Journal of Life Sciences Volume 6 Number (6)
J LS
Journal of Life Sciences
Volume 6, Number 3, March 2012 (Serial Number 47)
Contents
Microbiology and Biochemical Pharmacy
Phytochemical Screening and Antimicrobial Evaluation of the Aqueous Extracts of Ammoides verticillata , an Endemic Species
Oumessaad Toubal, Abdelghani Djahoudi, Chérifa Henchiri and Mohamed Bouazza 248
The Survey of Microbiological Contamination of Pitcher Cheese in West Azarbayjan Province, Iran
Ehsan Barati, Mona Daneshgar Moghaddam, Navab Ghobadi, Hamid Reza Shafieian and Abbas Barin
253 Detection of “Candidatus Phytoplasma asteris” in Brussels Sprout and Its Possible Association
with Flower Bud Failure in Poland
Maria Kami ńska, Hanna Berniak and Piotr Kamiński
260 The Role of Heat Shock Protein 70, IgE and MMP-9 in Detecting Early Minor Myocardial Damage and Evaluating the Efficacy of Coronary Artery Bypass Grafting (CABG)
Amal A. Baalash, Hala E. Hamouda, Ghada M. Ismail, Ibrahim K. Yassein and Bedir M. Ibrahim
Molecular Cloning of PhoR Sensor Domain from Mycobacterium tuberculosis for Structure-Based Discovery of Novel Anti-Tubercular
Aldina S. Suwanto, Ihsanawati and Ernawati Arifin Giri-Rachman
Botany and Zoology
Effect of Cement Dust Pollution on the Yield and Quality of Ficus carica L. Fruits
Amal Mohamed Abdel-Rahman 283
The Effect of Drought Occurring at Different Growth Stages on Productivity of Grain Amaranth Amaranthus cruentus G6
Silva Grobelnik Mlakar, Martina Bavec, Manfred Jakop and Franc Bavec
Structural Changes of Lignified Tissues from Sugarcane Leaves Induced by Smut (Sporisorium scitamineum ) Virulence Factors
Borja Alarcón, Rocío Santiago, Carlos Vicente and María Estrella Legaz 300
The Contribution of the Dromedary in the Spontaneous Plant Seeds Transfer in the Northern Algerian Sahara
Trabelsi Hafida, Senoussi Abdelhakim, Chehma Abdelmadjid and Faye Bernard 304
Biological Status of Captive Crane in Southern Districts of Northern Pakistan
Farzana Perveen 312
Effect of the Variety of Fig Tree on Some Biological Parameters of Ceratitis capitata Wied. 1824 (Diptera: Trypetidae) in Some Orchards in the Kabylie
Sadoudi-Ali Ahmed Djamila, Nabila Rezoug, Ferroudja Saiki and Noreddine Soltani
Interdisciplinary Researches
320 Community-Based Health Insurance: An Evolutionary Approach to Achieving Universal
Coverage in Low-Income Countries
Hong Wang and Nancy Pielemeier
330 Comparison of GFR by Creatinine Clearance with Estimated GFR by Various Prediction Equations in a Bangladeshi Population
Muhammad Saiedullah, Muhammad Rezwanur Rahman, Md. Aminul Haque Khan, Shoma Hayat and Shahnaj Begum
The International Research Group in Geophysics Europe Africa: A Laboratory without Borders in the Earth Science and Environment
Christine Amory-Mazaudier
The Effect of Anthropogenic Increase on the Earth as a Life-Support System for Mankind
Nickolay Pechurkin and Lydia Somova 348
A Note on the Current Status of Arin, a Yoruba Traditional Game Played with the Seeds of
Dioclea reflexa Toye Ekunsanmi
Journal of Life Sciences 6 (2012) 243-247
Phytochemical Screening and Antimicrobial Evaluation of the Aqueous Extracts of Ammoides verticillata, an Endemic Species
1 2 3 Oumessaad Toubal 4 , Abdelghani Djahoudi , Chérifa Henchiri and Mohamed Bouazza 1. Department of Biology, Badji Mokhtar University, Annaba 23000, Algeria
2. Department of Pharmacy, Badji Mokhtar University, Annaba 23000, Algeria 3. Department of Biochemistry, Badji Mokhtar University, Annaba 23000, Algeria 4. Department of Botany, Aboubekr Belkaid University, Tlemcen 13000, Algeria
Received: February 10, 2012 / Accepted: February 29, 2012 / Published: March 30, 2012.
1973) of the aerials parts wealth in polyphenol compounds: flavonoids, saponins, leucoanthocyanes, terpens and steroids and tannins; there is no alkaloids. There is an important quantity of essential oils in the flowers; the interest of this study is that this species remains until then it is not very known. The results of Aromatogram method by incorporation of Müller-Hinton on solid medium, showed a significant antimicrobial activity (method of Duraffourd, 1987) of the infusion and the ethanolic extract; the infusion of stems and flowers is indeed much more active on Echerichia coli, Citrobacter, Enterobacter, Staph aureus and Staph epidermidis, such as flowers extracts demonstrate an important antimicrobial activity on Staph aureus, Staph epidermidis, Streptococcus, Pseudomonas and Acinetobacter recognized as antibiotic resistant. This could give opportunities for using this species in the treatment of diverse infections and as a disinfecting additive on nosocomial area. The valorization, preservation and sustainable use of Ammoides verticillata require the protection of its habitats.
Key words: Ammoides verticillata, aqueous extracts, phytochemical screening, antimicrobial activity, disinfection.
