Vol.72, Issue 4, July-September 2013
Transcription
Vol.72, Issue 4, July-September 2013
ROMANIAN ARCHIVES OF MICROBIOLOGY AND IMMUNOLOGY Founded by PROFESSOR ION CANTACUZINO in 1928 VOLUME 72 - Issue 4 October - December 2013 Published quarterly by CANTACUZINO INSTITUTE BUCHAREST TOTAL PUBLISHING HOUSE ROMANIAN ARCHIVES OF MICROBIOLOGY AND IMMUNOLOGY ISSN 1222-3891 Editor-in-Chief: Maria DAMIAN Deputy Editor: Aurora SĂLĂGEANU Editorial Board: Viorel ALEXANDRESCU, Antonios ANTONIADIS, Jean-Marc CAVAILLON, Ana CĂLUGĂRU, Cornelia CEIANU, Carmen CHIFIRIUC, Irina CODIțĂ, Radu COJOCARU, Nicolae CORCIONIVOSCHI, Lidia CREMER, Angel GALABOV, Steliana HUHULESCU, Luminiţa Smaranda IANCU, Gabriel IONESCU, Anca ISRAIL, Emilia LUPULESCU, Roxana MOLDOVAN, Geza MOLNAR; Marian NEGUŢ, Adrian ONU, Hervé PELLOUX, Graţiela PÎRCĂLĂBIORU, Dorel Lucian RADU, Alexandru RAFILA, Lila SHUNDI, Constantin SPÎNU, Demetrios SPANDIDOS, Dan STERIU, Cătălina-Suzana STÎNGU, Galina TSENEVA, Codruţa-Romaniţa USEIN, Hans WOLF, Imola ZIGONEANU Editorial Team: Felicia RAPILAT, Monica TRĂISTARU TOTAL PUBLISHING HOUSE INDEXED IN MEDLINE CNCSIS Category B+ Subscription orders: Orders can be placed directly with the publisher: „Cantacuzino“ National Institute of Research-Development for Microbiology and Immunology C.P. 1-525, splaiul Independentei 103, 050096, Bucureºti, România Fax: (40.21)306.93.07 e-mail: [email protected] www.roami.ro Copyright © 2013 CANTACUZINO INsTITUTe Bucharest ROMANIAN ARChIves OF MICROBIOlOgy AND IMMUNOlOgy CONTeNTs MICROBIOlOgy 225 PheNOTyPIC PROFIles OF vIRUleNCe IN DIFFeReNT Candida sPeCIes IsOlATeD FROM vUlvOvAgINAl INFeCTIONs Ionela sârbu, Diana Pelinescu, Ileana stoica, luminiþa Mãruþescu, Tatiana vassu 234 seleCTION OF BACTeRIAl sTRAINs eFFICIeNT IN DeCOlORIZATION OF ReMAZOl BlACK-B Maulin P. shah, soniya sebastian, hemangi M. Mathukiya, A.M. Darji, Jigna Patel, Kavita Patel 242 sINgle AND MUlTIPAThOgeN vIRAl INFeCTIONs IN hOsPITAlIZeD ChIlDReN WITh ACUTe ResPIRATORy INFeCTIONs Cristina Þecu, Maria e. Mihai, viorel I. Alexandrescu, Dumitru Orãşeanu, Carmen Zapucioiu, Alina e. Ivanciuc, gheorghe Necula, emilia lupulescu, Daniel Chiriþã and Daniela Piþigoi 250 seROPRevAleNCe OF ANTIBODIes TO DIPhTheRIA, TeTANUs AND PeRTUssIs AMONg heAlThy ADOlesCeNTs AND ADUlTs IN IRAN Babak Pourakbari, Behnaz Moradi, Farin Mirzaee, shima Mahmoudi, Mostafa Teymuri, setareh Mamishi IMMUNOlOgy 255 ResveRATROl MODUlATes APOPTOsIs IN 5-FlUOROURACyl TReATeD COlON CANCeR Cell lINes Dan hotnog, Mirela Mihãilã, Iulia virginia Iancu, georgiana gabriela Matei, Camelia hotnog, gabriela Anton, Marinela Bostan, lorelei I. Braşoveanu 265 sUBJeCT INDeX 270 AUThOR INDeX vOlUMe 72 IssUe 4 OCTOBeR - DeCeMBeR 2013 223 INsTRUCTIONs TO AUThORs Aims and Scope Romanian archives of Microbiology and immunoloy, an international journal dedicated to original research work, publishes papers focusing on various aspects of microbiology and immunology. Romanian archives of Microbiology and immunology is indexed in MeDlINe. The frequency of the Journal is currently four issues per year. Categories of manuscripts Full-length articles are full-length descriptions of original research (up to 10 printed pages). Reviews are comprehensive appraisals of research in a field of current interest. All reviews are subject to the normal review process (up to 12 printed pages). Rapid Communications are brief, definitive reports of highly significant and timely findings in the field (up to 5 printed pages). 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Romanian archives of Microbiology and immunology PheNOTyPIC PROFIles OF vIRUleNCe IN DIFFeReNT Candida sPeCIes IsOlATeD FROM vUlvOvAgINAl INFeCTIONs Ionela Sârbu, Diana Pelinescu*, Ileana Stoica, Luminiþa Mãruþescu, Tatiana Vassu University of Bucharest, Faculty of Biology, Bucharest, Romania ABsTRACT In order to establish an infection, pathogenic microorganisms have to colonize, survive, multiply, evade the immune system and spread to other tissues [1, 2, 3]. Although some Candida species are normally commensal in humans, in the last decades the frequency and the severity of nosocomial diseases due to Candida strains have increased dramatically [4]. The aim of the present study was to characterize some Candida strains isolated from vulvovaginal infections by determining the virulence and pathogenicity profile. The assessment of the in vitro expression of virulence cell wall associated factors (hyphal formation, adherence to HeLa cell line, biofilm development), soluble secreted enzymes (aspartyl protease, lipase, phospholipase, DN-ase) and Fe3+ accumulation was achieved by phenotypic methods on 13 yeast strains belonging to five Candida (C.) species (C. albicans, C. krusei, C. parapsilopsis, C. catenulata and C. kefyr). Candida sp. strains isolated from vulvovaginal infections showed species/ strain specific virulence profile. ReZUMAT Colonizarea, capacitatea de a supraviețui și de a se multiplica a microorganismelor patogene în organismele gazdă sensibile reprezintă etape esențiale ale procesului infecțios [1, 2, 3]. În ultimele decenii au crescut frecvenţa şi severitatea infecțiilor nosocomiale produse de specii ale genului Candida, deși în mod normal aceste microorganisme sunt comensale [4]. Scopul acestui studiu a fost reprezentat de caracterizarea profilului de virulență și patogenitate al unor tulpini de drojdii izolate din infecții vulvovaginale. Studiile in vitro privind expresia factorilor de virulență, asociați peretelui celular (capacitatea de a filamenta, de a adera la celule epiteliale și la substrat inert), capacitatea de a secreta enzime solubile (aspartil proteaze, lipaze, fosfolipaze și DNaze), capacitatea de a lega Fe3+, dar și rezistența la compuși cu acțiune antifungică au fost evaluate prin metode fenotipice. Testele au fost realizate pe 13 tulpini aparținând genului Candida (C. albicans, C. krusei, C. parapsilosis, C. catenulata și C. kefyr). Tulpinile izolate din infecții vulvo- vaginale au prezentat un profil de virulență specie/tulpină specific. Keywords: vulvovaginal infections, Candida, virulence factors, pathogenic yeasts INTRODUCTION Candida sp. strains are commensal in humans and part of normal vaginal microbiota, but in particular host conditions they are able to cause a broad spectrum of mucosal, skin, nail and bloodstream infections [5, 6, 7]. Vulvovaginal candidiasis (VVC) is a mucosa-associated infection and represents one of the most common clinical problems in women at the reproductive age. Clinical reports estimated that 75% of women will experience at least one episode in their lifetime, with a recurrence rate of over 50%. VVC is favored by different conditions, such as: hormonal disbalances, depressed immune status, stress and antibiotic therapy. C. albicans has been identi- fied as the main pathogenic agent implicated in VVC, in approximately 85-90% of cases, but there are numerous reports of increasing incidence of infection in which non-albicans species, like: C. glabrata, C. parapsilosis, C. inconspicua and C. krusei have been involved [8, 9]. The pathogenicity of Candida species is due to a large number of virulence factors, such as adhesion to epithelial cells, morphological dimorphism, enzymes secretion, biofilm development and antimicrobial resistance [3, 10]. A recent report estimates that Candida species are the fourth leading cause of nosocomial bloodstream infection in the United States. One reason of increasing frequency of invasive yeast infections is *Corresponding author: Diana Pelinescu, University of Bucharest, Faculty of Biology, Bucharest, Romania, e-mail: [email protected] 225 SÂRBU et al. the recent progress in medical techniques, transplantology and antimicrobial treatment [11, 12, 13]. Despite extensive research conducted in the last past years, the invasive molecular mechanisms of yeast infections are not fully understood [10, 14]. The main objective of our study was the phenotypic characterization of virulence profiles in yeast strains isolated from vulvovaginal infections in order to determine the pathogenic potential of different Candida strains. MATERIALS AND METHODS Yeast strains and growth conditions The yeast strains used in this study were isolated from women with vulvovaginal infections and stored at -80°C with 20% glycerol. The strains were previously taxonomically identified according to their biochemical characteristics using Biolog System YT MicroPlate (Biolog, USA) as C. albicans (eight strains), C. krusei (two strains), C. parapsilopsis (one strain), C. catenulata (one strain) and C. kefyr (one strain). The yeast strains were grown in YPG (Yeast Peptone Glucose - g/L yeast extract 10, peptone 10, glucose 20, agar-agar 20) broth for 24h, in aerobic conditions (150 rpm) at 37 oC. Virulence factors assay Hyphal formation Hyphal induction was carried out in liquid RPMI broth supplemented with 10% (vol/vol) fetal bovine serum (FBS). The broth was inoculated with cells from overnight cultures in order to achieve a starting density of 5×106 cells/ml and incubated at 37 °C for 3h. The germ tubes or hyphae formation of each strain was microscopically assessed using Micros Pink MC50 Microscope. Percentage of hyphal forms was determined by counting the number of budding and hyphal/pseudohyphal cell, on ten randomized microscopic fields. Adhesion to HeLa cell line assay HeLa cells were grown up to 80% confluence in 75 cm2 tissue culture flasks using Dulbecco’s modified Eagle’s medium supplemented with 10% FBS at 37 °C, in a humidified atmosphere with 5% CO2. On 80% confluence cells were trypsinized and seeded onto 6-well polyvinylidene difluoride (PVDF) plates (Corning, USA) (105 cells/ well) and were grown up to 70% confluence. Fresh culture of pathogenic yeast strains grown in YPG broth for 18 h at 37 oC were centrifuged 5 min 226 at 6000 rpm and pellet was resuspended in phosphate buffered saline (PBS) (pH 7.2) to a density of 0.5 McFarland. 300 µl of microbial strains were used to infect HeLa cells for 2h at 37 oC. After incubation at 37 ºC, 5% CO2 for 2h the plates were washed three times with PBS (pH 7.2), then fixed with methanol 95% for 5 minutes at room temperature. The plates were air dried and Giemsa stained. Yeast adhesion was detected microscopically by counting the number of yeast cells attached to eukaryotic cells in ten randomized microscopic fields per well. Each determination was carried out in triplicate [15]. Biofilm development on the inert substratum Biofilm formation assay was performed on 96 wells polystyrene plates (Corning, USA) according to the method proposed by Jin Y. and col., slightly modified. Yeast strains were grown at 37 oC for 18 h in Sabouraud’s dextrose agar (SDA). Cell pellet obtained after 10 min spin at 6500 rpm was washed twice with PBS and resuspended in yeast nitrogen base (YNB) supplemented with 50 mM glucose. 200 µl of cell suspension (105 cells/ml) in YNB were added in each well and incubated for 72h at 37 oC without shaking. After incubation wells were washed gently 4 times with PBS in order to remove non-adherent cells and fixed with methanol for 5 min. Methanol was removed and 200 µl of crystal violet (2mg/ml) were added in each well and incubated for another 15 min at room temperature. After staining wells were washed with PBS for 4 times in order to remove the excess dye and destained with 220 µl of 33% acetic acid. The biofilm formation was evaluated by measuring absorbance at 490 nm using a microtiter plate reader Apollo LB 911 (Berthold Technologies). Assays were carried out in triplicate and were repeated three times [16]. Yeast aspartyl protease assay Yeast aspartyl protease production was determined according to the method proposed by Mohan Vinitha using bovine serum albumin medium (dextrose 2%, KH2PO4 0.1%, MgSO4 0.05 %, agar 2%, mixed after cooling to 50 ºC with 0.5 % bovine serum albumin fraction V). Protease activity was detected by spotting 10 μl of overnight microbial suspension (108 cells/ml), resuspended in PBS onto the surface of the medium. The plates were incubated at 37 ºC for 7 days. After incubation, the plates were fixed with 20% trichloracetic acid, stained with 1.25% amidoblack and destined with 15% acetic acid. Clear zone, corresponding to proteolysis around spots that could not be stained with amidoblack, indicated degradation of the protein. Phenotypic profiles of virulence in different Candida species isolated from vulvovaginal infections The diameter of unstained zones around the culture spot well was considered as a semiquantitative assay of the proteases amount production. The proteases production (Pz) was determined by the ratio between the total diameter (culture spot diameter plus diameter of the proteolytic unstained zone) and the culture spot diameter. When the Pz > 1, protease activity was detected [17]. Phospholipase detection Extracellular phospholipase activity testing was performed according Vinitha Mohan method. Extracellular phospholipase production was detected by spotting 10 μl of overnight microbial suspension (108 cells/ml), resuspended in PBS, onto the surface of the egg-yolk medium. Egg-yolk medium consisted of 13 g/L Sabouraud dextrose agar supplied with 11.7 g/L NaCl, 0.111g/L CaCl2 and 10% sterile egg yolk. The egg yolk was added into the media after sterilization when the media reached 50 oC. The plates were incubated at 37 ºC for 48h. The precipitation zone around the colony was corresponding to the phospholipase production (PLz) and was calculated as previously mentioned for the yeast aspartyl protease production method [17]. Lipase detection Tween 80 was used as a substrate for lipase detection according to the method proposed by Rudek W. The Sabouraud agar was supplemented with 1% Tween 80 which was added after autoclaving at 121°C for 15 min when the medium reached 50 oC. 10 μl of overnight microbial suspension (108 cells/ml), resuspended in PBS, were spotted onto the surface of the medium. The plates were incubated at 37 °C for 48h. Detection of lipolytic activity (Lz) was performed by observing zones of precipitation around the culture spot. The precipitate is represented by the insoluble calcium salts formed as a result of chemical reaction of calcium ions with fatty acids released by Tween 80 hydrolysis. Lipase production (Lz) was calculated as we mentioned at yeast aspartyl protease production method [18]. Hemolytic activity Hemolytic activity was evaluated using Sabouraud agar broth supplemented after sterilization when the medium reached 45 ºC with 5% defibrinated sheep blood. 10 μl of overnight microbial suspension (108 cells/ml), resuspended in PBS were spotted onto the surface of the medium. The plates were incubated at 37 °C for 24h. The presence of a distinct translucent/clear zone around the colony indicated positive hemolytic activity (Hz). The assay was performed in triplicate on three separate expe- riments for each isolate strain and calculated according to the methods presented above. Extracellular deoxyribonuclease (DN-ase) production DN-ase activity was quantified on DNase test agar (Difco) supplemented with 0.01% toluidine blue solution (v/v). 