Acta Microbiologica Bulgarica

108-113

Authors: Nehra, C., Marwal, A., Gaur, R. K.

Begomoviruses are plant-infecting viruses, which are transmitted by the whitefly vector Bemisia tabaci and have been known to cause extreme yield reduction in a number of economically important vegetables around the world. They are abundant in tropical and subtropical environments, where insects that transmit these viruses are abundant. Identification of plant viruses, monitoring for new viral diseases, understanding the vectors that transmit viruses, and determining viral and vector impacts on the growth and development of crop cultivars and lines is vital to managing and controlling these diseases. In addition to damaging crops and causing yield losses, plant viruses interact with vectors and other diseases to increase the damage from the diseases/pests. The crops and weeds growing close to the crop fields are potential reservoirs of begomoviruses, but it is not known whether the same viruses infect several host species or co-infect any of the hosts. This increases the difficulty of controlling both the plant virus and the interacting pathogen or vector. In a survey carried out in 2011-14 we observed several cultivated crops, ornamental plants and weeds with leaf curling, vein yellowing and growth stunting - typical symptoms of begomovirus. The DNA was isolated from the infected samples and the presence of begomovirus was confirmed by PCR using component specific primers. Further, rolling circular amplification (RCA) was performed to get the full-length sequence of the isolates. The full length sequence showed genome arrangement typical of monopartite/ bipartite begomovirus. Some of the infected samples showed association of a betasatellite responsible for the epidemic diseases in the case of monopartite. Phylogenetic analysis confirmed the diversity of begomovirus in India. Our result also indicates that begomovirus has broaden its host range by recombination process. Expression of various full length or truncated or defective proteins of the virus has been effective in accomplishing pathogen-derived resistance. We analyzed the recombination parmeters of begomovirus strains and developed the RNAi strategy for the disease management.

121-125

Authors: Stoev, A.

The paper deals with the particularities in the manifestation of aggressiveness by phytopathogenic viruses and the methods of its tracing out and evaluation. Aggressiveness is a property of phytopathogens. We present some examples of viral pathogens which cause economically important diseases in the agroecosystems of Bulgaria. We have considered the manifestation of virus aggressiveness in association with other properties which characterize plant pathogens.

133-138

Authors: Dimitrova, A., Mileva, M., Krastev, D., Gegova, G., Galabov, A. S.

Influenza is a highly contagious viral infection of the respiratory system. Many studies provide compelling evidence that the abnormal production of reactive oxygen species is a crucial mediator of acute lung injury in influenza A virus infection. Therefore, antioxidants are potentially useful against this ongoing clinical problem. Our studies showed that S-adenosyl-L-methionine (SAM) has a protective effect in a model of influenza infection in mice. This substance converts into glutathione - the main antioxidant in the body, through a multistep biochemical cycle. In the present study, we report the effect of combined treatment with SAM and the antiviral agent oseltamivir in infected with influenza A virus mice. SAM was given as a single daily dose of 50 and 100 mg/kg in different mice groups, starting from 5 days before infection until day 4 after infection. Oseltamivir was given in a dose of 2.5 mg/kg daily in two intakes for 5 days, starting from 4 h before infection. End-point evaluation was 14 - day survival. Survival was 70% in the treatment with oseltamivir and rose to 90% in the treatment with oseltamivir and SAM in both doses. SAM alone did not show any antiviral activity. The present findings suggest that therapy with molecules converted into antioxidants in the body increases survival by modulating the host defense mechanisms and by a direct antioxidant effect against oxidative stress associated with viral infections. This study demonstrated the effectiveness of combining agents that act through different mechanisms - antiviral drug oseltamivir as a specific neuraminidase inhibitor of influenza virus, and SAM as a precursor of the most important antioxidant - glutathione.

142-147

Authors: Simeonov, K. B., Sirakov, I. N., Gergova, R. T., Koburova, K., Todorov, T. G.

