Antimicrobial Activity of Chitosan Scaffold Loaded with Soluble Factors of Different Probiotic Strains Against Multidrug Resistant Pseudomonas aeruginosa.

Background: Bacterial infection remains the most frequent complication of burn injury, which can lead to sepsis, even if antibiotics are used topically and systemically. Pseudomonas aeruginosa (P. aeruginosa) is the main causative agent in many cases. The emergence of antibiotic-resistant strains in recent years has increased the need to find novel alternative therapies, such as probiotics. Therefore, this study aimed to examine the antimicrobial properties of probiotic cell-free supernatant (CFS), along with the potential use of a chitosan scaffold both as an antimicrobial agent and as a carrier for the delivery of these complexes. Objective: Evaluation of the antimicrobial properties of cell-free soluble factors of probiotic bacteria both alone and in combination with chitosan scaffolds. Materials and Methods: Nine isolates of P. aeruginosa previously identified by standard diagnostic tests were investigated. The antimicrobial effects of probiotics in the form of Pedilact® oral drop which contained three probiotic strains, Kidilact® sachet, which contained seven probiotic strains, and strains of Lactobacillus casei (L. casei) and Lactobacillus acidophilus (L. acidophilus) isolated from yogurt were studied by an agar well diffusion assay and by using CFS harvested at various growth stages, without pH neutralization. Chitosan with different concentrations of glutaraldehyde (GA) as a crosslinking agent was fabricated to produce a suitable scaffold for loading cell-free supernatants of probiotic strains. The scaffolds were then characterized using scanning electron microscopy. The antimicrobial properties of the CFS, chitosan, and chitosan scaffolds loaded with CFS were analyzed against MDR P. aeruginosa. Results: In the agar well diffusion assay, CFS obtained from probiotic strains effectively inhibited the growth of a clinical strain of P. aeruginosa. This effect was observed when CFS was assessed without pH neutralization. Kidilact® was the most promising synbiotic formulation based on its inhibitory activity. The chitosan scaffold was successfully fabricated, as shown by SEM, and its structure was not affected by acidic CFS. The fabricated scaffolds were able to deliver CFS and, interestingly, antibacterial activity against P. aeruginosa when CFS was loaded on the chitosan scaffold was enhanced significantly. Conclusion: The results of this study showed chitosan scaffold loaded with cell-free probiotics metabolites can be considered to be a promising antimicrobial dressing in wound healing applications.

Production and Evaluation of Ag85B:HspX:hFcγ1 Immunogenicity as an Fc Fusion Recombinant Multi-Stage Vaccine Candidate Against Mycobacterium tuberculosis.

An urgent need is to introduce an effective vaccine against Mycobacterium tuberculosis (M.tb) infection. In the present study, a multi-stage M.tb immunodominant Fcγ1 fusion protein (Ag85B:HspX:hFcγ1) was designed and produced, and the immunogenicity of purified protein was evaluated. This recombinant fusion protein was produced in the Pichia pastoris expression system. The HiTrap-rPA column affinity chromatography purified and confirmed the fusion protein using ELISA and Western blotting methods. The co-localisation assay was used to confirm its proper folding and function. IFN-γ, IL-12, IL-4, and TGF-ß expression in C57BL/6 mice then evaluated the immunogenicity of the construct in the presence and absence of BCG. After expression optimisation, medium-scale production and the Western blotting test confirmed suitable production of Ag85B:HspX:hFcγ1. The co-localisation results on antigen-presenting cells (APCs) showed that Ag85B:HspX:hFcγ1 properly folded and bound to hFcγRI. This strong co-localisation with its receptor can confirm inducing proper Th1 responses. The in vivo immunisation assay showed no difference in the expression of IL-4 but a substantial increase in the expression of IFN-γ and IL-12 (P ≤ 0.02) and a moderate increase in TGF-ß (P = 0.05). In vivo immunisation assay revealed that Th1-inducing pathways have been stimulated, as IFN-γ and IL-12 strongly, and TGF-ß expression moderately increased in Ag85B:HspX:hFcγ1 group and Ag85B:HspX:hFcγ1+BCG. Furthermore, the production of IFN-γ from splenocytes in the Ag85B:HspX:hFcγ1 group was enormously higher than in other treatments. Therefore, this Fc fusion protein can make a selective multi-stage delivery system for inducing appropriate Th1 responses and is used as a subunit vaccine alone or in combination with others.