1. Introduction demonstrated that essential oils are active on all the tested strains, but their activity is more important on
Ammoides verticillata (Desf.) Briq., a flowering mushrooms than on bacterias. Bekhechi et al. [3]
grassy plant in the Apiaceae, is an endemic of demonstrated that isothymol was the major northwestern Algeria and southern Mediterranean component (51.2%) of this plant with y-cymene Europe; this species was traditionally used for its (14.1%), thymol (13.0%), limonene (11.9%) and culinary and medicinal properties (against flu, y-terpinene (6.8%); Av seed oil is known to be a rich intestinal parasites, as laxative and as poultice against source of natural thymol. The antifungal properties boil) and it is often used as decoction and infusion; it against Aspergillus niger and Fusarium sp. studied by is rich on thymol, a phenolic compound known for its Chaker et al. [4]; whose test results showed that the antimicrobial properties [1]. The antimicrobial activity polar (hexane/dietyl ether 2/1) and non polar (hexane) of Ammoides verticillata (Av) essential oils was extracts had moderate antifungal activity, and the Av studied by Abdelouahid and Bekhechi [2] who
extracts were more efficient.
Corresponding author: Oumessaad Toubal, Ph.D., The antifungal potential of Av essential oils was professor, research fields: ecology and plant biotechnology.
studied by Mohammedi et al. [5], who showed a great E-mail: oumessaad2000@yahoo.fr.
Phytochemical Screening and Antimicrobial Evaluation of the Aqueous
Extracts of Ammoides verticillata, an Endemic Species
activity against Aspergillus flavus (rate of inhibition = The antimicrobial activity was made on raw plant 100%) which seems to be related to a high content of
extracts (10%) and infusion (10%) of stems and thymol; they found the highest yield of Av essential
flowers; the extraction was made by the soxlhet with oils (3.09%).
ethanol solvents. These extracts are cleared of solvents The aim of this work is to study the phytochemical
by evaporation (using a rotavapor) before screening and the antimicrobial evaluation of the
incorporation against bacterial strains ATCC aerial parts (stems, flowers) of Av towards various
(American Type Culture Collection): gram+ and bacterial strains. This study wasn’t made before. A
gram-oxidative and fungus as follows: Escherichia similar study was conducted on Genista numidica by
coli , Klebsiella pneumonia, Proteus mirabilis, Toubal et al. [6]; recently, the acceptance of traditional
Staphylococcus aureus , Pseudomonas aeruginosa, medicine as an alternative to the available antibiotics
Acinetobacter baumanii , Streptococcus faecalis, has led authors to investigate the antimicrobial activity
Proteus vulgaris , of medicinal plants [7]. Moreover, the increasing use
Staphylococcus epidermidis ,
Enterobacter aerogenes and Candida albicans which of plant extracts in food, cosmetic and pharmaceutical
were obtained from the Bacteriology Laboratoy, industries suggests that, in order to find active
Faculty of Medicine, HCU of Dorban in Annaba. compounds, a systematic study of medicinal plants is
The antibacterial activities were carried out by very important [8].
Aromatogram by diffusion method of Müller-Hinton
2. Materials and Methods on solid medium; the strains were reactivated using a
20 h culture growth at 37 °C and adjusted to 10 8
2.1 Plant Material CFU/mL. The bacterial strains are sowed on the The plant was collected on April 2010 [9] at
surface of the agar in radial spots form by means of Tlemcen Mountain (1,000 m) on calcareous soil [10].
swab and suspensions of young bacterial cultures Nomenclature is about its vernacular name which is
prepared according to the CLSI (committee for Nunkha, Ajowan [11]. The harvesting, drying, laboratory standards institute [14]). The application grinding, and chemical and microbial tests were
is made by sterile filters paper disks (6 mm diameter, conducted on May 2011.
06/limp) which were placed on the inoculated agar surfaces and impregnated with 10 µL of each
2.2 Different Analysis solution (10% dilution); the plates were incubated
The microscopic analysis was made in Botany during 24 h at 37 °C [15]. The reading of the results Laboratory, Faculty of Sciences, Annaba; a small
is made by the measurement of the inhibition quantity of powder is put down on the blade of
diameter around the disk.
microscope with lactic reagent [12] and colored with
3. Results and Discussions
iodine, covered with a small strip and warmed. The chemical screening consisted on preliminary
The aerial parts of the plant should be treated tests on the infusion (10%), searching for saponins,
separately because the antimicrobial activity is linked anthocyanes, tannins and leucoanthocyanes, and the
to the origin of the extract (stem, flower), the solvents preliminary tests on the powder searching for alkaloids,
and the used strains [16].
flavonoids, terpenes and steroids; the notation is (+) for
3.1 Microscopic Analysis
a low presence, (++) for a middle presence, and (+++) for a high presence, all these tests were carried out
The originality of this study was to identify Av by according to Harborne [13] methods. We determined
microscopic method; the diagnosis will relay on also the proteins % by Kheldjal method.
microscopic characters enough numerous and
Phytochemical Screening and Antimicrobial Evaluation of the Aqueous
Extracts of Ammoides verticillata, an Endemic Species
recognizable to differentiate this plant species; Table 1 Phytochemical screening of Av.
representative elements of flowers and stems powder Components Stems Flowers of Av were observed; in the powder of flowers, there
Flavonoids +++ ++ Saponins - are abundant pollen grains with exine, intine and a + Anthocyanes +++ ++
thickening of cellulose; there are also secretor canal of
+ essential oils (Fig. 1a) which constitute a discriminate
Terpenes & steroids
Tannins +++ +++ character allowing of family recognition.
Leucoanthocyanes ++ - Alkaloïdes - -
3.2 Chemical Composition
Table 2 Bacterial inhibition zone diameter (mm) by Av
The phytochemical screening (Table 1) of Av
flowers.
showed a wealth in polyphenolics compounds:
Infusion flavonoids, anthocyanes and tannins with a great
Bacterial strains
Extract
Escherichia coli
quantity in stem and flowers; leucoanthocyanes don’t <6
Proteus vulgaris
11.9 exist in flowers; saponins don’t exist in stem. There
Citrobacter freundii
<6 are no alkaloids in the stem and flowers; terpenes and
Proteus mirabilis
9.1 < 6 steroids exist in stems and flowers but with a little
Klebsiella pneumoniae
Enterobacter aerogenes
quantity. The present investigation showed essential 18.7 9.5
Staphylococcus aureus
17.2 10.5 oils in the flowers.