10 μl of overnight microbial suspension (108 cells/ml), resuspended in PBS, were spotted onto the surface of the medium, and plates were incubated for 48h at 37 oC. The DNA hydrolysis is indicated by production of pink zones in toluidine blue medium. Siderophore-like production Bile esculine agar (BEA) medium is used for the differentiation of Enterococcus species from Streptococcus species but is also used to reveal the capacity of some strains to produce compounds implicated in the iron chelation, resulted from the esculin hydrolysis. Iron chelating agents (esculetin) were detected by spotting 10 μl of overnight microbial suspension (108 cells/ ml), resuspended in PBS, onto the surface of the BEA medium. The plates were incubated at 37 ºC for 24h. Positive reaction was observed after black pigmentation of the media around the culture spot by interaction between esculetin with iron ferric citrate salt added to culture media. The amount of siderophore-like production (Iz) was determined reporting the total diameter (culture spot diameter plus diameter of the black zone) to culture spot diameter. RESULTS Hyphal formation An important virulence attribute in Candida species is the ability to switch between budding to pseudohyphal or hyphal forms. The filamentous form is the most virulent and has an important role in colonization and penetration of epithelial human cells [19, 20, 21]. The ability of yeast strains to switch between budding to filamentous form was assessed after 3h of incubation, high number of filamentous forms after 6 or 12 h making impossible to count the percentage of budding form and to differentiate among strains. The comparative analysis of the hyphal form generation revealed the highest ability to generate germination tubes for four C. albicans strains (CMGBy 1, CMGBy 9, CMGBy 12, CMGBy 18) and the lowest for two C. krusei strains (CMGBy 5, CMGBy 8). Most of C. albicans strains generated approximately 40% hyphal forms after 3 h of incubation. In the same conditions for C. krusei CMGBy 8, 227 SÂRBU et al. Fig. 1. Candida sp. hyphal/pseudohyphal formation in RPMI 1640 medium supplemented with 10% FBS, quantified at 3h the filamentation rate was less than 20%. Compared with the rest of the strains C. albicans CMGBy 1 exhibited the highest rate of filamentation in RPMI 1640 medium supplemented with 10% FBS (Fig. 1). In the case of C. parapsilopsis CMGBy 3 and C. catenulata CMGBy 7 strains true hyphae forms were not observed only pseudohyphal forms. C. kefyr CMGBy 11 strain did not present any pseudohyphal or hyphal forms. Biofilm formation It is well known that Candida sp. strains are able to develop biofilms in the human host by adhering to different implanted medical devices, like: catheters, endoprosthesis, artificial joints s.o. [7, 22, 23, 24]. Biofilms formed by fungal organisms are associated with drastically enhanced resistance against most antimicrobial agents, contributing to the persistence of the fungi despite antifungal therapy [25]. Although C. albicans is the predominant etiologic agent of candidiasis, other Candida species that tend to be less susceptible to the commonly used antifungal drugs such as C. krusei, C. glabrata, C. lusitaniae, and the newest Candida species, C. dubliniensis, have emerged as substantial opportunistic pathogens [26]. The assay of biofilm formation by different Candida strains isolated from vaginal infections indicated a higher degree of biofilm development by C. krusei strains compared with C. albicans strains. The most virulent strain in terms of biofilm formation was C. krusei CMGBy 8, followed by C. krusei CMGBy 5, C. catenulata CMGBy 7 also presented a high ability to form biofilm (Fig. 2). This ability explains the more and more frequent reports on non- Fig. 2. Quantification of the 72 h biofilm developed by different Candida strains 228 Phenotypic profiles of virulence in different Candida species isolated from vulvovaginal infections Fig. 3. Candida sp. adherence rates (%) to HeLa cell line albicans Candida species implicated in different types of clinical infections. Adhesion to HeLa epithelial cell line The adherence to epithelial host cells and tissues involves the capacity of microorganisms to express different molecules generically called adhesins, which allow microbial binding to host epithelial cell. This process is essential for pathogenic strains to develop an infection process [20, 27, 28]. The highest ability to adhere to HeLa vaginal epithelium cell line was observed for C. albicans CMGBy 4 (Fig. 3). A low level of adhesion was detected for strains belonging to C. krusei and C. kefyr CMGBy 11 species. Adhesion to HeLa cell line started after 2h of the cellular line infection, suggesting that colonization occurs slowly after the initial contact between the epithelium cells and yeast strains. The microscopic examination allowed us to establish not only the adherence rates, but also a certain pattern of adherence. Three adherence patterns have been previously established for E. coli: localized adherence (LA), in which bacteria attach to eukaryotic cell and form micro-colonies in distinct regions of the surface; diffuse adherence (DA), in C. albicans CMGBy 6 C. catenulata CMGBy 7 which bacteria adhere uniformly to the whole cell surface, and aggregative adherence (AggA), in which aggregated bacteria attach to the cell or inert support in a stacked-brick arrangement [29]. For example, C. albicans CMGBy 6 and C. catenulata CMGBy 7 exhibited a localized or mixed localized-aggregative adherence pattern, while C. parapsilopsis CMGBy 3 and C. krusei CMGBy 8 a diffuse one (Fig. 4). Hydrolytic enzymes biosynthesis Hydrolytic enzymes have an important role in different stages of infection process, such as invasion, nutrient accumulation and also in cell defense by inactivating some molecules of the immune system. Phenotypic quantification of enzymes/compounds expression of these will allow us to appreciate the pathogenic potential of each strain [30, 31, 32]. Previous studies have established that hemolysins are important in infection processes caused by different pathogens by accumulating iron from hemoglobin and different nutrients important in metabolism. In case of Candida species, red cells and hemoglobin lysis ability are induced by a variety of hydrolytic enzymes, such as proteases, lipases, phospholipases, esterases, and phosphatases [33]. C. parapsilosis CMGBy 3 C. krusei CMGBy 8 Fig. 4. Optic microscopy images showing the adherence patterns of different Candida strains to HeLa cell line (Giemsa staining, x 100) (from left to right: LA, AggA-LA, DA, DA) 229 SÂRBU et al. Twenty-four hours post inoculation, a greenish zone which corresponded to alpha hemolysis was observed for all Candida strains. C. albicans strains exhibited the highest hemolytic activity comparing to non-albicans strains. The highest hemolytic activity was observed for C. albicans CMGBy 4 and CMGBy 6 strains and the lowest for C. kefyr CMGBy 11 strain (Table 1). The ability of pathogenic microorganisms to acquire iron from the environment during infection is another important virulence attribute. One of the strategies used by pathogens in scavenging the host iron is by production of low molecular weight Fe3+ specific ligands called siderophores, that chelate iron from host iron-binding proteins, followed by uptake of the ferric siderophore via specific cell surface receptors [20, 34, 35]. Iron accumulation was observed by cultivating the strains on BEA media. The yeast strains hydrolyze esculin to glucose and produce esculetin (siderophore-like compound). Esculetin combines with ferric citrate and produce a black complex. Most strains showed this ability to produce siderophore-like molecules. However, the highest activity was observed for C. krusei CMGBy 5 and C. kefyr CMGBy 5. C. albicans ATCC 10231, C. albicans CMGBy 6 and C. parapsilopsis CMGBy 3 strains were not able to hydrolyse esculin (Table 1). The secreted aspartyl protease (Saps) are responsible for the adhesion, tissue damage and invasion of host immune responses. These enzymes are expressed at different stages of a systemic infection and are responsible for membrane protein degrada- tion [5, 36, 37, 38]. Aspartyl proteases secretion was detected for all C. albicans, C. parapsilopsis and C. catenulata isolated strains, in comparable amounts (Pz value: 1.57-1.09). The highest level of the enzymatic activity was observed for C catenulata CMGBy 7 (Table 1). Aspartyl protease secretion was not detected for C. krusei and C. kefyr strains. These strains probably do not express Sap2 proteins which seem to be induced by the presence of BSA as the sole nitrogen source in the medium [39, 40]. Lipase catalyzes the hydrolysis of fatty acid ester bond in the triacylglycerol (Tween 80) and release free fatty acids [20]. All C. albicans strains and C. parapsilopsis CMGBy 3 strain exhibited the ability to hydrolyze Tween 80. The strains belonging to C. krusei (CMGBy 5, CMGBy 8), C. catenulata (CMGBy 7) and C. kefyr (CMGBy 11) are not able to hydrolyze Tween 80 after 48 h (Table 1). The phospholipases, in general, catalyze the hydrolysis of phospholipids, which are the major components of all cell membranes. The activity of phospholipases is very high during tissue invasion, because these enzymes are responsible for hydrolysis of one or more ester linkages of cell membrane glycerophospholipids [41, 42]. In case of phospholipase assay, strains belonging to C. catenulate, C. krusei and C. kefyr were negative for this activity assessed on egg yolk medium (Table 1). All strains with lipase activity also presented the ability to degrade phospholipids from egg yolk, however, in case of some strains, the results were different. For example, C. albicans CMGBy 12 Table 1. The level of hydrolytic enzymes production by different Candida sp. strains Hemolysins Yeast strains C.a. ATCC10231 C.a. CMGBy 1 C.a. CMGBy 2 C.a. CMGBy 4 C.a. CMGBy 6 C.a. CMGBy 9 C.a. CMGBy10 C.a. CMGBy12 C.a. CMGBy13 C. p. CMGBy 3 C. c. CMGBy 7 C. k. CMGBy 5 C. k. CMGBy 8 C. kf.CMGBy11 Hz. 1.67 1.80 1.50 2.00 2.00 1.70 1.73 1.73 1.73 1.42 1.40 1.38 1.22 1.10 Std. dev. 0.01 0.03 0.10 0.20 0.14 0.19 0.14 0.21 0.30 0.41 0.23 0.21 0.10 0.21 Iron accumulation Std. Iz. dev. 1.00 0.00 1.66 0.05 1.46 0.09 1.76 0.15 1.00 0.00 1.46 0.16 1.53 0.14 1.33 0.08 1.50 0.06 1.00 0.00 2.04 0.14 2.53 0.16 1.50 0.02 2.22 0.07 Aspartyl protease Std. Pz. dev. 1.49 0.02 1.35 0.14 1.55 0.01 1.09 0.16 1.47 0.08 1.49 0.14 1.38 0.1 1.24 0.09 1.47 0.05 1.57 0.13 1.50 0.15 1.00 0.00 1.00 0.00 1.00 0.00 Lipase Lz. 1.18 1.23 1.36 1.42 1.55 1.55 1.42 1.17 1.55 1.55 1.00 1.00 1.00 1.00 Std. dev. 0.15 0.01 0.14 0.13 0.20 0.18 0.15 0.09 0.16 0.05 0.00 0.00 0.00 0.00 Phospho lipase Std. PLz dev. 1.36 0.09 1.29 0.18 1.24 0.07 1.10 0.12 1.43 0.07 1.38 0.01 1.53 0.08 1.53 0.15 1.25 0.07 1.38 0.13 1.00 0.00 1.00 0.00 1.00 0.00 1.00 0.00 Value 1.0 and - means negative reaction; values 1.0 and + means positive reaction C.a.-C. albicans; C. p.-C. parapsilosis; C. c.-C. catenulata; C. k.-C. krusei; C. kf.-C. kefyr 230 DNase + + + + + + + + + + + - Phenotypic profiles of virulence in different Candida species isolated from vulvovaginal infections strain presented the lowest lipase activity on Tween 80, but the highest activity on egg yolk (Table 1). The presence of extracellular DN-ase activity helps pathogenic strains to fight against neutrophil extracellular traps produced by leukocytes which are composed of DNA and histones [43, 44]. The DNA release in the extracellular space induces the occurrence of a viscous secretion which impaired the microbial pathogen dissemination. The ability of the pathogenic strains to produce DN-ase helps them escape from the inflammatory lesion. The production of extracellular deoxyribonuclease was examined after 48 h. DN-ase production could not be assessed quantitatively, due to the weak color signal around the colony. All C. albicans, C. parpapsilopsis and C. catenulata isolated strains present DN-ase activity, which was absent in case of C. krusei and C. kefyr strains (Table 1). DISCUSSION Phospholipases, lipases, haemolysins, aspartyl proteases, DN-ase, iron accumulation, adhesins, biofilm and morphological dimorphism are considered to be important virulence factors for Candida sp. strains. The absence or lower expression of these enzymes/compounds may indicate the less virulent nature of Candida species [17, 45, 46, 47]. Previous experiments showed that both yeast and hyphal cells of C. albicans are found in infected host tissues, but it is widely believed that hyphal formation is important for the invasive properties of the yeast strains [48]. The newly isolated C. albicans strains presented a high capacity to produce hyphal forms after 3 h of incubation compared with C. krusei strains. C. parapsilopsis CMGBy 3 and C. catenulata CMGBy 7 produced just pseudohyphae. Biofilm quantification results obtained by crystal violet microtiter method are confirming the data reported by Parahitiyawa N. B. and col., which state that Candida krusei exhibit the largest biofilm mass comparing with C. albicans, C. glabrata, C. dubliniensis and C. tropicalis. In our case C. krusei CMGBy 8 (O.D.490 0.57) capacity to form biofilm was 19 times higher than C. albicans CMGBy 12 (O.D.490 0.03) [49]. C. albicans, C. parapsilopsis and C. catenulata presented a high capacity to adhere to vaginal epithelium HeLa cell line. Instead C. krusei and C. kefyr presented only a low level of adhesion. The expression of hemolysins was higher in case of C. albicans strains (Pz 1.5-2) as compared with non-albicans strains (Pz 1.1-1.42) and the results are different from the iron accumulation where to non-Candida strains present a higher capacity to accumulate iron (C. krusei CMGBy 5 Pz 1.53). All of C. albicans, C. parapsilopsis and C. catenulata tested strains were aspartyl proteases producers and showed almost similar protease activity. C. krusei and C. kefyr do not present any protease, lipase, phospholipase and DN-ase activity. Lipase and phospholipase profile were very similar for C. albicans and C. parapsilopsis CMGBy 3. Hemolysis activity of C. krusei and C. kefyr isolates is not correlated with lipase, phospholipase or protease activity, being probably induced by other type of enzymes. CONCLUSION The virulence of Candida species is attributed not only to a single factor but to a combination of several factors like: hyphal formation, adherence, enzyme biosynthesis and resistance to antimicrobial compounds. Evaluation of the phenotypic virulence profile of C. albicans, C. parapsilopsis, C. krusei, C. catenulata and C. kefyr strains isolated from vaginal infection revealed a strain - dependent virulence profile. Strains belonging to C. albicans species presented the ability to produce filaments and different type of hydrolytic enzymes, and also presented a high capacity to adhere to HeLa epithelial cell line. C. parapsilopsis strain appeared to be the most prolific producer of aspartyl protease, hemolysins, lipase, and phospholipase and DN-ase. 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Acta Pathologica Microbiologica et Immunologica Scandinavica 2006. 114: 298–306. 