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.

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

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 ß-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 blaCTX-M and blaTEM 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.

The Effect of Cytotoxicity and Antimicrobial of Synthesized CuO NPs from Propolis on HEK-293 Cells and Lactobacillus acidophilus.

Background: Drug resistance is currently possible anywhere in the world. Due to the discovery of antimicrobials, medicine, and health have made tremendous advances over the past several decades. Aim: This research evaluated the antimicrobial and cytotoxicity effects of green synthesis of copper oxide nanoparticles (CuO NPs) on Lactobacillus acidophilus and human embryonic kidney 293 cells (HEK). Method and Materials. Propolis was sampled and extracted. Green synthesis of CuO NPs was synthesized and characterized using SEM, TEM, DLS, BET, and zeta potential methods. L. acidophilus (ATCC 4356) was used, and the antimicrobial tests were carried out at different concentrations (10≥ mg/ml). Moreover, the cytotoxicity was evaluated using an MTT assay on human embryonic kidney 293 cells (HEK). Results: Synthesized CuO NPs using propolis extracts from Khalkhal (sample 1) and Gillan (sample 2) showed -13.2 and -14.4 mV, respectively. The hydrodynamic sizes of well-dispersed samples 1 and 2 were 3124.9 nm and 1726.7 nm, respectively. According to BET analysis, samples 1 and 2 had 5.37 and 8.45 m2/g surface area, respectively. The surface area was decreased due to the addition of propolis extract, and the pore size was increased. CuO NPs of samples 1 and 2 were visible on SEM images with diameters ranging from 75 to 145 nm and 120 to 155 nm, respectively. Based on TEM analysis, the size of CuO particles was increased in samples 1 and 2. CuO NPs particles had narrow size distributions with evenly dispersed NPs on all sides. The cell viability of the CuO NPs of samples 1 and 2 after 24, 48, and 72 hours was greater than 50%. As a result of the MIC and MBC tests, it was determined that samples 1 and 2 had the same effect against L. acidophilus (0.0024 mg/ml). Biofilm formation and degradation of sample 1 were more efficient against L. acidophilus. Conclusion: There was no evidence of cytotoxicity in the samples. In addition, results showed that the green synthesized CuO NPs from Khalkhal propolis were effective against L. acidophilus. Thus, the green synthesized CuO NPs from Khalkhal propolis were the best candidates for clinical application.

Whole-genome sequencing of foot-and-mouth disease virus serotype O/PanAsia-2/QOM-15 and comparison of its VP1-encoding region with two vaccine strains.

Despite widespread vaccination against foot-and-mouth disease, many outbreaks still occur in endemic areas. We attempted to determine the genetic and antigenic properties of the O/PanAsia-2/QOM-15 foot-and-mouth disease virus new vaccine strain. Thus, whole-genome sequencing was used to identify vulnerable pinpoint sites across the genome. The VP1 sequence (1D gene) of the O/PanAsia-2/QOM-15 viral genome was then compared to the VP1 sequences of two previously used vaccine strains, O/PanAsia (JQ321837) and O/PanAsia-2 (JN676146). The antigenic relationship of these three viruses was calculated by the two dimensional-virus neutralization test. At the nucleotide level, 47 single variants were identified, of which 19.00% were in the 5' untranslated region (UTR), 79.00% in the polyprotein region, and 2.00% in the 3' UTR region. Approximately half of the single nucleotide polymorphisms that have occurred in 1D gene resulted in amino acid (AA) substitutions in the VP1 structure. The single nucleotide polymorphisms also caused AA substitutions in other structural proteins, including VP2 and VP3, and some non-structural proteins (Lpro, 2C, and 3A). The O/PanAsia-2/QOM-15 shared higher sequence similarity with O/PanAsia-2 (91.00%) compared to O/PanAsia (87.30%). Evaluating r-value showed that the antigenic relationship of O/PanAsia-2/QOM-15 with O/PanAsia-2 (29.00%) was greater than that of the O/PanAsia (24.00%); however, all three viruses were immunologically distinct. After 10 years, the alteration of virus antigenicity and the lack of detectable adaptive pressure on VP1 sequence suggest that studying genetic dynamics beyond the VP1 region is necessary to evaluate FMDV pathogenicity and vaccine failure.

