Acta Microbiologica Bulgarica

105-110

Authors: Vilhelmova-Ilieva, N., Galabov, A. S.

Since ancient times, folk medicine has used the potency of a wide range of plants for treatment of various diseases. In recent years, the development of technology has made it possible to identify the active ingredients determining these properties of plants. One of the largest group of substances isolated from plants are polyphenols. Tannins are a type of polyphenols that have already proven to possess different biological activities important to human health, such as antioxidant, antitumoral, antimicrobial, antiviral, etc. As an integral part of many food products, tannins are included in the daily diet of many people suffering from various diseases. Tannins can naturally enhance the body's ability to cope with various infections and diseases, improving the immune response and also having a positive effect on patients with heart disease and high blood pressure, which makes them promising therapeutic agents. All of the activities described above allow tannins to be considered as promising therapeutic agents.

115-118

Authors: Boyanova, L., Nikolov, R., Markovska, R., Yordanov, D., Mitov, I.

Propolis possesses many beneficial biological activities and 13C urea breath (13C UBT) test is considered to be the best non-invasive test for the diagnosis of Helicobacter pylori infection. The aim of this study was to evaluate propolis effects on H. pylori infection status and 13C UBT results of two untreated symptomatic adults. The first patient with chronic gastritis and gastroesophageal reflux was tested by 13C UBT for H. pylori several times over >1 year. The subject used only non-antibiotic agents involving propolis. All 13C UBT results of the patient were positive. The second patient had taken propolis on the day before the test and the UBT result was negative, while the culture was positive. In conclusion, although propolis alone or with other non-antibiotic agents may be a good adjunct in the infection therapy, the long-term bee glue use did not lead to H. pylori eradication in the first patient. Conversely, the use of propolis several days before the test may result in false negative UBT results, raising the question of the need to avoid propolis use at least several days before the UBT. The optimal dosage and duration of bee glue use should be further evaluated.

125-130

Authors: Chiurciu, C., Chiurciu, V., Danes, M., Stoica, O. G., Frunzareanu, B., Banciu, D., Lupu, E., Uluitu, C., Oprisiu, D., Stefan, P. C., Ionescu, I., Filip, V., Tudoran, C., Nicolae, I., Diaconu, L., Stanculescu, S., Radu, A., Chitonu, P.

Starting from ONE HEALTH concept of integrated medicine in which human, animal and environmental health are approached together, this paper presents the attempts of ROMVAC Co. to reduce the risk of the occurrence and spread of antibiotic-resistant germs (ARGs) originating from the digestive tract of broiler chickens and mammary glands of dairy cows. The purpose is to demonstrate the efficacy of alternative veterinary medicines to antibiotic therapy in avian enteritis and dairy cow mastitis. Enterococcus faecium NCIMB 11181 culture at a minimum concentration of 1x108 CFU / ml; Ross 308 broiler chickens (250,000 heads); automatic dosing system in water flow - Dosatrom. The product was administered during the first 3 days of life and the body weight evolution was monitored at 0, 7, 14, 21, 28 and 35 days. Oils with strong antibacterial and antiviral effects (oregano, thyme, pine etc.) were used by ROMVAC to produce three medicines: a) Greenvet mastirat - gel, curative, b) Ugeroclean - S, liquid soap and c) Ugeroclean - gel applied after milking. The products were administered locally to mammalian animals (cattle, sheep, goats) daily for 7 days. Udder edema, milk production, dermal reaction to the product (erythema, nodules, pustules, etc.) and clinical status of the animals were daily monitored. Chickens that received probiotic had a weight gain of 68 grams / chicken (over 6 tons per 100,000 chickens) and a lower mortality rate of 0.78% (1794 chickens). E. coli and Salmonella germs have completely disappeared from the digestive tract. Vegetable oil products improved clinical symptoms of mastitis, were well tolerated by animals, decreased the intensity of erythema, improved the clinical condition of animals, maintained milk production, very few cases requiring antibiotic treatment. The product is also an insect repellent. The in vitro antiviral activity of vegetable oils on CDV paramyxovirus has also been demonstrated. Replacement of antibiotic treatment in veterinary medicine with alternative therapies avoids the occurrence and transmission to the human consumer of ARGs via chicken and milk.