In this study, the CPsittF(R) and Or1(2) primers, targeting the ompA gene of Chlamydia psittaci, were used for PCR detection of Chlamydia abortus DNA in clinical samples from aborted animals. The sensitivity and specificity of the reaction were compared with those achieved by C. abortus-specific primers CpsiA(B). The comparative analysis of the results showed that all samples that were positive for C. abortus in PCR, performed with primers CpsiA(B) also gave a positive result in the PCR using the CPsittF(R) primers, generating a 1041 bp specific amplification product. PCR amplification using the Or1(2) primers was uncertain and produced an amplification product of 212 bp, which was different from the expected length of 245 bp. In an attempt to improve these primers, their sequence was modified at the 11th and the 21st nucleotide. Although the sensitivity of the reaction performed with the modified primers was improved, it was still lower compared to that achieved with the original C. abortus-specific primers CpsiA(B) and primers CPsittF(R). The results show that the primer pair CPsittF(R), developed for the detection of C. psittaci could be successfully used in the diagnosis of abortions, induced by C. abortus, while the primer set Or1(2) is less effective.

10-25

Authors: Kroumov, A. D., Módenes, A. N.

The objective of this review article was to highlight the authors’ more than 30 years’ experience in the field of model development procedure of ethanol biosynthesis. During all these years a powerful system analysis theory and its decomposition principles were applied not only for studying and description of microbial kinetics but for the needs of bioprocesses and bioreactors/photobioreactors optimization and scale-up. Special attention was given to the description of phenomena and techniques involved in ethanol synthesis on single and two substrates utilization processes by wild and genetically modified strains. The method of simultaneous saccharification and fermentation of starch to ethanol (SSFSE) by genetically modified microbial Saccharomyces cerevisiae YPB-G strain is presented to fully illustrate the strength of the applied theory. Another modern method for extractive fermentation and ethanol production is presented as well. Diauxic growth phenomena in ethanol synthesis is the example showing a modeling approach by connecting knowledge from molecular and population hierarchic levels. This modeling work helped tremendously to find new insights on the control of internal metabolic mechanisms of the cells and how the processes can be guided to an optimal trajectory. A parameter identification procedure was performed by using the latest achievements in global search methods. The applied strategy of model development was extremely successful in obtaining new knowledge about the microbial systems’ behavior as well as for fast and robust bioprocesses development. The review article can be very useful for modelers and young scientists working in the fields of biotechnology, bioengineering, metabolic and chemical engineering.

42-46

Authors: Bumbarov, V., Tzymanis, A., Hristov, M., Peshev, R., Eligulashvili, R.

An immunohistochemistry method on frozen tissue cryostat sections based on new polyclonal monospecific affinity-purified rabbit antibodies (PMAP) against 19-residue synthetic peptides (154-171), originated from the group-specific peptide P-32 for sheep poxvirus (SPV) was developed. PMAP were used as primary antibodies in an immunohistochemistry test on frozen ovine tissue for rapid laboratory diagnosis of SPV infection. This method is highly specific, reproducible, simple and inexpensive. The total time of the procedure was within 2 hours of receiving the samples in the laboratory.

56-64

Authors: Atanasova-Pancevska, N., Popovska, I., Davalieva, K., Kungulovski, D.

New antimicrobial compounds are continuously required to combat antibiotic-resistant bacteria and pathogenic yeast as such resistance increasingly limits the effectiveness of current antimicrobial drugs. New screening approaches, including the search for novel targets and exploration of non-conventional places as sources of producer microorganisms, are needed. Rotten apples compost samples from the composting plant in Resen, Macedonia, were screened for microbial colonies which form a clear zone of inhibition. The isolated bacterial strains were further screened for their antimicrobial activity against different bacteria and fungi by diffusion agar method. The isolates were later identified as Paenibacillus alvei and Bacillus subtilis by morphological studies and sequencing of the 16S rRNA bacterial gene. The maximum growth of P. alvei and B. subtilis for different cultivation media, pH, temperature, time of incubation and glucose concentration were determined. Maximum growth of P. alvei and B. subtilis was observed in MHB, at pH 6 after 24 hours of incubation at 30 °C for P. alvei, and at pH 7 after 48 hours at 40 °C for B. subtilis. In conclusion, the identified strains with antimicrobial activity in this study are a useful addition to the worldwide effort for new antibiotic discovery.

74-80

Authors: Zheleva, P., Vasileva, T., Bivolarski, V., Iliev, I.

