Iranian Journal of Microbiology
Volume:6 Issue: 5, Oct 2014

Pseudomonas aeruginosa is responsible for devastating nosocomial infections among severely burn patients. Class C of cephalosporinase (AmpC-β-lactamases) is important cause of multiple β-lactam resistance in P. aeruginosa. The aim of this study was to detect the AmpC-β-lactamases producing isolates among carbapenem resistant P. aeruginosa isolated from burn patient. a total of 100 isolates of carbapenem resistant P. aeruginosa isolates from different burn patients were investigated. Three phenotypic methods were selected for identification of the AmpC-β-lactamases producing isolates. Fifty four isolates were AmpC producer as detected by AmpC disk test. Seventeen isolates were identified as AmpC producer using combined disk method. Fifty two isolates showed a twofold or threefold dilution difference between the minimum inhibitory concentration of imipenem or ceftazidime and the minimum inhibitory concentration of imipenem or ceftazidime plus cloxacillin. One isolate was identified as AmpC producer using three methods. Three isolates produced AmpC as detected by both AmpC disk test and combined disk methods and 19 isolates were found as AmpC producer using both AmpC disk test and minimum inhibitory concentration methods. Six isolates were AmpC producer as shown by the MICs of both imipenem and ceftazidime. According to the results of this study, AmpC- β-lactamase looks to be the main mechanism of resistance of Pseudomonas aeruginosa to cephalosporins and carbapenems in the study hospital.

Klebsiellapneumoniae (K. pneumoniae) is an opportunistic microorganism. This study aimed to investigate the presence of magA gene and antimicrobial susceptibility in K. pneumoniae. 195 clinical specimens were collected from hospitals of Shahrekord, Iran. Bacterial culture, biochemical diagnostic standard test, determination of antibiotic sensitivity, phenotypic testing hypermucoviscosity (HV) and polymerase chain reaction (PCR) was performed for isolation and characterization of K. pneumoniae. 173 samples were positive for K. pneumoniae. The highest and lowest rates of resistance were related to amoxicillin 79.19% and ciprofloxacin 15.60%, respectively. Also 4 samples were positive for magA gene. Based on our results, K. pneumoniae strains were resistant to different antibiotics. Knowing how to identify strains of K. pneumoniae, spreading of its virulence and also antimicrobial resistance genes can be useful in treatment of infection caused by this bacterium.

Treatment of Pseudomonas aeruginosa infections is greatly hampered by innate and acquired antibiotic resistance. The goal of this study was to compure the immunogenicity of conjugatesof P. aeruginosadepolymerized alginate-diphtheria toxoid (D-ALGDT) and P. aeruginosadetoxified lipopolysaccharidediphtheria toxoid (D-LPSDT) in mouse model. Alginate and LPS were purified from P. aeruginosastrain PAO1. The resulting depolymerized alginate (D-ALG) and detoxified LPS (D-LPS) were covalently coupled to diphtheria toxoid (DT) as a carrier protein with adipic acid dihydrazide (ADH) as a spacer molecule and carbodiimide as a linker. Sterility, safety and pyrogenicity tests were performed. 30 mice in two groups were immunized intraperitoneally on days 0, 14 and 28 with 10 µg of D-ALGDT and D-LPSDT. Conjugates specific antibody levels were also determined by enzyme-linked immunosorbent assay (ELISA). The conjugates were non-toxic and non-pyrogenic. Conjugates of D-ALGDT and D-LPSDT were shown to be safe and to elicit total IgG, IgM, IgA, IgG1, IgG2a, IgG2b and IgG3 antibodies in mice. ELISA results indicated that antibodies titer of D-ALGDT was more than D-LPSDT. Immunization with D-ALGDT showed significant increase in all types of antibodies titers in versus D-LPSDT, suggesting D-ALGDT as a vaccine candidate against P. aeruginosainfections.