137-142

Authors: Gotova, I., Dimitrov, Zh., Najdenski, H.

In vitro tests for establishing the anti-inflammatory potential of twenty-two previously selected Lactic acid bacteria (LAB) strains were carried out. The aim was to compare the antiinflammatory potential of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus isolated from authentic homemade yogurt in non-industrial mountain villages in Bulgaria with selected strains originated from the intestinal tract of healthy volunteers. The assessed signal peptide associated with inflammation was IL-8, with and without stimulation of TNF-alpha, in order to simulate inflammatory shock in vitro. To establish the antiinflammatory potential of the strains, two cytokines with predominant anti-inflammatory effects, IL-10 and TGF-beta were evaluated as well. The different cytokines were evaluated using three different analytical models, including mice splenocytes. Among the presented strains of intestinal origin only two Lactobacillus gasseri, one Enterococcus faecium and one Bifidobacterium longum possessed simultaneously the desirable optimal cytokine profile, where the highest induction of IL-10 was 1670.2 pg/ml (E. faecium 2092/2); the strongest reduction of IL-8 was 46.9 pg/ml (B. longum 10/48) and the induction of TGF-ß varied between 548.75 and 834.53 pg/ml. Among the strains of yogurt origin, three Lactobacillus bulgaricus and three Streptococcus thermophilus possessed concurrent or even better anti-inflammatory profiles, where the highest induction of IL-10 was 1584.1 pg/ml (L. bulgaricus b58); the strongest reduction of IL-8 was 46.0 pg/ml (Str. thermophilus T43) and the induction of TGF-ß varied between 499.96 and 841.50 pg/ml.

49-51

Authors: Kayacan, Z. C.

Humans co-evolve with their microbiota. It is becoming better understood today that health and disease are determined by microbiota in many cases. The gut microbiota constitutes the main part of the current research on the subject and is already found to be related to many disorders in humans. Diet and gut microbiota seem to have a stable relation.

59-64

Authors: Atanasova-Pancevska, N., Kungulovski, D.

Microorganisms are widespread in the environment. Most of them are not dangerous to human health, but some produce toxins which are toxic, carcinogenic, mutagenic or teratogenic for humans and animals. Their fast reproduction leads to contamination in food and damage to material goods. Within this experimental research, the effects of nine laboratory- obtained antimicrobial agents of different chemical compositions and concentrations are determined against foodborne pathogenic microorganisms. The following microorganisms were used in the test: Escherichia coli ATCC 8739, Bacillus subtilis ATCC 6633 и Staphylococcus aureus 6538, isolate FM2 (yeast from pickled peppers), isolate FM20 (yeast from pickle), isolate FM3 (mold from bread), isolate FM4 (mold from jam), isolate FM18 (mold from tangerine) and isolate FM15 (mold from meat). The experiment was based on the macrodilution method. The microorganisms were exposed to different compositions and different concentrations of components of antimicrobial agents (designated from No1 to -No9), and the minimum inhibitory concentration (MIC) was determined. The results showed that all antimicrobial agents possess antimicrobial activity against the tested microorganisms, depending on the type of microorgasnisms (procaryiots or eucaryots) and the compositions of the antimicrobial agents. The lowest MIC of 3.125% for both procaryots and eucaryots was shown by antimicrobial agent No2 the antimicrobial agent with no or some weak activity for eucaryots was No4, and for procaryots agents No7 and No8. Each of the tested agents has a different potential impact on the growth and multiplication of the test organisms. These differences result from the composition of the laboratory-obtained antimicrobials and the resistance of the test microorganisms to them.

74-78

Authors: Jovičić-Petrović, J., Mihajlović, M., Tanović, B., Radić, D., Karličić, V., Raičević, V.

