Metagenomic investigation of bacteria associated with dental lesions : a cross-sectional study

Dental caries is considered as one of the most significant global health problem over the world. Dental caries initiates from bacterial shifts within the supragingival biofilm, then a polymicrobial biofilm is formed on the surface of tooth, and finally various bacterial species aggregate in a complex-organized manner. The exploiting variability in 16S rRNA gene sequence has been considered as a cost-efficient high-throughput characterization approach in human oral microbiome investigations. The aim of this study is to characterize bacterial species associated with superficial dental biofilm, underlying carious dentine and root caries lesion by16S rRNA gene-based metagenomic analysis. Herein, the bacterial communities in carious dentin lesion, biofilm and root canal samples of 30 subjects (aged 4?76 years) admitted to a clinic in Tehran during 2017 were investigated using a culture independent approach. Total genomic DNA of each tissue was subjected to metagenomic identification of bacteria using a nested PCR assay and 16S rRNA library construction method. 31 samples collected from 30 consenting patients (29 samples from 29 patients ant two biofilm samples from one patient). Bioinformatics analyses of a-800bp sequences of the second step of Nested-PCR revealed presence of 156 bacterial isolates in carious (n=45), biofilm (n=81) and root canal (n=30) specimens. Prevotella spp., Lactobacillus vaginalis, and streptococcus spp. showed higher prevalence in carious dentin, root and biofilm samples, respectively. Exploring the dental microbiota and comparing them in health or diseased conditions is critical step in the determination of human general health. The method applied in this study could identify bacteria related to the three dental lesions. However, due to lack of data for comparison in Genbank or because of the sequence similarity lower than 98% for most identified bacteria, the use of more powerful approaches like NGS platforms or typing of multiple loci (MLST) in future studies is recommended

Ekspresija i pročišćavanje površinskog lipoproteinskog antigena Lipl41 iz patogenih bakterija roda Leptospira

Pathogenic species of Leptospira lead to a zoonotic disease called leptospirosis, which is spread worldwide. A major topic of investigation is to detect the antigens that induce an immune response and to utilize them in diagnostic kits or vaccine development. The outer membrane proteins (OMPs) of Leptospira are potential candidates for this purpose. Lipl41 is an OMP that is conserved among pathogenic Leptospira. The aim of this study was to express and purify the Lipl41 recombinant protein in Iranian isolates. All collected Lipl41 protein sequences were compared and analyzed using bioinformatics tools from NCBI databases. Complete codon sequences of the Iranian pattern of Lipl41 recombinant protein were codon optimized and sub-cloned into a pET32a+ expression vector, and transformed into Escherichia coli BL21 (DE3). Optimal expression of recombinant Lipl41 (47kDa) was achieved post-induction with IPTG within the inclusion body. It was then purified by denaturation using serial concentrations of urea, and the recombinant protein was confirmed by western blot. In this study, sufficient amounts of Lipl41 were expressed and purified to be used for the development of a diagnostic kit and subunit vaccine.Patogene vrste Leptospira uzrokuju zoonotsku bolest leptospirozu koja je raširena u cijelom svijetu. Cilj istraživanja bio je dokazati antigene koji potiču imunosni odgovor i mogu se primijeniti u dijagnostičkim kompletima ili za razvoj cjepiva. Vanjska proteinska membrana (OMPs) leptospira potencijalni je kandidat za tu svrhu, a gen Lipl41 je dobro očuvan među patogenim leptospirama. Ekspresija i pročišćavanje rekombinantnog proteina Lipl41 provedeno je u izolatima iz Irana. Uspoređene su sve prikupljene sekvencije proteina Lipl41 i analizirane bioinformatičkim alatom iz baze podataka NCBI. Kompletne sekvencije kodona rekombinantnog proteina Lipl41 u izolatima su optimizirane, zatim subklonirane u pET32a+ vektor i pretvorene u bakteriju Escherichia coli BL21 (DE3). Optimalna ekspresija rekombinantnog Lipl41 (47kDa) postignuta je post-indukcijski pomoću isopropyl β-d-1-thiogalactopyranoside (IPTG). Zatim je pročišćena denaturacijom što je uključilo primjenu serijskih koncentracija ureje. Rekombinantni protein je potvrđen metodom western blot. Istraživanje potvrđuje mogućnost ostvarenja dostatne količine i ekspresije pročišćenog Lip141 da se može upotrijebiti za razvoj dijagnostičkih kompleta i subjediničnih cjepiva

