BioRisk 18: 105-1 13 (2022) Apeer-reviewed open-access journal BO ae Gan & BioRisk https://biorisk.pensoft.net A study of the microbiology of the intestinal tract in different species of Teleost fish from the Black Sea Sevginar Ibryamova', Stephany Toschkova', Borislava Pavlova', Elitca Stanachkova', Radoslav Ivanov', Nikolay Natchev', Nesho Chipev’, Tsveteslava Ignatova-Ivanova' | Department of Biology, Shumen University, Shumen, Bulgaria, Shumen University “Konstantin Preslavski” Department of Biology 115 Universitetska Str, Shumen, Bulgaria 2 Laboratory of Free Radical Processes, Institute of Neurobiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str. 23, Sofia, Bulgaria Corresponding author: Tsveteslava Ignatova-Ivanova (ts.ignatovaivanova@shu.bg) Academic editor: Josef Settele | Received 10 January 2022 | Accepted 15 July 2022 | Published 4 August 2022 Citation: Ibryamova S, Toschkova S, Pavlova B, Stanachkova E, Ivanov R, Natchev N, Chipev N, Ignatova-Ivanova T (2022) A study of the microbiology of the intestinal tract in different species of Teleost fish from the Black Sea. BioRisk 18: 105-113. https://doi.org/10.3897/biorisk. 18.80357 Abstract This paper presents a study on the microbial status of different fish species and their habitats in the Bul- garian Black Sea area. The samples were collected in the period of January 2021 until March 2021. The fish species we used in this study were Black Sea turbot (Scophthalmus maximus), round goby (Neogobius melanostomus), shore rockling (Gaidropsarus mediterraneus) and European anchovy (Engraulis encrasicolus). The BIOLOG system was used for microbiological determination. From the different fish species, differ- ent species of microorganisms were isolated (using selective nutrient media). From the torbut, we isolated species Enterococcus villorum with 24 x 10° cells in 1 ml, Moraxella nonliquefaciens with 70 x 10° cells in 1 ml and Pseudomonas synxantha with 123 x 10° cells. Pseudomonas putida was isolated from the round goby with 20 x 10° cells in 1 ml. The species Streptococcus entericus with 123 x 10° cells in 1 ml was isolated from the shore rockling. Pseudomonas fulva with 60 x 10° cells in 1 ml was isolated from the European anchovy. A total of 223 x 10° cells in 1 ml of Pseudomonas agarici were isolated from Trachinus draco. Pseudomonas tolaasii with 145 x 10° cells in 1 ml were isolated from Merlangius merlangus. A different species of bacteria of the genus Pseudomonas was found for each of the investigated species of Black Sea fish. Apparently, the species Pseudomonas is characteristic of marine Teleostei and is important for the life and metabolism of these vertebrates. These microorganisms probably are resident species and developed not as result of pol- lution or environmental change. Keywords marine fish, microbial status, intestinal tract Copyright Sevginar Ibryamova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 106 Sevginar Ibryamova et al. / BioRisk 18: 105-113 (2022) Introduction Fish are consumed in large quantities throughout the world and are considered one of the main sources of protein. According to FAO (2007), fish are the most important single source of high quality protein providing about 16% of the animal protein con- sumed by the world’s human population. Fish have high nutritional values and are a good source of saturated fats, omega-3 fatty acids that cannot be synthesised by the human body. Fish are known to be low in fat and cholesterol and are easily digested and suitable for infants, children and the elderly. Unlike other local products, the price of fish is much lower and more affordable for most people. The microbiology of fish is closely related to human health because it can represent a risk depository, as fish carry inside and on their surface different types of microorganisms. On the other hand, the microbiology of fish is closely dependent on the state of the environment and can be used as an indicator of the water quality. Several bacterial genera, such as Escherichia, Listeria, Pseudomonas, Klebsiella and Salmonella were isolated from fish and can indi- cate multisource pollution (Sichewo et al. 2014; Manhondo et al. 2018). Banquero et al. (2008) described aquatic systems as genetic reactors or hotspots for AMR genes where significant genetic exchange and recombination can occur and can shape the evolution of future resistance profiles. Bacteria very quickly acquire resistance either through gene transfer or through mutations. In this way, antibiotic resistance genes can also be passed on. These processes occur mainly in the intestinal tract of fish (Levy and Marshall 2004). Random use of antimicrobials is associated with increased selection pressure on bacterial populations and favours the survival and reproduction of resist- ant bacteria. In Bulgaria, Stratev et al. (2015) conducted a study of 161 samples of frozen fish products collected from retail markets. High numbers of total viable counts (TVCs) were found in Black Sea roach (7.28 log cfu/g) and horse mackerel (5.90 log cfu/g). High amounts of Aeromonas spp. were found in the Black Sea horse mackerel (2.68 log cfu/g). Aeromonas spp. was not found in the Black Sea roach. After their death, fish undergo rapid bacterial and microbiological changes, which are determining factors in