[A new method for the disruption of cell walls of gram-positive bacteria and mycobacteria on the point of nucleic acid extraction: sand method]. / Nükleik asit ekstraksiyonunda kullanilmak üzere, gram-pozitif bakteriler ve mikobakterilerin hücre duvarlarinin ortadan kaldirilmasinda yeni bir yöntem: Kum yöntemi.

Nowadays molecular methods are widely used in the rapid diagnosis of infectious agents. Polymerase chain reaction (PCR) is the most preferred method for this purpose. Obtaining sufficient and pure DNA or RNA is important for the PCR. Different DNA extraction protocols such as phenol-chloroform, proteinase K, glass beads and boiling have been used successfully for DNA isolation from gram-negative bacteria. However since gram-positive bacteria have a thicker layer of peptidoglycan and mycobacteria have complex glycolipids in their cell walls, for the isolation of DNA or RNA from these microorganisms, the complex cell wall structure must be eliminated. For this purpose, the bacterial cell wall must be completely or partially removed forming sferoblast using lysostaphin in the Staphylococcus genus as gram-positive bacteria and using a chemical like cetyltrimethyl ammonium bromide for the Mycobacterium genus. In this study, we planned to use sand particles for the mechanical elimination of the cell wall without any need for chemicals and we called this procedure as "sand method". For the purpose of DNA extraction, the fine-grained sand was washed with ddH(2)O without losing small particles and then sterilized by autoclaving. For the purpose of RNA extraction; the sand was washed with ddH(2)O, incubated for 30 minutes with 10% HCl, and then autoclaved. A methicillin-resistant Staphylococcus aureus (MRSA) strain previously isolated and identified from a clinical specimen was mixed in 100 µl Tris-EDTA buffer with 100 mg sand. The mixture of bacteria and sand was vortexed at the maximum speed for 5 minutes. The MRSA-sand mix was treated with proteinase K and phenol-chloroform, and ethanol precipitation protocol was then followed for obtaining DNA. For comparison of the sand method with the other methods, the same amount of bacteria used in the sand method was incubated for one hour with lysostaphin, and then the proteinase K DNA extraction method were completed in the same way used in the sand method. For obtaining RNA from M.tuberculosis H37Rv ATCC 25618, M.tuberculosis H37Ra ATCC 25177 and M.tuberculosis H37Rv Pasteur Institute RSKK 598 standard strains, bacteria were dissolved in 20 µl Tris-EDTA buffer with 100 mg sand. The mixture of bacteria and sand was vortexed at the maximum speed for 5 minutes. After that, the classic RNA extraction protocol using guanidinium thiocyanate-phenol-chloroform (GTPC) was completed. To investigate the usefulness of the obtained DNA, a PCR was performed with specific primers for staphylokinase and enterotoxin genes that were shown in the genome of the chosen MRSA strains from our previous studies. To investigate the usefulness of the obtained RNA from the sand method; first cDNA synthesis is completed. The PCR efficiency was then tested using primers specific to the efflux pump genes of M.tuberculosis including Rv1410c, Rv2333c, and DrrA genes. To compare the effect of the sand method, GTPC protocol was applied in the same amount of mycobacteria without the sand treatment. The DNA obtained from MRSA with the application of lysostaphin and the DNA obtained from MRSA by the sand method were run in agarose gel electrophoresis. The amount and purity of DNAs were measured with a spectrophotometer. The same amount and purity of the DNAs were approximately the same in both of the extraction methods. The existence of non-inhibitors of DNA in the sand method was shown with the PCR, which have worked efficiently with the DNAs obtained from the sand method. RNA was obtained efficiently from the Mycobacterium strains by the sand method, but no RNA could be obtained from the mycobacteria with the other methods. It was shown that the RNA obtained using the sand method worked effectively in both cDNA synthesis and PCR in which synthesized cDNA was used. The sand method described in the study worked effectively to obtain sufficient amount of pure DNA and RNA from the bacteria containing rigid cell walls that are difficult to obtain the nucleotide. It was concluded that, using the sand method instead of relatively expensive lysostaphin or other chemicals, has important advantages such as decreasing the cost and the shortening of the DNA extraction period.

[Molecular characterization of Salmonella enterica serovar Typhimurium strains isolated from humans in Turkey]. / Türkiye'de insanlardan izole edilen Salmonella enterica serovar Typhimurium suslarinin moleküler özellikleri.

