‌گیری: در طول توالی ژن کد کننده زیر واحد اصلی آنتی ژن فاکتور کلونیزاسیون کدون های نادر پشت سرهم وجود دارد که باعث کاهش بیان این پروتئین می شود

An LTB-entrapped protein in PLGA nanoparticles preserves against enterotoxin of enterotoxigenic Escherichia coli

Objective(s): Enterotoxigenic Escherichia coli (ETEC) is known as the most common bacterial causes of diarrheal diseases related to morbidity and mortality. Heat-labile enterotoxin (LT) is a part of major virulence factors in ETEC pathogenesis. Antigen entrapment into nanoparticles (NPs) can protect them and enhance their immunogenicity.Materials and Methods: In the present study, recombinant LTB protein was expressed in E. coli BL21 (DE3) and purified by an Ni-NTA agarose column. The protein was entrapped in PLGA polymer by the double emulsion method. NPs were characterized physicochemically and the protein release from the NPs was evaluated. ELISA assay was performed for investigation of raised antibody against the recombinant protein in mice. The anti-toxicity and anti-adherence attributes of the immune sera against ETEC were also evaluated.Results: It showed the successful cloning of a 313 bp DNA fragment encoding LTB protein in the pET28a vector. Over-expression in BL21 (DE3) led to the formation of corresponding 15.5 kDa protein bands in the SDS-PAGE gel. Western blotting by using anti-CTX confirmed the purified LTB. Protein-entrapped NPs had a spherical shape with the size of 238 nm mean diameter and 85% entrapment efficiency.  Immunological analyses showed the production of a high titer of specific IgG antibody in immunized animals. The neutralizing antibody in the sera of immunized animals was approved by GM1 binding and Ileal loop assays.Conclusion: The results indicate the efficacy of the entrapped LTB protein as an effective immunogen which induces the humoral responses

Immunogenicity evaluation of rBoNT/E nanovaccine after mucosal administration

Objective(s): The Botulism syndrome is caused by types A to G of botulinum neurotoxins. The binding domains of these neurotoxins are immunogenic and considered as appropriate candidate vaccines. Due to the low immunogenicity of recombinant vaccines, there have been many studies on the use of biocompatible carriers such as chitosan nanoparticles for the delivery of these vaccines. The aim of this study was evaluating the efficiency of chitosan nanoparticles as carriers for a candidate vaccine, binding domain of BoNT/E, through oral and intranasal routes.Materials and Methods: Chitosan nanoparticles containing rBoNT/E binding domain, were synthesized via ionic gelation. After administration of the nanoparticles to mice through oral and intranasal routes, antibody titers were assessed by ELISA and, finally, all groups were challenged by active botulinum neurotoxin type E.Results: The groups that received nanoparticles containing the antigen, through oral and intranasal routes, and the group that received the bare antigen orally, were able to tolerate 5×102 folds of MLD. The intranasally immunized mice by the bare antigen were able to tolerate 2×103 folds of the toxin’s MLD. Conclusion: It seems that the use of chitosan nanoparticles has no significant effect on the protective immunization of the mice against botulinum BoNT/E in either route (P>0.05), even intranasal administration of the bare antigen gives better mice immunization against the toxin

Biodegradation of Chlorpyrifos and Diazinon Organophosphates by Two Bacteria Isolated from Contaminated Agricultural Soils

