Posters

 

P1. INHIBITION OF GROWTH AND BIOFILM FORMATION OF PSEUDOMONAS AERUGINOSA BY NEWLY ISOLATED LACTOBACILLI FROM HUMAN BREAST MILK

D. Bratanova1, D. Borisova1, Ts. Paunova-Krasteva1, T. Strateva2, S. Danova1, S. Stoitsova1

1Institute of Microbiology, Bulgarian academy of Sciences; 2Department of Medical Microbiology, Medical University, Sofia

 

Pseudomonas aetuginosa is an opportunistic pathogen. It invades, as a biofolm, burn wounds and skin damages of patients with diabetes. It can contribute to the morbidity and mortality in immunocompromised patients and patients with chronic lung infections and cystic fibrosis (CF). The global problem with antibiotic resistance relates to this bacterium as well, which necessitates the search of alternative approaches to its eradication. One prospective development is the exploration of bacterial inter-species antagonistic relations. In this regard, probiotic bacteria represent one major target group.

The aim of the present study is to estimate the effect of supernatants of exponential lactic acid cultures in MRS broth on the growth and biofilm development of a P. aeruginosa CF isolate. The tested 16 Lactobacillus sp. are newly isolates from human breast milk and are identified to the genus level with classical phenotypic characteristic. A preliminary test showed antagonistic activity of selected for this study strains against E. coli ATCC 25927 and Bacillus subtilis ATCC6633. The cell-free supernatants supplement (5% v/v) to TSB were used to test their antagonistic activity against P. aeruginosa. Bacterial growth was registered as spectrophotometrically (OD 600nm) and the biofilm biomass was quantificated by the CV assay. It was shown that 6 of the supernatants suppress to some extent bacterial growth. With the biofilm, 12 of the CFS suppressed significantly the biofilm, while 2 others had stimulation effects. The results show that most of the CFS may contain active metabolites, which are prospective for the control of P. aeruginosa biofilm. Further characterization of naturally produced inhibitors, however is needed and it is still in progress.

 

 

 

P2. POLYMETHYLGALACTURONASE BIOSYNTHESIS BY ASPERGILLUS NIGER 26 CELLS CO-IMMOBILIZED IN CALCIUM ALGINATE WITH PECTIN

Ekaterina Krumova*, Jeni Miteva-Staleva, Vladislava Dishlijska, Maria Angelova

The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,

Academician G. Bonchev 26, 1113 Sofia, Bulgaria

*Corresponding author: ekrumova@abv.bg , Tel. +359 2 9793126;

 

Polymethylgalacturonase (PMG) catalizes the cleavage of a-1,4-glicosidic linkages of highly esterified pectin. This enzyme can be applied in food and textile industry, as well as in waste management and animal feed. PMG is one of the least studied pectinolytic enzymes. Aspergillus niger 26 was selected for the present work since it demonstrated significant activity of PMG during submerged cultivation. Immobilization of fungal spores in Ca-alginate resulted in an increase in PMG productivity and realisation of semicontinuous cultivation. This study was desighned to examine the effect of co-immobilization on the regulation of the enzyme synthesis. Fungal spores were entraped in the carier containing Na-alginate and pectin. Although the included cells were into direct contact with the inductor, increase in PMG production was not found. These results could be due to the participation of the feedback control by accumulated enzyme protein in the culture filtrate. In contrast, the co-immobilization with pectin reduced the effect of catabolite reprression in the presence of glucose in the nutrient medium.

 

 

 

P3. PECTINASE PRODUCTION AND ACTIVITIES OF KEY METABOLIC PATHWAYS IN FREE AND IMMOBILIZED CELLS OF ASPERGILLUS NIGER 26

Ekaterina Krumova*, Radoslav Abrashev, Nedelina Kostadinova, Maria Angelova

The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,

Academician G. Bonchev 26, 1113 Sofia, Bulgaria

*Corresponding author: ekrumova@abv.bg , Tel. +359 2 9793126;

  

