Antimicrobial effect of silver and gold nanoparticles in combination with linezolid on Enterococcus biofilm.

Background and Objectives: In the past few years, application of new antimicrobial e.g. nanoparticles (NPs) to treat infection caused by drug-resistant bacteria has increased. This study aimed to determine antimicrobial property of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) in combination with linezolid on Enterococcus biofilm. Materials and Methods: A total of forty-eight isolates of Enterococcus spp. were collected and confirmed by PCR method. The synthesis of biocompatible AgNPs was performed, then analyzed by Fourier Transform Infrared spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy. We carried out minimum inhibitory concentration (MIC) and biofilm forming capacity of AgNPs and AuNPs with linezolid. Results: Twenty-two E. faecium isolates and twentysix E. faecalis investigated in this study. Strong biofilm formation was seen in 12 (25%) of isolates, and others isolates (75%) formed moderate biofilm. AgNPs and Au-NPs size were 26 nm and 20 nm respectively. The MIC of AgNPs was 23.2 µg/ml, and AuNPs were 92.1 µg/ml and the lowest MIC was obtained 2 µg/ml in linezolid. Biofilm formation inhibitory activity by AuNPs + Linezolide and AgNPs + Linezolide 70 to 80 percent increased in average. Conclusion: The antibiofilm activity of AgNPs and AuNPs increased when both agents were used in combination with linezolid in comparison with each agent alone.

Evaluating the relationship between Helicobacter pylori infection and carotid intima-media thickness a cross sectional study.

INTRODUCTION: Helicobacter pylori is a gram-negative spiral bacterium that is frequently found in the human stomach. Significant association has been reported between Cytotoxin associated gene A (CagA)- positive Helicobacter pylori strains and coronary heart disease. The aim of the present study is to investigate the carotid intima-media thickness as an indicator of atherosclerosis in people with Helicobacter pylori infection. METHODS: This study was done on patients who underwent upper GI endoscopy and biopsy, and after obtaining conscious consent underwent ultrasound of the right and left carotid arteries for measuring carotid intima-media thickness (CIMT) and blood tests. RESULTS: In this study, 90 patients who underwent upper GI endoscopy were examined in three groups: negative H. pylori negative, positive cagA and negative cagA. The right, left and average of CIMT in cagA-positive group were significantly higher than the other two groups (p < 0.05). Howerver, the average of CIMT was not significantly different between men and women. Also, the hsCRP average level in positive cagA group was significantly higher than other groups (p < 0.05). CONCLUSION: Our findings suggest that there is an increase in CIMT values in patients with H. pylori infection, especially in cases of positive cagA. The positive cagA group showed significantly higher levels of hs-CRP, as a marker of elevated inflammatory response. Therefore, H. pylori infection, especially cagA-positive strains and its associated systemic inflammatory response can be considered as a contributing factor in atherosclerosis and cardiovascular disease.

The combined effect of stressful factors (temperature and pH) on the expression of biofilm, stress, and virulence genes in Salmonella enterica ser. Enteritidis and Typhimurium.

Salmonella enterica is a major food borne pathogen that creates biofilm. Salmonella biofilm formation under different environmental conditions is a public health problem. The present study was aimed to evaluate the combined effects of stressful factors (temperature and pH) on the expression of biofilm, stress, and virulence genes in Salmonella Enteritidis and Salmonella Typhimurium. In this study, the effect of temperature (2, 8, 22.5, 37, 43 °C) and pH (2.4, 3, 4.5, 6, 6.6) on the expression of biofilm production genes (adr A, bap A), virulence genes (hil A, inv A) and the stress gene (RpoS) of S. Enteritidis and S. Typhimurium was evaluated. The response surface methodology (RSM) approach was used to evaluate the combined effect of the above factors. The highest expression of adr A, bap A, hil A, and RpoS gene for S. Typhimurium was at 22 °C-pH 4.5 (6.39-fold increase), 37 °C-pH 6 (3.92-fold increase), 37 °C-pH 6 (183-fold increase), and 37 °C-pH 3 (43.8-fold increase), respectively. The inv A gene of S. Typhimurium was decreased in all conditions. The adr A, bap A, hil A, inv A, and RpoS gene of S. Enteritidis had the highest expression level at 8 °C-pH 3 (4.09-fold increase), 22 °C-pH 6 (2.71-fold increase), 8 °C pH 3 (190-fold increase), 22 °C-pH 4.5 (9.21-fold increase), and 8 °C-pH 3 (16.6-fold), respectively. Response surface methodology (RSM) indicated that the temperature and pH had no significant effect on the expression level of adr A, bap A, hil A, Inv A, and RpoS gene in S. Enteritidis and S. Typhimurium. The expression of biofilm production genes (adr A, bap A), virulence genes (hil A, inv A) and the stress gene (RpoS) of S. Enteritidis and S. Typhimurium is not directly and exclusively associated with temperature and pH conditions.

