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Background and Aim: Oral infections are common among people of any age and can trigger systemic inflammation. The microbiota is a diverse group of microorganisms that play important roles in metabolism, immune function, and homeostasis. Oral microbiota in human atherosclerotic plaques has been identified using various techniques. Therefore, the focus of this study was to determine the correlation between metabolic parameters and oral microbiota composition in patients with atherosclerosis using Denaturing Gradient Gel Electrophoresis (DGGE) assays. Materials and Methods: In this case-control study, saliva samples were collected from 139 patients with atherosclerosis and healthy individuals from Imam Ali Cardiovascular Hospital, Kermanshah, Iran. After DNA extraction, PCR products were examined and evaluated using DGGE assays. Results: The study included 89 (36%) patients with a history of atherosclerosis and 50 (36%) healthy individuals. There was a significant relationship between the mean total cholesterol, Low-Density Lipoprotein (LDL), Fasting Blood Sugar (FBS), and Blood Urea Nitrogen (BUN) in the two groups. However, there was no significant difference in the mean high-density lipoprotein (HDL) and Triglyceride levels between the study groups. Conclusion: Our results showed a relationship between metabolic parameters and oral microbiota composition in patients with atherosclerosis. Additionally, our results indicated that the DGGE assay is a useful method for diagnosing and comparing the oral microbiota of people with atherosclerosis and healthy individuals. Therefore, further examination of the oral microbiota is necessary to determine its potential as a biomarker for atherosclerosis. [ABSTRACT FROM AUTHOR]

Background and Aim: Entamoeba histolytica and Giardia intestinalis are two common parasites associated with gastrointestinal infections and diarrheal diseases worldwide. This study investigated the prevalence, molecular characterization, and phylogenetic relationships of E. histolytica and G. intestinalis among patients with diarrhea and intestinal disorders in hospitals across Iraq. Materials and Methods: Stool samples were collected from individuals presenting with gastrointestinal symptoms, and a microscopic examination was performed to screen for the presence of E. histolytica and G. intestinalis. DNA was extracted from positive samples, and PCR amplification targeting the 16S rRNA gene for E. histolytica and the triose phosphate isomerase (TPI) gene for G. intestinalis was carried out. The resulting PCR products were subjected to agarose gel electrophoresis, purified, and sequenced. Phylogenetic trees were constructed using the Maximum Likelihood method based on the Tamura-Nei model, and bootstrap analysis was performed. Results: Among the studied samples, 27.8% (n=85) were positive for E. histolytica, while 19.6% (n=60) were positive for G. intestinalis. Sequence analysis confirmed the presence of E. histolytica and G. intestinalis by matching the obtained sequences with those available in the NCBI gene bank. The phylogenetic analysis revealed the genetic diversity and relatedness of the E. histolytica and G. intestinalis isolates. Clustering patterns specific to the Iraqi population were observed, suggesting potential regional transmission dynamics. Conclusion: This study provides valuable insights into the prevalence, molecular characterization, and phylogenetic relationships of E. histolytica and G. intestinalis in Iraqi patients with diarrhea and intestinal disorders. [ABSTRACT FROM AUTHOR]

Background and Aim:. Pseudomonas aeruginosa exotoxins are responsible for pathogenicity and antibiotic resistant. The aim of this study was to investigate the effects of the Ammi. visnaga (A. visnaga) extract on the P. aeruginosa exotoxin A and S genes expression. Materials and Methods: Antibacterial effects of the A. visnaga ethanolic extract were performed using the micro dilution technique in sequential dilutions. Then, the Real-Time- RT PCR method was used to analyze the level of expression of the exotoxin A and S genes of the bacterium. Real-Time PCR data were analyzed using Graph Pad Software Inc. San Diego (GRAPHPAD PRISM 6CA, USA). Results: Results showed that, the expression of Exotoxin A and S genes was significantly (P <0.001) reduced in comparing the expression level of the reference gene and control. It had a significant effect on the inhibition of bacterial growth at different concentrations but the lowest inhibitory concentration was observed at 3.125 µg/well. Conclusion: Accordingly, the extract of A. visnaga can decrease the expression of the Exotoxin A and S genes of P. aeruginosa and consequently it may decrease the pathogenicity of bacteria. These phenomena (exoA and exoS expression inhibition) may involve reducing the antibiotic resistance of P. aeruginosa. [ABSTRACT FROM AUTHOR]

