Acta Microbiologica Bulgarica

3-8

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

The review presents the basic data on the chemical compounds manifesting pronounced activity against SARS-CoV-2, the causative agent of the COVID-19 pandemic. The compounds are characterized with their chemical structure, and experimental anti-coronavirus activity in vitro (in cell cultures) and in vivo (in laboratory animals). The existing data on their clinical use in the treatment of patients with COVID-19, double-blind trials included, occupies an important place and are object of discussion on the therapeutic effectiveness of antivirals.

19-29

Authors: Krumova, E., Najdenski, H., Dishlijska, V., Abrashev, R., Stoyancheva, G., Gocheva, Y., Kostadinova, N., Miteva-Staleva, J., Spasova, B., Angelova, M.

The amount of waste generated on manned long-duration space missions away from Earth orbit creates the daunting challenge of how to manage the waste through reuse, rejection, or recycling. Microbial degradation, for both economic and ecological reasons, has become an increasingly popular alternative for the treatment of cellulose containing organic waste in the space stations. This approach offers several advantages: low energy, mild operation conditions, control on biological hazard of the wastes, etc. Fungi are the main cellulose-degrading microorganisms in the nature and their cellulolytic features are object of intensive studies. This review describes the ability of anaerobic, aerobic, and microaerophilic fungi to degrade organic wastes generated during manned space flight.

164-171

Authors: Ivanova, E., Zdravkova, A., Peshev, R.

African swine fever (ASF) is a highly contagious and devastating disease in feral and domestic swine. The infection spreads rapidly through swine farms with clinical signs of high fever 42°C 4-5 days after initiation of the infection followed by dullness, coughing, ocular and nasal discharge, breathing difficulty, cyanosis of the extremities, vomiting, and abortions in pregnant sows. The causative agent of ASF is a large, double-stranded DNA virus of the Asfarviridae family named African swine fever virus (ASFV). ASFV causes haemorrhagic fever in domestic pigs and wild boar, resulting in mortality rates of up to 100%. The disease spreads over a short period of time in many regions of the country. In Bulgaria, the first outbreak of the disease in domestic pigs (backyard, village of Tutrakantsi, Provadiya, Varna district) appeared in August 2018. Later in the same year, 20 new outbreaks in wild boars were recorded in Silistra and Dobrich regions. In 2019, fourteen new cases of ASF in wild boars (WBs) were found, with the last event recorded at the beginning of June 2019 in Alfatar, region Silistra. Later in that year, in July, 16 new outbreaks were found in the Northern part of the country – Pleven, Burgas, V. Tarnovo, Vratca, Russe, Vidin. The situation in Russe region was the worst. The disease was detected in three industrial pig farms – Nikolovo, Brashlen and G. Vranovo. In order to stop the spread of the disease in industrial pig farms, large numbers of pigs were destroyed. The clinical symptoms observed in diseased pigs were typical of ASF infection. Samples investigated by real time PCR were strongly positive (Ct < 30), which was evidence of high quantity of ASFV DNA. Currently, there is no vaccine against ASF and biosecurity measures are the key to prevent the spread of ASF within and among domestic pig farms.

176-181

Authors: Kaftandjieva, A., Stefkova, M., Ivanovska, E., Cekovska, Z.

The aim of the present study was to assess the time-related changes in the microflora of burn wounds in our setting and to determine the susceptibility pattern to commonly used antibiotics. In a period of 8 months a total of 90 burn wound samples (swabs) from 23 hospitalized patients at the Burn Unit were sent for microbiological examination. Swabs were taken weekly in a period of four weeks or until the patients were dismissed from the hospital. Isolation, identification of bacteria and determination of the antimicrobial susceptibility were according to standard microbiological techniques. In the first and second week of hospitalization, the predominant organism was Acinetobacter spp. By the end of the third week, Pseudomonas aeruginosa had become more predominant. In a period of four and more weeks of hospitalization, 7 samples from two patients were positive (yielding double and triple isolates) with further prevalence of Acinetobacter spp. Most of Gram-negative isolates were multidrug-resistant. Acinetobacter spp isolates were resistant as follows: 100% to amoxicillin-clavulanic acid and to cephalosporins, 86.4% to cefepime, 91% to both amynoglycosides, 88.6% to ciprofloxacin and co-trimoxazole, and 84% to piperacillin/ tazobactam and carbapenems. P. aeruginosa isolates were resistant as follows: 100% were resistant to AMC, cefuroxime and cefixime, 95% to co-trimoxazole, from 9% to 40% to cephalosporins, 13.6% to gentamicin,9% to ciprofloxacin, and 4.5% to amikacin and carbapenems. In conclusion, knowledge of the responsible bacterial flora of burn wounds, its prevalence and bacterial resistance, is of crucial importance for fast and reliable therapeutic decisions.

