Аннотация и ключевые слова
Аннотация (русский):
На фоне возрастающей резистентности патогенов аквакультуры к антибиотикам особое значение приобретает поиск новых подходов к контролю инфекционных заболеваний. Перспективным подходом представляется разработка пробиотических препаратов на основе бактерий-антагонистов. Изучена антагонистическая активность бактерий р. Bacillus, перспективных для создания пробиотиков для рыб. Штаммы выделяли из донных отложений р. Дон в районе Донского рыбного заповедника, а также из кишечника карповых рыб природных популяций, а именно от серебряного карася (Cyprinus gibelio), тарани (Rutilus heckelii), леща (Abramis brama). Ингибирующее действие бактерий р. Bacillus в отношении возбудителей аэромоноза, псевдомоноза и бактериальной геморрагической септицемии исследовали методом отсроченного антагонизма. Патогенные для рыб бактерии р. Aeromonas и р. Pseudomonas были выделены от осетровых, карповых и лососевых рыб, выращиваемых в условиях прудовых хозяйств и установках замкнутого водоснабжения Краснодарского края, Ростовской области и Республики Адыгея. Показано, что чувствительность к действию бацилл является не только видовым, но и штамм-специфичным признаком среди бактериальных патогенов рыб. Выделено 5 штаммов, проявляющих максимальную антагонистическую активность в отношении исследуемых патогенов, проведена их видовая идентификация молекулярно-биологическим и масс-спектрометрическим методами. Установлено, что наиболее выраженный антагонизм проявляют штаммы бацилл, выделенные из донных отложений. Рост всех изученных штаммов патогенных для рыб видов р. Pseudomonas подавлял только штамм B. subtilis R4. Таким образом, выделены и охарактеризованы штаммы аэробных спорообразующих бактерий, подходящие для создания на их основе пробиотических препаратов для объектов аквакультуры.

Ключевые слова:
: аэромоноз, пробиотики, заболевания рыб, бациллы-антагонисты, аэромонады, псевдомонады, объект аквакультуры
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Introduction

