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A study of the microbiology of the intestinal tract in different species of Teleost fish from the Black Sea
expand article infoSevginar Ibryamova, Stephany Toschkova, Borislava Pavlova, Elitca Stanachkova, Radoslav Ivanov, Nikolay Natchev, Nesho Chipev§, Tsveteslava Ignatova-Ivanova
‡ Shumen University, Shumen, Bulgaria
§ Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bulgaria
Open Access

Abstract

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.

Keywords

marine fish, microbial status, intestinal tract

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 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.

Collection of the samples

The fish individuals were randomly sampled from trawl catches using pelagic Midwater otter trawl (7 × 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).

Microbial Identification Databases for the “BIOLOG” System

The microbial identification was performed by the BIOLOG Microbial Identification System VIO45101AM. The isolated strains were screened on BL4021502 Tryptic 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 bacteria. The 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 provides 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 identify the bacterium from its phenotypic pattern in the GEN III MicroPlate. The BIOLOG 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 addition 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 microplate. 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 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.

Table 1.

Number of cells in 1 ml on the different media.

Media/ fish Pseudomonas agar Streptococcus selective agar Chromokult agar MacConkey agar strain BIOLOG
T26F1 Scophthalmus maximus –turbot–Varna 24.103 Enterococcus villorum
Scophthalmus maximus – turbot – Varna 70.103 Moraxella nonliquefaciens
Scophthalmus maximus – turbot – Varna 123.103 Pseudomonas synxantha
T26F3 Neogobius melanostomus – round goby – Varna 20.103 Pseudomonas putida
20T29-30F7 Platichthys flesus – shore rockling – Varna 123.103 Streptococcus entericus
Engraulis encrasicolus – European anchovy Sozopol 60.103 Pseudomonas fulva
20T10-11F2 Trachinus draco – greater wever – Varna 223.103 Pseudomonas agarici
20T10-11F3 Merlangius merlangus – whiting–Varna 145.103 Pseudomonas 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 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.

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 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.

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 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.

Acknowledgements

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.

