Publications by authors named "Hadi Farsiani"

22 Publications

  • Page 1 of 1

Practical Methods for Expression of Recombinant Protein in the Pichia pastoris System.

Curr Protoc 2021 Jun;1(6):e155

Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

One of the most critical challenges of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems from bacteria to mammalian tissue culture cells are available for the production of recombinant proteins for medical and industrial purposes. Among various choices, yeast expression systems such as Pichia pastoris are promising candidates. The P. pastoris expression system is a standard tool for the production of biopharmaceuticals and industrial enzymes. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Using P. pastoris as an expression system for heterologous proteins, this article provides detailed basic protocols for cloning of a recombinant cassette into a suitable expression vector, the transformation of foreign vector DNAs into the yeast by electroporation, and expression and purification of desired recombinant protein. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Cloning of a recombinant cassette into a suitable expression vector Basic Protocol 2: Transformation of P. pastoris and selection of transformants Basic Protocol 3: Optimization and large-scale expression of recombinant proteins Basic Protocol 4: Purification of recombinant proteins.
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http://dx.doi.org/10.1002/cpz1.155DOI Listing
June 2021

Corrigendum to "Helicobacter pylori infection and autoimmune diseases; Is there an association with systemic lupus erythematosus, rheumatoid arthritis, autoimmune atrophy gastritis and autoimmune pancreatitis? A systematic review and meta-analysis study".

J Microbiol Immunol Infect 2021 Jun 24;54(3):540. Epub 2020 Oct 24.

Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Microbiology and Virology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

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http://dx.doi.org/10.1016/j.jmii.2020.10.006DOI Listing
June 2021

Response surface methodology optimized electrochemical DNA biosensor based on HAPNPTs/PPY/MWCNTs nanocomposite for detecting Mycobacterium tuberculosis.

Talanta 2021 May 11;226:122099. Epub 2021 Jan 11.

Antimicrobial Resistance Research Center, Department of Medical Bacteriology and Virology, Qaem University Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

An important issue in the prognosis of tuberculosis (TB) is a short period between correct diagnosis and start the suitable antibiotic therapy. So, a rapid and valid method for detection of Mycobacterium tuberculosis (M. tb) complex is considered as a necessity. Herein, a rapid, low-cost, and PCR-free DNA biosensor was developed based on multi-walled carbon nanotubes (MWCNTs), polypyrrole (PPy), and hydroxyapatite nanoparticles (HAPNPs) for highly sensitive and specific recognition of M.tb. The biosensor consisted of M.tb ssDNA probe covalently attached to the HANPs/PPy/MWCNTs/GCE surface that hybridized to a complementary target sequence to form a duplex DNA. The M.tb target recognition was based on the oxidation signal of the electroactive Methylene Blue (MB) on the surface of the modified GCE using differential pulse voltammetry (DPV) method. It is worth to mention that for the first time Plackett-Burman (PB) screening design and response surface method (RSM) based on central composite design (CCD) was applied as a powerful and an efficient approach to find optimal conditions for maximum M.tb biosensor performance leading to simplicity and rapidity of operation. The proposed DNA biosensor exhibits a wide detection range from 0.25 to 200.0 nM with a low detection limit of 0.141 nM. The performance of designed biosensor for clinical diagnosis and practical applications was revealed through hybridization between DNA probe-modified GCE and extracted DNA from sputum clinical samples.
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http://dx.doi.org/10.1016/j.talanta.2021.122099DOI Listing
May 2021

A case of multidrug-resistant in an elderly woman.

Respirol Case Rep 2021 Mar 1;9(3):e00715. Epub 2021 Feb 1.

Antimicrobial Resistance Research Center Mashhad University of Medical Sciences Mashhad Iran.

is an emerging and spreading pathogen in Iran and little data about its drug susceptibility test (DST) and no standard treatment regimen are available. We report a case of multidrug-resistant . respiratory infection in a 65-year-old woman with a history of previous infection. The patient was treated with clarithromycin, levofloxacin, and cotrimoxazole for one year and eventually died while still suffering from respiratory problems. For DST, broth microdilution method was used according to the Clinical and Laboratory Standards Institute guidelines as well as molecular DST in clinical isolate. was resistant to streptomycin, moxifloxacin, clarithromycin, and cotrimoxazole antibiotics and was sensitive to clofazimine and amikacin antibiotics. Inappropriate use of antibiotics without determining the pattern of antibiotic resistance increases the likelihood of resistance and, for resistant specimens, the need to review the treatment protocol and replace antibiotics. Effectiveness based on antibiotic resistance pattern is essential.
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http://dx.doi.org/10.1002/rcr2.715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848708PMC
March 2021

Detection of efflux pump genes in multiresistant Acinetobacter baumannii ST2 in Iran.

Acta Microbiol Immunol Hung 2021 Feb 2. Epub 2021 Feb 2.

11Department of Clinical Psychology, Zahedan University of Medical Sciences, Zahedan, Iran.