Prevalence of Gardnerella vaginalis infection and antibiotic resistance pattern of isolates of gynecology clinic patients at Shahriar Noor Hospital from January to June 2020 by PCR and culture methods

Background and Objectives: Gardnerella vaginalis is one of the most important causes of prevalent genital infections that pose serious risks. This study aimed to determine the prevalence of Gardnerella vaginalis and antibiotic resistance pattern of isolates of patients referred to the gynecology clinic of Shahriar Noor Hospital by PCR and culture methods. Materials and Methods: The study was conducted on 500 patients who had suffered from a vaginal infection. The demographic data of patients were studied. For diagnosis of Gardnerella vaginalis isolates, cultivation in anaerobic conditions, biochemical tests, PCR and Gardnerella vaginalis antibiotic susceptibility test to metronidazole and clindamycin were performed. Data analysis was performed utilizing SPSS statistical software version 19 and the Chi-square test. Results: Among the 500 patients, 173 were diagnosed with Gardnerella vaginitis. There was a significant relationship between age group, level of education, and contraceptive method with Gardnerella vaginosis incidence. Performing antibiotic susceptibility tests showed that the resistance of Gardnerella vaginalis isolated strains to metronidazole and clindamycin was 86.12% and 17.34%, respectively. Conclusion: The high prevalence of Gardnerella vaginalis infections confirms the critical role of the bacterium in the occurrence of bacterial vaginosis. Therefore, it is necessary to check the prevalence of bacterial infections to recommend the correct medical treatment in different societies

Molecular Detection of Shigella spp. Contamination in Ready-to-Eat Salad Samples in West of Tehran

Background: Shigella bacteria can infect human body by taking contaminated food and water and are transmitted from person to person. Human body is the only natural host for these bacteria. Objective: The aim of this study was to detect Shigella contamination in pre-packed samples of salads at restaurants in western regions of Tehran city using PCR method. Methods: To conduct this research, 90 samples were purchased from the restaurants during the period of June to November 2016. The samples were cut into very small pieces, homogenized and a 25g portion of these samples was added to 225 ml of Shigella broth media containing novobiocin and incubated for 24 hours. Then DNA of cultured samples was extracted using DNPTM kit (CinnaGen, Iran) and PCR method was optimized for amplification of 613 bp segment of ipaH gene and performed on extracted DNA of all samples (before and after enrichment in Shigella broth). Results: Shigella contamination was detected in 7(7.8%) and 20(22.2%) of the tested samples before and after the enrichment, respectively. Conclusion: The results showed the contamination with Shigellabacteria in remarkable percentage of the samples and revealed the necessity of more attention and supervision in the processes of production and distribution of pre-packed salads. Besides, the findings points to the importance of enrichment in increasing the sensitivity of the PCR method to detect the bacteria in food samples

The combined use of recombinant and pVAX-omp31 DNA Vaccine for immunological protection against pathogenic Brucellas melitesnsis in an experimental model of BALB/c Mice

Background and Aim: Brucellosis is still an important zoonotic infection and evaluation of immunologic properties of various bacterial antigens along with different vaccination strategies helps in designing efficient vaccines against the disease. The aim of this study is to immunological evaluate the eukaryotic vector pVAX1, carrying the outer membrane protein gene of 31 kDa (Omp31) B.melitensis. Materials and Methods: In this study which was carried out in 2014, whole sequence of omp31 of B. melitensis was inserted between BamHI and XhoI of pVAX1 plasmid vector. Female BALB/c mice aged 6-8 weeks (purchased from Pasteur Institute of Iran) were immunized intra-muscularly with 100μg of the construct, followed by either protein or plasmid boosters separately. The level of IL-4, IL-12, IFN-γ, total serum IgG, and specific IgG1 and IgG2a against recombinant Omp31 were evaluated. Finally the protective immune response following exposure to B. melitensis 16M was evaluated. Results: DNA-vaccine omp31 career with protein reminders Omp31, stimulate higher levels of IFN-γ, IL-12 and IgG2a compared to groups of DNA-vaccine or recombinant protein. Protective immunity was also significantly higher in mice which immunized with DNA vaccine– protein regimen. Conclusions: Mice which immunized with DNA vaccine–protein regimen showed a significantly higher levels of IL-12 and IFN-γ along with serum IgG2a which together imply augmentation of T cell-mediated immune responses against Omp31. The latter was confirmed by significant protective response to B. melitensis 16M challenge

FMD virus type Asia-1 irradiated vaccine and evaluation of immune response on guinea-pig model