233 seleCTION OF BACTeRIAl sTRAINs eFFICIeNT IN DeCOlORIZATION OF ReMAZOl BlACK-B Maulin P Shah*, Soniya Sebastian, Hemangi M Mathukiya, A M Darji, Jigna Patel, Kavita Patel applied & Environmental Microbiology Lab, Enviro Technology Limited (CETP) Plot no: 2413/2414, GidC, ankleshwar- 393 002, Gujarat, india ABsTRACT Azo dyes are released into wastewater streams without any pretreatment and polluted water and soil environments. To prevent contamination of our vulnerable resources, removal of these dye pollutants is of great importance. For this purpose, wastewater samples were collected from dye-contaminated sites of Ankleshwar, Gujarat, India. About 50 bacterial isolates were isolated through enrichment and then tested for their potential to remove Remazol Black-B azo dye in liquid medium. Three bacterial isolates capable of degrading Remazol Black-B azo dye efficiently were screened through experimentation on modified mineral salt medium. Isolate ETL-1 was able to completely remove the Remazol Black-B dye from the liquid medium in 18 h. Further, the isolate showed the best performance at the dye concentration of 100 mg L-1 medium (pH 7) and at temperature 35oC. Similarly, yeast extract proved to be the best carbon source for decolorization purpose. The results imply that the isolate ETL-1 could be used for the removal of the reactive dyes from textile effluents. ReZUMAT Coloranţii azoici sunt eliberaţi în apele reziduale fără tratament prealabil, poluând astfel apele şi solul. Pentru a preveni contaminarea resurselor noastre vulnerabile, este foarte importantă îndepărtarea acestor coloranţi. În acest scop, au fost prelevate mostre de ape reziduale din zonele contaminate din Ankleshwar, Gujarat, India. Aproximativ 50 de tulpini bacteriene izolate prin îmbogăţire au fost testate pentru a se determina potenţialul lor de a îndepărta colorantul azoic Remazol Black-B din mediul lichid. Aceste izolate bacteriene capabile să degradeze în mod eficient colorantul Remazol Black-B au fost analizate folosind un mediu cu un conţinut mineral şi salin modificat. Izolatul ETL-1 a fost capabil să îndepărteze complet colorantul din mediul lichid în decurs de 19 ore. Mai mult, izolatul a prezentat o capacitate maximă de acţiune la o concentraţie de colorant de 100 mg-l (pH 7) şi o temperatură de 35° C. În mod similar, extractul de drojdie s-a dovedit a fi cea mai bună sursă de carbon spentru decolorare. Rezultatele arată că izolatul ETL-1 poate fi folosit pentru îndepărtarea coloranţilor reactivi rezultaţi din industria textilă. Keywords: azo dyes, Remazol Black-B, wastewater, yeast extract INTRODUCTION Due to rapid industrialization, a lot of chemicals including dyes are manufactured and used in day to day life [1]. Synthetic dyes are extensively used in textile, dyeing, paper printing, colour photography, food, cosmetic and other industries. Approximately 10,000 different dyes and pigments are used industrially and over 0.7 million tons of synthetic dyes are produced annually, worldwide [2]. Explosion of population coupled with industrial revolution results in pollution of water, air and soil. The discharge of pollutants from various industries poses threat to the biodiversity of the earth. The textile finishing generates a large amount of dyes containing wastewater from dyeing and subsequent steps that forms one of the largest contributions to water pollution [3]. The traditional textile finishing industry consumes about 100 liters of water to process about 1 kg of textile material. The new closed-loop technologies such as the reuse of microbial or enzymatic treatment of dyeing effluents could help reducing this enormous water consumption [4]. It was already reported that 10-15% of dyes are lost in the effluent during dyeing process [5]. Azo dyes have been used increasingly in industries because of their ease and cost effectiveness in synthesis compared to natural dyes. However, most azo dyes are toxic, carcinogenic and mutagenic [6]. Azo bonds present in azo dyes are resistant to breakdown, with the potential for the per- *Corresponding author: Maulin P. shah, Applied & environmental Microbiology lab, enviro Technology limited (CeTP), Ankleshwar - 393 002, gujarat, India e-mail: [email protected] 234 Selection of bacterial strains efficient in decolorization of Remazol Black-B sistence and accumulation of high levels of dye in the environment [7]. These dyes cannot be easily degraded, while some are toxic to higher animals [8]. Azo dyes are very stable in acidic and alkaline conditions and are resistant to temperature and light. However, they can be degraded by bacteria under aerobic and anaerobic conditions [9]. The complex aromatic substituted structures make conjugated system and are responsible for intense color, high water solubility [10]. Their discharge into surface water also leads to aesthetic problems, obstructing light penetration and oxygen transfer into water bodies [11-12]. Several physicochemical techniques have been proposed for treatment of colored textile effluents. These include adsorption on different materials, oxidation and precipitation by Fenton’s reagent, bleaching with chloride or ozone, photo degradation or membrane filtration [13]. The economic and safe removal of the polluting dyes is still an important issue. All these physicochemical methods are very expensive and results in the production of large amount of sludge, which creates the secondary level of land pollution. In this situation bioremediation is becoming important, because it is cost effective and environmentally-friendly and produces less sludge [14]. Therefore, in such situations, biological treatment may be a real hope. These methods have the advantages of being environment friendly. Microorganisms have developed enzyme system for the decolorization and mineralization of azo dyes under certain environmental conditions [15-17]. Thus, this study was designed to isolate efficient azo dye decolorizing bacterial strains from the textile effluents. Since the bacterial isolates originated from the dye contaminated textile wastewater of local industry, they can easily adapt to the prevailing local environment. Therefore, such bacteria can be used to develop an effective biological treatment system for the wastewaters contaminated with azo dyes. MATERIALS AND METHODS Sampling Water and sludge samples were collected from Ankleshwar Industrial Estate, Ankleshwar, Gujarat, India around which many textile processing units are situated. Samples were taken from drain at different locations and sampling sites were selected on the basis of the allocation of outlet from textile units. Electrical conductivity (EC) and pH were determined to assess the presence of Total Suspended Solids (TSS) and acidity or alkalinity of the collected samples (Table 1). Isolation of azo dye decolorizing bacteria Bacterial strains were isolated from wastewater and sludge samples of dye industry units. Isolates from each inoculum source were first enriched using MSM medium amended with an azo dye Remazol Black-B as the sole source of C and N [18]. Dye was added at a concentration of 150 mg L-1. The cultures containing 200 mL of MSM broth with dye in 500 mL Erlenmeyer flasks were inoculated with 10 mL volume of wastewater or sludge suspensions. The flasks were incubated at 32°C for 7 days under static conditions. After incubation, cell suspensions from each flask were plated onto MSM agar medium and incubated at 32°C for 24 h. Microbial colonies that appeared on the agar medium were washed gently with sterile water and resuspended into the flasks containing fresh MSM broth spiked with the Remazol Black-B dye. About 50 actively growing colonies were selected for purification. Purification of bacterial isolates Selected isolates were purified by streaking on MSM medium containing agar at the concentration of 20 g L-1. Streaking was done thrice in zig-zag manner. The purified cultures were preserved in a refrigerator for subsequent study. Screening of azo dye decolorizing efficiency of bacterial isolates Screening was done to find out the efficient bacterial strains capable of decolorizing the Remazol Black-B azo dye using modified MSM. For this purpose, 50 isolates having the ability to decolorize Remazol Black-B from all samples were selected. After that decolorization, ability of each isolate was tested in the liquid medium. Media inoculated with the re- Table 1. Total soluble salts (TSS) and pH of the dye contaminated textile effluent and sludge Sampling Site Near K Patel Dye Unit Near Dynamic Dye Unit Near Harpal Dye Unit Near Chemcrux Unit Near Suyog Dye Unit TSS 58 78 152 118 98 pH 8.2 8.5 10.3 9.4 8.7 Notes Effluent Effluent Sludge Effluent Sludge 235 SHAH et al. % Decolorization 120 100 80 ETL-1 60 ETL-2 ETL-3 40 20 0 6 12 18 24 Time (Hrs) Fig. 1. Biodecolorization efficiency of Remazol Black –B spective inocula were incubated at 35ºC for 24 h. After 24 h, the respective cells were harvested by medium centrifugation at 10,000 rpm (REMI R-23, India) for 10 minutes. Then decolorization was determined with the help of a spectrophotometer (SHIMADZU, Japan) at 597 nm. Uninoculated blanks were run to determine abiotic decolorization. The three most effective bacterial isolates (ETL-1, ETL2 & ETL-3) from the final screening were further examined for their decolorization potentials in test tubes at different time periods. Ten milliliters of the sterilized MSM broth containing Remazol Black-B at the concentration of 100 mg L-1 was added to autoclaved test tubes supplemented with 0.5% yeast extract as a co-substrate. The medium was inoculated with the respective bacterial strains by adding inocula of uniform cell density (OD: 0.6) at 597 nm. The test tubes were tightly sealed and incubated at 35°C under static conditions. Uninoculated test tubes with MSM containing azo dye plus yeast extract were incubated under similar conditions to check for abiotic decolorization of dye. Decolorization was measured after 6, 12, 18 and 24 h at 597 nm by spectrophotometer as described by Khalid et al. (2008). Optimization of environmental factors for efficient decolorization Factors like substrate concentration, temperature and pH were optimized during the experimentation for different carbon sources (glucose, yeast extract, Mannitol and maltose) at the concentration of 4 g L-1 were also tested as co-substrate in the decolorization process. Optimization studies included various concentration of dye (50, 75, 100, 125, 150, 200 and 250 mg L-1), pH values (5, 6, 7, 8, 9) and 236 temperatures (25, 30, 35, 40, 45oC). All the bacterial isolates ETL-1, ETL-2 & ETL-3 were tested to optimize their decolorization efficiency. While culture conditions were the same as used in decolorization experiment i.e., MSM was used along with the 100 mg L-1 of Remazol Black-B azo dye. Uninoculated blanks were run to check the abiotic decolorization during the experimentation. Statistical analysis Data were entered in a Microsoft® Excel 2007 spreadsheet. RESULTS Efficiency of the bacterial isolates to decolorize Remazol Black-B was examined by measuring the color intensity in liquid medium. Based upon the relative decolorization efficiency of different isolates, the most efficient isolates (ETL-1, ETL-2 & ETL-3) with more than 80% decolorizing efficiency were selected for further experiments (data not shown). Biodecolorization of Remazol Black-B by selected bacterial isolates Biodecolorization of Remazol Black-B by the selective bacterial isolates (ETL-1, ETL-2 & ETL3) was confirmed by conducting another experiment in liquid medium at different time periods (Fig. 1). It was found that the different bacterial isolates had variable potential to remove Remazol Black-B in the growing cultures. The most efficient bacterial isolate to decolorize the Remazol Black-B was ETL-1 with 98% color removal efficiency in 18 h incubation Selection of bacterial strains efficient in decolorization of Remazol Black-B % Decolorization 120 100 80 ETL-1 ETL-2 60 ETL-3 40 20 0 0 50 100 150 200 250 300 Dye Concentration Fig. 2. Effect of substrate concentration period while the remaining isolates displayed maximum decolorization in 24 h. Isolate ETL-2 was the second most efficient bacterial isolate and it decolorized the Remazol Black-B up to 94% in 24 h. Similarly, ETL-3 isolate had a decolorization potential of 80%. Factors affecting biodecolorization of Remazol Black-B in liquid medium Potential of selected isolates (ETL-1, ETL-2, and ETL-3) was further investigated for the optimization of various incubation/environmental conditions for decolorizing the azo dye in liquid medium. It was evident (Fig. 2) that Remazol BlackB azo dye decolorization sharply increased up to 100 mg L-1 of substrate concentration and maximum decolorization was observed at 100 mg L-1 of substrate concentration. Then, there was a gradual decrease in the azo dye decolorization. Isolate ETL-1 was the most efficient azo dye decolorizing strain with more or less complete removal of the color i.e., 100% decolorization at 100 mg L-1 and minimum decolorization was recorded at 50 mg L-1 while after 100 mg L-1 substrate concentration, again ETL-1 showed a decreasing trend. Isolate ETL-2 was the second at the rank with 90% decolorization at 100 mg L-1. But, ETL-3 showed different trend from the other isolates, it indicated enhanced decolorization up to 200 mg L-1 (82%). Types of carbon sources Effects of different carbon sources such as maltose, mannitol, glucose and yeast extract were evaluated on Remazol Black-B decolorization by bacterial isolates (Fig. 3). It was found that the maximum decolorization occurred with 4% yeast extract in all selected strains (85 to 95%) followed by glucose in which decolorization occurred in the range of 20 to 25%. However, least decolorization was observed in the case of mannitol (10 to 15%). Similarly, maltose application also showed decolorization in the lower range (up to 18%). Effect of pH For studying effect of pH value, different levels of pH ranging from 5 to 9 were used and incubation of all selected isolates was done at these levels (Fig. 4). Initially with the increase in pH value from 5 to 7, decolorization increased and maximum occurred at 7 pH. Similarly, further increase in pH from 7 to 9 had negative effect on decolorization capacity of various isolates. The maximum decolorization was observed with the isolate ETL-1 (98%) at pH 7 while minimum decolorization occurred at pH 9. Similar trends in remaining isolates ETL-2, and ETL-3 were observed at pH 7. Overall, it was noted that all the bacterial isolates showed optimum decolorization from pH 5 to 7. Effect of incubation temperature Five levels (25, 30, 35, 40, and 45oC) of temperature were used for assessing optimal biodecolorization of Remazol Black-B by selected bacterial isolates. It is evident (Fig. 5) that when the temperature rose from 25 to 35oC there was inconsistent trend in decolorization by different isolates. The ETL-1 and ETL-2 isolates showed gradual increase in decolorization, while one isolate ETL-3 displayed maximum decolorization at 25oC. The remaining two bacterial isolates (ETL-1 and ETL-2) with a 237 SHAH et al. 4% Yeast Extract 95 Glucose 25 Mannitol 15 Maltose 18 0 20 40 60 80 100 % Decolorization Fig. 3. Effect of different sources of carbon on decolorization of Remazol Black-B gradual rise from 25 to 35oC showed maximum decolorization at 35oC. As the temperature increased further from 35oC to 45oC, a sharp decline in the decolorization capacity was noticed in all the isolates. It was also observed that with temperature rise, abiotic decolorization also increased. Maximum decolorization was observed with the isolate ETL-1 (98%) at 35oC and was followed by ETL-2 (94%) at the same temperature. Least decolorization was observed at 45oC in all the selected isolates. DISCUSSION Industrial effluent is not stable and it varies often in a wide range depending upon the practiced treatment process. South Asian countries are experiencing severe environmental problems due to rapid industrialization. This phenomenon is very common where the polluting industries like textile dyeing, leather tanning, paper and pulp processing, sugar manufacturing, etc. thrive as clusters. Among these the textile industries are large industrial consumers of waters as well as producers of wastewater. The effluent discharged by this industry leads to serious pollution of groundwater and soils and ultimately affects the livelihood of the poor [19]. During the dying process a substantial amount of dyes other chemicals are lost in waste water. Estimates put the dye loses between 10 - 15% [20]. Dye is generally not toxic to the environment but the color water bodies may hinder high penetration there by affecting the aquatic life and limiting the utilization [21]. Color removal of industrial effluent has been a major concern in wastewater that originates from textile %Decolorization 100 90 80 70 60 50 40 30 20 10 0 ETLͲ1 ETLͲ2 ETLͲ3 5 6 7 8 9 pH Fig. 4. Effect of pH on decolorization of Remazol Black-B 238 Selection of bacterial strains efficient in decolorization of Remazol Black-B 120 % Decolorization 100 80 ETL-A ETL-B 60 ETL-C 40 20 0 25 30 35 40 45 Temperature (in celcius) Fig. 5. Effect of incubation temperature and dye stuff plant with a continuous discharge of great quantity of remaining dyes to the environment. The efficient treatment of the effluent is an ecofriendly method for treatment of textile effluent. The degradation of molecules of dyes in the environment by microorganisms is likely to be slow, which means that it is possible for high levels of dye to persist and potentially accumulate. Due