The immobilization of microbial cells resulted in enhanced production of valuable compounds but the effect on their metabolism has not yet been studied in detail. Comparison between physiological activity of the free and immobilized fungal cells on the basis of key enzymes of the glycolytic pathway and the tricarboxylic acid (TCA) cycle (hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucose isomerase, succinate dehydrogenase, isocitrate dehydrogenase, and malate dehydrogenase) was performed. Cells of Aspergillus niger on pectin-containing medium were used as a model systems. Our investigations showed a re-routing of carbon metabolism from glycolysis into the pentose phosphate pathway (PPP) and activation of TCA cycle after immobilization. Moreover, the established trend remains stable during repeated-cycle batch cultivation (6 cycles, 96 h). Probably, these changes in both metabolite pathways are a part of cellular stress-resistance mechanism

 

 

 

P4.CHARACTERISTICS OF BACTERIOCIN-PRODUCING LACTOBACILLUS CRISPATUS G4 STRAIN

Galina Stoyancheva1*, Elena Chorukova2

1Department of Microbial Genetics, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev str. bl. 26, Sofia, Bulgaria,

2Department of Applied Microbiology, The Stephan Angeloff Institute of Microbiology

*Corresponding author: galinadinkova@abv.bg

 

According to World Health Organization probiotics are "Live microorganisms which when administered in adequate amounts confer a health benefit on the host". Production of bacteriocins is one of the most important properties of the bacterial probiotic strains. These are antimicrobial proteinaceous substances secreted by some bacteria against other microorganisms.

The present work describes the antimicrobial activity and molecular identification of a bacterial strain isolated from the Human Microbiota. The strain was isolated from vaginal sample of healthy reproductive-age woman. Lactobacillus crispatus G4 strain inhibit the growth of human pathogen microorganisms and the physicochemical nature of the active substance was investigated. The detection of the gene related to bacteriocin production was determined by PCR and sequencing analysis.

After additional researches Lactobacillus crispatus G4 strain can use in developing probiotic products for restore and maintain a healthy vaginal flora which can help reduce the risk of infections.

 

 

 

P5.MICROBIOLOGICAL ANALYSIS OF SOIL AROUND THE BUTCHERY DURING SPRING SEASON 2008

Idriz Vehapi*,  Kemajl Kurteshi

Department of Biology, Faculty of Natural Sciences, University of  Prishtina, Kosovo

*Corresponding author: vehapiidriz@yahoo.com

 

The microbial population in soil contaminated with butchery  effluent was investigated using the spread plate method. The results revealed a high mean count of 4.70±0.01×106 cfu/g for bacteria  and 1.40±0.04×104 cfu/g for  fungi respectively. The counts were higher than that of the control soil sample with 2.40±0.02×104 cfu/g and 1.10±0.02×103 cfu/g for the bacteria and fungi respectively. Among the bacteria isolated, Escherichia coli had the highest frequency of occurrence of 21.0 %, followed by Streptococcus faecalis and Staphylococcus aureus with 17.0 % .

 

Key words: microbiological, analysis, soil , butchery 

 

 

 

P6.MICROBIOLOGICAL ANALYSIS OF WATER USED IN BUTCHERY

Ilmije Vllasaku* ,  Kemajl Kurteshi

1State advisor in Ministry of Angriculture of  R.Macedonia

2Department of Biology, Faculty of Natural Sciences, University of Prishtina, Kosovo

*Corresponding author:  ilmijevllasaku@yahoo.com

 

Bacteriological analysis of water used in two butchery in Gjilani city,  Kosovo. The samples collected   during the spring season 2008.

Results from the bacteriological analysis indicated that all the samples collected were highly contaminated with pathogenic organisms, Escherichia coli having the highest observed prevalence (27%) while Enterobacter spp.  had the least observed prevalence (7.6 %) among the encountered isolates. Highest mean bacterial count observed during the study was 6.4 x 106 cfu/ml.

 

Key word : microbiological, analysis,water, butchery

 

 

 

 

P7. MICROBIAL EVALUATION OF COMMERCIAL BOTTLED WATERS

Kemajl Kurteshi*,  Idriz Vehapi

Department of Biology, Faculty of Natural Sciences, University of  Prishtina, Kosovo

*Corresponding author: kemajlkurteshi@yahoo.com

 

 

Continuous increase in the sale and indiscriminate consumption of packaged drinking waters in Kosovo is of public health significance.

