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IDeNTIFICATION OF TRePONeMA DeNTICOLA IN sUBGINGIVAL sAMPLes BY PCR TeCHNOLOGY
AND ITs CORReLATION WITH CLINICAL DIAGNOsIs
Bogdan Dabu, Magdalena Mironiuc - Cureu, Dumitru Jardan and Camelia szmal
In vItro AND In vIvo ANTIBACTeRIAL ACTIVITY OF ACORN HeRBAL exTRACT AGAINsT sOMe
GRAM-NeGATIVe AND GRAM-POsITIVe BACTeRIA
Reza Mohebi, sobhan Ghafourian, Zamberi sekawi, Afra Khosravi, elham Abouali Galehdari, Reza Hushmandfar,
Reza Ranjbar, Abbas Maleki, Mona Mohammadzadeh, Mohammad Rahbar, Nourkhoda sadeghifard
THe ROLe OF BLAOxA-LIKe CARBAPeNeMAse AND THeIR INseRTION seQUeNCes (Iss) IN THe INDUCTION
OF ResIsTANCe AGAINsT CARBAPeNeM ANTIBIOTICs AMONG ACInEtoBACtEr BAUMAnnII IsOLATes
IN TeHRAN HOsPITALs
Khairollah Asadollahi, eshrat Alizadeh, Mehdi Akbari, Morovat Taherikalani, Mohammad Niakan, Abbas Maleki,
Parisa Asadollahi, setareh soroush, Mohammad-Mahdi Feizabadi, Mohammad emaneini
MeTHODOLOGY OPTIMIZATION AND DIVeRsIFICATION FOR THe INVesTIGATION OF VIRULeNCe
POTeNTIAL IN HAEMoPHILUS InFLUEnZAE CLINICAL sTRAINs
Mihaela Cristina Giucã, Monica strãuþ, Maria surdeanu, Maria Nica, Vasilica Ungureanu, Grigore Mihãescu
ANTIBODY AND sPLeNOCYTe PROLIFeRATION ResPONse TO WHOLe INACTIVATeD StrEPtoCoCCUS
PnEUMonIAE seROTYPe 1, 3 AND 6B IN MICe
Marina Panã, Rasid Orhan, Leontina Bãnicã, Adina Daniela Iancu, Crina stãvaru
GLUTeN sCReeNING OF seVeRAL DIeTARY sUPPLeMeNTs BY IMMUNOCHROMATOGRAPHIC AssAY
simona Oancea, Adriana Wagner, elena Cîrstea, Mirela sima
ROMANIAN exPeRIeNCe IN CHILD CeLIAC DIseAse DIAGNOsIs
Gabriel samaşca, Mihaela Iancu, Adrian Bãican, Manuela Bruchental, Paraschiva Cherecheş-Panþa,
Otilia Fufezan, Angela Butnariu, Dorin Farcãu, Lucia Burac, Tudor Pop, Alexandru Pîrvan, Lucia slãvescu,
Miu Nicolae, Andreica Mariana, Matinca Doina, Gheban Dan, Nechit Romulus, Michaela Ponta,
Ileana Constantinescu, Doru Dejica, Victor Cristea
VOLUMe 70 NO. 4 OCTOBeR - DeCeMBeR 2011
European Social Fund Investing in PEOPLE!
SECTORAL OPERATIONAL PROGRAMME HUMAN RESOURCES DEVELOPMENT 2007- 2013
Project: “Professional training system for medical personnel in new technology
for health care system (molecular diagnostic) “ - POSDRU/81/3.2/S/58819
Project cofinanced from the European Social Fund by Sectoral Operational Programme Human Resources Development 2007- 2013
European training for the Romanian Health Care System
As announced early this year, we have started a European Structural Funded teaching project: “Professional training system for
medical personnel in new technology for health care system (molecular diagnostic)“ TDM, Project cofinanced from the European
Social Fund by Sectoral Operational Programme Human Resources Development 2007- 2013, and we have chosen to inform you
about the development of this complex project in a journal dear to us. There are several reasons for publishing this brief report. The
first reason is the appropriate use of European funds engaged in the medical personal teaching for implementing state-of-the-art
technologies in order to improve Romanian Health Care. The second reason is that, our trainees, medical personnel involved in
diagnosis, are more open to learning in Romania the international recognized technologies for implementing up-to-date techniques.
Last but not least, the anonymous evaluation of this course, brought great satisfaction to the management team, thus bypassing the
inherent difficulties of implementing a project of such endeavour.
In an ever growing unsympathetic world, a few researchers with an internationally recognized scientific background have sacrificed
their time and strength to put together the First National Proteomics Course intended to improve the diagnostic of the Romanian
patients in their own country.
In the framework of project POSDRU/81/3.2/S/58819 „Victor Babeş” National Institute of Pathology, Bucharest, organized in 2011
the course entitled Proteomics from research to clinic - basic course and hands-on applications.
For 6 months, 40 trainees, physicians, biologists, biochemists and chemists involved in Health Care System have gone through
a round of theoretical lectures, followed by a series of applications of the latest proteomic technologies valid for implementation in the
The Proteomic course aimed the introduction of the trainees in the current proteomic technologies for laboratory investigations,
hands-on applications for these methodologies that can improve diagnosis, prognosis and therapy monitoring in oncology, immunology,
cardiology, rheumatology and so on.
80% of our trainees were women and were distributed between the development regions of Romania as follows: N-E was
represented by 12.5% of the trainees, South - 17.5%, Centre - 10%, N-V - 5% and 55 % from Bucharest - Ilfov. We had 42.5% physi-
cians, 22.5% biochemists, 17.5% biologists and 17.5% chemists.
