SPECIFIC MICROBIOLOGY SPECTRUM OF WHITE SAND MUSSELS FROM KEY SAMPLE SPOTS FROM BULGARIAN BLACK SEA AQUATORY

The “white sand mussels” are edible bivalves inhabiting the littoral shores usually buried in the sand. They are invasive species for the Bulgarian waters of the Black Sea. The samples for this study were collected from different points on the northern and southern Black Sea Bulgarian coast in the period January 2020 to December 2020. The study of different types of microorganisms was performed by using the microbial identification system model: MicroLog M® BIO45101 BiologInc and the software product GEN III. The physic-chemical parameters of the waters – temperature, pH, salinity and dissolved O2 were also determined. In the different species, we had detected specific microbiological complexes. The species Pseudomonas viridilivida and Citrobacter farmer were isolated only from Donax trunculus. The species Escherichia hermannii was found only in Mya arenaria, and Acinetobacter johnsonii was detected only in Chamelea gallina. The isolated species Acinetobacter gyllenbergii and Acinetobacter johnsonii are related to humans and are indicators for pollution of the water with channel waste waters. Our results demonstrated an increase of the quantity of the coliforms in the region of Sveti Vlas from August, where they were 50 the norms. In the region of Arkutino in July and Ahtopol in August, the quantity of the fecal coliforms is 190 and 30 times the norms prescribed in the Ordinance No. 4 from 20.10.2000 for the quality of fisheries water and the breeding of shellfish (the amount of fecal coliforms in the intershell content should be less than 300 NVB). We noticed also a serious pollution of the Varna lake even months after an accident with a leaky pipe.


INTRODUCTION
Òhe World Nutrition Organization recognized as edible species of the white sand mussels such as Cardium, Pecten, Tapes, Donax, Mactra, Chamelea and others, which are also found in the Black Sea. The term "white sand mussel "refers to four species of bivalve mollusks: Chamelea gallina, Donax trunculus, Mya arenaria and Anadara kagoshimensis (National Marine Fisheries Service, 2003) [1]. Their main habitats along the Bulgarian Black Sea coast are the sublittoral sands from 0.5 to 15-20 m depth. The "white sand mussel "M. arenaria L. 1758 is a bivalve shellfish found along the Danish coast, Mediterranean, Black and Azov seas. It inhabits littoral shores, usually buried in the sand (the name "arenaria" means sandy). It has a smooth, dirty-white to the yellowish shell, which is rather fragile. It grows to 130 mm in length and is, in fact, allochton/invasive species for the Bulgarian Black Sea coast [2]. In countries like France, Italy and Spain, the species is known under the name "telline" and is widely used as food. A very similar shellfish in Australia is locally known as "Pippies". The name Chamelea of the third species of white mussel was introduced in 1952. From 2016, the two known subspecies-the Mediterranean (C. g. gallina) and the Atlantic (C. g. striatula), were united in one species [3].
The species is often found on East Atlantic coast, Mediterranean and Black Sea. It is very typical for Adriatic Sea. It has a whitish, creamy or pale yellow color and has three characteristic reddish-brown stripes [3]. The habitat of C. gallina is below the surface of the sand, at a depth of five to twenty meters. It serves as a filter of the sea and is collecting and storing bacteria, microalgae and small particles of detritus. According to Regulation No. 1967No. /(2006 of the European Union [4], trade in mussels with a length of less than 25 millimeters is prohibited. The full list of prohibited marine species is available as an annex to the Habitats Directive. The "white sand mussels" are considered a delicacy in many countries and are consumed especially during the summer season. In recent years, the mass production of white sand mussels has started on the Bulgarian coast, which has led to a sharp decrease in its population. To date, there is no literature on the microbiology of sand mussels in the Bulgarian Black Sea aquatory. In the Bulgarian section of the Black sea can be found three other mussels which are already an object of consummation: the Black mussel (Mytilus galloprovincialis), the Striped venus clam (C. gallina) and the Wedge clam (D.x trunculus) [5].
The present work is reporting on our results concerning the microbiology and habitat specifics in the "white sand mussel" from the species M. arenaria, C. gallina and D. trunculus.

Collection of the samples
The mussels were harvested from the Bulgarian Black Sea aquatory. After collection, the samples (about 2 kg) were immediately refrigerated (4°C) and transported to the laboratory for the further analyses.
In this study, we examined mussels of similar size, weight, and shape to ensure maximal uniformity in the applied methods [6]. The average length of mussels used in the study was 2.2 ± 0.43 cm.
Physico-chemical analysis of the inhabited waters During the mussel sampling, we measured in situ the temperature, total salinity (by using YSI Model 33 salinity meter), and pH (by using ATC Piccolo HI1280 pHmeter).

Microbiological analysis
Three subsamples (each of about 1 kg of mussels) were used for the microbiological analyses. The mussels were scrubbed free of dirt, washed in hypochlorite solution (20 mg l-1), rinsed with sterile distilled water, and shucked with a sterile knife. Tissue liquor samples (about 100 g) were homogenized [7]. Fecal coliforms were enumerated through a five tubes per dilution most probable number (MPN) series [8]. After 3 h at 37°C plus 21 h at 44°C, gas positive tubes were recorded for F.C. From each F.C. gas positive tubes, 0.1 ml were transferred in tubes with 10 ml of Tryptone Water (Oxoid, Basingstoke, U.K.) and then incubated for 24 h at 44 °C. E. coli was enumer-

Microbial Identification Databases for the "Biolog" Systems
The microbial identification was performed by a manual microbial identification system Biologist 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 stereomicroscope OPTIKA (Italy) with a DinoEye, Eyepiece camera with 5 megapixels. The photographs were performed by using a Canon EOS 60D camera.

