Optimization of a pulsed-field gel electrophoresis for molecular typing of Proteus mirabilis

Objective: For the detection of outbreaks caused byProteus mirabilis, strains clonal relations are determinedmethods as “pulsed-field gel electrophoresis (PFGE)”.The aim of this study was optimization of a pulsed-fieldgel electrophoresis for molecular typing of P. mirabilis.Methods: In this study, PFGE’ protocol is optimized foruse in molecular typing of P. mirabilis. Phylogenetic analyzesof strains were evaluated with Bionumerics softwaresystem (version 6.01; Applied Maths, Sint-Martens-Latem, Belgium).Results: This protocol compared with Gram-negativebacteria PFGE protocols, NotI enzyme is suitable for thisbacterium. Electrophoresis conditions should be revealedas; - block 1: initial pulse duration 1 sec, ending pulseduration 30 sec, striking angle 120°, the current 6 V/cm2,temperature 14°C, time 8 hours; - block 2: initial pulseduration 30 sec, ending pulse duration 70 sec, strikingangle 120°, the current 6 V/cm2, temperature 14°C, time16 hours; - TBE, pH=8.4.Conclusion: P. mirabilis strains were typed by PFGE andBionumerics analysis program were determined clonal relationships.The procedure was simple, reproducible andsuitable for these bacteria. Also it was evaluated, becauseof reducing time, the solution volumes and enzymes canbe economically. Outbreaks of nosocomial infections dueto bacteria studied assessment and the potential to provideuseful information about the degree of prevalence.This optimized protocol is allowed different centers’ PFGEresults to compare with other laboratories results. J ClinExp Invest 2013; 4 (3): 306-312Key words: Proteus mirabilis, molecular typing, pulsedfieldgel electrophoresis

Mycobiome in the Middle Ear Cavity with and Without Otitis Media with Effusion

Objective:No data have yet been published revealing the composition and the diversity of fungal communities (mycobiome) in the human middle ear cavity. The presented study investigated the mycobiome in the middle ear cavities of individuals with healthy middle ears and patients with otitis media with effusion.Methods:A total of 77 middle ear and four adenoid samples were collected from 47 individuals (35 children and 12 adults) in Group 1 and from 20 children in Group 2. The mycobiome profile was analyzed with nuclear ribosomal internal transcribed spacer 2 (ITS2) based metabarcoding using an Illumina MiSeq metagenomics kit.Results:ITS2-based metabarcoding detected 14 different genera and 17 different species with a mean relative abundance of ≥1% in the samples analyzed. Mycobiome profile was similar between the adenoid tissue and the middle ear cavity, between Groups 1 and Group 2, and between children and adults. Fusarium, Stemphylium, Candida, and Cladosporium were the most abundant genera detected in all samples. The mean relative abundances of the genera Candida and Fusarium were remarkably higher in Group 2 compared to Group 1.Conclusion:The species Candida glaebosa, Candida cretensis, Aspergillus ruber, Penicillium desertorum, and Rhizopus arrhizus were significantly more abundant in patients with otitis media with effusion (OME), raising the possibility that they affect the pathogenesis of OME

Phenotypic and molecular characterization of Rhizobium vitis strains from vineyards in Turkey

Crown gall-affected grapevine samples were collected during 2009–2010 from major vineyards, located in different Turkish provinces. One hundred and three bacterial strains were obtained from 88 vineyards and 18 grapevine varieties; they were tumorigenic when inoculated in tobacco, sunflower and Datura stramonium plants and were identified as Rhizobium vitis using biochemical and physiological tests as well as PCR and specific primers. Nineteen R. vitis strains presented a number of anomalous biochemical and physiological characters. PCR and opine-specific primers revealed the presence of octopine/cucumopine-type plasmid in 82 R. vitis strains, nopaline-type plasmids in 18 strains and vitopine-type plasmids in three strains. Clonal relationship of strains was determined using Pulsed Field Gel Electrophoresis following digestion of genomic DNA with the restriction endonuclease PmeI. The greatest genetic diversity was found for the strains from Denizli, Ankara and Nevşehir provinces. Nopaline and vitopine-types of Rhizobium vitis were detected for the first time in Turkey

Investigation of Molecular Mechanisms of Carbapenemase Producing Acinetobacter baumannii complex Isolates Isolated from Blood Cultures