Experts from „Victor Babeş” National Institute Laboratories and highly renowned speakers lectured during the courses. The trainees
had the opportunity to attend the lectures of Prof. Gary Coulton, Head of Omics Centre, St. George’s University of London (United
Kingdom) and Prof. Ştefan Constantinescu, Head of Intracellular Signalling Department, Ludwig Institute for Cancer Research, Christian
de Duve Institute, Bruxelles (Belgium). All the lectures were highly appreciated as seen in the anonymous evaluation of the course.
All the theoretical courses had a homogenous structure: principles of proteomic technology, methodology description and application
possibilities in clinical laboratories appropriate for Health Care improvement. After completion of the theoretical course, and in relation
with their option, the trainees attended the practical modules in five series:
? Advanced electrophoretic techniques
? Post-electrophoresis analysis - Western blot;
? Immune-based detection - from ELISA to Protein microarray;
? Liquid phase multiplexing methods
? SELDI-TOF - Mass spectrometry for biomarker discovery.
The proteomic technology presented at the theoretical courses is available in the laboratories that hosted the applications modules.
The hands-on courses lasted for one week. In the first day all the proteomic technologies were presented to the trainees, followed
by 6 days when they actually used their chosen technology. Each of the trainees has been applying the technology, individually per-
forming the technique on pathological/normal samples and ending with data interpretation and results conveying to a specific diag-
After the final evaluation, all our trainees had good and very good grades and were entitled to receive a graduation diploma with
credits from the Bucharest College of Physicians and the Order of Biochemists, Biologists and Chemists in the Health System in Ro-
The highly professional milieu in which the course has been developing for 6 months was created both by the lecturers and by the
trainees that could accumulate international level scientific information in a short period of time. The human and professional bonds
that were created during this time, the positive feed-back gave us confidence in the importance of this project. Sometimes the trainees’
overall positive response was the only incentive that motivated us to bypass the inherent difficulties encountered during the project
development. We do really hope that our trainees will be our future research collaborators.
What lies in our future? 2012 will open with two new series of proteomic courses, basic and applications. Related to the inter-
nationally evolving domain and connected to our trainees demands coming from the Health Care System, the course will be upgraded
towards the fast implementation of these technologies in clinic, having always in mind the wellbeing of the patient and the improvement
of their diagnostic.
Sometimes too optimistic, but always professional, we are eagerly awaiting our new trainees in 2012.
Monica Neagu - Proteomics Area Coordinator
Cristiana Tănase - Project Assistant Manager
Mihail Hinescu - Project Coordinator
Recently there has been a remarkable expansion
in the knowledge of the relationship between the
large number of bacterial species in both the healthy
sulcus and the inflamed periodontal pocket and of
their potential role in the progression of the healthy
sulcus to gingivitis and ultimately to periodontal dis-
ease with bone loss and tissue destruction . The
complexity of subgingival physiological and patho-
logical inhabitants has hindered the identification of
the precise microbial etiology of periodontitis,
although very strong correlations between the
amount and composition of the dental plaque biofilm
and disease have been described .
Initial observations indicate a positive relation-
ship between the presence and number of spirochetes
in the oral environment and between oral health and
subgingival profile has been identified by seve -
ral methods: anaerobic culture, microscopy, enzyme
reaction, immunohistology, DNA-probes and poly-
merase chain reaction. Culture techniques require
complex media and microaerophilic or strictly anae -
robic growth conditions. The rapid development of
mo lecular biology methods has facilitated the use of
PCR for bacterial detection .
treponema denticola is present at significantly
elevated levels in deep-pocket sites of patient with
severe periodontitis [6, 7]. treponema denticola is
often found in close proximity to Porphyromonas
gingivalis and tannerella forsythia forming together
the “red complex”. These three bacterial species are
generally found in the subgingival plaque of active
periodontal lesions [1, 8].
The aim of this study was to identify treponema
denticola in subgingival samples using PCR and
corre late it with clinical diagnosis of subjects.
treponema denticola has been associated with gingivitis and chronic periodontitis. The aim of this
study was to identify treponema denticola in subgingival samples using PCR technology and to
correlate it with clinical diagnosis of subjects. The study was carried out on seventy patients (20-
84 years of age; mean age, 45.06±12.58) of which 22 individuals with no detectable gingivitis or
periodontitis, 4 subjects with chronic gingivitis and 44 subjects with chronic periodontitis.
subgingival plaque samples were collected from five sites in each patient. DNA was extracted from
the samples using QIAamp® DNA Mini Kit (QIAGeN®). treponema denticola and other four peri-
odontopathogens were found using multiplex polymerase chain reaction followed by a reverse
hybridization. The relationship between clinical diagnoses and detection of treponema denticola
was determined with Fisher exact test. The results showed significant differences between diag-
nostic groups regarding subject proportion. treponema denticola was detected in 2 out of 22 sub-
jects with no detectable gingivitis or periodontitis, 2 out of 4 subjects with chronic gingivitis, and
40 out of 44 subjects with chronic periodontitis.
Our findings suggest that treponema denticola is closely connected to the initiation and pro -
gression of periodontal disease.
Bogdan Dabu1*, Magdalena Mironiuc - Cureu2, Dumitru Jardan3and Camelia Szmal3
1Dpt of Microbiology, Faculty of Dentistry, UMPh “Carol Davila” Bucharest; 2Dpt of Periodontics, Faculty of Dentistry,
UMPh “Carol Davila” Bucharest; 3Molecular Biology Laboratory, Medlife Bucharest
Keywords: treponema denticola, periodontitis, polymerase chain reaction
* Corresponding author: Bogdan Dabu, Department of Microbiology, Faculty of Dentistry, UMPh “Carol Davila” Bucharest; Dimitrie Gerota
street 19-21, Bucharest, sector 1; Tel. +4021.3180757; firstname.lastname@example.org.