We analyzed 4 different brand bottled water. Total heterotrophic bacteria plate counts (HPC) ranged from 2 to 150 for bottled water. One brand of bottled water had mean HPC greater than 100 per ml millilitre water and thus fell below the United States Environmental Protection Agency (USEPA) and World Health Organisation (WHO) drinking water standard of 100 HPC per ml water. Total coliforms and Escherichia coli not detected in all brand of bottled water.

 

Key words: microbial, evaluation, bottled, waters

 

 

 

P8.CHEMICAL COMPOSITION AND ANTIBACTERIAL ACTIVITY OF GEUM URBANUM EXTRACTS

Lyudmila Dimitrova1*, Milena Popova2, Iva Tsvetkova1, Vassya Bankova2, Hristo Najdenski1   

1The Stephan Angeloff Institute of Microbiology, BAS

2Institute of Organic Chemistri with Centre of Phytochemistry, BAS

*Corresponding author: Lus22@abv.bg

 

Object of Research. Pathogenic bacteria have always been considered as a major cause of human and animal infections. Even though pharmaceutical companies have produced new antibacterials in the last years, resistance to these drugs has increased and has now became a global concern. Many plants used in traditional medicine are valuable source of new substances with therapeutic potential. In the recent study the chemical composition of different extracts of the herb Geum urbanum and its antibacterial activity were investigated.

Materials and Methods: Dry mass of aerial and underground parts of the herb was extracted with methanol and re-extracted with petroleum ether, ethyl acetate and butanol MIC and MBC were determined by using liquid broth microdillution method..

Results. All extracts displayed varying antibacterial activity against Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Bacillus subtilis, Listeria monocytogenes). No activity was found against Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium) and Candida albicans as well. The most sensitive bacteria are Staphylococcus aureus and Staphylococcus epidermidis. The ethyl acetate extracts of aerial parts and roots were the most active. We isolated and identificated six chemical substanses with different types of chromatography, NMR and GC/MS – catechine, gein, rhamnose, glycoside of ellagic acid, wich is new for the species and other acetyl derivatives of ellagic acid, which are new for the genus Geum.

Conclusions. Geum urbanum possesses antibacterial activity against Gram-positive bacteria. The activity of individual substances will further be studied which will contribute to the sustainable use of Bulgarian natural resources and treatment of various skin sequelae, food infections, etc., which are related with the corresponding bacterial pathogens.

 

 

 

P9.MOLECULAR DIAGNOSTIC APPROACH TO QUICK AND SIMULTANEOUS DETECTION OF DIARRHEA-CAUSING PATHOGENS

Miglena Koprinarova1, 2*, Kremena Slavova2

Institute of Molecular Biology “Roumen Tsanev”, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria1

Genetic Medico-Diagnostic Laboratory “Genica”, Sofia, Bulgaria2

*Corresponding author: mkoprinarova@obzor.bio21.bas.bg , phone: +359 2 979 2638

 

Purpose: Diarrhea caused by viral and bacterial infections is still a health problem in many parts of the world including the most developed countries. The aim of this study is that a rapid and reliable approach be developed for detection of some of the most common causes of diarrhea in Bulgaria.

Methods: Nucleic acid extraction and reverse transcription were carried out using commercial kits. Multiplex end-point PCR was used for pathogen detection. The amplified DNA products were detected on 3% agarose gel.

Results: We developed a multiplex PCR method for simultaneous detection of the following bacterial pathogens: Shigella, EIEC, EHEC, EPEC, Salmonella, Campylobacter jejuni and Yersinia and pathogenic viruses: norovirus, rotavirus, astrovirus, bocavirus and adenovirus. A total of 42 samples were tested with our in-house method and the results received were compared with commercial real-time PCR kit. The sensitivity of the multiplex PCR was 30-150 copies/reaction. The specificity was 93-100%.