Staphylococcus epidermidis
Streptocoque faecalis
3.3 Antimicrobial Activity
Pseudomonas aeruginosa
11.7 8.7 The various tests of sensitivity of Av demonstrate a
Acinetobacter bauannii
considerable antimicrobial activity of its diverse about 80% of sensitivity essentially for Staph. a extracts towards tested strains; this antibacterial
17.2 mm and Staph. ep. 18.7 mm (Fig. 1); Proteus v, evaluation was not reported previously; it is about Proteus m and Citrobacter are resistant, with the
cultures of bacteria gram+ and gram-oxidative and infusion inhibition zone diameter 8.7-11.9 mm, fungus cited before. Citrobacter and Staph ep are the most sensitive
3.3.1 Sensitivity to the Flowers bacterias; efficiency is about 60%; Proteus m, Proteus The results are listed in Table 2; the extract v , Pseudomonas a, and Streptococcus faecalis are the
inhibition zone diameter is 8.3-18.7 mm; there is
very resistant ones.
3.3.2 Sensitivity to the Stems The extract inhibition zone diameter is ranged from
7.1 to 10.6 mm; extract is active only against E. coli
8.4 mm and Staphylococcus epidermidis 10.6 mm; then there is about 70% of resistant strains, and the
a. Pollen grain, Gr: 60 × 10. b. Citrobacter freundii. efficiency is about 30%; the infusion inhibition zone diameter is 9.5-11.2 mm; the infusion act on E. coli, Staph. ep , Enterobacter, Staph. aureus and Citrobacter,
then its efficiency is about 50% (Fig. 2).
3.3.3 Comparison between Stems and Flowers
Antimicrobial Activity
c. Staphylococcus epidermidis. d. Staphylococcus aureus.
Fig. 1 Pollen grains (a) and bacterial inhibition zone
More relevant results were obtained with the
diameter by flower infusion (b) and extract (c, d).
flowers extract which is active on 80% of the strains
Phytochemical Screening and Antimicrobial Evaluation of the Aqueous
Extracts of Ammoides verticillata, an Endemic Species
10 r (mm 8 6
nhibition zone diamete I
Flowers infusion
Stems infusion
Fig. 2 Bacterial inhibition zone diameter by Av stems and flowers infusion.
Inhibition zone dia
0 strains
Fig. 3 Bacterial inhibition zone diameter by Av stems and flowers extract.
and showed the maximal activity 18.7 mm, within
4. Conclusions
the stems extract 11.2 mm act only on 30% of them
A chemical and microbial studies were realized on (Fig. 3). Proteus v and Proteus m seem to be the most
Av , an endemic species; chemical screening of its resistant bacteria within Klebsiella, Streptococcus and
aerials parts indicates a wealth in polyphenolics Pseudomonas that have a medium resistance to all the
compounds; the results of aromatogram of raw plant extracts. Stems and flowers infusion: a similar
extract as well as the infusion, showed a significant antimicrobial activity excepted for Acinetobacter
antimicrobial activity towards bacterial tested strains, which is sensible to flowers infusion but resistant to
especially bacilli gram of enterobacteriaceae stems one; then oxidative and fermentative gram–
recognized as antibiotic resistant, particularly in the bacilli show an increase sensitivity (8.4-11.9 mm),
case of urinary infections. It seems that it is the flower which seems to be significant, whereas gram + cocci
extract which has more effect essentially on Staphyl. show a good sensitivity (7.1-18.7 mm) which doesn’t
aureus and Staphyl. epidermidis and the stem extract seem to be significant.
which has the low one. For the rest, flower extract and
Phytochemical Screening and Antimicrobial Evaluation of the Aqueous
Extracts of Ammoides verticillata, an Endemic Species
infusion seem to have a complementary effect. antiaflatoxinogenic potential of essential oils from an endemic thymus fontanesii boiss and reut, Les
This could give opportunities for using this species Technologies de Laboratoire 5 (19) (2010) 10-15.
in the treatment of urinary, respiratory, intestinal and [6] O. Toubal, A. Djahoudi, A. Bouzabata, Preliminary cutaneous infections and as a disinfecting additive on
studies and antimicrobial evaluation of the aerials parts nosocomial area. Flavonoids must be tested because of Genista numidica ssp. numidica of Genista numidica ssp. Numidica, Journal of Life Sciences 5 (2011)
the antimicrobial activity is principally due to
954-959.
flavonoids and terpenes; free fatty acids can be [7] M. Maoz, I. Neeman, Antimicrobial effects of aqueous regarded as potential bactericidal [17, 18], then it will
plants extracts on the fungi Microsporum canis 2193-2199
be interesting to test Av ones. The investigations to and Trichophyton rubrum and 03 bacterial species, Letters
en Applied Microbiology 26 (2002) 61-63. determine the degree of Av toxicity are in progress.
[8] A. Nostro, M.P. Germano, V. D’Angelo, A. Maino, M. We also have to test the effect of different dilutions of
Caunaelli, Extraction methods and bioautography for the plant extracts and determine its inhibitory
evaluation of medicinal plant antimicrobial activity, minimum concentration. Letters in Microbiol. Appl. 30 (5) (2000) 379-384. [9] O. Toubal, A. Djahoudi, C. Henchiri, Phytochemical
All these results constitute only a first step in the study and antimicrobial activity of Ammoides verticillata, research of natural substances biologically active;
an Algerian endemic species, Abstract in Current Opinion complementary trials will be necessary to confirm the
in Biotechnology, 2011, p. 143. [10] M. Felidjo, M. Bouazza, T. Ferouani, Short paper about the
revealed performances, because several variables can fliristic community and the interest of Ammoides verticillat, a influence the results, such as the environmental and
medicinal plant from Mounts of Tlemcen Natural Park climatic conditions of the plant, the choice of the
(Western Algeria), Geo-Eco-Trop 34 (2010) 147-154. [11] P. Quezel, S. Santa, Nouvelle flore d’Algérie et des
extraction methods and of the antimicrobial tests. The régions désertiques méridionales (New Flora of Algeria
valorization, preservation and sustainable use of Av and Southern Desert Areas), Vol. I & II, C.N.R.S., Paris, require the protection of its habitats.