190-199

Authors: Afolabi, F. T., Obisesan, O., Igbinadolor, R. O.

This study aimed at isolating fungi from cocoa pod husks, screen for their ability to degrade cellulose, optimize, produce and purify cellulase. Cocoa pod husks were obtained from Badeku area of Oyo State. The isolates that showed consistent zone of hydrolysis and negative for aflatoxin production were used for cellulase production. Cellulase activity was measured in terms of FPUase and CMCase. A total of 23 fungal isolates were obtained from cocoa pod husk out of which 12 showed a consistent zone of hydrolysis. The fungal isolates were: Aspergillus niger CH2, A. niger CH4, A. flavus CH5, Fusarium oxysporium CH8, A. niger CH10, A. flavus CH13, A. niger CH15, Phytophthora sp. CH16, A. niger CH18, A. niger CH20, A. flavus CH21, A. niger CH23. A. niger CH4 and F. oxysporium CH8 were the isolates used in this study. Incubation time, pH, temperature and substrate concentrations were the parameters used for the optimization of cellulase. The preferred optimized conditions determined for the production of cellulase by A. niger CH4 were 96 hrs, pH 5.5, 30°C and 5% CPH concentration while for F. oxysporium CH8 they were 72 hrs, pH 5.9, 30°C and 4% CPH concentration. Endoglucanase activity was higher in F. oxysporium CH8 while exoglucanase was higher in A. niger CH4. The purification of the ammonium sulphate purified cellulase enzyme using Sephadex G-200 gel filtration increased A. niger CH4 cellulase activity by 1.49-times while that of F. ox¬ysporium CH8 increased by 1.63-times. A. niger CH4 and F. oxysporum CH8 can be used for the production of cellulose.

125-132

Authors: C. Daccò, M. E. E. Temporiti, L. Nicola, S. Tosi

Lignocellulosic residues, deriving from the production of biofuels, represent a precious source and being able to purify and exploit them is crucial for a more sustainable supply of lignin. The aim of this work was to increase the efficiency of the biological treatment of lignocellulosic residues to obtain purified lignin which can be used in biotechnological applications. The study evaluated the activity of two Ascomycota fungal strains on a bioethanol production waste material rich in lignocellulosic residues (LRR). The tested fungi were Trichoderma asperellum 1020, isolated from a hydroponic plant culture and Paecilomyces variotii 1030, isolated from a sample of lignin purified by chemical methods. The degradation ability of the selected fungi was verified by measuring their consumption of reducing sugars over 3 months, while the purification was assessed by measuring the degradation of reducing sugars and the activity of the secretome (endo-β-1,4-glucanase, β-glucosidase, β-1,3-glucanase, endo-1,4-β-xylanase, pectinase). Both strains appear to use almost all the available reducing sugars in the first 4 days after inoculation and a decrease of the reducing sugars up to 70% was obtained in the samples treated with T. asperellum 1020. The quantification of enzymes secreted revealed that P. variotii 1030 has greater potential in the degradation of cellulose and pectin, while T. asperellum 1020 degrades preferably pectin and carbohydrate residues. These strains act on the components of LRR and are particularly promising to processes lignocellulosic residues.

141-146

Authors: Aljicevic, M., Omerika, A., Rebic, V., Mahmutovic, S., Abduzaimovic, A.