Anticancer Effect of Enterocin A-Colicin E1 Fusion Peptide on the Gastric Cancer Cell.

Cancer is one of the most causes of death all over the world, although improvements in its treatment and recognition. Due to the limitations of common anticancer methods, including surgery, chemotherapy, and radiotherapy, attention has been drawn to other anti-cancer compounds, especially natural peptides such as bacteriocins. In this study, we used a combination of two bacteriocins, colicin E1 and enterocin A, against AGS gastric cancer cell lines. In order to evaluate anticancer properties of fusion peptide, we applied MTT assay, real-time PCR, and flow cytometry tests. This is the first report to show the cell growth inhibitory activity of the enterocin A in combination with colicin E1 against AGS human cancer cells. The results of this study showed that this fusion peptide at a concentration of 60.4 µg/mL and 24 h was able to kill half of the tested cancer cells, and treatment of the cells with this concentration increased the expression of bax and caspase 3 genes and reduced the expression of bacl-2 in 24 h. Flow cytometry analysis of annexin V-FITC/propidium iodide results also showed that our peptide was able to induce apoptosis in treated cells compared with control. Taken together, enterocin A-colicin E1 (ent A-col E1) can be considered as a good candidate for anticancer therapies.

Molecular analysis and the toxin, MSCRAMM, and biofilm genes of methicillin-resistant Staphylococcus aureus strains isolated from pemphigus wounds: A study based on SCCmec and dru typing.

INTRODUCTION: Pemphigus is a chronic autoimmune blistering disease. Pemphigus blisters can damage the natural skin barrier and increase the risk of life-threatening conditions. Colonization of pemphigus wounds with methicillin-resistant Staphylococcus aureus (MRSA) prolongs wound healing and increases mortality rate. Assessing MRSA prevalence, types, and toxin and adhesion genes can facilitate the detection of MRSA strains which cause infections, selection of appropriate treatments, and healing of pemphigus wounds. This study aimed to determine the SCCmec, the direct repeat unit (dru) types (dts), and the toxin, MSCRAMM, and biofilm genes of MRSA strains isolated from pemphigus wounds. METHODS: In this cross-sectional study, 118 S. aureus isolates were gathered from 118 patients with pemphigus. MRSA detection was performed using the mecA gene. Using the polymerase chain reaction method, all MRSA isolates were assessed for the presence of the sea, seb, sec, tst, eta, pvl, hla, hlb, MSCRAMM, and ica genes. Typing and subtyping were performed through respectively SCCmec typing and dru typing methods. The Bionumerics software was used for analyzing the data and drawing the minimum spanning tree. FINDINGS: From 118 S. aureus isolates, 51 were MRSA. SCCmec typing revealed the prevalence of SCCmec II with a prevalence of 64.7% (33 out of 51 isolates) and SCCmec III with a prevalence of 35.3% (18 out of 51 isolates). Dru typing indicated seven dts, namely dts 10a, 10g, 10m, 13i, 8h, 8i, and 9ca in two main clusters. The dt9ca was a new dru type and was registered in the dru-typing database (www.dru-typing.org). The prevalence rates of the hla, sea, and sec genes in MRSA isolates were respectively 54.9%, 27.4%, and 1.9%, while the hlb, seb, eta, and pvl genes were not detected at all. Only one MRSA with SCCmec III and dt10a carried the tst encoding gene. MSCRAMM gene analysis revealed the high prevalence of the eno (31.3%) and the fib (21.5%) genes. The prevalence rates of the icaA and icaD biofilm formation genes were 3.9% and 5.8%, respectively. There were no significant differences between the two detected SCCmec types and between the two detected dts clusters respecting the prevalence of the encoding genes of virulence factors and MSCRAMMs. CONCLUSION: The toxin genes hla and sea are prevalent among MRSA strains with SCCmec II and III isolated from pemphigus wounds. The most prevalent dts are dt10a and dt10g among MRSA with SCCmec III and dt8h and dt8i among MRSA with SCCmec II.