France, 1962-1963, p. 1170. (in French) [12] P. Belty, Jackson, W. Derek, Atlas of Microscopy Plants
Acknowledgments
Culinary Herbs and Spices Press, Adivision of Pinter Public, London, 1990.
This study was partly supported by MESRS [13] J.B. Harborne, Methods of plant analysis, in: (Ministry of Scientific Research) of Algeria (Project
Phytochemical Methods Chapman and Hall, London, 1973, CNEPRU, No. F-0112008001). p. 132. [14] J.A. Kiehlbauch, G.E. Hannet, M. Salfinger, W. Archinal,
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[1] K. Ettayebi, J.El. Yamani, B.D. Rossihasani, Synergistic (Chemical Treaty of Phytotherapy), Edition Masson, effects of nisin and thymol on antimicrobial activities
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Listeria nonocytogenes and Bacillus subtilis, FEMS [15] L. Duraffourd, Traité de Phytothérapie Chimique Microbial Lett. 183 (2000) 191-195.
(Chemical Treaty of Phytotherapy), Edition Masson, [2] D.E. Abdelouahid, C. Bekhechi, Pouvoir antimicrobien
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de l’huile essentielle d’Ammoides verticillata (Nûnkha), [16] F. Bssaibis, N. Gmira, M. Megrane, Activité Biol et Santé 4 (2) (2004) 1-10. antibactérienne de Dittrichia viscosa (L.) W. Qreuter, [3]
C. Bekhechi, J.B. Boti, F.A. Bekkara, D.E. Abdelouahid, J. Casanova, F. Tomi, Isothymol in ajouan essential oil, Nat.
Microb. Indu. San. et Envir. Maroc 3 (1) (2009) 44-55. Prod. Com. 5 (7) (2010) 1107-1110.
[17] J.Y. Lee, Y.S. Kim, D.H. Shin, Antimicrobial synergistic [4] A.N. Chaker, H. Laouer, M.M. Zerroug, Antifungal
effect of linolenic acid and monoglyceride against activity of three Apiaceae (Ammoides verticillata Desf.)
Bacillus cereus and Staphylococcus aureus, J. Agric. Briq., Magydaris pastinaceae (Lamk.) Paol. and
Food Chem. 50 (7) (2002) 2193-2199. Bupleurum plantagineum Desf.) organic extracts, Revue
[18] C.Q. Sun, O’ Connor, A.M. Roberton, Antibacterial of des Régions Arides (Tunis) (2007) 420-422.
fatty acids and monoglycerides against Helicobacter [5] Z. Mohammedi, S. Bachik, N. Bekaroube, Antifungal and
pylori , FEMS Immunol. Med. Microbiol. 36 (2003) 9-17.
Journal of Life Sciences 6 (2012) 248-252
The Survey of Microbiological Contamination of Pitcher Cheese in West Azarbayjan Province, Iran
1 1 2 3 Ehsan Barati 4 , Mona Daneshgar Moghaddam , Navab Ghobadi , Hamid Reza Shafieian and Abbas Barin 1. Islamic Azad University, Tehran Branch, Tehran 6516935897, Iran
2. Payame Noor University, Bahar Branch, Bahar 6531855584, Iran 3. Shahid Maghsoodi, Teachers Training College, Hamedan, Iran 4. Department of Microbiology, Veterinary Medicine Faculty, Tehran University, Tehran 645314155, Iran
Received: August 11, 2011 / Accepted: September 21, 2011 / Published: March 30, 2012.
Abstract: Milk acts as a suitable peripheral culture for growth and propagation of different kinds of micro organisms. During the process of producing cheese, some micro organisms such as Escherichia coli, Coliform, Staphylococcus, Mold and Yeast may cause its contamination. In respect to the fact that pitcher cheese is produced in traditional way in different regions in West Azarbayjan, the aim of this research is examining the rate of contamination of pitcher cheese in West Azarbayjan. About 42 samples of pitcher cheese were gathered under strill condition from different parts of West Azarbayjan. In order to study microbes contamination, the samples were examined by standard microbiologic ways in laboratory from the 42 samples of pitcher cheese, four samples were contaminated by Staphylococcus aureus coagulase positive, 16 samples were contaminated by E. Coli, 21 samples by Coliform, 17 samples by mold and yeast. The producing and delivering should be controlled because of the rate of contamination in pitcher cheese and this kind of cheese should be produced in half industrial way by controlling and making special facilities for pitcher cheese producers.
Key words: Pitcher cheese, E. coli, coliform, mold and yeast, staphylococc.
1. Introduction because of producing unstrilled cheese in rural areas of Iran and not caring for health cases.
The pitcher cheese has the biggest consumption in Staphylococcus bacteria are the most important rural and citizen areas of West Azarbayjan, East spieces of micrococcus family. It is the factor of more Azarbayjan and more or less in other parts of Iran. It is than 80% of pustular infections. Food poisioning from produced from sheep milk, goat milk and sometimes the poision of Enterotoxin, Staphylococcus is widely from cow milk. To get the cheese with better flavor and spread all over the world that causes gastroenteritis and more stature, the cheese is produced without heating is caused mainly because of contamination with and by adding some natural starter. After clotting or different biotypes, Staphylococcus aureus. Human or refilling, the clot is salted and laid in pitches and the cattle are a potential resource of milk contamination openings are covered and fastened with cloths and the and its products during breast pumping, producing and pitchers are laid under wet soil. The cheese is used after delivering milk products. Rate of death by this six months. poisoning is very rare, although 1,500 children who This research has been done in different areas of West were poisoned by dried milk, died [1]. Azarbayjan in order to show the rate of contamination of Forty percent of spread of gastroenteritis were coliform, E. coli, Staphylococcus aureus, mold and yeast caused by food factors in Hungary. This percent is
reported less in Japan (10%-25%). The cause of food Corresponding author: Ehsan Barati, D.V.M., research
poisoning in rice was the time of its producing [1]. In field: microbiology. E-mail: ehsanbarati@yahoo.com.
