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ROMANIAN ARCHIVESOF

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ROMANIAN ARCHIVES OF MICROBIOLOGY AND IMMUNOLOGY

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193

K-RAS EXPRESSION - MARKER OF DYSPLASIA AND CANCER

Coralia Bleotu, Adriana Pleºa, Anca Botezatu, Cristina Goia, Roxana Drãguþel,

Demetra Socolov, F. Corniþescu, S. Teleman, Gabriela Anton

CURDLAN DERIVATIVES ABLE TO ENHANCE CYTOSTATIC DRUGS ACTIVITY ON TUMORAF CELLS

Maria-Mihaela Bãdulescu, Natalia Simona Apetrei, Andreea-Roxana Lupu, Lidia Cremer,

G. Szegli, M. Moscovici, Georgeta Mocanu, Doina Mihai, Ana Cãlugãru

THE INFLUENCE OF SOME PROBIOTIC SUPERNATANTS ON THE GROWTH AND VIRULENCE

FEATURES EXPRESSION OF SEVERAL SELECTED ENTEROAGGREGATIVE E. COLI CLINICAL STRAINS

Veronica Lazãr, Yoshibumi Miyazaki, Tomoko Hanawa, Lia-Mara Diþu,

Luminiþa Mãruþescu, Mariana-Carmen Chifiriuc, Coralia Bleotu and Shigeru Kamiya

IN VITRO STUDY OF THE INHIBITORY ACTIVITY OF USNIC ACID ON DENTAL PLAQUE BIOFILM

Mariana Carmen Chifiriuc, Lia Mara Diþu, Eliza Oprea, Sanda Liþescu,

Marcela Bucur, Luminiþa Mãruþescu, Gerard Enache, Crina Saviuc, Mihai Burlibaºa,

Teodor Trãistaru, Gabriela Tãnãse, Veronica Lazãr

IN VITRO SUSCEPTIBILITY OF ERWINIA AMYLOVORA (BURRILL) WINSLOW ET AL.

TO CITRUS MAXIMA ESSENTIAL OIL

Luminiþa Mãruþescu, Crina Saviuc, Eliza Oprea, Bogdan Savu, Marcela Bucur,

Gheorghe Stanciu, Mariana Carmen Chifiriuc, Veronica Lazãr

ANTIBIOTIC RESISTANCE OF GRAM NEGATIVE BACILLI STRAINS ISOLATED

FROM THE INTENSIVE CARE UNIT IN FUNDENI CLINICAL INSTITUTE, BUCHAREST, ROMANIA

Elvira Borcan, Camelia Mihaela Ghiþã, Mariana Carmen Chifiriuc,

Luminiþa Mãruþescu, Cãtãlina Isar, Veronica Lazãr

GROUP B STREPTOCOCCUS COLONIZATION OF ROMANIAN WOMEN:

PHENOTYPIC TRAITS OF ISOLATEDS FROM VAGINAL SWABS

Codruta R. Usein, Anca Petrini, Raluca Georgescu, Laura Grigore, Monica Strãuþ, Vasilica Ungureanu

INVESTIGATION OF HELICOBACTER PYLORI INFECTION IN JORDAN PATIENTS USING SIX ENZYME

IMMUNOASSAYS FOR IMMUNOGLOBULIN G (IGG) AND IGA TESTING

Hasan Abusini, Khaled Abuelteen, Ali Elkarmi, Faris Q. Alenzi, Mahmoud Lotfy

SUBJECT INDEX

AUTHOR INDEX

CONTENTS

IMMUNOLOGY

MICROBIOLOGY

VOLUME 68 NO. 4 OCTOBER - DECEMBER 2009

207

215

223

228

235

240

247

251

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Aims and ScopeRomanian Archives of Microbiology and Immunoloy , an internation-al journal dedicated to original research work, publishes papers focus-ing on various aspects of microbiology and immunology. Romanian

Archives of Microbiology and Immunology is indexed in MEDLINE.The frequency of the Journal is currently four issues per year.

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INTRODUCTION

Human carcinogenesis is a multistage process

that involves activation of proto-oncogenes and/or

inactivation of tumor suppressor genes [1]. It was

demonstrated that Ras oncogene has a dual role in

cell transformation and apoptosis. Ras is a cellular

GTPase that signals through MAPK, PI3K, and Ral-

GEF pathways [2]. Ras activation is involved in

human carcinogenesis through apoptosis inhibition

and cell cycle promotion.

Wild-type Ras gene possesses antioncogenic pro-perties; there are several genes, differently expressed

depending on the tissue. Normal H-ras1 gene expres-

sion suppresses transformed phenotype induced by

mutant H-ras [3], or mutant N-ras [4] present in the

tumor cell lines. The most frequent alterations of ras

gene are point mutations in codons 12, 13 and 61,

which constitutively activate ras [5]. Oncogenic ras

mutations were found in 30% of human cancers indi-

cating the implication of this gene in cell prolifera-

tion, as demonstrated also in cell cultures, when ras

oncogene induced the cells immortalization. In pri-

mary fibroblasts, Ras expression induces proliferation

and stops premature senescence [6]. Moreover, the

epithelial cells surface from human ovary, murine

keratinocytes and human oesophageal keratinocytes

react similarly.

It is believed that ras-mediated senescence is

determined by the activation of ARF-p53 pathways and

p16INK4a that target cell cycle arrest. Unlike mice

cells, human diploid fibroblasts disturbances require

both p53 and p16INK4a pathways to overcome

senescence [7]. On the other hand, the constitutiveactivation of ras signalling pathway is an important

component of malignant progression in various types

of cancers. It is clear that different modes of action are

determined by the functioning cell cycle regulators.

Molecular and epidemiological data indicated

that the presence of HPV virus is not sufficient to

induce transformation, suggesting that several cellu-

lar factors also play an important role [8]. The ras

gene is one of the most important genes involved in

cancer development. Ras gene activation in HPV

infections has been described in particular in cervical

ABSTRACT

Molecular and epidemiological data indicated that the presence of HPV virus is not sufficient to induce

transformation, suggesting the implication of other several cellular factors. Constitutive activation of the

Ras signaling pathway is an important component of malignant progression for a number of different can-

cers. In this context, the objectives of our study were: the quantitative assessment of the K- ras gene

expression changes in the development of the HPV positive cervical cancers. We observed that the K-ras

mRNA expression levels did not gradually increase with the severity of injury. The mRNA expression inthe ASCUS increased 2.02 times as compared with the control group, while in LSIL group only 1.76

times. However ras expression was increased in the HSIL / cancer group by 2.27 times when was repor-

ted to the control group. The presence of low risk HPV infection (lrHPV) does not lead to increased ras

expression, remaining at baseline, but K-ras expression was increased in the presence of high risk HPV

infection (hrHPV). In addition, we noted that in hrHPV single infections ras expression is increased

(0.96±0.48) comparing with hrHPV co-infections. Our findings indicate that high expression of ras

among hrHPV infection can be a marker of cervical cancer development.

K-RAS EXPRESSION -

MARKER OF DYSPLASIA AND CANCER

Coralia Bleotu1, Adriana Pleºa1, Anca Botezatu1, Cristina Goia1, Roxana Drãguþel1, Demetra Socolov2, F.Corniþescu3, S. Teleman2, Gabriela Anton1

1ªtefan S. Nicolau Institute of Virology, 285, Mihai Bravu Ave., 030304, Bucharest, Romania; 2UMF Iaºi; 3UMF Craiova

Key words: marker of transformations, HPV, K-ras

* Corresponding author: Coralia Bleotu, ªtefan S. Nicolau Institute of Virology, 285 Mihai Bravu Ave., 030304, email: [email protected]

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196

carcinomas, but the information on involvement in

premalignant lesions is limited. In this context, the

objective of our study was: the quantitative assess-

ment of the change in K-ras gene expression in the

development of the HPV positive cervical cancers.

MATERIALS AND METHODS

Patients: For K-ras expression, there were inves-

tigated 50 women aged from 17 to 55 years coming

back for regular gynaecological control, some of them

underlying suggestive pathology for HPV infection.

The study was conducted on the following groups:

NILM: Negative for Intraepithelial lesion. Includes

reactive changes, infection; ASCUS: Atypical Squa-

mous Cells of Undetermined Significance; LSIL: Low-

Grade Squamous Intraepithelial Lesions; HSIL: High-

Grade Squamous Intraepithelial Lesions. Groups mean

age was: NILM (31.9 ± 8.1) (range 19-46); ASCUS

(37 ± 9.2) (range 25-55); LSIL (35.8 ± 8.2) (range 24-

51); HSIL / cancer (41.5 ± 8.3) (range 30-42)]. Infor-

med consent was obtained from each participant.

The project received the approval of the Ethics Com-

mittee of Stefan S. Nicolau Institute of Virology. Cer-

vical specimens for molecular analysis were taken

during colposcopic examination using a combination

of plastic spatula and cyto-brush. Cervical specimens

were collected in Copan transport medium.

Cytology: Harvested vaginal secretions have

been spread and fixed on the blade by spraying withalcohol (not to dry before fixing). Subsequently, pre-

paration has been colored with Harris hematoxilin (3-

5 minutes) and orange-G (3-4 minutes). The diagnos-

tic elements were isolated cell morphology and any

significant changes in terms of diagnosis. The test

may reveal nonspecific inflammatory lesions caused

by various forms of vaginitis, or some significant issues

for vaginal infections. It was essential to identify those

cellular characteristic changes induced by human

papilloma virus. Results were reported according to

Bethesda classification.DNA extraction: Exfoliated cells obtained from

female patients were transported in Copan medium.

DNA extraction was done with Qiagen kit according

to the manufacturer’s procedures. For handling bio-

chemical processes in molecular diagnostics, DNA

was resolubilized in TE-1, pH 7.4-8.0. The amount of

DNA was determined both in spectrophotometry

(UV absorber in the micro-titer plate) and in fluori-

metry with Hoechst 33258, using the TECAN Genius

device.

HPV detection / genotyping: viral testing was

done with IINNOLIPA kit (Innogenetics), according

to manufacturer instructions. Briefly, a fragment of L1

HPV region was amplified in a PCR reaction using

biotinilated nucleotides; denatured amplicons were

hybridized with specific oligonucleotide probes that

are fixed on the nitrocellulose membrane. After hybri-

dization and stringent washing, the membranes were

incubated with streptavidin conjugated with alkalinephosphates and chromogen, the positive sample oc-

curring as a purple precipitate. Interpretation of the

results was done with the specific read card of the

INNOLIPA HPV Genotyping kit. A test is considered

positive when a specific line of type or a control line

is HPV positive. The kit allows the identification of

17 different HPV genotypes.

RNA extraction: A 0.5 ml aliquot of the sample

was mixed with 0.5 ml phosphate buffered saline

(PBS) and centrifuged at 60000 x g for five minutes.

The cell pellet was lysed with 1 ml Trizol reagent (In-

vitrogen). RNA extraction was carried out according

to the manufacturer’s instructions. The pellet was

vacuum dried for 30 seconds and the RNA dissolved

in ultra pure water by heating at 55°C for 10 minutes.

Any contaminating DNA was removed by incubating

with 1 U of DNAse I (Life Technologies) at room tem-

perature for 15 minutes. The enzyme was denatured

at 65°C for 10 minutes and reverse transcription was

performed. An aliquot of this sample was stored at -

20°C as a control to test for DNA contamination.

Reverse transcription: 2 mg RNA were mixed

with 1 mM dNTP and 1.5 pmoles of random primers,and incubated at 70°C for 10 minutes, allowed to cool

and the reverse transcription (RT) reaction carried out

using 200 units Superscript reverse transcriptase

(Promega), 1 mM DTT and 1 mM RNAse inhibitor and

incubation for 60 minutes at 37°C and 15 minutes at

70°C. The cDNAs were stored at -20 °C until use.

RNA PCR analysis: The efficiency of cDNA syn-

thesis from each sample was estimated by PCR using

GAPDH-specific primers. The expression of ras mRNA

was semi-quantitatively evaluated after RT-PCR am-

plification. Briefly, 2 µl aliquots of the reverse-transcribed cDNA were amplified for 33 cycles in

PCR in a final volume of 50 µl containing buffer (10

mM Tris-HCl (pH 8.3), 2.5 mM MgCl2, and 50 mM

KCl) containing 1 mM each of dATP, dCTP, dGTP,

and dTTP, 2.5 units of Taq DNA polymerase (Pro-

mega), and 0.2 µM primers (FW: 5’-ATGACT-

GAATATAAACTTGTGGTAG-3’ and R: 5’- TTA-

CATAATTACACACTTTGTCTTTGAC-3’). Each cycle

consisted of denaturation at 95°C for 40 s, annealing

at 60°C for 50 s and extension at 72°C for 50 s. The

PCR products were resolved by electrophoresis in

2% agarose gels and analysis of the bands intensity

was performed using ImageJ 1.33u.

BLEOTU et al.

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K-ras expression - marker of dysplasia and cancer

197

Statistical analysis: The Student t test was perfor-

med to compare differences between two groups for

quantitative variables. Double-sided P values of =0.05

were considered significant.

RESULTSWe observed that the K-ras mRNA expression

levels did not gradually increase with the severity of

injury (Table 1). The mRNA expression in the ASCUS

increased 2.02 times as compared with the control

group, while in LSIL group only 1.76 times (Figure 1).

However ras expression was increased in the

HSIL / cancer group by 2.27 times when was reported

to the control group. Even in these conditions, varia-

tion of ras expression in different study groups was

statistically significant as comparing with the control

group: ASCUS: NILM (p = 0.013); LSIL: NILM (p =0.045); HSIL: NILM (p = 0.003). At the reassessment

of smears, it was observed an increase in the number

of lymphocytes, probably as a result of inflammation,

this increase being correlated with the ras overex-

pression in 3 cervical specimens classified as having

inflammatory issue.

In our groups, we detected HPV in 84% of the

studied cases. The group with NILM cytology / infla-

mmatory aspect comprises: a negative HPV (5.9%), 4

cases with single infection of HPV low risk (23.5%),

3 cases HPV undetectable using INNOLIPA (17.65%)

and 9 cases in which there were involved at least one

of the high risk genotypes (52.9%). In ASCUS group

we detected only high risk genotypes in single infection

(HPV45 - one case and HPV53 - one case) and only 2

cases HPV- negative (20%). In 30.8% (3 / 13) LSIL

cases, HPV has not been detected, and 7.7% (1 / 13)cases, HPV could not be genotyped. 61.5% (8 / 13)

cases LSIL experienced hrHPV in single or co-infec-

tion. In LSIL, lrHPV in single infection was not detec-

ted, but associations with hrHPV were observed.

hrHPV was detected in 90% of HSIL cases, in single

infections (77.7%) and co-infection (22.3%). lrHPV

was not detected in HSIL.

Analyzing the expression of ras depending on

the presence or absence of HPV DNA (Figure 2) we

noted that:

- The presence of lrHPV infection does not lead toincreased ras expression, remaining at baseline

(0.375±0.215);

- Ras expression was increased in the presence of

hrHPV (0.992±0.16); in addition, we noted that in

hrHPV single infections ras expression is increased

(0.96±0.48) comparing with hrHPV co-infections

(0.842±0.48);

- HPV: X cases had low expressions, which indicates

that this group is not included hrHPV genotypes.

Ras expression in HPV16 single infection (n = 10)

was higher compared with co-infection cases

(n=13), 0.96±0.48 vs 0.82±0.41 (Figure 3). This

may be due to the fact that HPV16 single infections

group included in particular advanced forms of the

disease (HSIL/ CIS-7 cancer cases).

Fig. 1. Analysis of K-ras expression depending on the severity of the disease.

Table 1. K-ras expressions in 50 cervical lavage

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198

BLEOTU et al.

DISCUSSION

Evaluation of ras expression in cancer and pre-

cancer lesions of the cervix was performed in order

to analyse its clinical utility as a tumoral marker playing

a role in diagnosis (the differentiation between malig-

nant and benignant disease), and in the early stage

detection (evaluating the risk or the liability for dis-

ease in cases of healthy suspects or detecting the dis-

ease in preclinical stages). The results are in agree-

ment with other studies indicating an association

between ras overexpression or mutation and high

degree lesions. In addition, a series of studies have

stated increased ras levels as compared to the control

group, in late stage cancer lesions [9]. It is obvious

that the overregulation of the ras gene activity offers

carcinogenic properties. Comparing the normal tis-

sues with the low-risk dysplasia, the overexpression

of ras was observed in CIN3 and in invasive carcino-

mas, with a IHC (immunohistochemistry) positivity

ranging between 39-100% [10]. In the present study,

K-ras expression was evaluated in 50 cervical speci-

mens by RT-PCR. Our data show an overexpression

of ras gene in cervical cancers and suggest that high

levels reported in immunohistochemical examina-

tions could be due especially to high levels of ras

gene activation.

Few studies were dedicated to ras expression at

mRNA level in HPV infections. The studies performed

by Mammas et al., (2004) claim that H-ras and N-ras

expression significantly increase in cases of cancer,

as compared to the normal cervical tissues, but the K-

ras levels of expression remain similar to those of the

normal tissues [11]. The above - mentioned authors

have shown that the value of the ras mRNA/ b-actin

mRNA ratio are high in H-ras cancers (cancers - 5.17;

CIN - 0.430; control - 0.73) and N-ras (cancers - 3.53;

CIN - 0.87; control - 1.44), and low for K-ras (cancers

- 0.2; CIN - 0.14; control - 0.38). It is important to

note that their set of patients was smaller than ours.

On the other hand, in this particular study, HPV was

detected using a single set of primers GP5/6 without

genotyping in 4 (36%) normal subjects, 11 (73%)

CIN and 8 (89%) cervical cancers.

Our study proves that the HPV genotype is very

important for the evaluation of K-ras expression, HPV

being detected in 84% of the studied cases. The nor-

mal group included one HPV negative case (5.9%), 4

cases of exclusive low-risk HPV infection, 3 cases of

undetectable HPV through INNOLIPA (17.65%) and

9 cases which implicated at least one high risk geno-

type (52.9%). Among the ASCUS cytodiagnosis

group, we only detected high risk HPV genotypes in

exclusive infections (HPV 45 one case and HPV 53

one case) and 2 HPV negative cases (20%). In 30.8%

of the LSIL cases, HPV was not detected, and in 7.7%

(1/13) of cases, HPV could not be genotyped. 61.5%

Fig. 2. Analysis of K-ras expression (the expression K-ras /GAPDH)depending on the presence or absence of HPV genotypes oncogene.

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(8/13) of the LSIL cases showed high risk HPV in

unique infections or in co-infections. In LSIL, no cases

have been found to be exclusively low risk HPV

infections, but only associated to high risk HPV.

Among the HSIL group, the presence of high risk was

found in 90% of the cases in unique infections

(77.7%) and in co-infections (22.3%). Low risk HPV

has not been detected in HSIL. Unlike Mammals et

al., (2004) who detected HPV by help of a single

primers pair, which makes the HPV identification

imprecise, we found HPV by using the InnoLipa kit

that detects and genotypes 27 HPV types. In this way,

we were able to observe that the presence of lrHPV

does not lead to an increase in ras expression, whose

values remain at a basal level (0.375 ± 0.215), and

that ras expression is amplified in the presence of

hrHPV (0.992 ± 0.16), especially in unique hrHPV

infections (1.1841±1.2376) in comparison with cases

of co-infection (0.842±0.48).