Background and Aim: Otitis media (OM) or middle ear inflammation is an inflammatory disease caused by a bacterial infection such as Pseudomonas aeruginosa (P. aeruginosa), provided with genetic virulence factors. This study aims to detect nan1 and tox A genes of P. aeruginosa isolated from chronic OM patients. Materials and Methods: One hundred thirty OM patients were recruited from Al-Sader medical city and Al-Hakim general hospital in Najaf province from September to January 2021 to collect ear swaps. The Primary identification depends on Gram stain and biochemical tests. We also used Vitek 2 system to make another identification and examine resistance to antibiotics. A polymerase chain reaction (PCR) was used to amplify the required genes. Furthermore, gel electrophoresis was performed to show the amplified genes. Results: The results have revealed that 115 samples (88.46%) give a positive result, while 15 samples (11.54%) are negative for culture bacteria. Only 49 (42.76 %) of the 115 clinical specimens showed positive tests for P. aeruginosa. However, the toxA gene is found in 67.3% of P. aeruginosa isolates, while the nan1 gene is found in 38.77 % of the isolates. Conclusion: Our findings suggest that P. aeruginosa is one of the most common causes of OM infection in Iraqi patients. Most bacterial isolates show the presence of nan1 and tox A genes, with a high prevalence of tox A than nan1. The presence of these genes is one of the virulence factors and probably partly explains the resistance to antibiotics. [ABSTRACT FROM AUTHOR]

Background and Aim: The healthy people’s fecal flora in the community represents a large potential reservoir. Therefore, the current study aimed to detect antibiotic resistance patterns and virulence factors in Klebsiella pneumoniae isolated from healthy volunteers’ feces. Materials and Methods: Three hundred and fifty stool specimens were collected from sales rep healthy individuals referring to the Northwest Tehran Health Centers to get a health card. Bacterial isolation, identification, and antimicrobial susceptibility testing were conducted according to the routine instructions. In addition, polymerase chain reaction (PCR) was used to detect the genetic factors responsible for producing extended-spectrum β-lactamases (ESBLs: SHV, TEM, and CTX-M) blaKPC and other virulence genes. Results: Among fecal samples analyzed, 60 (17.1%) K. pneumoniae were isolated. The results demonstrated that the highest resistance rate was related to piperacillin-tazobactam (n=25, 41.6%), followed by and meropenem (n=17, 28.8%) and cotrimoxazole (n=11, 18.3%), respectively. Also, all strains were susceptible to amikacin, gentamicin, and imipenem. The PCR results of the virulence gene showed that 95% (n=57) of isolates were positive for fimH gene, 93.33% (n=56) for BssS gene, 27 (45%) for rmpA gene. The PCR results for antibiotic resistance genes showed that 41.66% (n=25) had blaTEM gene, 38.33% (n=23) blaCTX-M gene, 35% (n=21) blaSHV gene and 3.33% (n=2) isolates had blaKPC gene, and none of these isolates carried magA gene. Conclusion: Antibiotic resistance was common among K. pneumoniae isolated from healthy volunteers’ feces who participated in this study. Transmission of resistant bacteria and plasmids through oral-fecal sources threats to the public, which could complicate treatment options for community-acquired infections caused by K. pneumoniae. [ABSTRACT FROM AUTHOR]

