This paper presents a study on the microbial status of different fish species and their habitats in the Bulgarian 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, different species of microorganisms were isolated (using selective nutrient media). From the torbut, we isolated species Enterococcus villorum with 24 × 10³ cells in 1 ml, Moraxella nonliquefaciens with 70 × 10³ cells in 1 ml and Pseudomonas synxantha with 123 × 10³ cells. Pseudomonas putida was isolated from the round goby with 20 × 10³ cells in 1 ml. The species Streptococcus entericus with 123 × 10³ cells in 1 ml was isolated from the shore rockling. Pseudomonas fulva with 60 × 10³ cells in 1 ml was isolated from the European anchovy. A total of 223 × 10³ cells in 1 ml of Pseudomonas agarici were isolated from Trachinus draco. Pseudomonas tolaasii with 145 × 10³ 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 pollution or environmental change.

marine fish, microbial status, intestinal tract

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 consumed 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 indicate 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 resistant 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 Aeromonasspp. were found in the Black Sea horse mackerel (2.68 log cfu/g). Aeromonasspp. 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 (Velioğlu 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, Bulgaria, 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).

Figure 1.

Sampling locations at the Black sea coast.

Using some classical microbiological methods, we investigated probes from the gastrointestinal tract of different species of fish from the Bulgarian Black Sea aquatory. 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.

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 stereomicroscope OPTIKA (Italy) and DinoEye, Eyepiece camera, USB, 1.3 megapixsel, up to 5 megapixels.

For the present study, fresh Black Sea fish were used, which were dissected in laboratory 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) – E. villorum species (Fig. 2a) were isolated from S. maximus with 24 × 10³ cells in 1 ml, M. nonliquefaciens with 70 × 10³ cells in 1 ml and P. synxantha with 123 × 10³ cells in 1 ml. The species P. putida was isolated from the Round goby with 20 × 10³ cells in 1 ml. The species S. entericus with 123 × 10³ cells in 1 ml was isolated from P. flesus. P. fulva with 60 × 10³ cells in 1 ml was isolated from Sardine. A total of 223 × 10³ cells in 1 ml of P. agarici (Fig. 2b) were isolated from T. draco and P. tolaasii with 145 × 10³ cells in 1 ml were isolated from M. merlangus.

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 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 (Özogul 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), immunological condition (Pérez et al. 2010), but also lifestyle (Stephens et al. 2016). Exogenous 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 microbiota 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-microbial 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.

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 life and metabolism of these vertebrates. These microorganisms are probably resident species and have developed not as result of pollution or environmental change. The presence of the species Enterococcus villorum and Moraxella nonliquefaciens, which are pathogenic species, may be due to pollutants of seawater, which settled in the sediment and infested the turbot in its natural habitat.

This study was financially supported by the National Research Fund of the Bulgarian Ministry of Education and Science (Grant – KP-06-PH41/2 28.09.2020) and the project by Shumen University project 08-80 / 09.02.2022 Department of Biology.