to the low degradability of the dyes, conventional biological treatment process is inefficient in treating dye waste waters. Biological decolorization is employed under either aerobic or anaerobic environment. A number of reports discourage the azo dye decolorization by microorganism under anaerobic conditions as it leads to the formation of corresponding aromatic amines. The effectiveness of microbial decolorization depends on the adaptability and the activity of selected microorganisms. Over the past decades, many microorganisms are capable of degrading azo dyes, including bacteria, fungi and yeast. The release of colored wastewater into water streams by textile industry represents a serious environmental problem and a public health concern. Major portion of this wastewater contains azo dyes which are increasingly used in industries because of their ease and cost-effectiveness in synthesis compared to natural dyes. Relative effectiveness of the isolated bacteria for the decolorization of Remazol Black-B clearly implies that these can be effectively used for the removal of Remazol Black-B from contaminated industrial wastewater. Azoreductase is reported to be the key enzyme expressed in azo-dye-degrading bacteria and catalyses the reductive cleavage of the azo bond [17, 21]. Azoreductase activity had been identified in several species of bacteria recently, such as Staphylo- coccus aureus, Shewanella putrefaciens, Shewanella strain J18 143 and Pseudomonas spp. [17, 21, 2223]. It was indicated that increase in substrate concentration from its optimum level had negative effect on decolorization capacity of isolates. Investigations with different dye concentrations in other experiments also reported higher net color removal efficiencies at lower dye concentrations [24-26]. Decrease in decolorization ability at high substrate concentration might be due to the toxicity of the dye (and co-contaminants) [27]. Azo dyes generally contain one or more sulphonic-acid groups on aromatic rings, which might act as detergents to inhibit the growth of microorganisms [27]. Another reason of the toxicity at higher concentration may be due to the presence of heavy metals (metal-complex dyes) and/or the presence of nonhydrolyzed reactive groups which may retard the bacterial growth (reactive dyes) [26]. Similarly, reduction in decolorization at low concentration of the substrate might be due to the decrease in enzyme ability to recognize the substrate efficiently. Whereas in case of different carbon sources tested yeast extract proved to be the best amongst tested carbon source. Our results were in agreement with the research conducted by Guo et al. (2008), in which the bacterial strains grew well and completely decolorized K-2BP where either yeast extract or peptone was present in the medium; however, glucose, glycerol, sucrose, lactose and starch resulted in lower rates of growth and decolorization of these dyes. Other studies also reported the maximum decolorization of azo dyes in the presence of yeast extract by bacteria [29]. In case of pH as a variable, decolorization was on higher side at pH 7. Whereas higher pH values (alkaline conditions) 239 SHAH et al. decreased the decolorization efficiency of all the tested isolates. So, from this study, it could be concluded that neutral pH supported bacterial activity to decolorize Remazol Black-B in liquid medium [3031]. Temperature is another very important parameter for anaerobic treatment of wastewater. Selected isolates were mesophilic bacteria because they all showed better decolorization in the temperature range of 25 to 35°C. Similar results were also reported by Guo et al. (2008). The mesophilic range is traditionally used [32] since it is generally thought that maintaining high temperature would be uneconomical, while degradation within the psychrophilic range is too slow. Overall, one of the selected isolate (ETL-1) of bacteria was able to completely remove color of the dye in 18 h. 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Ivanciuc1, Gheorghe Necula1, Emilia Lupulescu1, Daniel Chiriþã1 and Daniela Piþigoi1,3 1Cantacuzino niRdMi, Respiratory Viral infections department, Bucharest, Romania; alexandrescu Pediatrics Hospital, Bucharest, Romania; 3Carol davila University of Medicine and Pharmacy, Bucharest, Romania 2Grigore ABsTRACT We aimed to describe the viral etiology of acute respiratory tract infections in children aged 0 – 8 years admitted to Grigore Alexandrescu Hospital, the largest pediatric hospital in Romania. The patients had clinical diagnosis of pneumonia, bronchiolitis or viral respiratory infections and had been hospitalized between September 2010 and September 2011. The study was part of the “Molecular investigations of acute respiratory infections caused by non-influenza viruses, to assess the implications of infant and young child pathology” (2008-2011), a National Project II – 42-164 (MIRVI). We included in the study 241 children that were swabbed in the first 8 days of the onset with the following symptoms during the previous 7 days: fever > 38 oC, AND cough or sore throat, and shortness of breath or difficulty breathing .We identified by RT-PCR 131 (54.4%) positive samples: 112 (85.5%) for a single pathogen, 18 (13.7%) for coinfection with two pathogens and 1(0.8%) for coinfection with three pathogens. The most frequent pathogen identified was Respiratory Syncytial Virus (RSV) (40.18%), followed by Rhinovirus (RhV) (20.54%) and human Metapneumovirus (hMPV) (12.50%). We extrapolated our data to the National program of surveillance of SARI (Severe Acute Respiratory Infections). In this program, 191 children aged one month-8 years, were hospitalized in the same period, in which the highest percentage of positivity was due to Influenza viruses (62.65%), but RSV was identified with almost the same percent like in MIRVI (32.53%). It should be noted that among patients with pneumonia, bronchiolitis or respiratory viral infections were identified as the causal agent RhV. ReZUMAT Autorii au avut ca scop principal al lucrării descrierea etiologiei virale a infecţiilor acute ale tractului respirator la copiii cu vârste cuprinse între 0 şi 8 ani şi care au fost spitalizaţi la Spitalul de Pediatrie Grigore Alexandrescu cu diagnosticul clinic de pneumonie, bronşiolită sau viroză respiratorie, în perioada septembrie 2010 – septembrie 2011. Studiul a făcut parte din Proiectul Naţional PN II 42-164, acronim MIRVI, „Investigaţii moleculare ale infecţiilor respiratorii acute determinate de virusuri non-gripale cu implicaţii în patologia sugarului şi copilului mic”. S-au inclus în studiu 241 copii de la care s-au prelevat exsudate nazale în cursul primelor 8 zile de la debutul bolii. S-au detectat prin metoda RT-PCR cu Kit-ul Seeplex RV 15 OneStep ACE Detection Seegene, 131 (54,4%) probe pozitive: 112 (85,5%) pentru un singur patogen, 18 (13,7%) pentru coinfecţii cu 2 patogeni şi 1 (0,8%) pentru coinfecţia cu 3 patogeni. Cel mai frecvent patogen detectat a fost Virusul Respirator Sinciţial (VRS) (40,18%), urmat de RhinoVirus (RhV) (20,54%) şi MetaPneumoVirusul uman (hMPV) (12,50%). Datele obţinute au fost extrapolate la datele furnizate de Programul Naţional de Supraveghere SARI (Severe Acute Respiratory Infections). Cu ocazia acestui program, au fost lucrate 191 probe prelevate de la copii cu vârste cuprinse între 1 lună şi 8 ani şi spitalizaţi în aceeaşi perioadă ca aceia din proiectul MIRVI şi la care cel mai mare procent de pozitivitate a fost datorat Virusurilor Gripale (62,65%), dar VRS a fost detectat cu aproape acelaşi procent ca în MIRVI (32,53%). Ar trebui remarcat faptul că RhV nu este un „banal” virus care provoacă o simplă răceală, deoarece din toate cele 23 de probe care au fost pozitive, 8 au fost prelevate de la pacienţi cu pneumonie, 10 cu bronşiolită şi numai 5 de la cei ce prezentau o simplă răceală. Keywords: multipathogen viral infections, RT-PCR, bronchiolitis, pneumonia *Corresponding author: Cristina Þecu, Cantacuzino NIRDMI, spl. Independentei 103, CP 1-525, 050096, sector 5 Bucharest, e-mail: [email protected] 242 Single and multipathogen viral infections in hospitalized children with acute respiratory infections INTRODUCTION Lower respiratory tract infection (LRTI) is the most frequent cause of hospitalization worldwide [1, 2, 3, 4, 5, 6, 7]. In developed countries, the incidence of LRTI is high and causes 19% to 27% of hospitalizations in children under the age of 5 years in the USA [8, 9]. The correlation between clinical symptoms and respiratory infections has been discussed frequently in the literature, but viral detection provides more specific information on the correlation between clinical symptoms and specific infections [10, 11, 12]. The aim of this study was to describe the viral etiology of acute respiratory tract infections presenting to hospital with clinical diagnosis of LRTI (pneumonia, bronchiolitis) or viral respiratory infections in the children aged 0-8 years admitted to Grigore Alexandrescu, the largest pediatric hospital in Bucharest, Romania. The main idea of this study is to compare the data from the study MIRVI and SARI surveillance system. MATERIALS AND METHODS The study was part of the “Molecular investigations of acute respiratory infections caused by noninfluenza viruses, with implications in the pathology assessment of infant and little child” (MIRVI). Also, a SARI (Severe Acute Respiratory Infections) surveillance system has been set up in Romania since 2009. During the season 2010-2011, the surveillance system comprised 26 hospitals (in Bucharest and 8 counties). Case definition used for SARI- surveillance system [13] were patient ≥ 5 years old, onset of the symptoms during the previous 7 days, fever > 38 oC, AND cough or sore throat, AND shortness of breath or difficulty breathing. Case definition used for SARI in children < 5 years old is the IMCI case definition for pneumonia and severe pneumonia. The SARI system was developed under the coordination of the National Centre for Surveillance and Control of Communicable Diseases and Cantacuzino National Institute of ResearchDevelopment for Microbiology and Immunology (Cantacuzino NIRDMI) which performed the laboratory confirmation. We conducted a study between September 2010 and September 2011 to describe the viral etiology of acute respiratory tract infections in children aged 0 – 8 years admitted to Grigore Alexandrescu Hospital. The patients were clinically diagnosed with pneumonia, bronchiolitis or viral respiratory infections (Table 1 – case definition). The etiologic diagnosis of LRTI caused by viruses was assessed using qualitative and not quantitative methods (infectious agents and no viral load in patients admitted to hospitals) The samples from patients were nasal swabs within 8 days from onset. The samples were transported to the laboratory in viral transport medium, on the same day and immediately processed or stored at minus 80°C and subsequently processed. We included in the MIRVI study 241 samples. Diagnostic methods: Since the early season, the first 11 samples were analyzed using duplex OneStep RT-PCR Qiagen kit (QIAGEN GmbH, D40724 Hilden, Germany) and the other 230 samples were analyzed using multiplex kit Seeplex RV15 OneStep ACE Detection (Seegene Inc. Taewon Bldg., 65-5, Bangyi-Dong, Songpa-Gu, Seoul 138050, Korea). Duplex RT-PCR was the method used for investigation of infections caused by RSV, hMPV, Para influenza viruses (PIV) 1, 2, 3, Corona viruses (CoV) 229E and OC43. Qiagen kit was used for extractions and One Step Qiagen kit for RT-PCR, according to manufacturer’s instructions. The work protocol was: mix for multiplex - RNase free water 7.6 µl, PCR 5X Buffer 5 µl, Q Solution 3 µl, deoxynucleotide triphosphate (dNTP) (10 mM each) 1 µl, Qiagen Enzyme one-step Mix 1 µl, Primer forward (Fw) (10 µM) 1.2 µl (for RSV) +1,2 µl (for hMPV), Primer reverse (Rev) (10 µM) 1.2 µl +1,2 µl, Total volume 20 µl, RNA template /RNA(PC)/NC 5 µl. Machine for amplification: BioRad Termocycler. Amplification reaction program: Reverse transcription 30 s at 50°C, initial PCR activation 15 min at 94°, followed by 40 cycles of denaturation 30 s at 94°, annealing 30 s at 55°, extension 1 min at 72°, and a final extension for 10 min at 72°. Electrophoresis was carried out in 2% agarose gel and TBE 1X (Bio-Rad Laboratories, 2000 Alfred Nobel Drive Hercules, CA 94547, US). The size of the amplicons was estimated using Benchtop G753A/Ladder100 bp (2800 Woods Hollow Rd. Madison, Wisconsin US) as molecular weight. The primers used for detection of the viruses and the size of amplicons are shown in Table 2. The work protocol for the Multiplex RT-PCR with Seeplex RV 15 One Step ACE Detection kit was according to manufacturer’s instructions (Seegene’s Product User Manual 12/2010 V2.02.) 243 ÞECU et al. Table 1. Case definitions of pneumonia [13] and bronchiolitis [14] Age Children below 2 months Children 2 months up to 8 years old Severe Pneumonia - symptoms: cough or difficult breathing and - signs: 60 or more breaths per minute, or severe chest indrawing - and no general danger signs (the sign ‘stopped feeding well’ in young infants replaces ‘unable to drink’ of the older children as a danger sign), wheezing, stridor in calm child or fever or low body temperature Pneumonia - symptoms: cough or difficult breathing - and signs: 50 or more breaths per minute for infants aged 2 months up to 1 year, or 40 or more breaths per minute for children aged 1 up to 8 years - and no chest indrawing, general danger signs, stridor in calm child or severe malnutrition Diseases Very Severe Pneumonia - symptoms: cough or difficult breathing - and signs: general danger signs, wheezing, stridor in a calm child or fever or low body temperature Severe pneumonia - symptoms: cough or difficult breathing - and signs: chest indrawing - and no general danger signs, stridor in a calm child or severe malnutrition Bronchiolitis of infancy - clinically diagnosed respiratory condition presenting with breathing difficulties, cough, poor feeding, irritability and, in the very young, apnea. These clinical features, together with wheeze and/or crepitations on auscultation combine to make the diagnosis. - bronchiolitis most commonly presents in infants aged three to six months Table 2. Primers used for the detection of the viruses and the size of the resulted amplicons No. Viruses 1 RSV 2 hMPV 3 PIV1 4 PIV2 P 5 PIV3 6 CoV 229E 7 CoV OC43 RESULTS Primers Fw: 5’ GGA ACA AGT TGT TGA GGT TTA TGA ATA TGC 3’ Rev: 5’ CTT CTG CTG TCA AGT CTA GTA CAC TGT AGT 3’ Fw: 5’GCT TCA GTC ATT CAA CAG 3’ Rev: 5’ CCT GCA GAT GTT GGC ATG T 3’ Fw: 5’CCG GTA ATT TCT CAT ACC TAT G 3’ Rev: 5’ CCT TGG AGC GGA GTT GTT AAG 3’ Fw: 5’ AAC AAT CTG CTG CAG CAT TT 3’ Rev: 5’ ATG TCA GAC AAT GGG CAA AT 3’ Fw: 5’ CTC GAG GTT GTC AGG ATA TAG 3’ Rev: 5’ CTT TGG GAG TTG AAC ACA GTT 3’ Fw: 5’ TGG CCC CAT TAA AAA TGT GT 3’ Rev: 5’ CCT GAA CAC CTG AAG CCA AT 3’ Fw: 5’ GGC TTA TGT GGC CCC TTA CT 3’ Rev: 5’ GGC AAA TCT GCC CAA GAA TA 3’. In MIRVI study a total of 241 patients were enrolled between week 38/2010 and week 30/2011. Weekly distribution of positive cases from MIRVI collection and SARI surveillance system is shown in Figure 1. 