In another study, it was proved that patients ha-

ving HPV16/18 associated cancers have high levels

of H-, K- and N-ras expression unlike pacients with-

out HPV, this idea confirming our results [9]. But

unlike us, they found that the levels of H-, K- and N-

ras expression were higher in case of patients with

multiple infections. For patients with SIL we have not

found a statistically significant relationship between

ras expression and the HPV status.

In our study, ras expression in single HPV geno-

type infection (HPV16) cases was higher in compari-

son to HPV co-infection cases, phenomenon corre-

lated by us with the composition of the groups (the

group of exclusive HPV16 infection especially inclu-

ded late stages of the disease) and with the preva-

lence of integrated and mixed forms, while HPV16

co-infections were especially detected in samples

from NILM patients or with cytodiagnosis showing

mostly episomal forms. So, the K-ras levels increase

in advanced cervical lesions. Moreover, it is obvious

that ras is able to stimulate malignant transformation

of human cells that contain integrated hrHPV forms.

The frequency of integration was higher in severe

lesions and in cancers, while in low degree lesions spe-

cial episomal forms were found. In HSIL, HPV16

integrated forms were detected in one case, while

mixed forms were found in 4/5 cases (44.4%) (data

not shown).

The presence of HPV is a necessary, but not suf-

ficient condition in order to induce carcinogenesis,

the regression of the lesion occuring in many cases.

In vitro studies have shown that ras gene activation

may produce tumorigenic conversion in HPV immor-

talized cervical keratocites, indicating a cooperant

effect in cell transformation between ras genes and

HPV E6/E7. Our study can not declare a strict corre-

lation between the presence of HPV and ras expres-

K-ras expression - marker of dysplasia and cancer

199

Fig. 3. 2D cartesian plot analysis of K-ras expression in the HPV infectionwith one genotype or co-infection with more genotypes.

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BLEOTU et al.

sion, indicating that overexpression of ras might be

an important event in cervical carcinogenesis, regardless

of HPV infection. Ostor et al., (1993) and Duggan et

al., (1998) demonstrated that only approx. 1% of the

CIN1 lesions and approx. 10% of the CIN3 lesions

lead to cancer [12, 13]. The exact mechanism throughwhich ras gene supression becomes involved in cer-

vical cancer is yet to be clarified.

CONCLUSION

Because the presence of HPV infection in low

risk patients does not lead to increased ras expres-

sion, remaining at basal level, but increases especially

in infections with single hrHPV K-ras expression we

consider that high expression of ras among hrHPV

infection can be a marker of cervical cancer develop-

ment.

BIBLIOGRAPHY

1. Lehman, T.A., Reddel, R., Pfeifer, A.M.A., Spillare, E.,

Kaighn, M.E., Weston, A., Gerwin, B.I., Harris, C.C.,1991, Oncogenes and tumor-suppressor genes,Envirom. Health Persp., 93, p.133-144.

2. Katz, ME; McCormick, F., 1997, Signal transductionfrom multiple Ras effectors. Curr Opin Genet Dev., 7,p.75-79.

3. Spandidos, D.A., Wilkie, N.M., 1988, The normalhuman H-ras1 gene can act as an onco-suppressor. Br. J.

Cancer , S9, p.67-71.

4. Spandidos, D.A., Frame, M., Wilkie, N.M., 1990,Expression of the normal H-ras1 gene can suppress thetransformed and tumorigenic phenotypes induced bymutant ras genes. Anticancer Res., 10, p.1543-1554.

5. Pronk G.J., Bos J.L., 1994, The role of p21ras in recep-tor tyrosine kinase signalling. Biochim Biophys Acta,1198, p.131-147.

6. Serrano, M., Lin, A.W., McCurrach, M.E., Beach, D.,

Lowe, S.W., 1997, Oncogenic ras provokes premature

cell senescence associated with accumulation of p53and p16INK4a. Cell, 88, p.593-602.

7. Mason, D.X., Jackson, T.J., Lin, A.W., 2004, Molecularsignature of oncogenic ras-induced senescence,Oncogene, 23, p.9238-9246.

8. Hanahan, D; Weinberg, RA., 2000, The hallmarks of cancer. Cell, 100, p.57-70.

9. Mammas, I.N., Zafiropoulos, A., Spandidos, D.A.,2005, Involvement of the ras genes in female genitaltract cancer, Int. J. Oncol., 26, p.1241-1255.

10. Busmanis, I., 1998, Biomarkers in carcinoma of cervix:emphasis on tissue-related factors and their potentialprognostic factors, Ann. Acad. Med. Singapore, 27,p.671-675.

11. Mammas, I.N., Zafiropoulos, A., Koumantakis, E.,

Sifakis, S., Spandidos, D.A., 2004, Transcriptional acti-vation of H- and N-ras oncogenes in human cervicalcancer. Gynecol. Oncol., 92, p.941-948.

12. Ostor, A.G., 1993, Natural history of cervical intrae-pithelial neoplasia: a critical review. Int. J. Gynecol.

Pathol., 12, p.186-192.

13. Duggan, M.A., McGregor, S.E., Stuart, G.C., Morris,

S., Chang-poon, V., Schepansky, A, Honore, l., 1998,The natural history of CIN I lesions. Eur. J. Gynaecol.

Oncol., 19, p.338-344.

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INTRODUCTION

Cancerous tumors are characterized by cell divi-

sion, which is no longer controlled as it is in normal

tissue. Normal cells stop dividing when they come

into contact with like cells, a mechanism known as

contact inhibition. Cancerous cells lose this ability

and they no longer have the normal checks and bal-

ances in place that control and limit cell division. The

process of cell division, whether normal or cancerous

cells, is through the cell cycle. The cell cycle goes from

the resting phase, through active growing phases, and

then to mitosis (division). The ability of chemothera-

py to kill cancer cells depends on its ability to halt

cell division [1].

Unfortunately, chemotherapy does not know the

difference between the cancerous cells and the nor-

mal cells. Chemotherapy will kill all cells that are ra-

pidly dividing. The normal cells most commonly

affected by chemotherapy are the blood cells, the

mouth cells, stomach and bowel, and the hair folli-

cles resulting in low blood counts, mouth sore, nau-

sea, diarrhea, and/or hair loss. Different drugs may

affect different parts of the body. Taking into account

the mode of action and the degree of toxicity of com-

pounds with cytostatic effects on tumor cells, we

selected three substances with cytostatic effects, com-monly used in chemotherapy [1].

Doxorubicin (also called Adriamycin, Rubex) is

an anthracycline antibiotic with cytostatic activity [2],

isolated from cultures of Streptomyces peucetius var.

caesius [3]. Actinomycin D (also called Dactinomy-

cin) is a polypeptide antibiotic isolated from soil bac-

teria of the genus Streptomyces. It was the first antibi-

otic shown to have anticancer activity [4], but it has a

highly toxic action and can cause damage to the ge-

netic material [5]. Cyclophosphamide (the generic

name for Endoxan, Cytoxan, Neosar, Procytox, Re-

* *Corresponding author: Ana Calugaru, NIRDMI „Cantacuzino”, 103 Splaiul Independentei, 050096, Bucharest, Romania,Tel.: +40(21)3069251, Fax: +40(21)3069298; E-mail: [email protected]

ABSTRACT

The chemotherapy success to kill cancer cells depends on its ability to stop cell division. The faster

the cells are dividing, the more likely it is that chemotherapy will kill the cells, causing the tumor

to shrink. Taking into account the severe side effects of chemotherapy, drugs producers also focus

on natural products obtained either from medicinal plants, or from microorganisms. The complex

polysaccharides named b-glucans are active compounds with immune activity. b-glucan polymers

belong to a class of drugs with effects on the immune system, such as: anti-tumoral, anti-infectious,protection against fungi, bacteria and viruses infections. The correct selection of b-glucans is essen-

tial to identify compounds with favorable clinical effects. The aim of this study was to investigate

the capacity of six Curdlan (b-glucan) derivatives to up-regulate the Doxorubicin, Actinomycin D

and Cyclophophamide cytostatic drug activity on tumor cells (murine B16 melanoma and human

HEp-2 laryngeal carcinoma cell lines). Our results demonstrated that Palm SP derivative, as well as

SP and Palm CM/SP derivatives were able to potentiate Doxorubicin action or Actinomycin D

effect on B16 tumor cells. SP derivative significantly enhanced cytostatic activity of Cyclophos-

phamide on B16 cells.

All the investigated Curdlan derivatives (SP, Palm CM/SP, CM/SP, Palm CM, Palm SP and CM) were

able to inhibit HEp-2 tumor cell growth, by up-regulating Doxorubicin and Actinomycin D cyto-

static activity.

CURDLAN DERIVATIVES ABLE TO ENHANCE CYTOSTATIC

DRUGS ACTIVITY ON TUMOR CELLS

Maria-Mihaela Bãdulescu1, Natalia Simona Apetrei1, Andreea-Roxana Lupu1, Lidia Cremer1,G. Szegli1, M. Moscovici2, Georgeta Mocanu3, Doina Mihai3, Ana Cãlugãru1*

1Cantacuzino National Institute for Research/Development in Microbiology and Immunology,Immunomodulation Laboratory, Bucharest, Romania; 2National Institute for Chemical Pharmaceutical

Research/Development, Bucharest, Romania; 3Petru Poni Macromolecular Chemistry Institute, Iaºi, Romania

Key words: cytostatic drugs, Curdlan derivatives, tumor cells

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202

vimmune), also known as cytophosphane, is a nitro-

gen mustard alkylating agent, used to treat various

types of cancer [6] and some autoimmune diseases,

being converted in the liver to active forms with

chemotherapeutic activities.

The polysaccharides named b-glucans havebeen implicated in the initiation of anti-microbial

immune response. b-glucans act on several immune

receptors including Dectin-1, complement receptor

(CR3) and TLR-2/6 and trigger a group of immune

cells including macrophages, neutrophils, monocytes,

natural killer cells and dendritic cells. As a conse-

quence, both innate and adaptive response can be

modulated by b-glucans and they can also enhance

opsonic and non-opsonic phagocytosis [7]. b-glucans

of different sizes and branching patterns may have

significantly variable immune potency. Careful selec-tion of appropriate b-glucans is essential if we wish to

investigate the effects of b-glucans clinically.

The b-(1,3)-glucan known as Curdlan is a linear

nonionic homopolymer of D-glucose with b-(1®3)

glucosidic linkages [8]; its synthesis was reported by

Harada in 1966 as a polysaccharide produced by the

microorganism Agrobacterium biovar through glu-

cose fermentation and has been commercially pro-

duced since 1989 year, in Japan. The literature has

shown that Curdlan sulfopropyl derivatives may have

anticoagulant [9] or anti-tumor [10] activity and car-

boxymethyl derivatives [11] presented anti-tumor

activity. Some studies noticed the immunostimulating

activity [12], [13] and anti-tumorigenic effects [14],

[15] of Curdlan derivatives [16].

In this paper we studied the effect of six Curdlan

derivatives alone or in combination with three cyto-

static drugs on B16 and HEp-2 growth inhibition cell

lines. Our results demonstrated that Curdlan deriva-

tives were able to differently enhance the action of

Actinomycin D, Cyclophosphamide and Doxorubi-

cin cytostatic drugs on B16 and HEp-2 cell lines.

Curdlan derivatives used in our experiments had nocytotoxic activity.

MATERIALS AND METHOD

Cell lines/Cell culture

Murine melanoma B16 cell line and human lar-

ynx epidermoid carcinoma HEp-2 cell line were

maintained in culture at 37°C in a humidified atmos-

phere with 5% CO2, in Dulbecco’s Modified Eagle’s

Medium Nutrient Mixture F-12 HAM - DMEM (SIGMA)

supplemented with 10% heat-inactivated fetal calf

serum (FCS) (SIGMA), 50 IU/ml sodium penicillin-G,

500 mg/ml streptomycin sulphate and 2 mM L-gluta-

mine (SIGMA).

Curdlan derivativesSix Curdlan derivatives (from Curdlan - Agrobac-

terium biovar , Takeda - Kirin Food Corporation, Japan)

synthesized and purified in Petru Poni Macromole-

cular Chemistry Institute, Iasi, Romania were investi-

gated [16]: Carboxymethyl curdlan (CM), Sulfopropyl

curdlan (SP ), Carboxymethyl/Sulfopropyl curdlan

(CM/SP ), Palmitoyl/Carboxymethyl curdlan (Palm

CM), and Palmitoyl/Sulfopropyl curdlan (Palm SP ),

Palmitoyl/Carboxymethyl/Sulfopropyl curdlan (Palm

CM/SP ). Stock solutions (1 mg/ml) of tested biopoly-

mers have been prepared in phosphate bufferedsaline (PBS).

Cytostatic drugs

Stock solutions of Doxorubicin, Actinomycin D

and Cyclophosphamide (SIGMA) drugs (1 mg/ml)

have been prepared in PBS.

MTT viability test

5x104 B16 or HEp-2 cells/well/100 µl were cul-

tured at 37oC and 5% CO2 humidified atmosphere in

culture complete medium (DMEM supplemented

with 10% FCS, 50 IU/ml sodium penicillin-G, 500

mg/ml streptomycin sulphate and 2 mM L-glutamine)

for 4 hrs in flat-bottomed 96-well tissue culture plates

for adherence. Then cytostatic drugs and Curdlan de-

rivatives alone or in combination were added in dif-

ferent concentrations. Each sample was tested in trip-

licate. After 72 hours, the plate was centrifugated at

800 g for 5 minutes and the supernatants were taken

out. 50 ml/well MTT [3-(4, 5-Dimethylthiazol-2-yl)-2,

5-diphenyltetrazolium bromide] (SIGMA), 1 mg/ml in

PBS stock solution, were added to each well and the

plate was incubated for 4 hours at 37oC and 5%CO2.Then 50 ml/well Sodium Dodecyl Sulfate solu-

tion (10% SDS) (BIO-RAD) were added to each wells.

The plate was kept over night at 37oC and then ab-

sorbance was measured at 540 nm by using an ELISA

microplate reader (Thermo Multiskan). Average values

for triplicate samples were calculated.

RESULTS AND DISCUSSION

Our study was focused on the capacity of six

Curdlan derivatives to enhance the inhibitory effect

BÃDULESCU et al.

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Curdlan derivatives able to enhance cytostatic drugs activity on tumor cells

Figure 1. The action of Curdlan derivatives on B16 (a) and HEp-2 (b) tumor cells

of three cytostatic drugs (Doxorubicin, Actinomycin

D, and Cyclophosphamide) on B16 and HEp-2 tumor

cell growth.

Literature data pointed out the antitumor activity

of b - glucans. The success of these treatments is

dependent on a variety of factors, also including the

type of tumor. In our experiments we used several

Curdlan derivatives (SP; Palm CM/SP; Palm SP; Palm

CM; CM/SP; CM) with possible cytostatic activity. Forthis reason, we selected the six Curdlan derivatives

mentioned above and we treated the tumor cells with

these products at different concentrations from 5

mg/100 ml/sample to 50 mg/100 ml/sample. By analysis

of data presented in Figures 1 (a, b) we can notice

that the six Curdlan derivatives had slow cytostatic

activity (from 2% to 35%), not being cytotoxic for cell

lines used in our experiments. The data represent the

results of two experiments.

Cytostatic drugs and Curdlan derivatives testing

has been made in variable doses, aiming to select the

optimum interval where their effect was only cytosta-

tic and not cytotoxic. Following preliminary studies

on the selected tumor cell lines (data not shown), we

selected the following optimal ranges: Doxorubicin

and Actinomycin D (0.001 - 1 mg/100 ml) and Cyclo-

phosphamide (1 -50 mg/100 ml).

After B16 tumor cells treatment with Doxoru-

bicin or Actinomycin D, we obtained the results pre-

sented in Figure 2.

The inhibition of B16 tumor cell growth was

dose-dependent and reached 84% inhibition with

Actinomycin D, 1 mg/100 ml concentration. Doxoru-

bicin had inhibitory activity on B16 cell growth only

when higher concentrations than 0.1 mg/100 ml were

used, reaching 77% inhibition.

Then we tested the ability of Curdlan derivatives

to potentiate the effect of selected chemotherapeutic

drugs. Doxorubicin was used together with selected

Curdlan derivatives, applied in a single dose (20

mg/100 ml) (Figure 3).

It can be observed that cell growth is not influ-

enced by Palm CM/SP and SP derivatives (20 mg/100ml) together with Doxorubicin, while 20 mg/100 ml

Palm SP was able to potentiate the effect of Doxoru-

bicin starting the concentration of 0.1 mg/100 ml Do-

xorubicin, producing 26% cell growth inhibition.

B16 tumor cells treated with Palm SP derivative alone

at concentrations from 5 mg/100 ml to 50 mg/100 ml

Figure 2. The effect of Doxorubicin or Actinomycin Don B16 tumor cell growth. The concentration of cyto-static drugs is expressed as mg/100 ml. The results are

representative for three or more independent experi-ments with similar results.

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204

produced a slow antiproliferative effect of these cells.

The percent of growth inhibition was between 4-8 %

(see Fig. 1a). We can say that Palm SP plays can po-

tentiate Doxorubicin cytostatic action on B16 tumor

cells.

Palm CM/SP and SP derivatives had not cytosta-

tic activity in combination with Doxorubicin. The

growth inhibition was below 5%.

In Figure 4 are presented the results obtained by

using a combined treatment of Curdlan derivatives and

Actinomycin D cytostatic drug on B16 tumor cells.

Actinomycin D is a drug that acts on B16 tumor

cell growth. In our experiments, Curdlan derivatives

have been used in a single concentration of 20

mg/100 ml. By analysis of presented results (Figure 4),

we noticed that Actinomycin D produced a growth

inhibition of tumor cells between 54 - 84%. A com-

bined treatment of tumor cells with Actinomycin D

and Palm CM/SP or SP, demonstrated that both deri-

vatives, especially Palm CM/SP, enhanced cytostatic

activity of Actinomycin D by producing a cell growth

inhibition between 69 - 90%. We mentioned that SP

and Palm CM/SP derivatives, only used as cytostatic

treatment of B16 tumor cells, had no inhibitory effect

on cell growth - below 10% (see Fig. 1a)

The results obtained using a combined treatment

of Cyclophosphamide with Curdlan derivatives on

B16 tumor cells are presented in Figure 5.

As in previous experiments, the selected Curdlan

derivatives have been used in a single dose (20

BÃDULESCU et al.

Figure 3. The effect of combined treatment Doxo-rubicin and Curdlan derivatives on B16 tumor cellgrowth. The concentrations of Doxorubicin andCurdlan derivatives are expressed as mg/100 ml. The

results are representative for three or more inde-

pendent experiments with similar results.

Figure 4. The effect of combined treatment of Acti-

nomycin D cytostatic drug and Curdlan derivatives on

B16 tumor cell growth. The concentrations of Ac-

tinomycin D and Curdlan derivatives are expressed as

mg/100 ml. The results are representative for three or

more independent experiments with similar results.

Figure 5. The effect of combined treatment of Cyclo-phosphamide and Curdlan derivatives on B16 tumorcell growth. The concentrations of Cyclophosphamideand Curdlan derivatives are expressed as mg/100 ml.

The results are representative for three or more inde-pendent experiments with similar results.

Figure 6. The action of Doxorubicin and Actinomy-cin D on HEp-2 cell growth. The concentration ofcytostatic drugs is expressed as mg/100 ml. The results

are representative for three or more independentexperiments with similar results.