INTRODUCTION: It was aimed to determine the presence of metallo-beta-lactamases (MBL) and oxacillinases (OXA) enzymes in Acinetobacter baumannii complex (ABC) isolates, which show decreased sensitivity to carbapenems isolated from blood cultures and to investigate the relationships of isolates among each other and with European clones (EU) I, II, III. METHODS: The study included ABC isolate which has reduced sensitivity to at least one of either imipenem or meropenem which was isolated from blood samples obtained from 74 patients who were admitted to the hospital between 2008 and 2009. Identification of isolates and their antimicrobial susceptibilities were performed using BD Phoenix Automated System (Becton-Dickinson, USA). OXA and MBL genes were investigated by polymerase chain reaction (PCR). The clonal relationship of the isolates was determined by pulsed-field gel electrophoresis (PFGE) method. RESULTS: MBL genes and blaOXA-24-like gene were not determined in any of the isolates. blaOXA-51-like was detected in all isolates, blaOXA-58-like gene in 32 isolates and blaOXA-23-like gene in 26 isolates. Using PFGE method, it was detected that fifty-five blood isolates carrying the blaOXA23-like and/or blaOXA-58-like gene were clustered under six clusters. The similarity of EU clones with clinical ABC isolates in the clusters was found to be over 90%. DISCUSSION AND CONCLUSION: ABC isolates producing oxacillinase in our hospital; The presence of association with EU clone III, which is reported very rarely in our country, and the detection of possible related isolates with EU clones I and II show the potential for the spread of these clones in our hospital and in our country

Antimicrobial Activity of Garlic Derivatives on Common Causative Microorganisms of the External Ear Canal and Chronic Middle Ear Infections

Objective:Today, antibiotic resistance is increasing and evolving into an important health problem. Therefore, it is important to research on alternative therapies to antibiotics. This study aimed to investigate the inhibitory effect of four garlic derivatives on microorganisms commonly isolated in ear infections.Methods:The antimicrobial activities of allicin, s-allyl cysteine (SAC), diallyl disulfide (DADS), and s-allyl mercaptocysteine (SAMC) were investigated on standard strains of commonly isolated microorganisms using the broth microdilution method. The test strains were selected among the microorganisms responsible for chronic suppurative otitis media and otitis externa. These microorganisms were Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium, Candida albicans, and Candida tropicalis.Results:Minimum inhibitory concentration (MIC) values of allicin and SAC ranged from 0.125 to 20 μg/ mL for fermentative bacteria (E. coli and K. pneumoniae), 20 to 80 μg/mL for non-fermentative bacteria (P. aeruginosa and A. baumannii), 5 to 10 μg/mL for gram-positive cocci (S. aureus and E. faecium), and 40 to 80 μg/mL for yeasts (C. albicans and C. tropicalis). MIC values of DADS ranged from 40 to 80 μg/mL for fermentative bacteria, 40 to 160 μg/mL for non-fermentative bacteria, 40 to 80 μg/mL for gram-positive cocci, and 20 to 40 μg/mL for yeasts. The MICs of SAMC were >640 μg/mL for the tested bacteria and yeasts.Conclusion:Both allicin and SAC showed antimicrobial activity against the tested microorganisms, even at low concentrations. These two derivatives may be used to treat infections in the future

Optimization of isothermal amplification method for Mycobacterium tuberculosis detection and visualization method for fieldwork

Background/aim: Tuberculosis is still one of the most contagious diseases around the world. Key factors of tuberculosis control are rapid diagnostic, efficient treatment, and prevention of contamination by surveillance and monitoring. However, culture is the gold standard method for laboratory diagnosis of tuberculosis; the results are several weeks to obtain. In order to prevent contamination of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. The aim of this study is to optimize the loop-mediated isothermal amplification (LAMP) method, which provides a much faster and more sensitive result than the polymerase chain reaction (PCR) method and allows the replication of target nucleic acid sequences under isothermal conditions without the need for laboratory infrastructure. Materials and methods: Sputum samples were homogenized with 5% trypsin solution in CaCl2 to obtain DNA. DNA was purified using QIAGEN QIAamp DNA mini kit. LAMP primers were design using Primer explorer V5 program according to IS6110 gene of Mycobacterium tuberculosis. NEB Bst 3.0 DNA polymerase kit was used for LAMP reactions. Besides, LAMP reactions were compared with TaqMan based RT-PCR method using NEB’s Taq polymerase kit. Finally, for visualization of LAMP products, lateral flow dipsticks that produced by Milenia Biotec, colorimetric 2X LAMP master mix that produced by NEB and 2% w/v agarose gel electrophoresis methods were used. Results: Optimum amplification temperature for LAMP was found to be 71.4 °C. The detection limit of the method was 102 CFU/mL and sensitivity was determined 100% compared to five different Mycobacterium species. Conclusion: The current study indicated that the LAMP-LFD and colorimetric LAMP protocol optimized with sputum samples can be reliable used as a rapid, sensitive and specific assay in the diagnosis of tuberculosis in the field