IDeNTIFICATION OF TRePONeMA DeNTICOLA
IN sUBGINGIVAL sAMPLes BY PCR TeCHNOLOGY
AND ITs CORReLATION WITH CLINICAL DIAGNOsIs
MATERIALS AND METHODS
seventy patients (20-84 years of age; mean age,
45.06±12.58) of which 22 with no detectable gin-
givitis or periodontal disease, 4 with chronic gingivi-
tis and 44 with chronic periodontitis, visiting two pri-
vate dental clinics were followed up in this study
between 2010 and 2011. experimental protocol was
approved by local ethics committee and informed
consent was obtained from each patient.
Inclusion criteria: at least two sites with charac-
teristic clinical findings for chronic gingivitis, exten-
sive and generalized types of chronic periodontitis,
periodontal pockets >4 mm, abnormal tooth mobi -
lity and characteristic radiographic findings.
exclusion criteria: periodontal therapy and anti -
microbial agents in the previous 3 months, any sys-
temic condition which could influence the progres-
sion or treatment of periodontitis, localized or aggres-
Clinical Diagnostic Criteria
The subjects were divided into three groups: pe -
riodontal health status (lot 1), chronic gingivitis (lot 2)
and chronic periodontitis (lot 3).
Periodontal health status means no detectable
clinical findings of gingivitis or periodontal disease
and no characteristic radiographic aspect. Chronic
gingivitis is considered in the presence of sponta-
neous bleeding on probing, gum discoloration (bright
red or purple red), gingival enlargement, no true peri-
odontal pockets on probing and no alveolar bone
loss on radiographs. Diagnostic criteria for chronic
periodontitis include gum discoloration (from pale
pink to red and purple red), abnormal gingival vo -
lume (retraction or enlargement), the presence of true
or false periodontal pockets, abnormal dental mobi -
lity grade I, II or III, alveolar bone changes on radio -
graphs from various halisteresis types to both hori-
zontal and vertical bone loss .
Collection of Subgingival Plaque Samples
Prior to sampling, the supra-gingival plaque was
carefully removed; the sample sites were isolated with
sterile cotton rolls and gently air-dried. each sterile
paper point was inserted into pre-defined sites down
to the base of periodontal pocket for 10 seconds. The
probes were similarly gathered from gingival sulci of
periodontally healthy and chronic gingivitis subjects.
Five paper points were collected from each patient.
samples were stored in a refrigerator until assayed as
HAIN Lifescience’s protocol indicates.
Detection of periodontal pathogens
After DNA extraction from the samples by using
QIAamp® DNA Mini Kit (Qiagen®) according to ma -
nu facturer’s recommendations, treponema denticola
and several other periodontopathogens were determi -
ned by microIDent® test (Hain Lifescience, Ger many).
This kit identified five periodontopathogenic bacteri-
al species (Aggregatibacter actinomycetemco mitans,
Porphyromonas gingivalis, Prevotella intermedia,
tannerella forsythia and treponema denticola).
PCR amplification was performed in a reaction
volume of 50μL consisting of 5 μL of template DNA
and 45 μL of master mix containing 35 μL of primer-
nucleotide mix (provided in microIDent kit), 5μL of
10 x polymerase incubation buffer, 3 μL of 25
mmol/L MgCl2,and 1U (0,2 μL) Hot start Taq DNA
polymerase (Qiagen). PCR cycling was performed on
GeneAmp PCR system 9700 (Applied Biosystem).
The cycling conditions comprised an initial denatura-
tion step at 95°C for 15 min; 10 cycles at 95°C for
30 s and at 58°C for 2 min; 20 cycles at 95° for 25
s, at 53°C for 40s and at 70°C for 40 s; and a final
extension step 1 cycle at 70°C for 8 min.
The subsequent reverse hybridization was per-
formed according to manufacturer’s instructions on
Beeblot (BeeRobotics). Briefly, the biotinylated am -
plicons were denatured and incubated at 45°C with
hybridization buffer and strips coated with 2 control
lines and 5 species-specific probes. After PCR pro -
ducts had bound to their respective complementary
probe, a highly specific washing step removed any
nonspecific bound DNA. streptavidin-conjugated
alkaline phosphatase was added, the samples were
washed and hybridization products were visualized
by adding a substrate for alkaline phosphatase. Inter -
pretation of the banding pattern was ensured by a
standard template. In order to validate the correct
performance of the test and the proper functioning of
reagents, two controls, one for amplification and the
second for hybridization were included.
The relationship between clinical diagnostic
groups and detection of treponema denticola was
de termined with Fisher exact test.
In our study the methods we used enabled the
detection of treponema denticola in 44 out of 70
tested patients. treponema denticola was detected in
2 out of 22 subjects with no detectable gingivitis or
DABU et al.
periodontitis, in 2 out of 4 patients diagnosed with
chronic gingivitis, and 40 out of 44 patients diag-
nosed with chronic periodontitis. (Table 1)
In 42 out of 48 patients with marginal periodon-
tium disease, treponema denticola was detected. The
results showed significant differences between groups
as regards the proportion of subjects with tre ponema
denticola. The microIDent assay also detected anoth-
er four species of bacteria (Aggregatibacter actino-
mycetemcomitans, Porphyromonas gingivalis, Pre vo -
tella intermedia, tannerella forsythia). The pa tients
found with any or all these 4 bacteria, not forming
the subject of the present study, were referred to the
Our results showed a positive association bet -
ween diagnosis and treponema denticola (statistica lly
significant p<0.001 by Fisher exact test).
The periodontal microbiota is heterogeneous;
over 700 species have been described in the oral ha -
bitat. A large number of reports have unequivocally
established that the progress from health to gingivitis
and hence to periodontitis is marked by a shift in the
resident microbiota from a “healthy” biofilm to a “dis-
eased” microbial ecology . examination of poten-
tial virulence characteristics shared by red complex
bacteria demonstrated their ability to disrupt peri-
odontal innate defense functions facilitating unto-
ward host interactions with the entire dental plaque
community which has been found to be potent acti-
vators of Toll-like receptor-2 (TLR-2) and Toll-like
receptor-4 (TLR-4) .
