Acta Microbiologica Bulgarica

221-223

Authors: Galabov, A. S.

The review considers the compound N1, N1-anhydro-bis(β-hydroxyethyl)-biguanide hydrochloride (abitylguanide, ABOB) as the first applied in the clinical practice anti-flu drug, which later did not prove efficacious, was officially rejected and classified as an agent whose merit only deserved a place in the history of antivirals. The compound manifested some activity against other RNA-containing viruses (paramyxo) but with contradictory effects, strongly dependent on the test methods used and on the virus inoculation dose (multiplicity of infection).

232-236

Authors: Rahimipour, F., Roodbari, F., Ghazvini, K., Youssefi, M.

The increasing number of methicillin-resistant Staphylococcus aureus (MRSA) infections strains is a global health threat. Vancomycin is one of the very limited options in treating such infections. The emergence of vancomycin-resistant S. aureus (VRSA) is therefore a great concern in clinical settings. During recent years, the incidence of vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus has increased in various parts of the world, which have been identified based on criteria defined by the Clinical and Laboratory Standards Institute (CLSI). We have recently shown a high resistance rate to methicillin in S. aureus isolates from two main university hospitals in northeastern Iran. Here we expanded the study to reveal the frequency of vanA and mecA genes in the isolated MRSA strains. We selected 45 MRSA isolates, which were shown phenotypically methicilin- resistant to further genotypic investigation of the mecA and vanA genes. DNA was extracted from bacterial suspension and mecA and vanA genes were identified using PCR technique. The majority of MRSA isolates, 42 out of 45 (93%), were positive for the mecA gene. None of the MRSA isolates were positive for the vanA gene. The mecA gene is frequently circulating among phenotypically identified MRSA isolates, which confirms the phenotypically resistant strains and explains the resistance mechanism. The high frequency of circulating mecA gene highlights the need for policies to overcome the MRSA problem in clinical settings. Though none of the isolates showed vancomycin-resistance based on phenotypic tests, we also evaluated the isolates for possible vanA gene positivity and none of the isolates were shown to be positive for the vanA gene.

10-19

Authors: Budzanivska, I. G., Shevchenko, T. P., Shevchenko, O. V., Polishchuk, V. P.

The availability of analytical approaches allowing fast deciphering of nucleotide sequences enables phylogenetic analysis of viruses and establishing homology among their strains/isolates for unveiling the history of virus evolution. This work was focused on phylogenetic analysis of the most important viruses infecting vegetable crops in Ukraine. ‘Field’ isolates of different plant RNA viruses were collected and analyzed from various agroecosystems. These included Potato virus Y (PVY), Cucumber mosaic virus (CMV), Watermelon mosaic virus 2 (WMV-2), Tomato mosaic virus (ToMV), Pepper mild mottle virus (PMMoV). Using MEGA6 software, phylogenetic trees were constructed, based on sequenced viral cDNAs corresponding to parts of the coat protein genes of the studied pathogens. Differing evolutionary trajectories were shown for some of the Ukrainian virus isolates. The study of the molecular properties of viruses, and especially those of viral genomes, has fundamental importance (tracing phylogenetic relationships of a given virus with other members of the kingdom, its evolutionary history and trends), and is also of practical significance, as such data may further be used both for developing highly specific PCR-based diagnostic techniques as well as for forecasting possible alterations and novel properties of circulating viruses for prevention of disease spread and minimizing the aggressiveness of viruses.

27-32

Authors: Stoykova, Z., Ivanova, L. I., Todorova, T. T., Kostadinova, T., Tsaneva-Damyanova, D.

The prevalence of cytomegalovirus (CMV) infections is between 50% and 85% in adults in different parts of the world. Its epidemiology varies on socioeconomic and age groups. The present retrospective study has been performed to determine the seroprevalence of CMV among the population in North-Eastern Bulgaria. For the period 2003–2015, the prevalence of individuals with antibodies to CMV was estimated, using indirect enzyme-linked immunosorbent assay (ELISA) to detect virus-specific IgG and IgM. The population sample included 7879 randomly chosen hospitalized patients of both sexes and different ages. The total seroprevalence of CMV was determined to be 78.4% (CI 95% 77.5; 79.3), and the relative proportion of acute CMV infections 22.9% (CI 95% 21.9; 23.7). The proportion of CMV IgG and IgM by sex and by age was also analysed. The results of the study reveal that CMV infection is highly prevalent among the population and occurs mainly in the first years of life.

39-44

Authors: Hinkov, А., Valchovski, H., Shishkov, S., Nedyalkova, K., Donkova, R.

The coelomic fluid of four earthworm species, Aporrectodea rosea, Eisenia fetida, Lumbricus terrestris and Octolasion lacteum, was investigated against Gram-negative bacteria strains: Aeromonas hydrophila, Pseudomonas fluorescens and Pseudomonas luteola and human pathogen Herpes simplex virus type 2 (genital herpes). The research revealed that only studied material from Eisenia fetida acted selectively on pathogenic bacteria. The greatest growth-inhibiting effect was observed on Aeromonas hydrophila. No antiviral potential was demonstrated against Herpes simplex virus. The metabolic components of the coelomic fluid have a strong cytolytic effect, but lack any activity against the tested virus pathogen.

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.