Multidrug resistant (MDR) Salmonella infections, especially infections due to Salmonella Typhimurium DT104 phage type strains are an important public health issue in many parts of the world. S.Typhimurium is the most common serotype isolated from clinical samples in Turkey but we have limited data about the phage types of these isolates. The aims of this study were to find out whether these MDR S.Typhimurium isolates are DT104 phage type isolates and have class 1 integrons and to investigate the relationships of these characteristics between plasmid and pulsed field gel electrophoresis (PFGE) profiles. A total of 66 S.Typhimurium stock strains selected from Enterobacteria Laboratory culture collections of Ankara University School of Medicine, Department of Medical Microbiology were investigated by plasmid profile analysis (PPA) and PFGE with the use of XbaI and SpeI enzymes. The presence of class 1 integrons and the phage type 104 were investigated by polymerase chain reaction (PCR). The strains used in the study were sporadically isolated cases from seven provinces after year 2000 with ACSSuT (63), ACGSSuTT/S (1), ACSSuTT/S (1) and ASSuTT/S (1) resistance types [ampicillin (A), chloramphenicol (C), gentamicin (G), streptomycin (S), sulphonamide (Su), tetracycline (T), trimethoprim/sulfamethoxazole (T/S)]. Of the isolates 65 were found as DT104 phage type. Forty-three S.Typhimurium DT104 isolates that carry class 1 integrons had five different bands between 350-1600 base pairs (bp); all of the isolates harbored 1-4 plasmids with sizes ranging from 1.0-180 kbp and 62 isolates had 90 kbp plasmid which was serotype specific and virulence related. S.Typhimurium DT104 isolates were grouped into five (X1-X5) and seven (S1-S7) profiles with XbaI and SpeI enzymes, respectively. When the profiles of the two enzymes were evaluated, 58 of the 65 (89.2%) isolates showed similar (X1.S1) profile. The molecular characteristics of the most S.Typhimurium isolates were clustered in similar groups when class 1 integron, plasmid and PFGE types were analyzed together. In this study we showed that nearly all S.Typhimurium isolates with five drug resistance pattern (ACSSuT) were DT104 isolates. PFGE profiles of these sporadic isolates suggested that they were epidemiologically related.

Identification of Malassezia species from pityriasis versicolor lesions with a new multiplex PCR method.

Despite the fact that a range of molecular methods have been developed as tools for the diagnosis of Malassezia species, there are several drawbacks associated with them, such as inefficiency of differentiating all the species, high cost, and questionable reproducibility. In addition, most of the molecular methods require cultivation to enhance sensitivity. Therefore, alternative methods eliminating cultivation and capable of identifying species with high accuracy and reliability are needed. Herein, a multiplex polymerase chain reaction (PCR)-based method was especially developed for the detection of eleven Malassezia species. The multiplex PCR was standardized by incorporating a consensus forward primer, along with Malassezia species-specific reverse primers considering the sizes of the PCR products. In the method, the multiplex-PCR primer content is divided into three parts to circumvent the problem of increased nonspecific background resulting from the use of a large number of primers. DNA extraction protocol described by Harju and colleagues was modified using liquid nitrogen instead of -80 °C to break down the yeast membrane. By a modified extraction procedure followed by multiplex PCR and electrophoresis, the method enables identification and differentiation of Malassezia species from both of the samples obtained directly from skin and yeast colonies grown in culture. Fifty-five patients who were confirmed with pityriasis versicolor were enrolled in the study. Multiplex PCR detected and differentiated all 55 samples obtained directly from the patients' skin. However, 50 out of 55 samples yielded Malassezia colony in the culture. In addition, eight of 50 colonies were misdiagnosed or not completely differentiated by conventional methods based on the sequence analysis of eight colonies. The method is capable of identifying species with high accuracy and reliability. In addition, it is simple, quick, and cost-effective. More importantly, the method works efficiently for the diagnosis of Malassezia species obtained directly from patient samples.

[Investigation of seven different RNA viruses associated with gastroenteritis in children under five years old]. / Bes yas alti çocuklarda gastroenterite neden olan yedi farkli RNA virusunun arastirilmasi