Technological advances in microbial identifica-
tion and classification based on the introduction of
non-culture methods such as: DNA hybridization,
PCR, sanger sequencing, and the more recent deve -
lopments of in high-throughput pyrosequencing-
based analyses and metagenomics have allowed
detection of microorganisms that are difficult or even
impossible to culture, especially in case of spiroche-
tes . PCR methods have been increasingly used in
the investigation of periodontal flora and are able to
detect the presence of genomic DNA bacteria from
subgingival sulcus [11, 12].
subjects with severe periodontitis displayed sig-
nificantly higher levels of treponema denticola in
their deep pockets compared to healthy periodontal
subjects or those diagnosed with moderate periodon-
Literature suggests that a high salivary level of
matrix metalloproteinase-8 (MMP-8) detected con-
comitantly with treponema denticola in subgingival
plaque displays a robust characteristic in predicting
periodontal disease severity. Thus, monitoring these
bacteria may prove to be an important tool in the
assessment of periodontitis patients .
The results of this study indicate that the rela-
tionship between the presence of treponema denti-
cola and periodontitis patients was statistically signi -
ficant. Detection rate of treponema denticola was
lower in chronic gingivitis group than in chronic peri-
odontitis group. some researchers (Ito et al. 2010,
Mineoka et al. 2008) using molecular methods found
treponema denticola the most prevalent pathogen in
both shallow and deep sites with increased numbers
in sites with higher probing depths and bleeding on
probing, which supports our result [15, 16].
early identification of bacteria associated with
the development of periodontitis using PCR techno -
logy may be helpful for screening periodontitis and
healthy patients providing important information for
diagnosis and treatment. As it also allows bacterial
detection following antibiotic treatment, this method
is able to detect residual bacterial load, helpful for
patient monitoring. On the other hand, detection of
residual bacteria can be a sign of inadequate treat-
ment prescriptions or poor patient compliance.
In our study the low incidence of treponema
denticola in periodontally healthy subjects indicated
Identification of Treponema denticola in subgingival samples by PCR technology and its correlation with clinical diagnosis
Table 1. Relationship between Treponema denticola prevalence and subject clinical status
of T. denticola
of T. denticola
No gingivitis or
periodontitis (lot 1)
20 subjects 2 subjects 22 subjects
2 subjects 2 subjects 4 subjects
4 subjects 40 subjects 44 subjects
Total 26 subjects 44 subjects 70 subjects
DABU et al.
12. Jervøe-Storm PM, AlAhdab H, Koltzscher M, Fimmers
R, Jepsen S. Quantification of periodontal pathogens
by paper point sampling from the coronal and apical
aspect of periodontal lesions by real-time PCR. Clin
oral Investig 2010. 14(5): 533-41.
13. Riviere GR, Smith KS, Carranza NJr, Tzagaroulaki E,
Kay SL, Dock M. subgingival distribution of t. denti-
cola, t. socranskii, and pathogen-related oral spiro-
chetes, prevalence and relationship to periodontal sta-
tus of sampled sites. J Periodontol 1995. 66: 829-837.
14. Ramseier CA, Kinney JS, Herr AE, Braun T, Sugai JV,
Shelburne CA et al. Identification of pathogen and
host-response markers correlated with periodontal
disease. J Periodontol 2008. 80: 436-446.
15. Ito KA, Ishihara K, Tomita S, Kato T, Yamada S.
Investigation of subgingival profile of periodontopathic
bacteria using polymerase chain reaction. Bull tokyo
Dent Coll 2010. 51(3):139-144.
16. Mineoka T, Awano S, Rikimaru T, Kurata H, Yoshida
A, Ansai T, Takehara T. site-specific development of
periodontal disease is associated with increased levels
of Porphyromonas gingivalis, tannerela forsythia and
tremonema denticola in subginigival plaque. J Perio -
dontal 2008. 79: 670-676.
that its presence alone was not responsible for the
Based on our test results and data analysis, we
may conclude that the presence of treponema denti-
cola is associated with chronic periodontitis diagnosis.
The identification method used in this study
proved to be a reliable and sensitive assay.
We are grateful to Dr. Monica Teleman (Uni ver -
sity of Medicine and Pharmacy “Carol Davila” Bu -
charest) for her assistance in data analysis and to Prof.
Magdalena Cuturicu for language corrections.
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england 2006, pp 323-356.
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5. Keyes PH, Rams TE. A rational for management of perio-
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6. Sela MN. Role of treponema denticola in periodontal
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7. Yashida A, Kawada M, Suzuki N, Nakano Y, Oho T et
al. TaqMan real-time polymerase chain reaction assay
for the correlation of treponema denticola numbers
with the severity of periodontal disease. oral Microbiol
Immunol 2004. 19: 196-200.
8. Wara-aswapati N, Pitiphat W, Chanchaimongkon L,
Tawechaisupapong S, Bach JA, Ishikawa I. Red bacte-
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9. Masunaga H, Tsutae W, Oh H, Shinozuka N, Kishimoto
N, Ogata Y. Use of quantitative PCR to evaluate met-
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Taipale L, Skurnik M, Könönen E, Pussinen PJ.
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Innate Immun 2009. 15(4): 195-204.
The search continues for safe and effective
antimicrobial agents for treating, therapeutically and
prophylactically, a wide variety of bacterial infec-
tions. This need has been urged recently by the emer-
gence of many antimicrobial-resistant organisms like
methicillin resistant staphylococci, multidrug, exen-
dended drug and pandrug resistant microorganisms
. The best therapeutic antimicrobial agents cause
virtually no adverse reactions, have a wide spectrum
of activity, and are not likely to select resistance.
Medicinal herbs are being increasingly studied
by pharmacological researches, and many such herbs
have a long history of medicinal use in Asia .