A β-Galactosidase preparation from ten strains Lactobacillus plantarum was evaluated as a biocatalyst for the synthesis of oligosaccharides. Among them, the enzymes from L. plantarum S26 and L. plantarum S30 were selected for further studies. The correlation between organic acid production, α-galactosidase, β-galactosidase and lactate dehydrogenase activity and the initial concentration of lactose and lactulose was studied. It could be suggested that the studied β-galactosidase catalyzes the trans-glycosylation reaction at 20% lactose and 20% lactulose concentrations.

85-93

Authors: Trigueros, D. E. G., Kroumov, A. D.

The paper presents the modeling approach to aerobic biodegradation of 1,2-dichloroethane (1,2-DCE) by the microorganism Xanthobacter autotrophicus GJ10, and of trichlorethylene (TCE) by the microorganism Pseudomonas cepacia PR131 in a bioreactor with an ultrafiltration (UF) membrane unit. The aim of the study was to estimate the kinetics of the bioprocess and to perform a parameter identification procedure. The specific growth rate was assumed to be a function of 1,2-DCE and TCE and oxygen substrate concentrations. The parameter values were estimated based on experimental data of continuous and discontinuous systems published elsewhere. For this purpose, the Particle Swarm optimization algorithm, coded in Maple 15® software, was applied. Simulations with the developed kinetic model showed new highlights and an optimal trajectory that can help to increase organochlorine wastewater treatment efficiency.

93-106

Authors: Stoyanova, A., Galabov, A. S.

Millions of people, mostly children, are infected annually by enteroviruses with a wide variety of clinical manifestations. Most enteroviral infections are inapparent (Modlin et al., 2001), i.e. these viruses circulate in the human population without being “spotted”. In a number of cases they cause clinically manifested illnesses of varying severity, including diseases affecting the central nervous system, heart, endocrine pancreas, etc. The high morbidity, the mortality rates in children and the high-risk groups (immunocompromised patients and newborns), as well as the lack of effective vaccines (excluding polioviruses) determine chemotherapy as the primary means of control of enteroviral infections. Up to now, no effective chemotherapeutic agents for specific treatment of such infections have been registered. A multitude of compounds have been tested, but only few of them are active in vivo. The substances that have reached clinical trial stages have adverse side effects, low selectivity, high toxicity or unfavorable pharmacokinetic properties, and are, therefore, not suitable for use in clinical practice. The main reason for the failure of the anti-enteroviral therapy is that enteroviruses become drug-resistant and even drug-dependent. This is due to the extremely high levels of mutation and recombination in these viruses, leading to heterogeneous mutant populations. One way to combat resistance is to develop approaches for combined application of new or already existing substances with different mechanisms of action. Our team was the first to introduce analysis of the combined effects of selective inhibitors of enterovirus replication with different mechanisms of action (Nikolaeva and Galabov, 1999, 2000, 2011). Furthermore, the process of development of resistance to the most effective enterovirus inhibitors – the WIN compounds – was studied. A panel of phenotype markers was also presented for characterization of drug-dependent mutants (Nikolova and Galabov, 2003; Nikolova et. al., 2011). In addition, a new highly effective approach has been developed for combined administration of anti-enteroviral substances for chemotherapy of coxsackievirus neuroinfection in newborn mice, which could serve as the basis for new therapeutic strategies. It consists in consecutive, alternating, not concomitant, application of the compounds in the combination (consecutive alternating administration treatment course, CAA course). Double, triple and quadruple regimens with some of the best studied anti-enteroviral inhibitors were tested. The highest activity was manifested by the triple combinations in consecutive administration of inhibitors with different mechanisms of action, applied in a strict order (Vassileva-Pencheva and Galabov, 2010; Stoyanova et.al., 2015).

115-117

Authors: Raynova, Y., Marchev, A., Doumanova, L., Pavlov, A., Idakieva, K.