Acinetobacter baumannii, as a nosocomial pathogen has become a worldwide concern in recent years. In the current study, the resistance to tetracyclines and colistin were assessed in the isolates from different provinces of Iran.During the timeline of this study, a number of 270 isolates of A. baumannii were collected from tracheal aspirates, wounds, urine and blood cultures. The minimum inhibitory concentration (MIC) for tetracycline, doxycycline, minocycline, tigecycline and colistin were evaluated. Tetracycline resistance genes were assessed by PCR. The mean expression level of adeB, adeJ and adeG were assessed using semi quantitative Real-Time PCR. The clonal relationship of the isolates was evaluated by the repetitive extragenic palindromic PCR (REP-PCR), International Clonal (IC) Lineage Multiplex PCR and multilocus sequence typing (MLST) (Pasteur scheme) methods.The MIC by microdilution method showed that 87.5, 51.4, 28, 0.74 and 0% of the isolates were resistant to tetracycline, doxycycline, minocycline, tigecycline and colistin respectively. The prevalence of tetracycline resistance genes was 99.2, 99.2, 98, 86.7, 10, 3.33, 0.37, 0% for adeB, adeJ, adeG, tetB, tetA(39), tetA, tetM and tetH in tetracycline-resistant isolates. Moreover, the expression level of adeB, adeJ, adeG genes in tigecycline-nonsusceptible A. baumannii (TNAB) strain was higher compared to the tigecycline-susceptible A. baumannii (TSAB). A broad genomic diversity was revealed, but ST2 was the most prevalent ST. Our results indicated that tetracycline resistance in Iran is mediated by resistance-nodulation-cell division (RND) and tetB efflux pumps.
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http://dx.doi.org/10.1556/030.2021.01314DOI Listing
February 2021

extracellular vesicles: exploitation for vaccine technology and diagnostic methods.

Crit Rev Microbiol 2021 Feb 12;47(1):13-33. Epub 2020 Oct 12.

Antimicrobial Resistance Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.

Tuberculosis (TB) is a fatal epidemic disease usually caused by (). Pervasive latent infection, multidrug- and extensively drug-resistant tuberculosis (MDR- and XDR-TB), and TB/HIV co-infection make TB a global health problem, which emphasises the design and development of efficient vaccines and diagnostic biomarkers. Extracellular vesicles (EVs) secretion is a conserved phenomenon in all the domains of life. Various cargos such as nucleic acids, toxins, lipoproteins, and enzymes have been recognised in these nano-sized vesicles that may be involved in bacterial physiology and pathogenesis. The intrinsic adjuvant effect, native immunogenic cargo, sensing by host immune cells, circulation in all body fluids, and comprehensive distribution of antigens introduce EVs as a promising tool for designing novel vaccines, diagnostic biomarkers, and drug delivery systems. Genetic engineering of the EV-producing bacteria and the subsequent production of proper EVs could facilitate the development of the EV-based therapeutic applications. Recently, it was demonstrated that thick-walled mycobacteria release EVs, which contain immunodominant cargos such as lipoglycans and lipoproteins. The present article is a comprehensive review on the recent findings of EVs biology and the exploitation of EVs for the vaccine technology and diagnostic methods.
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http://dx.doi.org/10.1080/1040841X.2020.1830749DOI Listing
February 2021

The overview and perspectives of biosensors and Mycobacterium tuberculosis: A systematic review.

J Cell Physiol 2021 Mar 15;236(3):1730-1750. Epub 2020 Sep 15.

Medical Toxicology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

Tuberculosis (TB) is referred to as a "consumption" or phthisis, which has been a fatal human disease for thousands of years. Mycobacterium tuberculosis (M. tb) might have been responsible for the death of more humans than any other bacterial pathogens. Therefore, the rapid diagnosis of this bacterial infection plays a pivotal role in the timely and appropriate treatment of the patients, as well as the prevention of disease spread. More than 98% of TB cases are reported in developing countries, and due to the lack of well-equipped and specialized diagnostic laboratories, development of effective diagnostic methods based on biosensors is essential for this bacterium. In this review, original articles published in English were retrieved from multiple databases, such as PubMed, Scopus, Google Scholar, Science Direct, and Cochrane Library during January 2010-October 2019. In addition, the reference lists of the articles were also searched. Among 109 electronically searched citations, 42 articles met the inclusion criteria. The highest potential and wide usage of biosensors for the diagnosis of M. tb and its drug resistance belonged to DNA electrochemical biosensors (isoniazid and rifampin strains). Use of biosensors is expanding for the detection of resistant strains of anti-TB antibiotics with high sensitivity and accuracy, while the speed of these sensory methods is considered essential as well. Furthermore, the lowest limit of detection (0.9 fg/ml) from an electrochemical DNA biosensor was based on graphene-modified iron-oxide chitosan hybrid deposited on fluorine tin oxide for the MPT64 antigen target. According to the results, the most common methods used for M. tb detection include acid-fast staining, cultivation, and polymerase chain reaction (PCR). Although molecular techniques (e.g., PCR and real-time PCR) are rapid and sensitive, they require sophisticated laboratory and apparatuses, as well as skilled personnel and expertise in the commentary of the results. Biosensors are fast, valid, and cost-efficient diagnostic method, and the improvement of their quality is of paramount importance in resource-constrained settings.
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http://dx.doi.org/10.1002/jcp.30007DOI Listing
March 2021

Helicobacter pylori infection and autoimmune diseases; Is there an association with systemic lupus erythematosus, rheumatoid arthritis, autoimmune atrophy gastritis and autoimmune pancreatitis? A systematic review and meta-analysis study.