Herbs have many potential clinical and therapeutic
applications in the modern medical setting, as
numerous studies have revealed that they contain
bioactive components, and have resulted in a better
understanding of their physiological, therapeutic and
clinical actions . Antimicrobial agents can also be
derived from herbs, and over 1000 plants exhibit
antimicrobial effects . Traditionally, these herbs
are said to provide safe and effective treatments
against many diseases.
surface of acorn is traditionally used in Iran to
treat gastrointestinal infections.
* Corresponding author: Nourkhoda sadeghifard, email@example.com
Background: The search for safe and effective antimicrobial agents, which treat, therapeutically and
prophylactically, a wide variety of bacterial infections still represents a top priority for the biome -
dical field. This study was undertaken to investigate the antimicrobial properties of herbal extract
(acorn) against bacterial pathogens in intestinal tract infections in in vitro and in vivo conditions and
to study the effect of herbal extracts against bacteria in comparison with current antibiotics.
Findings: ethanol extraction of acorn herb (Jaft) were evaluated against Klebsiella pneumoniae,
Escherichia coli, Staphylococcus aureus, Salmonella typhi and Pseudomonas aeroginosa in in vitro
and in vivo conditions. Minimal Inhibitory Concentration (MIC) was 10 g/ml, 10 g/ml, 5 g/ml,
15 g/ml and 15 g/ml for K. pneumoniae, E. coli, S. typhi, S. aureus and P. aeroginosa, respec-
tively. The in vivo results showed that the experimental infection produced by K. pneumoniae, E.
coli, S. typhi and P. aeroginosa was totally inhibited in rats treated by the acorn extraction, while
positive control rats died after five days.
Conclusion: The finding revealed that acorn extract has great potential as antimicrobial compounds
against pathogenic microorganisms. Thus, acorn extract can be used in the treatment of infectious
diseases caused by resistant bacteria.
In vItro AND In vIvo ANTIBACTeRIAL ACTIVITY
OF ACORN HeRBAL exTRACT AGAINsT
sOMe GRAM-NeGATIVe AND GRAM-POsITIVe BACTeRIA
Reza Mohebi1, Sobhan Ghafourian1,2, Zamberi Sekawi2, Afra Khosravi3,
Elham Abouali Galehdari1, Reza Hushmandfar4, Reza Ranjbar5, Abbas Maleki1,
Mona Mohammadzadeh6, Mohammad Rahbar7, Nourkhoda Sadeghifard1*
1Clinical Microbiology research Center, Ilam University of Medical Sciences, Ilam, Iran;
2Department of Medical Microbiology & Parasitology, Faculty of Medicine and Health Sciences,
University Putra Malaysia, 43400 Serdang, Selangor, Malaysia; 3Department of Immunology, Faculty of Medicine,
Ilam University of Medical Sciences, Ilam, Iran; 4Faculty of veterinary Medicine, Ilam University, Ilam, Iran;
5Biology research Center, Baqiyatallah University of Medical Sciences, tehran, Iran;
6Department of Microbiology, Milad Hospital, tehran, Iran;
7Department of Microbiology, Iranian reference Health Laboratories, tehran, Iran
Keywords: acorn extract, antimicrobial activity, pathogenic bacterial strains
MOHEBI et al.
period, it was possible to observe the growth inhibi-
tion zone. Overall, cultured bacteria with growth
inhibition area diameters equal to or greater than 7
mm were considered susceptible. DMsO and Tween
80 to 2% were used to dissolve the extracts in the
culture media when necessary .
Confirmation for the antimicrobial potential of
the plant extracts:
Minimal Inhibitory Concentration (MIC) for each
bacterial sample has been performed. For this pur -
pose, 100 μL of microbial suspension of 108cells/mL
density were inoculated in tubes with nutrient broth
supple mented with different concentrations (1-100
μg/mL) of the tested extracts. After 24 h of incubation
at 37 °C, the MIC of each sample was determined by
measu ring the optical density using a spectropho-
tometer (620 nm), comparing the sample turbidity
with that of non inoculated nutrient broth .
Cell culture: The extract was tested for its cyto-
toxicity effect on vero Cell Line. The percentage via-
bility of the cell line was carried out by using Trypan
blue dye exclusion method and the MTT assay.
toxicity assay: The cells were plated in 96- well
flat bottom plates at a density of 5000-1000 cells/well
and were allowed to adhere to the wells overnight.
Then the cells were treated with different concentra-
tions of the tannin ethanol herbal extracts (1-100
g/mL) with a maximal final ethanol concentration of
1%. After 24 h, MTT assay was used to monitor cell
growth. The absorbance of converted dye was meas-
ured at a wavelength of 570 nm with a background
subtraction at 630 nm .
Data interpretation: Absorbance values lower
than the control cells indicated a reduction in the rate
of cell proliferation. Conversely, a higher absorbance
rate indicated an increase in cell proliferation. Rarely,
an increase in proliferation or morphological changes
may be offset by cell death.
1 – (OD observed / OD control) ×100 = %
In vivo assay: eighteen female rats aged between
4 and 5 weeks were used for all in vivo experiments.
Rats were divided into five study groups, the positive
control (infected rat without extract) and negative
control. Throughout each experiment rats were given
water containing streptomycin (5 mg/ml) to reduce
the level of facultative anaerobic bacteria that nor-
mally colonize the intestine .