Bacteriocins: New Potential Therapeutic Candidates in Cancer Therapy.

Cancer is one of the most important disorders which is associated with high mortality and high costs of treatment for patients. Despite several efforts, finding, designing and developing, new therapeutic platforms in the treatment of cancer patients are still required. Utilization of microorganisms, particularly bacteria has emerged as new therapeutic approaches in the treatment of various cancers. Increasing data indicated that bacteria could be used in the production of a wide range of anti-cancer agents, including bacteriocins, antibiotics, peptides, enzymes, and toxins. Among these anti-cancer agents, bacteriocins have attractive properties, which make them powerful anti-cancer drugs. Multiple lines evidence indicated that several bacteriocins (i.e., colcins, nisins, pediocins, pyocins, and bovocins) via activation/inhibition different cellular and molecular signaling pathways are able to suppress tumor growth in various stages. Hence, identification and using various bacteriocins could lead to improve and introduce them to clinical practices. Here, we summarized various bacteriocins which could be employed as anti-cancer agents in the treatment of many cancers.

Evaluation of antibacterial activity of enterocin A-colicin E1 fusion peptide.

OBJECTIVES: Bacterial resistance to most common antibiotics is a harbinger of the requirement to find novel anti-infective, antimicrobials agents, and increase innovative strategies to struggle them. Numerous bacteria produce small peptides with antimicrobial activities called bacteriocin. This study aimed to investigate the antibacterial properties of the fusion protein of Enterocin A and Colicin E1 modified against pathogens. MATERIALS AND METHODS: Analysis of recombinant bacteriocin Enterocin A and Colicin E1 (ent A-col E1) was performed to assay the stability and antibacterial activity of this fusion protein. The pET-22b vector was employed to express the coding sequence of the ent A-col E1 peptide in Escherichia coli BL21 (DE3). Minimum inhibitory concentration (MIC), disk diffusion, and time-kill tests were performed to evaluate the antibacterial activity of the ent A-col E1 against Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 10536), Enterococcus faecalis (ATCC 29212), and Staphylococcus aureus (ATCC 33591). RESULTS: The suggested recombinant peptide had good antibacterial activity against both Gram-negative and Gram-positive pathogens. It has also good stability at various temperatures, pH levels, and salt concentrations. CONCLUSION: Because bacteriocins are harmless compounds, they can be recommended as therapeutic or preventive supplements to control pathogens. According to the obtained results, the ent A-col E1 peptide can serve as an efficient antibacterial compound to treat or prevent bacterial infections.

Bacterial Etiology and Antibiotic Susceptibility of Conjunctivitis Patients' Isolates in Kashan, Iran.