Phylogenetic analysis of BKV genetic variations, based on the whole sequence of the genome and different genomic sections.

BK polyomavirus (BKV) primarily infects humans in their early life stages, and in later life stages, immunosuppressed patients may develop asymptomatic infections. The nucleotides 1744-1812 in the VP1 gene are traditionally used to determine this virus's genotype. The complete genome of the BKV samples from patients referred to Masih Daneshvari Hospital's Virology Research Center was amplified by previously known primer sets. The phylogenetic diversity of the whole genome, different genomic sections, and the noncoding control region of BKV samples were investigated. Using software Mega X and references, the samples' genotype was determined in separate genomic fragments and the whole genome. The samples were classified into two genotypes (I and IV) and five subtypes (Ia, Ib-1, Ib-2, IVc-1, and IVc-2), but none of the isolates belonged to genotypes II, III, V, or VI. The large T antigen-based phylogenetic tree provided 100% bootstrap values for these divisions, which were superior to those (96%-100%) used in the VP1 sequence. Among the genomic segments, large tumor antigen and VP1 had the most mutations. The noncoding control area contained mutations at the O41 position in the granulocyte/macrophage stimulus gene and the P31 position in the NF-1 gene. The validity of the phylogenetic analysis was supported by sequence analysis, which found single-nucleotide polymorphisms (SNPs) that could be useful for subclassifying isolates. More research with a large number of samples and in the wider geographical areas is needed to understand the genetic diversity of the BKV in Iran and also to determine these SNPs' clinical significance in terms of patient outcome and viral load dynamics.

In vivo murine breast cancer targeting by magnetic iron nanoparticles involving L. GG cytoplasmic fraction.

OBJECTIVES: Use of chemical anti-cancer drugs frequently creates serious side effects. However, probiotics are natural and treat different kinds of cancer without undesired effects. MATERIALS AND METHODS: In this study, a nano delivery system was planned to transport the Lactobacillus rhamnosus GG (L. GG) cytoplasmic fraction (Cf) to cancerous tissue in a mouse model. Magnetic iron nanoparticles (MINPs) were synthesized and loaded with L. GG-Cf(0, 0.312, 0.625, 1.25, and 2.5 mg/ml) and were administrated for three weeks to treat experimentally induced murine breast cancer in a constant magnetic field. At the end of the trial, the treating efficacy of this complex molecule was evaluated via western blotting, immunohistochemistry, and qPCR. RESULTS: Results showed that MINPS can deliver and accumulate L. GG-Cf in cancer tissue, and reduce the size and volume of the tumors. Additionally, in cancer tissues of treated mice with 2.5 mg/ml of Cf-MINPs, significantly induced apoptosis was seen compared with untreated mice (control), and our data proved that this induction may be due to the caspase-3 pathway. CONCLUSION: L. GG-Cf could treat murine breast cancer, and MINPs are a suitable candidate for drug delivery because of their safety, uniformity, and magnetic properties.

Efficient Immunization of BALB/c Mice against Pathogenic Brucella melitensis and B. ovis: Comparing Cell-Mediated and Protective Immune Responses Elicited by pCDNA3.1 and pVAX1 DNA Vaccines Coding for Omp31 of Brucella melitensis.