The Survey of Microbiological Contamination of Pitcher Cheese in West Azarbayjan Province, Iran
Britain 15% of 359 cases of food poisoning from were covered with aluminium foils. Each sample with a enterotoxin staphylococcus that was reported in
completed form containing information about the site 1969-1990 were from red meat, poultries and their
of sampling was sent.
products [2]. Other cases in Britain were related to milk
2.2 Chemical Analysis
contamination of the cheese which was transferred by chest swelling [3]. In Turkey the study in 50 samples of
The rate of the pH in all samples which was done Cara cheese showed that the reason of high existence of
with pH meters (sartarius) was sent to the lab. Staphylococcus aureus was the use of raw milk and
2.3 Microbial Analysis
unsuitable conditions while saving and producing the cheese [4]. Haegbert et al. had a 2-continuous-year
The cheese samples were homogenized by study (1999-2000) of poisioneal food with mechanical mixer and complete care was done for the
Staphylococcus aureus in France, which showed that test by sodium sitrate solution 2%. 32% of its contamination were related to milk products
specially cheese [5]. 2.4 Method of the Test
E. coli is the most common aerobic organism in
2.4.1 Coliform Culture
digestive system in men and most animals, and the
A plate was considered for each sample and the existence of this organism in water and nutrients is also
characteristics of the samples were written on the plate. the rate of contamination index. Consideration of
1 mL of the sample was transferred to the plate while health conditions in the process of producing and
caring for sterilization cases.
keeping, and also the quality of raw milk is necessary. About 15-20 mL of the culture violet red blood agar The entrance resources were the consumed water and
(VRBA), whose temperature was 45 °C, was poured instruments.
in each plate and the culture and the sample were Turanta et al. [6] did experiments on 38 samples of
mixed well with a rotation movement. After it got into white Turkish cheese and they declared the existence of
solid form, about 5-10 mL culture was added to it again coliform in this cheese. The researches were done on
and it got into solid form again. The cultured plates Orgu cheese in Turkey by Turkoglue et al. who showed
were kept upside down in the hot-house 31 °C for 24 that coliform was separated from this cheese [7].
hours.
Fungi in our surroundings are microorganisms that
2.4.2 The Confirming Test
spread widely in our surroundings and because of this One fifth of the suspicious colonies was chosen and spreading we can consider them as a part of natural floor injected to the testing tube containing Dorham tube and of a nutrient. In an experiment that Grasi et al., did on common BGb culture. The tubes were kept in the Manura cheese, they separated the mold and yeast from hot-house 31 °C for 24 hours. The existence of gas at the samples that its average was 7.41 log CFU/g [8]. the end of incubation emphasized the coliform [9].
2. Materials and Methods 2.4.3 Method of Searching E. coli
2.1 Sampling This method showed that E. coli is based on
producing gas because of lactose fermentation and Forty two samples of pitcher cheese (each samples
producing indole from tryptophan analysis at 44 °C. 200 g) were gathered randomly from nine areas of
1 mL of the confirmed positive coliform (BGb tube West Azarbayejan in autumn-winter in 2010-2011. All
containing Dorham) was added to the culture Brilliant the samples were transferred to the laboratory in
lactose Broth and another 1 mL added to peptone conical flask and under fridge conditions. The glasses
water.
The Survey of Microbiological Contamination of Pitcher Cheese in West Azarbayjan Province, Iran
The testing tubes were laid in the incubator 44 °C for had staphylococcus contamination [11]. In a study that
48 hours. At the end of the incubation period the was done on 25 samples of Irish rural cheese by sample would have been positive showing E. coli if the
Convey et al., 10 samples having positive gas had been produced in the tube containing the
Staphylococcus aureus were reported [12]. And also in Brilliant lactose Broth. Then 5 mL Coax were added to
a research that was done on three samples of Italian the tube containing peptone water after incubation and
cheese by Dicargo et al., the rate of staphylococcus was it was mixed well. The result was studied after one
estimated 4.2-4.4 log CFU/g [13]. minute. The tubes whose surfaces got red showed
In this study that, performing on 42 samples of positive reaction of endol and the sample was positive
pitcher cheese gathered from different parts of West of E. coli [9].
Azarbayjan four samples, showed contaminated by
2.4.4 Staphylococcus Culture Staphylococcus aureus Coagulase positive and the
25 g of each samples of the cheese were separated in most contaminated sample was observed in Mahabad. strilled form for testing the existence of staphylococcus
Sample number six of Bookan area had the most and the meat was added to the 70 mL and they were
contamination with Staphylococcus areus (3.8 × 10 laid at 37 °C for 24 hours after hemogenation. Then the
CFU/g) that was higher than the usual level. This samples were cultured on Barid Parker and they were
shows the unsuitable health conditions while producing, incubated at 37 °C for 48 hours. Gram coloring was
keeping, transporting cheese by personal and if the done on the colonies suspicious of having initial numbers of this cheese are high, even the staphylococcus. The catalase test would have been
reduction of pH in the time of clotting can’t prevent the done if the gram positive cocci had been observed.
production of toxin and occurring staphylococcus Coagulase test was done in the tube method for positive
poisoning.
gram bacteria and positive catalase. The positive In different traditional cheese, Staphylococcus coagulase bacteria were interpreted as Staphylococcus
aureus disappeared while producing and it is caused aureus [10].