In modern conditions, aquaculture is developing in several directions, which have significant differences. These are pasture, pond and industrial aquaculture (in pools, in recirculating aquaculture systems, in fish farms using cages), as well as artificial reproduction including sturgeon, salmon hatcheries and spawning farms. For each of these directions the risk of diseases in breeding objects is quite high [1]. Diseases can be associated with a violation of biotechnological, sanitary standards for growing and quarantine measures of imported aquaculture objects. In addition, a high risk of morbidity is caused by the environmental problems due to the unsatisfactory quality of the aquatic environment, especially in cage farms concentrated in coastal zones and in freshwater reservoirs, which are more often exposed to anthropogenic pollution. As a result, the resistance of the fish organism sharply decreases, which causes diseases from the opportunistic bacteria that constantly circulate in the aquatic environment [2, 3]. Bacterial diseases all over the world cause significant economic damage to fish farms, both due to fish death, the need for quarantine measures, and from a decrease in the consumer attractiveness of raw materials and, accordingly, the cost of fish. Bacterial diseases of fish in ponds and farms in the Russian Federation rank second after the parasitic ones among infectious diseases. The most common are aeromonosis of salmon and cyprinid fish species, pseudomonosis
of carp, myxobacteriosis of sturgeon and trout fish species [4, 5]. For the period 2014–2018 80.55% of all detected outbreaks of diseases of bacterial etiology accounted for aeromonoses of cyprinids and salmon fish [4]. It should be noted that cyprinid and salmon aeromonoses are included in the list of especially dangerous quarantine diseases [6]. In addition, many fish diseases (vibriosis, aeromonosis, pseudomonosis, citrobacteriosis) are characterized by natural foci [7]. Certain environmental factors are favorable for the persistence of certain types of pathogens. The most common pathogens for the water bodies of the North Caucasus and the southern region as a whole are aeromonads. It should be noted that bacteria of the genus Aeromonas are constantly present in the aquatic environment, and their epizootic significance is determined by abundance and virulence [8]. Mild climatic conditions contribute to the long-term persistence of virulent aeromonads in the aquatic environment. A major factor for the high incidence of aeromonosis in fish is an increase in water temperature above 14°C. At the same time, the percentage of death (10-100%) depends on the conditions of detention and the load per unit area of the reservoir of each particular farm. Outbreaks of aeromonosis can occur in the form of epizootics, affecting species of all age groups. However, in the acute form, the disease is more often recorded in fry and yearlings of sturgeon, salmon and some species of cyprinids (crucian carp, grass carp, mirror carp) [4]. For the Rostov region, cyprinid aeromonosis is the most dangerous because it spreads not only in pond farms, but also in natural reservoirs. Aeromonosis cases were recorded in the Don, Bolshaya Kalancha, Mius and Aksai rivers. A growing number of aeromonads in natural water bodies, especially strains with pathogenicity factors in the summer–autumn period, increases the risks for the development of infection or colonization of aquatic organisms by these bacteria [8]. Pseudomonas bacteria are the causative agents of pseudomonosis and this disease is rather rarely detected in aquaculture objects in the farms of the southern region. However, recently the incidence of diseases, caused by the associations of gram-negative microorganisms (aeromonads, pseudomonads, enterobacteria, etc.) has increased. In particular, a more severe course of the disease is observed and an acute form of the disease develops more often if the clinical signs of aeromonosis are accompanied by the presence of pseudomonads, enterobacteria, moraxella and other opportunistic bacteria [4]. In this case, it is difficult to choose effective drugs to combat the disease. 
The existing complex of veterinary-sanitary and fish-breeding and reclamation measures cannot fully provide protection against bacterial infections in aquaculture objects. This is largely due to the resistance of pathogens to drugs used in fish farming. The use of probiotics based on spore-forming bacteria of the Bacillus genus is a modern trend in the prevention and treatment of infectious diseases in aquaculture. The presence of probiotic effects in spore-forming microorganisms has led to the development of preparations based on them, belonging to the group of “self-eliminating antagonists”. Probiotic preparations offered on the market differ not only in price, but also in composition, quality, method and dose of application. Bacillus based probiotics are suitable candidates for the development of preparations for use in aquaculture. They exhibit antagonistic activity against pathogenic microorganisms and are also nontoxic to fish [9, 10]. At the same time, the ability of probiotic bacilli to manifest antagonism is a rather variable trait and depends predominantly not on the species, but on the spectrum of antimicrobial metabolites secreted by a particular strain [11]. Therefore, to combat fish diseases, it is necessary to find species-specific bacterial antagonists. In addition, it is important to consider their ability to multiply effectively and exhibit probiotic properties under suboptimal temperature conditions. The effectiveness of the prevention and treatment of bacterial diseases in aquaculture objects is associated with the development of new probiotics, including those based on spore-forming bacteria. This approach will reduce the economic damage from the death of aquaculture objects and reduce the prevalence of antibiotic-resistant strains of pathogens. In this regard, the aim of the work was to search for new natural probiotic Bacillus strains and evaluation of their antagonistic activity against pathogens of bacterial diseases relevant to aquaculture objects (pseudomonosis, aeromonosis, bacterial hemorrhagic septicemia) by the method of co-cultivation.

 

Materials and methods

The object of the study were Bacillus strains isolated from silver carp (Cyprinus gibelio), roach (Rutilus heckelii), bream (Abramis brama) as well as from bottom sediments of the lower reaches of the Don River in the area of the Donskoy Fish Reserve. Inoculation of bacteria from bottom sediments and imprints of samples of fish gills and intestines was carried out on wort-agar. The species were identified by MALDI-TOF MS (matrix-associated laser desorption/ionization time-of-flight mass spectrometry) on an Autoflex speed III device with Biotyper software (Bruker Daltoniss, Germany). For more precise species identification, DNA was isolated from bacterial cells and the 16S rRNA gene was sequenced. The conditions for DNA isolation and purification, as well as for sequencing, are similar to those described in our previous works [12]. The strains with no hemolytic and DNAse activity were included in the study. 7 species of bacteria were used as test cultures. Species of Aeromonas genus - Aeromonas salmonicida (7 strains), Aeromonas veronii (30), Aeromonas caviae (14), Aeromonas eucrenophila (4), Aeromonas ichthiosmia (3), Aeromonas bestiarum (3), Aeromonas hydrophila (5), of which 14 are deposited in collections of microorganisms of Federal State Budgetary Institution “The Russian State Center for Animal Feed and Drug Standardization and Quality” (FGBU “VGNKI”) (Table 1); 3 species of Pseudomonas genus - P. putida (2 strains), P. fluorescens and P. aureofaciens by 1 strain from each were аlso used to assess the antagonistic activity of bacilli.