BACKGROUND: Conjunctivitis is a very common ocular disease, which can be caused by a wide variety of microorganisms. This study was aimed to assess the bacterial etiology and antibiotic susceptibility of conjunctivitis patients' isolates from Central Iran. MATERIALS AND METHODS: This study was performed in 180 patients referred to the Department of Ophthalmology in Kashan University with symptoms of conjunctivitis from July 2017 to December 2017. To detect of different bacteria, Gram staining, morphological characterization, pigment production, biochemical characteristics, coagulase test, optochin and PYR tests, oxidase test, and culture on specific media were used. Antibiotic susceptibility of the bacteria isolated was done using the Kirby-Bauer method. Methicillin resistance in staphylococci isolated from the patients was identified using polymerase chain reaction technique. RESULTS: Of the 195 bacteria isolated, about 81.5% were Staphylococcus epidermidis and Staphylococcus aureus and the remaining 19.5% included other species. In the present study, Pseudomonas aeruginosa was most resistant to ampicillin. In the case of S. epidermidis and S. aureus, the highest resistance was observed against erythromycin and the least resistance was against rifampicin and linezolid. CONCLUSION: In this study, S. aureus and S. epidermidis are the most common causes of conjunctivitis in all age groups, however, this condition decreases with age and is also influenced by other factors such as season and weather conditions. The results of this study can be helpful in planning more prudent treatment strategies for patients with conjunctivitis in Kashan.

Molecular Epidemiology of Antimicrobial Resistance of Uropathogenic Escherichia coli Isolates from Patients with Urinary Tract Infections in a Tertiary Teaching Hospital in Iran.

To characterize the resistance patterns of uropathogenic Escherichia coli (UPEC) in a Tertiary Teaching Hospital in Iran, we conducted a descriptive epidemiology study using molecular techniques. The subjects consisted of patients having acute urinary tract infection, who were enrolled in the study from 2014 to 2017. The antimicrobial susceptibility profile of 101 UPEC isolates was determined by Kirby-Bauer disc diffusion method. Extended spectrum ß-lactamase (ESBL) was detected by the double-disk synergy test. Biofilm formation was done using microtiter plates. The presence of virulence genes (pai, pap, hly, traT, pai, cnf-1, sfa, and afa) was evaluated by a PCR. Molecular typing of UPEC E. coli isolates was performed with fimH and multilocus sequence typing (MLST). 70.3% of isolates were multidrug-resistant. 37.6% of isolates were Extended spectrum ß-lactamases (ESBLs) producer. Strong biofilm formation was seen in 27.7%. Forty-seven different fimH allelic variants were identified. Among identified fimH allelic variants, the most common types were f1 (18.8%) and f14 (18.8%). ST131 (54.5%) was the most prevalent clonal group significantly correlated with the pai gene. Seven sequence types (STs) were detected only once (ST405, ST410, ST450, ST636, ST648, ST1193, and ST6451). Clonal groups showed no significant differences in terms of antibiotic resistance patterns. There was no significant difference between virulence genes and antibiotic resistance patterns in the studied clonal groups. To our knowledge, the present study is the first study in Iran that investigated the genotypic diversity of UPEC isolates by MLST and fimH typing methods. The two methods might serve as a useful molecular test for surveillance and epidemiological studies of isolates.

Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain.

Introduction: The bacterial enzyme has gained more attention in therapeutic application because of the higher substrate specificity and longer half-life. L-asparaginase is an important enzyme with known antineoplastic effect against acute lymphoblastic leukemia (ALL). Methods: Novel L-asparaginase genes were identified from a locally isolated halo-thermotolerant Bacillus strain and the recombinant enzymes were overexpressed in modified E. coli strains, OrigamiTM B and BL21. In addition, the biochemical properties of the purified enzymes were characterized, and the enzyme activity was evaluated at different temperatures, pH, and substrate concentrations. Results: The concentration of pure soluble enzyme obtained from Origami strain was ~30 mg/L of bacterial culture, which indicates the significant improvement compared to L-asparaginase produced by E. coli BL21 strain. The catalytic activity assay on the identified L-asparaginases (ansA1 and ansA3 genes) from Bacillus sp. SL-1 demonstrated that only ansA1 gene codes an active and stable homologue (ASPase A1) with high substrate affinity toward L-asparagine. The Kcat and Km values for the purified ASPase A1 enzyme were 23.96s-1 and 10.66 µM, respectively. In addition, the recombinant ASPase A1 enzyme from Bacillus sp. SL-1 possessed higher specificity to L-asparagine than L-glutamine. The ASPase A1 enzyme was highly thermostable and resistant to the wide range of pH 4.5-10. Conclusion: The biochemical properties of the novel ASPase A1 derived from Bacillus sp. SL-l indicated a great potential for the identified enzyme in pharmaceutical and industrial applications.