Urinary tract infections (UTIs) are considered to be the most common bacterial infection. Escherichia coli is the most common uropathogen in uncomplicated upper and lower urinary tract infections (70–95% of cases). The aim of this study was to examine the presence of antimicrobial susceptibility/resistance among E. coli's strains in outpatients. This retrospective study was carried out from January1st to December 31th 2017, at the Department of Microbiology, Faculty of Medicine, University of Sarajevo, in cooperation with the Microbiological laboratory of the Cantonal Hospital „Dr. Safet Mujić“, Mostar. During the research, a total of 3 148 urine samples of outpatients were examined. Our study showed that E. coli had the highest resistance to ampicillin (58%), followed by trimethoprim-sulfamethoxazole (38.4%) and amoxicillin-clavulanic acid (38.4 %), and the lowest resistance to nitrofurantoin (2.7%) and cefuroxime (4.1%). The isolated strains of E. coli showed the highest resistance to ampicillin, and the highest susceptibility to nitrofurantoin. Gender distribution of positive E. coli isolates showed statistically significant differences in favor of females.

155-161

Authors: Afolabi, F. T., Oduokpaha, G. E., Onilude, A. A.

This study was carried out to investigate the tolerance of yeasts isolated from some Nigerian traditional fermented foods to formic acid during laboratory-scale fermentation of ethanol. A total of 27 yeast strains were isolated from burukutu, ogi, kunu and palm wine. The yeasts were screened for formic acid tolerance using spot plate technique on two culture media. One strain was selected based on its ability to tolerate up to 15 mM concentration of formic acid on Yeast Extract Peptone Dextrose Agar and was further identified as Candida tropicalis strain IFM 63517. C. tropicalis was used for fermentation of ethanol with varying concentrations of formic acid, ethanol and residual glucose concentrations which were monitored at intervals. The total viable cell count was determined using plate count technique. The highest ethanol yield of 8.36% (v/v) with a residual glucose concentration of 0.33 g/L was obtained from 0 mM formic acid (control fermentation vessel) with a total viable cell count of 8.7x109 cfu/ml, while the lowest ethanol yield of 8.00% (v/v) with a residual glucose concentration of 0.14g/L was obtained from 15 mM concentration of formic acid with a total viable cell count of 6.1x109 cfu/ml. The yeast strain used in this work exhibited a high ethanol yield despite the presence of an inhibitory compound (formic acid) when comparing the ethanol yield at its tolerance threshold (15 mM of formic acid) to the control fermentation vessel without formic acid.

41-45

Authors: Masalski, N., Najdenski, H.

Pest des petits ruminants (PPR) is a disease first described in 1941 in West Africa. It is caused by a virus belonging to the family Paramyxoviridae. The virus is highly virulent for goats and sheep. There is no virus carriage. The incubation period is 5-6 days. Mortality is high. Transmission of infection is through close contact between sick and healthy animals – infection occurs mainly through the respiratory tract. In pathogenesis, the lymphatic system plays a major role. The infection predominantly affects small ruminant flocks. Clinical symptoms are mainly lesions on the muzzle, nose and urogenital tract. Diagnosis is based on characteristic clinical and pathological anatomical evidence. Stringent measures are necessary to isolate and quarantine risky flocks and farms. In connection with a PPR epizootic in Syria, the import of lambs from our country was terminated. The team of Bulgarian specialists sent to Syria proved that Bulgaria had nothing to do with the epizootic in that country. In 2018, the dangerous disease of sheep and goats was detected in Bulgaria. Data reported in the press and other media seriously differed from the well-known characteristics of the Pest des petits ruminants infection.

53-59

Authors: R. Peshev, I. Sirakov

Bovine herpesvirus 4 (BoHV 4) is a member of the Herpesviridae family in the Gammaherpesvirinae subfamily, Radinovirus genus. The purpose of this study was to isolate BoHV 4 from calves, heifers and cows presenting various clinical entities – respiratory, conjunctivitis, pneumonia, skin damage, genital lesions and abortion, and to characterize its molecular biological features. Primary and permanent bovine cell cultures were used for virus isolation and culturing. Polymerase chain reaction (PCR) was used to confirm the gB and TK genes and the PCR products were sequenced. After infecting the primary bovine and permanent cell lines, a cytopathic effect typical of BoHV 4 was observed and 10 cytopathic viral agents were isolated. Amplification products with molecular weights of 567 and 615 bp for the Tk and gB genes, respectively, were found after PCR. After the BoHV 4 isolates had been sequenced, 473 and 476 nucleotide sequences were analyzed for the Tk and gB genes, respectively. The phylogenetic trees constructed based on the Tk and gB genes of Bulgarian BoHV 4 strains fall within the same branch with the reference BoHV 4 European strains deposited in the genomic bank and their genomic features were similar to European sequenced genomes of other Radinoviruses.

