78 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2018 Vol. 4 issue 1 DOI 10.11603/IJMMR.2413-6077.2018.1.8643 OPPORTUNIsTIc BAcTERIA IN AGROEcOsysTEms Of UKRAINE L. M. Butsenko, L. A. Pasicnhyk DANyLO ZABOLOTNy INSTITUTE OF MICROBIOLOGy AND VIROLOGy OF THE NATIONAL ACADEMy OF SCIENCES OF UKRAINE, KyIV, UKRAINE Background. Recently, the number of diseases caused by opportunistic bacteria has been increasing all over the world. Opportunistic microorganisms are characterized by ubiquitous proliferation, flexibility in adaptation to the conditions of the environment, lack of specific relationship with the macroorganism. Phytopathogenic bacteria are also able to infect both plants and animals. Objective. The purpose of the research was to detect the Pantoea agglomerans and Pseudomonas fluorescens bacteria in various ecological niches and establish their virulence. Methods. Classical microbiological, biochemical, serological methods were used in the research. The identification of bacteria was carried out according to their phenotypic properties. Results. It has been established that opportunistic bacteria species P. agglomerans and P. fluorescens are present in wheat agrophytocenoses. The bacteria isolated from the agrophytocenoses are virulent for wheat, rye and weed plants. Antibodies to opportunistic bacteria, which are spread in agrophytocenoses of cereals crops, have been found in the blood of healthy rabbits. Conclusions. Thus, we have established that virulent strains of opportunistic bacteria P. agglomerans and P. fluorescens are spread in agrophytocenoses of cereals. The presence of antibodies to these bacteria in the blood of healthy rabbits proves that opportunistic bacteria from plant material get into animals and humans. Knowledge of biology and the spread of opportunistic pathogens in agrophytocenoses is necessary for prevention of infections that these bacteria cause in humans. KeY WoRdS: opportunistic bacteria; Pantoea agglomerans; human health. International Journal of Medicine and Medical Research 2018, volume 4, Issue 1, p. 78-82 copyright © 2018, TSMU, All Rights Reserved Corresponding author: Liudmyla Butsenko, Department of Phytopathogenic Bacteria, Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine, 154, Akademika Zabolotnoho Str., Kyiv, 03680, Ukraine E-mail: plant_pathol@ukr.net Introduction Recently, the number of diseases caused by opportunistic bacteria has been increasing all over the world. Opportunistic microorganisms are characterized by ubiquitous proliferation, flexibility in adaptation to the conditions of the environment, lack of specific relationship with the macroorganism. Such bacteria are cha- racterized by the ability to cause nonspecific toxic infections in weakened people and animals. It has been established that strains of certain species of microorganisms can cause damage to plants, insects, animals and humans [1]. This phenomenon is known as polybiotrophy [2], which is particularly spread among oppor- tunistic microorganisms. for example, con- ditionally pathogenic bacteria for humans Pseudomonas aeruginosa cause an internal of putrefaction onion during storage [3]. Strains P. aeruginosa, isolated from sick people, under experimental conditions affect plants, nema- todes and insects [4]. Phytopathogenic bacteria are also able to infect both plants and animals [1, 2]. Bacteria of the genus Erwinia, which are well known exclusively as pathogenic to plants, are often isolated in pathological processes in humans and animals [5]. In this case isolates isolated from humans and animals are patho- genic to plants [2]. The causative agent of vascular bacteriosis of Erwinia toxica cucumbers, in intraperitoneal administration to mice, leads to sepsis in animals. Infected with these bacteria fruits of cucumbers cause poisoning in people [6]. A high degree of similarity established by DNA hybridization analyses and phenotypic data between strains of Erwinia herbicola, Enterobacter agglomerans and Erwinia milletiae led Gavini et al. [7] to unite them as a single species, namely Pantoea agglomerans (Beijerinck 1888) comb. nov. P. agglomerans is widespread in numerous diverse natural habitats