Down regulation of sidB gene by use of RNA interference in aspergillus nidulans

Introduction of the RNA interference [RNAi] machinery has guided the researchers to discover the function of essential vital or virulence factor genes in the microorganisms such as fungi. In the filamentous fungus Aspergillus nidulans, the gene sidB plays an essential role in septation, conidiation and vegetative hyphal growth. In the present study, we benefited from the RNAi strategy for down-regulating a vital gene, sidB, in the fungus A. nidulans. The 21-nucleotide small interfering RNA [siRNA] was designed based on the cDNA sequence of the sidB gene in A. nidulans. Transfection was performed through taking up siRNA from medium by 6 hour-germinated spores. To evaluate the morphologic effects of siRNA on the fungus, germ tube elongation was followed. Moreover, total RNA was extracted and quantitative changes in expression of the sidB gene were analyzed by measuring the cognate sidB mRNA level by use of a quantitative real-time RT-PCR assay. Compared to untreated-siRNA samples, a significant inhibition in germ tube elongation was observed in the presence of 25 nM of siRNA [42 VS 21 microM]. In addition, at the concentration of 25 nM, a considerable decrease in sidB gene expression was revealed. Usage of RNAi as a kind of post-transcriptional gene silencing methods is a promising approach for designing new antifungal agents and discovering new drug delivery systems

Immune reactivity of Brucella melitensis-vaccinated rabbit serum with recombinant Omp31 and DnaK proteins

Brucella melitensis infection is still a major health problem for human and cattle in developing countries and the Middle East. In this study, in order to screen immunogenic candidate antigens for the development of a Brucella subunit vaccine, a cytoplasmic protein [DnaK] and an outer membrane protein [Omp31] of B. melitensis were cloned, expressed in E.coli BL21 and then purified using Ni-NTA agarose. Immunized serum was prepared from a rabbit inoculated with attenuated B. melitensis. It was proved that immunized serum contains antibodies against recombinant Omp31 [rOmp31] and DnaK [rDnaK] by Western blot and ELISA assays. The results may suggest the importance of these proteins as subunit vaccines against B. melitensis as well as targets for immunotherapy

Relation between expression of the las quorum-sensing system in clinical isolates of pseudomonas aeruginosa and expression of efflux pump and ampC

Quorum-sensing systems regulate expression of several virulence factors in Pseudomonas aeruginosa. This study investigated the relation between expression of the las quorum-sensing system and expression of mexY and ampC in 35 clinical isolates of P. aeruginosa. Antibiotic susceptibility was determined by the disc diffusion method according to the Clinical and Laboratory Standards Institute [CLSI] guidelines. Expression of genes including lasl, lasR, mexY, ampC and ampR was assessed by real time RT-PCR. Twenty eight isolates with elevated expression of mexY and ampC [compared to P. aeruginosa PAO1] were resistance to ceftazidime, imipenem, ciprofloxacin and amikacin. Also these isolates demonstrated increased expression of lasl and lasR. The remaining seven isolates showed intermediate resistance to ciprofloxacin and amikacin. These seven isolates with elevated ampR expression -demonstrated decreased expression of mex Y, ampC, lasl and lasR. In contrast to previous study, current study demonstrated that the expression pattern of ampC was identical to the lasl expression pattern among clinical isolates. Furthermore according to previous study we expected that AmpR positively regulated ampC expression but in our study, some of the isolates with elevated ampR expression showed decreased ampC expression. The expression pattern of lasR and mexYwas identical in all of the isolates. It seems there was a direct or indirect relation between expression of lasR and mexY expression in P. aeruginosa

Frequency and antimicrobial resistance patterns of methicillin-resistant staphylococcus aureus in Tehran

Methicillin-Resistant Staphylococcus aureus [MRSA] is the one of most commonly isolated organisms from clinical samples which can cause life-threatening infections. The emergence and spread of antibiotic resistance makes the treatment of these infections more complicated. In this study, we aimed to determine the patterns of antibiotic resistance among MRSA isolates from Tehran, Iran. From December 2012 to April 2014, 120 clinical samples were collected. MRSA was identified by cefoxitin disc diffusion. Antimicrobial susceptibility testing was performed on MRSA isolates for eight other antibiotics by disc diffusion method according to CLSI [2013] recommendations. Also, the minimum inhibitory concentration [MIC] was determined for vancomycin by MIC test strips. According to disc diffusion, 60 [50%] isolates showed resistance to cefoxitin. Among these isolates, the rate of resistance to nitrofurantoin, vancomycin, teicoplanin, doxycycline, trimethoprim, erythromycin, clindamycin, and ciprofloxacin were 0%, 0%, 0%, 28.3%, 28.3%, 58.3%, 63.3%, and 70%, respectively. All isolates were susceptible to vancomycin according to disc diffusion and MIC. Compared to other reports from Iran, our study indicated a moderate rate for MRSA. However, the rates of resistance to generally prescribed antibiotics in these isolates were high. In this situation, it is recommended to monitor the antibiotic resistance in these hospitals