Viruses are the most frequently detected etiologic agents of gastroenteritis seen in small children. In addition to classical gastroenteritis viruses namely rotavirus, norovirus, adenovirus type 40/41, astrovirus and sapovirus, some novel picornaviruses (Aichi virus, parechovirus, enterovirus) that have been identified in parallel to the developments in molecular diagnostic methods, thought to be associated with diarrhea in humans. However, the data are not enough to prove their actual roles in the pathogenesis of gastroenteritis. The aim of this study was to investigate the presence of rotavirus, norovirus, sapovirus, astrovirus, Aichi virus, parechovirus and enterovirus in the stool samples of children with diarrhoea by reverse-transcriptase polymerase chain reaction (RT-PCR). A total of 50 samples from children admitted to our hospital with diarrhoea between June-December 2012 were included in the study. All the patients were under 5 years of age. Routine bacteriological and parasitological examinations of the patients' stool samples were negative. Total RNAs were extracted from each of the samples and cDNAs were obtained by reverse transcription. All cDNAs were investigated first with the internal control (IC) using PCR. Thirty-one of the 50 cDNAs (62%) were found IC positive. Those 31 samples were further investigated in terms of rotavirus group A and C, norovirus (NoV) genogroup GI and GII, sapovirus, astrovirus, Aichi virus, parechovirus and enterovirus by PCR using specific primer pairs. The predicted sized PCR products obtained were cloned into the pBSK cloning vector and were sequenced. Sequences obtained were subjected to a BLAST search with registered sequences in the GenBank database for the confirmation of the PCR product. Out of 31 RNA positive stool specimens, 12 (38.7%) were found positive for five types of the target viruses. NoV GII (6/31, 19.3%) were detected as the most prevalent virus, followed by NoV GI (2/31, 6.5%), rotavirus group A (2/31, 6.5%), astrovirus (1/31, 3.2%) and sapovirus (1/31, 3.2%). The results of this study revealed that the most frequently detected agents were noroviruses (8/50, 16%) followed by rotavirus (2/50, 4%), astrovirus (1/50, 2%) and sapovirus (1/50, 2%). It was concluded that RT-PCR performed with the primers used in this study could be applied effectively for the molecular epidemiological analysis of RNA viruses leading to gastroenteritis. Larger scale studies conducted in different areas for longer time periods and with a larger population size will provide data for the epidemiological properties of agents of viral gastroenteritis.

Complications of therapeutic apheresis in children.

UNLABELLED: Since the equipment of therapeutic apheresis is prepared for adults, the use of it in children may lead to higher complication risks and there are little data in children undergoing therapeutic apheresis. METHODS: In this study the complications experienced during therapeutic apheresis in children between April 2010 and May 2012 at our center are analyzed retrospectively. There were 14 patients who had undergone a total of 50 sessions of therapeutic apheresis. The ages of patients' ages ranged from 20months to 16years. The procedures were plasma exchange and leukodepletion. RESULTS: Complications were observed in four patients. One of them was vascular access complication because of insufficient flow. Urticeria was observed in two patients. Abdominal pain and chilling were other complications. Our patients, who underwent TA, did not experience major complications. Minimal or mild allergic reactions were observed and treated by medications. For extracorporeal volume erythrocyte prime is useful. TA will be performed more successfully with correct planning and close examination of the patient with an experienced team.

Identification of methicillin-resistant Staphylococcus aureus carrying an exfoliative toxin A gene encoding phage isolated from a hospitalized patient in Turkey.

From the four known isoforms of the staphylococcal exfoliative toxins (ETs), only ETA and ETB are the major causative agents. General knowledge is that the gene for ETA is located on the chromosome, whereas that for ETB is located on a large plasmid. Yoshizawa and co-workers (2000, Microbiol. Immunol. 44(3): 189-191) isolated, for the first time, a temperate phage (φETA) that carried the structural gene for ETA from an ETA-producing strain of Staphylococcus aureus. In this study, we presented eta gene encoding temperate phages isolated from methicillin-resistant S.aureus (MRSA) isolates obtained from patients in a Turkish hospital. Molecular analysis of the phage genome revealed that the eta gene is located upstream to amidase and holin genes, the same as in the φETA genome. However, partial sequence analysis of amidase and holin genes revealed polymorphic variation. In addition to polymorphic variation, restriction fragment length polymorphism (RFLP) analysis of all of the phage genomes showed that the ETA-containing phage is different from the rest of the phage genomes. The phylogenetic dendrogram of pulsed field gel electrophoresis (PFGE) analysis showed that the ETA-carrying MRSA is quite different from the rest of the MRSA strains. This is the first report showing that a MRSA strain carries an ETA-encoding phage.

HHV-6 is ubiquitously found using Western blot in tonsils and adenoid tissues of healthy people.

Few studies have examined the prevalence and cellular proclivity of latent human herpesvirus 6 (HHV-6) in healthy populations. Difficulties in detection of HHV-6 genome in different tissues using polymerase chain reaction (PCR) and immunohistochemistry (IHC) techniques have been reported by various researchers. We examined tonsils and adenoid tissues of 54 patients who had undergone tonsillectomy or adenoidectomy without any evidence of acute infection for the presence of latent HHV-6 infection. While we were investigating the prevalence of HHV-6, we tested the efficiency of PCR, IHC and Western Blot (WB) for detection of HHV-6 in tonsil tissues. We found that 100% of tonsil tissues were positive for HHV-6 with WB, 40% of tonsils were positive with PCR and no tonsil was positive with IHC. This result correlates well with most studies claiming HHV-6 is a ubiquitous organism in various populations and tissues. Western blot may be a good choice for detecting HHV-6 in tissues. Expression of the HHV-6 gp60/110 envelope protein disclosed by WB may indicate that HHV-6 does not have true latency. To our knowledge, this is the first report to use WB to test for HHV-6 in tissues.