and is particularly associated with many different plants as a common epiphyte and endophyte [8]. Additionally, it has been also isolated from seeds, water, humans (e.g., wounds, blood, L. M. Butsenko et al. 79 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2018 Vol. 4 issue 1 urine and internal organs) as well as from animals [7, 9-12]. Pantoea species are ubiquitous in nature and occasionally are associated with infections caused by contaminated clinical material. Hence, Pantoea agglomerans is considered as an opportunistic pathogen of humans [13]. The Pseudomonas fluorescens bacteria, under conditions favorable to their development, can cause diseases of agricultural crops [14]. For some human diseases, antibodies to P. fluorescens lipopolysaccharide have been identified in the patients’ blood. Therefore, the purpose of the research was to detect the Pantoea agglomerans and Pseudo- monas fluorescens bacteria in various ecological niches and establish their virulence. Methods Materials for research were the plants with symptoms of damage of rye, wheat and weeds that grew in wheat agrocenoses. Bacteriological analysis and isolation of bacteria were carried out using generally accepted methods [15]. virulent properties were investigated on the host: plant, aggressiveness of the pathogen was determined by a 4-point scale. The bio- logical properties were investigated by the methods described by Klement et al [15]. The bacteria were identified by comparing their properties with the characteristics of strains collection, and according to the Bergey’s Manual of Systematic Bacteriology [16]. Results We have found out that yellow-pigmented bacteria, flat or with a conical center, are opaque, wavy edges; and the oxidase-positive gray-colored isolates with wavy edges were isolated from all investigated plant materials. It was established that a part of the investigated isolates of bacteria was avirulent for plants. In all other cases, the isolated bacteria caused diseases of rye, wheat and weeds. The yellow pigmented bacteria are poly- morphic, short rods, single arranged, sometimes in pairs in the form of short chains. Bacteria are mobile, Gram negative, spores are not formed, oxidase negative. facultative anaerobes (Table 1). On the meat-peptone broth they grow with the formation of uniform turbidity, ring, film and sediment. the bacteria are utilized glucose (anaerobic), reduce nitrates, acidify litmus serum. Strains differ in the use of rafinose, sorbitol and inulin. all investigated strains do not use dulcitol, cause coagulation or peptonization of milk. The isolates obtained from the affected plants according to the physiological and Table 1. Physiological and biochemical properties of isolates Test yellow-pigmented isolates P.аgglomerans [16] Unpigmented isolates P.fluorescens [16] Gram’s staining – – – – Motility + + + + Nitrate reduction + + – – oxidase – n/i + + Formation of H2S –/+ n/i – n/i Formation of indole – – – n/i Gelatinase +/- + + n/i OF-test Facultative anaerob Facultative anaerob Aerob Aerob utilization: D-glucose, l-arabinose, d-mannitol, d- xylose + + + + Fructose, galactose + + + n/i Salicin + + – – lactose + d – – Inositol + + +/– + Raphinose +/– d +/– n/i Dulcitol – – +/– n/i Sorbitol +/– – +/– + Inulin +/– n/i Note: n/i – not investigated. L. M. Butsenko et al. 80 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2018 Vol. 4 issue 1 biochemical properties are similar to each other and do not differ from the characteristics of the described P. agglomerans species [16]. according to this they were identified as P. agglomerans. gray colored oxidase-negative bacteria grow on the meat-peptone broth, form a film, ring and precipitate, and use glucose (aerobic), mannitol, xylose, fructose, arabinose, and do not utilize lactose, inulin, salicin, as a single source of carbon. Most of the isolates utilize raffinose, mal- tose and dulcitol. All isolates hydrolyze gelatin, alkalinize the litmus serum, and do not reduce nitrates (Table 1).The isolates of bacteria obtained from the affected cereals and weeds on the morphological and cultural-biochemical properties did not differ from the described strains of