Sustain-release lipid-liquid crystal formulations of pexiganan against Helicobacter pylori infection: in vitro evaluation in C57BL/6 mice.

INTRODUCTION: The Gram-negative bacterium Helicobacter pylori, H. pylori, is associated with significant digestive disorders. However, the effectiveness of bacterial eradication is declining due to drug resistance. A potent anti-H. pylori activity is shown by the natural antimicrobial peptide pexiganan. OBJECTIVE: The current study aimed to evaluate the effectiveness of pexiganan and its lipid-liquid crystals (LLCs) in inducing Helicobacter pylori in mice. METHODS: In this experimental study, H. pylori infection was first induced in C57BL/6 mice. Secondly, the antibacterial efficacy of pexiganan and its LLCs formulations was investigated to eliminate H. pylori infection. RESULTS: The H. pylori infection could not be completely eradicated by pexiganan peptide alone. However, incorporating pexiganan within the LLC formulation resulted in an increased elimination of H. pylori. Under the H&E strain, the pexiganan-LLCs formulation revealed minimal mucosal alterations and a lower amount of inflammatory cell infiltration in the stomach compared to the placebo. CONCLUSION: Clarithromycin was more effective than pexiganan at all tested concentrations. Furthermore, the pexiganan-loaded LLCs exhibited superior efficacy in curing H. pylori infection in a mouse model compared to pexiganan alone. This formulation can enhance H. pylori clearance while mitigating the adverse effects, typically associated with conventional drugs, leading to a viable alternative to current treatment options.

Helicobacter pylori virulence factors: subversion of host immune system and development of various clinical outcomes.

Helicobacter pylori (H. pylori) is a worldwide spread bacterium, co-evolving with humans for at least 100 000 years. Despite the uncertainty about the mode of H. pylori transmission, the development of intra-gastric and extra-gastric diseases is attributed to this bacterium. The morphological transformation and production of heterogenic virulence factors enable H. pylori to overcome the harsh stomach environment. Using numerous potent disease-associated virulence factors makes H. pylori a prominent pathogenic bacterium. These bacterial determinants are adhesins (e.g., blood group antigen-binding adhesin (BabA)/sialic acid-binding adhesin (SabA)), enzymes (e.g., urease), toxins (e.g., vacuolating cytotoxin A (VacA)), and effector proteins (e.g., cytotoxin-associated gene A (CagA)) involved in colonisation, immune evasion, and disease induction. H. pylori not only cleverly evades the immune system but also robustly induces immune responses. This insidious bacterium employs various strategies to evade human innate and adaptive immune responses, leading to a life-long infection. Owing to the alteration of surface molecules, innate immune receptors couldn't recognise this bacterium; moreover, modulation of effector T cells subverts adaptive immune response. Most of the infected humans are asymptomatic and only a few of them present severe clinical outcomes. Therefore, the identification of virulence factors will pave the way for the prediction of infection severity and the development of an effective vaccine. H. pylori virulence factors are hereby comprehensively reviewed and the bacterium evasion from the immune response is properly discussed.

Characterization of carbapenem-resistant Escherichia coli and Klebsiella: a role for AmpC-producing isolates.