[Identification of a novel lytic bacteriophage obtained from clinical MRSA isolates and evaluation of its antibacterial activity]. / Klinik MRSA Izolatlarindan Elde Edilen Yeni Bir Litik Bakteriyofajin Tanimlanmasi ve Antibakteriyel Etkisinin Degerlendirilmesi.

Multidrug-resistant bacteria particularly MRSA is well known as a worldwide problem. Since the rate of development of novel antimicrobial agents has been slowed down during the last years, there have been a need for the exploration of alternative solutions for the treatment of resistant bacterial infections. Treatment of infections by bacteriophages (phages) that specifically kill the infecting pathogen, i.e. by the process known as phage therapy, is considered as a possible approach to treat multidrug resistant bacteria. Phage treatment has also been considered to treat Staphylococcus aureus infections. This study was aimed to evaluate the antibacterial and cytotoxic activities of a new lytic phage obtained from clinical MRSA strains. This lytic phage named as f LizAnk was obtained during the phage infectivity studies performed with 13 lysogenic phages against MRSA strains. The antibacterial activity of the f LizAnk phage was determined in vitro in BHI (Brain Heart Infusion) and LB (Leuria Bertani) broths and the in vivo antibacterial activity against MRSA strains and possible cytotoxic effect against mammalian cells were tested on fibroblastic cell cultures (3T3). This study was conducted using 20 MRSA strains isolated from hospitalized patients. Identification of the isolates was performed by conventional methods and methicillin resistance was detected with oxacillin disk diffusion test and mecA gene detection by PCR. The method described by Kaneko et al. [Biosci Biotechnol Biochem 1997; 61(11): 1960-2] was used with some modifications, for induction and isolation of the phages. In vitro studies indicated that this phage killed the six different MRSA strains (in 107 cfu/ml concentrations) in 8 hours, and this powerful lytic effect was similar in both of the liquid media. In vivo studies were performed by using cell cultures prepared in microplates, and the wells have been inoculated with only phage, phage + MRSA mixture, and only MRSA. The cells were then evaluated microscopically as well as by MTT assay which detected alive cells colorimetrically, at 2nd and 24th hours. In our study, the f LizAnk phage did not cause any toxic effect on fibroblast cell cultures, in addition it was observed that the antibacterial effect of the phage against MRSA has proceeded in the cell culture. In conclusion, since the fLizAnk phage described in this study exhibited strong antibacterial activity against MRSA strains and no cytotoxic effect was detected against mammalian cells, it might be safely used alone or in a phage cocktail to treat skin infection caused by MRSA.

[Characterization of Shigella strains isolated in ankara, Turkey by antimicrobial resistance models, plasmid profile analysis and pulsed-field gel electrophoresis]. / Ankara'da Izole Edilen Shigella Kökenlerinin Antibiyotik Direnç Modelleri, Plazmid Profil Analizi ve Degisken Alanli Jel Elektroforezi ile Incelenmesi.

Shigella is one of the most important causative agents of diarrhea especially in childhood. Since man is the main reservoir of Shigella and human to human transmission is possible, Shigella can easily spread in public and cause outbreaks. In this study, a total of 60 Shigella strains isolated in Ankara, Turkey by years 2001, 2008 and 2009 were investigated by their antimicrobial susceptibility profiles, plasmid profile analysis (PPA) and pulsed-field gel electrophoresis (PFGE). For epidemiological investigation, the results obtained by antibiotic resistance typing (ART) which was the phenotyping method, was compared to the results of the genotyping methods which were PPA and PFGE. Of the isolates 49 (81.6%) were S.sonnei, 10 (16.6%) were S.flexneri and one was (1.6%) S.dysenteriae. Antimicrobial susceptibilities were evaluated by disc diffusion method and the highest resistance rates were found against trimethoprim/sulfamethoxazole (91.6%), followed by tetracycline (68.3%) and ampicillin (26.6%). Resistance against ampicillin, chloramphenicol and amoxycillin/clavulanic acid were found higher in S.flexneri isolates than S.sonnei (p< 0.001). All isolates were found to be susceptible to ciprofloxacin, gentamicin and ceftazidime. S.sonnei demonstrated 12 and S.flexneri demonstrated 4 antibiotic resistance models. All isolates were carrying plasmids with varying sizes and varying numbers between 1 to 7. S.sonnei isolates demonstrated 27 and S.flexneri isolates demonstrated 8 plasmid profiles. S.sonnei isolates were clustered in 4 patterns and S.flexneri were clustered in 5 patterns by PFGE. This method demonstrated obvious clonal similarity among S.sonnei strains isolated in Ankara and discriminative power (DP) was calculated as 0.26. PPA and ART demonstrated higher DP among S.sonnei strains (0.97 and 0.75, respectively). In this study gain or loss of instable genetic mobile elements were thought to be responsible for higher discriminative powers of PPA and ART methods. These typing methods were found to be appropriate for the epidemiological investigation of strains collected in a short time period. PFGE was found to be convenient for the evaluation of clonal relatedness of the strains, however, in such geographical areas where the same clone was in circulation, use of ART and/or PPA together with PFGE would be useful for precise discrimination of Shigella strains.