P. fluorescens and from the P. fluo- rescens characteristic given in Bergey’s Manual of Systematic Bacteriology [16]. All bacterial strains tested were virulent for wheat, rye and weed plants (Table 2). It was established that the strains of the isolated bacteria are more aggressive on weeds than on agricultural crops. Consequently, pathogenic bacteria for plants of the species Pantoea agglomerans and Pseudomonas fluorescens are able to come into contact with humans and animals and cause their diseases. This is evidenced by the fact that we detected antigens to the strains Pantoea agglomerans and Pseudomonas syringae pv. syringae in the serum of non-immunized rabbits (true phytopathogen) (Table 3). Thus, the study of phytopathogenic and opportunistic bacteria is important not only for the development of plant protection methods, but also from the point of view of studying their effects on human health, since these bacteria are widespread in nature and can be ingested by humans. Discussion Our research has found that rye, wheat and weeds that grow in the agroecosystem of wheat are affected by opportunistic bacteria Pantoea agglomerans and Pseudomonas fluorescens. Our previous studies proved that strains of P. agglomerans and P. fluorescens were more commonly found in agricultural crops as epiphytes and did not cause plant diseases [17]. With the change of environmental factors and the influence of agronomic techniques, these opportunistic bacteria in recent years increa- singly acquire virulent properties. for example, the bacteria of P. agglomerans were isolated from the affected locales of cotton bolls collec_ ted in a field in the uSa and were able to cause comparable disease symptoms in greenhouse grown cotton fruit [18]. P. agglomerans, gram negative bacteria of Enterobacteriaceae family, were isolated from feculent material, plants and soil. Soft tissue and bone-joint infections due to P. agglomerans following penetrating trauma by vegetation and bacteraemia in association with intravenous fluid, total parenteral nutrition, blood products and anesthetic agent contamination were reported [19]. Some authors on the basis of their studies suggested that, independent of their origin, all P. agglomerans strains might possess indistinguishable virulence potential [13]. P. agglomerans was also proved to be an antibiotics producer [20]. Some researchers reported on isolation of P. agglomerans in two cases of septic mono ar- thritis after plant thorn and wood sliver injuries [21]. This indicated the transfer of P. agglomerans from the infected plant material to humans. Table 2. Virulence properties Species aggressiveness (marks) on plant: Rye Wheat Weed Pantoea agglomerans 1–2 1–3 2–3 Pseudomonas fluorescens 1–2 2 2–3 Table 3. Results of agglutination reaction Species, strains Titres of agglutination reaction with sera of non-immunized rabbits Serum 1 Serum 2 Pantoea agglomerans, 116 50 100 Pseudomonas syringae pv. syringae, NCPPB 281 200 200 Erwinia amylovora, 2024 0 0 L. M. Butsenko et al. 81 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2018 Vol. 4 issue 1 When studying the effect of bacteria of the genus Klebsiella (K. pneumoniae, K. hinosclero- matis, K. ozaenae), which, like the Erwinia phytopathogenic genus, belonged to the Entero bacteriaceae family, on leaves of potatoes, horse beans, beans, cabbage, cucumber, pumpkin and apple fruits Jonathan found out that four strains of K. pneumoniae were capable to affect horse beans and potatoes. The phy- topathogenic properties of Klebsiella species did not correlate with their ability to produce pectinases [22]. At the same time, phytopathogenic pro- perties can be presented by bacteria, which are traditionally pathogens of animals and humans. It is proved that 15% of the strains of bacteria of genera Escherichia, Citrobacter, Enterobacter, Proteus, Pseudomonas, isolated from urological patients, have phytopathogenic properties. The most pronounced these properties were on fruits of tomatoes [23]. Among the species of the genus Pseudo- monas, which produce pigments (P. aeruginosa, P. fluorescens, P. aureofaciens), it is proved that the strains have phytotoxic and entomocidal