because of skin, nose, mouth contamination of the
2.4.5 Yeast and Mold Culture related personals. So if the heating control and health
1 mL of the samples was cultured briefly in the cases are not considered, contamination with chosen site. The plates were laid in an aerobic
staphylococcus will occur.
condition at 25 °C about 3-5 days. Then the mold and As a result of the experiments that were conducted yeast colonies were counted separately and their rates
on 38 samples of white Turkish cheese by Turantus et in gram were reported from the samples.
al., the amounts of coliform and E. coli were separated from these samples [6]. Hayaloglu et al. researches on
2.5 Statistical Analysis Tulum cheese showed coliform as much as 5.01 log
Statistical computing was done by software SPSS CFU/g [14]. In another study that was done on nine copy 12 (SPSS Inc. and Chicago, IL, USA) by using K
samples of Anevato cheese by Hatzikamari et al., square.
contamination of this cheese with coliform is as much as 7.01 log CFU/g [15].
3. Results and Conclusions
Convey et al. reported the contamination of most In the studies done by Turanta et al. on 38 samples
samples with coliform as much as 10 × 10 CFU/g [12]. of Turkish cheese, a few staphylococcus were
In a study that was performed by Deker et al. on the separated [6]. And also Deluca et al. in Bolonia Italy
cheese that was gotten from unstrilled milk, the rate of and Araujo et al. in Brazil illustrated that the cheese
contamination with E. coli was reported as much as 10
The Survey of Microbiological Contamination of Pitcher Cheese in West Azarbayjan Province, Iran
CFU/g. In a research that was done on Fossa cheese by [7]. In another study that was done on Tulum cheese by Gobbeti et al., the samples were negative considering
Hayaloglu et al., the rate of contamination to mold and contamination with E. coli [16].
yeast was about 5.2-6.041 log CFU/g [7]. In the present study that was done on 42 samples of
In this study that has been done on 42 samples of pitcher cheese, 21 samples showed contamination with
pitcher cheese, 17 samples had contamination with coliform and 16 samples showed contamination with E.
mold and yeast and the most contamination was coli where these contaminations were more than the
observed in Urumia and the sample number 42 of other studies that had been done on other kinds of
Mahabad area had the most contamination by mold and cheese. The most contamination with coliform and E.
yeast (3.8 × 10 CFU/g). It seems that the reason of the coli was observed in Urimia and the most rate of
contamination of cheese with mold and yeast is the contamination with coliform was observed on sample
existence of surrounding contaminations while filling number 12 in Bookan that was about 2.1 × 10 CFU/g. It
the pitchers, the pots related to micro organisms, don’t seems that the existence of coliform and E. coli in the
care for health cases by personal and the existence of samples is because of the percent of the added salt to
moisture in these products while keeping and clotting the cheese and not reducing pH in the products as well
of the cheese under the soil. And of course no suitable as not caring for health conditions in producing and
lidding over the pitchers provides enough oxygen for clotting. The coliforms can be checked in order to
yeast and mold growings.
control intestine E. coli in the cheese because the In Table 1, the spread of contamination of pitcher method of producing, and clotting can be main
cheese in Azarbayjan has been shown separately based resource of contamination and coliform growing.
on the place of sampling.
During producing soft cheese and half soft cheese, It must be noted that the findings were performed reducing pH factor is the cause of controlling E. coli,
statistically with SPSS software copy 13, and no so pH will increase when the salting percent is high,
meaningful difference was observed in relation to therefore it must be avoided to add salt in order to
microbial contaminations of the studied areas. prevent E. coli growth in the cheese again. The study
The rate of pH in all samples was about 4.05-5.20 shows the pathogen intestine E. coli growth in the
and no relation was observed between the rate of pH cheese having pH less than five.
and got positive of the samples.
In the studies that Turkuglue et al. did on Orgu This study showed that the microbial contamination Turkish cheese, the mold and yeast were separated
of pitcher cheese had been provided from different parts from all samples that the rate was about 5.5 log CFU/g
of Azarbayjan, indicating very bad health conditions
Table 1 Spread of contamination of pitcher cheese in Azarbayjan places.
City Samples Staphylococus E. coli Coliform Mold & yeast Bookan 15 6.6% 46.6% 66.6% 53.3% Piranshahr 5 20% 20% 20% 20% Khoi 2 - - - 50%
- Mahabad 4 50% 50% 50% 75% Total
The Survey of Microbiological Contamination of Pitcher Cheese in West Azarbayjan Province, Iran
during producing, clotting, keeping transferring of [8] E. Gerasi, E. Litopoulov, N. Tzanaetakis, Microbiological stody of manura, ahard cheese made from raw ovine milk
these kinds of cheese. In order to improve the in the Greek islaan sifnos, International J. of Dairy
microbiologic qualities of these kinds of cheese, it is
Technology 156 (2) (2003) 117-122.
recommended to use strilled milk or enough heated [9] E.J. Baron, S.M. Finegold, Enyerobacteriaceae, in: Bailey milk instead of raw milk, and these kinds of cheese
and Scott’s Diagnostic Microbiology, 11th ed., Mosby, St. Louis, Missouri, USA, 2002, pp. 481-487.
should be produced under good producing conditions, [10] E.J. Baron, S.M. Finegold, Staphylococcus, micrococcus,
good clotting and keeping, good transferring and it is and similar organisms, in: Bailey and Scott’s Diagnostic recommended to study more to get more information
Microbiology, 11th ed., Mosby, St. Louis, Missouri, USA, about the process of contaminating the cheese.
2002, pp. 218-229. [11] J.A. Centeno, J.L. Rodrigves, A. Cepeda, Microbiological
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of the microbiological status of Irish farmhouse cheeses pp. 343-351.
