Vol.72, Issue 4, July-September 2013

Transcription

ROMANIAN ARCHIVES
OF
MICROBIOLOGY
AND
IMMUNOLOGY
Founded by
PROFESSOR ION CANTACUZINO
in 1928
VOLUME 72 - Issue 4
October - December 2013
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CANTACUZINO INSTITUTE BUCHAREST
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ROMANIAN ARCHIVES
OF
MICROBIOLOGY
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IMMUNOLOGY
ISSN 1222-3891
Editor-in-Chief: Maria DAMIAN
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Roxana MOLDOVAN, Geza MOLNAR; Marian NEGUŢ, Adrian ONU, Hervé PELLOUX,
Graţiela PÎRCĂLĂBIORU, Dorel Lucian RADU, Alexandru RAFILA, Lila SHUNDI,
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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
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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
vulvovaginale 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: diana_smarandache@yahoo.com
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
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
+
+
+
+
+
+
+
+
+
+
+
-
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. The expression
of these enzymes was lower or absent in C. krusei,
C. kefyr CMGBy 11 and C. parapsilopsis CMGBy
3 strains, but they presented the ability to form
biofilms on the inert substratum and to adhere to
HeLa epithelial cell line. C. krusei strains exhibited
the highest ability to form biofilm. The less virulent
strain was C. kefyr CMGBy11 which produced just
hemolysins and siderophore-like compounds.
Our results also indicate that even though all the
isolated strains were pathogenic, not all strains of
Candida sp. are able to produce all the virulence
markers involved in pathogenesis.
231
SÂRBU et al.
ACKNOwLEDGEMENTS
This work was supported by: CNCSIS Human
Resources
Project
66/2010
and
POSDRU/88/1.5/S/61150 “Doctoral Studies in the
field of life and earth sciences”, project co-financed
through Sectorial Operational Program for the Development of Human Resources 2007-2013 from
European Social Fund.
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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: shahmp@uniphos.com
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.
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
% 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
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. However, these isolates
should be tested at large scale treatment system to
examine their potential for bioremediation of dyepolluted wastewaters.
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241
sINgle AND MUlTIPAThOgeN vIRAl INFeCTIONs IN hOsPITAlIZeD
ChIlDReN WITh ACUTe ResPIRATORy INFeCTIONs
Cristina Þecu1*, Maria E. Mihai1, Viorel I. Alexandrescu1, Dumitru Orãşeanu2, Carmen Zapucioiu2,
Alina E. 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: tecucristina@yahoo.com
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.
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
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
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.
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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: smamishi@sina.tuns.ac.ir
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. However, before these decisions,
evaluation of the health benefits, risks, costs and cost
effectiveness of pertussis vaccination is necessary.
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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: luli_brasoveanu@yahoo.com
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.
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
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 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
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
NT
RSV 25 PM
RSV 50 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
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-
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. Nevertheless, any
concurrent supplementation of RSV during the
course of 5-FU-based chemotherapy should be carefully controlled to avoid limiting the efficacy of
treatment [22].
ACKNOwLEDGEMENTS
The study was funded from R&D Romanian
Project PNII 42-173/2008 of Ministry of Education
and Research.
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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
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
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
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
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
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
Ţecu Cristina
Ulea Irina
Ţ
U
242
101
Vagu Codruţa
Vassu Tatiana
Zapucioiu Carmen
V
Z
5
225
242
271
272

Vol.72, Issue 4, July-September 2013

Transcription

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