PREGNANCY OUTCOMES OF 115 CASES WITH MATERNAL HEART DISEASE

Objective: To evaluate the outcomes of pregnancies with maternal heart disease

A Prospective Analysis of Blood Cultures with Regarding to Clinical, Epidemiological and Bacteriological Features in One Year Period

In this study we aimed to determine epidemiological, clinical and the microbiological characteristics of the bloodstream infections. A total of 8730 blood cultures collected from 3459 patients in one year period have been assessed. Total 890 episodes were determined among the 782 patients showing positive blood culture. Of these episodes 216 (24.3%) were community acquired and 674 (75.7%) were hospital acquired infection. There was no growth in 7039 (80.6%) of the 8730 culture. Of the growing microorganisms, 682 (87.2%) were monomicrobial and 100 (12.8%) were polimicrobial. The prevalences of gram-positive, gram-negative and yeast were 79.1%, 16.7% and 4.2% respectively. Although coagulase-negative staphylococci (CNS) were isolated as the most prevalent bacteria, only 12.8% of these isolates were evaluated as causative agent, the remaning 51.6% were contaminant. The order of the microorganisms frequently encountered community acquired bacteremia were as follow; Brucella spp. (12.0%), Escherichia coli (9.5%), Staphylococcus aureus (7.5%) and CNS (6.5%) and the microorganisms isolated in hospital acquired infections were S. aureus (13.9%), CNS’s (6.3%), Enterococcus spp. (4.8%), E. coli (4.6%), Candida spp. (4.5%), Klebsiella spp. (3.6%), Pseudomonas spp. (2.9%). The frequency of contamination was 5.7% among whole blood cultures, it was estimated as high as 30.5% among positive blood cultures. Predisposing factors were detected in 74.4% of the patients. Both in community and hospital acquired infections, the most prevalent risk factor was found as the intravenous catheter (sequentially; 39.5% and 70.6%), followed by previous antibiotic use. In order to define the isolates as pathogen or contaminant that is derived from positive blood bottles, we need to evaluate; the course of patient, predisposing factors and sampling technique all together

Bir Yoğun Bakım Ünitesinde Hastane Enfeksiyonuna Neden Olan Pseudomonas aeruginosa ve Acinetobacter baumannii İzolatlarının Çapraz Taşınımı ve Antimikrobiyal Direncinin Analizi

Pseudomonas aeruginosa and Acinetobacter baumannii ciddi hastane enfeksiyonlarına neden olur. Bu çalışmada, Üçüncü basamak bir hastanenin, yoğun bakım ünitesinde bu bakterilerin hastalar arasında çapraz geçişini ve antibiyotik duyarlılığını araştırdık. GGeerreeçç vvee YYöönntteemmlleerrPseudomonas aeruginosa and Acinetobacter baumannii cause nosocomial in— fections in intensive care units. We investigated the antimicrobial susceptibility and cross—transmis— sion of these bacteria amongst patients in an intensive care unit. Material and Methods: Thirty—three P. aeruginosa (from 26 patients) and 48 A. baumannii isolates (from 41 patients) responsible for noso— comial infections were isolated from patients between October 2009 and June 2010. Pulsed field gel electrophoresis was used to investigate clonal relationship among isolates. Susceptibility to amikacin, ceftazidime, gentamycin, imipenem, cefepime, piperacillin/tazobactam, aztreonam, and meropenem was examined using the disk diffusion method. Results: P. aeruginosa isolates formed 18 pulsotypes; five of these were clusters including or more strains having indistinguishable PFGE patterns and the re— maining 13 were unique. After excluding the repeated samples of the same patients, the clustering rate was estimated as 38.5%. The 48 A. baumannii isolates formed 13 pulsotypes; eight pulsotypes were clusters including totally 41 strains of which five were from repeated samples of five patients. The clustering rate was 87.8% for the isolates obtained from 41 different patients. The antimicrobial resis— tance rates of P. aeruginosa ranged from 27—39%, but were 45.5—91% for A. baumannii isolates. Conclusion: Despite an implemented infection control program, P. aeruginosa and A. baumannii isolates showed cross—transmission among patients, and the antimicrobial resistance rate of A. bau— mannii isolates was very high. These findings indicate that the current infection control programs should be reassessed and modifications should be made according to the specific hospital and staffing conditions