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cheese, J. Food Engineering 66 (2005) 401-404. [14] A.A. Hayaloglu, S. Cakmakci, E.Y. Brechany, [5] S. Haeghebaert, F. Le Querrec, A. Gallay, Les
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[6] F. Turanta , A. Unluturk, D. Goktan, Microbiological and [15] M. Hatzi kamari, E. Litopovlov-tzanetaki, M. Tzunetakis, compositional status of Turkish white cheese international,
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Applied Microbiology 87 (1999) 595-601. [7]
H. Turkoglv, Z. Ceylan, K.S. Dajisolyv, The [16] M. Gobbetti, Microbiology and biochemistry of micribiological and chemical quality of Orgv cheese
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Detection of “Candidatus Phytoplasma asteris” in Brussels Sprout and Its Possible Association with Flower Bud Failure in Poland
1 1 Maria Kami 2 ńska , Hanna Berniak and Piotr Kami ński 1. Laboratory of Virology, Department of Plant Protection, Research Institute of Horticulture, Skierniewice 96-100, Poland
2. Department of Genetics, Breeding and Biotechnology, Research Institute of Horticulture, Skierniewice 96-100, Poland
Received: October 07, 2011 / Accepted: November 16, 2011 / Published: March 30, 2012.
Abstract: Severe growth abnormalities including shoot stunting, leaf blade reduction and flower bud failure of Brussels sprout were observed in Poland. The presence of phytoplasma in diseased as well as in healthy looking plants, was demonstrated by nested polymerase chain reaction assay employing phytoplasma universal rRNA primer pairs-P1/P7 followed by R16F2n/R16R2. Products of PCR primed by R16F2n/R16R2 primer pair from naturally infected Brussels sprouts were sequenced. Comparison of the obtained 16S rDNAs revealed high nucleotide sequence identity between analyzed phytoplasma isolates (99.8%-100%). They were also nearly identical with the sequences of other phytoplasmas isolates from sub-group 16SrI-B, and they were classified as members of “Candidatus Phytoplasma asteris”.
Key words: Brussels sprout, flower bud failure, phytoplasma, 16S rDNA.
1. Introduction amplified by means of PCR technology, have been used. These molecular methods have been used to
Phytoplasmas are non-culturable, wall-less detect and characterize phytoplasmas in a number of prokaryotes (class Mollicutes) causing yellows and
plant and insect hosts.
witches’ broom type diseases. The diseases have been In the Brassicae family, retarded growth, shoot distributed worldwide in several hundred plant species. proliferation and flower virescence associated with During the past two decades, phytoplasmas have been phytoplasma infection, have been reported from found in many herbaceous and woody plant species with Europe, North America and Iran. In Europe several types of symptoms using polymerase chain phytoplasma infection has been observed in diseased reaction (PCR) for detection and identification [1, 2]. cabbage (Brassica oleracea var. capitata) [3, 4], In the past, numerous reports on such disorders were sprouting broccoli (Brassica oleracea var. Italica) based merely on symptom expression and [4-6], turnip (Brassica rapa var. rapifera), kale pathogen/vector relationships. Some of these disorders (Brassica oleracea var. Palmifolia), wild radish are now clearly assigned to identified phytoplasmas. (Raphanus raphanistrum) [4, 5], Brussels sprout
Recently, molecular methods based on sequence and (Brassica oleraceae L. var. gemmifera DC) [6] and
restriction fragment length polymorphism (RFLP) oilseed rape (Brassica napus) [7-10]. Since the 1980s,
analysis of ribosomal genes (rDNA) specifically in Alberta, Canada, symptoms of canola yellows
Corresponding author: Maria Kami ńska, Prof., research similar to those of green petal of oilseed rape, have fields: plant virology, virus and phytoplasma diseases of horticulture and agriculture crops. E-mail: been observed in canola plants (Brassica napus and
maria.kaminska@insad.pl.
Detection of “Candidatus Phytoplasma asteris” in Brussels Sprout
and Its Possible Association with Flower Bud Failure in Poland
Brassica rapa ) [11, 12]. In 2000, in southwestern Institute of Horticulture, Skierniewice, Poland. Texas, about 5% of cabbage plants displayed
Plants of four Brussels sprout lines obtained from symptoms of purple leaf discoloration and sprout
seeds were cultivated in the field in 2008. At the end of proliferation characteristic of phytoplasma [13, 14].
October healthy looking plantings with desired Very recently in Iran, similar cabbage disease yellows,
morphological characters for the breeding purposes damaged cabbage up to 50% in certain fields [15].
were taken for the generative propagation. Plantings On the basis of molecular analyses, phytoplasmas
were potted and rooted in sterile medium and grown in associated with shoot proliferation, flower virescence
15 °C - 22 °C for five weeks. All genotypes were and malformation of plants belonging to the Brassica
vernalized for three months at 5 °C - 7 °C and natural family in Europe and USA were identified as
photoperiod. The vernalized plants were maintained in members of the “Ca. phytoplasma asteris”, subgroup
the greenhouse at a temperature of 12 °C - 24 °C. 16SrI-B [6, 8, 13, 14]. Most phytoplasmas associated
Fertilization and pest and disease control followed the with yellows-type symptoms of canola plants in
current requirements and recommendations for Canada belong to the subgroup 16SrI-A or 16SrI-B
Brussels sprouts.
[11, 12], while phytoplasma associated with Iranian For PCR amplification, samples of leaves were cabbage disease is related to “Ca. phytoplasma
collected from 12 symptomatic plants (line CAP) with trifolii”, subgroup 16SrVI-A [15].
stunted growth and leaf malformation and from 10 Brussels sprout, as well as other brassica vegetables,
apparently healthy looking Brussels sprout plants is an economically important vegetable crops in Poland.
(lines AP, P5 and DE) without disease symptoms. They are grown worldwide in cool regions of Europe
Samples of leaves of Catharanthus roseus inoculated and America for their content of valuable vitamins,
by grafting with the reference strains of aster yellows folate, and phenolic compounds together with vitamin
phytoplasma (AY1, 16SrI-B, kindly supplied by Dr.