Immunization with recombinant FliD confers protection against Helicobacter pylori infection in mice.

Nearly half of the world's population is infected with Helicobacter pylori. Clinical manifestations of this infection range from gastritis and peptic ulcers to gastric adenocarcinoma and lymphoma. Due to the emerging of antibiotic resistant strains and poor patient compliance of the antibiotic therapy, there is increasing interest in the development of a protective vaccine against H. pylori infection. The bacterial protein FliD forms a capping structure on the end of each flagellum which is critical to prevent depolymerization and structural degradation. In this study, the potential of FliD as a prospective H. pylori subunit vaccine was assessed. For this purpose, immunogenicity and protective efficacy of recombinant FliD (rFliD) from H. pylori was evaluated in C57BL/6 mice. Purified rFliD was formulated with different adjuvants and administered via subcutaneous or oral route. Subcutaneous immunization with rFliD elicited predominantly mixed Th1 and Th17 immune responses, with high titers of specific IgG1 and IgG2a. Splenocytes of immunized mice exhibited strong antigen-specific memory responses, resulting in the secretion of high amounts of IFN-γ and IL-17, and low levels of IL-4. Immunization with rFliD caused a significant reduction in H. pylori bacterial load relative to naïve control mice (p < 0.001), demonstrating a robust protective effect. Taken together, these results suggest that subcutaneous vaccination with rFliD formulated with CpG or Addavax could be considered as a potential candidate for the development of a subunit vaccine against H. pylori infection.

Recombinant Endolysins as Potential Therapeutics against Antibiotic-Resistant Staphylococcus aureus: Current Status of Research and Novel Delivery Strategies.

Staphylococcus aureus is one of the most common pathogens of humans and animals, where it frequently colonizes skin and mucosal membranes. It is of major clinical importance as a nosocomial pathogen and causative agent of a wide array of diseases. Multidrug-resistant strains have become increasingly prevalent and represent a leading cause of morbidity and mortality. For this reason, novel strategies to combat multidrug-resistant pathogens are urgently needed. Bacteriophage-derived enzymes, so-called endolysins, and other peptidoglycan hydrolases with the ability to disrupt cell walls represent possible alternatives to conventional antibiotics. These lytic enzymes confer a high degree of host specificity and could potentially replace or be utilized in combination with antibiotics, with the aim to specifically treat infections caused by Gram-positive drug-resistant bacterial pathogens such as methicillin-resistant S. aureus. LysK is one of the best-characterized endolysins with activity against multiple staphylococcal species. Various approaches to further enhance the antibacterial efficacy and applicability of endolysins have been demonstrated. These approaches include the construction of recombinant endolysin derivatives and the development of novel delivery strategies for various applications, such as the production of endolysins in lactic acid bacteria and their conjugation to nanoparticles. These novel strategies are a major focus of this review.

Sensitivity of levofloxacin in combination with ampicillin-sulbactam and tigecycline against multidrug-resistant Acinetobacter baumannii.

BACKGROUND AND OBJECTIVES: The selection of alternative treatment options with antibiotic combinations may be used for successful managing of multidrug-resistant Acinetobacter baumannii. The aim of this study was to determine the synergistic effects of ampicillin-sulbactam combined with either levofloxacin or tigecycline against MDR A. baumannii. MATERIALS AND METHODS: A total 124 of A.baumannii isolates collected from clinical samples of hospitalized patients which assessed for antibiotic susceptibility using disk diffusion method. E-test was used on 10 MDR A. baumannii isolates to determine the minimum inhibitory concentration (MIC) of ampicillin-sulbactam, levofloxacin and tigecycline. Any synergistic effects were evaluated at their own MIC using E-test assay at 37°C for 24 hours. Synergy was defined as a fractional inhibitory concentration index (FICI) of ≤0.5. RESULTS: Levofloxacin plus ampicillin-sulbactam combination was found to have synergistic effects (FIC index: ≤0.5) in 90% of the isolates, but there was no synergistic effect for ampicillin-sulbactam/tigecycline and tigecycline/levofloxacin combination. The antagonist effect in 50% of isolates (FIC index: >2) showed in combination of levofloxacin/tigecycline. CONCLUSION: The emergence of multidrug A. baumannii isolates requires evaluating by combination therapy. The combination of levofloxacin plus a bactericidal antibiotic such as ampicillin-sulbactam is recommended. Results should be confirmed by clinical studies.