References

  • FAO (2007) Social issues in small-scale fisheries: how a human rights perspective can contribute to respective fisheries in Proceedings of the Presentation to FAO Committee of Fisheries No 27, Rome, Italy.
  • Fengou LC, Lianou A, Tsakanikas P, Gkana EN, Panagou EZ, Nychas GJE (2019) Evaluation of Fourier transform infrared spectroscopy and multispectral imaging as means of estimating the microbiological spoilage of farmed sea bream. Food Microbiology 79: 27–34. https://doi.org/10.1016/j.fm.2018.10.020
  • Ignatova-Ivanova TS, Ibrjmova S, Stanachkova E, Ivanov R (2018) Microbiological characteristic of microflora of (Mytilus galloprovincialis Lam.) in the Bulgarian Black Sea aquatory. Research Journal of Pharmaceutical, Biological and Chemical Sciences 9(1): 199–205. https://www.rjpbcs.com/pdf/2018_9(1)/[29].pdf
  • Jorquera MA, Silva FR, Riquelme CE (2001) Bacteria in the culture of the scallop Argopecten purpuratus (Lamarck, 1819). Aquaculture International 9(4): 285–303. https://doi.org/10.1023/A:1020449324456
  • Levy SB, Marshall B (2004) Antibacterial resistance worldwide: Causes, challenges and responses. Nature Medicine 10(S12): S122–S129. https://doi.org/10.1038/nm1145
  • Llewellyn MS, McGinnity P, Dionne M, Letourneau J, Thonier F, Carvalho GR, Creer S, Derome N (2016) The biogeography of the Atlantic salmon (Salmo salar) gut microbiota. The ISME Journal 10(5): 1280–1284. https://doi.org/10.1038/ismej.2015.189
  • Llewellyn MS, Leadbeater S, Garcia C, Sylvain FE, Custodio M, Ang KP, Powell F, Carvalho GR, Creer S, Elliot J, Derome N (2017) Parasitism perturbs the mucosal microbiome of Atlantic Salmon. Scientific Reports 7(1): 43465. https://doi.org/10.1038/srep43465
  • Maffei M, Vernocchi P, Lanciotti R, Guerzoni ME, Belletti N, Gardini F (2009) Depuration of Striped Venus Clam (Chamelea gallina L.): Effects on microorganisms. sand content and mortality. Journal of Food Science 74(1): M1–M7. https://doi.org/10.1111/j.1750-3841.2008.00971.x
  • Manhondo P, Gono RK, Muzondiwa J, Mafa B (2018) Isolation and identification of pathogenic bacteria in edible fish: A case study of bacterial resistance to antibiotics at Lake Chivero, Zimbabwe. African Journal of Agricultural Research.
  • Miyake S, Ngugi DK, Stingl U (2015) Diet strongly influences the gut microbiota of surgeon-fishes. Molecular Ecology 24(3): 656–672. https://doi.org/10.1111/mec.13050
  • Orozova P, Chikova V, Najdenski H (2010) Antibiotic resistance of pathogenic for fish isolates of Aeromonas spp. Bulgarian Journal of Agricultural Science 16(3): 376–386. https://www.agrojournal.org/16/03-17-10.pdf
  • Özogul Y, Özogul F, Kuley E, Özkutuk AS, Gökbulut C, Köse S (2006) Biochemical, sensory and microbiological attributes of wild turbot (Scophthalmus maximus), from the Black Sea, during chilled storage 2006. Food Chemistry 99(4): 752–758. https://doi.org/10.1016/j.foodchem.2005.08.053
  • Pérez T, Balcázar JL, Ruiz-Zarzuela I, Halaihel N, Vendrell D, de Bla I, Múzquiz JL (2010) Host-microbiota interactions within the fish intestinal ecosystem. Mucosal Immunology 3(4): 355–360. https://doi.org/10.1038/mi.2010.12
  • Stephens WZ, Burns AR, Stagaman K, Wong S, Rawls JF, Guillemin K, Bohannan BJ (2016) The composition of the zebrafish intestinal microbial community varies across development. The ISME Journal 10(3): 644–654. https://doi.org/10.1038/ismej.2015.140
  • Stratev D, Vashin I, Daskalov H (2015) Microbiological status of fish products on retail markets in the Republic of Bulgaria. International Food Research Journal 22(1): 64–69.
  • Stratevaa M, Pencheva G, Stratev D (2021) Histological, physicochemical and microbiological changes in the carp (Cyprinus carpio) muscles after freezing. Journal of Aquatic Food Product Technology 30(3): 323–337. https://doi.org/10.1080/10498850.2021.1882633
  • Sylvain FE, Derome N (2017) Vertically and horizontally transmitted microbial symbionts shape the gut microbiota ontogenesis of a skin-mucus feeding discus 646 fish progeny. Scientific Reports 7(1): 14. https://doi.org/10.1038/s41598-017-05662-w
  • Sylvain FÉ, Cheaib B, Llewellyn M, Gabriel Correia T, Barros Fagundes D, Luis Val A, Derome N (2016) pH drop impacts differentially skin and gut microbiota of the Amazonian fish tambaqui (Colossoma macropomum). Scientific Reports 6: 64310. https://doi.org/10.1038/srep32032
  • Sylvain FÉ, Holland A, Bouslama S, Audet-Gilbert É, Lavoie C, Val AL, Derome N (2020) Fish skin and gut microbiomes show contrasting signatures of host species and habitat. Applied and Environmental Microbiology 86(16): e00789–e20. https://doi.org/10.1128/AEM.00789-20
  • Velioğlu HM, Temiz HT, Boyaci IH (2015) Differentiation of fresh and frozen-thawed fish samples using Raman spectroscopy coupled with chemometric analysis. Food Chemistry 172: 283–290. https://doi.org/10.1016/j.foodchem.2014.09.073
  • Zotta T, Parente E, Ianniello RG, De Filippis F, Ricciardi A (2019) Dynamics of bacterial communities and interaction networks in thawed fish fillets during chilled storage in air. International Journal of Food Microbiology 293: 102–113. https://doi.org/10.1016/j.ijfoodmicro.2019.01.008