Introduction: The organophosphate insecticides such as chlorpyrifos and diazinon have been widely used to control insects in home, agriculture, and veterinary. These compounds are a threat to public health and the environment; on the other hand, they have low degradation rate and therefore can be stable for a long time in soil. A major factor determining the fate of organophosphate insecticides in soil and water is biodegradation. Materials and methods: Two diazinon and chlorpyrifos degrading bacterial strains were isolated from pesticides contaminated soils. The isolated bacterial strains were identified by 16S ribosomal RNA gene and fatty acid methyl ester analysis. Results: Strong correlation was seen between microbial growth and the two organophosphates degradation. On average, bacterial strain 1 and 2 degraded 88.27% and 82.45% of initial applied diazinon in medium and degraded 81.07% and 88.35 % of initial applied chlorpyrifos during 20 days, respectively. The isolated bacterial strains were identified as Acinetobacter and Pseudomonas sp. The highest diazinon degradation were found by Acinetobacter and the highest chlorpyrifos degradation were found by Pseudomonas when cultivated in the mineral salt medium. Discussion and conclusion: The identified pure bacterial strains utilized chlorpyrifos and diazinon as a source of carbon. They were able to degrade most of the parental molecule in 20 days. Therefore, the isolated bacterial strains may have the potential for use in the bioremediation of diazinon and chlorpyrifos-contaminated soils

Presenting a rapid method for detection of Bacillus cereus, Listeria monocytogenes and Campylobacter jejuni in food samples

Objective(s): Listeria monocytogens, Bacillus cereus and Campylobacter jejuni are three toxin producing bacteria over the world, especially in Iran, and it is essential to find a certain, rapid procedure to identify these microorganisms. In this research, these bacteria were simultaneously detected by multiplex PCR technique in foods. Materials and Methods: The primary approval of bacterial strains was performed by biochemical tests. PCR primers were designed based on the nucleotide sequences of the NHEB/NHEC gene of B. cereus, the hly gene of L. monocytogenes and the C gene of C. jejuni. The specificity of Multiplex PCR method was determined using seven food poisoning bacteria including Salmonella typhi, Shigella dysentery, Yersinia pestis, Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum and Vibrio cholerae. To confirm the reaction, DNA extraction was performed from 30 food samples (milk), and gene amplification was performed by PCR. The length of amplified fragments was 300 bp, 210 bp and 160 bpfor NHEB/NHEC, hly and C genes, respectively. Results: The detection limits of the PCR method were 5, 4 and 3 pg for L. monocytogenes, B. cereus and C. jejuni, respectively. Specifisity test showed that this reaction is spesific to these 3 bacteria. Conclusion: In this study, we  introduced a new multiplex PCR method for simultsnus detection of L. monocytogens, B. cereus and C. jejuni. These results can be used  for detection of other toxin producing bacteria in food

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.

Molecular Typing Isolates of Salmonella Enterica Serovar Infantis Using Eric-PCR Method

Background: Salmonella is the important bacteria of the Enterobacteriaceae family that are very diverse biochemically and serologically, and mainly transmitted through food. The spread of non-typhoid Salmonella is one of the challenging issues in current research. Therefore rapid diagnosis and typing of the pathogensprovide efficient information about detection and controlling of infection. The aim of this study is typing the clinical strains of Salmonella Infantis. Materials and Methods: In this study, strains of Salmonella Infantis were isolated from different health centers. All of the strains were identified by standard microbiology, biochemical and molecular methods. Genetic relationship between strains was analyzed by ERIC-PCR method. Results: In this study, from 842 stool and blood sample of patients with diarrhea, 48 different strains were isolated which related to Salmonella Infantis. Strains categorized into 14 different groups by genotyping using ERIC-PCR method that highest number of the strains were placed in group 5 (20%, 10 strains). Conclusion: Results of this study demonstrated that strains of Salmonella Infantis which are examined genetically were diverse which can be due to the prevalence of polyclonal strains in human samples. It was also shown that ERIC-PCR method has great differential power for the molecular typing. &nbsp

Genotyping of Clinical Isolates of Salmonella enterica serovar Typhimurium from Medical Centers of Kerman Province Using ERIC-PCR Method