[Oropharyngeal tularemia: a case report]. / Orofarengeal tularemi: Olgu sunumu.

Tularemia is a zoonotic disease caused by the gram-negative, aerobic coccobacillus Francisella tularensis. It transmits with the body secretions of the infected rodents, ingestion of the food contaminated with these fluids and bites of infected insects. Ulceroglandular, glandular, oculoglandular, oropharyngeal, typhoidal and pneumonic types may be observed based on the entrance route to the body and location of the bacteria. Although the clinical presentation may vary, oropharyngeal tularemia is the most commonly seen clinical form in Turkey. Otolaryngologists generally experience tularemia with membranous tonsillopharyngitis and cervical lymphadenopathy. Oropharyngeal tularemia should be considered in the differential diagnosis of patients with tonsillopharyngitis who are refractory to penicillin therapy. In this article, we present a 42-years-old female case of oropharyngeal tularemia who was initiated with penicillin therapy due to tonsillopharyngitis and cervical lymphadenitis and remained unresponsive to the treatment. Clinical, laboratory and radiological findings of the patient were evaluated and discussed in the light of similar cases in the literature.

[Investigation of Salmonella serotype Enteritidis isolates by plasmid profile analysis and pulsed field gel electrophoresis]. / Salmonella Serotip Enteritidis Izolatlarinin Plazmid Profil Analizi ve "Pulsed Field" Jel Elektroforezi ile Incelenmesi.

In this study a total of 122 Salmonella serotype Enteritidis stock strains selected from the culture collection of Enterobacteriaceae Laboratory of Ankara University Faculty of Medicine, Department of Medical Microbiology, were investigated by plasmid profile analysis with the method defined by Kado and Liu and pulsed field gel electrophoresis (PFGE) according to World Health Organization protocols using SpeI and XbaI macrorestriction enzymes, for better understanding of the molecular epidemiology of S. Enteritidis. The study strains were selected from a collection of previously isolated epidemic (n= 13) and sporadic (n= 109) strains (103 stool, 16 blood and one each bile, urine and cerebrospinal fluid) obtained from 10 different cities after the year 2000. PFGE patterns were analyzed with Gene Directory software (Syngene, UK) and a similarity index was determined by using Dice coefficient and the unweighted pair group method with mathematical averaging (UPGMA). Plasmid-carrying 110 (90%) strains that harbored 1-4 plasmids with sizes ranging from 2.0 to 100 kb were separated into patterns more than 14 (p1-p14). A total of 85 (69.7%) isolates harbored the 57 kb plasmid solely or in combination with other plasmids. By PFGE, 11 distinct patterns were shown with each enzyme SpeI and XbaI. S. Enteritidis strains after digestion with macrorestriction enzyme SpeI generated 11 different PFGE patterns (A to K), whereas XbaI generated also 11 different PFGE patterns (a to k). PFGE pattern A consisted of 93 strains (76.2%) after digestion with macrorestriction enzyme SpeI, while PFGE pattern a consisted 53 (43.4%) and PFGE pattern b 42 strains (34.4%) after digestion with macrorestriction enzyme XbaI. Using two macrorestriction enzymes two PFGE cluster profiles Aa (50 strains, 40.9%) and Ab (42 strains, 34.4%) were found to be predominating among 17 different PFGE clusters. Our results confirmed the clonal nature of S. Enteritidis strains in Turkey. The use of two enzymes in PFGE analysis appeared to increase the discriminatory power of PFGE, leading to greater diversity among strains. PFGE analysis performed by SpeI and XbaI enzymes combined with plasmid profiling could be established as a useful tool for detection of genetic relationship between isolates.

[Molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae strains isolated between 2004-2007 in Ankara University Hospital, Turkey]. / Ankara Universitesi tip fakültesinde 2004-2007 yillari arasinda izole edilen karbapeneme dirençli Klebsiella pneumoniae suslarinin moleküler epidemiyolojisi.