BACKGROUND: Brucella spp. are intracellular pathogens, therefore cell-mediated immunity is the main response to inhibit survival and growth of the bacteria in vertebrate host. OBJECTIVE: Many eukaryotic plasmid vectors are being used in setting up DNA vaccines which may show different efficiencies in same conditions. This is important in designing the vaccines and immunization strategies. We looked into the probable differences of immune responses induced by different eukaryotic DNA plasmid vectors (pcDNA3.1 and pVAX1) harboring the same Omp31 gene of B. melitensis. MATERIALS AND METHODS: Female BALB/c mice were immunized with pcDNA -omp31 and pVAX-omp31 and further boosted with recombinant Omp31. Subclasses of specific serum IgG against the rOmp31 were measured by ELISA. Cytokines responses to rOmp31 in Splenocyte cultures of the immunized mice were evaluated by measuring the production of IL-4, IL-10, IL-12 and IFN-γ. Protective responses of the immunized mice were evaluated by intraperitoneal challenge with pathogenic Brucella melitensis 16M and Brucella ovis PA76250. RESULTS: Both DNA vaccine candidates conferred potent Th1-type responses with higher levels of cytokines and immunoglobulins observed in mice immunized with pVAX-omp31. Although pcDNA-omp31 and pVAX-omp31 both elicited protective immunity, mice immunized with the latter showed a higher protection against both B. melitensis and B. ovis PA76250. CONCLUSION: The results of this study highlight the significant differences between efficiency of diverse plasmid backbones in DNA vaccines which code for an identical antigen. Comparing various plasmid vectors should be considered as an essential part of the studies aiming construction of DNA vaccines for intracellular pathogens.

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

BACKGROUND: 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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

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First Occurrence of Saffold Virus in Sewage and River Water Samples in Karaj, Iran.

Saffold virus as a newly discovered virus, which seems to be related to acute gastroenteritis as with other enteric viruses and to human airway diseases in children belongs to Cardiovirus genus in picornaviridae family with 11 genotypes. Saffold virus initially was detected in America from infant stool sample. Saffold virus has also been detected in environmental water samples. Until now, two reports have demonstrated that sewage water sources are contaminated with Saffold viruses. Molecular detection of Saffold virus mostly depended on reverse transcription PCR methods and RT-qPCR, which had targeted 5'UTR region of the viral genome. The present study aims to evaluate the molecular detection and quantity of Saffold virus in sewage water and river water specimens by RT-qPCR assay in Karaj, Iran. Fifty samples collected from environmental waters containing treated and untreated sewage water and river water samples were included in this study. After viral RNA extraction, the Real-time PCR was developed to amplify the 5'UTR sequence of Saffold virus genome and viral load was assessed. Out of the 50 samples tested (consisting 28 river water samples and 22 sewage water samples), the Saffold virus genomic RNA was identified in 10/28 (35.7%) of river water samples and in 4/12 (33.3%) of treated and 4/10 (40%) of untreated sewage samples. The maximum viral load was 6.8 × 106 copies/l in untreated sewage water sample in December, and the lower viral load was 1.2 × 106 copies/l related to treated sewage water taken in October. Our results for the first time indicate that Saffold virus has apparently been circulating among Iranian peoples. Also, the viral prevalence of Saffold virus in each of the three sets of tested samples was within moderate to high in range.

Implementation of a novel self-induced promoter for the expression of pharmaceutical peptides in Escherichia coli: YY(3-36) peptide.

Background Increasing the expression rate of recombinant mammalian hormones in Escherichia coli by combining efficient promoters and signal sequences is a never ending process. A self-induced promoter will have some beneficial gains compared to the classical T7 promoter or its variants with isopropyl ß-D-1-thiogalactopyranoside (IPTG) as the inducer. Obesity is the prime suspect in widespread frequency of diabetes type II and cardiovascular diseases worldwide. YY (tyrosine-tyrosine) peptide is a local acting hormone, controlling appetite. Excitingly, it was has been shown that a truncated version of the YY peptide, YY(3-36) peptide, has potential as a worthy biopharmaceutical agent in the fight against obesity. Materials and methods To develop an economical expression system for the large scale production of the peptide in Gram-negative bacteria, we introduced a promoter sequence upstream of a chimeric gene for the extracellular expression of this peptide with the assistance of a signal sequence of asparaginase II from E. coli. This system has the advantage of producing a complete sequence of a truncated YY peptide, YY(3-36), without any extra tags that would require further removal with the assistance of expensive specific proteases and reduced the downstream steps, significantly. Results Recombinant production of YY(3-36) peptide under a self-induced promoter proves the efficacy of the asparaginase II signal sequence as a communicator of foreign peptides and proteins into the extracellular space of E. coli. Conclusions The application of fusion protein expression of biopharmaceuticals, especially mammalian hormones, in prokaryotic systems with the help of native signal sequences makes some common tags with expensive proteases for the removal of the attached protein Tag redundant.