An outer membrane protein (OMP) of Helicobacter pylori namely OipA, is an important virulence factor associated with peptic ulcer and gastric cancer risks. The purpose of this study was to isolate the 34 KDa OMP of H. pylori and evaluate its immunogenicity in experimental animals for rapid detection of more virulent H. pylori isolates. Sarcosine insoluble fraction of membrane proteins (OMPs) were prepared from 15 clinical isolates of H.pylori and their profiles were analyzed by SDS-PAGE. Two out of 15 isolates which demonstrated higher expression for apparent 34 KDa proteins were selected. Under optimal conditions, 34 KDaprotein was recovered from 5% SDS-Agarose gel, purified and injected into the New Zealand white rabbits with Fruend′s adjuvant in multiple stages with two weeks intervals. Collected antiserum was purified through affinity chromatography with Sepharose columnand its titer was determined by ELISA. Specific immune response was demonstrated by Dot blot and western blotting methods. The titer of antibody was determined about 1/3000 and western blotting demonstrated a 34 KD-protein.Screening of various strains by Dot blot method for its presence showed that its expression was more frequent in strains isolated from the patients with more severe pathology. High titer obtained for pAbs antibody, suggested the high immunogenicity of this protein in experimental animals. Detection of 34 KDa OMP in strains isolated from the patients with more severe pathology proposes the possible application of this pAbs in detecting more virulent strains of H. pylori.

Molecular epidemiological studies have shown that certain genotypes of Mycobacterium tuberculosis (MTB) are over-represented in limited geographical regions, suggesting of evolution of certain genotypes with increasing virulence and pathogenicity. Beijing strain of MTBwas initially described by its potential to cause outbreaks worldwide and its association with drug resistance.Due to tuberculosis (TB)-related mortality which is associated with Beijing genotype, this study was designed with the aim to detect the MTB Beijing genotype in the region of study. A total of 170 clinical isolates of MTB were collected from the TB reference laboratory of Khuzestan province, Iran, over one year period from February 2010 to February 2011. Phenotypic tests were used for preliminary detection of MTB. Culture positive MTB isolates were confirmed by multiplex PCR based on IS6110 gene with subsequent screening for resistance to isoniazid (INH), and rifampin (RIF) by PCR using relevant primers. Three set of primers were used to differentiate Beijing from non-Beijing strains by using Deletion- Targeted Multiplex (DTM) PCR. From 160 PCR-confirmed MTB isolates, 18 (11.25%) showed mutation in katG gene related to INH resistance and 20 (12.5%), associated with mutation in rpoB gene related to RIF resistance, and 8 (5%) were detected as Beijing strain using multiplex PCR. The majority of detected Beijing strains (6/8[75%]) comprised mutation in katG gene with the prevalent mutation specifically in codon 315. In 4 Beijing strains (2.5%), mutation in rpoB gene were also detected. Using DTM- PCR, the rate of Beijing strains in the region of study was determined as 5%. Although for detection of MTB antimicrobial resistance, it is advised to use a combination of conventional antimicrobial susceptibility testing and molecular techniques, however for time saving, it seems that DTM-PCR, is a simple technique for use in areas of the world where Beijing strains are highly prevalent.

We developed and evaluated the utility of a quadruplexTaqman real-time PCR assay that allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. The specificity of the assay was tested using reference strains of vancomycin-resistant and susceptible enterococci. In total, 193 clinical isolates were identified and subsequently genotyped using a QuadruplexTaqman real-time PCR assay and melting curve analysis. Representative QuadruplexTaqman real-time PCR amplification curve were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis. Phenotypic and genotypic analysis of the isolates gave same results for 82 enterococcal isolates, while in 5 isolates, they were inconsistent. We had three mixed strains, which were detected by the TaqMan real-time PCR assay and could not be identified correctly using phenotypic methods.Vancomycin resistant enterococci (VRE) genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using TaqMan real-time multiplex real-time PCR assay.

Methicillin-resistant Staphylococcus aureus (MRSA) is a well-known pathogen with a worldwide distribution. Given the increasing rate of MRSA infections, implementing of reliable, accurate and rapid testing for diagnosis of MRSA is necessary. The aim of this study was to compare four diagnostic methods for detection of MRSA isolates. From December 2012 to April 2014, 120 S. aureus isolates were collected from three hospitals affiliated with Tehran University of Medical Sciences. MRSA isolates were detected by four different methods including cefoxitin disc diffusion test, oxacillin disc diffusion test, minimum inhibitory concentration (MIC) of oxacillin as determined by MIC test strip, and mecAdetection by PCR. Out of 120 S. aureusisolates, cefoxitin disc diffusion test, oxacillin disc diffusion test and MIC test strip identified 60 (50%), 48 (40%), 55 (45.83%) isolates as MRSA, respectively. The sensitivity and specificity for oxacillin disc diffusion, cefoxitin disc diffusion and MIC of oxacillin were 80% and 100%, 100% and 100%, and 91.6% and 100%, respectively. Cefoxitin disc diffusion test is reliable substitute for detection of MRSA in clinical laboratory where MIC detection and molecular methods are not accessible.