The prevalence of carbapenem-resistant gram-negative bacteria in the hospital setting is in an increasing trend worldwide. Since most of the carbapenem-resistant Enterobacteriaceae are resistant to all antimicrobial agents except polymyxins and tigecycline, the emergence of carbapenem resistance in Klebsiella pneumoniae strains requires careful monitoring. This study was conducted to analyse the epidemiological relatedness between the carbapenem-resistant isolates of K. pneumoniae collected from different wards (intensive-care, surgery, hematology, neurology, internal medicine, emergency services) of Ankara University Hospital. A total of 26 carbapenem-resistant K. pneumoniae isolates (13 blood, 6 urine, 2 bronchoalveolar lavage, 1 abscess, 1 tissue, 1 catheter tip, 1 drainage fluid, 1 tracheal lavage fluid) were identified and antibiotic susceptibility tests were performed with API 20E System or VITEK 2 Compact (Bio-Merieux, France) at the Central Laboratories of Ankara University Hospital between February 2004 and April 2007. MICs of imipenem and meropenem were also confirmed using E-test (AB Biodisk, Sweden). The clonal relationship between the isolates was studied by pulsed-field gel electrophoresis (PFGE). After digestion of total genomic DNA with restriction endonuclease Xbal, the 26 isolates generated 7 PFGE profiles. PFGE pattern B consisting of different antibiotic susceptibility profile was seen only in 2006. Carbapenem-sensitive strains isolated at the same time from the same wards which carbapenem-resistant isolates were recovered, generated different PFGE patterns. The predominant carbapenem-resistant isolates in our hospital were found clonally related. Interhospital transmission of carbapenem-resistant K. pneumoniae strains which have a particular epidemic potential, is likely to occur during patient transfer between wards. It is likely that intensive efforts, similar to those used to control vancomycin resistant enterococci, are needed to identify and control the spread of resistant Klebsiella species. Therefore, active surveillance and strict infection control measures for this multidrug-resistant microorganism should be implemented at local and national basis.

Molecular investigation of salmonella choleraesuis and salmonella hadar strains isolated from humans in Turkey.

Eleven Salmonella Choleraesuis and seven Salmonella Hadar strains isolated from various clinical humand samples were investigated by plasmid profile analysis, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and pulsed-field gel electrophoresis (PFGE) in order to obtain information at a molecular level on the epidemiology of S. Choleraesuis and S. Hadar, which are significantly present in Turkey. Plasmid profile analysis showed that 10 (90.9%) of 11 S. Choleraesuis isolates harbored one to two plasmids with sizes of 2.0, 5.0 or 6.5 kb; and 5 (71.4%) of 7 S. Hadar isolates harbored one to three plasmids ranging from 2.5 to 70 kb. ERIC-PCR was performed using ERIC-2 primers; since isolates within each serotype showed similar band models, we concluded that ERIC-PCR is not suitable for differentiating isolates within the same serotype and for grouping into clusters. In PFGE using the AvrII enzyme, S. Choleraesuis isolates formed three clusters, and S. Hadar isolates formed three clusters; using the XbaI enzyme, S. Choleraesuis isolates formed two clusters, and S. Hadar isolates formed four clusters. These results showed that plasmid profile analysis and PFGE are reliable and discriminative methods that would complement antibiograms, and could contribute to the investigation of outbreak epidemiology. This is the first report on S. Choleraesuis and S. Hadar isolates from Turkey investigated by plasmid profile analysis, ERIC-PCR and PFGE methods.

A new, simple, rapid test for detection of DNase activity of microorganisms: DNase Tube test.

The DNase test is a simple, economical method that has traditionally been used as a supplemental test to identify pathogenic Staphylococcus. This test also aids in the differentiation of closely-related genera within the Klebsiella-Enterobacter-Serratia division of Enterobacteriaceae and several other pathogens, including screening of C. diphtheriae. Currently DNase activity of microorganisms was tested using DNase agar plate methods. These tests have some drawbacks including the necessity of the extensive time to see the results of DNase activity of bacteria. In here, we developed a new method which is simple, rapid, inexpensive and applicable to examine DNase activity of any bacteria. In this method, simply, bacteria is added to the broth medium containing DNA and followed for the DNA degradation caused by the DNase of bacteria by running in agarose gel. This method we called DNase Tube test showed DNA degradation as fast as in half an hour depending on the DNase activity of the bacteria.

[A novel Salmonella Typhimurium plasmid, pAnkS: an example for plasmid evolution in antibiotic resistance]. / Yeni bir Salmonella Typhimurium plazmidi, pAnkS: antibiyotik direnç gelisiminde plazmid evriminin bir örnegi.

In this study, a plasmid, carrying ampicillin resistance (ampR) gene, isolated from a clinical isolate of Salmonella enterica serotype Typhimurium presenting ACSSuT (ampicilin, chloramphenicol, streptomycin, sulphonamide, tetracycline) resistance phenotype, was defined. The length of complete sequence of this plasmid was 8271 base pairs (bp), and it was named as pAnkS owing to its isolation place (plasmid-Ankara- Salmonella). The plasmid was analyzed for potential reading frames and structural features indicative of transposons and transposon relics. The Xmnl enzyme restriction fragments of pAnkS were cloned into E. coli plasmid vectors (pBSK), sequenced and analyzed with the BLAST programs. Plasmid pAnkS has contained a previously defined enterohemorrhagic E. coli (EHEC) plasmid p4821 as a core region and also contained a complete Tn3-like transposon of 4950 bp consisting of the left terminal repeat, Tn3-related tnpR and tnpA genes for transposition functions, ampicillin resistance gene bla(TEM), and the right terminal repeats, pAnkS showed strong homology with another Salmonella plasmid, pNTP16, for sequences that belong to p4821 and partial Tn3 segments. It was found that pNTP16 also carries kanamycin resistance gene (kanR) in addition to ampR gene. Plasmid pAnkS is one of the few completely sequenced plasmids from Salmonella Typhimurium and is in the middle of the pathway of evolution of plasmid from p4821 to pNTP16. The identification of pAnkS might help better understanding of plasmid evolution.