A Novel Chimeric Endolysin with Antibacterial Activity against Methicillin-Resistant Staphylococcus aureus.

Cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) and amidase are known as catalytic domains of the bacteriophage-derived endolysin LysK and were previously reported to show lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). In the current study, the in silico design and analysis of chimeric CHAP-amidase model was applied to enhance the stability and solubility of protein, which was achieved through improving the properties of primary, secondary and tertiary structures. The coding gene sequence of the chimeric CHAP-amidase was synthesized and subcloned into the pET-22(+) expression vector, and the recombinant protein was expressed in E. coli BL21 (DE3) strain. Subsequent affinity-based purification yielded ~12 mg soluble protein per liter of E. coli culture. Statistical analysis indicated that concentrations of ≥1 µg/mL of the purified protein have significant antibacterial activity against S. aureus MRSA252 cells. The engineered chimeric CHAP-amidase exhibited 3.2 log reduction of MRSA252 cell counts at the concentration of 10 µg/mL. A synergistic interaction between CHAP-amidase and vancomycin was detected by using checkerboard assay and calculating the fractional inhibitory concentration (FIC) index. This synergistic effect was shown by 8-fold reduction in the minimum inhibitory concentration of vancomycin. The chimeric CHAP-amidase displayed strong antibacterial activity against S. aureus, S. epidermidis, and enterococcus. However, it did not indicate any significant antibacterial activity against E. coli and Lactococcus lactis. Taken together, these findings suggest that our chimeric CHAP-amidase might represent potential to be used for the development of efficient antibacterial therapies targeting MRSA and certain Gram-positive bacteria.

Antibodies raised against divalent type b flagellin and pilin provide effective immunotherapy against Pseudomonas aeruginosa infection of mice with burn wounds.

Burn wound infections caused by multidrug-resistant Pseudomonas aeruginosa strains are a serious challenge to therapy because of the complex pathogenesis and paucity of new effective antibiotics. Therefore, there is renewed interest in developing antibody-based therapeutic strategies. Immunotherapy strategies typically target selected virulence factors that are expressed by the majority of clinical strains of P. aeruginosa, particularly because virulence factors mediate infection. Here we used a murine model of burn wound infection to evaluate the efficacy of antibodies raised against the divalent type b flagellin and PilA (flagellin b + PilA), as acute virulence factors, to prevent and treat infection. Antibodies to flagellin b + PilA exhibited superior synergistic effects that improved opsono-phagocytosis and cell invasion compared with antibodies to each monovalent flagellin b or PilA. Further, when used for prophylaxis, the antibodies against flagellin b + PilA and combined therapeutic and prophylactic regimens markedly improved the survival of mice infected with disparate P. aeruginosa strains from 91.6% to 100% compared with treatment using imipenem. Therefore, antibodies against flagellin b + PilA interfere with the activities of their respective cognate individual target antigens and enhance coverage against clinical strains of P. aeruginosa that may not express one of these two virulence factors.

Purification of Antibacterial CHAPK Protein Using a Self-Cleaving Fusion Tag and Its Activity Against Methicillin-Resistant Staphylococcus aureus.