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Curdlan derivatives able to enhance cytostatic drugs activity on tumor cells

mg/100 ml). The analysis of experimental data showed

that Cyclophosphamide had no inhibitory effect on

cell growth (below 5%). By applying a combined

treatment of Cyclophosphamide together with each

tested Curdlan derivative, it was observed that only

SP derivative significantly potentiated cytostatic

activity of Cyclophosphamide, producing a cell

growth inhibition between 13 and 27%.

Other experiments have been performed on

human larynx epidermoid carcinoma HEp-2 cell line

using the same Cytostatic drugs in similar conditions

as on murine B16 tumor cell line. Figure 6 shows the

cytostatic activity of the 2 drugs tested on HEp-2 cells.

Analyzing the results presented in Figure 6, we

noticed that by comparing with Actinomycin D, Do-

xorubicin induced a different inhibition on HEp-2

cell growth. Thus, at low concentrations, Doxorubi-

cin was a weak cell growth inhibitor (2-8%), but it

reached 57% inhibition when 1 mg/100 ml was used.

Actinomycin D had a superior cytostatic activity than

Doxorubicin, producing a cell growth inhibition in

the range 17-59%.

Figure 7 presents the results obtained using a

treatment with Doxorubicin (single dose) and Curdlan

derivatives (different doses) on HEp-2 cells.

According to the data presented in Figures 7 (a, b),

a complex treatment consisting in Doxorubicin (1

mg/100 ml) and Curdlan derivatives (5 - 50 mg/100 ml)

on HEp-2 cells induced a cell growth inhibition

between 58 - 78%. These results attest again the abi-

lity of Curdlan derivatives to potentiate Doxorubicin

cytostatic activity.

Figure 7. The effect of Doxorubicin and Curdlan derivatives on HEp-2 tumor cell growth: a) SP, Palm CM/SP,

CM/SP; b) Palm SP, Palm CM, CM. The concentration of cytostatic drugs and Curdlan derivatives are expressedas mg/100 ml. The results are representative for three or more independent experiments with similar results.

Figure 8. The effect of Actinomycin D and Curdlan derivatives on HEp-2 tumor cell growth: a) SP, Palm CM/SP,

CM/SP; b) Palm SP, Palm CM, CM. The concentration of cytostatic drugs and Curdlan derivatives are expressedas mg/100 ml. The results are representative for three or more independent experiments with similar results.

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The effects of combined treatment of Actino-

mycin D (1 mg/100 ml) and Curdlan derivatives (5 - 50

mg/100 ml) on HEp-2 tumor cells growth are illustra-

ted in Figure 8.

As it can be seen in Figure 8 (a and b), Curdlan

derivatives enhance the cytostatic effect of Actino-mycin D on HEp-2 tumor cell growth, when they

were applied together with Actinomycin D, inducing

a cell growth inhibition between 75 - 89%. These re-

sults, compared with HEp-2 cells growth inhibition

produced by Actinomycin D alone (17 - 59%), support

the hypothesis that the cytostatic effect of Actino-

mycin D was potentiated by Curdlan derivatives.

CONCLUSIONS

All together our data suggested that the tested

Curdlan derivatives differently enhance the cytostaticactivity of some drugs such as Actinomycin D, Cyclo-

phosphamide and Doxorubicin on tumor cells (B16

and HEp-2).

Acknowledgment

This study was supported by the National

Research Program PN II, Grant No. 61006/2007.

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3. Hutchinson CR, Colombo AL. Genetic engineering of doxorubicin production in Streptomyces peucetius: areview. J. of Industrial Microbiology and Biotechnology ,1999, 23: 647 - 652.

4. Kleeff, J, et al. Actinomycin D induces apoptosis andinhibits growth of pancreatic cancer cells. Int. J. Cancer ,2000, 86: 399-407.

5. Henry M. Sobell. Actinomycin and DNA transcription.Proc. Natl. Acad.Sci., 1985, 82: 5328 – 5331.

6. Shanafelt TD, Lin T, Geyer SM, et al. Pentostatin,cyclophosphamide and rituximab regimen in olderpatients with chronic lymphocytic leukemia. Cancer,

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8. Laroche C, Michaud P. New Development and Perspec-tives Applications for b-(1,3) Glucans, Recent Patents on

Biotechnology. 2007; 1: 59-73

9. Lee KB, Bae JH, Kim JS, Yoo YC, Kim BS, Kwak ST, Kim

YS. Anticoagulant activity of sulfoalkyl derivatives of curdlan. Arch. Pharmacol. Res., 2001, 24: 109 - 113.

10. Demleitner S, Kraus J, Franz G. Synthesis and antitu-mour activity of sulfoalkyl derivatives of curdlan andlichenan. Carbohydr. Res., 1992, 226: 247 - 252.

11. Jin Y, Zhang H, Yin Y, Nishinari K. Comparison of curdlan and its carboxymethylated derivative by meansof Rheology, DSC and AFM. Carbohydr. Res., 2006,

341: 90 - 99.

12. Ferencik M, Kotulova D, Master L, Bergendi L, San-

dula J, Stefanovic J. Modulatory effects of glucan onthe functional and biochemical activities of guinea-pigmacrophages. Methods Find. Exp. Clin Pharmacol.,1986, 8(3): 163 - 166.

13. Sandula J, Machoya E, Hribova V. Immunomodulatoryactivity of particulate yeast b - 1,3-D-glucan and water-soluble derivatives. Int. J. Biol. Macromol., 1995, 17:232 - 326.

14. Rost GD, Vetvicka Y, Yan J, Xia Y, Vetvikova J. Thera-peutic intervention with complement and b-glucan in

cancer. Immunopharmacology , 1999, 42: 61 - 74.15. Ohno N, Asada N, Adachi Y, Yadomae T. Enhance-

ment of LPS triggered TNF-alpha (tumor necrosis factor-alpha) production by (1-3)-beta-D-glucans in mice.Biol. Pharm. Bull., 1995, 18: 126 - 133.

16. Mocanu G, Mihai D, Moscovici M, Picton L, LeCerf D.

Curdlan microspheres. Synthesis, characterization andinteraction with proteins (enzymes, vaccines). Int. J.

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INTRODUCTION

Despite other pathogenic bacteria which areone-disease organisms, E. coli strains can cause an

impressive variety of diseases, including diarrhea,

disenthery, haemolytic uremic syndrome, pneumo-

nia, meningitis and septicemia [1-3]. The versatility of

E. coli strains is due to the fact that different strains

have acquired different sets of virulence genes by

horizontal gene transfer, genes which allow bacteria

to better adapt to different micro-environments of the

human body [4].

Enteroaggregative Escherichia coli (EAggEC)

pathotype is a group of pathogenic E. coli strains cha-

racterized by the ability to adhere to cultured cell

monolayers with an aggregative or “stacked brick”

adhesion phenotype [5]. These E. coli strains are also

distinct from other congeneric strains by their patho-

genicity, the EAggEC being an emerging pathogen

and a significant cause of acute diarrhoea in children

and in immunocompromised patients [6-8], as well as

a cause of bacterial gastroenteritis in adults, both in

developing and industrialized countries (9-11). The

true incidence of EAggEC infection is underestimated

because the standard assay for EAggEC demonstrating

the aggregative attachment of organisms to human

epithelial (HEp-2) cells, is time-consuming to set up

and prone to contamination [12].

The gastrointestinal tract is the principal target of

available probiotic cultures and most definitions of

probiotics refer to the intestinal site of action. A num-

ABSTRACT

The purpose of this study was the assessment of the influence of three probiotic supernatants

(Bifidobacterium breve ATCC 15700, Enterococcus faecium ATCC 19434, Lactobacillus casei subsp.

casei ATCC 393) on the growth (quantified by viable cell counts) and virulence features expression

(adherence ability to HEp-2 cells and inert substratum- slime test) of several selected EAggEC diarrho-eagenic strains as well as the cytotoxicity of the respective supernatants on HEp-2 cells.

Results: Our in vitro studies are demonstrating that the selected supernatants, when added simultaneously

with the bacterial culture are generally opposing to the adherence to the cellular substratum by the

EAggEC strains. When added after the pre-adherence period, the supernatants did not change the adhe-

rence indexes of the EAggEC strains, but induced slight changes in the adherence pattern, reducing the

frequency and size of bacterial aggregates. Only in few cases, the bacterial growth rate was slightly

increased or sustained by the probiotic supernatants, a possible explanation being that we used super-

natants obtained from 24 hrs fresh cultures, which are probably still containing some nutrients and pro-

bably also other growth factors.

Conclusion: Our results are demonstrating that soluble probiotic metabolites accumulated in culture

supernatants may interfere with the first step of adherence and colonization of the cellular and inert sub-strata by EAggEC strains, probably by the cross-talk between probiotic soluble molecules and quorum-

sensing mediators of opportunistic strains; so, direct contact between the probiotic and pathogenic bac-

teria are not always necessary for the occurrence of a protective effect, that could be partly mediated by

the soluble molecules secreted by specific probiotic strains.

THE INFLUENCE OF SOME PROBIOTIC SUPERNATANTS

ON THE GROWTH AND VIRULENCE FEATURES EXPRESSION OF SEVERAL

SELECTED ENTEROAGGREGATIVE E. COLI CLINICAL STRAINS

Veronica Lazãr1, Yoshibumi Miyazaki2, Tomoko Hanawa2, Mariana-Carmen Chifiriuc1,Lia-Mara Diþu1, Luminiþa Mãruþescu1, Coralia Bleotu and Shigeru Kamiya2

1)Department of Microbiology & Immunology, Faculty of Biology - University of Bucharest, Romania

Center of Research, Formation and Consultancy in Microbiology, Genetics and Biotechnology;2)Division of Medical Microbiology, Department of Infectious disease, Kyorin University School of Medicine, Tokyo, Japan

Key words: Enteroaggregative Escherichia coli, probiotic supernatants, virulence

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ber of factors are believed to be important for lacto-

bacilli to colonize the intestinal tracts and reduce the

risk of infection. Of these, adhesion to cells and mu-

cus is believed to be one of the main factors when it

coincides with survival of the organism and inhibi-

tion of growth and adhesion of pathogens. Since pre-sent, the most important microorganisms used as pro-

biotics are included in few genera belonging to the

large group of lactic acid bacteria: Lactococcus, Lac-

tobacillus, Pediococcus, Leuconostoc, Carnobacte-

rium, Streptococcus, Enterococcus, Bifidobacterium.

The purpose of this study was the assessment of

the adherence capacity and biofilm development of

some EAggEC clinical strains on inert and cellular

substrata and of the influence of some probiotic su-

pernatants on the growth and virulence features ex-

pression (adherence ability) of several selectedEAggEC diarrheagenic strains.


Bacterial strains. In this study there were tested

three enteroaggregative E. coli (EAggEC) clinical strains

isolated from patients with diarrhea, resident in Japan

(encoded in our study by TN-1, TN-2 and respectively,

TN-3) and two reference strains of enterotoxigenic E.

coli (ETEC 1321-4) and E. coli MC 4160 un-patho-

genic strain, respectively. The EAggEC strains have

been selected from patients exhibiting chronic diarrhea

without other possible etiology and the aggregative

pathovar has been established exclusively by pheno-

typic tests, evidencing the specific colonization pat-

tern of HEp-2 cells, in which aggregated bacteria

attach to the cell in a stacked-brick arrangement.

Probiotic strains: three reference strains (Japan

Collection of Microorganisms, Catalogue, Eighth Ed.,

2002, Riken, Japa, Bussines Center for Academic So-

cieties, Tokyo, Japan) - SN1- Bifidobacterium breve

ATCC 15700 (source of isolation: infant faeces), SN2-

Enterococcus faecium ATCC 19434 (source of isola-tion: intestine); SN3- Lactobacillus casei subsp. casei

ATCC 393 (source of isolation: cheese); the su-

pernatants used in this study were obtained from 24

hours liquid cultures on appropriate media, centri-

fuged and sterilized by membrane filtration (45 µm).

Cellular cell line: we used HEp-2 cell line, the

most used epithelial cell line for testing the adher-

ence ability of enterobacterial strains as 80% conflu-

ence monolayers.

Bacteriological media: The E. coli strains were

taken from preservation medium and cultivated on

MacConkey agar and subcultured into LB broth me-dium with overnight incubation without shaking at

37°C.

HEp-2 cell-adherence assay was performed

using the method of Cravioto [13-14], in two variants,

qualitative and quantitative; HEp-2 cells were grown

for 48 h to 80% confluency in 6/24flat-bottoms tis-

sue-culture plates in Eagle’s medium (MEM), supple-

mented with 10% fetal calf serum, 2% L-glutamate

and 1% non-essential amino acids (Sigma-Aldrich).

Prior to inoculation with bacterial strains, the culture

medium was removed and the HEp-2 cells were

washed three times with (phosphate buffered saline)

PBS (1ml/well each time); the inoculation was done

by the addition of 1ml of bacterial suspension/well,

adjusted to 0.5 MacFarland density scale, the plates

being incubated at 37°C in atmosphere of 95 %

O2/5 % CO2 for 2 h. The HEp-2 cells were washed

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LAZÃR et al.

Fig. 1. Adherence control - EAggEC TN-2 strain

(Giemsa staining, 1000x)

Fig. 2. Adherence of EAggEC TN-2 strain in the pres-ence of Bifidobacterium breve ATCC 15000 super-

natant (SN1), after direct incubation with the infect-ed monolayer (Giemsa staining, 1000x)

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thoroughly with PBS (3 times) to remove non-adher-

ent bacteria and fixed with 80% cold methanol for

5 min. After fixation, the coverslips were washed

three times with PBS, stained with 10% Giemsa

(BDH) for 15 min, rinsed and air-dried, prior to light-

microscopy examination using an I.O. objective. Theadherence patterns were assessed and recorded.

For quantifying the adhered viable cells, after

incubation, the monolayers grown in multiplates

without coverslips were washed three times with PBS

to remove non-adherent bacteria, the eukaryotic cells

were detached and permeabilized with 1% Triton

100x for 5 minute, the lysates were homogenized,

and ten-fold dilutions were spotted on solid media

(each dilution was inoculated in duplicate), incuba-

ted at 37°C for 24h, the optimal dilution being cho-

sen for viable cell counts expressed in CFU/ml.The qualitative and quantitative study of the

influence of LAB supernatants on the adherence ca-

pacity of the pathogenic EAggEC strains to the cellu-

lar substratum was performed by the same method,

each variant in other two experimental models: the

simultaneous addition of 50 µl of LAB supernatants

and the microbial suspensions on the cellular sub-

stratum and respectively, the addition of the LAB

supernatants after 2 hrs incubation of microbial sus-

pensions and the cellular substratum.

Adherence to the inert substratum was quanti-

fied by slime test using a simple microtiter, colori-

metric method for the assessment of bacterial adher-

ence capacity to the plastic multi-well plate. In this

purpose, the bacterial cells were cultivated in liquid

medium distributed in microplates. After 24 hr of in-

cubation, the wells were emptied, washed three times

with PBS. The biofilm formed on the plastic wells

was fixed for 5 min with cold methanol, colored for

15 min by violet crystal solution and resuspended by

33% acetic acid solution. The absorbance of the

colored solution was measured at 490 nm using an

ELISA reader (Apollo LB 911), the results being pro-portional with the degree of biofilm development on

the inert substrate.

Cytotoxicity assay was performed using the Cell

Counting kit (Roche), by quantifying the cell mass of

the surviving cells of the monolayer based on a co-

lorimetric method (staining with the basic dye methy-

lene blue) (31, 40). At 1-h intervals the monolayers

were washed with PBS and stained for 10 min with

methylene blue (1% in 10 mM borate buffer). Excess

staining was removed by five washing steps with bo-

rate buffer, and plates were dried overnight at roomtemperature. Bound methylene blue was extracted

with 200 µl of 0.1 M HCl and quantified measuring the

optical density at 620 nm in a microtiter plate reader.

RESULTS AND DISCUSSION

The first stage of infectious process is the adher-

ence of pathogenic microorganisms to epithelial cells

or extracellular matrix molecules, which will lead to

the colonization at the entrance gate.

Host colonization involves complex cell to cellcommunication systems, both between bacterial cells,

and cross communication between bacterial and host

cells. A very well known and studied example is that

of Escherichia coli; eight E. coli pathovars have been

well characterized, six responsible for intestinal in-

Effect of probiotic cultures soluble fraction on EAggEC adhesive properties

209

Fig. 3. Adherence of EAggEC TN-1 strain in the pres-ence of Enterococcus faecium ATCC 19434 super-

natant (SN2) after direct incubation with the infect-ed monolayer (Giemsa staining, 1000x)

Fig. 4. Adherence of EAggEC TN-2 strain in the pres-ence of Bifidobacterium breve ATCC 15000 super-

natant (SN1), added after 2h of pre-incubation ofthe infected monolayer (Giemsa staining, 1000x)

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LAZÃR et al.

infectious process [17]. All EAggEC investigated strains

exhibited the ability to colonize the cellular and inert

substratum, showing a specific aggregative pattern

and 100% adherence indexes (the adherence index

was expressed as the ratio between the number of the

eukaryotic cells with adhered bacteria: 100 eukaryo-tic cells counted on the microscopic field) (Fig. 1). We

have also used one ETEC strain, as negative control

for the adherence to the HEp- 2 cells and at the same

time as a positive control in order to appreciate the

level of the cytotoxic effect exhibited by the EAggEC

strains upon the HEp-2 cells monolayer. The ETEC

1321-4 strain has indeed shown the lowest capacity

of adherence, as expected, having in view that its

main virulence factor is the cytotoxicity.

The reference unpathogenic E. coli MC4160

strain showed a high ability to adhere to the inert sub-stratum and also to the cellular substratum, this ability

being preserved in the presence of probiotics.

Concerning the EAggEC strains, the selected lac-

tic acid bacteria (LAB) supernatants of Bifidus breve

(SN1) P1, Enterococcus faecium (SN2) P2 and Lacto-

bacillus casei (SN3) P3, when added simultaneously

with the bacterial suspension to the cellular substrate

are generally opposing to the adherence to the cellu-

lar substratum by the enteroaggregative strains (10 -

1000 fold decrease of the viable cells number and

the decrease of the adherence indexes from 100% to

10-15%). All tested LAB supernatants also induced

changes in the adherence pattern, from the typical

aggregative to a diffuse or localized one (Fig. 2-3).

fections (enterotoxigenic E.coli- ETEC , enteroinvasive

E.coli- EIEC, enteropathogenic E.coli- EPEC , entero-

hemorrhagic E.coli-EHEC, enteroaggregative E. coli -

EAEC/EaggEC , diffuse adhering E.coli- DAEC) and two

of extraintestinal ones (urinary tract infections, sepsis

and meningtis) called ExPEC (from extratestinal ente-

ropathogenic E. coli) (uropathogenic E. coli - UPEC,

septic E. coli- SEC) [15-16].

Each pathovar uses a large arsenal of virulence

factors to subvert host cellular functions to potentiate

its virulence, but all of them possess the ability to

adhere to the epithelial cells in order to initiate the

Fig. 5. Adherence of EAggEC TN-3 strain in the pre-sence of Enterococcus faecium ATCC 19434 super-natant (SN2), added after 2h of pre-incubation of the

infected monolayer (Giemsa staining, 1000x)

Fig. 6. Quantitative results of the influence of Bifidobacterium breve ATCC 15000 super-natants (SN1) on the adherence capacity of the pathogenic EAggEC strains

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211

When added after the 2 h pre-adherence period,

the supernatants did not change the adherence in-

dexes of the EAggEC strains, but induced slight chan-

ges in the adherence pattern, reducing the frequency

and size of bacterial aggregates (Fig. 4-5).