Hemocyanins (Hcs) are oligomeric copper-containing proteins that function as oxygen carriers in the hemolymph of several mollusks and arthropods. In this study we present an investigation on the antioxidant activity of the Hc isolated from snails Helix aspersa maxima (HaH), using various experimental models. The free radical scavenging activity was determined against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and [2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] free radical cation (ABTS). Cupric ion reducing antioxidant capacity (CUPRAC) and Ferric-reducing antioxidant power (FRAP) assay also were carried out. The results obtained show that HaH exhibits good radical scavenging activity by 59.54% reduction of the DPPH and 62.31% inhibition of ABTS radicals. Furthermore, HaH demonstrated a strong chelating effect on copper ions measured through CUPRAC method. In conclusion, the present study revealed for the frst time the antioxidant properties of molluscan Hc. The antioxidative activity of HaH could probably involve quenching of reactive oxygen species and metal ion chelation, thereby reducing the potential of prooxidants to attack cellular components.

122-126

Authors: Gergova, R. T., Markovska, R. D., Mitov, I. G.

The following Moraxella catarrhalis strains were used in the experiments: eight beta-lactamase-producing clinical isolates and four isolates including a control strain 353 CCUG without beta-lactamase. The other strains used as controls in this procedure were two isolates of Staphylococcus aureus and two of Escherichia coli: beta-lactamase producers and non-producers control strains of S. aureus: ATTC 29213 and E. coli ATCC 25922. All of the microbial strains were tested with ampicillin alone and with ampicillin in combination with murine monoclonal antibody (MAb) 219 against lipooligosaccharide of M. catarrhalis in a 96-well plate to determine the minimal inhibitory concentration (MIC). The testing was performed by broth dilution method and minimal bactericidal concentration (MBC) on brain heart infusion agar (BHI). The effect of the combination of aminopenicillin with MAb219 was manifested as a fourfold reduction of the MIC and the MBC of the tested beta-lactamase producing M. catarrhalis. The effect over the control strains and moraxellae without beta-lactamase was either missing or there was a slight reduction of MIC and MBC. The conclusion of the study is that MAb219 in combination with ampicillin in in vitro testing can decrease resistance to aminopenicillins in M. catarrhalis after the linking of MAb219 with lipooligosaccharide of the outer membrane in the antigen-antibody complex.

132-140

Authors: Abrashev, R., Dolashka, P., Angelova, M.

The protective effect of pretreatment with low oxidative stress factors is little known in relation to the growth and development of filamentous fungi. The acquisition of new knowledge can be particularly useful for industrially important fungal strains. In the present study, the adaptive response of the filamentous fungus Aspergillus niger 26 to oxidative stress has been examined. The findings indicate that pretreatment with a sub-lethal temperature leads to the development of resistance to lethal concentrations of paraquat (PQ), H2O2 and extremely high temperatures. Fungal cultures subjected to 35°C followed by exposure to high doses of stress agents showed a higher amount of biomass accumulation compared to the single treated cells. The pre-exposure strategy exerted a protective effect with respect to the amount of oxidatively damaged proteins in A. niger cells, which was accompanied by a corresponding increase in the intracellular protein content and induction of antioxidant enzymes compared to the non-adapted cultures. At the same time, the results demonstrated different responses in the temperature-adapted cells. Pretreatment makes cells more resistant to both PQ and temperature than to H2O2.

145-148

Authors: Stoev, A., Kostadinov, G.

The paper presents an investigation aimed at identifying the zones with regular incidence of pear rust. Such zones are appropriate for testing of cultivar susceptibility to the pathogen and fungicidal efficacy. The investigation data indicate that the disease is common on the monitored pear trees grown in the skirts of the north slopes of mountain Vitosha. Further away from the mountain, in Sofia Valley, the rate of pear rust attack is lower or the infection is missing.

12-20

Authors: Galabov, A. S., Gesheva, V., Negoita, T.

The review describes the spreading of viruses in Antarctic ecosystems including terrestrial, marine, and freshwater habitats. The great attention is paid to virus infections in Antarctic animal and plant populations. Observation on viruses in Antarctic ecosystems might contribute for their diversity and systematics, as well as for viral ecology. Comparison of the detected viruses with the well-known viruses, using modern methods, reveals some structural differences in their nucleic acids and proteins. Several new virus species unknown in temperate geographic regions were described. Distribution of viruses in Antarctica may elucidate their migration, potential reservoirs, and hosts. Observations on viruses would explain the processes of virus infections, survival, pathogenicity, and medicine.

32-36

Authors: Baykushev, R., Raykova, V., Mitov, I.