244 Amplicon size (bp) Reference 278 [15] 150 [16] 317 [17] 517 [17] 189 [17] 573 [17] 335 [17] According to the case definition 115 (47.7%) of the patients presented pneumonia, 94 (39%) bronchiolitis and 32 (13.3%) respiratory viral infections. We identified 131 (54.4%) positive samples: 112 (85.5%) for a single pathogen, 18 (13.7%) for two pathogens and 1(0.8%) for three pathogens. The Single and multipathogen viral infections in hospitalized children with acute respiratory infections Table 3. Distribution of viral infectious agents on age groups Age group 0<1 year 1-4 years 5-8 years RSV 45 (40.18) 31 (68.89) 13 (28.89) RhV 23 (20.54) 12 (52.17) 11 (47.83) hMPV 14 (12.50) 7 (50) 5 (35.71) 1 0 2 RSV 4 RSV A + RSV B RSV B + RhV 2 2 0<1 year 1-4 years 5-8 years Viral pathogens n (%) Single infections N = 112 PIV IV 13 10 (11.61) (8.93) 9 3 (69.23) 4 5 (30.77) 0 ADV* 4 (3.57) BoV** 1 (0.89) CoV 1 (0.89) EV*** 1 (0.89) 2 0 1 0 1 1 0 1 1 0 0 0 2 Double infections N = 18 (13.74%) ADV PIV 3 IV 4 5 2 ADV + ADV + PIV 3 + PIV 3 + IV A IV B + RhV hMPV RhV RSV B + EV RSV A 2 2 4 1 1 1 hMPV 2 hMPV + RhV 2 BoV 1 BoV + RhV 1 1 1 2 1 2 1 1 1 2 0 1 0 0 0 2 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0 Triple infection N=1 (0.76%) IV + RhV + BoV 1 1-4 years most frequent pathogen identified was Respiratory Syncytial Virus (40.18%), followed by Rhinovirus (20.54%) and human Metapneumovirus (12.50%) (Table 3). According to the data obtained in MIRVI study, (Table 3), the largest share of respiratory infections caused by major respiratory viruses were identified in descending order in the following age groups: the group 0- < 1 year, followed by group 1-4 years and 5-8 years. For respiratory infections caused by Influenza viruses (IV) most cases have been recorded in 1-4 years age group. Clinical symptoms displayed by the patients with viral infections and acute respiratory infections – ARI and coinfections admitted to the Grigore Alexandrescu Hospital are shown in Table 4. According to the SARI surveillance program 191 children, aged one month-8 years, were hospitalized between September 2010 – July 2011 in 26 infectious diseases pediatric hospitals in 8 counties and Bucharest. For these inpatients, the following results were obtained: 85 (44.50%) were positive for all the tested viruses while 106 (55.50%) were negative. 1 Weekly distribution of the positive cases is shown in Figure 1. Eighty-three out of 85 (97.64%) infections were caused by a single pathogen and 2 (2.36%) infections were produced by two pathogens. Regarding respiratory infections caused by IV, most cases have been recorded in 1-4 years age group. Distribution of viral infectious agents on age groups recorded by SARI surveillance system is shown in Table 5. DISCUSSION In the MIRVI project we detected infections caused by single viral pathogen in 85.5% of the cases while in SARI program the percent was 97.64% The greater percentage recorded by the latter system is due to the fact that the samples were collected from patients hospitalized in the intensive care units (ICUs). We detected more than one agent in 14.5% of children with a clinical diagnosis of LRTI. The most frequent combination was coinfection with two agents, mostly PIV type 3 plus RhV. One case presented a coinfection with three agents (Table 3). 245 ÞECU et al. Table 4. Clinical symptoms of patients admitted to Grigore Alexandrescu Hospital 20 0 3 Digestive disorders Expectoration 60 15 Otitis 0 Cough Headache Wheezing 4 0 109 Pharyngitis 15 50 2 Rhinitis / coryza Viral Respiratory Infections (total 28) 80 62 Maximum temperature Bronchiolitis (total 85) 88 Dyspnea Pneumonia (n = 109) Fever Disease Clinical symptoms of single and ARI 69 82 > 38 oC (75.23%) 27 afebrile (24.77%) 42 13 0 1 38 80 > 38 oC (94.12%) 5 afebrile (5.88%) 18 0 0 0 2 15 > 38 oC (53.57 %) 13 afebrile (46.43%) 20 2 0 0 Clinical symptoms of multipathogen viral infections Pneumonia (total 6) 6 3 0 6 0 6 6 > 380 C (100%) 3 1 0 0 Bronchiolitis (total 9) 9 6 0 8 0 5 9 > 38 oC (100%) 3 0 0 0 0 2 > 38 oC (50%) 2 afebrile (50%) 2 0 1 1 Viral Respiratory Infections (total 4) 2 1 0 3 1 The prevalence of coinfection reported in the previous studies is 11-27% in young children. Diverse types of respiratory tract infections were found in young children and multiple respira- tory tract infections were seen in the hospitalized patients or in patients addressing the emergency department units [18, 19, 20, 21, 22, 23, 24]. Fig. 1. Positive cases per weeks, from MIRVI and SARI projects 246 Single and multipathogen viral infections in hospitalized children with acute respiratory infections Table 5. Distribution of viral infectious agents on age groups according to SARI Program Age group 0<1 year 1-4 years 5-8 years 0<1 year 1-4 years 5-8 years Viral pathogens n (%) Single infections N=83 IV RSV hMPV PIV 52 27 2 2 (62.65) (32.53) (2.40) (2.40) 7 13 0 1 (13.46) (48.15) 26 13 2 1 (50) (48.15) 19 1 0 0 (36.54) (3.70) Double infections N=2 RSV + PIV 2 1 1 0 In most published studies concerning dual respiratory viral infections, more than one viral diagnostic technique was used to identify respiratory viruses [20, 21] When only one diagnostic method was used, the overall rate of these infections was 1.8%, whereas when two virus detection methods were used the rate was 9.9% and when three methods were used the rate was 8.4 % [25]. Figure 1 shows the weekly positive cases registered in MIRVI and SARI projects. The absence of positive cases at the beginning and end of the epidemic season is due to the few cases enrolled in SARI project in weeks 36-43/2010 (0 cases in weeks 39-43) and in weeks 21-26/2011(0 cases in weeks 22, 23, 25). Increased values recorded in the project MIRVI during the weeks 43-47/2010 and in week 50 of the SARI project are due to the increased number of infections caused by RSV and RhV (MIRVI) and RSV (SARI); data also presented in the specialized literature about RSV peak circulation in November, December. During weeks 3-11/2011 the percent of influenza infection cases was high in both programs. The period of weeks 22-25/2011 is characterized by a large number of positive cases caused by hMPV, samples which were taken in the project MIRVI and not in SARI program. This fact demonstrates the circulation of the virus during the summer. RSV is the most common cause of bronchiolitis and pneumonia in infants and young children [26]. In the MIRVI project, the percent of bronchiolitis caused by RSV in single pathogen infections was 25.8% and the percent of pneumonia caused by the same virus was 14.68%. In case of hospital overcrowding, over infection with RSV may occur (2 children were hospitalized with infection with RSV type A and over infected with RSV type B after hospitalization). Our study revealed that there are not clinical differences between bronchiolitis caused by RSV and hMPV on the first days of illness, but bronchiolitis caused by hMPV does not cause prolonged wheezing, while RSV infection is associated with wheezing that can persist from 1 month to 12 months. The small number of infections caused by ADV (4) was due to the fact that most children tested were aged less than 2-5 years, group where the incidence is the highest [5, 7]. It should be noted that RhV is not a “trivial” virus that causes common cold, since out of the 23 positive samples for this virus, 8 were collected from patients with pneumonia, 10 from individuals with bronchiolitis and only 5 with cold [20]. Clinically, in 14 cases, infection with RhV was characterized by rapid onset within very few hours, ascending to severe respiratory failure and rapid improvement with corticosteroid treatment, adrenaline or salbutamol aerosol and oxygen. All patients with pneumonia and bronchiolitis caused by multipathogen had fever unlike patients with simple infection. Of the hospitalized patients with pneumonia (109), 27 were afebrile, and among those hospitalized with bronchiolitis (85) 5 were afebrile (Table 4). One of the patients diagnosed with pneumonia who had on admission to hospital symptoms of enterocolitis had infection caused by ADV and one of the patients diagnosed with viral respiratory infection had digestive disorders caused by ADV (this patient had coinfection ADV + RhV). We have noted a significant correlation between viral coinfections and increased disease severity of bronchiolitis and pneumonia and the dual infection was a risk factor for admission to the pediatric intensive care unit, independent of the host’s condition. The same rate was described by ParanhosBaccala et al. [27]. 247 ÞECU et al. Thus, out of 19 patients admitted to ICU, 14 (74%) had viral coinfection and 5 (26%) simple infections. Of the 19 patients with coinfection, 14 (74%) were admitted to ICU, and from 112 with simple viral infections 5 (4.46%) were admitted to ICU. CONCLUSIONS The percent of the single pathogen viral infections in MIRVI were 85.50% and for SARI programs were 97.64%. The percent of the coinfections with two pathogens were 13.74% in MIRVI collection. It should be noted that among patients with pneumonia, bronchiolitis or respiratory viral infections were identified as the causal agent RhV. MIRVI project data mainly coincide with those of the national surveillance program for SARI, and the delay in detecting positive cases recorded in SARI program is due to the small number of cases taken at the beginning of the season. We have noted a significant correlation between viral coinfections and increased disease severity of bronchiolitis and pneumonia and the dual infection was a risk factor for admission to the pediatric intensive care unit, independent of the host’s condition. The study has been carried out in Cantacuzino NIRDMI, Respiratory Viral Infections Department: Spl. Independentei 103, C.P. 1-525, Postal Office: 050096, Sector 5, Bucharest. REFERENCES 1. 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Macfarlane JT, Colvile A, Guion A, Macfarlane RM, Rose DH. “Prospective study of aetiology and outcome of adult lower-respiratory-tract infections in the community”. Lancet 1993, 341:511-514. 6. Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, Anderson LJ, Fukuda K. ”Mortality associated with influenza and respiratory syncytial virus in the United States”. JAMA 2003, 289:179-186. 7. Ekici M, Ekici A, Akin A, Atinlaia V, Bulcun E. “Chronic airway diseases in adult life and childhood infections”. Respiration, 2008, 75:55-59. 8. Henrickson KJ, Hoover S, Kehl KS, Hua W. “National disease burden of respiratory viruses detected in children by polymerase chain reaction”. Pediatr.Infect.Dis.J. 2004, 23:11-18. 9. Peck AJ, Holman RC, Curns AT, Lingappa JR, Cheek JE, Singleton RJ, Carver K, Anderson LJ.”Lower respiratory tract infections among American Indian and Alaska native children and the general population of U.S. children”. Pediatr.Infect.Dis.J. 2005, 24:342-351. 10. 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Stempel HE, Martin ET, Kuypers J, Englund JA, Zerr DM. “Multiple viral respiratory pathogens in children with bronchiolitis”. Acta Paediatr.2009, 98:123-126. 23. Cilla G, Onate E, Perez-Yarza EG, Montes M, Vicente D, Perez-Trallero E. “Viruses in communityaquired pneumonia in children aged less than 3 years old. High rate of viral coinfection”. J.Med. Virol. 2008, 80:1843-1849. 24. Falchi A, Arena C, Andreoletti L, Jacques J, Leveque N, Blanchon T, Lina B, Turbelin C, Dorleans Y, Fiahault A, Amoros JP, Spadoni G, Agostini F, Varesi L.”Dual infections by Influenza A/H3N2 and B viruses and by Influenza A/N3N2 and A/H1N1 viruses during winter Corsica Island, France”. J. Clin. Virol.2007, 41:148-151. 25. Drews AL, Atmar RL, Glezen WP, Baxter BD, Piedra PA, Greenberg SB. “Dual respiratory virus infections”. Clin. Infect. Dis. 1997, 25: 1421-1429. 26. Hemming VG.”Viral respiratory diseases in children classification, etiology, epidemiology, and risk factors”. J. Pediatr., 1994, 124:513-516. 27. Paranhos-Baccala G, Komurian-Pradel F, Richard N, Vernet G, Lina B, Floret D. ”Mixed respiratory virus infections”. J. Clin. Virol. 2008, 43:407-410. 249 seROPRevAleNCe OF ANTIBODIes TO DIPhTheRIA, TeTANUs AND PeRTUssIs AMONg heAlThy ADOlesCeNTs AND ADUlTs IN IRAN Babak Pourakbari1, Behnaz Moradi2, Farin Mirzaee2, Shima Mahmoudi1, Mostafa Teymuri1, Setareh Mamishi1,3* 1Pediatrics infectious diseases Research Center, Tehran University of Medical Sciences, Tehran, iran 2School of Medicine, Tehran University of Medical Sciences, Tehran, iran 3department of Pediatric infectious diseases, School of Medicine, Tehran University of Medical Sciences, Tehran, iran ABsTRACT Serologic data on diseases that are preventable by vaccine are useful to evaluate the success of immunization programs. In this study we evaluated the serologic levels of antibodies to diphtheria, tetanus, and pertussis. In a cross sectional study, a total of 360 people aged 10-25 years were randomly selected and classified by sex and age (10-14, 15-20, 21-25 years). Overall, 78.8% of people aged 10-25 years had fully protected levels of diphtheria antibody (≥ 0.1 IU/ML), and 89.7% had fully protected levels of tetanus antibody (≥ 0.1 IU/ML), 94.3% of women aged 15-25 years had anti tetanus antibody sufficient to protect against neonatal tetanus (≥ 0.1 IU/ML). Antibodies to Pertussis toxin (PT) were found in 44.2% samples but only 1.4% had fully protective levels. Antibodies to PT increased with age, ranging from 33.5% in aged 10-14 years to 54.6 % in aged 21-25 years. No differences were found between male and female, except for diphtheria in age group 21-25 years. Results of this study reveal that diphtheria and tetanus (dT) are efficient between booster doses. About pertussis, most people are susceptible to pertussis and increased PT antibodies with age suggest acquired asymptomatic Bordeella pertussis infection. Also B. pertussis infections in adolescents and adults are of concern, as they are the most important source of transmission of pertussis to young, unprotected infants. So one booster dose in adolescents and adults (as CDC recommended), to reduce mortality and morbidity in infants, is therefore suggested. ReZUMAT Datele serologice privind bolile prevenibile prin vaccinare sunt utile în evaluarea reuşitei programelor de imunizare. În studiul de faţă, evaluăm titrul anticorpilor anti difterie, tetenos şi pertussis. Într-un studiu transversal, au fost selectate aleator şi clasificate în funcţie de sex şi vârstă un număr total de 360 de persoane, cu vârste cuprinse între 10-25 ani (10-14, 15-20, 21-25). 