properties. The greatest number of such strains is found among the bacteria of P.fluorescens species. The cultural fluid of these strains suppresses the germination of seeds of radish, lettuce and, to a lesser extent, wheat, and also causes the death of 100% of mosquito larvae. Entomopathogenic strains of P. fluorescens have antagonistic effects on some saprophytic bac- teria (Bacillus subtilis, B. megaterium, Sarcina lutea, Escherichia coli, Mycobacterium sp.). Thus, the toxins of pigmented strains of the genus Pseudomonas are not narrowly specific and affect a wide range of organisms [24]. In some human diseases antibodies to the lipopoly- saccharide of P. fluorescens 7769, which were isolated from affected rye tissues, were iden- tified [25]. The adaptation of P. agglomerans to diverse microenvironments might suggest that this species maintain high genetic plasticity. P. agglomerans appears to be readily accessible to horizontal gene transfer driven by plasmids and other mobile elements [26], a trait that may explain its flexibility in adapting to different life styles. Some researchers noted that pathogenic microorganisms had a lot in common in me- chanisms of pathogenicity, regardless of which macroorganism they were infected with [1, 27, 28]. Conclusions Thus, in the agrophytocenosis of wheat, one of the most widespread agricultural crops in Ukraine, there are virulent strains of oppor- tunistic bacteria of P. agglomerans and P. fluo- rescens species. Present agroecosystems, which are overloaded with chemical pollutants, create conditions for increasing the aggressiveness of opportunistic bacteria. It has been established that in the blood of healthy rabbits antibodies to opportunistic bacteria, which are spread in agrophytocenoses, are present. It proves the intake by animals of opportunistic bacteria together with plant food. Since opportunistic bacteria can cause the infections processes in humans, animals, insects and plants, the control of their spread and investigation of peculiarities of virulent strains circulation is necessary. L. M. Butsenko et al. ОПОРТУНІСТИЧНІ БАКТЕРІЇ В АГРОЕКОСИСТЕМАХ УКРАЇНИ Л. М.Буценко, Л. А. Пасічник ІНСТИТУТ МІКРОБІОЛОГІЇ І ВІРУСОЛОГІЇ ІМЕНІ Д. К. ЗАБОЛОТНОГО НАН УКРАЇНИ, КИЇВ, УКРАЇНА Вступ. Останнім часом кількість захворювань, спричинених опортуністичними бактеріями, зростає в усьому світі. Опортуністичні мікроорганізми характеризуються значними темпами проліферації, гнучкістю в адаптації до умов навколишнього середовища, відсутністю специфічних зв’язків з макроорганізмом. Фітопатогенні бактерії також здатні інфікувати як рослини, так і тварини. Метою дослідження було виявлення бактерій Pantoea agglomerans і Pseudomonas fluorescens в різних екологічних нішах і встановлення їх вірулентності. Методи дослідження. У дослідженні використовувалися класичні мікробіологічні, біохімічні та серологічні методи. Ідентифікацію бактерій було здійснено за їх фенотиповими властивостями. Результати дослідження. Встановлено, що в агрофітоценозах пшениці присутні опортуністичні бактерії виду P. agglomerans і P. fluorescens. Виділені бактерії вірулентні для рослин пшениці, жита та бур’янів. Антитіла до опортуністичних бактерій, які поширюються в агрофітоценозах зернових культур, виявлені в крові здорових кроликів. Висновки. Встановлено, що вірулентні штами опортуністичних бактерій P. agglomerans та P. fluorescens поширюються в агрофітоценозах зернових культур. Наявність антитіл до цих бактерій у крові здорових кроликів доводить, що опортуністичні бактерії з рослинного матеріалу потрапляють в організми тварин і людини. Знання біології та поширення опортуністичних патогенів в агрофітоценозах необхідні для профілактики інфекцій, що викликаються цими бактеріями в організмі людини. КЛЮЧОВІ СЛОВА: опортуністичні бактерії; Pantoea agglomerans; здоров’я людини. 82 B io m e d ic a l S c ie n c e S iSSn 2413-6077. iJmmR 2018 Vol. 4 issue 1 9. Cruz AT, Cazacu AC, Allen CH. Pantoea agglo- merans, a plant pathogen causing human disease. J Clinical Microbiol. 2007;45:1989-1992. 10. Williams AJ, Scott RJ, lightfoot NF. Erwinia herbicola as a cause of bacterial endocarditis. 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