Aim: This study aimed to investigate the role of AmpC enzymes in carbapenem resistance among AmpC/extended-spectrum ß-lactamase (ESBL)-producing clinical isolates of Escherichia coli and Klebsiella spp. Methods: Fifty-six bacterial strains that were AmpC producers were examined. The antibiotic susceptibility test was performed by the disk diffusion and E-test. The prevalence of the plasmid carbapenemase was determined using PCR. Results: The resistance to meropenem in the AmpC+/ESBL+ group was 64%, higher than that reported for the AmpC-/ESBL+ group. Ten isolates of the carbapenem-resistant AmpC producers were negative for carbapenemase-encoding genes. Conclusion: Carbapenem resistance among AmpC-producing isolates with negative results for carbapenemase-encoding genes potentially demonstrates the role of AmpC enzymes among these isolates.

Antibacterial activity of Mangifera indica seed extracts combined with common antibiotics against multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii isolates

In this project, we employed ethanolic (EMI) and aqueous (AMI) extracts of mango (Mangifera indica L., Anacardiaceae) fruit seeds as a modulator of antibiotic resistance against multidrug-resistant (MDR) Pseudomonas aeruginosa and Acinetobacter baumannii to evaluate natural compounds isolated from by-products or waste of edible plants. We also investigated the effect of these extracts alone and in combination with standard classes of antibiotics in the desired strains. M. indica seeds were processed and exploited using ethanol and water. The minimum inhibitory concentrations (MICs) of clinical isolates were examined against EMI and AMI extracts, followed by seven antibiotics of ceftazidime, ciprofloxacin, penicillin, amikacin, meropenem, ampicillin, and colistin. The checkerboard method evaluated the synergistic action between mango kernel extract (EMI) and seven antibiotics. EMI extract significantly revealed antimicrobial properties against MDR A. baumannii and P. aeruginosa with synergistic effects with the applied antibiotics. The considerable antibacterial efficacy of ethanolic extract of M. indica seeds can have great curative value as antibacterial drugs against infections caused by MDR P.aeruginosa and A. baumannii.

Designing and development of epitope-based vaccines against Helicobacter pylori.

Helicobacter pylori infection is the principal cause of serious diseases (e.g. gastric cancer and peptic ulcers). Antibiotic therapy is an inadequate strategy in H. pylori eradication because of which vaccination is an inevitable approach. Despite the presence of countless vaccine candidates, current vaccines in clinical trials have performed with poor efficacy which makes vaccination extremely challenging. Remarkable advancements in immunology and pathogenic biology have provided an appropriate opportunity to develop various epitope-based vaccines. The fusion of proper antigens involved in different aspects of H. pylori colonization and pathogenesis as well as peptide linkers and built-in adjuvants results in producing epitope-based vaccines with excellent therapeutic efficacy and negligible adverse effects. Difficulties of the in vitro culture of H. pylori, high genetic variation, and unfavourable immune responses against feeble epitopes in the complete antigen are major drawbacks of current vaccine strategies that epitope-based vaccines may overcome. Besides decreasing the biohazard risk, designing precise formulations, saving time and cost, and induction of maximum immunity with minimum adverse effects are the advantages of epitope-based vaccines. The present article is a comprehensive review of strategies for designing and developing epitope-based vaccines to provide insights into the innovative vaccination against H. pylori.

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

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.

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

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.

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

A case of multidrug-resistant Mycobacterium simiae in an elderly woman.

Mycobacterium simiae 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 M. simiae respiratory infection in a 65-year-old woman with a history of previous Mycobacterium tuberculosis 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. Mycobacterium simiae 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.

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

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.

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.

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.

Mycobacterium tuberculosis extracellular vesicles: exploitation for vaccine technology and diagnostic methods.

Tuberculosis (TB) is a fatal epidemic disease usually caused by Mycobacterium tuberculosis (Mtb). 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 Mtb EVs biology and the exploitation of EVs for the vaccine technology and diagnostic methods.

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

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.

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

OBJECTIVE: The production of ß-lactamase enzymes such as AmpC ß-lactamases and extended-spectrum ß-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 (ß)-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 ß-lactamases was performed by a susceptibility test to a cefoxitin disc among ESBL producing isolates. A confirmatory test for AmpC ß-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.

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

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.

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

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. MIC50 and MIC90 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.

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

OBJECTIVES: The recent expansion of extended-spectrum ß-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.