78,8% dintre persoanele cu vârste cuprinse între 10-25 ani prezentau nivele protectoare de anticorpi anti difterie (≥ 0,1 UI/ml), 89,7% aveau nivele protectoare de anticorpi antitetanos (≥ 0,1 UI/ml) şi 94,3% dintre femeile cu vârste cuprinse între 15-25 ani prezentau nivele de anticorpi antitetanos suficiente să asigure protecţie împotriva tetanosului neonatal (≥ 0,1 UI/ml). Anticorpi anti pertussis au fost detectaţi în 44,2% dintre probe, dar numai 1,4% prezentau nivele protectoare. Nivelele anticorpilor anti pertussis au crescut la grupele cu vârstă mai mare, fiind cuprinse între 33,5% la persoanele din grupa de vârstă 10-14 ani și 54,6% la grupa 21-25 ani. Nu s-au găsit diferenţe între bărbaţi şi femei, cu excepţia titrului de anticorpi anti difterie la grupa de vârstă 21-25 ani. Rezultatele studiului arată că nivelul anticorpilor anti difterie și anti tetanos rămâne protector între dozele de rapel. În ceea ce priveşte pertussis, majoritatea indivizilor sunt susceptibili la infecţie, iar nivelele crescute de anticorpi anti pertussis la vârste mai mari sugerează infecţie asimptomatică cu B. pertussis. De asemenea, sunt de interes infecţiile cu B. pertussis la adolescenţi şi adulţi, deoarece sunt cea mai importantă sursă de transmitere la copii neprotejaţi. De aceea, se recomandă o doză de rapel la adolescenţi şi adulţi (conform recomandării CDC) pentru a reduce mortalitatea şi morbiditatea în rândul copiilor mici. Keywords: diphtheria, tetanus, pertussis, vaccination, immunization INTRODUCTION Vaccination against diphtheria and tetanus has markedly reduced the number of cases and deaths from these diseases. In the United States, during 1988-2006, seven cases of respiratory diphtheria were reported to CDC [1] but in Iran, during 1995-2006, 159 cases of respiratory diphtheria were reported [2]. Exposure to diphtheria remains possible during travel to coun- *Corresponding author: Setareh Mamishi, Department of Pediatric Infectious Diseases; Children Medical Center Hospital, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran, E-mail: [email protected] 250 Seroprevalence of antibodies to diphtheria, tetanus and pertussis tries where diphtheria is endemic or epidemic, following exposure to toxin producing strains of Corynebacterium ulcerans, contact with dairy animals or consumption of unpasteurized dairy products [1]. Tetanus rarely occurs in people who have received a complete vaccination course. During 19952006, 81 cases of tetanus and one case of neonatal tetanus were reported in Iran [2]. Neonatal tetanus occurs as the result of Clostridium tetani infection of umbilical stump of an infant born to mother with a maternal tetanus antitoxin level insufficient to protect infants from neonatal tetanus [1, 3-6]. Only three cases of neonatal tetanus were reported to CDC during 1990-2004 in the United States. Since 1980 the number of reported pertussis cases has been steadily increasing especially among adolescents and adults, despite high immunization rates in childhood [1, 7]. Protection against pertussis wanes approximately 5-10 years after completion of childhood pertussis vaccination [1, 3]. Neither natural immunity nor vaccine-induced immunity to Bordetella pertussis is long lasting [8]. B. pertussis infection in adolescents and adults is often not typical so real incidence of pertussis is markedly underestimated [8]. Also adolescents and adults are the most important source of transmission of pertussis to infants [7, 9]. In this study we report serosurvey immunity to diphtheria, tetanus and pertussis in Iran to evaluate the efficiency of vaccination against these diseases and finding susceptible persons especially to pertussis. METHODS Study population and design: The survey was conducted during 2007 at Children Medical Center. People with history of immune deficiency (primary or secondary), unvaccinated or with uncertain history of administration of three doses of combined diphtheria, tetanus and pertussis (DTP) vaccine in childhood, and who did not receive booster vaccination at 15 years old, were excluded. Recruitment was done by randomized routes system. Questionnaires about demographic and history of cough more than two weeks were filled for each participant. Serum samples were collected and then frozen and stored at -70ºC until assayed at the same time. This study was designed as cross-sectional study on a population aged 10-25 years. Cluster sampling by sex and age was performed and distributed in three age groups (10-14, 15-20, 21-25). Sample size: Blood samples were obtained from 360 healthy adolescents and adults aged 10-25 years in Tehran city, Iran January - June 2007. Serum sample size for this study was determined on the basis of expected levels of immunity to diphtheria, tetanus and pertussis in each of the age groups 10-14, 15-20 and 21-25 years. Laboratory tests: IgG antibodies against diphtheria and tetanus were detected using enzyme linked immunosorbent assay (ELISA) (IBL, Hamburg, Germany) and IgG antibodies to Pertussis toxin (PT) were determined by ELISA, (Pertusscan, Euro-Diagnostica, Malmö, Sweden). Determination of each antibody was in accordance with the instructions provided in the package insert. For diphtheria and tetanus assay, sera were classified as negative (susceptible) when the antitoxin level was < 0.01 International Unit (IU)/ml and positive (immune) when it was ≥ 0.1 IU/ml. Anti toxin levels in the range of 0.01-0.1 IU/ml were classified as low positive (partially immune). Childbearing aged females (15-25 years of age) with anti-tetanus antitoxin level ≥ 0.1 IU/ml could protect against neonatal tetanus. About pertussis, according to manufacturer, absorbance above 0.3 indicates antibodies. Absorbance values of IgG to PT above 1 indicate previous, current/recent pertussis infection or immunization. The ≥ 0.1 limit represents approximately 100 EU/ml. Statistical analysis: Differences in antibody prevalence were determined by disease, sex and age group, and compared using the Chi-square test and P-values less than 0.05 were considered statistically significant. Statistical calculations were performed with SPSS statistical software (version 13.0; SPSS Inc., Chicago, IL, USA). RESULTS Tetanus: The proportion of subjects susceptible to tetanus and partially immune was higher in aged 10 -14 years than in other age groups (Table 1) but did not differ significantly between age groups. No differences between males and females were detected when comparing the overall prevalence and the prevalence by age groups (Fig. 1). 94.3 % of women aged 15-25 years had anti tetanus antibody sufficient to protect against neonatal tetanus. Diphtheria: As for tetanus, the proportions of subjects susceptible to diphtheria and partially immune were higher in aged 10-14 years than in other age groups (Table 1) and were statistically significant (P = 0.001). Anti diphtheria antibodies were lower than anti tetanus antibody in all age groups especially in 10-14 years of age (Table 1). Proportions 251 POURAKBARI et al. Table 1 - Immunity to diphtheria, tetanus and pertussis by age group Age group (years) Total 10-14 15-20 21-25 360 130 109 121 18 (5%) 13 (10%) 2 (1.9%) 3 (2.5%) Partially immune² 58 (16.1%) 33 (25.4%) 13 (11.9%) 12 (9.9%) Immune³ 284 (78.9%) 84 (64.6%) 94 (86.2%) 106 (87.6%) 360 130 109 121 Susceptible¹ 4 (1.1%) 3 (2.3%) 1 (0.9%) 0 (0%) Partially immune² 33 (9.2%) 22 (16.9%) 6 (5.5%) 5 (4.1%) 323 (89.7%) 105 (80.8%) 102 (93.6%) 116 (95.9%) 350 128 103 119 Susceptible* 195 (55.7%) 85 (66.4%) 56 (54.4%) 54 (45.4%) Partially immune† 50 (42.9%) 42 (32.8%) 45 (43.7%) 63 (52.9%) 5 (1.4%) 1 (0.8%) 2 (1.9%) 2 (1.7%) Diphtheria Number tested Susceptible¹ Tetanus Number tested Immune³ Pertussis Number tested Immune‡ ¹Susceptible : < 0.01 IU/ml, ²Partially immune: 0.01 to < 0.1 IU/ml, ³Immune 0.1 IU/ml *Susceptible: Absorbance value < 0.3, † Partially immune: Absorbance value 0.3 to < 1, ‡ Immune: Absorbance value 1 of males and females with immunity did not differ significantly in each age group except in 21-25 years of age (83.3% of female v 95.3% of males, P= 0.03, had levels ≥ 0.1 IU/ml) Pertussis: Of the 360 serum samples tested, 44.3% had antibodies to PT but only 1.4% were immune against pertussis. Prevalence of subjects seropositive for anti PT increased with age, ranging from 33.6 % in the 10-14 years of age to 54.6% in the 21-25 years of age (Table 1). Chi-square test for linear trend was highly significant (p = 0.0001) with rates peaked at aged 21-25 years. A total of 195 (55.7%) subjects were seronegative. Percentage of anti PT increasing with age in women is higher than in men (Fig. 1), but these differences were not significant statistically (p > 0.05). The prevalence of cough for more than two weeks was found to be 5%. Sixty six percent of them had antibody to PT. DISCUSSION In Iran, little is known about seroprevalence of antidiphtheria, tetanus and pertussis antibodies Fig. 1. Percentage of sera with antitoxin levels 0.01 IU/ml for diphtheria and tetanus and percentage of seropositive sera for anti PT by sex and age group 252 Seroprevalence of antibodies to diphtheria, tetanus and pertussis among healthy adolescents and adults. The Iranian Advisory Committee on the immunization practices has implemented a primary course of combined DTP vaccine in childhood (at age 2, 4 and 6 months, with two additional doses at the age of 18-24 months and 4-6 years), plus booster doses containing only diphtheria and tetanus (dT) every 10 years thereafter. Also to prevent neonatal tetanus pregnant women whose vaccination state is unknown or who have not received three primary doses of tetanus vaccination series during their lives should receive two doses of dT vaccine at 4th and 6-7th month of pregnancy and those who complete their childhood vaccination courses, but receive last tetanus toxoid containing vaccines ≥ 10 years previously, should receive one dose dT during their pregnancy. Diphtheria and tetanus: In this study over 90% of children had tetanus and diphtheria antitoxin levels ≥ 0.01 IU/ml. For both diphtheria and tetanus the lowest antitoxin levels were seen in persons aged 1014 years whereas subjects aged 21-25 years had the highest levels. Diphtheria antitoxin levels were lower than tetanus antitoxin in all age groups and compatible with other studies [10, 11]. In one study in Razi Institute in Iran, the immunity level against tetanus and diphtheria 2-4 weeks after DTP vaccination was evaluated; thus, 99.4% of children were immune against both tetanus and diphtheria, so lower diphtheria antitoxin level than tetanus in this study shows that people lose their immunity against diphtheria sooner than tetanus. Also 94.3 % of women aged 1525 years had sufficient tetanus antitoxin to protect against neonatal tetanus compatible with CDC recommendation [3]. Our results are comparable with those of serosurvey in other countries with similar vaccination histories, such as Australia, Thailand and the United States [11-13], and reflect the efficiency of the vaccination program in Iran. To reduce the risk of reintroducing diphtheria to Iran, travelers to endemic or epidemic regions like Afghanistan need to be fully immunized; also, maintaining high childhood vaccination rates is necessary to achieve herd immunity to diphtheria and to protect individuals against tetanus. Insufficient maternal anti tetanus concentration is the most important reason for neonatal tetanus, so screening and vaccination of eligible women during pregnancy is essential. In addition to vaccination, promotion of clean deliveries and improvement of surveillance are important to reduce neonatal tetanus cases. Pertussis: Over half of the subjects did not have any antibody to PT and the percentage of immune subjects was very low, compatible with the fact that immunity to pertussis wanes approximately 5-10 years after completion of childhood pertussis vaccination [1, 13, 14] and may be because of lower efficiency of pertussis vaccines produced in Iran [15]. These results were similar to other studies, estimate the antibody to PT in Spain and Australia [8,16] but antibody to PT was lower than in two studies reported in Turkey [17, 18]. In our study the percentage of people who had antibody to PT increased with age and the highest was in 21-25 years of age, which was not compatible with routine vaccination against pertussis in Iran. This increase reflects the acquisition of natural immunity from asymptomatic B. pertussis infection [8, 14, 17, 19]. These adolescents and adults can then become a source of infection for neonatal and young infants who have not yet completed their vaccination schedules. Also the proportion of persons with cough more than two weeks was 5% which may be the result of subclinical infections. In the United States, two Tdap products (Adacel and Boostrix) were licensed for use in adolescents and adults (11-64 years of age) [1, 20], and in many studies efficiency and safety of these vaccines were evaluated [1, 10, 21-23]. Because of the high proportion of persons without antibody to PT, increasing subclinical B. pertussis infection, and high prevalence of mortality and hospitalization of infants due to pertussis in Iran, the best way to reach higher immunity in adults and adolescents and potentially reduce the incidence of pertussis in infants, is addition of pertussis vaccine in Iranian vaccination schedule for one dose as CDC recommended. 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J Adoesc Health 2005; 37:517. 23. Pichichero MF, Blatter MM, Kennedy WA, Hedrick J, Descamps D, Friedland L. Acellular pertussis vaccine booster combined with diphtheria and tetanus toxoids for adolescents. Pediatrics 2006;117:1084-93. ResveRATROl MODUlATes APOPTOsIs IN 5-FlUOROURACyl TReATeD COlON CANCeR Cell lINes Dan Hotnog1, Mirela Mihãilã1, Iulia Virginia Iancu2, Georgiana Gabriela Matei1, Camelia Hotnog1, Gabriela Anton2, Marinela Bostan1, Lorelei I. Braşoveanu1* Ştefan S. nicolau institute of Virology, 1Center of immunology and 2Viral Genetics dept., 285 Mihai Bravu ave, S3, 030304, Bucharest, Romania ABsTRACT Since cancer is a cellular disease, it is essential to identify the development stages and use the information in the prediction, prevention, early detection and design of drug targets. Colon cancer represents a malignancy with high incidence and mortality throughout the world, its etiology involving many genetic, immunological and biochemical factors. 5-fluorouracyl (5-FU) is one of the most effective anti-cancer agents used in the treatment of colorectal cancers, but tumor chemoresistance is a major limiting factor of its use. In order to choose the most effective chemotherapeutic doses of 5-FU, and thereby diminish the side-effects, we tried to modulate the anticancer properties of 5-FU by adding dietary natural compounds. The study focused on the role of natural compounds as resveratrol (RSV) in sensitization of LoVo human colon adenocarcinoma cell line to 5-FU action. Real-time cell analysis (RTCA) by xCELLigence System was used to continuously monitor the cytotoxic effects of drug treatments on LoVo cells. RTCA allowed us to choose the proper concentrations for further end-point assays, such as flow-cytometry techniques used for the evaluation of apoptotic events, progression through cell cycle phases or nuclear antigen expression of compound-treated LoVo cells. Data obtained showed additional effects of RSV to 5-FU treatments on the increase of apoptotic events, and suggested alternative approaches to obtain a stronger antitumor response, and diminished side-effects when low concentrations of anti-cancer drugs are used. Modulation of the mechanisms of programmed cell death process seem to be of great importance for malignant transformation, and therefore for anti-cancer therapeutic approaches. ReZUMAT Deoarece cancerul este o boală a celulei, este esenţial să se identifice stadiile de dezvoltare şi folosirea informaţiilor în predicţia, prevenţia, depistarea precoce şi conceperea ţintelor pentru medicamente. Cancerul de colon reprezintă o malignitate cu incidenţă şi mortalitate crescute în întreaga lume, etiologia sa implicând numeroşi factori genetici, imunologici şi biochimici. 