Acta Microbiologica Bulgarica

151-156

Authors: Muhtarova, A., Gergova, R., Setchanova L., Mitov, I.

Streptococcus pyogenes, or Group A streptococcus (GAS), is the major pathogen of bacterial tonsilo-pharyngitis tonsillopharyngitis and of skin and soft-tissue infections, which can also cause severe invasive disease or dangerous post-streptococcal autoimmune complications. The differences in the pathogenesis are due to the existing variety in the GAS genome, which often occurs as a result of the cumulative effect of a greater number of various virulence genes. The aim of this study was to determine the frequency and distribution of genes encoding super-antigens and toxins in Bulgarian GAS isolates according to their source and kind of infection. Clinical isolates (n=238) from throat samples, wounds, punctures from peri-tonsilar peritonsillar abscesses, middle ears and sinuses, blood cultures and cerebrospinal fluid, identified as GAS, were screened for the presence of 21 virulence genes, using multiplex polymerase chain reaction (PCR). All of the tested strains were shown to carry the typical of GAS genes: slo, speB, sypCEP, sdaB; more than 80%, mac and smeZ; followed by speF, speG, speJ and genes for DNA-ses sdc, sdaD, Spd3 (between 50% and 75%). An attempt was made to seek an association between the PCR-detected GAS virulence genes, the kind of infection and the source of isolates. A large genetic diversity was found in the studied strains. The genes speA, speF, speL and speM or their combinations were detected more often in invasive isolates (p < 0,05) than in non-invasive ones.

167-175

Authors: Năşcuţiu, A-M., Ionescu, M. D., Ionescu, G.

Mycoplasma hominis and Ureaplasma spp. are frequently found in the genital tract, especially in sexually active women, pregnant or not, with a negative impact on health and outcome of pregnancy. The retrospective study investigates the prevalence of colonization/infection with genital mycoplasmas in women and the antibiotic susceptibility profile of the isolated strains. From August 2011 till July 2017, 931 endo-cervical samples were analyzed. Mycoplasma IST2 kit (Biomerieux) was used for culture, identification, indicative enumeration and antibiotic susceptibility testing. Culture was positive in 38.5% of the samples. Ureaplasma predominated, Mycoplasma being detected mostly in association with Ureaplasma, seldom alone. Overall, the level of resistance to quinolones was high (77% for ciprofloxacin, 55% for ofloxacin), whilst that of resistance to tetracyclines only started to rise. Some Ureaplasma strains showed resistance to three classes of antibiotics. For erythromycin, a particular trend was noticed, with a drop of resistance level during the study, but with recent re-emergence of resistant Ureaplasma strains. Pristinamicin resistance was not encountered. Tetracyclines are still the antibiotics of choice for these infections. As the rising trend of resistance to several classes of antibiotics might become problematic over the next decades, antibiotic susceptibility of the strains should be assessed prior to the initiation of treatment.

178-181

Authors: Sarzhanov, F., Dogruman-Al, F.

In molecular diagnosis, the detection of protozoa in fecal samples is hampered by poor recovery of DNA and the presence of PCR inhibitors. In this regard, a reliable extraction of DNA is a key step in the molecular diagnosis of parasitic infections. Currently, there are many commercially available kits for isolation of DNA from feces. Each of these includes a method for removing PCR inhibitors from samples. The purpose of the study was to determine a suitable kit for isolating Blastocystis sp. DNA from fecal samples and then use it in PCR to detect Blastocystis sp. in the samples. Three commercially available kits were used to isolate the DNA of Blastocystis sp. using human feces samples. The study included stool samples of patients referred for routine laboratory testing for parasites. The samples in which Blastocystis sp. was detected by microscopic examination (a native lugol preparation and staining with trichrome) and cultivation in Jones’ medium containing 10% horse serum were utilized. QIAamp DNA Stool Mini kit (Qiagen, Germany), ZR Fecal DNA kit (Zymo Research, USA) and ExgeneTM Stool DNA mini (GeneAll, Korea) were used for the extraction of Blastocystis DNA. The quality and purity of total genomic DNA was measured with a spectrophotometer. Specific primers were employed for the amplification of Blastocystis sp. DNA. In this study, it was observed that ExgeneTM Stool DNA mini could not detect Blastocystis sp. DNA in gel electrophoresis, while the QIAamp DNA Stool Mini kit and ZR Fecal DNA kit outperformed for isolation of DNA.

189-193

Authors: Popova, G., Ilievska, T., Blagoevska, K.

The present study focuses on antimicrobial resistance among the most important respiratory pathogens. The study population were children with respiratory tract infections (RTIs), treated in ambulatory and hospital settings at the Institute for respiratory diseases in children –Skopje, in the period from 01.04.2016 to 30.09.2016. Standard microbiological procedures were used for isolation and identification of bacteria. Disc diffusion test was used for antibiotic susceptibility testing. Oxacillin-resistant pneumococcal isolates were subjected to MIC determination by gradient E-test. Of all respiratory pathogens, Streptococcus pneumoniae was the most frequently isolated with 35% (362/1027). Ampicillin nonsusceptibility rate (MIC>2mg/l) was 28.2%. Macrolide-resistant were 46.7%, with cMLSb as dominant phenotype (80.15%). The rate of MRSA was 34.9% (73/209) and gentamycin resistance in Staphylococcus aureus in 90.7% was connected with MRSA. 26.5% were macrolide-resistant with iMLSb as dominant phenotype. Ampicillin resistance rates among Moraxella catarrhalis and Haemophilus influenzae were 100% and 34.9%, respectively. There was no resistance to β-lactams or to macrolides among Streptococcus β haemolyticus. Tracing bacterial resistance profiles plays a critical role in preparing guidelines for appropriate antimicrobial treatment.

198-201

Authors: Stoev, A.

The investigation was aimed at the receptivity and possible reactions of apricot (Prunus armeniaca) to plum pox virus (sharka virus, PPV) widely spread not only in the orchards but in the uncultivated terrains on the territory of Bulgaria, where fruit trees of the genus Prunus grow. Field observations of manifestation of plum pox (sharka) disease were carried out during the period of 2015–2017. They covered trees of plum (P. domestica), wild plum (P. cerasifera), peach (P. persica) and apricot (P. armeniaca). Apricot seedlings grown in the yard nearby the Plant Protection Department of ISSAPP were also included in the investigation. The seedlings originated from pits with and without sharka symptoms. A double antibody sandwich enzyme linked immunosorbent assay (DAS ELISA) was used for detecting the viral pathogen. The spread of plum pox disease was visually identified in all investigated orchards. Plum trees were the most contaminated. Single cases of sharka on wild plum, peach and apricot trees were also observed. During the period of investigation apricot seedlings remained healthy and the results of DAS ELISA were negative. Apricot seedlings were not receptive to PPV in field conditions when the insects (aphids) were natural vectors of the viral infection. The apricot trees could have been infected through infected rootstock (P. cerasifera). The results confirmed that apricot generative posterity remained free of PPV.

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.