Introduction: Foot and Mouth Disease (FMD) is the most contagious disease in cloven-hoofed animals which causes a lot of economical losses. FMD virus belongs to Picornaviridae family and Aphthovirus genus. The aim of the study is evaluation of humoral and cellular immune responses triggered by a Gamma-irradiated FMD vaccine in the guinea-pig model. Materials and Methods: FMD virus type Asia-1 was multiplied on BHK21 cell line and irradiated by gamma ray in different doses. According  to dose/survival curve, D10 value and optimum dose of virus inactivation were calculated 7.69 and 50 kGy, respectively. Antigenic characteristic of irradiated and un-irradiated virus samples were evaluated by complement fixation test (CF test) and safety test was done by four blind passages cell culture on IBRS2 cell line. The inactivated virus sample was formulated as Radio- vaccine and immune responses were evaluated in three groups of ginea-pigs; the first group Radio-vaccine, the second group conventional vaccine and the last one was negative control. Results: The results of neutralizing antibody titration for two vaccinated groups were significant (

High prevalence of Mucosa-Associated extended-spectrum β-Lactamase-producing Escherichia coli and Klebsiella pneumoniae among Iranain patients with inflammatory bowel disease (IBD)

Abstract Background Several pieces of evidence suggest that certain pathobionts belonging to Enterobacterales are associated with the development and progression of inflammatory bowel diseases (IBD). Extended-spectrum β-lactamases (ESBLs) ESBLs are frequently found in the Enterobacterales members, particularly in Escherichia coli and Klebsiella spp., and might trigger antibiotic-induced perturbations of the intestinal microbiota and led to more severe disease activity in IBD. Therefore, the severity of IBD could be influenced by ESBL-producing Enterobacterales, and hence, this study aimed to investigate the presence of ESBLs and carbapenemases among mucosa-associated E. coli and Klebsiella pneumoniae isolated from colonic biopsies of Iranian patients with IBD. Methods In this cross-sectional study, E. coli and K. pneumoniae were isolated from inflamed ileum and/or colon tissue of patients with IBD, including Ulcerative colitis (UC) and Crohn’s disease (CD), during colonoscopy. Demographic data and clinical characteristics were recorded, and UC and CD disease activity and extent were evaluated according to the full Mayo score and Crohn’s disease activity index (CDAI), respectively. Phenotypic and molecular detection of ESBL- and carbapenemase-producing E. coli and Klebsiella pneumoniae were carried out. Disease activity and other clinical and microbial features were compared in patients with and without gut colonization with ESBL producers. Results A total of 83 IBD patients, including 67 UC and 16 CD, were enrolled in the initial analysis. Intestinal colonization with ESBL-producing E. coli and/or Klebsiella pneumoniae was found in 37 (55.2%) of UC and 9 (56.2%) of DC patients – mostly harbored E. coli containing the bla CTX−M and bla TEM genes. UC patients with intestinal colonization with ESBL-producers had more severe disease compared with patients without colonization. Moreover, 10.2% of tested E. coli and 34.8% of K. pneumoniea were recognized as potential carbapenemase producers. Conclusion Intestinal colonization with ESBL producers could arise disease activity in IBD patients. Further large-scale case-control studies should be performed to investigate the possible confounding factors that could contribute to this outcome

First Molecular Detection of Saffold Virus in Children with Acute Gastroenteritis in Iran: Detection of Saffold Virus in Children

Background: Saffold virus as a new member of cardiovirus genus in picornaviridae family has been suggested to be related to diarrheic cases and human airway diseases. However, relationship between Saffold virus and human diseases is unclear. In order to establish an investigation for the occurrence of Saffold virus among pediatric patients involved to acute gastroenteritis, we implemented a RT-PCR assay for detection and quantification of Saffold virus in stool specimens. Materials and Methods: In this study, a total of 160 stool samples from September 2018 to May 2019 were collected from presenting pediatric patients with acute gastroenteritis in a Karaj hospital, Iran. After viral RNA extraction, the RT-PCR was performed to amplify the 5’UTR region of Saffold virus genome. Results: Out of the 160 samples tested, the Saffold virus genomic RNA was detected in 26/160 (16.2%) of stool samples. The high Saffold virus detection rate was related to February (6/26 or 23%). The co-infection of Saffold virus with Aichivirus and Salivirus as other new emerging viruses was also assessed, among which high double or triple mixed-infections were determined. Conclusion: This is the first documentation of Saffold virus detection in stool samples that demonstrates Saffold virus has been circulating among Iranian pediatric patients. Our results indicated that Saffold virus in association with Aichivirus and Salivirus may be possibly considered as causative agent of acute gastroenteritis