J Microbiol Immunol Infect 2021 Jun 28;54(3):359-369. Epub 2020 Aug 28.

Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Microbiology and Virology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

Autoimmune diseases are considered as one of the most important disorders of the immune system, in which the prolonged and chronic processes eliminate self-tolerance to the auto-antigens. The prevalence of autoimmune diseases has been increasing worldwide in the recent years. According to the literature, biological processes such as the host genome, epigenetic events, environmental condition, drug consumption, and infectious agents are the most important risk factors that make the host susceptible to the development of autoimmune diseases. In the recent years, the role of Helicobacter pylori in the induction of autoimmune diseases has attracted extensive attention. Via molecular mimicry, epitope spreading, bystander activation, polyclonal activation, dysregulation in immune response, and highly immune-dominant virulence, such as cagA, H. pylori causes tissue damage, polarity, and proliferation of the host cells leading to the modulation of host immune responses. Moreover, given the large population worldwide infected with H. pylori, it seems likely that the bacterium may develop into autoimmune diseases through dysregulation of the immune response. The frequency and relationship between H. pylori infection and systemic lupus erythematosus, rheumatoid arthritis, autoimmune atrophy gastritis, and autoimmune pancreatitis were evaluated using the data from 43 studies involving 5052 patients. According to statistical analysis it is probable that infection with more virulent strains of H. pylori (such as H. pylori cagA positive) can increase the risk of autoimmune diseases. In addition, it was shown that infection with H. pylori can prevent the development of atrophic gastritis by stimulating inflammation in the gastric antrum. However, future studies should confirm the validity of this study.
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http://dx.doi.org/10.1016/j.jmii.2020.08.011DOI Listing
June 2021

High rate of resistance to ceftriaxone and azithromycin among Shigella spp. isolates at three children's referral hospitals in Northeast Iran.

J Infect Chemother 2020 Sep 20;26(9):955-958. Epub 2020 May 20.

Department of Pediatric Infectious Disease, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

Acute dysentery is a prevalent case of hospital admission in developing countries, whose most common cause is believed to be Shigella species. Treatment failure employing oral or intravenous antibiotics is an increasing problem among children with dysentery. This is a prospective descriptive study that aims to find the antibiotic resistance pattern of Shigella spp. isolates from children with acute diarrhea in three children's referral hospitals in Mashhad, northeast-Iran. Between February 2018 to September 2019, a total of 233 stool samples were collected from children with inflammatory diarrhea. Shigella spp. were identified by culture and biochemical standard tests. Moreover, polyvalent Shigella antisera were used for serogrouping. The antibiotic susceptibility was performed by disk diffusion method. During the 9-month study period, a total of 94 non-duplicate clinical Shigella spp. were identified by culture and biochemical tests. Based on slide agglutination with appropriate group-specific polyvalent antisera, Shigella sonnei (70.2%) was found to be the most prevalent Shigella spp. followed by S. flexeneri (23.4%), S. dysentery (1%). Among isolates, S. boydii was not detected and five isolates (5.3%) were nonserotypable isolates. The resistance rate of Shigella spp. to azithromycin, ceftriaxone, ciprofloxacin, co-trimoxazole, nalidixic acid, gentamicin, amoxicillin, ampicillin, doxycycline and cefixime was 25.5%, 43.6%, 3.8%, 82.9%, 15.9%, 26.6%, 40.4%, 57.4%, 41.4%, 22.3%, respectively. The results revealed that the resistance of Shigella spp. to the three most commonly utilized antibiotics (azithromycin, ceftriaxone and, cefixime) is too high to recommend them as empirical therapy for children with acute dysentery in this city.
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http://dx.doi.org/10.1016/j.jiac.2020.04.022DOI Listing
September 2020

High prevalence of bla AmpC beta-lactamase in ESBL co-producing Escherichia coli and Klebsiella spp. clinical isolates in the northeast of Iran.

J Glob Antimicrob Resist 2020 09 1;22:477-482. Epub 2020 Apr 1.

Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

Objective: The production of β-lactamase enzymes such as AmpC β-lactamases and extended-spectrum β-lactamases (ESBLs) is among the main mechanisms for resistance to expanded-spectrum cephalosporins. The present study was conducted to investigate the prevalence and molecular epidemiology of plasmid-mediated AmpC beta (β)-lactamase in ESBL co-producing Escherichia coli (E. coli) and Klebsiella spp. (Klebsiella pneumoniae and Klebsiella oxytoca) clinical isolates in the northeast of Iran.

Methods: A total of 602 E. coli and Klebsiella spp. clinical isolates were collected from three hospitals in Mashhad (northeast of Iran). A combination disk test (CDT) was performed for the phenotypic detection of ESBLs. Screening for the detection of AmpC β-lactamases was performed by a susceptibility test to a cefoxitin disc among ESBL producing isolates. A confirmatory test for AmpC β-lactamases was performed using the Mast® D68C test. Identification of plasmid-mediated AmpC cluster genes was done by multiplex polymerase chain reaction (PCR).

Results: Among 336 ESBL-producing strains, 230 (68.4%) isolates were resistant to cefoxitin. Results of the Mast® D68C test showed that 30% (69/230) of cefoxitin-resistant isolates simultaneously exhibited ESBL and AmpC activity and 22% (51/230) of isolates probably showed multi-drug resistant (MDR) phenotype. Results of multiplex PCR among ESBL-positive isolates showed that, 16.7% (56/336) of isolates were positive for plasmid-borneampC cluster genes, and CMY (38%) was the most frequent genotype of plasmid mediated AmpC.

Conclusion: Findings of the study revealed that an increase in the prevalence of ESBL and AmpC co-producer in E. coli and Klebsiella spp. strains may become an important public health issue. Therefore, there is a vital need for surveillance of spread of these clinical isolates.
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http://dx.doi.org/10.1016/j.jgar.2020.03.011DOI Listing
September 2020

Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins.

J Cell Physiol 2020 09 14;235(9):5867-5881. Epub 2020 Feb 14.

Mashhad University of Medical Sciences, Antimicrobial Resistance Research Center, Mashhad, Iran.

One of the most important branches of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems are used for the production of recombinant proteins including bacteria, yeasts, molds, mammals, plants, and insects. Yeast expression systems such as Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris) are more popular. P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology. Overall, the benefits of protein production by P. pastoris system include appropriate folding (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins to the external environment of the cell. Moreover, in the P. pastoris expression system due to its limited production of endogenous secretory proteins, the purification of recombinant protein is easy. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Although P. pastoris expression systems are impressive and easy to use with well-defined process protocols, some degree of process optimization is required to achieve maximum production of the target proteins. Methanol and sorbitol concentration, Mut forms, temperature and incubation time have to be adjusted to obtain optimal conditions, which might vary among different strains and externally expressed protein. Eventually, optimal conditions for the production of a recombinant protein in P. pastoris expression system differ according to the target protein.
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http://dx.doi.org/10.1002/jcp.29583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228273PMC
September 2020

Diversity of aminoglycoside modifying enzymes and 16S rRNA methylases in Acinetobacter baumannii and Acinetobacter nosocomialis species in Iran; wide distribution of aadA1 and armA.

Infect Genet Evol 2018 12 4;66:195-199. Epub 2018 Oct 4.

Antimicrobial Resistance Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

Purpose: Acinetobacter baumannii-calcoaceticus complex (ABC) make a great burden on health-care systems due to hospital-acquired infections and antibacterial resistance. Aminoglycoside in combination with other antibacterials used as treatment options. However, ABC species overcome this class of antibacterials in different ways. This study provides a comprehensive report on the distribution of aminoglycoside modifying enzymes (AMEs) and 16S rRNA methylase in Acinetobacter baumannii and Acinetobacter nosocomialis isolated from various provinces in Iran.

Methods: During six month of study, from eight referral centers in seven provinces across the country, Iran, 178 A. baumannii and 43 A. nosocomialis isolates were collected. The minimum inhibitory concentration of amikacin, gentamicin, netilmicin, kanamycin and tobramycin were measured by microbroth dilution method. AMEs and 16S rRNA methylase variants were sought by PCR.

Results: High rates of resistance were seen in all centers. MIC and MIC for all A. baumannii and A. nosocomialis isolates from different centers were > 512 mg/L. The most frequent AME was ant(3″)-Ia (aadA1) in both of A. baumannii (74.1%) and A. nosocomialis (86%). armA was detected in A. baumannii and A. nosocomialis at the frequency of 41.6% and 67.4%, respectively. rmtA, B, C, D, aac(3)-Ia (aacC1) and aac(6')-Im were not detected, neither in A. baumannii nor A. nosocomialis. Moreover, aac(6')-Ih was only found in A. baumannii isolates. The distribution of some of the ARGs was limited to a definite center.

Conclusion: The overall high-level carriage of ARGs in Acinetobacter species may limited usage of this class of antibacterials as a treatment option.
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http://dx.doi.org/10.1016/j.meegid.2018.09.028DOI Listing
December 2018

High prevalence of sequence type 131 isolates producing CTX-M-15 among extended-spectrum β-lactamase-producing Escherichia coli strains in northeast Iran.