 

Table 1

Deposited test strains of aeromonads from the collection of microorganisms
of the
FGBU “VGNKI”

Bacterial species
of Aeromonas genus

Registration number at depositing

Fish species / sampling area

Aeromonas caviae

VKSHM-B-297М

Acipenseridae, Seversky district
of the Krasnodar Region

VKSHM-B-300М

Sterlet, Krasnodar

Aeromonas veronii

VKSHM-B-296М

Сyprinids, Dinskoy district
of the Krasnodar Region

VKSHM-B-299М

Cyprinids, Krasnodar

VKSHM-B-305М

Cyprinids, Adygea

VKSHM-B-302М

Cyprinids, Krasnodar

Aeromonas salmonicida

VKSHM-B-295М

Koi carp, Krasnodar

VKSHM-B-293М

Russian sturgeon, village Starominskaya,

the Krasnodar Region

VKSHM-B-307М

Acipenseridae, village Bryukhovetskaya,

the Krasnodar Region

Aeromonas eucrenophila

VKSHM-B-294М

Acipenseridae, village Starominskaya,

the Krasnodar Region

VKSHM-B-298М

Acipenseridae, Korenovsky district
of the Krasnodar Region

Aeromonas iсhthiosmia

VKSHM-B-303М

Cyprinids, Dinskoy district
of the Krasnodar Region

VKSHM-B-304М

Acipenseridae, village Starominskaya

the Krasnodar Region

VKSHM-B-306М

Cyprinids, Shcherbinovsky district
of the Krasnodar Region

 

Strains of Aeromonas and Pseudomonas were isolated from parenchymal organs and skin ulcers of fish with bacterial hemorrhagic septicemia and aeromonosis. The study included fish from 3 families: cyprinids (Cyprinidae) – common carp (Cyprinus carpio), koi carp (Cyprinus carpio haematopterus), silver carp (Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idella); sturgeons (Acipenseridae) – Russian sturgeon (Acipenser gueldenstaedtii), sterlet (Acipenser ruthenus), stellate sturgeon (Acipenser stellatus); salmonids (Salmonidae) – rainbow trout (Salmo gairdneri irideus) and brook trout (Salmo trutta morpha fario) grown in pond farms and recirculating aquaculture system in the Krasnodar Region, Rostov Region, Republic of Adygea. Pathological anatomical autopsy, primary bacteriological inoculation of samples of fish organs and tissues, and isolation of bacteria were carried out in accordance with regulatory documents [13, 14]. To assess the antagonistic activity of Bacillus strains the method of delayed antagonism was used (method of perpendicular streaking) in accordance with MUK 4.2.2602-10. Inoculation of antagonist strains on the rich nutrient medium (nutrient agar is used for cultivating microorganisms) was carried out using a loop with a diameter of (3.5 ± 0.5 mm) with a straight streaking along the diameter of a Petri dish. It was incubated for 3 days, after which a suspension of the overnight test culture (not less than 109 CFU/ml) was inoculated perpendicularly to the grown streak of the antagonist strain using a loop with a diameter of 2 mm. The cultures were incubated for 24 h at a temperature of (26 ± 1°C), followed by measurement of the growth inhibition zone of the test cultures

 

Results and discussion

As a result of the screening of 28 strains, 5 promising strains of Bacillus were selected, of which 3 strains (R1, R4, R5) were isolated from cyprinids of natural populations and 2 strains (G5, G6) from bottom sedimentsThe results of mass spectrometric analysis showed that strains R4, G5, G6 belong to Bacillus subtilis, R1 to Bacillus mojavensis, R5 to Bacillus sp. To clarify the species identification, an analysis of the 16S rRNA gene was carried out, as a result of which strain R5 belonged to the species Bacillus velezensis

It was shown that the level of antagonistic activity of Bacillus strains varies not only in relation to the species of aeromonads, but also in relation to the strains of these species (Table 2).