[Urinary tract infection in renal transplant patients in Sina university hospital]

Renal transplantation is the treatment of choice in patients with end-stage renal disease. Urinary tract infection [UTI] is one of the most common complications after renal transplantation and it has serious consequences. The aim of this study was assessing UTIs in renal transplanted patients and evaluation of risk factors associated with post-transplant UTI. In this prospective study, 173 patients [48 hospitalized patients and 125 outpatients] were enrolled in this study. These renal transplant recipients evaluated for bacterial urinary tract infection in urology research center at Sina Hospital. After collecting urine samples from symptomatic and asymptomatic patients, urinalysis and colony count were performed. Identification of bacteria was performed by routine microbiological tests in the Department of Pathobiology, School of Public Health, Tehran, Iran, in 2011. UTI was observed in 47 patients and the most prevalent microorganism was Escherichia coli [E.coli] 18 [38.2%]. Nearly 71% of UTI cases were diagnosed during the first three months post transplantation. Risk factors for post transplant UTI were female gender, age, length of hospitalization and diabetes mellitus. Female patients were more susceptible than males [OR=0.50 and P=0.047] to infection. There were no significant difference between diabetes mellitus and UTI. Most of the isolated bacteria were susceptible to imipenem and resistant to tetracycline and trimethoprim-sulfamethoxazole. Our study confirmed that bacterial infections remain as the most common infectious complication in the early post-transplant period, and antibiogram rather than empirical treatment is needed to find the best effective antibiotics. Moreover, risk factors such as female gender, increased age and length of hospitalization are predisposing factors to increased urinary tract infection in renal transplantation.

[Minimum inhibitory concentration of ciprofloxacin in combination with hexahydroquinoline derivatives against Staphylococcus aureus]

Staphylococcus aureus is the most common pathogen responsible for skin and soft tissue infections worldwide. Methicillin-resistant S. aureus is a major cause of both nosocomial and community acquired infections. The emergence of antimicrobial-resistant S. aureus is of global concern. Fluoroquinolone antimicrobials including ciprofloxacin, levofloxacin, and moxifloxacin are used to treat skin and soft tissue infections due to S. aureus. Emergence of ciprofloxacin resistance has increased in community acquired methicillin-resistant S. aureus strains. The aim of this study was to evaluate the minimum inhibitory concentration of ciprofloxacin and hexahydroquino-line derivatives against methicillin- and ciprofloxacin-resistant S. aureus. Identification of S. aureus was performed by routine microbiological tests in the Department of Pathobiology in Winter 2012. The susceptibility of S. aureus strains to both methicillin and ciprofloxacin was examined by the Kirby-Bauer disk-diffusion method. The minimum inhibitory concentration of ciprofloxacin, hexahydroquinoline derivatives and their combination were separately determined by broth microdilution method against methicillin- and ciprofloxacin-resistant S. aureus. The minimum inhibitory concentration of ciprofloxacin decreased in the presence of hexahydroquinolinein derivatives in comparison with ciprofloxacin alone. This study showed that hexahydroquinoline derivatives enhance the antibacterial effect of ciprofloxacin against methicillin- and ciprofloxacin-resistant S. aureus. Therefore, these derivatives could be used as inhibitors of antibiotic resistance in combination therapies. This enhancement may be related to the inhibitory effect of hexahydroquinoline derivatives on the expression of antibiotic efflux pump in the bacteria. However, the structural features of a fluoroquinolone that determine whether it is affected by efflux transporters are not fully defined.