The results concerning the influence of LAB

supernatants on the adherence ability of the tested

bacterial strains demonstrate that the probiotic strains

are secreting soluble molecules which are opposing

to the adherence and colonization of HEp-2 cells by

the EaggEC strains isolated from chronic diarrhea, de-

monstrating their potential use in the porphylaxis/treat-

ment of gastro-intestinal disorders, as an alternative or

in association with antibiotics. The quantitative assay

of the LAB supernatants influence on the adherence

ability of the tested EAggEC strains showed that the

level of the inhibitory activity of the three LAB super-

natants was different for the three EAggEC strains.

Fig. 7. Quantitative results of the influence of Enterococcus faecium ATCC 19434 (SN2)supernatants on the adherence capacity of the pathogenic EAggEC strains

Fig. 8. Quantitative results of the influence of Lactobacillus casei susp. casei ATCC 393superntants (SN3) on the adherence capacity of the pathogenic EAggEC strains

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However, the E. coli TN-1 strain proved to be the most

sensitive to all tested supernatants (Fig. 6-8). The strain

TN-3 was less inhibited by the SN3, and TN-2 less

inhibited by SN 1 and SN2 (Fig. 6-8).

Taken together, our results are demonstrating that

soluble probiotic metabolites accumulated in LAB

culture supernatants may interfere with the first step

of adherence and colonization of the cellular sub-

strata by EAggEC strains, probably by the cross-talk

between the probiotic soluble molecules and quo-

rum-sensing mediators of opportunistic strains, which

are regulating the coordinated virulence features ex-

pression of opportunistic pathogens.

In case of the ETEC and non-pathogenic referen-

ce strains, the bacterial growth rate was slightly in-

creased or sustained by the Lactobacillus casei su-

pernatant (Fig. 8), when the supernatant was added

later, after the 2 hrs pre-adherence period; a possible

explanation could be the fact that during this study

there were used supernatants of 24 hrs cultures, which

are probably still containing some nutrients and pro-

bably also other growth factors, not only inhibitory

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LAZÃR et al.

Fig. 9. Quantitative results of the influence of Bifidobacterium breve ATCC 15000 supernatants (SN1) onthe adherence capacity of the pathogenic EAggEC strains to the inert substratum, quantified by measu-ring the A 490 nm of the colored cells adhered to the plastic wall and resuspended with acetic acid

Fig. 10. Quantitative results of the influence of Enterococcus faecium ATCC 19434 (SN2) supernatantson the adherence capacity of the pathogenic EAggEC strains to the inert substratum, quantified by mea-suring the A 490 nm of the colored cells adhered to the plastic wall and resuspended with acetic acid

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substances, which are promoting the bacterial growth

during the incubation time with the eukaryotic cells.

These results lead us to conclude that for the assay of

antimicrobial activities of the probiotic supernatants,

48 hrs cultures could be more appropriate.

Concerning the influence of the tested probiotic

supernatants on the ability of EAggEC strains to colo-

nize the inert substratum, our results showed a ge-

neral slight stimulatory effect on the slime production

(Fig. 9-11).

Our results come into agreement with other stu-

dies suggesting that the soluble molecules secreted by

probiotics and prebiotics could reduce the adherence

to the cellular substrate mainly by inhibiting the ex-

pression of adherence proteins and not by changing

the physical features of bacterial cells [18, 19]. In ex-

change, in the case of adherence to the inert substrate

we quantified the slime production, this slime being

an exo-polysachharide produced by the bacterialcells to protect themselves against desication and

other limiting conditions, this behavioral structure

seeming not to be inhibited by probiotics. Moreover,

the opportunistic bacterial strains, especially EAggEC

are hydrophobic, this feature favoring the strong phy-

sical interaction with the inert substratum that could

hardly be affected by the soluble molecules secreted

by probiotics.

One of the most important conditions to be ful-

filled by a probiotic is the absence of side effects on

the host organism, such as cytotoxicity. During this stu-

dy, the reduced cytotoxicity is representing an impor-

tant advantage in the selection process of probiotic

products to be administered in humans (Table no. 1).

CONCLUSION

Our results proved that the anti-infectious probi-

otic effect is partly due to soluble factors accumulated

in liquid cultures, probably by the interference of

these substances with the intercellular signaling me-

chanisms implicated in the adherence of pathogens

to intestinal mucosa. It remains to investigate whe-

ther the soluble molecules are accumulating only in

probiotic cultures or are also produced in the intes-

tine and are producing similar in vivo effects. The

selected supernatants exhibited low cytotoxicity levels,

this representing an advantage in the case of further

use in humans, especially in immuno-depressed pa-

tients or children, often infected with EaggEC strains.

Taken together, our results are suggesting that thetested probiotic strains could be used mainly in the


213

Fig.11. Quantitative results of the influence of Lactobacillus casei susp. casei ATCC 393 superntants (SN3)on the adherence capacity of the pathogenic EAggEC strains to the inert substratum, quantified by mea-suring the A 490 nm of the colored cells adhered to the plastic wall and resuspended with acetic acid

Table no 1. The effect of the probiotic strains supernatantson HEp-2 cells quantified by Cell Counting kit

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prophylaxis (the inhibitory effect being more evident

when the probiotics are administered before the

pathogens adherence is settled, but even in the treat-

ment of gastro-intestinal chronic disorders, as an

alternative or in association with antibiotics.

ACKNOWLEDGEMENTS

This study was accomplished in the Division of

Medical Microbiology, Department of Infectious Di-

seases, Kyorin University School of Medicine, Tokyo,

Japan, due to a JSPS ( Japanese Society for the Promo-

tion of Science) grant for an experience exchange

program of a Romanian researcher in this department

conducted by Prof. Shigeru Kamiya.

REFERENCES

1. Ghosh A R, Nair GB, Nalik TN, Paul M, Pal SC, Sen D.

Entero-adherent Escherichia coli is an important diar-rhoeagenic agent in infants aged below 6 months inCalcutta, India. J. Med. Microbiol. 1992. 36:264-268.

2. Oberhelman RA, Laborde D, Mera R, Starszak E, Saun-

ders P, Mirza A, Bessinger GT, Hull A. Colonizationwith enteroadherent, enterotoxigenic and enterohemor-rhagic Escherichia coli among day-care center attendeesin New Orleans, Louisiana. Pediatr. Infect. Dis J.

1998.17:1159-1162.3. Glandt M, Adachi J A, Mathewson J J, Jiang ZD,

DiCesare D, Ashley D,. Ericsson CD, DuPont HL. En-teroaggregative Escherichia coli as a cause of traveler’sdiarrhea: clinical response to ciprofloxacin. Clin. Infect.

Dis. 1999. 29:335-338.

4. Salyers Abigail A, Whitt Dixie D. Bacterial Pathogenesis.A molecular approach. ASM Press, Washington D.C.2002, 407-421.

5. Pizzaro-Cerda J, Cossart P. Bacterial adhesion and entryinto host cells. Cell. 2006. 124: 715-727.

6. Mathewson J J, Johnson PC, DuPont HL, Morgan DR,

Thornton SA, Wood LV, Ericsson CD. A newly recogni-

zed cause of travelers’ diarrhea: enteroadherent Esche-richia coli. J. Infect. Dis. 1985. 151:471-475.

7. Mathewson JJ, Oberhelman RA, DuPont HL, de la

Cabada FJ, Garibay EV. Enteroadherent Escherichia coli

as a cause of diarrhea among children in Mexico. J. Clin.

Microbiol. 1987. 25:1917-1919.

8. Bhatnager S, Bhan MK, Sommerfelt H, Sazawal S, Kumar

R, Saini S. Enteroaggregative Escherichia coli may be anew pathogen causing acute and persistent diarrhea.Scand. J. Infect. Dis. 1993. 25:579-583

9. Adachi JA, Ericsson CD, Jiang ZD, DuPont MW, Palle-

gar SR, DuPont HL. Natural history of enteroaggregativeand enterotoxigenic Escherichia coli infection amongU.S. travelers to Guadalajara, Mexico. J. Infect. Dis.

2002. 185:1681-1683.

10. Adachi JA, Jiang ZD, Mathewson JJ, Verenkar MP,

Thompson S, Martinez-Sandoval F, Steffen R, Ericsson

CD, DuPont HL. Enteroaggregative Escherichia coli asa major etiologic agent in traveler’s diarrhea in 3regions of the world. Clin. Infect. Dis. 2001. 32:1706-1709.

11. Wakimoto N, Nishi J, Sheikh J, Nataro JP, Sarantuya J,Iwashita M, Manago K, Tokuda K, Yoshinaga M,

Kawano, Y. Quantitative biofilm assay using a micro-titer plate to screen for Enteroaggregative Escherichia

coli. Am. J. Trop. Med. Hyg . 2004.71(5): 687-690.

12. Spencer J, Chart H, Smith HR, Rowe B. Improveddetection of enteroaggregative Escherichia coli usingformalin fixed HEp-2 cells. Lett. Appl. Microbiol. 1997.5:325-326.

13. Cravioto A, Gross R J, Scotland SM, Rowe B. An adhe-sive factor found in strains of Escherichia coli belon-ging to the traditional infantile enteropathogenic se-rotypes. Curr. Microbiol. 1979. 3:95-99

14. Nataro JP, Steiner T, Guerrant RL. EnteroaggregativeEscherichia coli. Emerg. Infect. Dis. 1998. 4:251-261.

15. Law D, Chart H. Enteroaggregative Escherichia Coli. J. Appl. Microbiol. 1998. 84: 685 - 697.

16. Law DA. Review: virulence factors of Escherichia coli

O:157 and other shiga toxin-producing E. coli. J. Appl.

Microbiol. 2000. 88: 729 - 745. Croxen M, Finlay BB.Molecular mechanisms of Escherichia coli pathogeni-city. Nature Reviews Microbiology , advance onlinepublication, 2009.Published online 7 December 2009| doi:10.1038/nrmicro2265

17. Shoaf K, Mulvey GL, Armstrong GD, Hutkins RW.

Prebiotic Galactooligosaccharides Reduce Adherenceof Enteropathogenic Escherichia coli to Tissue CultureCells. Infection and Immunity 2006. 74 (12): 6920-6928,

18. Vanmaele RP, Heerze LD, Armstrong G D. 1 Role of lactosyl glycan sequences in inhibiting enteropatho-genic Escherichia coli attachment. Infect. Immun. 999.67:3302-3307

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INTRODUCTION

A microbial biofilm is considered to be the most

successful and competitive expression of the proka-

ryotic genome - the cells of the biofilm being meta-

bolically more efficient and well protected, exhibiting

resistance to different stress factors, including host

defense mechanisms and antibiotics (Costerton,

1995; Fux et al., 2005).

The behavioral or phenotypic resistance mecha-

nisms (different from the genetic resistance mecha-

nisms) of the biofilm forming cells towards antimi-

crobial activity of some substances are determined by

many factors: the adherence on the substratum and

bacterial aggregation, which explain the physiologi-

cal changes, including growth rate modification and

dormant/persistent cells appearance; secretion of

exopolymeric substances protecting the cell wall;

accumulation of antibiotic degrading enzymes in

high concentrations or synergic activity of the enzy-

mes produced by different species from the biofilm;

changes in gene expression (activation/inhibition) of

the bacterial strains grown in biofilms (Lazar, 2003).Quorum sensing mechanism plays an important

role in cell density-responsive regulation during inter-

actions between bacteria and plant or animal hosts

(Fuqua, 1994). Many photosynthetic organisms

(algae, lichens, some superior plants, especially

aquatic) have developed defence mechanisms

against bacterial colonization that involve the pro-

duction of metabolites which inhibit intercellular sig-

nalling - these are termed “Quorum sensing inhi-

bitors (QSI)” . For example, some species of algae

such as Delisea pulchra produces halogenated fura-

nones which prevent bacterial colonization and bio-

film formation (Manefield, 1999).

215

ABSTRACT

The vegetal extracts are used as an ecological alternative to classical anti-infectious treatments based on

antibiotics, exhibiting the advantage of reduced secondary effects. Most of these compounds are secon-

dary metabolites, especially aromatic substances synthesized by plants in a reduced concentration. The

aim of this study was to investigate the influence of usnic acid against quorum sensing and response

mechanisms involved in the initiation and development of the dental plaque biofilm and its tolerance toantimicrobials. Three samples of super-gingival dental plaque were treated for different time intervals

with usnic acid at 200 ìg/ml in dimethyl-sulfoxide, representing the MIC value. Each dental plaque sam-

ple was inoculated in Brain Heart Infusion medium to establish the microbial growing curve by viable

cells counts using the tenfold microdilutions method. For strains identification there were used the

microtest galleries: API 20Strep, API Staph, API 20NE, API 20E. MIC value for usnic acid was determined

by twofold microdilution technique. Usnic acid selectively inhibited the biofilm development by Gram

positive bacteria and the expression of haemolytic properties of strains isolated from the dental plaque.

The growth curve of the isolated strains was affected by usnic acid, the changes consisting of the lag phase

extension to 6-10 h (this time interval being considered as the persistence time of antimicrobial activity)

and the significant decrease of the viable cell number and consecutively, the prolongation of the genera-

tion time. These effects are demonstrating the interference of the usnic acid with the intra- and inter-species signalling mechanisms based on quorum sensing and response and dependent on cell density,

giving the possibility to use them as an active principle in some new pharmaceutical formula intended

for the prevention and treatment of gingival and periodontal pathologies.

IN VITRO STUDY OF THE INHIBITORY ACTIVITY

OF USNIC ACID ON DENTAL PLAQUE BIOFILM

Mariana Carmen Chifiriuc1, Lia Mara Diþu1, Eliza Oprea2, Sanda Liþescu1,Marcela Bucur1, Luminiþa Mãruþescu1, Gerard Enache1, Crina Saviuc1, Mihai Burlibaºa3,

Teodor Trãistaru3

, Gabriela Tãnãse3

, Veronica Lazãr1

1)University of Bucharest, Faculty of Biology, Microbiology Immunology Department, Aleea Portocalelor, 1-5,

Bucharest, Romania; 2)Bucharest, Faculty of Chemistry, Organic Chemistry Department, Bucharest, Romania;3)“Carol Davila” University of Medicine and Pharmacy, B-dul Eroilor Sanitari 8; Bucharest, Romania

Key words: usnic acid, dental plaque, anti-biofilm agents

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The vegetal extracts are used as an ecological

alternative to classical anti-infectious treatments ba-

sed on antibiotics, exhibiting the advantage of secon-

dary effects absence. The superior plants are the

resources for a high number of antimicrobial sub-

stances with the same potency as the antibiotics. Inaddition, lichens which are symbiotic organisms,

composed of a fungal partner, the mycobiont, and

one or more photosynthetic partners, the photobiont,

which is most often either a green alga or cyanobac-

terium. Lichens produce many metabolites; for in-

stance, Usnea barbata produces usnic acid and bar-

batic acid. Most of these compounds are secondary

metabolites, especially aromatic substances synthesi-

zed by plants in a reduced concentration. One of the

high therapeutic important secondary metabolite syn-

thesized by Usnea barbata is usnic acid, isolated forthe first time, in 1844 by Knop. Usnic acid, a sec-

ondary lichen metabolite, exhibits antimicrobial

activity against a number of planktonic bacteria, and,

as many other secondary lichen metabolites, offers

protection to lichen communities against adherent

microorganisms (Shibata et al., 1948; Sharma et al.,

1966; Ghione et al., 1988; Grasso et al., 1989; Laut-

wewein et al., 1995; Francolini, 2004; Nash, 2008).

Usnic acid is a yellowish pigment produced by seve-

ral lichen species. Usnic acid has been documented

to have antihistamine, spasmolytic, antiviral, and an-tibacterial activities. Two biologically active natural

enantiomers of usnic acid, differing in the orientation

of the methyl group at 9b, otherwise rigid molecule,

have been identified as showing different biological

activities and mechanisms of action (Lauterwein et

al., 1995). Proska et al. (1996) reported that (-)-usnic

acid inhibited urease and arginase activity. Several

reports revealed that the (+)-enantiomer is a more

effective antimicrobial agent, although no specific mode

of action was determined (Cardarelli et al., 1997).

Our previous studies demonstrated the inhibi-

tory activity of usnic acid towards biofilms developed

by Staphylococcus aureus and Pseudomonas aerugi-

nosa strains, being active as an inhibitor of quorum

sensing mechanism by interference with the coordi-

nated expression of virulence factors, including

adhesines synthesis (Lazar et al., 2008). The effects of

some QSI on P. aeruginosa virulence factor produc-

tion and biofilm sensitivity suggested that bacterial

virulence can be controlled by means of substancesthat specifically block cell-to-cell communication.

QSI compounds may find applications in many dif-

ferent settings, such as medicine, agriculture and food

technology. Chemical attenuation of bacterial viru-

lence, rather than bactericidal or bacteristatic strate-

gies, is a highly attractive concept because such an-

tipathogenic agents are less likely to pose a selective

pressure for development of resistant mutants. The

concept of direct targeting virulence is promising as

an early prophylactic treatment of individuals with P.

aeruginosa infections. QSI drugs might prevent theformation of detrimental biofilms in the lung, on

implants or in wounds (Hentzer at al., 2003).

Dental plaque forms naturally on teeth and is of

benefit to the host by helping to prevent colonization

by exogenous species. Modern molecular biological

techniques have identified about 1000 different bac-

terial species in the dental biofilm, twice as many as

can be cultured. The bacterial composition of plaque

remains relatively stable despite regular exposure to

minor environmental perturbations. This stability

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CHIFIRIUC et al.

Fig. 1. Aspect of microbial culture recovered on blood agar from the dental plaque sample

Usnic acid structure

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control. 10 ml of bacterial suspension at the standard

density of 0.5 Mc Farland was added in each well.

RESULTS

After inoculation of dental plaque samples on

blood agar, there were obtained colonies with different

morphologies, from small, rough, self-aggregative and

non-hemolytic colonies to big, hydrated, smooth,

b-hemolytic colonies (fig. 1).

The identified strains are represented in tableno. 1. For Staphylococcus epidermidis strain (sample

M), the treatment with usnic acid (at MIC value)

induced a switch from Gram positive to Gram nega-

tive affinity. The staining affinity change was probably

due to the influence of usnic acid on the cell wall

structure.

The treatment of all samples with usnic acid (at

200 mg/ml in DMSO) for different intervals (1 min., 2

min., 5 min., 15 min.) induced the exclusive selection

of Gram negative, non-hemolytic bacterial strains (fig.

2), independently from the exposure time to the usnic

acid or the analyzed dental plaque sample.

Instead, untreated or DMSO treated samples de-

veloped both Gram positive hemolytic and Gram

negative non-hemolytic bacterial strains (fig. 2-3).

The assessment of the growing curve by esta-

blishing the VCC (at 3 h, 6 h, 16 h and 24 h) for the

selected strains after usnic acid treatment for different

intervals (1 min., 2 min., 5 min., 15 min.) showed that

these strains exhibited a slower growth rate in the first

6-10 h from inoculation, with a drastic decrease of

VCC value (fig. 4). The contact of the samples with

DMSO did not influence the growing curve.For all samples the exposure to DMSO did not

influence the growing curve, irrespective to the time

of exposure. The most evident inhibitory effect to-

wards the bacterial growth was exhibited in the first

6 h, after 3 minutes treatment of dental plaques with

usnic acid (at 200 mg/ml representing the MIC value)

(fig. 5).

MIC value for usnic acid was determined for

each bacterial strain isolated from supra-gingival den-

tal plaque samples. In the most cases, the inhibition

of cell growth was observed at 12.5 mg/ml concen-

tration of usnic acid, the DMSO control not influen-

cing this value (figure 6-10).