5-fluorouracilul (5-FU) este unul dintre cei mai eficienţi agenţi anti-canceroşi utilizaţi în tratamentul cancerelor colorectale, dar chimiorezistenţa tumorală este un factor major de limitare a utilizării sale. În scopul alegerii celei mai eficiente doze chimioterapeutice de 5-FU, diminuând, astfel, efectele secundare, am încercat să modulăm proprietăţile anticancerigene ale 5-FU, prin adăugarea de compuşi naturali alimentari. Studiul s-a axat pe rolul compuşilor naturali, cum ar fi resveratrolul (RSV), în sensibilizarea liniei celulare de adenocarcinom de colon uman LoVo la acţiunea 5-FU. Analiza celulară în timp real (RTCA) prin sistemul xCELLigence a fost utilizată pentru a monitoriza continuu efectele citotoxice ale tratamentelor medicamentoase asupra celulelor LoVo. RTCA ne-a permis să alegem concentraţiile adecvate pentru alte teste “end-point”, cum ar fi tehnicile de citometrie în flux, folosite pentru evaluarea evenimentelor apoptotice, progresiei prin fazele ciclului celular sau expresiei antigenice nucleare în celulele LoVo tratate. Datele obţinute au arătat efecte adiţionale ale tratamentelor cu RSV asupra evenimentelor apoptotice, comparativ cu cele induse de 5-FU, şi au sugerat abordări alternative pentru a obţine un răspuns anti-tumoral puternic, precum şi diminuarea efectelor secundare atunci când sunt folosite concentraţii scăzute de medicamente anti-cancer. Modularea mecanismelor procesului de moarte celulară programată pare a fi de mare importanţă pentru transformarea malignă, şi, prin urmare, pentru abordările terapeutice anti-canceroase. Keywords: apoptosis, resveratrol, 5-fluorouracyl, cytotoxicity, xCelligence, flow-cytometry, colon cancer cells *Corresponding author: lorelei I. Braşoveanu, Ştefan s. Nicolau Institute of virology, Center of Immunology, 285 Mihai Bravu Ave, s3, 030304, Bucharest, Romania, phone/fax: +40-21-3241471, e-mail: [email protected] 255 HOTNOG et al. INTRODUCTION Comprehension of the cellular and molecular pathology of malignant transformation is incomplete. Cancer is a disease of the cells, so it is essential to identify the stages of development and use of basic information in the prediction, prevention, early detection and development of drugs targeted action [1]. Colorectal carcinoma is one of the most common human cancers in the world and is frequently diagnosed at late stages that require chemotherapy. Therefore, as a principle strategy in colon cancer treatment it has been established the multi-drug combination chemotherapy, the reason being the potential additive or synergistic tumor cytotoxicity produced [2]. Advances in the molecular explanations of growth dysregulation, metastasis formation, extension of life span, and loss of maintenance of genomic and epigenetic integrity in cancer suggest models for their causal connection. The mechanisms of growth control, senescence, and anchorage dependence are linked on the molecular level. The etiology of colon cancer involves a multitude of genetic, immunological and biochemical modifications [1]. Transformation of normal cells, tumor progression and advanced metastasis involve a complex series of events such as genetic alterations, aberrant progression of the cell cycle, inhibition of apoptosis, modification of cell adhesion and induction of angiogenesis [2, 3]. One of the main obstacles to a successful treatment of many malignant diseases, including colon cancer, is the development of resistance to chemotherapy, that is responsible for the failure in eradication of the entire tumor cell population [4-6]. Since various plants and their bioactive compounds have been shown to have anti-carcinogenic and anti-proliferative effects towards colon cancer cells [7, 8], many studies focused on the discovery of new chemotherapeutic agents among natural products. Studies have also reported positive correlation between antioxidant activities of plants and their anti-proliferative effects, suggesting the potential action of antioxidants in inhibiting cancer cell growth [9, 10]. Over 5,000 flavonoids that represent a class of plant secondary metabolites, known for their antioxidant properties, might be found in vegetables and fruits, grains, teas, herbs, wines, seeds, and nuts [11]. These dietary compounds could interfere with specific stages of the carcinogenic process, and can inhibit cell proliferation and induce apoptosis in different types of cancer cells [12]. In vivo studies on animal models revealed that flavonoids might exert positive preventive effects in carcinogenesis 256 and neurodegenerative disorders, essentially because of their antioxidant activity, the capacity to affect the expression of several detoxifying enzymes and their ability to modulate protein signaling cascades [10, 13]. Resveratrol (trans-3,4’,5-trihydroxystilbene), a naturally occurring polyphenol phytoalexin, is abundant in a wide variety of plant species, such as grapes, mulberries, peanuts, seeds, and red wine [14]. Resveratrol exhibited anticancer properties by inhibiting cell proliferation, inducing apoptosis, decreasing angiogenesis, and causing cell cycle arrest in several cancer cell lines [15-17]. Apoptosis, also known as “programmed cell death”, is the most potent defense against cancer, and represents a cellular mechanism that allows the control of cell number from tissues and elimination of cells that present DNA mutations or have an aberrant cell cycle, predisposed to malignant transformation [18]. Apoptosis, the cellular intrinsic death program, plays a crucial role in the regulation of tissue homeostasis; an imbalance between cell death and proliferation may result in tumor formation [19,20]. Also, killing tumor cells by various cytotoxic approaches such as anticancer drugs, -irradiation, suicide genes or immunotherapy, is predominantly mediated through induction of apoptosis in tumor cells [9, 20]. Some anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction [20, 21]. Many cancer therapeutic approaches involve restoration of cellular mechanisms responsible for elimination of premalignant/malignant cells, inducing cell death (apoptosis) in tumor cells [19, 20]. Several studies showed that 5-FU inhibits DNA proliferation in colon cancer cells by inhibiting the enzyme thymidylate synthase, leading to apoptosis, a mechanism of active cell death characterized by rapid loss of plasma membrane integrity, DNA fragmentation, and altered expression of numerous genes [22-26]. By combining flavonoids with anti-cancer drugs, it might be obtained an increase of the effects, specifically in highly invasive cancer cells, while in nontumoral cells the cytotoxic side effects could be reduced [7, 27]. During the last years it was developed a non-invasive way to continuously monitor the cellular behavior that utilizes the inherent morphological and adhesive characteristics of the cell. Real-Time Cell Analysis (RTCA) by xCELLigence System (ACEA Biosciences) consists of a cell-based label-free platform technology with good sensitivity and reproducibility in monitoring an entire cell population. Therefore, it is possible to distinguish between different perturbations of cell viability, such as Resveratrol modulates apoptosis in 5-fluorouracyl treated colon cancer cell lines senescence, cell toxicity (cell death), and reduced proliferation (cell cycle arrest) [28]. The present study focused on investigation of the possible mechanisms of interaction between RSV and 5-FU regarding induction of apoptosis and cell cycle progression of colon cancer cells. Moreover, the study aims to establish the possible modulation of several molecules associated to apoptosis in RSV and/or 5-FU treated colon cancer cells. MATERIALS AND METHODS Reagents: Resveratrol (3,5,4’-trihydroxystilbene) and 5-fluorouracyl were purchased from Sigma Aldrich, St. Louis, Mo, USA. The stock solutions were prepared in DMSO and preserved at –20 oC. Working drug concentrations were prepared from the stocks in culture medium before each experiment. Ribonuclease A (RNase A) from bovine pancreas, propidium iodide (PI), ethylenediaminetetraacetic acid (EDTA), dimethyl sulfoxide (DMSO) were purchased from Sigma. Annexin V-FITC Apoptosis Detection kit was purchased from Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA (BD). The mouse anti-human 6A7 anti-Bax, 4D7 anti-Bcl-2, 2H12 anti-Bcl-x, SMP14 anti-Mdm2 or DO-7 anti-P53 monoclonal antibodies (mAb) were provided from BD Biosciences. FITClabelled Fc specific secondary rabbit anti-mouse polyclonal antibodies were purchased from Sigma. Cell Cultures and Treatments: LoVo cancer adenocarcinoma cell line was purchased from American Type Culture Collection (ATCC) and routinely maintained in culture in RPMI-1640 medium added with 2mM L-glutamine and 10% fetal calf serum (Sigma Aldrich, St. Louis, Mo, USA) and incubated at 37oC/5% CO2 humidified atmosphere. After 24h cells were treated with different concentrations of (RSV) and/or 5-FU for various periods of time. Then cells were detached with a nonenzymatic solution of PBS/1mM EDTA, washed twice in PBS and immediately used for the evaluation of apoptosis events or fixed in ice-cold ethanol/PBS (70:30) and kept until use at 4oC for cell cycle analysis. Real-Time Cell Analysis (RTCA): The xCELLigence System allows label-free and real-time monitoring of cellular processes, such as cell proliferation, cytotoxicity, adhesion, viability, invasion, and migration, using electronic cell sensor array technology. RTCA-DP instrument used is composed of three parts: RTCA DP Analyzer for concomitant or independent operation of three E-Plates, RTCA Control Unit and E-Plates with 16 wells. Cellular events are monitored in real time without the incor- poration of labels by measuring the electrical impedance across inter-digitated micro-electrodes integrated on the bottom of special tissue culture plates (E-Plates (ACEA Biosciences)) [28]. The electrode impedance, which is displayed as cell index (CI) values, was used to provide quantitative information about the biological status of the cells, including cell number, viability, and morphology. Changes in a cell status, such as cell morphology, cell adhesion, or cell viability led to a change in CI, which is a quantitative measure of cell number present in a well. Briefly, LoVo colon cancer cells, cultivated in RPMI-1640 medium added by 2mM L-glutamine and 10% FCS, were seeded for 24 h in E-Plates 16 cell and growth curves started to be automatically recorded on the xCELLigence System in real time. Then, scalar concentrations of drugs or dietary natural compounds were added and live cells monitored. Cell Cycle Analysis: Ethanol fixed LoVo cells were washed twice in PBS and cell pellet was resuspended in 350 l of PBS, and added with 50 l of 10 mg/ml Rnase A (1 mg/ml final concentration). After 10 min incubation at 37°C, 100 l of 100 g/ml PI solution were added (20 g/ml final concentration) and further incubated at 37°C for 10 min. The probes were kept in the dark at 4°C until data acquisition by flow-cytometry using a FACScan cytometer (Becton Dickinson (BD) Immunocytometry System, Mountain View, CA). Then cell cycle analyses were performed using ModFIT LT and CellFIT softwares to estimate the nuclear DNA content in 3x104 nuclei and study cell-cycle phases. Apoptosis Analysis: The apoptosis assay was carried out using the Annexin V-FITC kit and the manufacturer’s protocol from BD Pharmingen. The percentages of apoptotic cells were determined by double staining with Anexin V-FITC/ PI, followed by sample analysis by flow-cytometry using WinMDI 2.9 software. Analysis of Nuclear Antigen Expression: Intracellular antigen expression was estimated in LoVo cells cultured and compound-treated as described above. Then cells were treated for 1h/4oC in 2% PBS-PFA buffer for fixing the surface molecules, and permeabilized by additional incubation for 15 min/37oC in PBS-Tween-20 (0.2%) buffer. After two washings with PBS, 105 cells were sequentially stained by 0.5 g of specific mouse anti-human monoclonal antibodies, and 1:50 dilutions of FITClabelled pAb. Cell surface fluorescence data were acquired using a FACScan flow-cytometer (BD), while data analyses were performed using WinMDI software. 257 HOTNOG et al. RESULTS 1. Evaluation of Compound-Mediated Cytotoxicity in LoVo Cells The xCELLigence System of Real-Time Cell Analyzers (RTCA) allowed cell-based in vitro assays for the assessment of cell viability and cytotoxicity and are disruptive endpoint assays [28]. LoVo cancer cells were seeded at a density of 1.5x104 / well in EPlates for a dynamic monitoring of adherent cell proliferation, and cell growth curves were automatically recorded on the xCELLigence System in real time. In order to screen for the proper concentrations of natural compounds with cytotoxic or cytostatic potential, and continuously monitor the cell growth, proliferation and viability of LoVo cells, an RTCA assay was performed. The compound-mediated cytotoxicity by RTCA was performed for treated cells by 3.13 to 200 M of RSV or 5-FU. Results were expressed as normalized cell index (CI) after automatic comparison between the curves of viability for treated and non-treated cells (Fig. 1). Acquisition of real-time monitoring of cytotoxicity allowed for calculation of time-dependent IC50 values. Regarding timing of calculating IC50, internally, it was selected the time point where the highest concentration reached maximum response and before the controls started to crash. Therefore, we monitored compound-induced cytotoxicity to produce continuous compound- and concentrationdependent cell impedance profiles using xCELLigence System and LoVo cancer cell line as our in vitro models. We screened for the proper concentrations of compounds with cytotoxic or cytostatic potential (e.g. drugs as 5-FU, or natural compounds, as RSV) in killing adherent tumor cells, in order to be used in further end-point assays, such as apoptosis or cell cycle analyses. The IC50 values obtained by the xCELLigence System were comparable to the IC50 value obtained by colorimetric cytotoxicity assays as MTT or XTT (data not shown). 2. Analysis of the Effect of RSV and/or 5-FU-treatments on Apoptosis of LoVo Cells During the last years, apoptosis started to be considered as an ideal way to eliminate precancerous and/or cancer cells. Thus, chemopreventive agents that can modulate apoptosis may be able to affect the steady-state cell population, which may be useful in the management and therapy of cancer [7]. After automatic data acquisition from the homogenous assays of compound-mediated cytotoxicity, we determined the compound-specific profiling, the optimal point of compound treatment and calcu- RSV/ LoVo 5-FU CTRL ---- ; 200 PM ---- ; 100 PM ---- ; 50 PM ---- ; CTRL ---- ; 200 PM ----; 100 PM ---- ; 50 PM ---- ; 25 PM ---- ; 12,5 PM ---- ; 6,25 PM ---- ; 3.13 PM ----; 25 PM ----; 12,5 PM ----; 6,25 PM ---- ; 3.13 PM ---- ; 24 h ------ ; 48 h ------ ; 96 h ------ ; 12 h ------ ; 24 h ------ ; 36 h ------ ; 48 h ------; 72 h ------ Fig. 1. RTCA analysis of proliferation vs. cytotoxicity of RSV or 5-FU treated LoVo cells, using xCELLigence System 258 Resveratrol modulates apoptosis in 5-fluorouracyl treated colon cancer cell lines NT / 24h RSV 25 PM / 24h RSV 100 PM / 24h 5-FU 25 PM / 24 h RSV 25 PM / 5-FU 25 PM RSV 100 PM / 5-FU 25 PM Fig. 2. Effects of 24h treatments with RSV and/or 5-FU on induction of apoptosis in LoVo colon cancer cells, evaluated by Annexin-FITC/PI double staining followed by flow-cytometry acquisition and analysis lated the real-time IC50 values. In order to evaluate in an end-point assay the apoptotic events induced by 5-FU drug treatment, alone or in combination with RSV natural compound, LoVo cells were cultured for 24h. Then culture medium was changed, and cells were sensitized by 6h with fixed concentrations of 25, 50 or 100 M RSV, and then added by 25 M 5-FU. After 24-72h of treatment LoVo cells were detached, washed twice with PBS and apoptotic cells detected by using Annexin V-FITC/ PI double staining, followed by flow-cytometry analysis. Apoptotic events were expressed as percentage of positive cells. In each quadrant of Figs. 2 and 3 are represented the live (left down), early apoptotic (right down), late apoptotic (right up), and necrotic (left up) LoVo cells. Total apoptosis was calculated by summing early and late apoptotic events. Fig. 2 shows a significant experiment of modulated apoptosis by 24h treatments with RSV and/or 5-FU. Use of 24h treatment with 25 M RSV induced higher levels of early and late apoptosis (17%, and 18.34%, respectively) compared to control cells (2.69%, and 3.09% respectively) or 5-FU (12.11%, and 5.59% respectively) (Fig. 2, Table 1). The 25 M RSV/ 25 M 5-FU combined treatment induced a slight enhancement of the levels of early apoptosis, to 14.36% (Fig. 2, Table 1). Table 1. Modulation of apoptosis induced by 24h treatments with RSV and/ or 5-FU Treatment NT/24 h 5-FU 25 PM RSV 25 P0 RSV 100 PM RSV 25 PM/ 5-FU 25 PM RSV 100 PM/ 5-FU 25 PM Early Apoptosis (%) 2.69 12.11 17.05 14.92 14.36 12.58 Late Apoptosis (%) 3.09 5.59 18.34 8.51 4.24 5.72 Total Apoptosis (%) 5.78 17.7 35.39 23.43 18.6 18.3 259 HOTNOG et al. NT / 72h RSV 25 PM / 72h RSV 100 PM / 72h 5-FU 25 PM / 72 h RSV 25 PM / 5-FU 25 PM RSV 100 PM / 5-FU 25 PM Fig. 3. Effects of 72h treatments with RSV and/or 5-FU on induction of apoptosis in LoVo colon cancer cells, evaluated by Annexin-FITC/PI double staining followed by flow-cytometry acquisition and analysis When treatments were prolonged to 72h, stronger effects were observed both for single and combined treatments (Fig. 3). Treatments with 25 M and 100 M RSV induced 16.6%, and 24.45%, respectively, of early apoptotic events, comparing to nontreated cells (3.59%) (Fig. 3, Table 2). Also the total apoptosis increased from 9.74% for control cells to 35.21% and 49.33% for 25 and 100 M RSV concentrations. Combined treatments induced higher levels of early apoptosis, till 29.57% when 25 M RSV/ 25 M 5-FU combination was used, but determined also high levels of late apoptosis (19.08%) when 100 M RSV/ 25 M 5-FU combination was added (Fig. 3, Table 2). 