[Frequency of babA2 genotype in helicobacter pylori from patient with gastroduodenal diseases in Firoozgar Hospital in Tehran]

Blood group antigen binding adhesin [babA2], is essential for attachment of Helicobacter pylori [H. pylori] to the epithelial cell layer and is the most important adhesin of H. pylori. The prevalence rate of the babA2 gene varies in different geographic areas. The aim of this study is to determine the frequency of the babA2 gene in patients with different clinical outcomes. We obtained two gastric biopsy specimens from each patient who suffered from gastrointestinal disease. Rapid urease test [RUT] was performed using one biopsy and the remaining biopsy was delivered to the laboratory for DNA extraction. Prevalence of the babA2 gene was determined using gene specific primers. A total of 56 strains [68.3%] of H. pylori were babA2 positive. The prevalence of babA2 in gastric cancer patients [84.6%] was higher than seen with gastric ulcer [66.7%], duodenal ulcer [61%], and gastric patients [66.7%]. There was no correlation between the babA2 genotype and clinical outcomes. We found that babA2 gene was more prevalent in gastric cancer but no correlation was demonstrated. However, previous studies have demonstrated a correlation of babA2 with severe H. pylori-associated diseases such as duodenal ulcers and gastric cancer. This discrepancy may be related in part to geographic diversity or sample size.

[Prevalence of periodontopathogenic bacteria in patients suffering from periodontitis using culture and PCR methods]

Periodontitis is one of the most common oral diseases with the various incidence rates in different populations. A number of bacteria are considered as the major etiologic agents of periodontitis. The aim of the present study was to determine the prevalence of periodontopathogen bacteria in patients using both PCR and culture techniques. In this study, one hundred patients [including 62 females and 38 males with an average age of 49 +/- 11.5 years] with adult periodontitis referred to periodontics department of School of Dentistry/Tehran University of Medical Sciences were investigated. The samples were taken and sent immediately to the laboratory for culture and molecular evaluation. The PCR was performed using specific primers and the statistical analysis of data was performed using SPSS statistic software and McNemar test. The results demonstrated that the total detection rate in culture method was 64%. The rate of Aggregatibacter actinomycetemcomitans [Aa] was 28% which was significantly higher than that of Porphyromonas gingivalis [Pg] [6%] and Prevotella intermedia [Pi] [3%]. 27% of cases showed mixed bacterial growth. 65% of patients were positive using molecular method. The rate of Aa [30%] was significantly higher than that of Pg [7%] and Pi [5%]. The mixed PCR positive rate containing of Aa, Pg and Pi was [23%].In this study, it was found that most of the bacteria isolated using culture and molecular methods were Aa, Pg and Pi, respectively. Although the detection frequencies of both techniques were similar, the specificity, sensitivity and bacterial detection speed of the PCR technique is obviously higher. Therefore, the use of molecular techniques is strongly recommended. However, both techniques seem to be suitable for microbiological diagnostics.

Identification of class-1 integron and various b-lactamase classes among clinical isolates of Pseudomonas aeruginosa at Children's Medical Center Hospital

Pseudomonas aeruginosa is one of the most important opportunistic pathogens responsible for various types of infections. Children suffer significant morbidity and mortality due to nosocomial infections. The aim of this study was to investigate the presence of Class-1 integron, bla[BEL], bla[PER], bla[kpc], bla[VIM],bla[IMP] and bla[OXA] A-group-1 genes among P. aeruginosa isolates at Children's Medical Center Hospital in Iran and to determine phenotypic evidence of ESBL and MBL production. Antibiotic susceptibility tests were analyzed for 72 P. aeruginosa clinical isolates. Isolates were identified by using biochemical tests and confirmed by PCR assay for oprL gene. ESBL and MBL producer isolates were identified by phenotypic tests [double disc synergy tests]. Detection of beta-lactamase genes and class-1 integron were performed by PCR method. All of the isolates were susceptible to ceftazidime / clavulanate, me-ropenem, imipenem and ciprofloxacin. About 83.3% and 16.7% of isolates were resistant to ceftazidime and amikacin respectively. Approximately, 83.3% of isolates were considered as potential ESBL producers. None of the clinical isolates showed above P-lactamase genes. It seems that, the reason is the absence of class-1 integron in all of isolates. About 16.7% of strains were identified as multidrug resistant. Fortunately, all of the isolates were susceptible to meropenem and imipenem which are effective against ESBL producing strains. The absences of class-1 integron decreases the probability of acquired beta-lactamase especially MBL. Thus, absolute susceptibility to carba-penems and ciprofloxacin among P. aeruginosa isolates in pediatric hospital has important implications for empirical antimicrobial therapy. It seems that these properties help to decrease mortality of nosocomial infections within children

