Phylogeny and Antibiotic Susceptibility of Bacteria in Sediments of Aegean Sea

The bacterial diversity and antimicrobial resistance in coastal areas indicate the variability in the community structures and metabolic activities. In the present study, antibiotic susceptibility and phylogenetic analysis of bacteria isolated from stations with different depths and influenced by terrestrial and marine fluxes in eastern Aegean Sea were illustrated. Half of the isolates were found as resistant and 14 percent showed high MAR index indicating the high-risk sources of contamination in the environment. According to 16S rRNA gene analysis, the isolates were found as belonging to the phylum Firmicutes and the class Gammaproteobacteria with the genera Bacillus, Halomonas, Oceanobacillus, Photobacterium, Pseudoalteromonas, Psychrobacter and Vibrio. Approximately half of Bacillus strains which were dominant among all isolates were resistant. In addition to phylogenetically diverse bacteria, the variability in resistance, intermediate and high MAR index levels of the study area indicated the effect of geographical differences.

Studies in coastal sediments, especially beneath fish farms, near shore sediments and sandy beaches which are under high anthropogenic pressure, also demonstrated bacterial community changes and high levels of antibiotics in the sediments [6][7][8][9]. Unlike widespread antibiotic resistance in those areas, the susceptibility levels are expected to increase in the deep-basins. For instance, a recent study in coastal areas of Eastern Mediterranean Sea including Turkish coasts of Aegean Sea demonstrated the high multiple antibiotic resistance but no resistance in offshore North Aegean Sea [12].
In the present study, the phylogenetic analysis and antibiotic resistance of bacteria isolated from sediments of Aegean Sea were aimed.

Sampling
In the survey by RV/K Piri Reis research vessel in spring period, sediment samples were obtained from 5 stations (100-202 m depths) located between Lesvos Island and Karaburun in Aegean Sea ( Figure 1).
For the first sediment processing method, 10 ml wet sediment sample was dried overnight and 0.5gr dry sediment was aseptically spread in circular fashion onto the agar media. For the next method, 1ml wet sediment was diluted with sterile seawater (1:4) and 50µl was spread aseptically onto the agar media. And lastly, 0.5 g dry sediment was mixed with 4 ml sterile seawater and 50 µl was spread aseptically onto the agar media. Incubation was done at 26ºC for 2 -3 days. Then, isolates were cryopreserved with 50% glycerol at -20ºC.

Antibiotic Susceptibility
According to antibiotic tests of totally 51 isolates, approximately half of them were found as resistant (51%) and 41% were intermediate to at least one of eleven antibiotics. On the other hand, the resistance and the intermediate levels of the study area were 38 -60% and 11 -38%, respectively (Table 1). When multiple antibiotic resistance (MAR) index was calculated, 14% of all strains showed high MAR index indicating the high-risk sources of contamination in the environment. Furthermore, 10 -40% of isolates from only stations 3 -5 had this high index (Table  1).  The present study demonstrated the variability of antibiotic resistance in sediments of different stations and indicated multiple resistance in stations closer to coastal area like high multiple resistance seen in coastal environments [5][6][7][8][9][10][11]. In spite of scarcity in bacterial research at Turkish marine environment, high MAR index was also previously shown in Turkish coastal seawaters and sediments [12,[15][16][17]. In a previous study of Eastern Mediterranean Sea, the relation between antibiotic resistance and geographical differences was given as higher resistance in coastal areas of Syria than Turkey and Lebanon (48 %, 38 % and 31 % of total isolates, respectively) [12]. Moreover, limited number of studies in Turkish coastal environment have reported resistant bacteria for the coasts of Marmara Sea [15], Aegean Sea and North Levantin Sea [12]. Similarly, very high resistance and MAR index were obtained for isolates from coastal sediments of Izmir Bay in Aegean Sea influenced by river and sewage discharge, anthropogenic activities such as tourism-derived, industrial, agricultural and nautical activities [17]. On the other hand, in the present study, one of the lowest resistance without high MAR index was obtained in station 2 with 200 m depth and closer to open Aegean Sea which supported the previous findings as the decrease in resistance from coastal to offshore area in North Aegean Sea [12]. Moreover, compared to the present study in Aegean Sea, higher resistance and MAR index were obtained from coastal sediments of Izmir Bay [17] and there were lower resistance and MAR index in deep stations of Eastern Mediterranean Sea with different bacterial diversity levels [18].

Phylogeny and Resistance
According to 16S rRNA gene analysis, it was found that the isolates belonged to the phylum Firmicutes and the class Gammaproteobacteria. The phylogenetic tree constructed using nearly full 16S rRNA gene sequence of one representative isolate for each nearest type strain clearly supported those phyla forming two separate clades ( Figure  3).
The  Figure 4). In addition, Psychrobacter Photobacterium and Vibrio species isolated from the study area had no intermediate levels for all antibiotics (Figure 4).  Highly resistant and diverse Bacillus species belonging to the phylum Firmicutes were dominant among isolates in the study area. Similarly, resistant Bacillus species were dominantly seen beneath fish farms in Gulf of La Spezia, North Mediterranean Sea [6] and Gulf of Mannar, India [5]. Although lower bacterial diversity in higher taxa was obtained compared to phylogenetic composition of resistant isolates from near shore sediments in Gulf of Mannar, India [5], higher diversity in lower taxa was clearly seen in the present study.
Furthermore, the study area was in the middle of north Aegean Sea and west Levantin Sea in Eastern Mediterranean Sea and as supporting this geographical indication, the study area had the highest bacterial diversity in both lower and higher taxa compared to coastal sediments of Izmir Bay having terrestrial and anthropogenic effects and thus the highest resistance and bacterial diversity in higher taxa [17] and to deep and oligotrophic stations in Aegean Sea having higher antibiotic sensivity and phylogenetic diversity in lower taxa [18].

Conclusions
In addition to phylogenetically diverse bacteria, the variability in resistance, intermediate and high MAR index levels of the study area indicated the effect of geographical differences. Therefore, much more studies in bacterial diversity and antimicrobial resistance in coastal areas are needed to understand the variability in the community structures in association with environmental differences.