Background and Objectives: Salmonella enterica serovar Typhimurium is one of the most common Salmonella serotypes in gastroenteritis cases. ERIC-PCR method is used for genotyping studies and examinations in order to apply appropriate preventive and controlling conditions. This research was carried out with the objective of investigating the genotyping of Salmonella typhimurium strains in the treatment centers of Kerman province.   Methods: In this descriptive study, different strains of Salmonella Typhimurium, were isolated from different treatment centers. All strains were examined using standard methods of microbiology, biochemistry, and molecular biology. Genetic relationship between the strains was evaluated by ERIC-PCR method.   Results: In this study, Among 891 stool and blood samples of patients with diarrhea, 48 strains of Salmonella typhimurium, were isolated. The isolates were classified in terms of genotyping into 15 different groups using ERIC-PCR method, which the highest number of the strains was in the 14th group (25%, 12 strains).   Conclusion: The results of the present study showed high discrimination power of ERIC-PCR method for molecular typing and genetic diversity of Salmonella typhimurium strains isolated from human samples. Therefore, this method can be used for epidemiological studies in order to investigate the source of contamination, genetic diversity and its relationship with geographical distribution and drug resistance of the isolated strains

Evaluation of class 1, 2 and 3 integrons in clinical Salmonella enteritidis strains by PCR method

Background & Objective: Gastroenteritis is one of the most common Salmonella infections in human which is caused by Salmonella serotypes especially S.enteritidis and S.typhimurium. The spread of multi-drug resistant (MDR) Salmonella strains is a serious global issue. Obtaining integrons is considered as one of the most important factors in multi-drug resistance among gram-negative microorganisms, particularly in intestinal bacteria. The aim of this study was to investigate the molecular level of class 1, 2 and 3 integrons which are the most important integrons in Salmonella enteritidis isolated from patients using Multiplex PCR. Methods: In this study, 567 stool and blood samples were collected from patients with acute gastroenteritis and Salmonella enteritidis were detected using culture method, standard biochemical test, and PCR. After DNA extraction, the presence of class 1, 2, and 3 of integrons was analyzed by multiplex PCR. Results: From 567 samples, 48 strains were identified as Salmonella enteritidis. Of all 48 strains, 45 strains (95%) had the intI gene, 7 strains (14.5%) had the intII gene, and 2 strains (4%) had the intIII gene. Conclusion: In this study, high incidence of class 1, 2 and 3 integrons was detected. Screening integrons as a sign of obtaining and expansion of antibiotic resistance could be considered as an important mechanism to deal with antibiotic resistance in microorganisms

Identification and assessment of Salmonella typhimurium, infantis and enteritidis serotypes in clinical samples from medical centers of Kerman province

Background and Aim: Salmonellosis is one of the important infectious diseases and can be as spread disease between humans and animals that make it essential for identification and detection of Salmonella. Housekeeping genes are typically important genes which are necessary for maintenance and survival of basic cells and can be considered as a gene diagnostic screening bacterial agents. The aim of this study was analysis of Flic, Sdf1 and FljB, housekeeping genes for screening of typhimurium, infantis and enteritidis serovars isolated in Kerman’s hospitals. Materials and Methods: In a descriptive study from February 2015 to August 2015, 132Salmonella specimens were taken from patients with acute gastroenteritis referred to different medical centers and hospitals in Kerman. The specimens were transferred to microbiology laboratory for identification of Salmonella with serological and bacteriological standard methods. DNA of Salmonella genus strains were extracted by CTAB and, specific primers of housekeeping genes of Salmonella genus (invA) and typhimurium(fliC), infantis(fljB) and, enteritidis(sdfI) serotypes are used in PCR test. Results: Using PCR technique, the presence of Salmonella genus were confirmed by amplification of invA gene in 130 out of 132 specimens which are identified as a Salmonella by microbiological and biochemical methods (98%). Also results indicating the prevalence of 19% in infantis, 22% in Salmonella typhimurium and 32% in Salmonella enteritidis. Conclusions: Results showed that the prevalence of Salmonella enteritidis is more than any other serotypes in this region, but as the global statistics, the prevalence of typhimurium and Infantis are increasing