Designing a Novel Recombinant HN Protein with Multi Neutralizing Antigenic Sites and Auto Tag Removal Ability Based on NDV-VIIj for Diagnosis and Vaccination Application.

Hemagglutinin-neuraminidase (HN) protein besides its mediation in viral pathogenesis, is composed of various antigenic sites which stimulate production of host's antibodies. Thus, application of this protein in serological tests and vaccination plays a major role in biosecurity and control programs. In the present study, we designed a recombinant HN protein containing different neutralizing antigenic sites with velogenic patterns, and sub-cloned it into pET-43.1a+ expression vector. The expression of NusA-HN recombinant protein was induced. Affinity chromatography protein purification using HisPur™ Ni-NTA was then conducted. Moreover, we performed western-blot technique using HRP-conjugated Anti His-Tag. Results revealed that following induction of recombinant protein, two distinct bands of HN-61 kDa and NusA-63 kDa were purified and identified by western-blotting. We recommend further analysis should be carried out to determine the functional role of this recombinant protein in enzyme-linked immunosorbent assays for Newcastle disease diagnosis. This HN protein containing multi neutralizing antigenic sites might also be applicable in vaccination programs to increase vaccines potency.

The Molecular Study of Antibiotic Resistance to Quinolones in Salmonella enterica Strains Isolated in Tehran, Iran.

INTRODUCTION: Salmonella is known as one of the most important causes of gastrointestinal disease in the world. Quinolones and fluoroquinolones are used successfully in the treatment of salmonellosis particularly for infections that have become resistant to several antibiotics. But non-susceptible isolates to quinolones have been reported in several countries. The data are limited about the prevalence of quinolone-resistant isolates in our country. Therefore, this study investigated the plasmid-mediated quinolone resistance genes in Salmonella enterica isolated in Children's Medical Center in Tehran during 2014-2015. METHODS AND MATERIALS: Salmonella isolates were isolated and identified using standard microbiological methods. Antibiotic susceptibility testing and screening of Salmonella strains resistant to quinolones were performed according to the CLSI guidelines. The molecular investigation was done using specific primers for detection of qnr genes including: qnrA, qnrB and qnrS, by polymerase chain reaction. RESULTS: Overall, 92 (66.6%) strains were resistant to nalidixic acid. None of the strains showed resistance to ciprofloxacin. Out of the 92 nalidixic acid resistant strains, 52 (56.52%) harbored qnrS genes, 15 strains (16.30%) had both qnrA and qnrS genes. Two (1.1%) isolates were positive for qnrB gene. Twenty four (26.08%) nalidixic acid resistant isolates did not have any qnr qens. CONCLUSION: The results of this study show high prevalence of resistance to nalidixic and qnr genes in Salmonella isolates. Plasmid nature of this type of resistance poses an increased risk of dissemination of quinolone resistance between Salmonella and non-Salmonella isolates circulating in hospitals environments.

Identification of nontuberculous mycobacteria isolated from hospital water by sequence analysis of the hsp65 and 16S rRNA genes.

Nontuberculous mycobacteria (NTM) have emerged as an important cause of opportunistic nosocomial infections. NTM has frequently been isolated from hospital water distribution systems. The aim of this study was to survey the risk of NTM infections and determine the prevalence of NTM species in the hospital water distribution systems in Tabriz, Iran. One hundred and twenty samples of water from different sources of Tabriz hospitals were collected. The samples were filtered through 0.45-µm pore size membranes and decontaminated with 0.01% cetylpyridinium chloride. The sediment was inoculated onto Lowenstein-Jensen medium and incubated for 8 weeks. For identification to the species level, partial sequence analysis of the hsp65 and 16S rRNA genes were used. NTM were detected in 76 (63.3%) of 120 samples. Potentially pathogenic mycobacteria and saprophytic mycobacteria were isolated. Mycobacterium gordonae was the only single species that was present in all types of water. The prevalence of NTM in Tabriz hospitals' water compared with many investigations on hospital waters was high. This indicates that the immunocompromised patients and transplant recipients are at risk of contamination which necessitates considering decontamination of water sources to prevent such potential hazards.