DISCUSSIONS

Many secondary lichen metabolites, including

(+)-usnic acid, offer protection to lichen communi-

ties against microorganisms colonization, which

could interfere with the photosynthetic activity of the

algal component. The antimicrobial agent (+)-usnic

acid exhibits inhibitory activity against Gram-positive

bacteria and mycobacteria, but not against plankto-

nic Gram-negative bacteria and fungi (lichens are for-

med through symbiosis between fungi and algae

and/or cyanobacteria) (Nash, 2008). The mechanism

of action expressed by (+)-usnic acid is still unknown

(Francolini et al., 2004). However, experimental evi-

dence showed that its antiviral action is due to the

ability to inhibit RNA transcription (Campanella et

al., 2002). Due to its low solubility in water, the use

of (+)-usnic acid has been limited to oral care, topic

ointments, and cosmetic formulations. In addition,

(+)-usnic acid has been shown to be active against

clinical isolates of E. faecalis and E. faecium and cli-nical isolates of methicillin - or muporicin-resistant

218

CHIFIRIUC et al.

Fig. 3. Hemolytic colonies are still recoveredin the presence of DMSO (right part of the image)

Fig. 2. Non-hemolytic colonies selectionby the usnic acid (left part of the image)

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In vitro study of the inhibitory activity of usnic acid on dental plaque biofilm

219

Fig. 4. Aspect of the microbial colonies recovered from the dental plaque (patient 2) and treated with usnicacid (UA) for 1, 3, 5, 15 min. (left side of the Petri dishes), and with DMSO (the right side of the Petri dishes)

S. aureus. However, there is no published data concer-

ning its activity against microbial biofilms at this time.

It was demonstrated that (+)-usnic acid, which

belongs to the chemical class of dibenzofurandiones

may also influence QS in P. aeruginosa. This may be

important from a clinical perspective, since natural

and synthetic QS inhibitors have been found to atte-

nuate P. aeruginosa virulence and increase suscepti-

bility to tobramycin (Hentzer at all, 2003).

Dental plaque is the most common example of

microbial biofilms and the ethiological cause of cari-

ogenesis and periodontal disease. Our previous

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221

results obtained by CLSM (Confocal Laser ScanningMicroscopy) also revealed a very complex and highly

organized architecture of dental plaque (dense masses

of microorganisms embedded in a microbial mathrix,

biofilm thickness from 50 to 133 micrometers, pre-

sence of columns and canalicular system) (Lazar et al.,

2008).

It was proposed that the respective diseases can

be prevented or treated not only by targeting the

putative pathogens, but also by interfering with the

processes that drive the breakdown in homeostasis.

Our results have demonstrated that the inhibito-ry effect exhibited by usnic acid on dental plaque

development consisted of the total inhibition of the

Gram-positive strains growth and of the expression of

hemolytic properties of microbial strains isolated

from the supra-gingival dental plaque.

These effects demonstrate the potential interfer-

ence of the usnic acid with the intra- and inter-species

signaling mechanisms based on quorum sensing and

response, having in view that this regulatory mecha-

nism is dependent on cell density and is implicated

in the expression of virulence features.

In the presentexperiment, the growth rate of the isolated strains

was changed after the contact with usnic acid, with

the extension of the lag phase to 6-10 h. The prolon-

gation of the lag phase could be correlated with the

inhibition of bacterial cell multiplication by the usnic

acid, and despite the short contact time (few minutes)

this inhibitory activity persisted for many hours, these

results encouraging us to consider that this active

compound could be used for the design of new phar-

maceutical formula with anti-oral biofilm effect.

Taking in consideration that the dental-plaque is a

complex and multispecific community, the selection

by usnic acid of a simplified microbiota could pre-

vent the initiation, the development and the matura-tion of oral biofilm.

Many products of the dental plaque bacteria

reach the subepithelial tissue, causing inflammatory

responses such as increased vascularity and leuko-

cyte diapedesis. Both supragingival and subgingival

plaque may form a hard, mineralized mass called cal-

culus. The surface of calculus harbours bacteria,

which may exacerbate the inflammatory response

(Darveau et al., 1997; Bernimoulin, 2003). Similarly,

plaque accumulation around the gingival margin

leads to a chronic inflammatory host response and anincreased flow of gingival crevicular fluid. The inhi-

bition of the haemolytic properties of the bacterial

strains could be also considered a positive result, ha-

ving in view that the bacterial haemolysins are acting

as pore-forming toxins being are partly responsible

for the inflammatory response occurred in gingivitis

or periodontal disease.

CONCLUSIONS

The interference of the usnic acid with the intra-

and inter-species signalling mechanisms based on

quorum sensing and response and dependent on cell

density , raises the possibility to use usnic acid as an

active principle to be incorporated in some new

pharmaceutical formula intended for the prevention

and treatment of gingivitis or periodontal patholo-

gies. The use of vegetal extracts with QSI effects have

the great advantage of a simple and ecological way to

fight biofilm associated infections, so, it is important

to extend the knowledge of these natural inhibitors of

bacterial communication and of virulence factors

expression.

In vitro study of the inhibitory activity of usnic acid on dental plaque biofilm

Fig. 10. Graphic representation of the quantitative assay of the antimicrobial activityof usnic acid on Lactococcus lactis cremoris

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2. Bernimoulin JP. 2003. Recent concepts in plaque forma-tion. Journal of Clinical Periodontology 30 Issue S5.

3. Campanella L, Delfini M, Ercole P, Iacoangeli A,

Risuleo G. 2002. Molecular characterization and actionof usnic acid: a drug that inhibits proliferation of mousepolyomavirus in vitro and whose main target is RNAtranscription. Biochimie 84:329-334.

4. Cardarelli M, Serino G, Campanella L, Ercole P.1997.Antimitotic effects of usnic acid on different biologicalsystems. Cellular and Molecular Life Sciences 53 (8):667-672.

5. Costerton JW, Lewadowski Z, Caldwell DE, Korber DR,

Lappin-Scott HM. 1995. Microbial Biofilms. Ann. Rev.Microbiol. 49: 711 -745.

6. Darveau RP, Tanner A, Page RC. 2000. The microbialchallenge in periodontitis. Periodontology. 14:12-32

7. Davey ME, O’Toole GA. 2000. Microbial Biofilms: fromEcology to Molecular Genetics. Microbiol. Mol. Biol.Rev. 64: 847-867.

8. Davies, D.G., Parsek, M.R., Pearson, J.P., Iglewski,

B.H., Costerton, J.W., Greenberg, E.P., 1998. Theinvolvement of cell-to-cell signals in the development of a bacterial biofilm. Science, 280: 295-298.

9. Francolini I, Norris P, Piozzi A, Donelli G, Stoodley P.2004. Usnic Acid, a Natural Antimicrobial Agent AbleTo Inhibit Bacterial Biofilm Formation on PolymerSurfaces. Antimicrob Agents Chemother. 48(11),p.4360-4365.

10. Fuqua WC, Winans S.C., Greenberg E.P.1994.Quorum sensing in bacteria: the LuxR-LuxI family of

cell density-responsive transcriptional regulators, JBacteriol., 176:269-75.

11. Fux CA, Costerton JW, Stewart PS, Stoodley P. 2005.Survival strategies of infectious biofilms. Trends inMicrobiology 13 (1): 34-40.

12. Ghione, M., Parrello, D., Grasso, L. 1988. Usnic acid

revisited, its activity on oral flora. Chemioterapia7:302-305.

13. Grasso, L., Ghirardi, P.E., Ghione, M. 1989. Usnicacid, a selective antimicrobial agent againstStreptococcus mutans: a pilot clinical study. Curr. Ther.Res. 45:1067-1070.

14. Hentzer M, Wu H, Andersen JB, Riedel K, Rasmussen

TB, Bagge N, Kumar N, Schembri MA, Song Z,

Kristoffersen P, Costerton JW, Molin S, Eberl L,

Steinberg P. 2003. Attenuation of Pseudomonas aeru-

ginosa virulence by quorum sensing inhibitors. TheEMBO Journal 22 :380-385.

15. Knop W. 1844. Chemisch-physiologische Unter-suchung uber die Flechten. Justus Lieb. Ann. Chern 49:103-124.

16. Lauterwein M, Oethinger M, Belsner K, Peters T,

Marre R. 1995. In vitro activities of the lichen secon-dary metabolites vulpinic acid, (+)-usnic acid, and (-)-usnic acid against aerobic and anaerobic microorga--nisms. Antimicrobial Agents and Chemotherapy,39:2541-2543.

17. Lazar V, Chifiriuc C, Bucur M, Burlibasa M, Sfeatcu R,Stanciu G, Savu B, Traistaru T, Cernat R, Suciu I, Suciu

N. 2008. Investigation of dental- plaque formersbiofilms by optic and confocal laser scanningmicroscopy and microbiological tools, Rev Med Chir.

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18. Lazar V, Chifiriuc C, Oprea E, Bucur M, Iacob M,

Larion C. Osakwe Chiukwunomso E, Cercasov C.2008. The influence of usnic acid on the Pseudomonas

aeruginosa and Staphylococcus aureus biofilms devel-opment. Abstracts book of ThXXXI SOMED (Society of Microbial Ecology and Disease) Congress, 28 - 30 May2008, Stockholm, Sweden.

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21. Manefield, M,, de Nys, R., Kumar, N., Read, R.,

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22. Marsch PD. 2003. Are dental diseases examples of eco-logical catastrophes? Microbiology 149: 279-294.

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INTRODUCTION

Fire blight, a plant disease caused by the bac-

terium Erwinia amylovora, produces serious losses in

apple and pear orchards all over the world. This

pathogen, originally described in North America, is

spreading throughout Europe and the Mediterranean

countries with serious economic consequences [1].

Presently, it is considered a quarantine bacteriumwithin the European Union (EU) and in most South

American countries [2]. This disease was first reported

in Romania, in 1992 in two counties from the South-

east region: Piteºti (Mãrãcineni) and Braila [3]. Fire

blight disease spread rapidly in 1993 and since has

been reported in nine counties [4]. In 1994 fire blight

disease was detected in another five counties, the

total number of affected counties growing to eighteen

in 1995 [3]. In 1996, fire blight disease was reported

in tweenty-three counties [3]. In 1998, thrity-seven

counties were affected by fire blight [5] and in 2000

all counties accross Romania have reported the pres-

ence of the disease [6].

A large number of chemicals have been tested

against fire blight disease: cooper compounds, anti-

biotics, carbamates and miscellaneous compounds.

Two groups of chemicals that have the most impor-

tant role in controlling the disease on apples and

pears trees are cooper compounds and antibiotics.

The main disadvantage of cooper compounds is their

phytotoxicity on host plants, especially pears.

Although resistance of E. amylovora to copper has

not yet been detected we cannot rule out that it will

develop, since resistance to copper has been report-

ed in other phytopathogenic bacteria ( Xanthomonas

campestris pv vesicatoria, Pseudomonas syringae pv.

tomato, Pseudomonas syringae pv. syringae). Of the

large number of antibiotics evaluated against fire

blight, only streptomycin and to some extent, oxytet-

racycline and kasugamycin have met the necessary

requirements to be used in field applications. Alt-

hough streptomycin is considered the most effective

bactericidal agent to be used against fire blight, with

no real phytotoxic problems at the recommended

223

ABSTRACT

Regulatory constraints and environmental and human health concerns have promoted the search for

alternative bio-control strategies of fire blight, a destructive disease of rosaceous plants which pro-

duces serious losses in apple and pear orchards all over the world. The aim of this study was to

establish the antimicrobial activity of Citrus maxima essential oil against Erwinia amylovora. An agar

diffusion method was used for the screening of the inhibitory effect of Citrus maxima essential oil

on bacterial strains growth. The quantitative inhibitory effect of pomelo oil on in vitro biofilm deve-lopment was established by a microtiter colorimetric assay. In order to investigate the ability of

pomelo oil to interfere with bacterial adherence and subsequent biofilm development on leaves

obtained from different pomaceous fruit trees species and cultivars: Pyrus (Napoca, Williams) , Malus

(Golden Delicious) and Cydonia (Aromate), leaves were immersed in pomelo oil for 1, 2, 3, 5 and

10 minutes before exposing them to bacterial colonization. The architecture of bacterial biofilms

developed on leaf surface was analyzed using Confocal Scanning Laser Microscopy (CSLM). Our

results showed that Citrus maxima essential oil inhibited the development of bacterial biofilms on

leaves, pomelo oil being more active on Cydonia (Aromate) leaves when the leaves were treated for

5 minutes. The results obtained from this study may contribute to the development of new bio-con-

trol agents as alternative strategies to protect fruit trees from fire blight disease.

IN VITRO SUSCEPTIBILITY OF ERWINIA AMYLOVORA (BURRILL)

WINSLOW ET. AL. TO CITRUS MAXIMA ESSENTIAL OIL

Luminiþa Mãruþescu1, Crina Saviuc1, Eliza Oprea3, Bogdan Savu2, Marcela Bucur1,Gheorghe Stanciu2, Mariana Carmen Chifiriuc1, Veronica Lazãr1

1)University of Bucharest, Faculty of Biology, Microbiology Immunology Department, Aleea Portocalelor, 1-5, Bucharest, Romania; 2)Politechnical University of Bucharest, BUCHAREST, Romania;

3)University of Bucharest, Faculty of Chemistry, Organic Chemistry Department, Sos. Panduri, Bucharest, Romania

Key words: Erwinia amylovora, fire blight, Citrus maxima, biological control

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doses, its use in agriculture has been prohibited in

many countries, including Romania. The main reason

for this constraint is the development of resistance to

streptomycin not only by E. amylovora but also by

other microorganisms present on plant surface or in

soil or water, including potential human or animalpathogens [7].

During the last 20 years, biological control of

fire blight has advanced from a subject of basic re-

search to a component of an integrated disease con-

trol strategy in the United States of America, but later

on after dissemination of the pathogen under Euro-

pean conditions [8]. Increased research on this aspect

has resulted, in particular, with following factors: the

development of E. amylovora resistance to strepto-

mycin antibiotic, the greater desire of society for safe

production in agriculture and the prohibition of antibiotics for use in fruit growing in most countries

of the European community and other countries. Thus

several of these aspects have promoted the need for

alternative control strategies such as plant-based

essential oils and extracts [9]. Several essential oils

have been reported to possess antibacterial properties,

some of them being found to inhibit E. amylovora, the

causal agent of fire blight [10, 11]. Whether or not, any

of these compounds has any role in the strategy for

control of fire blight has yet to be determined. In this

study we investigated the antimicrobial activity of

Citrus maxima essential oil against E. amylovora.


Bacterial strains and growth conditions

Four strains of E. amylovora were used through-

out the study: Ea7 (isolated in Dambovita county

from apple orchard), Ea16 (isolated in Gorj county

from quince orchard) and Ea 34 (isolated in Ilfov

county from apple orchard). Maintenance media

used was represented by nutrient agar (NA) and NAwith 5% added sucrose (NSA). The inocula of the

bacterial strains were prepared from overnight broth

cultures and suspensions were adjusted to 0.5

McFarland standard turbidity.

Plant material

Fresh leaves from susceptible fire blight disease

pomaceous fruit trees species and cultivars: Pyrus

(Napoca, Williams) , Malus (Golden Delicious) and

Cydonia (Aromate) were used for testing and evalua-

tion of biofilm development. The leaves were sampled

in the autumn (september) from fruit trees orchards in

Ramnicu-Sarat city.

Assessment of the antimicrobial activity of

pomelo essential oil

In vitro antimicrobial tests were carried out by

an adapted agar-disc diffusion technique using 10 µL

of 0.5 McFarland suspension of bacteria directly distri-

buted on King’B medium. The inoculated plates wereincubated for 48 hours at 28°C. Antimicrobial activity

was assessed by measuring the growth inhibition zo-

nes diameters.

Bacterial adherence to leaves surface assays

Fresh leaves from different cultivars of apple, pear

and quince trees were disinfected with 70% ethanol

for several seconds. Leaf fragments were cut with a

borer and deeply immersed into an essential oil solu-

tion for 1, 2, 3, 5, and 10 minutes as pre-treatment, fol-

lowed by immersion into a 0.5 McFarland bacterialsuspension E. amylovora and incubated for two hours

to allow bacterial adherence. Positive control was

represented by the untreated strain and the negative

control by distilled sterile water. After incubation, the

leaf fragments were washed in sterile distilled water

for the removal of the non-adhered bacteria. The leaf

fragments were then immersed in 2 mL of distilled ste-

rile water and thoroughly homogenized at 150 rpm

for 30 minutes, thus the adhered bacteria were put in

suspension and quantified by viable cell counts.

Investigation of pomelo oil influence on bacte-

rial adherence was also qualitatively appreciated by

using the confocal laser scanning microscopy (CLSM)

technique. In this purpose, quince leaf fragments

were pre-treated with pomelo essential oil for 5 mi-

nutes and incubated for 2 hours at 28°C in the pre-

sence of bacterial suspension (108 cfu/mL) prepared

from 24 hour culture of Ea7. The negative control for

this experiment was represented by untreated leaf

fragments and, also by oil treated leaf fragments (for

5 minutes) immersed for 2 hours in the same experi-

mental conditions, i.e. 28°C in distilled sterile water.

The leaf samples were analyzed using a CSLM TGS-SP microscope equiped with PL FLUOTAR and Ar-Fe

laser set at 488 nm.

RESULTS

Effect of Citrus maxima essential oil on the

growth of E. amylovora

The results of the antimicrobial qualitative scree-

ning showed that pomelo essential oil exhibited anti-

bacterial activity against E. amylovora tested strain in

this study. The tested essential oil showed clear inhi-

bitory effect on bacterial growth, quantified by the

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MÃRUÞESCU et al.

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Antimicrobial properties of essential oils obtained

from various plants have been known for a long time,

but mostly reported in human medicine with some

practical applications against several diseases. Some

have been found to inhibit E. amylovora, the causal

agent of fire blight (12). First reports on the in vitro

antibacterial activity of essential oils against E.

amylovora come from Scortichini and Rossi (1989)

and refer to origanum (Origanum vulgare), savory

(Satureja hortensis), white thyme (Thymus vulgaris) and

other plants. Bactericidal activity has been reported

also for essential oils obtained from other thyme

species, Thymbria spicata var. spicata, coming from

the Taurus region of Turkey. Such oils act against se-

veral economically important plant pathogenic bac-

teria, including E. amylovora. Two main constituents:

thymol and carvacrol are suggested to be responsible

for the antimicrobial activity of this thyme oil [13].

In the present study we have investigated the

antimicrobial effect of pomelo essential oil against E.

amylovora. Our results showed that pomelo essential

oil inhibited both the growth rate and the adherence

of fire blight pathogen E. amylovora to fruits leaves.

This effect is exhibited in case of bacterial growth and

adherence. Previous studies concerning the chemical

composition of the pomelo essential oil have found

that the major compound is represented by limonene,

which antimicrobial effect against E. amylovora was

clearly demonstrated [14, 15].

The inhibitory activity exhibited by pomelo oil

on the bacterial adherence to leaves could be ex-

plained by the fact that pomelo oil acts as inhibitor of

226

MÃRUÞESCU et al.