Every obstetrician or microbiologist encounters bacterial vaginosis (BV) in his/her practice. 50% of the cases are asymptomatic. The percentage of affected women varies. The aim of this study is interpretation of microscopic and molecular diagnostic techniques for diagnosing fastidious microorganisms associated with BV. 234 women were tested. Vaginal secretions were collected, after medical history and detailed gynecological status were completed. Smears were prepared and stained by Gram. Identification of fastidious Gardnerella vaginalis, Atopobium vaginae, Megasphaera sp. type 1, and BV associated bacterium type 2 (BVAB2) was performed by PCR. 112 of the 234 tested women had microscopic evidence of some vaginal problem. 65 of them, had anaerobic infection found on the basis of the microscopic data. Materials from women suspected for anaerobic infection were subjected to PCR for proving of specific anaerobes. In 63 patients G. vaginalis was found, in 27– A. vaginae, in 18 - Megasphaera sp. type 1, and in 15 - BVAB2. The detected anaerobe in 25 swabs was single. Combination of several pathogens was demonstrated in 40 women. Gram stain is a fast, easily performed, and specific method for diagnosing BV. Methodology limitation is the inability to distinguish visually the different microorganisms involved in the condition. The PCR method provides possibilities for the sensitive, highly specific, and rapid identification of bacteria-specific markers for BV. Accurate determination of microorganisms involved in this syndrome provides possibilities for an adequate and complete treatment of the women affected.

40-47

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

The fungal strain Aspergillus fumigatus 32 was isolated from metal polluted soil (around the tailing Vlajkov vrah, Bulgaria). Effect of enhanced concentrations of cadmium ions (redox-inactive metal) on the growth and morphology, as well as the participation of oxidative stress in cadmium-induced toxicity, was reported. A. fumigatus demonstrated a high tolerance to cadmium, exhibiting remarkable growth in liquid and agar media. High metal concentrations affected fungal morphology and caused oxidative stress events such as changes in the level of reserve carbohydrates and oxidative damaged proteins. A sharp increase in trehalose content and accelerated consumption of glycogen in the presence of cadmium ions at concentration above 5 µg/ml were detected. In addition, a decrease in carbonylated proteins was measured, particularly pronounced at high concentrations (70 and 100 µg/ml). Cadmium ions exposure with 5, 20, and 50 µg/ml resulted in enhanced superoxide dismutase (SOD) level, but the higher concentrations (70 and 100 µg/ml) significantly inhibited this activity. Continuous decrease was also observed for catalase (CAT) activity. Probably, at higher concentrations of cadmium ions antioxidant enzymatic defence does not seem to be a major mechanism of cadmium tolerance in the strain A. fumigatus.

54-60

Authors: Tomov, A., Doycheva,, A., Mladenova P., Satchanska, G.

In recent years many antimicrobial agents became ineffective against bacteria. This lead to many studies of the antimicrobial effects among plants. Bulgarian vegetables are ingredients in many traditional Bulgarian foods. The aim of the study was to compare the antibacterial activity of organically grown parsley, tomatoes, cayenne peppers, onions and garlic against referent test bacteria - Bacillus subtilis No8751 and Escherichia coli No8752, supplied by the National Bank for Microorganisms and Cell Cultures. Sixteen vegetable samples were analyzed. Petri dishes were cultivated at 37oC for 24 hours. Our results demonstrated low antibacterial activity of tomato and parsley samples against the Gram(+) B. subtilis and from weak to no activity against the Gram (-) E. coli.Fresh garlic samples showed low antibacterial activity against Gram (+) bacteria with only fresh garlic leaves displaying wider sterile zones. No antibacterial activity was demonstrated by the fresh onion bulbs and leaves. In contrast, the mature garlic bulbs showed pronounced activity against the Gram (-) E. coli (30 mm zone d). Cayenne pepper tissue and seeds possess antibacterial properties against both Escherichia coli and Bacillus subtilis.Strong antimicrobial activity against B. subtilis was shown by the mature onion samples with 25 mm zone diameter for the red onion bulbs and 27 mm zone diameter for the white onion bulbs. Both mature onion samples demonstrated low activity towards E. coli.

71-72

Authors: Onderdonk, A. B., Tropcheva, R., Boleti, H., Petkov, P.

Short Communication