This study was undertaken to investigate the
antimicrobial properties of herbal extract (acorn) (that
locally was called Jaft and obtained in Ilam
Mountains) against bacterial pathogens isolated from
intestinal tract infections, using in vitro and in vivo
conditions and to compare their activity with that of
MATERIAL AND METHODS:
K. pneumoniae, E. coli, S. aureus, S. typhi, P. ae -
roginosa were analyzed against the acorn extraction
(kindly provided by Department of Microbiology,
Reference Laboratory of Ilam, Iran). The bacteria were
resistant to the antibiotics, as follows: i) K. pneumo-
niae to aztreonam, ceftazideme, ampicillin, ceftria -
xone, cotrimoxazol, ciprofloxacin, amikacin; ii) E.
coli to aztreonam, ampicilin, cefteriaxone, cotrimo -
xazol, amikacin; iii) S. aureus to oxacillin, tetracy-
cline, amikacin, ceftriaxone, cotrimoxazol, vanco -
mycin; iv) S.typhi to amikacin, ciprofloxacin, ceftria -
xone, imipenem, cotrimoxazol, tetracycline; v) P.
aeroginosa to oxacillin, amikacin, ciprofloxacin, cef-
triaxone, imipenem, cotrimoxazol, tetracycline.
Plant extracts: The acorn, or oak nut, is the nut of
the oaks and their close relatives (genera Quercus and
Lithocarpus, in the family Fagaceae). Acorns vary from
1–6 cm long and 0.8–4 cm broad. Acorns take between
about 6 and 24 months to mature the surface of acorn
were powdered and soaked in the ethanol for about
10-15 days then this cold extract is subjected to dis-
tillation at low temperature under reduced pressure
in rotary flash evaporator and concentrated on water
bath to get the crude extract. The powder was sub-
jected to soxhlation with ethanol for 48 hours. The
solvent was distilled off at lower temperature under
reduced pressure in rotary flash evaporator and con-
centrated on water bath to get the crude extract.
screening for the antimicrobial potential of the
The bacterial cultures were grown in Brain Heart
Infusion liquid medium at 37°C. After 6 h of growth,
each microorganism, at a density of 108cells/mL, was
inoculated on the surface of Mueller-Hinton agar
plates. subsequently, filter paper discs (6 mm in dia -
meter) saturated with herbal extract (50 μL) were pla -
ced on the surface of each inoculated plate. To eva -
luate the efficiency of the methodology, each extract
was distributed simultaneously in agar wells (50 μL).
The plates were incubated at 37°C for 24 h. After this
The bacteria were grown overnight in nutrient
broth, centrifuged, washed in phosphate-buffered
saline (PBs), and diluted to achieve the final suspen-
sion of 1×108CFU density, further diluted in 20%
sucrose and used to fed both control and study
groups. One hour after infection, study group ani-
mals were given orally 5mg of the herbal extract daily
whereas control animals were not.
Fecal samples were collected at 0, 1, 3, 4 days
after administering the bacterial suspensions and the
numbers of the bacteria per gram in feces were deter-
mined. For this purpose, aliquots (100 l) of fecal sus-
pensions were serially diluted in PBs and then were
plated on especial medium for each bacteria, which
were then incubated overnight at 37 °C, and typical
colonies were counted in plates containing between
30 and 300 colonies .
RESULTS AND DISCUSSION
The findings in screening stage revealed all that
the tested bacteria were susceptible to the herbal
extract of 10 g/ml concentration for the Gram-ne ga -
tive bacterial and of 5 g/ml for S. aureus. In the disk
diffusion assay, the inhibition zone diameters around
disks charged with the tested extract are presented in
The cytotoxicity study was carried out for plant
extract of surface of acorn (tannin). This extract was
screened for its cytotoxicity against vero cells line at
different concentrations to determine the IC50 (50%
growth inhibition) by MTT assay. It was found that
the percentage of growth inhibition increasing with
increasing concentration steadily up to 55 g/ml and
IC50 value of this assay was 0.245.
In the in vivo assay, the results showed the ex tract
could remove the K. pneumoniae, E. coli, S. typhi and
P. aeroginosa in rat faeces, thus that was effective
against Gram -negative bacteria, while control rat in -
fected by these bacteria died after 5 days. The control
rat infected with S. aureus died after 8 days.
The results showed that the tannin extract is more
effective against Gram-positive bacteria in in vitro
conditions but the in vivo experiments revealed an
increased tannin efficiency against the Gram-negative
The antimicrobial properties of plants have been
investigated by a number of researchers worldwide,
especially in Latin America. In Argentina, a research
tested 122 known plants species used for therapeutic
treatments . It was documented that among the
compounds extracted from these plants, twelve
inhibited the growth of S. aureus, ten inhibited E.
coli, and four inhibited Aspergillus niger and also
reported that the most potent compound was one
extracted from tabebuia impetiginosa. The antimicro -
bial properties of compounds obtained from Par the -
num argentatum against Candida albicans, toru lop -
sis, Hansemula, K. pneumoniae and P. aeruginosa
was detected . Another work showed that the
substances extracted from nine known plants in
Uruguay did not show any activity against C. albi-
cans and Saccharomyces cerevisiae, but inhibited the
growth of Bacillus subtilis, E. coli and P. aeruginosa
. Many studies have been conducted in Brazil.
The inhibitory activity of vatairea macrocarpa on
Klebsiella spp. and S. aureus was observed . Our
findings revealed that jaft extract was effective against
wide range of pathogenic Gram-negative bacteria
while this herb in in vitro conditions could inhibit the
growth of S. aureus but this result was not occurred
in in vivo. Our study performed in in vitro and in vivo
conditions, but the majority of the research in herbal
as mentioned above were only performed in in vitro
condition, thus our results can strongly suggest the
good effect of this extract against Gram negative bac-
The use of plants to heal diseases, including
infectious ones, has been extensively applied by peo-
ple. Our results reveal the great potential of plants for
therapeutic treatment, in spite of the fact that they
have not been completely investigated. Therefore,
more studies need to be conducted to search for new
compounds. Once extracted, and before being used
in new therapeutic treatments, they should be tested
for their toxicity in vivo. Bioassays have demonstra -
ted the toxicity of extracts from different plants.