Therapeutic LysK-CHAP is a potent anti-staphylococcal protein that could be utilized as an antibiotic substitute. Intein-mediated protein purification is a reasonable and cost-effective method that is most recently used for recombinant therapeutic protein production. Intein (INT) is the internal parts of the protein that can be separated from the immature protein during protein splicing process. This sequence requires no specific enzyme or cofactor for separation. INT sequence and their characteristic of self-cleavage by thiol induction, temperature, and pH changes are used for protein purification. The current study presents the expression of CHAPK262 domain of LysK gene that is fused with INT/chitin-binding sequence while evaluating its purification procedure and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The coding gene sequence of LysK-CHAP (CHAPK262) in pET22-b was amplified with polymerase chain reaction (PCR); the digested product was then cloned into the pTXB1 vector. Electrophoresis confirmed the cloning accuracy of the gene. The pTXB1-CHAPK262 plasmid was transformed to the Escherichia coli ER2566 (E. coli ER2566) expression strain and analyzed for expression of the recombinant protein by SDS-PAGE and Western blotting methods. Finally, CHAPK262 was purified by chitin affinity column using INT tag technology and confirmed by SDS-PAGE. Lytic activity of the purified protein was investigated by disk diffusion method. Cloning of CHAPK262 into the pTXB1 vector, which comprised INT/chitin-binding sequence, was successfully achieved. The SDS-PAGE data also revealed successful expression of the CHAPK262-INT fusion protein and Western blotting method validated the accuracy of the protein. Moreover, purification of CHAPK262 protein was induced by dithiothreitol (DTT) and confirmed by SDS-PAGE. Finally, inhibition zone in MRAS culture medium confirmed antibacterial activity of the protein. Application of intein-mediated antibacterial protein is an appropriate and streamlined method for one-step purification of CHAPK262 as a therapeutic and antibacterial protein. Self-cleaving tags like intein are cost-effective and could be used as a proper purification method for industrial purposes.

The Prevalence of S. aureus Skin and Soft Tissue Infections in Patients with Pemphigus.

Pemphigus vulgaris are autoimmune blistering diseases that may result in significant morbidity and death. Immunosuppressive therapy of pemphigus vulgaris would predispose the patients to infections. The aim of this study was to assess the prevalence of S. aureus infection and PVL gene in patients with pemphigus admitted to dermatology clinic. Materials and Methods. This descriptive study was conducted on 196 pemphigus vulgaris patients (119 males, 77 females) admitted to dermatology clinic between 2014 and 2015. In this study, the diagnosis of pemphigus vulgaris was made by histology, immunofluorescence pattern of perilesional skin, and indirect immunofluorescence testing of serum. Data were collected through a questionnaire. Results. 59.1% of pemphigus vulgaris patients had S. aureus infection. 49 out of 116 were methicillin-resistant. PVL gene was detected in 25 out of 116 S. aureus positive patients. Conclusion. This is the first report of S. aureus infection in pemphigus patients in Iran. More than forty percent of isolates were methicillin-resistant S. aureus. PVL gene carried by methicillin-resistant S. aureus was high in this study.

Emerging Carbapenem-Resistant Pseudomonas aeruginosa Isolates Carrying blaIMP Among Burn Patients in Isfahan, Iran.

BACKGROUND: Metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa is a significant pathogen in burn patients. OBJECTIVES: The aim of this study was to determine the prevalence of carbapenem-resistant P. aeruginosa isolates, including those resistant to imipenemase (IMP), in a burn unit in Isfahan, Iran. PATIENTS AND METHODS: One hundred and fifty P. aeruginosa isolates from burn patients were tested for antibiotic susceptibility by the disc diffusion method in accordance with CLSI guidelines. Production of MBL was identified with the EDTA disk method. DNA was purified from the MBL-positive isolates, and detection of the blaIMP gene was performed with PCR. RESULTS: Fifty-seven out of 150 (38%) isolates were multi-drug resistant (MDR), and 93 (62%) were extensively-drug resistant (XDR). Among all isolates, the resistance rate to ciprofloxacin, tobramycin, imipenem, meropenem, amikacin, ceftazidime, and cefepime was higher than 90%, while the resistance rates to piperacillin/tazobactam and aztreonam were 70.7% and 86%, respectively. Colistin and polymyxin B remained the most effective studied antibiotics. All of the imipenem-resistant P. aeruginosa isolates were MBL-positive, and 107 out of 144 (74.3%) of the MBL isolates were positive for the blaIMP gene. CONCLUSIONS: The results of this study show that the rate of P. aeruginosa-caused burn wound infections was very high, and many of the isolates were resistant to three or more classes of antimicrobials. Such extensive resistance to antimicrobial classes is important because few treatment options remain for patients with burn wound infections. blaIMP -producing P. aeruginosa isolates are a rising threat in burn-care units, and should be controlled by conducting infection-control assessments.