RESULTS
The microorganisms were isolated from M. arenaria, C. gallina and D. trunculus collected from two sample sites of North Bulgarian Black Sea aquatory. After 24 h of cultivation on different media, various microbial colonies were obtained. Data are represented on Table 1, Figures 2 and 3.  Figs. 3 B and C). The peak of E. cancerogenus was observed in January in the region of Sozopol. E. coli in largest quantities was detected in August in the region of Sveti Vlas; E. vulneris peaked in July in the Arkutino area; E. hermannii reached its peak in September and was found only in the species M. arenaria from the area of Varna Lake (indicating the presence of particular environmental conditions, which influenced the quality of the mussels harvested in that month). High amounts of Pseudomonas viridilivida and P. alcaligenes were found in March in the region of Sveti Vlas and in November and December in the region of Ahtopol. Probably this species develops at lower water temperatures. P. viridilivida was also reported in the region of Varna in June. Pseudomonas sp. were isolated only from D. trunculus. Citrobacter farmer discovered in September in the Arkutino area belongs to the family Enterobacteriaceae. This species is also found only in D. trunculus. Acinetobacter gyllenbergii and A. johnsonii were found in the autumn months of September and October. A. gyllenbergii is characteristic for the species D. trunculus, and the species A. johnsonii is characteristic for C. gallina.  Fecal indicator bacteria. i.e. total coliforms, fecal coliforms (thermo-tolerant coliforms) and intestinal enterococci (fecal streptococci) are excreted by humans, and warm-blooded animals pass through the sewage treatment plants and survive for a certain time in the aquatic environment [14]. The coliform bacteria differ considerably in their pathogenic properties. Aside from the intestines of vertebrates and invertebrates, they can also be present in the soil. Total coliforms indicate water pollution, but this does not have to directly correlate with an anthropogenic source, while fecal coliforms are used to indicate sanitary pollution. The fecal coliforms to enterococci ratio points to the origin of pollution. A ratio lower than 1.5 indicates pollution by the run off from agricultural surfaces. while a ratio higher than 4 is typical for anthropogenic pollution [15]. At all sampling locations, the number of fecal coliforms was higher than the number of enterococci indicating a great impact of human urban pollution [15]. The isolated coliform bacteria were identified as Enterococcus sp, Enterobacter sp. and Escherichia coli.
The high concentrations of F.C. detected in the warmer months may be related to the increased metabolic activity of the mollusks. The high metabolic activity of the mussels is directly related to the increase of the temperature of the sea waters from one side (Table 2) and their biological cycle from the other. In previous investigations

DISCUSSION
Mollusks such as bivalves are widely distributed sessile filter feeders that can serve as reliable bioindicators of the aquatic pollutants [9][10]. These animals represent a valuable tool for the screening of the environmental conditions. Due to the population growth worldwide, the increase of the need for protein results in the increase of consumption of food from animal origin. Therefore, seafood stands out as a way of providing animal source foods of high quality. Within seafood, the bivalves represent animals important food resource with its recently increasing consumption in Bulgaria. The United Nations and the World Food Program declared that seafood is going to have an important role in food supply in the future due to the population growth, which is estimated to increase up to 9 billion in 2050. Actually, it is assumed that the growth in the industry of seafood is going to continue on an accelerating way [11][12].
In the area of Varna Lake were detected very high amounts of E. hermannii and A. johnsonii. which are of human origin. These findings coincide with the time of spillage of fecal water as a result of a ruptured pipe in the area of Varna Lake. According to Ordinance No. 4 from 20.10.2000 for the quality of fisheries water and the breeding of shellfish [13], the amount of fecal coliforms in the inter-shell content should be less than 300 NVBin our samples from September 2020, the values were exceeded by 300 times. According to the same ordinance, there is an increase in the number of coliforms in the region of Sveti Vlas from August, where the increase is 50 Parallel to the performance of the microbiological experiments, we conducted a physicochemical analysis of the sea waters. The results are summarized in Table 2. of our working group, we demonstrated experimentally. that the black mussels can be isolated two stems of lactic acid bacteria belonging to L. plantarum, which show antifungal activities [16]. For the north section of the Bulgarian Black sea aquatory, for June 2018 were reported high levels of E. coli and Pseudomonas aeroginosa [8].
The temperature of the sea waters may affect the number of the microbes by concentration of the nutrients. Similar results were reported previously [17][18].

CONCLUSIONS
When studying the microbiota of populations of different species of mussels, it is very important to know their sanitary status, as well as to determine the pathobiological basis of periodic outbreaks of diseases affecting these populations. Our results demonstrated the presence of species of microorganisms in mussels, for which there was no data in the literature. With the worldwide growth of mussel consumption, we would like to point out on the possibility of the emergence of new diseases in the near future. Such diseases may result on the interaction between the pathogen, the host and the environment. The physiological and immunological status of the mussels as a host will play a leading role in the development of bacterial diseases in bivalves.