J Glob Antimicrob Resist 2018 12 25;15:74-78. Epub 2018 May 25.

Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Azadi Square, Medical Campus, Mashhad 9177948564, Iran. Electronic address:

Objectives: The recent expansion of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is a worldwide problem. The purpose of this study was to investigate the molecular characteristics of ESBL-producing E. coli strains in Mashhad, located in the northeast of Iran.

Methods: A total of 455 clinical E. coli isolates were collected at three hospitals in Mashhad between April-September 2015. Antimicrobial susceptibility was determined by the Kirby-Bauer disk diffusion test. The combination disk test was performed for phenotypic detection of ESBLs. PCR was used to screen isolates for ESBL typing. Phylogenetic groups and sequence type 131 (ST131) were determined by multiplex PCR.

Results: The prevalence of ESBL-producing E. coli among the collected strains was 51.6% (235/455). Among the 235 ESBL-producing strains, 222 (94.5%) tested positive for CTX-M type, whilst 115 (48.9%), 92 (39.1%) and 21 (8.9%) were positive for TEM, OXA and SHV, respectively. Moreover, CTX-M-15 (94.1%; 209/222) was the most common ESBL among E. coli. Based on multiplex PCR, phylogenetic group B2 was predominant (169/235; 71.9%), followed by D (32/235; 13.6%), A (21/235; 8.9%) and B1 (13/235; 5.5%). ST131 was the predominant clonal group among the phylogenetic group B2 isolates (151/169; 89.3%).

Conclusion: The results revealed that an urgent investigation of the source and transmission pathways of the CTX-M-15-B2-ST131 E. coli clone is needed to mitigate this emergent public-health problem.
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http://dx.doi.org/10.1016/j.jgar.2018.05.016DOI Listing
December 2018

Molecular characterization and genetic relatedness of clinically Acinetobacter baumanii isolates conferring increased resistance to the first and second generations of tetracyclines in Iran.

Ann Clin Microbiol Antimicrob 2017 Jul 19;16(1):51. Epub 2017 Jul 19.

Antimicrobial Resistance Research Center, Bu Ali Research Institute, Mashhad, Iran.

Background: The increasing resistance of Acinetobacter baumannii to antibiotics has recently been regarded as a notable therapeutic difficulty. Evaluating resistance rates of some A. baumannii isolates to tetracyclines had an impact on understanding the antibiotic resistance dissemination. By comparing genetic characteristics and relatedness of A. baumannii isolates, we are able to determine the transition dynamics of outbreak isolates.

Methods: A total of 72 non-duplicate isolates of A. baumannii were recovered in 2011 and 2015 and minimum inhibitory concentration (MIC) range distribution of the isolates to tetracyclines was performed by broth micro dilution (BMD) assay, and to determine the lineage relatedness of the outbreak isolates repetitive extragenic palindromic element based on polymerase chain reaction (rep-PCR) and international clonal (ICs) investigations were performed.

Results: Resistance rates to tetracycline, doxycycline and minocycline in 2011 were 73, 2 and 0%, while these rates in 2015 increased up to 90, 84 and 52%, respectively. The tetB existed in 100% of all the isolates of both years. tetA was not found in any of the isolates. According to the rep-PCR assays, up to 83% of all isolates clustered distinctly and only 6% of isolates had a common root. The percentage rates of IC1 decreased from 42% in 2011 to 22% in 2015, while those of IC2 increased from 28 to 36%, from 2011 to 2015.

Conclusions: Our data showed that resistance to the first and second generations of tetracyclines is on the rise and the clonal transition dynamics of isolates are in progress in our hospital.
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http://dx.doi.org/10.1186/s12941-017-0226-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518157PMC
July 2017

CFP10: mFcγ2 as a novel tuberculosis vaccine candidate increases immune response in mouse.

Iran J Basic Med Sci 2017 Feb;20(2):122-130

Antimicrobial Resistance Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Microbiology and Virology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Objectives: Despite treatment with antibiotics and vaccination with BCG, tuberculosis (TB) is still considered as one of the most important public health problems in the world. Therefore, designing and producing a more effective vaccine against TB seems urgently. In this study, immunogenicity of a fusion protein which consisting or comprising CFP-10 from Mycobacterium tuberculosis and the Fc-domain of mouse IgG2a was evaluated as a novel subunit vaccine candidate against TB.

Materials And Methods: The genetic constructs were cloned in pPICZαA expression vector and recombinant vectors (pPICZαA-CFP-10: Fcγ2a and pPICZαA-CFP-10:His) were transformed into Pichia pastoris. To evaluate the expression of recombinant proteins, SDS-PAGE and immunoblotting were used. The immunogenicity of recombinant proteins, with and without BCG were assessed in BALB/c mice and specific cytokines against recombinant proteins (IFN-γ, IL-12, IL-4, IL-17 and TGF-β) were evaluated.