Fig. 3. Essential oil inhibitory effect on the viable Ea 34 bacterial cells demonstratedby the decrease of viable cell count units recovered after adherence to pomacaous leaves

Fig. 4. Evidence of inhibitory effect exhibited by pomelo essential oil on the ability

of Erwinia strains to colonize the leaves surface by CLSM. A negative control (leaf

immersed into distilled sterile water); B. quince leaf incubated for 2 hrs in the presence

of Ea 7 bacterial suspension; C quince leaf immersed into essential oil for 5 minutes

and incubated for 2 hrs in the presence of distilled sterile water.

a b c

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quorum sensing and response mecanism, which in-

terferes with coordinated expression of virulence fac-

tors, including adhesin synthesis and biofilm develop-

ment.

Most microorganisms in nature are attached to

solid surfaces, including plants surfaces and soil par-ticules and to one another in a protective film of

excreted polymers. These so called biofilms are com-

plicated bacterial communities containing scores to

hundreds of different bacterial species that are coop-

erating and/or competing for resources. Biofilms are

constatly forming on leaf surface, adherence being an

essential step in colonisation procesess by epiphytic

and pathogenic microorganisms [16]. In case of bac-

terial species E. amylovora this adherence step is cor-

related with polysaccharide production, a major viru-

lence factor [17]. Several studies performed during

the past decade identified a number of epiphytic bac-

teria (belonging to several species like Pseudomonas

fluorescens, Erwinia herbicola or Bacillus subtilis)

that block colonization sites on the stigma, stimulate

host defense, and in some instances produce antibiot-

ic compounds that inhibit E. amylovora growth [18].

Quantitative analysis of the inhibitory effect on

bacterial adherence ability showed that this effect is

time-dependent, the best results being obtained in

the case of 5 minutes treatment of the leaf fragments.

Moreover, best anti-adherence effect was obtained in

case of quince leaf fragments, the inhibitory activitythus depending also on the plant species.

Confocal microscopy confirmed the results ob-

tained using the microdillution assay.

In conclusion, our study demonstrated that po-

melo essential oil extracted from Citrus maxima exi-

bited antimicrobial activity against E. amylovora plant

pathogen and could be used as a natural product in

the development of new biocontrol strategies against

fireblight disease.

REFERENCES

1. Van der Zwet T. Present worldwide distribution of fireblight. Acta Horticulturae. 2002. 590:33-34.

2. ***Anon. Council Directive 2000/29/EC of 8 May 2000on protective measures against the introduction into theCommunity of organisms harmful to plants or plantproducts and against their spread within the Community.Official Journal of the European Communities. L169. 10 July 2000. 2000. 43:1-112.

3. Severin V. Focul bacterian al rozaceelor (Erwiniaamylovora). 1996.

4. Constantinescu F, Severin V, Bibanu L, Chira E .Protectia Plantelor. 1994. 4 (14): 24-28.

5. Severin V, Constantinescu F, Jianu F. Apperance, expan-sion and chemical control of fire blight (Erwinia

amylovora) in Romania. Acta Hoticulturae. 1999.

6. Vlad F. Epidemiologia si prevenirea focului bacterian al

rozaceelor (Erwinia amylovora). Teza de doctorat. 2003.7. Psallidas P G, Tsiantos J. Chemical control of fire blight.

In Fire Blight: the Disease and its Causative Agent,Erwinia amylovora. Ed. J L Vanneste. Wallingford,United Kingdom: CAB International. 2000. pp. 199-234

8. Johnson KB & Stockwell VO. Biological Control of FireBlight. In Vanneste J.L. (Ed). Fire blight: the Disease andits Causative Agent, Erwinia amylovora. CABI PublishingWallingford. 2000. pp: 319-337.

9. Zeller W. Status of biocontrol methods against fireblight. Phytopathol. Pol. 2006. 39: 71-78.

10. Schortichini M & Rossi MP. In vitro activity of someessential oils toward Erwinia amylovora ( Burrill) Win-

slow ºi colab. Acta Phytopathologia et EntomologiaHungarica. 1989. 4:423-431.

11. Vanneste JL, Voyle MD. Genetic basis of streptomycinresistance in pathogenic and epiphytic bacteria isolatedin apple orchards in New Zeeland. Acta Horticulturae.1998. 489:671-672

12. Scortichini M, Rossi MP. In vitro activity of someessential oils towards Erwinia amylovora (Burill)Winslow et al. Acta Phytopathol. Entomol. Hung.1989. 4: 423-431.

13. Basim H, Yeðen O, Zeller W. Antibacterial effect of essential oil of Thymbra spicata L. var. spicata on someplant pathogenic bacteria. Z. Pflanzenkr. Pflanzensch.

2000. 279 (3): 279-284.14. Vanneste JL. Inhibition of Erwinia amylovora and

Potential Antagonistic Bacteria by Essential Oils andNatural Compounds. Proc. 9th Int. Workshop on FireBlight. Eds. C. Hale and R. Mitchell. Acta Hort. 2002.590.

15. Muhoho Njoroje S, Koaze H, Nyota Karanja P,

Sawamura M. Volatile constituents of redblush grape-fruit (Citrus paradisi) and pommelo (Citrus grandis)

peel essential oils from Kenya. J Agric Food Chem.2005.53:9790-9794

16. Zarnea G. Tratat de Microbiologie generalã. vol. V,Bucuresti, Edit. Academiei, Bucuresti. 1994.

17. Geider K. Exopolysaccharides of Erwinia amylovora:Structure, biosynthesis, regulation, role in pathogenici-ty of amylovoran and levan. In: Fire blight: The diseaseand its causative agent Erwinia amylovora (J. Vanneste,ed.) CABI Publishing. Wallingford Oxon/UK.-NewYork. 2000, pp. 117-140.

18. Olamedi JC. Fire blight (Erwinia amylovora) of rosa-ceous plants: pathogen virulence and selection andcharacterization of biological control agents, PhD the-sis. Univ. Girona Spain. 2005.

In vitro susceptibility of Erwinia amylovora (Burrill) Winslow et. al. to Citrus maxima essential oil

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INTRODUCTION

If prior to 1970, the frequency of infections

caused by opportunistic microorganisms was rela-

tively reduced, most often appearing subsequently to

surgical interventions at the urinary tract level [1],

currently such infections are more frequent and

affecting all organs. The Intensive Care Units (ICUs)

serve as a place for monitoring and care of patients

with potentially severe physiological instability

requiring technical and/or artificial life support. The

level of care in ICUs is greater than that available on

the floor or Intermediate Care Unit [2]. Life support

for vital functions in ICU has lead to an increase of

the survival rate of patients with more and more com-

plex pathology, but unfortunately, it has simultane-

ously induced an increase of risk for severe infec-

tions, the majority produced by antibiotic multiresis-

tant opportunistic microbial strains. This was initially

observed in the case of severe immunodepressed

patients (post-transplant or post-chimiotherapy in

oncological units); subsequently, it has been observed

that any patient admitted in ICU presents a risk for

occurrence and development of severe infections [3].

Factors such as prolonged stay in ICU, increased

number of invasive procedures and aggressive treat-

ments which can affect the host local defence mech-

anism, generate favourable conditions for coloniza-

tion and subsequent infections with selected micro-

bial strains by the “hospital environment”. Also,

“cross infection” - transmitted from patient to patient

via the hands of personnel, contributes to the

increase in the number of severe infections in ICU,

many of them expressed as nosocomial infections

with multiresistant microbial strains [4, 5].

During the last two years, the microbiological

incidence and aetiology at FCI showed an increase in

the number of severe respiratory infections, repre-

senting an important cause for mortality and morbi-

dity. In Fundeni Clinical Institute ICUs, the morbidity

rate by respiratory tract infections represents 27-28%,

228

ABSTRACT

The aim of this study was to investigate the antibiotic resistance profile of 58 Gram negative bacilli

strains (GNB): 36 non-fermentative GNB (NGNB), including 19 strains of Acinetobacter spp., 11 of

Pseudomonas aeruginosa, 6 of Stenotrophomonas maltophilia and 22 enterobacterial strains (14

strains of KEHSs, 6 belonging to the group Proteus-Providencia and 2 Escherichia coli) isolated

from nasal, pharyngeal exudates and also from bronchial secretions, from immuno-depressed

patients admitted in the Intensive Care Unit of Fundeni Clinical Institute. Methods: the antibioticsusceptibility testing was performed according to CLSI 2009 recommendations and the production

of beta-lactamases was investigated by ESBL chromogenic media, double disc diffusion test,

ESBL E-test, Amp C E-test and MBL E-test. Results: 68% of the enterobacterial strains produced

extended-spectrum beta- lactamases (ESBL), 13.63% of them expressing simultaneously the Amp

C enzyme. All enterobacterial strains were susceptible to carbapenems (Imipenem and Ertapenem).

Metallo-beta lactamases production among NGNB strains with resistance to Imipenem was high

(80%), these strains being also multi-resistant to the majority of tested antibiotics with the excep-

tion of colistin. Conclusions: our results showed that the majority of the analyzed strains were

multi-drug resistant. Antibiotic multi-resistance and the increasing number of severe infections

caused by these strains are a major issue for ICU, where patients with severe diseases and desta-

bilized physiological condition are often admitted.

ANTIBIOTIC RESISTANCE OF GRAM NEGATIVE

BACILLI STRAINS ISOLATED FROM THE INTENSIVE CARE UNITIN FUNDENI CLINICAL INSTITUTE, BUCHAREST, ROMANIA

Elvira Borcan1, Camelia Mihaela Ghiþã1, Mariana Carmen Chifiriuc2,

Luminiþa Mãruþescu2, Cãtãlina Isar1, Veronica Lazãr2

1)Bacteriology Laboratory of Fundeni Clinical Institute, Sos. Fundeni 258, Bucharest, Romania; 2)University of Bucharest,Faculty of Biology, Microbiology Immunology Department, Aleea Portocalelor, 1-5, Bucharest, Romania

Key words: Gram negative bacilli, ICU, antibiotic resistance, E-test ESBL, E-test Amp C, E-test MBL.

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whereas, the mortality rate is 50%. In this care unit,

oro-tracheal intubation and tracheostoma are fre-

quently practiced. In this respect, for intubated pa-

tients, (URT) colonization agents can become lower

respiratory tract (LRT) colonization agents. Further-

more, depending on the number of intubation days,the lower host’s immune defence, and the virulence

of the microorganisms involved, the LRT coloniza-

tion agents can produce severe infections. In the last

years the cases of Gram-negative bacilli (GNB) colo-

nization at the URT level has increased in patients

from ICUs, the majority of patients presenting multi-

ple colonisations with GNB.

The purpose of this study was to investigate the

antibiotic resistance profiles of GNB strains, the

major etiological agents of URT and LRT infectious

pathology, in mechanically ventilated patients inICUs of Fundeni Clinical Institute.


During the present study, 58 bacterial strains iso-

lated from URT and LRT secretions were analysed.

These secretions were represented by pharyngeal and

nasal exudates and bronchial secretions sampled

from ICU orotracheal intubated patients, most of

them immunodepressed. The microbial strains were

identified with the help of the BD Phoenix equip-ment. The investigation of antibiotic resistance pat-

tern was performed by the following methods: agar

disk diffusion method according to CLSI 2009 recom-

mendations [6], double disk test for revealing the syn-

ergisms, ESBL E-test, Amp C E-test for beta-lactamase

production and MBL E-test for metallo-betalactamase

production in case of GNB non-fermentative. The

beta-lactamase production was assessed by the follo-

wing methods:

Agar disc diffusion method: Muller-Hinton

plates were inoculated with bacterial suspensions of

0.5 McFarland standard turbidity (corresponding to107-108 CFU/mL) and aerobically incubated at 37oC

for 18-20 hours [7, 8]. The results were interpreted as

sensitive (S), intermediary (I) or resistant (R), accord-

ing to CLSI recommendation 2009. The following

antibiotics were used:

- for enterobacterial strains: ceftazidime (CAZ),

amoxicillin-clavulanic acid (AMC), cefotaxime (CTX),

ertapenem (ETP), imipenem (IMI), amikacin (AK), tri-

methoprim- sulfometoxazole (SXT), ciprofloxacin (CIP),

levofloxacin (LEV), piperacillin-tazobactam (TAZ),

gentamycin (G), cefoperazone+sulbactam (CES), to-bramicin (TOB), cefoxitin (FOX).

- for non-fermentative GNB, Pseudomonas aeru-

ginosa and Acinetobacter spp.: ticarcillin-clavulanic

acid (TIM), ceftazidime (CAZ), ceftriaxone (CRO), cefe-

pime (FEP), imipenem (IMI), amikacin (AK), gentamicin

(G), netilmicin (NET), piperacillin-tazobactam (TAZ),

ciprofloxacin (CIP), levofloxacin (LEV), cefopera-zone+sulbactam (CES) (Oxoid, England disks) and

colistin (CS), the last being confirmed by E-test. Ste-

notrophomonas maltophilia was tested to levoflo-

xacin (LEV), trimethoprim- sulfometoxazole (SXT), ti-

carcillin- clavulanic acid (TIM), ceftazidime (CAZ).

Double disc test: amoxicillin - clavulanic acid

(AUG) disk was placed in the middle of the plate and

around at 15 mm distance were placed: ampicillin

(AMP), a first (Cephalotin-KF), second (Cefuroxime-

CXM), third (Ceftriaxone-CRO) and fourth generation

cephalosporin (Cefepime-FEP).Chromogenic ESBL media (Biomerieux, France):

the samples were inoculated on a media that contains

a mixture of antiobiotics incuding Cepodoxime,

known as marker for this type of resistance mechan-

sims [9]. The chromogenic substrate allowed also the

rapid identification of enterobacterial strains produc-

ing ESBL, after 20-24 hours of incubation on this

medium the E. coli strains generating pink/purple, the

KEHS group green/blue colonies and the Proteus

group (Proteus, Providencia, Morganella) light/dark

brown colonies.

E-test ESBL: for the confirmation of extended-spectrum beta-lactamase production and quantitative

determination of antimicrobial susceptibility E-test

ESBL (AB Biodisk, Suedia) strips containing Ceftazi-

dime/ceftazidime + clavulanic acid (TZ/TZL) or Cefe-

pime/Cefepime + clavulanic acid (PM/PML) were used.

The strips consist of a thin, inert of non-porous plas-

tic carrier. One side of the strips is calibrated with mi-

nimum inhibitory concentrations (MIC) reading scale,

in µg/ml and the reverse surface carries two prede-

fined exponential gradients: TZ: 0,5-32 µg/ml and

TZ/TZL:TZ-0,064-4 µg/ml and AMC=4 µg/ml,respectively PM: 0,25-16 µg/ml and PM/PML: PM:

0,064-4 µg/ml and AMC 4 µg/ml.

E-test Amp C: all enterobacterial strains were tes-

ted for Amp C enzyme production. We used E-test

Amp C strips (AB Biodisk, Sweden) containing

Cefotetan (CN)/ Cefotetan+ cloxacillin (CNI).

E-test MBL: NGNB intermediary or resistant to

Imipenem on disc diffusion were tested for the pro-

duction of metallo beta-lactamases. E-test MBL (AB

Biodisk, Sweden) strips were used that incorporate

concentration gradients of two agents: imipenem (IP)

and imipenem + EDTA (IPI).

Antibiotic resistance of Gram negative bacilli strains isolated from the Intensive Care Unit in Fundeni Clinical Institute

229

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isolates have limited therapeutic options because of

an associate resistance to aminoglycosides and/or

quinolone [10, 11].

The routine susceptibility tests performed by

clinical laboratories can detect ESBL production

(double disk test or synergy) but fail to detect Amp C

production [12, 13]. This aspect could lead to unsuc-

cessful therapy of the patients, as the carbapenems

remains the only effective agents against these

strains.

The Amp C beta lactamases are enzymes capa-

ble of hydrolyzing all beta-lactams drugs, excepting

carbapenems. Amp C enzymes are seen in organisms

such as Citrobacter freundii, Enterobacter cloacae,

Morganella morganii, Hafnia alvei and Serratia

marcescens and are typically inducible by beta-lac-

tam antibiotics such as cefoxitin and imipenem, but

poorly induced (if at all) by the third- or fourth-gene-

ration cephalosporins [14]. These strains are resistant

to all marketed beta-lactamase inhibitors [15]. The

problems show up when both ESBL and Amp C are

produced in the same time, and then mask each

other. The initially 4GC-Cefepime recommended as

an alternative cephalosporine for ESBL detection inthe presence of Amp C beta lactamase, in parallel with

resistance to cefoxitin used as a screening test, does

not reliably indicate Amp C production [16, 17]. The

double disk test could detect synergy and ESBL pro-

ducers, but not Amp C. All enterobacterial strains were

tested for Amp C production (fig. 7) and 18.18% were

confirmed as Amp C-producing strains, 3 of them ex-

pressing simultaneously Amp C and ESBL enzymes

and one single strain producing only Amp C beta lac-

tamase and showing an inducible cephalosporinase

phenotype on disk- diffusion method (fig. 8).

NGNB were multidrug resistant (MDR), with ahigh level of resistance to carbapenems, aminoglyco-

Antibiotic resistance of Gram negative bacilli strains isolated from the Intensive Care Unit in Fundeni Clinical Institute

231

Fig. 6. Positive E-test ESBL in Klebsiella pneumoniae

Fig. 7. Negative E-test Amp-C and positive

E-test ESBL in Klebsiella pneumoniae.

Fig. 4,5. High level resistance to penicillins, first and second generation of cephalosporins

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sides and quinolones, 26.66% of tested strains being

susceptible to colistin only (Fig. 9-10).

The increasing resistance to the third generation

cephalosporins made these drugs not available as the-

rapeutic options. Instead, colistin, piperacillin-tazo-bactam and sulperasone are the most effective drugs.

Antibiotic susceptibility data for Stenotrophomo-

nas maltophilia are not representative for drawing a

conclusion because of the small number of tested

strains (Fig. 11).

All NGNB tested strains were susceptible to co-listin (fig 9). We have noticed an increasing rate of

232

BORCAN et al.

Fig. 8. Enterobacter sp. - inducible cephalosporinase aspect (left) andpositive test for Amp C production; E-test for ESBL-negative strain (right).

Fig. 10. Pseudomonas aeruginosa MDR (left), susceptible to colistin (right).

Fig. 9. The comparative levels (%) of antibiotic resistance/susceptibility inPseudomonas aeruginosa and Acinetobacter sp. 22, 66% of Pseudomonas aeruginosa and Acinetobacter spp. strains were susceptible to colistin only (fig 10).

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SELECTIVE REFERENCES

1. Carr S, Unwin N, Pless-Mulloli T. An Introduction toPublic Health and Epidemiology. Ed. Open UniversityPress, Berkshire, UK 2007.

2. Hall J, Schmidt G, Wood L. Principles of critical care.

3rd Ed. The McGraw-Hill Company Ic, USA. 2005

3. Richards M. Nosocomial infections in medical intensivecare units in USA. Journal of Critical Care Medicine

1999. 27: 887-892.

4. Goldmann DA. The role of barrier precautions in infec-tion control. The Journal of Hospital Infections. 1991. 18Suppl A:515-23.

5. Lazãr V, Herlea V, Bulai D, Ciolac-Russu A, Cernat R.

Some aspects related with health risk for human carriers of antibiotic resistant enterobacteria strains. Analele Univer-

sitatii din Bucuresti, Secþia Biologie. 1999. XLVIII: 3-10.

6. Clinical Laboratory Standard Institute. Performance stan-

dards for antimicrobial disk susceptibility tests. 18th

ed.Approved standard M2-A8. Wayne,Pa. Clinical La-boratory Standards Institute, 2004.

7. Centre de l’Enseignement de l’Institut Pasteur de Paris.

Milieux de culture et techniques. Cours de Bacteriologie

Medicale 2000.