Conclusions: The proposed method for simultaneous detection of viral and bacterial pathogens causing infectious diarrhea has several advantages. It is fast, sensitive, specific and cost-effective as well as easy to perform and interpret, therefore, applicable in different laboratories.

 

 

 

P10. MICROBIOLOGICAL ANALYSIS OF RIVER STANISHORKA DURING SUMMER SEASON 2008

Muharrem Ismaili1* ,  Kemajl Kurteshi2

1Master study,  Department of Biology, Faculty of Natural Sciences, University of Prishtina, Kosovo

2Department of Biology, Faculty of Natural Sciences, University of Prishtina, Kosovo

*Corresponding author: muharremismaili@yahoo.com

 

Bacteriological  analyses were carried out on water of river Stanishorka, which passed through Gjilani city Kosovo. The results obtained were compared with WHO and EPA

standards for drinking and recreational water.

None of the samples complied with bacteriological standards as Total coliform counts generally exceeded 1,600 MPN/ml, and pathogen count such as Salmonella-Shigella counts and total coliform counts were very high. The presence of pathogens in water for drinking and swimming purposes is of public health significance considering the possibility of the presence of other bacteria, protozoa and enteric viruses that are implicated in gastro-intestinal water borne diseases and the low infectious dose for these water borne pathogens.

 

Key words : Microbiological analysis, river Stanishorka

 

 

 

P11. MICROBIOLOGICAL ANALYSIS OF LAKE LIVOQI  DURING SUMMER SEASON 2012

Ram Kortoqi1* ,  Kemajl Kurteshi2

1Master study Department of Biology, Faculty of Natural Sciences, University of Prishtina, Kosovo

2Department of Biology, Faculty of Natural Sciences, University of Prishtina, Kosovo

*Corresponding author: rkortoqi@yahoo.com

 

            Investigations of composition and dynamics of bacterial community in lake Livoqi  during the summer season 2012. The purpose of this research is to determine the composition and dynamics of the bacterial community in lake Livoqi , and to give us a real picture about the quality of the water and biocoenose as a whole, on the basis of microbiological parameters significant as indicators of water quality. On the basis of results gained in this research, water in lake Livoqi  varies from relatively clean to moderately polluted water. Composition of bacterial community varies in the function of physico-chemical features, the state of other fact in biocenosis, eutrophication as well as anthropogenic influence. The total number of the bacterial community was 1,1х106-4,09х106 bacteria/cm3, changing during the season, in correlation with physical and chemical properties of the water as well as with other relevant factors in ecosystem.

Key words: microbiological, analysis,lake, livoqi

 

 

 

P12. Microflora of full cream home-made sheep and cow yogurt from Rodopa mountain, Bulgaria

Nikolai Dimov, Ana Doycheva and Galina Satchanska

New Bulgarian University, Department Natural Sciences, 1618 Sofia, 21 Montevideo str., Bulgaria

 

Abstract

 

Lactobacilli play a crucial role in probiotic features of yogurt. The aim of our study was to enumerate CFU of lactic acid bacteria in sheep and cow home-made yogurt produced in an industry-free Rodopa mountain region, near the town of Velingrad. Sheep yogurt possessed the typical light-yellow color while the cow yogurt was white in color. Sheep yogurt’s aroma was strongly emphasized compared to the aroma of cow yogurt. Analyses were conducted on solid MRS media by Koch’s method using serial dilutions. Cultivation was performed at 45 oC for 24-48h. Our results demonstrated that sheep yogurt possess higher count of lactobacilli4.4 x 107 CFU per g compared to the cow yogurt sample which contained 3.3 x 107 CFU lactobacilli per g. According to the Bulgarian State Standard - BDS 12:2010 “Bulgarian yogurt” the studied samples exceed the maximum standard requirements. In conclusion, the sheep yogurt which is typical with its increased lipid content showed higher alimentary value compared with the cow yogurt. According the lactobacilli concentration both yogurt could be evaluated as excellent food.