Staphylococcal food poisoning is a gastrointestinal disease, which is caused by consumption of contaminated food with enterotoxins produced by Staphylococcus aureus(SEs). Milk and its products are known sources of food borne diseases. This study was carried out to evaluate the prevalence of enterotoxigenicS. aureusstrains in organic milk and cheese in Tabriz - Iran. A total of 200 samples (100 milk samples and 100 cheese samples) were collected from farms and milk collection points in Tabriz - Iran. The samples were cultured and identified by standard bacteriological methods, then PCR was performed to detect sea gene.Results and Staphylococcus aureus was found in 27% of all samples (milk and cheese). Results of PCR showed that 12.96% of S. aureus isolates possessed sea gene. It suggested the potential public health threat of S. aureus resulting from contamination of dairy products. So, efforts are required to improve safety standards for preventing staphylococcal food poisoning.

Although hepatitis B vaccine immunogenicity is high, certain risk factors such as age, tobacco consumption, obesity and genetic background have been associated with low responsiveness to HBV vaccine. We aimed to evaluate the role of occult hepatitis B virus (HBV) infection in non-responder adults to HBV vaccine in a low endemic area for HBV. A total of 52 subjects who were non-responder to HBV vaccine were enrolled in the study. HBsAg, anti-HBs and anti-HBc were tested in all subjects. The presence of HBV-DNA was determined in plasma samples by real-time PCR. A total of 52 cases with median age 34 years were enrolled in the study. 63.5% of patients were male and 36.5% were female. Isolated anti-HBc (HBsAg negative, anti-HBs negative and anti-HBc positive) was detected in 3.8% of cases. HBV-DNA was not detected in our cases. This study showed no evidence of occult HBV infection in our HBV vaccine non-responders even in cases with isolated anti-HBc.

The genus Malassezia contains an expanding list of lipophilic yeasts involve in the etiology of various superficial fungal infections. Pityriasisversicolor (PV) is the most prevalent Malassezia-related infection distributed worldwide. In the present study, clinical and epidemiological features of the genus Malassezia are discussed with special focus on PV in Iran. During June 2012 to April 2013, among 713 confirmed cases of fungal infections, 68 (9.5%) were diagnosed as PVby positive direct microscopy results in 20% potassium hydroxide (KOH) preparation of skin scrapings. All the specimens were cultured on modified Dixon agar and incubated at 32ºC for 10 days. Identification of the isolated yeasts was carried out based on macro- and microscopic morphology, catalase test, utilization of Tweens, polyethoxylated castor oil (EL slant), and hydrolysis of esculin and utilization of Tween-60 (TE slant). Out of 68 skin scrapings, 55 (80.9%) yielded yeast colonies on mDixon’s agar which were finally identified as M. globosa (36.36%), M. pachydermatis (29.08%), M. furfur (23.65%), M. slooffiae (7.28%) and M. obtusa (3.64%). Results of the present study further indicate clinico-epidemiological importance of the genus Malassezia with growing importance of M. pachydermatis as a major species involve in the etiology of pityriasisversicolor. These findings are of major concern in management of Malassezia-related diseases.

Biodeterioration is an irreversible damage that is caused by colonization of microorganisms on the surface of different materials. Among all microorganisms, fungi play an important role in deterioration of materials. Fungi can colonize on stone surfaces and by secreting different enzymes, organic and inorganic acids and pigments, can cause bio-weathering and changing not only the substrate materials but the color of stones. Furthermore, fungal mycelia can penetrate into the internal surfaces of stones and change the interior chemical contents of stones. Pasargadae including Cyrus the Great Tomb is entitled by UNESCO as one of the World Heritage Sites. This study was focused on the identification of fungi that were colonized on tomb limestone monument. Sampling of stone was carried out to identify inhabiting molds and yeasts, biochemical and microscopic methods were used for isolated strains. In addition, the Polymerase Chain Reaction (PCR) and sequencing of the PCR products were done. Finally, phylogenic tree was constructed basde on the sequences of ITs region.Results and The common inhabiting fungi which isolated from the tomb limestone belong to Caldosporium sp., Embellisia sp., Cryptococcus sp., Candida sp., Meyerozyma sp., Arthirinium sp., Ulocladium sp., Montagnulaceae sp., Fusarium sp., Humicola sp. and Pseudozyma sp.. Stereomicroscopic images, Scanning Electron Microscope and XRD images, were taken from pieces of stone samples and indicated the severe pattern damages such as pitting, biomineralization, etching and sugaring on the surfaces of stones.