Excessive use of pesticides represents a growing problem in agriculture sustainability and food safety. The use of antagonistic fungal interactions represents a promising approach to achieve reduced pesticide input. Beneficial saprotrophic fungi from the genus Trichoredma are able to express different mechanisms of antagonistic activity and effectively suppress plant pathogens. The aim of the present research was to investigate the potential use of the antagonistic effect of T. longibrachiatum 10/5 strain against the plant pathogen Pythium aphanidermatum. The antagonistic activity of T. longibrachiatum 10/5 strain against P. aphanidermatum was tested in vitro by dual culture test. A glasshouse trial was conducted with cucumber plants grown on commercial substrate. Ten plants per container were inoculated at the cotyledon growth stage with P. aphanidermatum mycelium. The experiment was conducted in five replications. The number of inoculated plants without symptoms per treatment was recorded. P. aphanidermatum expressed asymmetrical growth in the dual culture test, and the colony diameter was reduced by 77% in comparison to the control. Sterile liquid culture filtrate of T. longibrachiatum 10/5 showed 50% efficiency in the suppression of cucumber damping-off caused by P. aphanidermatum. Metabolites produced by T. longibrachiatum 10/5 showed a suppressive effect on P. aphanidermatum damping-off, which has a potential in application in sustainable agriculture.

87-93

Authors: Radić, D., Karličić, V., Kljujev, I., Vujović, B., Lalević, B., Raičević, V.

The usage of fresh fruits and vegetables has risen over the past decades. Due to environmental problems, agricultural products can be contaminated during cultivation and other activities. The aim of this paper was to determine the microbial quality of soil, irrigation water and fresh vegetables in the central part of the Republic of Serbia. Sampling was performed in the summer of 2015 at the Agricultural high-school Svilajnac (AHS), Suburb of Svilajnac (SS), Village Gložane (GL), and Village Grabovac (GR). The microbial diversity of the soil and presence of potential human pathogens was determined using agar plate technique and MPN method, respectively. MPN method was also used for determination of total and fecal coliform bacteria in irrigation water samples. Nutrient agar was uses for determination of aerobic psychrophilic and mesophilic bacteria in irrigation water samples and vegetables and Cetrimide agar for Pseudomonas aeruginosa determination. Chemical characterization of irrigation water was performed using standard methodology. High microbial diversity as well as presence of pathogenic bacteria were noticed in the examined soil samples. The water cation and anion content was under the limits of permissible levels for irrigation waters. Although patogenic bacteria were detected in irrigation water samples, the obtained values were under the limits proposed by several international guidelines. In order to eliminate the possible contamination sources, principles of good agriculture practice must be implemented in production and manipulation processes.

3-9

Authors: Krumova, E., Abrashev, R., Kostadinova, N., Miteva-Staleva, J., Spasova, B., Angelova, M.

Copper is an essential element for the growth and development of the full range of living organisms, including filamentous fungi. At the same time, it is toxic for organisms when present in excess. The present study was conducted to assess the role of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) on the tolerance strategy of the fungal strain Humicola lutea 103 at different growth phases under enhanced Cu ions concentrations. We examined the changes in the growth, intracellular protein content and levels of antioxidant enzyme defense. The results revealed that the presence of Cu ions affected the duration of growth phases in a dose-dependent manner. The effect of Cu treatment (150 g/ml Cu ions) depends on the age of the treated culture. The spores and cells from the stationary growth phase demonstrated higher resistance compared to the corresponding control due to the enhancement of SOD and CAT activity. The increased total SOD activity was largely due to the Cu/Zn-SOD isoform. The non-growing cells taken from cultures of different growth phases demonstrated also up-regulation of both antioxidant enzymes in response to oxidative stress imposed by the ROS-generating heavy metal. During the stationary-phase, H. lutea cells demonstrated higher resistance to Cu-induced oxidative stress compared with the exponential phase cells.

17-23

Authors: Popova, R., Sirakov, I., Alexandar, I., Strateva, T., Daskalov, H., Mitov, I.

In this study, among five positive isolates of stx1 isolated from raw milk and chicken meat, one isolate showed a positive result for subtype stx1d and the other four isolates were positive for subtypes stx1d and stx1a. An isolate positive for stx2 isolated from pork was found to carry an stx2g subtype. Besides subtyping, we performed phylogenetic analysis based on 16s RNA gene sequencing data. Based on the mutations identified in the isolates, we sought for restriction enzymes to distinguish the strains from one another. The results for the combination of stx1 subtypes are in agreement with other reports, being the most common combination in Escherichia coli isolates with an origin from pigs. Interestingly, the pork isolate subtyped as stx2g by us is in contrast with studies according to which the most common subtype of this source is stx2e.