Key words: home-made yogurt, sheep milk, cow milk, lactobacilli

 

 

 

P13. ANTIFUNGAL ACTIVITY AND ENZYMOLOGICAL CHARACTERISTICS OF LACTOBACILLUS BULGARICUS GLB44 (PROVIOTIC®)

Rositsa Tropcheva1*, Yana Evstatieva1, Dilyana Nikolova1, Svetlana Saeva-Kondratenko2, Petko Petkov3, Kiril Petkov3

1Department of Biotechnology, Faculty of Biology, Sofia University St. Kliment Ohridski, 8, Dragan Tsankov Blvd., 1164, Sofia, Bulgaria;

2Genesis Laboratories LTD, Sofia, Bulgaria

3Proviotic LTD, Sofia, Bulgaria

*Corresponding author: tropcheva12@mail.bg

 

Moulds and yeasts are the main spoilage microorganisms, responsible for significant economic losses and several healthy risks in human food chain. The antimicrobial activity is an important criterion for the selection of bio-protective lactic acid bacteria (LAB). A limited data exists on the antifungal activity of Bulgarian LAB and their enzyme profile. With this aim, the activity of the commercially available probiotic Proviotic®, containing the strain Lactobacillus bulgaricus GLB44, against yeasts and moulds, and the presence of key enzymes, were studied.

Lactobacillus bulgaricus GLB44 (property of Genesis Laboratories LTD) was screened for antifungal activity against five mould species - Aspergillus flavus, Aspergillus niger, Fusarium graminearum, Trichoderma viride and Penicillium claviforme and three yeast species - Saccharomyces cerevisiae, Kluyveromyces marxianus and Rhodotorulla sp., using agar diffusion method. The enzyme profile of the L. bulgaricus GLB44 was determined using API ZYM miniaturized test (BioMerieux, France), following the manufacturer’s instructions.

Lactobacillus bulgaricus GLB44 possess a high amino-peptidase, acid-phosphatase and β-galactosidase enzymatic activity and a complete lack of the associated with the colon carcinogenesis β-glucuronidase activity.

The in vitro tests revealed a broad spectrum of antifungal activity. L. bulgaricus GLB44 completely (100%) suppress the growth of Aspergillus flavus, Fusarium graminearum, Trichoderma viride, Penicillium claviforme. With regard to Aspergillus niger,a lower inhibitory activity (66.6%), was observed. Proviotic®demonstrated a stronger inhibition against Saccharomyces cerevisiae, compared to the used as a control commercial antibiotic Fungostatin.

The demostrated strong proteolytic activity of Proviotic® makes the Lactobacillus bulgaricus GLB44 interesting for use in the production of antihypertensive and immuno-modulatory products and also in the manufacture of different dairy products.

The antifungal activity of Proviotic® is a promising advantage, suggesting its potential applications in different food technologies as a bio-preservative agent and a health promoting products against fungi.

 

 

 

P14.VIABILITY OF DIARY LACTOBACILLI IN SIMULATED MODEL OF GIT PASSAGE

Rositsa Tropcheva, Stoyanka Stoitsova, Svetla Danova

Department of General Microbiology, The Stefan Angeloff Institute of Microbiology, BAS

*Corresponding author: tropcheva12@mail.bg

                                                                                                                      

One important characteristic for the practical use of potential probiotic strains is their capacity to tolerate the hostile environment of the gastro-intestinal tract (GIT). We have previously isolated lactobacilli from traditional milk products. After extensive in vitro examination of their probiotic potential, three strains were selected as most prospective, i.e. L. brevis KR51, L. brevis KR53 and L. plantarum AC131. These strains were subjected to in vitro simulation of GIT passage, by sequential treatment with pepsin at pH 2, bile salts, pancreatin plus trypsin, and intestinal juice /colon. At different time-points, the viability of the lactobacilli was tested by plating, and cell morphology was examined by scanning electron microscopy. The results confirm that the three examined strains are characterised by good transit tolerance which indicates that they may be appropriate as probiotic adjuncts to functional foods.