A novel mutation pattern emerging during lamivudine treatment shows cross-resistance to adefovir dipivoxil treatment.

AIMS: This study was conducted to clarify the resistance profile of a novel mutation pattern emerging during lamivudine (3TC) therapy and showing cross-resistance to adefovir dipivoxil (ADV) in a patient with chronic hepatitis B. METHODS AND RESULTS: Successful suppression of hepatitis B virus (HBV) replication by sequential therapy of 9 MU thrice weekly interferon (IFN) and 3TC was followed by genotypical resistance detected at month 28 of therapy (month 19 of lamivudine treatment). ADV was added to 3TC therapy on month 44 of antiviral treatment. Neither alanine aminotransferase normalization nor a stable decrease in HBV viral load was observed, although ADV was used for more than 40 months. The HBV pol region was amplified from serum samples obtained before and after ADV treatment. The complete genome was cloned into a TA vector. PCR products and 7-10 clones from each cloned vector were sequenced. A novel mutation, A181S, in the reverse transcriptase gene leading to a conversion of W172C in the overlapping surface antigen gene was detected along with a M2041 mutation. The complete genome comprising the A181S+M2041 pattern was cloned into an expression vector and its in vitro susceptibility to 3TC, ADV, tenofovir (PMPA), clevudine (L-FMAU) and emtricitabine (FTC) were determined in transiently transfected Huh7 cells. This mutation pattern displayed more than 1000-fold resistance to the nucleoside analogues 3TC and FTC and approximately sixfold resistance to L-FMAU, while it confers 28.23- and 5.57-fold resistance for the nucleotide analogues ADV and PMPA, respectively. CONCLUSION: A new mutation pattern, A181S+M2041, arising under lamivudine treatment confers cross-resistance to ADV both in vivo and in vitro.

Detection of herpes simplex virus type 1 in addition to Epstein-Bar virus in tonsils using a new multiplex polymerase chain reaction assay.

HSV-1, HSV-2, CMV, EBV, which are the members of the herpes virus family colonize and establish latent infection in human. Although EBV is a well known virus most involved in recurrent bouts of acute tonsillitis, the role and possibility of HSV-1, HSV-2, and CMV for establishing infection in tonsils are not clear. The purpose of this study is to verify whether the tonsils might harbor the HSV-1, HSV-2, and CMV, in addition to EBV, in chronically hyperplastic nasopharyngeal lymphoid tissue. To accomplish the purpose, we developed a new Multiplex Polymerase Chain Reaction (M-PCR) assay using a single consensus forward primer and virus specific reverse primers for DNA polymerase gene of HSV-1, and 2, EBV, and CMV, and investigated its efficiency for detecting HSV1, HSV2, CMV, and EBV. The sample of 52 patients underwent tonsillectomy or adenectomy because of chronic lymphoid hyperplasia without any evidence of acute infections and were investigated for presence of HSV-1, HSV-2, CMV, and EBV. Of the 54 samples, 11 (20.4%) of them were positive for EBV, 4 of them (7.4%) were positive for HSV-1, and none of the samples were positive for HSV-2 and CMV. To the best of our knowledge, this is the first report that tonsils may be the reservoir for HSV-1 in addition to EBV, and HSV-1 may have a role in recurrent tonsillitis and systemic diseases. The MC-PCR assay presented in this study can provide a rapid, sensitive, and economical method for detection of HSV-1, HSV-2, EBV, and CMV in a single PCR tube.

LKB1 protein expression in human breast cancer.