C are the major antioxidants in Brussels sprouts. In I.M. Lee, Beltsville, USA) were also included in this 2009, a new disease seriously damaged some Brussels
study.
sprout plants in Poland. The affected plants were
2.2 DNA Extraction and PCR Amplification stunted, with severely malformed leaves and flower
bud failure [16]. To our knowledge, no information is Total DNA was extracted from fresh leaf midribs using DNeasy Plant Mini Kit (Qiagen, Biokom, Poland)
available on these types of symptoms in this crop. The according to the manufacturer’s recommendation.
aim was to describe the disease symptoms and to determine whether they were associated with
Extracted nucleic acids were used as templates for direct PCR with universal primers P1/P7 [17, 18].
phytoplasma infection, and if so, to identify the pathogen. In an attempt to identify the Brussels sprout
Products from the first PCR were diluted 25 times and then used in nested reactions as templates for
disease, standard diagnostic methods were used amplification with universal primers R16F2n/R16R2
including PCR-RFLP and sequence analysis of phytoplasma 16S rDNA.
[19]. All the PCR assays were run under parameters described previously [20].
2. Materials and Methods
The amplification products (5 μL) were analyzed by 1% agarose gel electrophoresis in 0.5 × TBE (45 mM
2.1 Symptom Observation and Plant Material Tris-borate, 1 mM EDTA, pH 8.3) buffer followed by
The observations were carried out on breeding lines staining with ethidium bromide (0.5 μg·mL -1 ) and of Brussels sprout plants growing in the greenhouse in
visualized with a UV transilluminator (Syngen Biotech, the former Research Institute of Vegetable Crops, now
Poland).
Detection of “Candidatus Phytoplasma asteris” in Brussels Sprout
and Its Possible Association with Flower Bud Failure in Poland
2.3 Sequencing and Computer Analysis PCR amplified products (obtained with primers
R16F2n/R16R2) from three samples (CAP, 218 and DE) were used for phytoplasma 16S rRNA gene sequence analysis. Sequencing was performed in an AbiPrism 3100 Genetic Analyzer apparatus (Applied Biosystems, USA), at the Maria Sk łodowska Memorial Cancer Center and Institute of Oncology, Warsaw, Poland.
Aligned nucleotide (nt) sequences of 16S rRNA gene fragments were analyzed and their identities determined using the Lasergene v. 7.1 software package (DNASTAR Inc., Madison, WI., USA). The sequences were then checked for the similarity to
known sequences, using the BLAST service available Fig. 1 Severe leaf malformation of phytoplasma affected
Brussels sprout.
at http://blast.ncbi.nlm.nih.gov/Blast.cgi [21]. A
phylogenetic relationship of tested isolates was No disease symptoms suggesting phytoplasma estimated using the neighbour-joining method in
infection were observed in Poland in other glasshouses MEGA software v. 4.0.2 [22].
or fields growing Brussels sprouts of several cultivars and lines.
3. Results
3.2 Phytoplasma Detection and Identification
3.1 Symptoms The affected glasshouse-grown Brussels sprouts in
Specific products were obtained in direct PCR with the second year of their growth developed conspicuous
the universal primer pair P1/P7 (expected length ~ 1.8 symptoms that included severely stunted growth, leaf
kb) only for the control samples of the reference strain malformation associated with severe leaf blade AY1. No visible product was amplified by the direct reduction, leaf chlorosis and flower bud failure (Fig. 1).
PCR from samples obtained from Brussels sprout and The roots of the diseased plants were reduced and
healthy periwinkle plants.
showed damage not related to fungus, bacteria or other However, in nested PCR with R16F2n/R16R2 pest infestation.
primer pair a specific product (1.25 kb) was obtained The occurrence of symptoms was noted on 25 plants
for DNA samples collected from leaves of two out of of the male sterile line CAP. The most severe
four symptomatic Brussels sprout plants, and from symptoms were observed in Brussels sprouts from the
three out of three symptomless Brussels sprout plants, beginning of April till the middle of May. At the end of
all collected in the first day of April. No specific May most of the affected plants recovered and the new
product was obtained for DNA samples collected at the developed leaves showed mild malformation. Healthy
end of April from leaves of eight symptomatic nor looking Brussels sprouts of the other lines (P5, AP and
eight healthy looking Brussels sprout plants. DE) grown in the same conditions remained free of
PCR products obtained using R16F2n/R16R2 symptoms. However, in the following months some of
primer pair for samples of Brussels sprout lines were them showed pods and seeds which were shriveled,
sequenced. Nucleotide sequence analysis of the often malformed and underweight.
PCR-amplified 16S rRNA gene fragment of the
Detection of “Candidatus Phytoplasma asteris” in Brussels Sprout
and Its Possible Association with Flower Bud Failure in Poland
isolated phytoplasmas revealed that they were closely Phytoplasma asteris”, reported from other brassicae. related to phytoplasma members of the 16SrI-B
Our results confirm the report of Marzachi et al. [6] that sub-group (Fig. 2). Sequences of two phytoplasmas
this taxon, “Candidatus Phytoplasma asteris”, is found in B. oleracea subsp. gemmifera plants with
implicated in the growth abnormalities in B. oleracea severe leaf malformation and flower bud deficiency
subsp. gemmifera plants. Based on the results of this (line CAP) and in asymptomatic plants (line 218 and
study and other reports from Europe and North DE) (GenBank accession numbers GQ240826, America, AY phytoplasma, mainly the subgroup GQ240827, HM480047) were identical, and they
16SrI-B, seems to be the most common yellows-type showed more than 98% identity to the sequences of
disease associated with various plant species. This ‘Candidatus Phytoplasma asteris’.
ranking is consistent with the top position of the AY group among other phytoplasma groups worldwide (1,