8. Courvalin P. Agents antimicrobiennes - Compte rendude la Conférence BioMerieux: Evolution et detection deresistance bacterienne. Bull. Soc. Franç. Microbiol. 1996.11(3): 239

9. Paterson DL, Bonomo RA. Extended-spectrum ß-lacta-mases: a clinical update. Clin Microbiol Rev . 200518(4):657-86.

10. Lazãr V, Herlea V, Bulai D, Ciolac-Russu A. Studiulfrecvenþei tulpinilor de enterobacterii rezistente la anti-biotice la bolnavi spitalizaþi. Analele Universitatii din

Bucureºti, Secþia Biologie. 1998. XLVII: 69-78.

11. Levy SB. The antibiotic paradox: How miracle drugsare destroying the miracle. Ed. Plenum Press, N.Y.1992, pp. 53-104.

12. Livermore D, Paterson D. Extended-spectrum ß-Lac-tamase in Resistance. Current Medicine Group Ltd.2006, pp. 18-19.

13. Philippon A, Arlet G, Jacoby GA. Plasmid-determinedAmp C-type beta-lactamase. Antimicrob Agents

Chemoter . 2002. 46 : 1-11.

14. Hanson N. Amp C beta-lactamase: what do we need toknow for the future? J Antimicrob Chemother . 2003.52: 2-42.

15. Tzelepi E, Gaikkoupi P, Sofianou D, Loukova V,

Kemeroglou A, Tsarikis A. Detection of extended spec-trum beta-lactamases in clinical isolates of Enterobactercloacae and Enterobacter aerogenes. J. Clin. Microbiol.2000. 38: 542-546.

16. Thomson KS. Controversies about extended-spectrum

and Amp C beta-lactamase. Emerg Infect Dis. 2001.7:333-336.

17. Walsh TR, Toleman MA, Poirel L et al Metallo-ß-lacta-mases: the quiet before the storm? Clin Microbiol Rev.

2005.18: 306-25.

18. Beesley T, Gascoyne N, Knott-Hunziker V, Petursson

S, Waley SG, Jaurin B. et al. The inhibition of class cbeta-lactamases by boronic acids. Biochem J. 1982.209: 229-233.

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Baquero F, Scoichet BK. The complexed structure andantimicrobial activity of a non- beta-lactam inhibitor of Amp C beta lactamase. Protein Sci. 1999. 8: 2330-7.

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INTRODUCTION

Traditionally, Streptococcus agalactiae or group

B streptococcus (GBS) was regarded as a neonatal

pathogen vertically transmitted to neonates from co-

lonized mothers. The active screening of maternal

GBS carriage and the intrapartum antibiotic use re-

sulted in a marked reduction of neonatal invasive in-

fections, with undeniable benefits for the so-called

early-onset disease (1). However, the epidemiology

of GBS disease underwent challenging modifications

reflected in the growing incidence of invasive infec-

tions in nonpregnant adults and the antimicrobial re-

sistance associated with the widespread intrapartum

chemoprophylaxis for GBS carriers. Thus, continuous

surveillance to monitor trends across various age

groups as well as efforts to identify suitable solutions

for each of them are needed.

In our country, streptococcal infections due to

GBS are included among the prioritized communica-

ble disease and their monitoring is mandatory to rely

on contemporary laboratory data regarding microbio-

logical aspects of locally circulating strains.

235

ABSTRACT

In the attempt to enrich the local contemporary laboratory data regarding the group B streptococ-

cus (GBS) colonization, isolates obtained from the vaginal swab cultures were characterized for

their serotype distribution and antibiotic susceptibility. The 100 GBS isolates analyzed were col-

lected during a four-month period of year 2009 from women screened in ambulatory for vaginal

carriage of GBS.

The GBS isolates were classified based on their capsular polysaccharide structures using

commercially available antisera. Susceptibility to penicillin, ampicillin, erithromycin, clindamycin,

tetracycline, ofloxacin, and chloramphenicol was initially tested using antibiotic disk diffusion tech-nique according to CLSI guidelines. Minimum inhibitory concentrations of erythromycin and tetra-

cycline for the isolates with reduced susceptibility were evaluated according to the CLSI criteria and

macrolide-lincosamide-streptogramin B (MLSB) resistance was investigated by a double-disk test

with erythromycin and clindamycin disks.

All the GBS isolates were serotypeable. Their distribution comprised six different serotypes of

which serotypes II (26%), III (26%), and Ia (19%) prevailed and no serotype VI, VII, and VIII iso-

lates were found. Overall, the GBS isolates were fully susceptible to penicillin and ampicillin, but

the rates of susceptibility to the other antimicrobial agents tested were decreased, ranging from

87% for chloramphenicol to 5% for tetracycline. Reduced susceptibility to clindamycin and eryth-

romycin was detected in 18% and 19% of isolates, respectively. For the latter, 84% displayed aconstitutive MLSB phenotype, 11% had an inducible MLSB phenotype, and M phenotype was

expressed by 5% of them. Erythromycin-resistant GBS isolates displayed concurrently resistance to

at least one more antibiotic.

In conclusion, according to our study the most frequent GBS serotypes isolated from the vaginal

microflora were II and III, followed by serotype Ia. While the GBS isolates remain susceptible to

beta-lactams, resistance to alternative antimicrobial drugs such as erythromycin and clindamycin

seems to be an increasing concern for our region. Further phenotypic and genotypic studies are

required to identify specific aspects of GBS strains colonizing or infecting the local population.

GROUP B STREPTOCOCCUS COLONIZATION OF ROMANIAN WOMEN:PHENOTYPIC TRAITS OF ISOLATES FROM VAGINAL SWABS

Codruþa-Romaniþa Usein*, Anca Petrini, Raluca Georgescu,Laura Grigore, Monica Strãuþ, Vasilica Ungureanu

National Institute of Research-Development for Microbiology and Immunology „Cantacuzino”

Key words: group B streptococcus, serotype distribution, antibiotic susceptibility

* Corresponding author: Codruþa-Romaniþa Usein; Molecular Microbiology Laboratory, N.I.R.D.M.I. „Cantacuzino”; e-mail: [email protected]

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236

We present some preliminary results regarding

the serotype distribution and antibiotic resistance of

GBS isolates collected during an ongoing study con-

ducted to obtain reliable information on vaginal GBS

carriage in Romanian women.


Female subjects and GBS strains

The GBS isolates were collected from May to

August 2009 in the Clinical Laboratory from National

Institute of Research-Development for Microbiology

and Immunology „Cantacuzino”, which mainly serves

demands of human microbiological investigations

from physician offices in the Bucharest metropolitan

area. The isolates originated in the vaginal swab cul-

tures of 100 women which were classified accordingto age and pregnancy status. The female population

with positive GBS cultures ranged in age from 19 to

74 years old. The reproductive age lot of females was

defined as aged between 19 to 40 years.

Isolates were identified to the species level by

Gram staining, colony morphology, catalase test, and

the use of a commercial latex agglutination kit (Slidex

Strepto B; Bio-Mérieux, France). Only one isolate per

vaginal specimen was included in the GBS collection

to be further characterized.

Serotype identification

Strains were serotyped with commercially availa-

ble antisera against GBS serotypes Ia, Ib, II-VIII (Sta-

tens Serum Institute Diagnostica, Denmark).

Antibiotic susceptibility testing

Antimicrobial susceptibility of all GBS isolates

was determined by Kirby-Bauer disk diffusion me-

thod and the results were interpreted according to the

guidelines of Clinical and Laboratory Standards In-

stitute (CLSI) (2). The following antimicrobials weretested: penicillin G (10 IU), ampicillin (10 mg), eryth-

romycin (15 mg), clindamycin (2 mg), ofloxacin (5 mg),

tetracycline (30 mg), and chloramphenicol (30 mg).

Any isolate that had reduced susceptibility to

erythromycin and/or tetracycline had minimum inhi-

bitory concentrations (MICs) determined by Etest (AB

BIODISK) or agar plate dilution method. The MIC va-

lues were interpreted according to CLSI guidelines (2).

Isolates defined as macrolide-resistant (interme-

diate or resistant to erythromycin) were further classi-

fied for macrolide-lincosamide-streptogramin B (MLSB)

resistance on the basis of a double-disk test with

erythromycin (15 mg) and clindamycin (2 mg) (2, 3).

Blunting of the clindamycin inhibition zone near the

erythromycin disk indicated an inducible type of

MLSB resistance (iMLSB), and resistance to both ery-

thromycin and clindamycin indicated a constitutive

type of MLSB resistance (cMLSB). Susceptibility to

clindamycin with no blunting indicated the M resis-tance phenotype (macrolide efflux).

RESULTS

Serotype identification

All the GBS isolates colonizing the women en-

roled in this study were typable based on their cap-

sular polysaccharide structures and six different sero-

types were identified (Table 1). Overall, more than

half (52%) of the isolates belonged to serotypes II and

III, and isolates of serotypes VI, VII, and VIII were not

found. Serotype Ib was the least frequent, accounting

for only 3% of the total number of GBS isolates.

Antibiotic susceptibility testing

All GBS isolates were susceptible to penicillin

and ampicillin. For the rest of antimicrobial agents

tested the prevalence of susceptible isolates ranged

between 5% for tetracycline to 87% for chloram-

phenicol (Table 2).

For the tetracycline-resistant isolates the MICs

range was 4 ->128 mg/ml, with MIC50=32 mg/ml andMIC90=64 mg/ml.

Overall, macrolide-resistance was expressed by

19 vaginal GBS isolates (19%) and their range of

MICs was 0.5 mg/ml - > 256 mg/ml (MIC50 and

MIC90 > 64 mg/ml). Of these isolates, 16 had cMLSB

phenotype, 2 isolates showed iMLSB phenotype, and

one expressed M phenotype. All the erythromycin-

resistant GBS isolates displayed concurrently resist-

ance to at least one more antibiotic. Two GBS iso-

lates expressed resistance to clindamycin and sus-

ceptibility to erythromycin.The evaluation of the relationship between se-

rotype and resistance to erythromycin and/or clin-

damycin revealed that none of serotype Ia isolates

were resistant, while the rates of susceptibility to these

antibiotics differed within the other GBS serotypes

(Table 3).

DISCUSSION

Within our GBS collection the prevalence of the

various serotypes was in accordance with other find-

ings showing that serotypes III, II and Ia are the most

USEIN et al.

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frequently identified among women in European

countries (4). When comparing pregnant and non-

pregnant women of reproductive age, the variations

of this distribution did not influence the overall pre-

dominance of serotypes II and III, but showed that

slightly more pregnant females were colonized with

GBS serotype II. As for the prevalence of serotype Ia

isolates, they were more frequently identified among

the nonpregnant subjects. Differences in serotype

distribution were also observed between reproduc-

tive age and non-reproductive age women, the latter

apparently carrying more frequently GBS strains be-

GBS vaginal isolates: phenotypic traits

237

Table 1. GBS serotypes by subjects’ age and pregnancy status

Table 2. Antibiotic susceptibility patterns of vaginal GBS isolates

Table 3. Erythromycin (E) and clindamycin (DA) resistance accordingwith the serotype distribution of vaginal GBS isolates

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longing to serotypes IV and V. However, as for the

pregnant women lot, the small sample size of the wo-

men classified in the non-reproductive age group pre-

vented us from making statistically significant com-

parisons, our preliminary findings requiring to be con-

firmed on an extended number of subjects of interest.In the attempt to update the knowledge regar-

ding GBS residing in female microflora, we also in-

vestigated the susceptibility to antibiotics expressed by

the vaginal isolates. All the women carried GBS strains

that were uniformly susceptible to betalactams.

However, other published reports have revealed the

existence of GBS isolates that acquired insusceptibil-

ity to betalactams, including penicillins. Their true

clinical significance as well as their characterization

on a molecular basis is yet to be clarified, but the

screening for reduced penicillin susceptibility in GBSisolates colonizing nonsterile body sites seems to

become very important (5).

According to our findings regarding the preva-

lence of resistance to eythromycin and clindamycin

among vaginal GBS isolates, 19% and 18% of the

GBS isolates studied were erythromycin and clin-

damycin resistant, respectively. In other words, in

case of allergy to betalactams the women colonized

with these strains would not benefit of these second

choice antibiotics. Similar recent results of preva-

lence for erythromycin-resistant GBS isolates were

found in Portugal for GBS isolates collected from co-lonized reproductive-aged women (6). However, in

our study less GBS isolates possessed iMLSB pheno-

type than the Portuguese ones (one vs. eight isolates).

In addition, among the Romanian GBS isolates we

found two resistant only to clindamycin and not to

erythromycin. This uncommon phenotype was previ-

ously observed in GBS strains (7-10). Its biochemical

and genetic basis remains obscure, but one explana-

tion could be clindamycin modification by lincosa-

mide nucleotidyltransferases, encoded by the lin(B)

gene, which can confer resistance to lincosamidesbut does not affect macrolides, which was first des-

cribed in Enterococcus faecium (11) and also found

in Streptococcus agalactiae (7). However, this gene

or other genes conferring resistance to macrolides

and lincosamides were not identified by others in

GBS isolates with a similar resistance (9).

When assessing erythromycin and/or clindamy-

cin resistance of GBS isolates and their serotype dis-

tribution, it seemed that serotypes Ib, V, and IV had a

higher rate of association with these resistance phe-

notypes. However, the small number of isolates pre-

vented us from considering these results as conclu-sive evidence for this trend.

In agreement with previously published studies

on GBS isolates from European countries such as

Spain (12), France (13), Czech Republic (14) we also

observed a very high prevalence (96%) of tetracy-

cline-resistant GBS isolates with a broad distribution

among the different serotypes.Tetracycline resistance mechanisms involve pro-

tection of the ribosome as an antibiotic target by ribo-

somal protection proteins or reduction of the intra-

cellular antibiotic concentration by efflux proteins

(15). The tetracycline resistance genes identified in

streptococci include tetK , tetL, tetM, tetO, tetQ, and

tetT (16). Considering the decline in tetracylines use

in streptococcal infections, the high rates of tetracy-

cline resistance seem to persist even when selection

pressure was reduced. In addition, another matter of

concern is the possibility of colocation of tet

geneson transposons with other antibiotic resistance genes,

any one of which if selected maintaining the rest.

Resistance to fluoroquinolones has already been

described in GBS isolates (17-20). Within our collec-

tion we also found ofloxacin non-susceptible (inter-

mediate and resistant) GBS isolates. The prevalence of

resistance to ofloxacin (15%) was comparable with the

prevalence of resistance to chloramphenicol (13%).

However, the first is more concerning taking into

account that unlike chloramphenicol, fluoroquino-

lones are among the most consumed antibacterial

agents worldwide, being used to treat a great varietyof infections in adults, GBS infections included.

In conclusion, according to our study the most

frequent GBS serotypes isolated from the vaginal

microflora were II and III, followed by serotype Ia.

While GBS isolates colonizing the local female popu-

lation are still fully susceptible to penicillin and ampi-

cillin, the changes in their susceptibilities to other an-

tibiotics, such as macrolides, lincosamides and fluroro-

quinolones are already significant.

More regional data obtained from surveillance of

GBS capsular antigenicity and resistance patterns areneeded to guide appropriate approaches to disease

prevention and therapy. Thereby, further phenotypic

and genotypic studies are required to identify speci-

fic aspects of GBS strains colonizing or infecting the

local population.

ACKNOWLEDGEMENTS

This study was supported by grant IDEI 721 from

the Romanian Ministry of Education and Research.

238

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INTRODUCTION

Helicobacter pylori is fastidious, small, curved,

microaerophilic and highly motile Gram negative bac-

teria. It is about 2.5 - 5.0 mm long and 0.5 - 1.0 mm

wide with 4-6 unipolar sheathed flagella [1]. H. py-

lori is identified on the basis of colony morphology(translucent colonies varying in size from barely de-

tectable with the naked eye to approximately 3 mm);

H. pylori are Gram-negative, curved rods that are ure-

ase, catalase and oxidase positive. The addition of

tetrazolium salts aids in the identification of H. pylori

colonies cultured on agar media [2]. H. pylori is the

etiologic agent of a variety of gastrointestinal disor-

ders including chronic active gastritis, peptic ulcer

and gastric cancer [3]. It is clear that virtually all H.

pylori infected subjects develop local and systemic

immune response against this organism [4], as with

most bacterial infections, H. pylori stimulates an im-

mune response and circulating antibodies appear in

the peripheral blood stream. This is the basis for se-

rology tests to diagnose H. pylori [5]. It has beenshown that the immune response to H. pylori at the

mucosal level is predominantly of the IgA type, while

the systemic immune antibody response essentially

consists of the IgG class [6]. Immunoglobulins of the

IgM class do not appear to differ between H. pylori

positive and negative patients [7].

Performance varies significantly between diffe-

rent commercial serologic kits, with the highest ex-

ceeding 90% in sensitivity and specificity and the lo-

240

ABSTRACT

Helicobacter pylori (H. pylori) is the etiologic agent of a variety of gastrointestinal disorders. The

rationale of the current study is to evaluate six enzyme immunoassays for detection of anti-H.

pylori immunoglobulin G (IgG) and IgA in Jordanian patients. Biopsy specimens and blood sam-

ples were obtained from patients underwent the endoscopy unit at Al-Bashir hospital in Jordan. The

serum samples were investigated for the presence of anti- H. pylori IgG and IgA antibodies inpatients with positive H. pylori biopsy samples. The results showed that IgG utilizing kits are more

sensitive than of IgA kits and the IgA kits are more specific than of that IgG kits. Moreover, the biop-

sy is seemingly the gold standard for diagnosis of H. pylori is followed by H. pylori culture on bru-

cella agar medium. An imperfect relation between the presence of H. pylori infection and the anti-

body response was existed that could be explained either because of the unsatisfactory sensitivi-

ties and specificities of the commercial kits used or because of weak immunological response in

our patients to H. pylori antigens. Collectively, the H. pylori diagnosis that depends on the detec-

tion of anti- H.pylori antibodies in the hospital setting and in the screening programs should con-

sider another test for confirmation the initial diagnosis.

INVESTIGATION OF HELICOBACTER PYLORI INFECTION

IN JORDANIAN PATIENTS USING SIX ENZYME IMMUNOASSAYS

FOR IMMUNOGLOBULIN G (IGG) AND IGA TESTING

Hasan Abusini1, Khaled Abuelteen2, Ali Elkarmi2, Faris Q. Alenzi3, Mahmoud Lotfy1,4

1)Department of Applied Medical Sciences, Jouf University, Qurayat, Saudi Arabia; 2)Department of Biology and Biotechnology, Faculty of Science, Hashemite University, Jordan; 3)Department of Clinical Laboratory Sciences,

College of Applied Medical Sciences, King Saud University, Al-Kharaj, Saudi Arabia; 4)Molecular and Cellular Biology Department, Genetic Engineering and Biotechnology Research Institute, Minufiya University, Sadat City, Minufiya, Egypt

Key words: Helicobacter pylori, IgA, IgG, Jordan

Conflict of interest: Non Declared

* Corresponding author: Dr. Mahmoud Lotfy - Current Address: Department of Applied Medical Sciences, Jouf University, Qurayat, P.O. 1300,Saudi Arabia. Permanent Address: Molecular and Cellular Biology Department, Genetic Engineering and Biotechnology Research Institute,Minufiya University, Sadat City, P.O 22857-79, Egypt. E-mail: [email protected]

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west having less than 70% in sensitivity and less than

50% in specificity [8]. The purpose of this study was

to evaluate six commercially available ELISA kits for

detection of anti-H. pylori IgG and IgA in patients

attending the Gastroenterology unit at Al-Bashir hos-

pital in Jordan. Moreover, to assess the culture on dif-ferent media for detecting H. pylori isolates.