High-level vancomycin-resistant Staphylococcus aureus [VRSA] in Iran: a systematic review

Staphylococcus aureus is a major human pathogen worldwide. Vancomycin has been used for decades to treat multidrug resistant S. aureus. Ten years has passed since the first report of vancomycin resistant S. aureus [VRSA]. The objective of this systematic review was to determine the total number of VRSA isolates that have been reported from Iran. Search terms reflected [Iran], [vancomycin] and [SI aureus] were searched in the ISI web of knowledge, PubMed, SciVerse, and Google scholar. Also two Persian scientific databases and 13 recent national congresses were investigated. Articles / abstracts working on S. aureus in Iran, evaluating vancomycin MIC and / or PCR of vanA/B were included in this systematic review. Out of the 3484 records found in mentioned resources, 13 related studies were included in the final analysis. The result showed that at least 24 VRSA isolates which have been reported from Iran up to September 2012. It seems that many Iranian researchers did not follow a spe-cific guideline for reporting and confirming VRSA. Establishing an Iranian reference center where studies on VRSA can be registered, evaluat-ed and confirmed is strongly recommended

Renal actinomycosis in presence of renal stones in a patient with end stage renal disease

Renal actinomycosis is a rare infection, and actinomycosis mostly acts as a normal flora in mouth, colon and vagina. We present a case of 56 years old man, who referred to our center for renal transplantation with kidney stone and diagnosed with renal actinomycosis. This case has risen possibility of rare infection that can be considered in the setting of renal transplantation

Prevalence of methicillin-resistant staphylococcus aureus carrying panton- valentine leukocidin gene in cutaneous infections in the city of Isfahan

Methicillin-Resistant Staphylococcus aureus [MRSA] is a major cause of Nosocomial and community infections that are becoming increasingly difficult to combat, because of emerging resistance to all classes of antibiotics. Moreover Panton-Valentine leukocidin [PVL] is an important virulence factor in S. aureus and causes white blood cell destruction, necrosis and accelerated apoptosis. The aim of this study was to determine the frequency of PVL-positive MRSA in cutaneous infections, for epidemiological purposes and also to determine antibiotic resistance of the isolates. Collectively, 56 isolates of S. aureus were obtained from Isfahan University of Medical sciences affiliated hospitals and confirmed with biochemical tests [coagulase, mannitol fermentation, and DNase]. Then polymerase chain reaction [PCR] was used to detect pvl gene. Coagulase gene was used as internal control. The antibiotic susceptibility of all isolates to methicillin was determined using disk diffusion method. Out of 56 isolates 14.3% were PVL positive that 37.5% were from abscess and 62.5% were from wound. Among all of these isolates 67.8% were MRSA and also 75% of PVL-positive isolates were MRSA. The prevalence of PVL positive MRSA in cutaneous isolates is high. Future works are necessary for a more complete understanding of distribution of these virulent isolates in nasal carriers to decrease the risk of infections.

[ identification of nocardia in BAL specimens of bronchoscopic patients by using classical and molecular methods]

Nocardiosis is a rare and potentially life-threatening infection caused by several species of the Nocardia genus. The objective of this study was to develop and evaluate a rapid and new method to clinically identify relevant Nocardia species. Rapid and accurate diagnosis of Nocardia species is essential for the treatment of severe infections and prevention of cerebral abscess. One hundred and eighty patients, 103 [57.22%] male and 77 [42.78%] female, with severe symptomatic pulmonary infection were studied in the course of a 12-month period in Dr. Shariati Teaching Hospital affiliated to Tehran University of Medical Sciences in 2010. The specimens were cultured and identified using microbiological and biochemical tests. Polymerase chain reaction [PCR] was used to directly identify the organism in the broncoalveolar lavage samples collected from the patients. NG1 and NG2 primers were used to amplify a Nocardia genus-specific 598-bp fragment of 16S rRNA. Nineteen samples [10.56%] were positive with PCR and 5 samples [2.78%] with conventional methods. All samples with positive cultures were also positive by PCR. The results of this study showed that PCR has a high sensitivity and accuracy for the detection of Nocardia compared with culture and biochemical tests. Considering the rapidity, precision, high sensitivity and specificity of molecular techniques, use of these techniques is suggested in conjunction with conventional methods for the detection of Nocardia phenotypes in clinical laboratories and research centers