30-35

Authors: Krumova-Valcheva, G., Kalinova, G.

Mussels are filter-feeding organisms that could be a potential source of food-borne diseases and poisonings. Consumption of molluscs, contaminated with bacterial contaminants or marine biotoxins, may lead to severe food-borne diseases. Considering the importance of the health problem connected with Escherichia coli and paralytic shellfish poisoning (PSP) toxins and also the requirements of the European legislation, the aim of this study was: to evaluate the level of E. coli and PSP contamination in cultivated mussels produced in farms along the Bulgarian Black Sea coast. The study was performed on 55 samples of live mussels (Mytilus galloprovincialis), from 20 aquaculture farms, during the period 2012 – 2015, in the warm season (May October) and in the cold season (November- April). The determined concentration of E. coli varied between < 20 MPN/100 g and 8.2 x 102 MPN/100 g. The level of E. coli in 5 samples tested in the warm season and in 2 samples tested in the cold season was higher than the permissible quantity. All samples met the health standards referring to the content of PSP toxins in bivalve molluscs. No connection was established between the concentration of E. coli and PSP toxins in the tested samples and the level of contamination with E. coli and PSP toxins in cultivated mussels, from the Bulgarian Black Sea coast, was minimal.

211-220

Authors: D. Dinkov

The literature shows contradictory results regarding the prevalence of microorganisms, as well as the contamination of flower bee pollen. Here is a summary of the species-level differentiation of Еnterobacteriaceae, Pseudomonadaceae, Staphylococcaceae, Micrococcaceae and Bacillaceae isolates obtained from investigations on fresh and dried flower bee pollen from different regions of Bulgaria.

224-231

Authors: Savov, E.

The spread of multidrug-resistant /MDR/ Gram-negative bacteria in the hospital setting is a worldwide problem. In this study we present data about the resistance to antimicrobials of some problematic for hospital infectious pathology bacteria – Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter baumannii and Pseudomonas aeruginosa on the model of a multiprofile hospital. There has been an increase in ESBLs-producing E. coli and K. pneumoniae strains, isolated especially in some so-called “risky units” at the Military Medical Academy /MMA/ for the last years. There were also registered the first strains E. coli, producing metallo-beta-lactamase NDM1. The data show specific association between the blaNDM-1 and rmtB genes conferring high-level resistance to all aminoglycosides in these strains. Non-fermenting A. baumannii and P. aeruginosa strains isolated usually are multiresistant with high-level resistance to carbapenems and other beta-lactams and quinolones, and the resistance to carbapenems in A. baumanni strains is associated with the production of Oxa 23, Oxa 58 and Oxa 72 carbapenemases, but not metallo-beta-lactamas.

237-242

Authors: Georgieva, I. L., Galabov, A. S.

The effect of several antiviral substances with different mode of action on the replication of human rhinovirus 14 (HRV-14) is the topic of the present study. Monolayer cultures of human cervical carcinoma (HeLa Ohio-I) cells in 96-well tissue culture plates were used. The antiviral effect against three different viral inoculation doses was tested by the neutral red uptake procedure in a CPE-inhibition setup. The following compounds were tested: ribavirin, arildone, disoxaril, S7, PTU-23, HBB and oxoglaucine (a newly characterized in this laboratory compound efficient against enteroviruses). Two of the compounds, HBB and oxoglaucine, showed the highest activity with a selectivity ratio (CC50/IC50) above 100 for the lowest viral inoculation dose of 100 CCID50. Ribavirin and disoxaril occupied intermediate position according to their antiviral effect, while the effect of arildone, PTU-23 and S-7 was not significant.

3-9

Authors: Mileva, M.

During influenza pandemics in the last few years many people have died from severe complications associated with this pandemic despite receiving intensive care. This suggests that a definitive medical treatment for severe influenza-associated complications has not yet been established. About the pathogenicity of flu, many studies have shown that superoxide anion produced by macrophages infiltrated into the virus-infected organs is implicated in the development of severe influenza-associated complications. Selected antioxidants, such as alpha-tocopherol, N-acetyl-L-cysteine, glutathione, ascorbic acid, 5,7,4-trihydroxy- 8-methoxyflavone, catechins, quercetin 3-rhamnoside, iso-quercetin etc. inhibit the proliferation of influenza virus and scavenge superoxide anions. The combination of antioxidants with antiviral drugs synergistically reduces the lethal effects of influenza virus infections. These results suggest that the combination of an agent with antiviral activities and an antioxidant could be a drug of choice for the treatment of patients with severe influenza-associated complications. Hopefully, this update of knowledge of antioxidant therapy of flu could be used as a potential approach to overcome the influenza-associated complications.