Results: The levels of IFN-γ and IL-12 in mice that received recombinant proteins was higher than the control groups (BCG and PBS). Thus, both recombinant proteins (CFP-10:Fcγ2a and CFP-10:His) could excite good response in Th1-cells. The Fc-tagged protein had a stronger Th1 response with low levels of IL-4, as compared to CFP-10:His. However, the highest level of Th1 response was observed in groups that were vaccinated with BCG (prime) and then received recombinant protein CFP-10: Fcγ2a (booster).

Conclusion: The results demonstrated that binding mice Fc-domain to CFP-10 protein can increase the immunogenicity of the subunit vaccine. Further studies, might be able to design and produce a new generation of subunit vaccines based on the Fc-fused immunogen.
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http://dx.doi.org/10.22038/ijbms.2017.8231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339651PMC
February 2017

Elevated prevalence of multidrug-resistant Acinetobacter baumannii with extensive genetic diversity in the largest burn centre of northeast Iran.

J Glob Antimicrob Resist 2017 03 21;8:60-66. Epub 2016 Dec 21.

Antimicrobial Resistance Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Microbiology and Virology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

Objectives: The emergence and spread of multidrug-resistant (MDR) Acinetobacter baumannii isolates is now frequently associated with nosocomial infections. The aim of this study was to evaluate the genetic relatedness and patterns of antimicrobial resistance amongst A. baumannii isolated from a burn centre at a teaching hospital in Iran.

Methods: A total of 54 A. baumannii isolates were collected from burn wound infections of hospitalised patients. Antimicrobial susceptibility of the isolates was determined, and genotyping analysis was performed by repetitive extragenic palindromic PCR (rep-PCR). PCR assay was performed to investigate the distribution of β-lactamase, aminoglycoside-modifying enzyme and efflux pump genes.

Results: Etest results revealed that the most active antimicrobial agent was colistin (100% susceptibility), followed by tigecycline (96.3%). The bla and bla genes were detected in all of the isolates, but bla was not detected. The prevalence of bla, bla, bla, bla and bla genes was 64.8%, 70.4%, 70.4%, 66.7% and 68.5%, respectively. ISAba1 was detected upstream of bla and bla in 66.7% and 77.8% of isolates, respectively. This study showed a high level of distribution of adeB (72.2%), aphA6 (81.5%), aacC1 (85.2%), aadA1 (59.3%), aadB (31.5%), tetB (70.4%) and aphA1 (29.6%) in A. baumannii strains. Based on rep-PCR analysis, four clusters (I-IV) were defined.

Conclusions: The elevated prevalence of MDR A. baumannii strains in this burn centre suggests that local antibiotic prescription policies should be precisely revised. Moreover, strict infection control procedures to prevent further dissemination need to be prioritised immediately.
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http://dx.doi.org/10.1016/j.jgar.2016.10.009DOI Listing
March 2017

Construction and immunogenicity of a new Fc-based subunit vaccine candidate against Mycobacterium tuberculosis.

Mol Biol Rep 2016 Sep 1;43(9):911-22. Epub 2016 Jun 1.

Antimicrobial Resistance Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.

As an ancient disease, tuberculosis (TB) is a major global health threat. Therefore, there is an urgent need for an effective and safe anti-TB vaccine. In the current study, a delivery system of Fc domain of mouse IgG2a and early secreted antigenic target protein 6 (ESAT-6) was evaluated for the selective uptake of antigens by antigen-presenting cells (APCs). Thus, it was based on the immunogenicity of a fusion protein. The study was initiated by the transfer of recombinant expression vectors of pPICZαA-ESAT-6:Fcγ2a and pPICZαA-ESAT-6: His into Pichia pastoris (P. pastoris). Recombinant proteins were assessed for immunogenicity following the immunoblotting analysis. High levels of IFN-γ and IL-12 were produced to induce Th1-type cellular responses through vaccination with both recombinant proteins [ESAT-6:Fcγ2a (EF) and ESAT-6:His (EH)]. The Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a low increment in IL-4 compared to PBS, BCG, and EH groups. Although in all the immunized groups, the ratio of IFN-γ/IL-4 was in favor of Th1 responses, the highest Th1/Th2 balance was observed in EF immunized group. Fc fragment of mouse IgG2a may induce a selective uptake of APCs towards the cross-presentation and formation of Th1 responses in favor of an appropriate protective anti-tuberculosis reaction. Thus, further research on Fc-fusion proteins is required to develop Fc-based TB vaccines.
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http://dx.doi.org/10.1007/s11033-016-4024-9DOI Listing
September 2016

Fc-based delivery system enhances immunogenicity of a tuberculosis subunit vaccine candidate consisting of the ESAT-6:CFP-10 complex.

Mol Biosyst 2016 06;12(7):2189-201

Inflammation and Inflammatory Diseases Research Center, Medical School, Mashhad University of Medical Sciences, Azadi-Square, Medical Campus, Mashhad, Iran.