66-70

Authors: Dikova, B., Djourmanski, A.

Symptoms of virus diseases on the leaves of the medicinal plant species Echinacea purpurea (L.) Moench. – purple coneflower were observed in the trial fields of the Institute of Rose, Essential and Medicinal Cultures (IREMC) near Kazanlak and private gardens in Kazanlak, Bulgaria. Virus diseases, caused by the following viruses were established on E. purpurea: Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV) and Potato virus Y (PVY). These viral pathogens existed in Echinacea plants in individual or most commonly in mixed infections. The symptoms caused by viral pathogens were spotting (chlorotic spots of different shape and size) on the leaves, sometimes typical mosaic, reduction in leaf size and flower head diameter and especially decrease in the height of the stems, called dwarfing. The results of the study have shown, that the most widely spread were СМV, АМV, ТМV - over 45 % and TSWV – nearly 30 %. Clear differences were detected in the yields of roots, seeds and leaves between Group I of symptomless Echinacea plants (practically healthy without symptoms or damage) and the spotted Echinacea plants (Group II – of spotted and dead plants and Group III - of spotted, but vital plants, in which the viruses were established. The spotted Echinacea plants were shorter (dwarfed) in comparison with the symptomless plants with fewer leaves and flower heads. The yield of the seeds, leaves and roots of the spotted plants was twice or several times lower in comparison with the symptomless plants.

3-8

Authors: Savov, E., Kovachka, K., Trifonova, A., Kioseva, E., Strateva, T.

Multidrug-resistant (MDR) Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii are of great concern and often related to the production of extended-spectrum beta-lactamases (ESBL) and carbapemenase - producing bacteria, which represent an increasing global threat. The therapy of nosocomial infections due to these MDR Gram-negative bacteria is challenging also because some of the new active drugs are not available in Bulgaria. In this paper we review the potential of some drugs (ceftolozane-tazobactam, ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, plazomicin, cefiderocol, eravactclin) and their action in vitro on Gram-negative bacteria that pose a major problem in hospital infection pathology. The recommendations to the use of these new drugs can be evaluated from a clinical point of view as an alternative for the treatment of severe life-threatening infections, but also as an opportunity to give an opinion in this field, while awaiting more definitive data after we have gained good practical experience.

19-28

Authors: Tomičić, R., Tomičić, Z., Dodić, S., Raspor, P.

The aim of this study was to assess the potential of Candida species and Pichia species to adhere to stainless steel (AISI 304) material with different degrees of surface roughness and polystyrene as most frequently used contact materials. Cell surface hydrophobicity (CSH) of Candida and Pichia strains was determined in order to assess correlation between the cell surface hydrophobicity and yeast adhesion to polystyrene. Candida albicans showed a higher ability to adhere to both surfaces compared with non-albicans Candida species. Regarding Pichia species, P. membranifaciens strains were less adherent to stainless steel than P. pijperi. Surface roughness of stainless steel was found to affect the adhesion of Candida and Pichia strains, whereas cell surface hydrophobicity was not correlated with adhesion. We also investigated the antimicrobial and antibiofilm activity of plant extracts such as Humulus lupulus, Alpinia katsumadai and Evodia rutaecarpa against C. albicans, C. glabrata and P. membranifaciens. According to the MIC values, all plant extracts were effective in inhibiting yeast strains. It was observed that biofilms of C. glabrata were more resistance to plant extracts as compared to C. albicans. However, extracts of A. katsumadai and E. rutaecarpa promoted the growth and development of a preformed biofilm of P. membranifaciens.