Background and Aim: Penicillinase plays diverse roles, such as inducing drug resistance by hydrolyzing the amide bond of beta-lactam factors, the metabolism of cephalosporin-attached prodrugs, as well as the metabolism of some antibiotics, and renal clearance. The present study aimed to evaluate the optimum conditions for extracting and purifying penicillinase enzyme. The enzyme source was Bacillus licheniformis 6346 obtained from the Iranian Research Organization for Science and Technology. Materials and Methods: B. licheniformis was cultured in a medium containing mineral salts, nitrogen compounds and carbon sources. Following the harvesting and preparation of bacterial suspension, the destruction of the bacterial cell wall and crude cell extraction was completed using a combination of lysozyme and weak ultrasonication. Afterward, enzyme amount was determined utilizing chromatography and electrophoresis. Furthermore, the optimum growth temperature and incubation time were assessed based on the absorbance of samples at 240 nm, enzyme activity, aerification intensity optimization, enzyme production time, and optimum pH. Results: Approximately 8 g of cells were retrieved in one liter of culture. The optimum pH was determined as 6.5. The heat stability evaluation of the enzyme at different times revealed that the enzyme was stable for more than 1 h at 20°C-45°C, while it is inactivated in 10 min at 60°C. Moreover, the best incubation time for this bacterium was obtained as 9 h at 30°C. In terms of aerification intensity, the highest enzyme production rate of 2394 U/mL was achieved in a 250 mL Erlenmeyer flask with a 50 mL culture medium at 200 RPM. More aerification led to the inhibitory effect of oxygen and reduced enzyme activity. Conclusion: Our findings showed that the bacterium B. licheniformis 6346 can be used for producing penicillinase enzyme in optimum conditions (30°C, 200 RPM, and pH: 6.5). [ABSTRACT FROM AUTHOR]

Background: Influenza viruses cause Avian Influenza (AI) is a serious infectious disease belonging to type A Orthomyxovirus. A viral RNA synthesis is due to an interaction of the nucleoprotein (NP) with the viral polymerase. In the present study, we have evaluated the immunogenicity of avian influenza virus nucleoprotein. Materials & Methods: An Influenza Virus N9H2 subtype A/Chicken Iran/259/2014 was selected. In order to perform electrophoresis and purification of nucleoprotein, the protein concentration of the samples was determined. SDS-PAGE and Native-PAGE electrophoresis on polyacrylamide gel were done and differences in gel bands in two methods were compared. Purification of the virus nucleoprotein performed by electroelution method and purified nucleoprotein were assayed by ELISA to obtain the produced antibody titers. Ouchterlony and western blot tests were used for final approval. Results: By determining the molecular weight of each polypeptides, the molecular weights of H9N2 proteins were ranged from 30 to 140kDa and the molecular weight of the nucleoprotein was 60kDa. The nucleoprotein was purified by electroelution method. Ouchterlony showed that in serum dilution of 1:2 and 1:4, the sediment lines were formed between the serum and the NP antigen. The NP-ELISA enables rapid serological diagnosis in 1:20. Finally, the western blot test confirmed the 60kDa nucleoprotein band. Conclusion: The nucleoprotein which was purified by electroelution retained its antigenic property and it could be applied in diagnostic kits [ABSTRACT FROM AUTHOR]

Background and Aims: Many bacteria including Escherichia coli may enter into a viable but non-culturable (VBNC) state under unfavorable stresses, which are unable to be detected by culture-based methods. In this study, the use of Reverse Transcription PCR (RT-PCR) for detection of VBNC state of E. coli O157:H7 was investigated. Materials and Methods: Escherichia coli O157:H7 was inoculated in distilled water and 30 percent salt water at room temperature. The cultivability of bacteria was determined using routine culture and colony counting on MacConkey agar. The RT-PCR of 16S rRNA gene involved direct extraction and purification of RNA, DNase I treatment for removing DNA contamination, cDNA synthesis and electrophoresis of PCR products of cDNA was used to detect viable E. coli O157:H7 under studied treatments and was compared with the results of RT-PCR of 16S rRNA gene of heat- killed bacteria. Results: The cultivability of bacteria was maintained during the study period in distilled water; however, the use of 30% NaCl caused the bacteria to be non-cultivable on day 4. The RTPCR of 16S rRNA showed the positive expression of this gene in cultivable and non-cultivable bacteria during the study period, whereas heat-killed bacteria were negative for this gene, which indicated the efficacy of RT-PCR of 16S rRNA in differentiation of alive from dead bacteria. Conclusions: Escherichia coli O157:H7 entered into the VBNC under 30 percent NaCl which can be associated with serious human health problems. RT-PCR can be used to detect bacteria in the VBNC state. [ABSTRACT FROM AUTHOR]