Tuberculosis (TB) remains a major global health threat despite chemotherapy and Bacilli Calmette-Guérin (BCG) vaccination. Therefore, a safer and more effective vaccine against TB is urgently needed. This study evaluated the immunogenicity of a recombinant fusion protein consisting of early secreted antigenic target protein 6 kDa (ESAT-6), culture filtrate protein 10 kDa (CFP-10) and the Fc-domain of mouse IgG2a as a novel subunit vaccine. The recombinant expression vectors (pPICZαA-ESAT-6:CFP-10:Fcγ2a and pPICZαA-ESAT-6:CFP-10:His) were transferred into Pichia pastoris. After SDS-PAGE and immunoblotting, the immunogenicity of the recombinant proteins was evaluated in mice. When both recombinant proteins (ESAT-6:CFP-10:Fcγ2a and ESAT-6:CFP-10:His) were used for vaccination, Th1-type cellular responses were induced producing high levels of IFN-γ and IL-12. However, the Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a small increase in IL-4 as compared to the BCG and ESAT-6:CFP-10:His groups. Moreover, mice primed with BCG and then supplemented with ESAT-6:CFP-10:Fcγ2a produced the highest levels of IFN-γ and IL-12 in immunized groups. The findings indicate that when Fcγ2a is fused to the ESAT-6:CFP-10 complex, as a delivery vehicle, there could be an increase in the immunogenicity of this type of subunit vaccine. Therefore, additional investigations are necessary for the development of appropriate Fc-based tuberculosis vaccines.
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http://dx.doi.org/10.1039/c6mb00174bDOI Listing
June 2016

Fused Mycobacterium tuberculosis multi-stage immunogens with an Fc-delivery system as a promising approach for the development of a tuberculosis vaccine.

Infect Genet Evol 2016 Apr 4;39:163-172. Epub 2016 Feb 4.

Inflammation and Inflammatory Diseases Research Centre, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address:

Tuberculosis (TB) remains a major health problem worldwide. Currently, the Bacilli Calmette-Guérin (BCG) is the only available licensed TB vaccine, which has low efficacy in protection against adult pulmonary TB. Therefore, the development of a safe and effective vaccine against TB needs global attention. In the present study, a novel multi-stage subunit vaccine candidate from culture filtrate protein-10 (CFP-10) and heat shock protein X (HspX) of Mycobacterium tuberculosis fused to the Fc domain of mouse IgG2a as a selective delivery system for antigen-presenting cells (APCs) was produced and its immunogenicity assessed. The optimized gene constructs were introduced into pPICZαA expression vectors, and the resultant plasmids (pPICZαA-CFP-10:Hspx:Fcγ2a and pPICZαA-CFP-10:Hspx:His) were transferred into Pichia pastoris by electroporation. The identification of both purified recombinant fusion proteins was evaluated by SDS-PAGE and immunoblotting. Then the immunogenicity of the recombinant proteins with and without BCG was evaluated in BALB/c mice by assessing the level of IFN-γ, IL-12, IL-4, IL-17 and TGF-β cytokines. Both multi-stage vaccines (CFP-10:HspX:Fcγ2a and CFP-10:HspX:His) induced Th1-type cellular responses by producing high level of IFN-γ (272 pg/mL, p<0.001) and IL-12 (191 pg/mL, p<0.001). However, the Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a low level of IL-4 (10 pg/mL) compared to the CFP-10:HspX:His group. The production of IFN-γ to CFP-10:HspX:Fcγ2a was markedly consistent and showed an increasing trend for IL-12 compared with the BCG or CFP-10:HspX:His primed and boosted groups. Findings revealed that CFP-10:Hspx:Fcγ2a fusion protein can elicit strong Th1 antigen-specific immune responses in favor of protective immunity in mice and could provide new insight for introducing an effective multi-stage subunit vaccine against TB.
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http://dx.doi.org/10.1016/j.meegid.2016.01.027DOI Listing
April 2016

APC targeting enhances immunogenicity of a novel multistage Fc-fusion tuberculosis vaccine in mice.

Appl Microbiol Biotechnol 2015 Dec 15;99(24):10467-80. Epub 2015 Sep 15.

Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.