Peutz-Jeghers syndrome is caused by germline mutations in the LKB1/STK11 gene. Peutz-Jeghers syndrome is associated with an increased risk of developing intestinal and extraintestinal cancers, including pancreatic, lung, and breast carcinomas. LKB1 gene inactivation has recently been demonstrated in a subset of sporadic pancreatic and lung carcinomas. The role of the LKB1 gene in sporadic breast carcinomas remains unclear, though recent studies suggest inactivation only within papillary carcinomas. Using a commercially available polyclonal antibody that has been shown to mirror LKB1 genetic status in gastrointestinal and pulmonary carcinomas, the authors performed IHC on a large series of breast cancers using tissue microarrays (TMAs). All abnormal TMA results were confirmed using whole sections; specifically, whole sections from the donor blocks of lesions demonstrating diminished or absent LKB1 protein expression on TMA were evaluated to compare labeling of the lesion with that of the surrounding normal breast. In all cases, normal breast epithelium demonstrated strong cytoplasmic labeling (providing an internal positive control), whereas the stroma was nonreactive. Luminal cells typically labeled more strongly than myoepithelial cells. Among 70 invasive ductal carcinomas, 3 (4.3%) showed complete loss of LKB1 labeling, whereas 6 others (8.6%) showed diminished labeling. Of the eight intraductal carcinoma lesions adjacent to these invasive carcinomas, one (12.5%) showed complete loss of LKB1 labeling and one other (12.5%) showed diminished labeling; these results were identical to those of the adjacent invasive carcinomas. One of 10 (10%) hematogenous metastases of mammary carcinoma showed loss of LKB1 labeling. Nine of the 10 invasive carcinomas and both of the ductal carcinoma in situ (DCIS) cases showing loss of or diminished LKB1 expression were of high grade. In contrast, all 13 pure nonpapillary DCIS lesions, all 5 invasive lobular carcinomas and 3 accompanying lobular carcinoma in situ lesions, all 7 papillary DCIS lesions, and all 3 papillomas evaluated showed intact LKB1 labeling. Therefore, although frequent methylation of the LKB1 gene has been reported in papillary carcinomas of the breast, the authors did not find loss of protein expression in these lesions. Instead, it was found that loss of LKB1 protein expression occurs in a subset of high-grade in situ and invasive mammary carcinomas. The authors found LKB1 gene methylation in several of these invasive carcinomas. Given recent Western blot results indicating that diminished LKB1 expression in breast carcinomas correlates with shorter relapse-free survival, LKB1 IHC merits evaluation as a potential prognostic marker for breast carcinoma.

Plasmid profiles and randomly amplified polymorphic DNA analysis of Salmonella enterica serotype Enteritidis strains from outbreaks and sporadic cases in Turkey.

The aim of the study was to investigate the characteristics of Salmonella serotype Enteritidis strains isolated from outbreaks and sporadic cases in Turkey by plasmid profiles and randomly amplified polymorphic DNA (RAPD) patterns. A total of 64 S. Enteritidis clinical strains were selected from the culture collection of the Enterobacteria Laboratory of Ankara University Medical School Department of Microbiology and Clinical Microbiology for molecular analysis using the plasmid profiles and RAPD method. Fifty-six isolates (88%) harbored one to four plasmids ranging in size from 2.5 to 100 kbp. 57 kbp plasmids were the most common plasmids, and forty-four strains (69%) carried 57 kbp plasmids alone or together with other plasmids. The outbreak strains carried the same plasmid profile: three plasmids sized 57, 40, 3.0 kbp. None of the strains analyzed displayed any RAPD bands with the primer OPB-17. By using primer p-1254, 42 strains (66%) were divided into fourteen RAPD patterns. Ten of the outbreak strains (77%) showed >80% similarity by cluster analysis program. Analysis of RAPD-PCR with primer p-1254 proved an easy, rapid and discriminative method complementing antibiogram and plasmid profiles in routine laboratories, and may contribute to the investigations of S. Enteritidis which still cause outbreaks in Turkey. This study presents the first report on S. Enteritidis isolates in Turkey investigated by plasmid profiles and RAPD methods.

[Investigation of the role of ribonuclease L gene in the response of tonsil tissues against viral infections]. / Tonsil dokusunun viral enfeksiyonlara karsi yanitinda ribonükleaz L geninin rolünün arastirilmasi.

Mutations or variants that impair function of ribonuclease L (RNase-L), particularly R462Q, have been proposed as susceptibility factors for the innate antiviral response. The aim of this study was to investigate and compare the expression levels of RNase-L and mutation of R462Q in the tonsils of tonsillectomy patients who were infected and not infected with herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV) and human herpes virus 6 (HHV-6). Six tonsils were included in the study. One tonsil was infected with all of these three viruses, two were infected with at least one of these viruses, and three were not infected with these viruses. The presence of viral DNAs in the tonsil tissues had been searched by polymerase chain reaction (PCR) in our previous study. Reverse transcriptase PCR method was used for RNase-L expression analyses, and single strand conformation polymorphism (SSCP) and direct sequencing methods were used for the mutation analyses. PCR products containing R462Q mutation site in the genomic DNA were used for SSCP analysis. In addition to SSCP analyses, partial sequencing of the cDNA PCR product containing R462Q mutation site were performed. As a result, no difference between the virus-infected and non-infected tonsils for the expressions of RNase-L were detected, and there were no mutations detected by SSCP and sequencing analyses. It was concluded that other factors than RNase-L protein, might be involved in the innate defense mechanisms of tonsil cells against viruses.