their consumption by humans (Stratevaa et al. 2021). The expiry period of fish is determined by the content of protein, fat, water (Velioglu et al. 2015) and the amount of microorganisms (Fengou et al. 2019). Fresh fish have a high water quantity and this may be the reason for their rapid spoilage (Zotta et al. 2019). To date, data on the microbiota of fish in the Black Sea are very limited. Gaining information in this field is important as the sea can be a source of species of bacteria that are potentially dangerous to human health. The main goal of the present work was to study the microbiological composition of the intestinal tract in different species of Teleost fish from the Black Sea. Place and duration of the study The study was conducted at the Department of Biology, University of Shumen, Bul- garia, January 2021 until March 2021. The samples were collected from the regions of Varna: 43.18 N, 27.91 E and Sozopol: 42.4317N, 27.70 E (Datum WGS 84) (Fig. 1). A study of the microbiology of fish from the Black Sea 107 Collection of the samples The fish individuals were randomly sampled from trawl catches using pelagic Midwa- ter otter trawl (7 x 7 mm mesh size of the codend) from two localities of the Bulgarian Black Sea aquatory (Fig. 1). On board, the fish samples were shock frozen for best preservation and transported to the laboratory for further analyses. Microbiological analysis The fish were scrubbed free of dirt, washed in hypochlorite solution (20 mg/l), rinsed with sterile distilled water and shucked with a sterile knife. Tissue liquor samples (about 100 g) were homogenised (Maffei et al. 2009). Faecal coliforms (FC) were enumerated through a five tubes per dilution most probable number (MPN) series (Ignatova-Ivanova et al. 2018). After 3 h at 37 °C plus 21 h at 44 °C, gas positive tubes were recorded for FC. From each FC gas positive tube, 0.1 ml were transferred in tubes with 10 ml of Tryptone Water (Oxoid, Basingstoke, UK) and then incubated for 24 h at 44 °C. E. coli were enumerated by MacConkey agar (Merck, Darmstadt, Germany). The plates very incubated aerobically at 35-37 °C for 18—24 hours. E. coli developed matte dark pink to tile red, surrounded by an opaque area due to the precipitation of bile salts in this environment. Pseudomonas sp. were enumerated by Cetrimide Agar (Merck KGaA, 64271 Darmstadt, Germany). Romania Black sea Bulgaria Turkey Figure |. Sampling locations at the Black sea coast. 108 Sevginar Ibryamova et al. / BioRisk 18: 105-113 (2022) Microbial Identification Databases for the “BIOLOG” System The microbial identification was performed by the BIOLOG Microbial Identifica- tion System VIO45101AM. The isolated strains were screened on BL4021502 Tryp- tic Soy Agar (TCA), cultured for 24 hours at 37 °C and then subjected to Gen III plaque identification to identify Gram positive and Gram negative aerobic bacte- ria. Ihe microscopic pictures were performed using the stereomicroscope OPTIKA (Italy) with a DinoEye, Eyepiece camera with 5 megapixels. The photographs were taken by using a Canon EOS 60D camera. The GEN III MicroPlate test panel pro- vides a standardised micromethod using 94 biochemical tests to profile and identify a broad range of Gram-negative and Gram-positive bacteria. BIOLOG’s Microbial Identification Systems software (e.g. OmniLog Data Collection) is used to iden- tify the bacterium from its phenotypic pattern in the GEN III MicroPlate. The BI- OLOG system allows us to quickly and accurately identify more than 2900 species of aerobic and anaerobic bacteria, yeasts and fungi. BIOLOG’s advanced phenotypic technology provides valuable information on the properties of the strains, in ad- dition to species-level identification. BIOLOG’s carbon technology identifies the environment and pathogenic microorganisms by producing a characteristic pattern or “metabolic fingerprint” of discrete test reactions performed in a 96-well micro- plate. The culture suspensions are tested with a panel of pre-selected assays, then incubated, read and compared with extensive data-bases. https://www.biolog.com/ productsportfolio-overview/microbial-identification. Results Using some classical microbiological methods, we investigated probes from the gastrointestinal tract of different species of fish from the Bulgarian Black Sea aqua- tory. After 24 h of cultivation on different media, the number of cells in 1 ml were obtained. The species of microorganisms were confirmed not only on selective media, but also by the results of the BIOLOG system. Data are represented in Table 1 and Fig. 2. For the present study, fresh Black Sea fish were used, which were dissected in labo- ratory conditions. The intestinal tract was used for microbiological analysis. Different species of microorganisms were isolated from the fish species using selective nutrient media (Table 1) — £. villorum species (Fig. 2a) were isolated from S. maximus with 24 x 10° cells in 1 ml, M. nonliquefaciens with 70 x 10° cells in 1 ml and P synxantha with 123 x 10° cells in 1 ml. The species P putida was isolated from the Round goby with 20 x 10° cells in 1 ml. The species S. entericus with 123 x 10° cells in 1 ml was isolated from P. flesus. P. fulva with 60 x 10° cells in 1 ml was isolated from Sardine. A total of 223 x 10° cells in 1 ml of P agarici (Fig. 