Numerous studies have demonstrated that targeting immunogens to FcγR on antigen-presenting cells (APCs) can selectively uptake and increase cellular immunity in vitro and in vivo. Therefore, the present study was conducted to evaluate immunogenicity of a novel multistage tuberculosis vaccine, a combination of an early and a dormant immunogenic protein, ESAT6 and HspX, fused to Fcγ2a fragment of mouse IgG2a to target all forms of tuberculosis. Codon-optimized genes consisting of ESAT6, a linker, and HspX fused either to mouse Fcγ2a (ESAT6:HspX:mFcγ2a) or 6× His-tag (ESAT6:HspX:His) were synthesized. The resulting proteins were then produced in Pichia pastoris. The fusion proteins were separately emulsified in dimethyldioctadecylammonium bromide(DDA)-trehalose-6,6-dibehenate(TDB) adjuvant, and their immunogenicity with and without bacille Calmette-Guérin (BCG) was assessed in C57BL/6 mice. Th1, Th2, Th17, and T-reg cytokine patterns were evaluated using the ELISA method. Both multistage vaccines induced very strong IL-12 and IFN-γ secretion from splenic cells; the Fc-tagged subunit vaccine induced a more effective Th1 immune response (IFN-γ, 910 pg/mL, and IL-12, 854 pg/mL) with a very low increase in IL-17 (∼0.1 pg/mL) and IL-4 (37 pg/mL) and a mild increase in TGF-β (543 pg/mL) compared to the BCG or ESAT6:HspX:His primed and boosted groups. The production of IFN-γ to ESAT6:HspX:Fcγ2a was very consistent and showed an increasing trend for IL-12 compared to the BCG or ESAT6:HspX:His primed and boosted groups. Fcγ2a used as a delivery vehicle supported the idea of selective uptake, inducing cross-presentation and forming a proper anti-tuberculosis response in context of Th1/Th2 and Th17/T-reg balances, which is important for protection and prevention of damage.
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http://dx.doi.org/10.1007/s00253-015-6952-zDOI Listing
December 2015

Limited genetic diversity and extensive antimicrobial resistance in clinical isolates of Acinetobacter baumannii in north-east Iran.

J Med Microbiol 2015 Jul 19;64(7):767-773. Epub 2015 May 19.

Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

This study determined the mechanisms and patterns of antimicrobial resistance among the isolates obtained from different wards of a teaching hospital in the city of Mashhad in north-east Iran. Between January 2012 and the end of June 2012, 36 isolates of Acinetobacter baumannii were collected from different wards of Ghaem Hospital. Antimicrobial susceptibility testing and epsilometer testing (E-test) were performed. The genetic resistance determinants of A, B and D classes of β-lactamases, aminoglycoside modifying enzymes (AMEs), efflux pumps and ISAba1 elements were assessed by PCR. Repetitive extragenic palindromic element (REP)-PCR was performed to find the genetic relatedness of the isolates. Colistin was the most effective antibiotic of those tested, where all isolates were susceptible. E-test results revealed high rates of resistance to imipenem, ceftazidime and ciprofloxacin. The majority of isolates (97  %) were multidrug-resistant. OXA-51, OXA-23 and tetB genes were detected in all isolates, but OXA-58, IMP and tetA were not detected. The prevalence of OXA-24, bla(TEM), bla(ADC), bla(VIM) and adeB were 64, 95, 61, 64 and 86  %, respectively. ISAba1 was found to be inserted into the 5' end of OXA-23 in 35 isolates (97  %). Of the AMEs, aadA1 (89  %) was the most prevalent, followed by aphA1 (75  %). The band patterns reproduced by REP-PCR showed that 34 out of 36 isolates belonged to one clone and two singletons were identified. The results confirmed that refractory A. baumannii isolates were widely distributed and warned the hospital infection control team to exert strict measures to control the infection. An urgent surveillance system should be implemented.
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http://dx.doi.org/10.1099/jmm.0.000090DOI Listing
July 2015

Association of myeloperoxidase -463 G/A polymorphism with clinical outcome of Helicobacter pylori infection in Iranian patients with gastrointestinal diseases.

Iran J Immunol 2007 Sep;4(3):155-60

Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.

Background: Polymorphisms in the immune related genes are important in the clinical outcome of Helicobacter pylori infection. Myeloperoxidase -463 G/A polymorphism has been shown to reduce enzyme expression and activity.

Objective: the aim of the present study is to investigate the association of myeloperoxidase G-463A polymorphism with clinical outcome of Helicobacter pylori infection.

Methods: two hundred and eighty five patients with positive culture of Helicobacter pylori from their gastric biopsies are included in this study. Human leukocyte DNA was extracted using salting out method and myeloperoxidase G-463A polymorphism was investigated by PCR-RFLP. All clinicopathological data were collected from individual records.

Results: When the patients were categorized according to the high (GG) and low + intermediate (AG+AA) genotypes of myeloperoxidase producers, there was a significant association between myeloperoxidase G-463A genotypes and clinical outcome of Helicobacter pylori infection (p=0.006). In search for combined effect of cagA status and myeloperoxidase genotypes on clinical presentations, only in cagA- Helicobacter pylori infected patients a significant association between myeloperoxidase genotypes and clinical outcome was found (p=0.0001). Also this association was found only in patients infected with vacA s1m1 genotype (p=0.008).

Conclusions: Our findings suggest that the myeloperoxidase G-463A polymorphism is a host genetic factor which determines the clinical outcome of Helicobacter pylori infection. Moreover, the combination of host and bacterial genetics could provide a better understanding of clinical outcome after infection with Helicobacter pylori.
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http://dx.doi.org/IJIv4i2A3DOI Listing
September 2007