Therefore, our results revealed the importance of
plant extracts when associated with antibiotics, to
In vitro and in vivo antibacterial activity of acorn herbal extract against some Gram-negative and Gram-positive bacteria
Table 1. Antimicrobial activity of tannin by disk diffusion method
5 ?g/ml 10 ?g/ml
0 20 mm
0 16 mm
0 15 mm
0 17 mm
15 mm 25 mm
MOHEBI et al.
11. Martinez MJ, Gonzalez BA, Jauregui A. screening of
some Cuban medicinal plants for antimicrobial activi-
ty. J. Ethnopharmacol 1996. 52: 171-174.
12. Alonso-Paz E, Cerdeiras MP, Fernandez J, Ferreira F,
Moyna P, Soubes M, Vazquez A, Veros S, Zunno L.
screening of Uruguayan medicinal plants for antimicro-
bial activity. J. Ethnopharmacology 1995. 45: 67- 70.
13. Matos FGA, Aguiar LMBA, Silva MGA. Chemical con-
stituents and antimicrobial activity of vatairea macro-
carpa Ducke. Acta Amazonica 1988. 18: 351-352.
control resistant bacteria, which are becoming a
threat to human health.
The results showed that acorn extract has great
po tential as antimicrobial compounds against micro -
organisms. Thus, they can be used in the treatment of
infectious diseases caused by resistant bacteria. This
extract was more effective against the Gram-negative
bacteria in the in vivo experimental model.
Ilam University of Medical sciences provided
partial support for the laboratory studies and inter-
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Acinetobacter baumannii (A. baumannii) is
recog nized as an important op portunistic gram-nega-
tive pathogen that is frequently associated with a vari-
ety of nosocomial infections worldwide .
Carbapenem is the first line drug against A. bau-
mannii infections; however, the emergence of car-
bapenem resistance to A. baumannii strains has been
reported worldwide [2,3].
The main mechanisms of resistance to carba -
penem drugs are mainly due to production of meta-
lo-beta-lactamase (molecular classes B) and OxA-type
carbapenemases (molecular classes D) .
Most studies have shown that blaOxA-likecarba -
penemase has frequently induced resistance to car-
bapenem drugs [5, 6].
Four types of blaOxA-likecarbapenemases inclu -
ding blaOxA-23, blaOxA-24, blaOxA-51and blaOxA-58have
been identified in A. baumannii isolates so far .
This study aimed to evaluate the occurrence and dissemination of blaOxA-likecarbapenemase genes
and their insertion sequences among Acinetobacter baumannii isolates, taken from different hos-
pitals in Tehran city and also their roles in the induction of resistance to carbapenem drugs.
A total number of 100 non duplicate Acine to bacter baumannii with different origins, were isola -
ted from patients with proved nosocomial infections at eight university hospital in Tehran city.
Antimi cro bial susceptibility of these strains was done by e-test against 7 antimicrobial agents
according to CLsI gui deline. PCR of blaOxA-51-like, blaOxA-23-like, blaOxA-24-like, blaOxA-58-like, ISABA-1,
IS1133was carried out by specia lized primers and then these strains were typed by ReP-finger-
printing. Colistin, imipenem and mero penem were the most sensitive antibiotics against
Acinetobacter baumannii isolates with 96%, 51% and 51% sensiti vity respectively. All the isolates
had a blaOxA-51-likeintrinsic to these species. The rates of blaOxA-23, 23and 58-likewere 38%, 32% and
Coexistence of blaOxA-51/23/24-likewas observed among 16% of these isolates. All blaOxA-23-like
carba penemase genes had only one ISABA1. ReP fingerprin ting showed 5 genotypes among car-
bapenem resistant isolates, 16 of them being genotype A.
This study emphasized on the major role of blaOxA-likecarbapenemase, particularly blaOxA-23-like
carbapenemase and their ISABA1, in the dissemination of carbapenem resistant Acinetobacter bau-
mannii. This study con firmed a presumptive role of IS element neighbo ring the carbapenemase
gene in the elevation of resistance to carbapenem drug among Acinetobacter baumannii isolates
for the first time in Iran.
THe ROLe OF BLAOxA-LIKe CARBAPeNeMAse AND THeIR INseRTION
seQUeNCes (Iss) IN THe INDUCTION OF ResIsTANCe
AGAINsT CARBAPeNeM ANTIBIOTICs AMONG
ACInEtoBACtEr BAUMAnnII IsOLATes IN TeHRAN HOsPITALs
Khairollah Asadollahi1, Eshrat Alizadeh2, Mehdi Akbari3, Morovat Taherikalani2,4*,
Mohammad Niakan3, Abbas Maleki4, Parisa Asadollahi4, Setareh Soroush2,4,
Mohammad-Mahdi Feizabadi5, Mohammad Emaneini5
1Department of Epidemiology and Biostatistics, School of Medicine, Ilam University of Medical Sciences;
2Department of Microbiology, School of Medicine, Ilam University of Medical Sciences; 3Department of Microbiology,
School of Medicine, University of Shahed, tehran, Iran; 4Clinical Microbiology research Center, Ilam University of
Medical Sciences, Ilam, Iran; 5Department of Microbiology, School of Medicine, tehran University of Medical Sciences
Keywords: A. baumannii, blaOxAgenes, Is element, carbapenem drugs
*Corresponding author: Dr. Morovat Taherikalani (PhD), Assistant Professor in Medical Microbiology, Department of Microbiology, Faculty
of Medicine, Ilam University of Medical sciences, Banganjab, Ilam, Iran. Tel: +98 0841-223-5747; Fax: +98 0841-222-7136; e-mail:
Apart from the intrinsic blaOxA-51-like[8, 9] resis -
tance to carbapenem drugs due blaOxA-23, blaOxA-24
and blaOxA-58enzymes was reported worldwide .
The genes encoding OxA-like enzymes have been
found to be linked to some insertion sequences (Is)
elements. Insertion sequence (Is) including ISAba1and
other Is may modulate the expression and transfer of
the OxA-type carbapenemases genes. Insertion
sequences are mobile genetic elements known to
affect the evolutionary pattern of bacterial genomes.