2b) were isolated from 7’ draco and P. tolaasii with 145 x 10° cells in 1 ml were isolated from M. merlangus. A study of the microbiology of fish from the Black Sea 109 Table |. Number of cells in 1 ml on the different media. Media/ fish Pseudomonas Streptococcus Chromokult MacConkey strain agar selective agar agar agar BIOLOG T26F1 Scophthalmus maximus — 24.10° Enterococcus turbot—Varna villorum Scophthalmus maximus — turbot 70.10° Moraxella — Varna nonliquefaciens Scophthalmus maximus — turbot 123.10° Pseudomonas — Varna synxantha T26F3 Neogobius melanostomus — 20.10° Pseudomonas round goby — Varna putida 20129-30F7 Platichthys flesus — 123.10° Streptococcus shore rockling — Varna entericus Engraulis encrasicolus — European 60.10° Pseudomonas anchovy Sozopol fulva 20T10-11F2 Trachinus draco — 223.10° Pseudomonas greater wever — Varna agarici 20T10-11F3 Merlangius merlangus 145.10° Pseudomonas — whiting—Varna tolaasii Figure 2. Stereomicroscope picture of the colonies of the isolated species in this case a E. villorum on media Chromocult agar and b P agarici on media Pseudomonas agar. The picture was taken using the ster- eomicroscope OPTIKA (Italy) and DinoEye, Eyepiece camera, USB, 1.3 megapixsel, up to 5 megapixels. Discussion The probable explanation for the difference in the species of microorganisms that were isolated from the intestine of the different fish species indicated that this may be due to the different habitats of fishes and their diet. In the winter months, the round goby lives at a depth of less than 60 m. The turbot is a predator and is found at a depth of 80 m buried in the sand. The flounder inhabits thin bottom layers at a depth of 50 m and feeds on crustaceans, worms and molluscs. The shore rockling feeds on “worms” and crustaceans, while the anchovy feed on plankton. According to Jorquera et al. (2001), some of the bacterial populations that inhabit the intestinal tract of fish include species 110 Sevginar Ibryamova et al. / BioRisk 18: 105-113 (2022) such as Vibrio, Pseudomonas, Acinetobacter, Photobacterium, Moraxella, Aeromonas, Micrococcus and Bacillus. The bacterial species that are part of the intestinal tract of fish depend, on the one hand, on the diet of the fish and, on the other hand, on the state of the marine environment. The microbiota found in marine organisms can be considered in two aspects — the so-called “resident” microbiota, which is stable and unaffected by the environment and the “transitional” microbiota, which depend on environmental conditions. ‘There are very little data in literature on the microbiological composition of the intestine of Black Sea fish. Some data were recently published (Orozova et al. 2010; Stratev et al. 2015; Stratevaa et al. 2021) and showed that these are mainly representatives of Aeromonas spp., which cause fish diseases, such as haemorrhagic septicaemia and ulcers. This may lead to large economic losses in fish farms. To date, mostly freshwater fish were studied, such as carp, trout and silver carp. A study on turbot conducted in Turkey showed that the total number of microorganisms isolated on day 0 was 3.3 log cfu/g, but no concrete species were identified (Ozogul et al. 2006). There are no data on the exact species composition of microorganisms in the intestinal tract of Black Sea fish to date. The microbiota of fish may be influenced by many factors, both endogenous and exogenous. Endogenous factors include the origin of the fish host (Miyake et al. 2015), genotype and diet (Wu et al. 2012), parasitic load (Llewellyn et al. 2017), immuno- logical condition (Pérez et al. 2010), but also lifestyle (Stephens et al. 2016). Exoge- nous factors are habitat specific and include: environment condition, physicochemical parameters of water (Llewellyn et al. 2016; Sylvain et al. 2016) and bacterioplankton composition (Sylvain et al. 2017). Usually in fish intestines, microorganisms play a key role in nutrition, facilitating the breakdown and absorption of specific compounds present in the diet of the host fish. According to data published very recently (Sylvain et al. 2020), the diverse mi- crobiota community in fish skin mucus is suitable for the development of microbial biomarkers indicating the conditions of the environment, while the more stable and persistent intestinal microbiome is more suitable for studying long-term host-micro- bial interactions. The different fish species and their habitats showed differences in the taxonomic structure of the microbial community and this was confirmed for all studied species. Conclusion The question whether host-related microbiomes are primarily species-specific or depend on environmental factors still remains open. Our study is one of the few to focus on the intestinal microbiome of Black Sea fish species. It is interesting to note that the phylogenetic structure of the microbial communities of different fish is formed by species and habitat-specific factors. A different species of bacteria of the genus Pseudomonas was found for each of the investigated species of Black Sea fish. Apparently, Pseudomonas species are characteristic of marine Teleostei and are important for the A study of the microbiology of fish from the Black Sea ia life and metabolism of these vertebrates. 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