3. Analysis of the Effect of RSV and/or 5-FUtreatments on Cell Cycle Phases in LoVo Cells Anticancer agents may alter regulation of the cell cycle machinery, resulting in an arrest of cells in different phases of the cell cycle and thereby reducing the growth and proliferation of cancerous cells [25]. In order to study the anti-carcinogenic effects of phytochemicals that may have inhibitory and/or chemopreventive potential, resveratrol was used in combination with cytotoxic drugs (5-FU). The influence of treatments upon the end-point assay of LoVo cell progression through cell cycle phases was studied, and cells were cultured and treated as in the experiment described above. Table 2. Modulation of apoptosis induced by 72h treatments with RSV and/or 5-FU Treatment NT/72 h 5-FU 25 PM RSV 25 PM RSV 100 PM RSV 25 PM/5-FU 25 PM RSV 100 PM/5-FU 25 PM 260 Early Apoptosis (%) 3.59 36.12 16.6 24.45 29.57 15.76 Late Apoptosis (%) 6.15 8.27 18.61 24.88 7.28 19.08 Total Apoptosis (%) 9.74 44.39 35.21 49.33 36.85 34.84 Resveratrol modulates apoptosis in 5-fluorouracyl treated colon cancer cell lines NT RSV 25 PM RSV 50 PM 5-FU 25 PM RSV 25 PM / 5-FU 25 PM RSV0 PM / 5-FU 25 PM Fig. 4. Flow cytometry analysis of the additional effects induced by RSV on proliferation through cell cycle phases of 5-FU treated LoVo colon tumor cells Increased concentrations of 5-FU induced a dose-dependent decrease in percentages of nuclei found in S cell cycle phase, from 29.7% for nontreated cells to 10.7% when 25 uM 5-FU was used (Fig. 4, Table 3). Additional treatments also induced a significant decrease of S phase: 20.1% when 10 M 5-FU was used or 8.1% when 50 M 5-FU was added (data not shown). Resveratrol also had a strong effect on diminishing percentages of nuclei in S phase of LoVo cells: 27.5% for 25 M, 13.2% for 50 M (Fig. 4, Table 3) or 9.5% for 100 M RSV. Combined treatments induced stronger effects than single ones: 25 M of 5-FU added by 25, 50 or 100 M RSV decreased S phase till 6% of nuclei. Single RSV treatments induced a G2M cell arrest, while combined treatments seem to block cells mainly in G0/G1 phase (Fig. 4, Table 3). The results suggested that cell cycle arrest in G0/G1 phase might represent an important cause for the anti-proliferative effect of RSV. The natural compound RSV, used at the concentrations tested in the present study, seemed to have a similar effect with 5-FU on the inhibition of tumor cells LoVo. Since the combined treatments of RSV with 5-FU induced a certain decrease of the S phase compared to the single treatments, they might be an alternative to improve the traditional therapeutical approaches, and revert the chemoresistance of colon cells to 5-FU. Table 3. Modulation of progression through cell cycle phases by RSV and/or 5-FU treatments of LoVo colon cancer cell line Treatment NT 72 h 5-FU 25 PM RSV 25 PM RSV 50 PM RSV 25 PM/5-FU 25 PM RSV 50 PM/5-FU 25 PM Go/G1 (%) 63.3 77.2 25 66 81.3 73 S (%) 29.7 10.7 27.5 13.2 6.1 6 G2M (%) 7 12.1 47.5 20.8 12.6 21 261 HOTNOG et al. NT 5-FU 25 M RSV 25 M RSV 50 M RSV 25 M 5-FU 25 M RSV 50 M 5-FU 25 M Fig. 5. Flow cytometry analysis of nuclear antigen expression associated to apoptosis in RSV and/or 5-FU treated LoVo cells 4. Modulation of nuclear antigen expression in LoVo cells by RSV and/or 5-FU The above results showed that resveratrol inhibited LoVo cell proliferation and effectively induced cell cycle arrest and apoptosis in a dose- and time-dependent manner. Therefore we further examined the potential role of RSV in modulation of nuclear antigens associated to apoptotic process in LoVo cells. The flow-cytometry analyses showed a differential expression of constitutive nuclear antigens (Fig. 5). Treatments with 25 M 5-FU or 50 M RSV slightly increased the expression of the proapoptotic molecules P53 and Bax expression. The combined treatments induced a stronger effect, 67.4% of the cells being P53 positive when cells were treated with 50 M RSV and 25 M 5-FU, compared to the nontreated cells (50.2%) (Fig. 5).The increase of Bax expression was much higher, from 55.2% in nontreated cells and 59.6% in 5-FU treated cells to 68.7%, and 75.5%, respectively for the combined treatments. Both RSV and 5-FU treatments seemed to decrease Bcl-2 expression, but the effect was stronger for the combined treatments (Fig. 5). The constitutive expression of Mdm-2 and Bcl-x was low in LoVo cells, therefore the effect of RSV and/or 5-FU treatments was not clearly observed. 262 DISCUSSION Contrast data are available on the anti-cancer effects of natural compounds in colon cancer. Flavonoids have drawn great attention in chemoprevention and for their potential clinical application when used in combination with anti-cancer drugs. The current interest in the potential effects of antioxidants from natural compounds in colon cancer treatment prompted us to investigate the cytotoxic vs proliferative capacity of resveratrol to modulate the chemo-sensitivity of 5-FU treated LoVo colon cancer cells, and reduce the undesirable side-effects. Real-time monitoring of cellular processes by the xCELLigence Systems offered distinct and important advantages over traditional end-point assays. A comprehensive representation of entire length of the assay was possible allowing us to make informed decisions regarding the timing of certain manipulations or RSV or 5-FU treatments. Real-time impedance data obtained by the xCELLigence System were used to generate compound-specific profiles both for RSV and 5-FU which were dependent on the biological mechanism of action of the compound used. The actual kinetic response of the cells within an assay prior or subsequent to certain manipulations provided important information regarding the bio- Resveratrol modulates apoptosis in 5-fluorouracyl treated colon cancer cell lines logical status of the cell such as cell growth, arrest, morphological changes and apoptosis. Cell proliferation and differentiation are mutually exclusive, with the former typically preceding the latter. Proliferation is incompatible with the expression of a genetic program of terminal differentiation. Therefore, irreversible arrest of cell division is a prerequisite for the expression of a terminally differentiated phenotype, and so differentiation protects from carcinogenesis. Perturbations in cell cycle progression may account for the anti-carcinogenic effects of flavonoids. Stimuli treatment of LoVo colon cancer cells differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of DNA changed. Nuclear antigen expression was differentially modulated by RSV as compared to 5-FU, but they seem to have similar effects. Combined treatments induced a higher increase of pro-apoptotic antigen expression, both for P53 and Bax, compared to single 5-FU treatment. The effect of modulatory agents on proliferation and apoptosis might be used in clinical departments in order to elaborate new therapeutic approaches and act as useful instruments in elaboration of individualized treatment schemes. 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ROMANIAN ARChIves OF MICROBIOlOgy AND IMMUNOlOgy sUBJeCT INDeX MICROBIOLOGY UNDERSTANDING THE MOLECULAR TARGETS FOR NEW THERAPEUTICAL AGENTS IN HEPATITIS C INFECTION Codruţa Vagu, Camelia Sultana, Simona Ruţă THE EFFECTIVENESS OF CYTOLOGICAL RESCREENING IN THE REDUCTION OF FALSE NEGATIVE / POSITIVE PAP REPORTS Elena Cernescu, Gabriela Anton, Simona Ruţă, Costin Cernescu THIN LAYER CHROMATOGRAPHY CHARACTERIZATION OF ELISA SPECIFIC GLYCOLIPIDS ANTIGENS IMMUNOMAGNETICALLY PURIFIED FROM ENVIRONMENTAL MYCOBACTERIA Irina Ulea and Henriette Stavri 5 93 101 HIV-1 CIRCULATING SUBTYPES IN ROMANIA Loredana Sabina Cornelia Manolescu, Aura Temereanca, Simona Ruţă 121 PHENOTYPIC PROFILES OF VIRULENCE IN DIFFERENT CANDIDA SPECIES ISOLATED FROM VULVOVAGINAL INFECTIONS Ionela Sârbu, Diana Pelinescu, Ileana Stoica, Luminiţa Măruţescu, Tatiana Vassu 225 SINGLE AND MULTIPATHOGEN VIRAL INFECTIONS IN HOSPITALIZED CHILDREN WITH ACUTE RESPIRATORY INFECTIONS Cristina Ţecu, Maria E. Mihai, Viorel I. Alexandrescu, Dumitru Orăşeanu, Carmen Zapucioiu, Alina E. Ivanciuc, Gheorghe Necula, Emilia Lupulescu, Daniel Chiriţă and Daniela Piţigoi 242 PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS STRAINS ISOLATED FROM A FAMILIAL FOODBORNE OUTBREAK Ileana Luminiţa Coldea, Elena Carmina Drăgulescu, Brânduşa Elena Lixandru, 210 Cristiana Cerasella Dragomirescu, Irina Codiţă SELECTION OF BACTERIAL STRAINS EFFICIENT IN DECOLORIZATION OR REMAZOL BLACK-B 234 Maulin P Shah, Soniya Sebastian, Hemangi M. Mathukiya, A.M. Darji, Jigna Patel, Kavita Patel SEROPREVALENCE OF ANTIBODIES TO DIPHTHERIA, TETANUS AND PERTUSSIS AMONG HEALTHY ADOLESCENTS AND ADULTS IN IRAN Babak Pourakbari, Behnaz Moradi, Farin Mirzaee, Shima Mahmoudi, Mostafa Teymuri, Setareh Mamishi 250 PARASITOLOGY GROWTH OF ENTAMOEBA INVADENS IN SEDIMENTS WITH METABOLICALLY REPRESSED BACTERIA LEADS TO MULTICELLULARITY AND REDEFINITION OF THE AMOEBIC CELL SYSTEM 25 Vladimir F. Niculescu TWO CASES REPORTS ON VISCERAL LEISHMANIASIS DIAGNOSED IN ROMANIA Marian Ghervan Gogoaşe, Irina Teodorescu, Carmen Preda, Simona Claudia Ionescu 49 265 ROMANIAN ARChIves OF MICROBIOlOgy AND IMMUNOlOgy IMMUNOLOGY RESVERATROL MODULATES APOPTOSIS IN 5-FLUOROURACYL TREATED COLON CANCER CELL LINES Dan Hotnog, Mirela Mihăilă, Iulia Virginia Iancu, Georgiana Gabriela Matei, Camelia Hotnog, Gabriela Anton, Marinela Bostan, Lorelei I. Braşoveanu 255 REVIEW ADENYLATE CYCLASES INVOLVEMENT IN PATHOGENICITY, A MINIREVIEW Adriana Costache, Nadia Bucurenci, Adrian Onu CLINICAL AND DIAGNOSIS CONSIDERATIONS OF LYME DISEASE Doris Ionescu, Ani Ioana Cotar, Daniela Bădescu, Silvia Dumitriu 63 135 SCIENTIFIC SESSION OF CANTACUZINO NATIONAL INSTITUTE OF RESEARCH-DEVELOPMENT FOR MICROBIOLOGY AND IMMUNOLOGY „VACCINE-PREVENTABLE DISEASES” Bucharest, November, 21-22, 2013 Event organized by CANTACUZINO NIRDMI in partnership with the Romanian National Commission for Unesco for the anniversary of 150 years since the birth of Professor Dr. ION CANTACUZINO (1863-1934) Abstracts Author Index 266 175 218 ROMANIAN ARChIves OF MICROBIOlOgy AND IMMUNOlOgy AUThOR INDeX scientific session of Cantacuzino National Institute of Research-Development for Microbiology and Immunology "vaccine-Preventable Diseases" Bucharest, November, 21-22, 2013 Abazaj E. Alexandrescu Viorel Badell Edgar Bajo R. Bahnarel I. Balabanski L. Bauer Michael Băicuş Anda Bănică Leontina Beba D. Berbecilă Laurenţiu Bichurina Maina Bino S. Birca I. Birca L. Bleotu Coralia Borcan Alina Maria Botea Smaranda Botuş Daniela Bucur Jenica Bucurenci Nadia Burghelea Victoriţa Bylykbashi E. A B C Caplan Dana Magdalena Caplan Marius Eduard Cavaillon Jean-Marc Chifiriuc Mariana Carmen Chiriţoiu Gabriela N. Chiriţoiu Marioara B. Chukuranovic R. Ciucă Viviana Ciulean Sonya Codiţă Irina Cojocaru Radu Coldea Ileana Luminiţa Coman Cristin Condei Maria Costache Adriana Z. 187 199 179 187 183 190 183 194 197 187 193 180 187 181 181 206 201 201 189 189, 208 178 195 187 201 201, 208 183 197, 204,205, 206, 207 209 209 190 195 197 196, 210 181, 183 210 193 186, 188 178 Cremer Lidia Cristea Maria Culcescu Marian Czobor Ilda 203 203 189 204 D Damian Maria 179 Daneş Mihai 189 Davlieva K. 190 Delcaru Cristina 205 Delpeyroux Francis 180 Denys A. 202 Dimitrov Jordan 183 Dimov T. V. 207 Dimova I. 190 Dinu Cristina 201 Dinu Sorin 179, 186, 199, 200 Djonov V. 190 Djordjevic V. 190 Djoumerska-Alexieva Iglika 183 Donos A. 181 Dorobăţ Olga 201 Dragomirescu Cristiana Cerasella 77, 179, 198, 210 Drăgulescu Elena Carmina 210 Duca Elena 201 Duma Vlad Mihai 189, 208 Eder V. Escobar-Ramirez A. Florian Paula E F G Galabov Angel Gheorghe Adelina Silvana Gheorghe Irina Gheorghiţa S. Ghiţă Maria 183 202 202 190, 191 193 204 181 201 267 ROMANIAN ARChIves OF MICROBIOlOgy AND IMMUNOlOgy Gorzo Gabriela Gostev I. Grama O. Grumezescu Alexandru Mihai Guiso Nicole Hadzhieva Maya Herold Aurora Hoedt E. Huvent I. Iancu Adina Daniela Iancu Alina Viorica Ilie Anamaria Felicia Ionescu Gabriel Israil Anca Michaela Isufi Sh. Ivanov S. Joffret Marie-Line Juravliov T. Kaveri Srini Kota M. Kuro M. Kusta G. H I J K 205 183 183 206 177, 179 183 203 202 202 197, 203 197 177, 198 189, 203 205 187 190 180 181 183 187 187 187 L Lazăr Mihaela 200 Lazăr Veronica 177, 197, 204, 205, 206 Lixandru Brânduşa Elena 210 Lorin Daniela 208 Lungu V. 181 Lupu Andreea Roxana 203 Lupulescu Emilia 193, 199 Lindig Sandro 183 M Malinov M. Marinaş Ioana-Cristina Măruţescu L. Mateescu Lorena Andreea Melnic A. 268 190 206 207 201, 207 181 Mihăescu Grigore Mihai Alexandra Mihai Maria Elena Militaru Georgeta Cristina Militaru Manuella Mitache M. M. Mitre A. Munteanu Cristian V. A. 200, 204 193 199 177 197 207 187 209 N Năşcuţiu Alexandra-Maria Necula George Necula Gheorghe Neguț Marian Nica Maria Nicolae Alexandru Nicolova Ivanka Nistor Irina Niţă Andreia Niţă Daniela Njouom Richard 186,194 199 200 175 201 189 191 201 193 195 180 Onu Adrian Oprea Eliza Oprea Mihaela Oprişan Gabriela Pană Marina Papagheorghe Raluca Pashov Anastas Pastramă Florin Petrescu Andrei-José Petrescu Ştefana-Maria Petroff Miliana Petrusevska G. Pierce A. Pistol Adriana Piţigoi Daniela Plasevska D. Polenakovic M. Popa Gabriela Loredana Popa Mihnea Gabriel Popa Mircea Ioan Popa Mirela Popa Virgilia Popârlan Niculai Popescu Nicoleta Popov Z. Popovici Odette O P 178, 193 206 188 186 185, 201 188, 201 183 209 209 209 188, 189 190 202 193, 201 199 190 190 193 193 193, 198 188, 189 189, 208 189, 208 201 190 177 ROMANIAN ARChIves OF MICROBIOlOgy AND IMMUNOlOgy R Radu Beatrice Mihaela Radu Mihai Rafila Alexandru Râpă Maria Raşid O. Razafindratsimandresy Richter Rista M. Romanenkova Natalia Roşeanu A. Roumenina Lubka Rousset Dominique Rozaeva Nadezhda Sadeuh-Mba Serge Sălăgeanu Aurora Samoilovich E. Sârbu Anca Scofertsa R. Shundi L. Sohoţchi V. Solomon Teodora Sorescu Ionuţ Spînu C. Spînu I. Stănescu Aurora Staneva R. Stăvaru Crina Stefanovic V. Sterio A. Stoica Petruţa S 203 203 201 206 197 180 187 180 202 183 180 180 180 193 181 201 183 187 181 193 188, 189 183 183 193 190 193, 197, 203 190 187 206 Stoimenova T. Stoyanova Adelina Străuţ Monica Szegli Geza Ştirbu Constantin Tedeschini A. Teleman Monica Delia Toncheva D. Ţecu Cristina Ţucureanu Cătălin 190 191 186, 188 203 Ș T Ţ U Ungureanu Vasilica Ursu E. Usein Codruta-Romaniţa V Vagarova R. Vassilev Tchavdar Vassileva-Pencheva Ralitsa Velichkovic L. Vila B. Vlase Ene 189 187 186 190 199 193 177, 198, 201 181 188 190 183 191 190 187 193 269 ROMANIAN ARChIves OF MICROBIOlOgy AND IMMUNOlOgy AUThOR INDeX Alexandrescu Viorel I. Anton Gabriela Bădescu Daniela Bostan Marinela Braşoveanu Lorelei I. Bucurenci Nadia Cernescu Costin Cernescu Elena Chiriţă Daniel Codiţă Irina Coldea Ileana Luminiţa Costache Adriana Cotar Ani Ioana A B C D Darji A.M. Dragomirescu Cristiana Cerasella Drăgulescu Elena Carmina Dumitriu Silvia G Gogoaşe Ghervan Marian Hotnog Camelia Hotnog Dan Iancu Iulia Virginia Ionescu Doris Ionescu Simona Claudia Ivanciuc Alina E. H I L Lixandru Brânduşa Elena Lupulescu Emilia 270 242 93, 255 135 255 255 63 93 93 242 210 210 63 135 234 210 210 135 49 255 255 255 135 49 242 210 242 M Mahmoudi Shima Mamishi Setareh Manolescu Loredana Sabina Cornelia Matei Georgiana Gabriela Mathukiya Hemangi M. Măruţescu Luminiţa Mihai Maria E. Mihăilă Mirela Mirzaee Farin Moradi Behnaz Necula Gheorghe Niculescu Vladimir F. Onu Adrian Orăşeanu Dumitru Patel Jigna Patel Kavita Pelinescu Diana Piţigoi Daniela Pourakbari Babak Preda Carmen Ruţă Simona Sârbu Ionela Sebastian Soniya Shah Maulin P. Stavri Henriette Stoica Ileana Sultana Camelia Temereanca Aura Teodorescu Irina Teymuri Mostafa N O P R S T 250 250 121 255 234 225 242 255 250 250 242 25 63 242 234 234 225 242 250 49 5, 93, 121 225 234 234 101 225 5 121 49 250 ROMANIAN ARChIves OF MICROBIOlOgy AND IMMUNOlOgy Ţecu Cristina Ulea Irina Ţ U 242 101 Vagu Codruţa Vassu Tatiana Zapucioiu Carmen V Z 5 225 242 271 272