Upon integration, Is elements may cause DNA inser-
tions/deletions, chromosomal rearrangement, modu-
late the expression of adjacent genes and thereby,
influence the phenotype of a bacterium [11, 12].
Because of the rise in the multiresistant strains of
A. baumannii in Iran [2, 13-14], nosocomial infec-
tions caused by this organism are difficult to be trea -
ted. Therefore, evaluation of antimicrobial resistance
of A. baumannii, especially carbapenem resistant
strains, and detection of genes conferring resistance
to carbapenems are necessary among different set-
tings and hospitals of various countries.
This study was carried to evaluate the occur-
rence and dissemination of blaOxA-likecarbapenemase
gene among A. baumannii isolates, detected from dif-
ferent hospitals in Tehran and their roles in the induc-
tion of resistance to carbapenem drugs.
MATERIAL AND METHODS
During a 6-month period, 100 non duplicate
Acinetobacter spp. were isolated from patients with
proved nosocomial infections at eight university hos-
pitals (showed as H1 to H8 respectively) in Tehran.
All positive samples were acquired from patients
with 72h of hospitalization. Patients had no bacterial
infections at the time of hospitalization. All the sam-
ples were then confirmed as A. baumanni by bio-
chemical and API 20Ne galleries and were enrolled
in this study.
Antimicrobial susceptibility tests
Using e-test, the susceptibility of all the isolates
was tested against the following antibiotics: imipen-
em, meropenem, ampicillin-sulbactam, cefepime, cef -
ta zidime, piperacillin-tazobactam and colistin.
The iso lates were then interpreted according to
the manu facturer’s instructions and CLsI guidelines
. E. coli ATCC 25922 and Pseudomonas aerugi-
nosa ATCC 27852 were used as qualitative standard
DNA Extraction and PCR Assay
DNA extraction was carried out by commercial
DNA extraction kit (Bioner, Republic of Korea). The
presence of different blaOxA-like carbapenemases
(blaOxA-51-like, blaOxA-23-like, blaOxA-24-likeand blaOxA-58-
like), ISABA1and IS1133was detected by PCR.
The PCR system (25 l) was composed of 1x
PCR buffer, 2 mM MgCl2, 0.2 mM dNTP, 10 pmol of
primers, 1U Taq DNA polymerase (Ferments, UK)
and 10ng DNA template. PCR amplification was then
carried out by thermal cycler (Bio-Rad, UsA). The
thermocycling parameters included an initial denatu-
ration at 94oC for 5 min, followed by 30 cycles of
denaturation at 95oC for 1 min, annealing according
to Table 1, extension at 72oC for 1 min, followed by
a final extension at 72oC for 8 min.
DNA extraction was carried out by DNA extrac-
tion kit (BioMerux, Republic of Korea) and 4 l of its
extract was used as the DNA template. The primer
pair ReP1, 5’- IIIGCGCCGICATCAGGC-3’ and ReP2,
5’-ACGTCTTATCAGGCCTAC-3’ was used to amplify
putative ReP-like elements in the genomic bacterial
Amplification reaction was performed in a final
volume of 25 l. each reaction mixture contained
2.5 l of 10x PCR buffer, 1.25 U Taq DNA polyme -
rase (Fermentase, UK), 0.8 l of 2mM mixed dNTPs
(Fer mentase, UK), 1.5 l of 25 mM MgCl2, 1 l of 10
pmol primers and 50 ng of bacterial DNA. Am pli -
fication reaction was carried out by thermal cycler
(ependorff, Germany) with an initial denaturation at
94oC for 10 min, followed by 30 cycles of denatura-
tion at 94oC for 1 min, annealing at 45oC for 1 min
and extension at 72oC for 1 min, followed by final
extension at 72oC for 16 min.
each sample aliquot was subjected to electro -
pho resis in 1.2% agarose gel. Amplified products
were detected by Geldoc apparatus after staining with
ethidium bromide (50 mg/L) and then photographed
for analysis. Photograph results, were then analy zed
Colistin, against which 96% of all the isolates
were susceptible, was the most effective antimicro-
bial agent. Among beta-lactam antibiotics, imipenem
and meropenem (46%) were the most effective
agents followed by ampicillin-sulbactam (40%). The
strains showed the lowest sensitivity rate against pi -
ASADOLLAHI et al.
155 Download full-text
pe racillin-tazobactam (11%), cefepime (8%) and cef-
tazidime (4%) respectively. The range of MIC to imi -
penem and meropenem was 0.004 to 32 g/ml;
while this range was 0.004 to 8 for colistin (Table 2).
Totally, 54 isolates (54%) were resistant to at least
five antimicrobial agents and were entitled as multi -
drug resistant A. baumannii. All A. baumannii strains
harbor blaOxA-51-like carbapenemase. The rate of
blaOxA-23, 24and 58-likewas 38%, 32% and 1% respec-
tively. There was coexistence between two and three
carbapenemase genes. A coexistence of blaOxA-
51/OxA-24, blaOxA-51/OxA-23/OxA-24 and blaOxA-51/OxA-24
carbapenemase was seen among 20%, 16% and
15% of the isolates, respectively.
Coexistence of blaOxA-51/OxA-23/OxA-58was ob ser -
ved only in one strain. Totally, 45% of the strains har-
bored only blaOxA-51-likecarbapenemase. ISABA1was
detected among 40% of the isolates; 38 isolates har-
Distribution of OXA-carbapenemase genes among Iranian A. baumannii strains
Table 1. Primer used for amplification of blaOXA-Likecarbapenemase
and ISs genes among Iranian Acinetobacter baumannii isolates
Table 2. Susceptibility testings of 100 A. baumannii isolates from Tehran Hospitals according to E-test results