 

Table 2

Species composition of aeromonad strains sensitive to antagonist bacilli

Strain

Bacillus species

Source of Bacillus strains isolation

Antagonism towards
aeromonad test strains

Proportion of test strains sensitive to antagonist
bacilli, %

R1

B. mojavensis

Intestines of carp fish

A. salmonicida

28

A. eucrenophila

62

R4

B. subtilis

Intestines of carp fish

A. salmonicida

28

A. caviae

100

A. eucrenophila

100

А. hydrophila

80

А. bestiarum

33

R5

B. velezensis

Intestines of carp fish

A. salmonicida

14

A. caviae

100

A. hydrophila

100

G5

B. subtilis

Sediments

A. salmonicida

86

A. eucrenophila

100

G6

B. subtilis

Sediments

A. salmonicida

86

A. caviae

100

A. eucrenophila

100

А. bestiarum

100

А. hydrophila

100

 

Thus, the antagonistic activity of bacilli differed significantly against the strains of immobile aeromonads Aeromonas salmonicida. The most pronounced antagonistic activity was found in bacilli isolated from the bottom sediments (more than 35 mm). Antagonistic bacilli isolated from fish exhibited a strong inhibitory effect only against 2 deposited strains (growth inhibition zone > 35 mm), the remaining 5 test cultures of aeromonads had a weak response (growth inhibition zone from 1 to 3 mm). During co-cultivation of bacilli with motile aeromonads A. eucrenophila, A. bestiarum, and A. ichthiosmia, their growth inhibition zones varied widely. All antagonist bacilli (except B. velezensis R5) showed a good inhibitory effect on A. eucrenophila test cultures (more than 35 mm). In relation to cultures of A. bestiarum, the effect of bacilli differed significantly (zones of growth inhibition from 1 to 35 mm). At the same time, strains of Bacillus subtilis R3 and G6 (more than 35 mm) showed the greatest antagonistic activity, while B. subtilis R4 and B. velezensis R5 inhibited the growth of the tested cultures to a lesser extent (growth inhibition zone 3-5 mm). The weakest antagonistic activity of bacilli was noted against A. ichtiosmia (from 1 to 3 mm)It is known that mobile species A. caviae and A. hydrophila are most often found as causative agents of aeromonosis and bacterial hemorrhagic septicemia. The inhibitory effect of bacilli to 5 cultures of A. hydrophila and 14 test cultures of A. caviae showed significant differences in the severity of antagonism (growth inhibition zones from 1 to 35 mm). However, the most effective antagonists were bacterial Bacillus strains R3, R4, R5, G6. The inhibitory effect of 4 strains of antagonist bacilli against pathogenic for fish Pseudomonas species was studied. 3 species of Pseudomonas (P. putidaP. fluorescens, P. aureofaciens) isolated from fish with bacterial hemorrhagic septicemia were used as test cultures. It was found that most of the tested antagonist bacilli (B. mojavensis R1, B. velezensis R5) did not show a pronounced inhibitory effect on P. fluorescens and P. aureofaciens species. The growth of Pseudomonas test cultures was suppressed only by B. subtilis R4. Although P. putida strains differed in a number
of biochemical properties and were isolated from different fish species (trout and Russian sturgeon), the antagonistic effect of bacilli against these strains was identical. B. subtilis R4 and Bacillus velezensis R5 strains suppressed the growth of P. putida test cultures. The results of the antagonistic activity of bacilli against Pseudomonas are presented in Table 3.