Prevalence of CTX-M-Type and PER Extended-Spectrum ß-Lactamases Among Klebsiella spp. Isolated From Clinical Specimens in the Teaching Hospital of Kashan, Iran.

BACKGROUND: Extended-spectrum ß-lactamases (ESBLs) is one of the most important mechanisms of resistance to ß-lactams especially among Enterobacteriaceae family including Klebsiella spp. Different types of extended-spectrum ß-lactamases including CTX-M-type and PER enzymes are identified among gram negative bacteria. OBJECTIVES: The current study aimed to determine the prevalence of CTX-M-type and PER extended-spectrum ß-lactamases among Klebsiella spp. isolated from clinical specimens in the teaching hospital of Kashan, Iran. PATIENTS AND METHODS: One hundred Klebsiella spp. were isolated from clinical specimens of hospitalized patients at Shahid-Beheshti hospital from December 2012 to November 2013. Disk diffusion method was used to determine the susceptibility of these isolates to 14 different antimicrobial agents; disks were purchased from MAST company (United Kingdom). The phenotypic double disk synergy confirmatory test was used to screen the isolates to produce extended-spectrum ß-lactamase. DNAs of isolates were extracted using boiling method and PCR assay was used to characterize the bla CTX-M type and bla PER genes. The purified PCR products were sent to Macrogen research company (Korea) for sequencing. RESULTS: Of the total 100 Klebsiella isolates, %93 was susceptible to imipenem. Resistance to ampicillin, ceftazidime, ceftriaxone, aztreonam and cefotaxime was (92%), (67%), (65%), (64%) and (59%), respectively. The phenotypic confirmatory test (PCT) confirmed that 35% (n = 35) of the isolates were ESBL-producing Klebsiella strains. The prevalence of bla CTX-M type and bla RER genes among Klebsiella isolates were 28% (n = 28) and 9% (n = 9), respectively. CONCLUSIONS: The prevalence of ESBL-producing Klebsiella strains in Shahid-Beheshti hospital in Kashan has increased. The study concluded that there was a high prevalence of the bla CTX-M type gene among ESBL positive isolates.

A strategy for soluble overexpression and biochemical characterization of halo-thermotolerant Bacillus laccase in modified E. coli.

An efficient method was introduced for soluble expression of recombinant laccase (rpCotA(SL-1)) from a newly isolated halo-thermotolerant Bacillus sp. SL-1 in modified Escherichia coli, trxB2/gor2 mutant (Origami™ B (DE3)). The yield of purified soluble laccase in Origami strain under micro-aerobic condition was ∼20mg/L of bacterial culture, showing significant improvement over the laccase produced in E.coli BL21 strain under aerobic condition. The specific activity of 13U/mg for purified laccase produced in micro-aerobic condition was higher than that of 1.07U/mg observed for the purified enzyme obtained in aerobic condition in Origami. The kinetic Km and kcat parameters for laccase-induced oxidation reactions were 46µM and 23s(-1) for ABTS (2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), and 19.6µM and 24s(-1) for SGZ (syringaldazine) substrates, respectively. The rpCotA(SL-1) displayed thermostability at 70°C and tolerance to specified concentrations of NaCl, NaN3, EDTA and SDS as inhibitors. The enzyme was relatively stable in the presence of different concentration of organic solvents, however the residual activity was adversely affected as the dipole moment of the solvents increase. Here we successfully report the production of soluble and functional laccase in Origami at the expression level suitable for industrial application.