20-26

Authors: Peshev, R., Hristov, M.

Wild and domestic animals inhabiting Sofia and Varna zoos showing clinical symptoms of malignant catarrhal fever (MCF) were investigated. Clinical and pathological studies were carried out with 16 wild and 8 domestic ruminants. Typical clinical symptoms for MCF were confirmed. Samples for virus isolation and molecular biological investigations were obtained from dead animals. Ten percent suspensions from the lymph nodes, spleens, lungs, brain samples, and buffy coats in phosphate buffered saline (PBS) were used for viral isolation. Primary and permanent cell cultures: rabbit kidney, Madin Darby bovine kidney (MDBK), embryonic bovine trachea (EBTR). Bovine trachea (TTr) and VERO cells were used. Viruses with peculiarities of herpesviruses were isolated from bison , yak, camel, Cameroon goats and hippopotamus on MDBK cell culture. The isolated strains were confirmed as MCF viruses by classical and nested polymerase chain reaction (PCR). The size of the amplicons after classical PCR was 422 bp, and 238 bp after nested PCR. Thirty seven serum samples originated from 17 sheep, 13 Cameroon goats, 5 mouflons and two bison were investigated serologically by a competitive-inhibition ELISA for spread of MCF. In 21.4% of sheep sera, 30.7% of Cameroon goat sera, and 50% of bison sera antibodies against MCF were found. The total distribution of MCF antibodies in wild and domestic ruminants was 27%.

33-38

Authors: Kostadinova, T., Ivanova, L., Raykov, T., Stojkova, Z., Tsankova, G.

Epstein-Barr Virus (EBV) is a widely spread member of the Herpesviridae family. Data show that in more than 90% of the adult population, specific anti-EBV can be found. In different Bulgarian studies the seroprevalence was set between 60% and 80%. The primary infection early in life is often asymptomatic or with nonspecific clinical symptoms, while in teenagers infectious mononucleosis develops. EBV is a proven agent of malignant, autoimmune and lymphoproliferative diseases, especially in immunocompromised patients. Tracking and analyzing the seroepidemiological status of the population in every region is of utmost importance. In our study, 5016 single serum samples from patients referred for EBV testing for various reasons were analyzed for the period 2010-2015. People aged between 1 month and 89 years old were divided into sixteen age groups. The samples were tested using indirect enzyme-linked immunosorbent assay (ELISA) for detection of anti-EBV (VCA) IgM and IgG. The proportion of EBV seropositive individuals and their corresponding confidence intervals (CI), chi-squared distributions and p-values were calculated. Our data indicated high, age-dependent EBV (VCA) IgG dissemination with 90% seroprevalence after age 26. Primary infections detected by anti-EBV (VCA) IgM occur in a bimodal model with peaks in age groups 1 – 5 and 11 - 20 years old.

45-48

Authors: Dikova, B., Balacheva, E., Moskova, I., Aleksieva, V.

The effect of the growth regulator β–monomethyl ester of itaconic acid (MEIA) on two economically important viruses, namely tomato spotted wilt virus (TSWV) and tomato mosaic virus (ToMV), was studied in tomato cultivars. Tomato plants of Keti and VK1 lines were infected separately with each virus: Keti with TSWV and ToMV, and VK1 with TSWV, respectively. The tomato plants were treated with MEIA in a concentration of 5x10-4 M after inoculation with TSWV in one variant, and with ToMV in another. An inhibiting effect was established on both viruses – TSWV and ToMV, when the tomato plants were infected and after that sprayed with MEIA solution. These initial results are a good reason to continue with the studies both for the establishment of the effect of growth regulators (MEIA in particular) on virus pathogens and the physiological indexes that prove the decrease in stress and injuries on tomato plants with virus diseases after treatment with MEIA..