  Table 3

Evaluation of the antagonistic activity of Bacillus bacteria against strains of Pseudomonas species pathogenic for fish

Test strains of pseudomonads

 

Bacteria Bacillus

Zone of Pseudomonas bacteria growth inhibition, mm

P. putida 100

P. putida 101

P. fluorescens 75

P. aureofaciens 107

R1 B. mojavensis

absent*

absent

not defined

absent

R4 B. subtilis

10

10

more than 40

5

R5 Bacillus velezensis

over 40

over 40

20

absent

 

*Zone of growth inhibition is absent.

 

The genus Bacillus is one of the most commonly used genera of probiotics in aquaculture due to its ability to produce bacteriocins, influence the growth rates, host immune system and resistance to pathogens [15].

It has been shown that in vivo conditions B. subtilis, B. velezensis, Bacillus amyloliquefaciens, Bacillus circulans, Bacillus thuringiensis and Bacillus aerius increase the resistance of aquaculture objects to pathogenic bacteria, including Streptococcus, Aeromonas, Vibrio, Enterococcus and Lactococcus [16]. Representatives of the genus Bacillus are a promising group for searching the new bacteria that inhibit quorum sensing in pathogens [17]. In addition, probiotics based on bacteria of the genus Bacillus, in particular B. subtilis and B. licheniformis, can be used to purify water and bottom sediments in fish ponds by removing toxic substances, such as ammonia, nitrites, nitrates and carbon dioxide, as well as by competing with opportunistic bacterial species [15, 18]. Bacillus strains also have a beneficial effect on the microbiome of the gastrointestinal tract of aquaculture objects, which leads to improved feed conversion and accelerates the growth of aquatic organisms [16]. Their mechanisms of action, however, are still the subject of active discussion and study. Probiotic strains of bacilli affect the process of intestinal colonization by other types of microorganisms, including pathogens, due to the mechanisms of both direct competition and influence on the adhesion process [15, 16]. Among the specific properties of bacillary probiotics, the immunostimulating properties are also worth mentioning. Thus, it was demonstrated that bacilli can modulate the mechanisms of the innate immune response of fish and molluscs, for example, phagocytic and lysozyme, antiprotease and peroxidase, superoxide dismutase and myeloperoxidase activities, and respiratory burst [15, 19]. In addition, probiotics based on Bacillus strains can cause changes in animal cell physiology, in particular, affect neutrophil migration, plasma bactericidal activity and increase the ability of neutrophils to attach, which ultimately can lead to an improvement in the immune response, for example, an increase in complement activity, immunoglobulin production in the gut-associated lymphoid fish tissues [16, 19–21]Spore-forming bacteria have the ability to synthesize a wide range of compounds that inhibit the growth of other microorganisms. In particular, B. velezensis was shown to be capable of synthesizing bacilizin, which shows activity against opportunistic Gram-negative bacteria [22]. The ability to synthesize a wide range of biologically active compounds, including peptides, lipopeptides, siderophores, etc., makes it possible to effectively use probiotics based on Bacillus strains against bacteria pathogenic for aquaculture objects, such as representatives of Aeromonas, Vibrio, Pseudomonas, and others [23]. In particular, it has recently been shown that the B. coagulans strain had a pronounced antagonistic effect against P. aeruginosa isolated from diseased carps and also reduced the death of fish in the in vivo experiment [24]. Similar data were obtained for B. velezensis WLY23 which effectively suppressed 20 fish pathogenic bacteria, including A. schubertii, A. jandaei, A. hydrophila, A. veronii, A. aquariorum, P. shigelloides, N. seriolae, S. agalactiae and S. iniae [25]. It should be noted that in our study, the B. velezensis R5 strain was also characterized by the widest spectrum of antimicrobial activity.

 

Conclusion

Thus, the conducted studies make it possible to identify a group of natural strains of bacilli with antagonistic activity against aeromonads and pseudomonads pathogenic for fish, which may be promising for developing the probiotic preparations for aquaculture. It has been found that sensitivity to the antagonistic action of Bacillus is not only a species, but also a strain trait among bacterial pathogens of fish. The strains selected in the course of this work can be used to develop drugs that are active against pathogens which are of current importance for aquaculture.

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