Background and Aims: Brucellosis is an infectious zoonotic disease responsible for reproductive disorders among animals and significant economic losses to the meat and milk supplychain. The purpose of this study was therefore to evaluate and diagnose the species and strains of Brucella isolates among cattles via multiplex polymerase chain reaction. Materials and Methods: In this study, blood samples were taken from 22 cows diagnosed as having brucellosis by PCR test. Blood samples were cultured in BACTEC vials and incubated at 37 degrees Celsius for 5 days following which each sample was cultured on Brucella agar and kept for 3 days under Co2 condition.The 16SrRNA gene was used as the target for the detection of Brucella genus and the bcsp31 gene was used for the detection of Brucella biovars. The PCR products were electrophoresed on %1 agarose and compared with standard strains. Results: The PCR results showed a total of 22 positive samples for Brucella among cattles. Out of the 22 cattle positive samples, 19 were identified as B.abortus (biovars 1, 2, 3, 4, 5) and 3 as B. melitensis (biovars 1, 2, 3). Conclusions: Brucella abortus biovars 1 and 3 and B .melitensis biovar 1 are the most prevalent biovars among cattles in Lorestan province, Iran. Moreover, multiplex PCR method could be used to improve the qualitative level and the Brucella diagnosis speed in laboratories. [ABSTRACT FROM AUTHOR]

Background and Aims: Due to its self-assembly properties on variety of surfaces and creating regular functional groups, surface layer protein isolated from bacteria, has significant applications in the field of nanobiotechnology such as biosensor production, targeting drug delivery systems and tissue engineering. In this research, optimization of discontinuous culture medium compositions for the production of HPI surface layer protein from Deinococcus radiodurans R1 strain was performed using the response surface (RSM) method. Materials and Methods: In 2016, culture medium for 16 designed experiments with fractional factorial analysis (FFA) was prepared and effective factors among six variables of the discontinuous culture medium was investigated for the production of surface layer proteins of D.radiodurans R1. Twenty experiments were then designed for the optimization of effective variables using central composite design (CCD) method. Surface protein purity was assessed using SDS-PAGE analysis and its concentration was calculated via Bradford method. Results: The optimized medium containing 13.36 g/L glucose, 5 g/L yeast extraction, 5 g/L tryptone, 2 g/L HEPES buffer, 0.55 MgSO4*7H2O, 0.0368 g/L MnCl2*4 H2O, was determined. Wet cellular mass was found as 16.87 g/L, which is 24% more than TGY basic medium and 74% much more than TYG culture medium including NaCl. Conclusions: Results of the Bradford method demonstrated that the concentration of surface layer protein HPI isolated from D. radiodurans R1 was 5.6 mg which was more than twice of that using 1liter of basic TGY medium. [ABSTRACT FROM AUTHOR]

Background and Aims: Cholera is a lethal diarrheal disease that cause by Vibrio cholerae. Cholera toxin and colonization factor pili (tcpA) are the major virulence factor in V.cholerae pathogenesis. The B subunit of the enterotoxin )ctxB) which is responsible for toxin binding to eukaryotic cells and toxin-coregulated pili A (tcpA) that is essential for V.cholerae colonization, have immunogenic properties. Chimeric proteins carrying epitopes, linkers or adjuvant sequences could increase immunogenicity for recombinant antigens and can also elicit broad immune responses. The aim of this study was to design am immunogen against adherence and toxicity of V.cholorae. Materials and Methods: ctxB and tcpA genes were analyzed for rare codons and gene optimization was performed using optimization software In 2017. The half-life and protein instability index was determined. Secondary and tertiary structure was predicted and evaluated. Linear and conformational epitopes were predicted. Recombinant pET28a/chimeric gene plasmid was transformed to E.coli BL21 DE3 and expression was induced with Isopropyl β-D-1- thiogalactopyranoside (IPTG). The protein expression was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Chimeric protein instability index was 24.04 and the codon adaptation index of chimeric constructs increased to 0.9. The tertiary predicted structure of the chimeric proteins confirmed and mRNA was stable. Conformational and linear epitopes were seen in both domains of the chimeric protein. Restriction analysis confirmed cloning of the gene into pET28a vector. Expression of recombinant protein in E.coli led to the production of chimeric protein with 37 k Da molecular weight. Conclusions: According to the results of bioinformatics and recombinant protein expression, the design chimeric protein could be used as an immunogen to evaluate the immunity against cholera. [ABSTRACT FROM AUTHOR]