 

 

 

P15. NEW FLUOROGENIC DYES FOR ANALYZES OF YOGURTS’ MICROFLORA

Rositsa Tropcheva1*, Stefka Kaloyanova 2*, Nedyalko Lesev 2, Svetla Danova 1

1 Department of Microbial Genetics, The Stephan Angeloff Institute of Microbiology, Bulgarian Acad. Sci.,26, Acad. G. Bontchev, str, 1113 Sofia, Bulgaria; e-mail: tropcheva12@mail.bg; stdanova@yahoo.com

2 Department of Applied organic chemistry, Faculty of Chemistry and Pharmacy,

University of Sofia, 1 James Bourchier Ave., 1164 Sofia, Bulgaria; e-mail: kaloianova@gmail.com

 

In the current conditions of a globalized market and technological innovations, the development of methods, that can provide quickly and adequately assessment of food products and to ensure an effective quality control and food safety, is becoming more significant. Analytical methods based on fluorescent tagging techniques offer excellent opportunities in this regard. Their adaptation of one of the most widely used foods and the search for new approaches for rapid microbiological quality control of dairy products is important in practice.

With this aim two new fluorogenic dyes are synthesized and their applicability for monitoring the microflora of the most popular dairy product - Bulgarian yogurt was estimated.

The goal is to develop a rapid fluorescence microscopy method for quantitative (live and dead cells) analysis ("live/dead" analysis) for the species Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius ssp. thermophilus, applicable to the control of the production standards for yogurt and to the functional assessment of starters for fermented products and other probiotic adjuncts. The method is based on the combination of monomethine cyanine dye and styryl dye, whose absorption maxima are in close spectral range (about 480 nm), which fluoresce in different regions of the visible spectrum, providing labeling in different colors. The styryl dye marks only the dead cells (red fluorescence) and cyanine dye - only the living ones (green fluorescence). Classical microbiological analyzes for the determination of this parameter in order to validate the results obtained by microscopic analyzes were conducted in parallel.

The studied dyes show improved properties and a higher sensitivity at low concentrations, compared to the widely used Ethidium bromide and Propidium iodide, where there is a slight increase in the fluorescence intensity of their complexes with the nucleic acids. These results and the data from the comparative,microbiological analyzes allow us to offer the novel dyes as suitable for an application in microbiological control and in particular "live/dead" analysis of the species Lactobacillus bulgaricus and Streptococcus thermophilus.

The work was supported by grant no. DMU 03-27/12.12.2011 from Bulgarian National Science Fund.

 

 

 

P16.ANTIBIOTIC ACTIVITY OF LACTOBACILLUS STRAINS ISOLATED FROM TRADITIONAL DAIRY PRODUCTS

Veronica Nemska1*, Nelly Georgieva1, Svetla Danova2

1Department of Biotechnology, Faculty of Chemical and System Engineering, UCTM, Sofia, Bulgaria

2Department of General Microbiology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Science, Sofia, Bulgaria

*Corresponding author: revn@abv.bg

 

Keywords: Lactobacilli, antibiotic susceptibility

 

Medical treatment with antibiotics causes non-selective elimination of a part of the intestinal microflora and makes it an easy target for pathogens. In order to prevent and restore the equilibrium in the gastrointestinal tract, consumption of functional foods containing probiotic bacteria during and/or after antibiotic intake is recommended. With this aim, the antibiotic susceptibility of newly isolated lactic acid bacteria from artisanal dairy products to different antibiotics (inhibitors of cell wall synthesis, the protein synthesis and the synthesis of nucleic acids) were tested.

A classical disc diffusion method was applied in analysis of the antibiotic resistance of the investigated strains. The test was performed with commercially available discs impregnated with antibiotics (BulBio, Bulgaria) and the sensitivity of lactobacilli was determined according to the zone of inhibition and clinical laboratory standards (NCCLS). No transferable antibiotic resistance has been found. The triplicate experiments established the Lactobacillus sensitivity to Penicillin, Meropenem, Tetracycline, Chloramphenicol, Rifampin, Sulfamethoxazole trimethoprim, Gentamicin and resistance to Cefotaxime and Pefloxacin.

Obtained results are only an initial step in characterization of the newly isolated lactobacilli. Further technological and functional evaluation is needed and is still in progress.