Patients

The study group included 71 individuals aged

13 to 83 years (37.7±1.5 years) who underwent en-

doscopy at the Gastroenterology unit. The study pro-

tocol respected the most recent Declaration of Hel-

sinki [9], and all of the patients gave consent to the use

of their sera and clinical data for research purposesafter being informed about the nature of the study.

Media

Brucella medium (HiMedia Laboratories, Mum-

bai, India), Columbia medium (Biomark, India) and

Brain Heart Infusion (BHI) medium (Liofil, Italy) were

prepared in accordance with the manufacturer’s in-

structions. The antibiotic supplement in each media

are identical; each medium contains 10 mg of van-

comycin per liter, 2.5 IU of polymyxin B per liter, 5 mg

of trimethoprim per liter, 2 mg of amphotericin B perliter and 5 mg of cefsulodin per liter. All media were

further supplemented with whole blood (5%) and 40

mg of 2,3,5-triphenyltetrazolium chloride (Sigma,

USA) per liter.

Biopsy Specimens

During upper endoscopy, gastric mucosal biop-

sy was taken for histopathology, formalin fixed, cut

into five mm sections, stained with hematoxylin and

eosin and viewed for the presence of H. pylori

(Fig.1). A positive result was considered indicative of active H. pylori infection as reported by us earlier

[10]. Additional two biopsies one from the antrum

and the other from the corpus were taken for micro-

biological studies as shown below.

The biopsy specimens were collected into tubes

containing three ml of brucella broth supplemented

with 0.5% (w/v) bovine serum albumin. The tubes

were transported to the laboratory on an ice box in

less than three hours.

Isolation and Identification

The biopsy specimens were finely minced with

a sterile scalpel blade in one ml of brucella broth in

Helicobacter pylori in Jordanian Patients

241

sterile conditions. One drop of the tissue homoge-

nate was inoculated onto each of the three selective

media plates. After incubation at 37°C under mi-

croaerophilic conditions (10% CO2, 5% O2, and 98%

humidity) for 10 days. Isolates were identified as H.

pylori on the basis of colonial morphology, in addi-

tion to the negativity for Gram staining, nitrate reduc-

tion, glucose and sucrose fermentation and indole

production, moreover, the positivity for catalase, oxi-

dase, and urease tests.

Enzyme Immunoassays:

Venous blood samples (5ml) were obtained from

each patient at the time of endoscopy. The serum was

separated, divided into aliquots, and stored at -20°C.

All sera were tested with six ELISA kits: Anti- Heli-

cobacter pylori (Anti-Hp) IgG ELISA (BioCheck Inc.

USA), Anti-Hp IgA ELISA (BioCheck Inc. USA), Anti-

Hp IgG ELISA (Biotest, Germany), Anti-Hp IgA ELISA

(Biotest, Germany), Anti-Hp ELISA IgG (Euroimmun,

Germany), and Anti-Hp ELISA IgA (Euroimmun, Ger-

many). The assays were performed in accordance

with the manufacturers’ instructions.

Statistical Analysis

Nominal data were analyzed using chi-square

test and P values < 0.05 were considered statistically

significant. Data were tabulated and analyzed using

the SPSS 17 statistical package (SPSS Inc. Chicago,

IL). The sensitivity, specificity and accuracy were cal-

culated as described by us earlier [11].

Figure 1: Shows a section from the corpus of aninfected patient. M: Mucus secreting cells. HP: H.

pylori (X 1000).

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RESULTS

Culture

Brucella agar medium gave the highest isolation

rate, Out of 71 positive biopsy specimens; isolation

of H. pylori was achieved in 61 antrum specimens

(86%). On the other hand, 57 out of 71 corpus biop-

sy specimens (80.3%) were culture positive. The num-

ber of patients from whom H. pylori was isolated

from either the corpus or antrum specimens was 70

out of 71 infected, thus H. pylori was recovered in

98.6% of the patients infected. Using Columbia agar

the organism was recovered from 54 antral speci-

mens (76%). On the other hand when corpus speci-

mens were cultured the recovery was achieved from

50 specimens (70.4%). The total number of patients

from whom H. pylori was isolated either from corpus

or antrum specimens was 63, thus H. pylori wasrecovered in 88.7% of the patients infected. BHI gave

the lowest isolation rate for H. pylori. Out of 71 pos-

itive antral biopsy specimens, the organism was iso-

lated only from 47 specimens (66.2%). In the oppo-

site direction when corpus specimens were cultured,

the number of specimens retreat the organism was 48

specimens (67.6%) (table 1).

Enzyme Linked Immunosorbent Assay (ELISA)

The serum samples were investigated for the

presence of anti-H. pylori

IgG and IgA antibodies inpatients with positive H. pylori biopsy samples. The

results showed that IgG utilizing kits are more sensi-

tive than of IgA kits and the IgA kits are more specif-

ic than of that IgG kits. Euroimmun IgG kit is the most

sensitive one, on the hand, BioCheck IgA is the most

specific kit. The sensitivities, specificities and accura-

cies of the six kits are illustrated in table (2).

DISCUSSION

H. pylori infection is prevalent in Jordan as evi-denced by several investigators [12-14]. In the current

study, we evaluated seventy one positive H. pylori

infected Jordanian patients with three different cul-

ture media and six EIA assays for anti-H. pylori anti-

bodies (IgG and IgA). It is well known that gastric

mucosal biopsy for H. pylori is the standard gold for

diagnosing that infection. Several histological stains,

one of them hematoxylin and eosin, was compared to

culture and the results revealed that all stains showed

high specificity ranging from 97 to 100% [15].

A wide variety of solid media have been used by

many investigators for the isolation of H. pylori [16-19]. In this study, different results were obtained by

using three selective media for isolation of H. pylori.

Brucella agar was considered the best medium and

showed the highest H. pylori recovery rate (98.6%),

while Columbia and BHI agar produce a lower recov-

ery rate, 88.7% and 81.7% respectively. The use of

selective media has been recommended in order toimprove the culture isolation of H. pylori from gastric

biopsy specimens and to prevent bacterial over-

growth by contaminants [20, 21].

These media (Brucella, Columbia and BHI Agar)

were used successfully by many investigators [18,

22]. In a study by Hachem et al., [23] proved that BHI

agar was the best one among the media tested and

gave 96% recovery rate. While the recovery rate was

78% with trypticase soy agar, 64% with egg yolk agar

and 32% with Columbia blood agar-cyclodextrin. In

this study, Brucella agar was the most accurate of themedia evaluated for culture isolation of H. pylori

from gastric biopsy specimens. The inclusion of

2,3,5-triphenyltetrazolium chloride gives H. pylori

colonies a specific golden yellow pigment which

helps in their identification, therefore reducing the

false identification of H. pylori. It is worth to mention

that all plates were incubated in an atmosphere of

10% CO2 and 98% relative humidity for up to 10

days to provide excellent growth conditions. Tee et

al., [20] stated that selective medium provides heav-

ier growth than non selective one. Their results

shows that Skirrow’s medium gave the highest isola-tion rate while growth on Dent’s and modified

Glupezyniski media were equal and less better than

the first one. Chocolate agar yielded a 76% positivity

rate. The addition of blood not only enhances the

growth of H. pylori, but also facilitated to distinguish

H. pylori colonies from those of other bacteria;

colonies of H. pylori appear distinctly clear [17].

In the present study, 100% of H. pylori infected

patients had a positive test result with Euroimmun

IgG. On the other hand the other two kits, Biocheck

IgG and Biotest IgG identified 98.6% and 88.7%respectively. On the other hand 60.6% of H. pylori

infected patients had a positive test result with

Euroimmun IgA. Only 21.1% and 48% of the infect-

ed patients were identified by Biocheck IgA and

Biotest IgA kits respectively. The sensitivities of the

three IgA based tests in comparison to IgG antibodies

confirm the results of previous studies. Best et al, [24]

report a sensitivity of only 50% for IgA, while a sen-

sitivity of 100% was found for IgG in the same cohort

in the same study. Also Kindermann et al, [25] sup-

port these results and found that the sensitivities were

<50% for the IgA kits and varied markedly betweenthe IgG kits between 70.7-92.7%. Biocheck IgA kit

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gave a high specificity (100%) while it is found lower

for the other two kits EuroImmun IgA and Biotest IgA

yielded a specificity of 48% and 80% respectively.

The results obtained in this study were similar to [26-

28]. A large number of false positive results were

observed in all IgG tests and the specificity was 52%,28% and 44% for Biotest IgG, Biocheck IgG and

Euroimmun IgG respectively. These results are lower

than those obtained by others [8, 25]. Positive sero-

logy results are evidence of contact with H. pylori but

do not necessarily indicate current infection, also

persistent antibodies after clearance of H. pylori in-

fection are the main reason for false positive results

[26, 28& 29], another cause of false positive results is

the cross reactions that could have occurred between

H. pylori antigens and other patient’s sera [30]. In a

study by Maciorkowska et al, [31] showed that IgG

antibodies were present in 31.1% of children and in16.1% of adults with normal antrum and corpus gas-

tric mucosa.

Finally, the imperfect relation between the pre-

sence of H. pylori infection and the antibody response

could be explained either because of the unsatisfac-

tory sensitivities and specificities of the commercial


243

Table 1: Relative Isolation Results of H. pylori Organism on the Three Culture Media (BA, CA, and BHI).

Table 2: Sensitivities, specificities and accuracies of the EIAs.

The results showed that the Euroimmun EIA IgG is more sensitive than other kits,while Biocheck EIA IgA is more specific than others.

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kits used or because of weak immunological res-

ponse in our patients to H. pylori antigens. Moreover,

H. pylori diagnosis that depends on the detection of

anti- Helicobacter pylori antibodies in the hospital

setting and in the screening programs should consi-

der another test for confirmation the initial diagnosis.Thus, it is important to submit that the combination

between the non-invasive (such as serology) and in-

vasive (such as culture and biopsy) methods may

markedly improve the detection of H. pylori.

ACKNOWLEDGMENTS

We sincerely would like to thank the staff of Al-

Bashir hospital, Amman, Jordan for their cooperation

in patient management and specimens’ collection

specially Dr Karim Lotfy and Dr Nasim Zyadat. Manythanks to Dr Nidal Al-Otibe for leading the pathology

team for assessment the Helicobacter pylori histo-

pathological examination.

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247

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Demetra Socolov, F.Corniþescu, S.Teleman, Gabriela Anton

CURDLAN DERIVATIVES ABLE TO ENHANCE CYTOSTATIC DRUGS ACTIVITY ON TUMOR CELLS

Maria-Mihaela Bãdulescu, Natalia Simona Apetrei, Andreea-Roxana Lupu,Lidia Cremer, G. Szegli, M. Moscovici, Georgeta Mocanu, Doina Mihai, Ana Cãlugãru

SUBJECT INDEX

5

63

69

80

119

125

136

195

201

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MICROBIOLOGY

COMPARISON OF TUBERCULIN SKIN TEST WITH A WHOLE/BLOOD INTERFERON

GAMMA ASSAY AND ELISA, IN HIV POSITIVE CHILDREN AND ADOLESCENTS WITH TB

Henriette Stavri, Luminiþa Ene, Gabriela Loredana Popa, Dan Duiculescu, Gheorghe Murgoci,

Constantin Marica, Irina Ulea, Gabriela Cus and Mircea Ioan Popa

NON-POLIO ENTEROVIRUSES ASSOCIATED WITH ACUTE FLACCID PARALYSIS (AFP)

AND FACIAL PARALYSIS (FP) CASES IN ROMANIA, 2001-2008

Ana Persu, Anda Bãicuº, Simona Stavri and Mariana Combiescu

SUBINHIBITORY CONCENTRATIONS OF PHENYL LACTIC ACID INTERFERE

WITH THE EXPRESSION OF VIRULENCE FACTORS IN STAPHYLOCOCCUS AUREUSAND PSEUDOMONAS AERUGINOSA CLINICAL STRAINS

Mariana-Carmen Chifiriuc, Lia-Mara Diþu, Otilia Banu, Coralia Bleotu, Olguþa Drãcea,

Marcela Bucur, Cristina Larion, Anca Michaela Israil, Veronica Lazãr

IN VIVO EXPERIMENTAL MODEL FOR THE STUDY OF THE INFLUENCE

OF SUBINHIBITORY CONCENTRATIONS

OF PHENYLLACTIC ACID ON STAPHYLOCOCCUS AUREUS PATHOGENICITY

Mariana-Carmen Chifiriuc, Coralia Bleotu, Lia-Mara Diþu, Diana Smarandache,

Elena Sãsãrman, Otilia Banu, Olguþa Drãcea, Cristina Larion, Lazãr Veronica

THE IN VITRO SUSCEPTIBILITY TO 7 ANTIBIOTICS OF NEISSERIA MENINGITIDIS STRAINSISOLATED LAST YEARS IN ROMANIA

Marina Panã, Maria Ghiþã, Ileana Leveneþ, Maria Nica, Smaranda Botea, T. Osz

COMPARATIVE STUDY OF PATHOGENICITY TESTS FOR SHIGELLA SPP.

AND ENTEROINVASIVE ESCHERICHIA COLI STRAINS

Daniela Cristea, ªtefania Ceciu, Dorina Tatu Chiþoiu, Coralia Bleotu,

Veronica Lazãr, Mariana Carmen Chifiriuc

INVESTIGATION OF THE INFLUENCE OF DIFFERENT PHYSICO-CHEMICAL PARAMETERS

UPON THE SUSCEPTIBILITY OF PLANKTONIC AND ADHERENT ESCHERICHIA COLI CELLS

TO BETA-LACTAMS AND QUINOLONESO. Drãcea, C. Iordache, M. Bucur, C. Bleotu, Banu O, C. Ungureanu, D. Cristea,

M.S. Lixandru, Cristina Larion, G. Necula, V. Lazãr, M.C. Chifiriuc

IDENTIFICATION OF PLASMID-MEDIATED QUINOLONE RESISTANCE QNR-LIKE GENES

IN ROMANIAN CLINICAL ISOLATES OF ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIAE

Codruþa-Romaniþa Usein, Andi-Marian Palade, Dorina Tatu-Chiþoiu, Simona Ciontea,

ªtefania Ceciu, Maria Nica, Maria Damian

GENETIC HETEROGENEITY OF GROUP B STREPTOCOCCI ISOLATED

FROM ROMANIAN PREGNANT WOMEN

Usein Codruþa-Romaniþa, Creþoiu Mariana-Silvia, Ungureanu Vasilica,Ghiþã Maria, Petrini Anca, Strãuþ Monica

14

27

34

38

44

50

55

83

20

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250


IN VITRO SUSCEPTIBILITY OF ERWINIA AMYLOVORA (BURRILL)

WINSLOW ET. AL. TO CITRUS MAXIMA ESSENTIAL OIL

Luminiþa Mãruþescu, Crina Saviuc, Eliza Oprea, Bogdan Savu, Marcela Bucur,

Gheorghe Stanciu, Mariana Carmen Chifiriuc, Veronica Lazãr

ANTIBIOTIC RESISTANCE OF GRAM NEGATIVE BACILLI STRAINS ISOLATED

FROM THE INTENSIVE CARE UNIT IN FUNDENI CLINICAL INSTITUTE, BUCHAREST, ROMANIA

Elvira Borcan, Camelia Mihaela Ghiþã, Mariana Carmen Chifiriuc,

Luminiþa Mãruþescu, Cãtãlina Isar, Veronica Lazãr

GROUP B STREPTOCOCCUS COLONIZATION OF ROMANIAN WOMEN:

PHENOTYPIC TRAITS OF ISOLATES FROM VAGINAL SWABS

Codruþa-Romaniþa Usein, Anca Petrini, Raluca Georgescu,

Laura Grigore, Monica Strãuþ, Vasilica Ungureanu

INVESTIGATION OF HELICOBACTER PYLORI INFECTION IN JORDANIAN PATIENTSUSING SIX ENZYME IMMUNOASSAYS FOR IMMUNOGLOBULIN G (IGG) AND IGA TESTING

Hasan Abusini, Khaled Abuelteen, Ali Elkarmi, Faris Q. Alenzi, Mahmoud Lotfy

VIROLOGY

THE RELATIONSHIP BETWEEN HPV16 AND HPV18 VIRAL LOAD AND CERVICAL LESIONS PROGRESSION

Anca Botezatu, Demetra Socolov, Cristina Daniela Goia, Iulia Virginia Iancu,

Carmen Ungureanu, Irina Huicã and Gabriela Anton

P16INK4A - POSSIBLE MARKER IN HPV PERSISTENCE SCREENING

Coralia Bleotu, Anca Botezatu, Cristina Goia, Demetra Socolov, F. Corniþescu,

S. Teleman, Irina Huicã, Iulia Iancu and Gabriela Anton

223

228

235

240

175

183

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L

Larion Cristina 27, 34, 50Lazãr Veronica 27,34,44,50,158,207,215,223,228Leveneþ Ileana 38Liþescu Sanda 215

Lixandru M.S. 50Lotfy Mahmoud 240Lupu Andreea Roxana 63, 119, 136, 201Lupulescu Emilia 80

M

Manda Gina 125Mareº M. 171Marica Carmen Maria 5, 69Marica Constantin 14Marinescu Bogdan 80

Matache Cristiana 5, 69Mavromara Penelope 151Mãrgãritescu Irina 125Mãruþescu Luminiþa 166, 207, 215, 223, 228Mihai Doina 119, 201Mitache Mihaela 158Miyazaki Yoshibumi 207Mocanu Georgeta 63, 119, 201Moscovici M. 63, 119, 201Motoi Magda 95Murgoci Gheorghe 14

N

Neagoe Ionela 89Neagu Monica 125Necula G. 50Nica Maria 38, 89, 555

O

Onu Adrian 80Oprea Eliza 215, 223Opriºan Gabriela 89, 145, 151

Opriºoreanu Ana-Maria 151Osz T. 38Oþelea Dan 151

P

Palade Andi-Marian 55, 89, 100Panait Mircea 151Panã Marina 38Penciu Alina 145Persu Ana 20, 89, 145Petrini Anca 83, 235

Pistol Gina 5, 69Pleºa Adriana 195

Pop Mariana 89Popa Gabriela Loredana 14, 95Popa Mircea Ioan 14, 95Popescu Maria 145Predeþeanu Denisa 5

Preoþescu Liliana 95

R

Radu Dorel Lucian 80

S

Saviuc Crina 215, 223Savu Bogdan 223Sãsãrman Elena 34Sesan Tatiana 166

Smarandache Diana 34Soare Alina 145Socolov Demetra 171, 175, 183, 195Stanciu Gheorghe 223Stavri Henriette 14Stavri Simona 20, 145Stãvaru Crina 5, 80Steriu Dan 89Strãuþ Monica 83, 235Streinu-Cercel A. 95Szegli G. 63, 119, 136, 201Szmal Camelia 89, 145, 151

ª

ªtefãnescu Maria 5, 69

T

Tamsulea Isabela 69Tatu-Chiþoiu Dorina 44, 55, 89, 100Tãnãse Gabriela 215Tãnãseanu Cristina 69Teleman S. 183, 195

Thiers Valerie 151Trãistaru Teodor 215

U

Ulea Irina 14Ungureanu Carmen 50, 175Ungureanu Vasilica 83, 235Usein Codruþa-Romaniþa 55, 83, 89, 100, 235

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