Multidrug-Resistant Epi-Endophytic Bacterial Community in Posidonia Oceanica Seagrass from Mahdia Coastal Seawater as Biomonitoring Factor for Antibiotic Contamination

Amel Jebara Universite de Monastir Wafa Hassen Université de Monastir: Universite de Monastir Amira Ouesleti Université de Monastir: Universite de Monastir Lot Mabrouk Université de Gafsa: Universite de Gafsa Ahlem Jaziri Université de Monastir: Universite de Monastir Guiseppa Di Bella University of Messina Faculty of Physical and Natural Sciences: Universita degli Studi di Messina Hedi Ben Mansour (  hdbenmansour@gmail.com ) Institut Superieur des Sciences Appliquees et de Technologie de Mahdia https://orcid.org/0000-0001-7936-9828


Introduction
Human activities have impacted ecosystems for several decades, posing real environmental problems with regard to biodiversity and resources, particularly in the marine environment, the nal receptacle for chemical pollutants. Indeed, various disturbances, generally of anthropogenic origin, combine and contribute to weakening the marine ecosystem, mainly at the coasts (Afsa et al., 2020). Antibiotics residue attracts more attention in many countries in the world. Veterinary and human antibiotics residues are continuously introduced into the environment by manufacturing industries, wastewater treatment plant (WWTP) and as well as through the aquaculture development, which can have an impact on the water quality, the ecosystem and the human and animal health (Afsa et  Most of these latest studies have shown that P. oceanica has the capacity to sequester chemical pollutants from the marine environment and accumulate them in its perennial tissues for several decades (Jebara et al., 2021b). Therefore, P. oceanica is a re ector of the ecological state of coastal ecosystems and its storage capacity of geochemical records of the marine environment could be used as an alternative to trace the history of contamination of coastal ecosystems (Tovar-Sánchez et al., 2010). In addition, Posidonia meadows promotes the development of species of economic interest (crustaceans, cephalopods, sh), including microorganisms that play important role in the marine environment (Torta et al., 2015). Many studies have described the diversity of endophytic bacterial communities associated with seagrasses meadows  Boontanom and Chantarasiri, 2020). The aim of the present work was to study the bacterial diversity in ve coastal seawater samples collected from Mahdia coastal (Tunisia) and to evaluate the antibiotic resistance patterns of isolated bacteria from Posidonia oceanica seagrass. These strains may be considered as bioindicator for biomonitoring the degree of contamination of pollution in Mediterranean seawater.
Material And Methods

Study area and sample collection
Mahdia coast is one of the most important aquatic ecosystems in Tunisia occupying a very strategic geographic territory with approximately 75 km of coastline, presenting a predominant shery, heritage and agricultural wealth. Despite its wealth, the Mahdia coast is subject to several sources of urban and industrial pollution, in addition, the development of shing activity. To cover the whole of this zone, Posidonia oceanica seagrass and seawater were collected in September 2019 from ve sites: (S1) the coastal zone of Mahdia, (S2) the coastal zone of Rejiche, (S3) the coastal zone of Salakta, (S4) the coastal zone of Chebba and (S5) the coastal zone of Mellouleche ( Figure 1).
Samples of Posidonia oceanica seagrass (leaves and epiphytes) were collected from each site at 2.5 m depth by scuba divers and placed in sterile polythene bags. In the same time, one liter of seawater was sampled aseptically from each site by Niskin bottles in previously sterilized glass bottles. The collected samples were immediately transported to the laboratory and kept at 4°C until analysis (within 24 h after collection). DGGE pro les were exploited to create matrices indicating the presence or absence of bands, and a dendrogram was built by the MVSP software (MultiVariate Statistical Package), which uses the algorithm Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and the Jaccard's coe cient. The Simpson's Diversity Index (D) and R richness index were determined. The band patterns of DGGE gel were further examined with an image analyzing system by using Image J software (version 1.46) and Principal component analysis (PCA) was produced using Microsoft Excel XLSTAT software (version 2021.2) which allows the conversion of the bands density and migration into numerical values.

3-Isolation of epi-endophytic bacteria from Posidonia oceanica seagrass
Epi-endophytic bacteria were isolated from leaves and epiphytes of Posidonia oceanica seagrass. The collected leaves were subjected to a surface-sterilization protocol adapted from Garcias-Bonet et al., (2012). Brie y, the leaves samples were rinsed with distilled water and then disinfected super cially in 70% ethanol for 1 min. After, they were sterilized for 4 minutes in sodium hypochlorite (2.5%) and nally washed ten times with sterile distilled water. Afterward, they were aseptically fragmented into smaller pieces and crushed with aqueous solution (0.9% NaCl) using a homogenizer. For epiphytes, they were scraped from two faces of the surface of Posidonia oceanica leaves and then recovered in sterile Eppendorf tubes with aqueous solution.
Isolation of pure bacterial strains from the enrichment cultures was performed by using serial dilution (10 -1 -10 -8 ) onto marine agar plates. The plates were subsequently incubated at 25°C for 5 days. Colonies were counted and expressed as Colony-Forming Unit (CFU) per mL.
The pure colonies were selected according to their morphology characteristics (color, size, shape), transferred to the same marine media and puri ed by repeated streaking to obtain pure cultures. All bacterial isolates were characterized by determining their Gram reaction, catalase and oxidase activity. The bacterial isolates were stored as 25% glycerol stocks at −80°C.

4-Antibiotic susceptibility testing
Antimicrobial susceptibility testing was performed on 43 isolates by the disk diffusion method on Mueller Hinton agar plates (Difco, Bectone Dickinson, Sparks, MD) according to the National Committee for Clinical Laboratory Standards guidelines (CLSI, 2017). Twelve antimicrobial agents were tested (Table 1). Results And Discussion

1-DGGE Analysis
To investigate the diversity and the dynamics of the dominating microbial communities, samples from ve coastal seawater sites of Mahdia were analyzed by denaturing gradient gel electrophoresis (DGGE), which is one of the most commonly used methods among the culture-independent ngerprinting techniques (Li et al., 2010). Moreover, DGGE analysis of 16S rRNA gene was applied to present an overview of culturable and unculturable bacterial assemblages. 16S rRNA fragments, obtained by targeting of the variable V3-V5 region by the PCR-DGGE, were separated on a polyacrylamide denaturing gel ( Figure 2). Samples from all sites were analyzed in triplicate. Visual analysis of DGGE band pro les highlighted a large variability in bacterial community structure of all the samples. A remarkable similarity was noted between the three replicates of each site. We detected several bands with variable intensities and migration distances. Some of the bands were speci c to a given site, while, other bands were found to be common to more than one sample. The obtained pro les showing a difference in migration, which allowed the bands to be divided into three migration levels: short migration bands (non-rich bands in GC), medium migration bands (moderately rich in GC) and long migration bands corresponding to high GC content sequences (Figure 2). A very similar variation was revealed in all pro les of different seawater coastal, which gave us a general idea of the abundance of the same bacterial community between the different sites. Similarity and variation among DGGE gel patterns of the various coastal seawater sites were also established based on analysis of Jaccard's coe cient with the UPGMA algorithm ( Figure 3). The cluster analysis showed a high similarity above 90%, indicating a stable community within S1 (Mahdia seawater coastal site), S2 (Rejiche seawater coastal site), S3 (Salakta seawater coastal site) and S5 (Melloulech seawater coastal site). Furthermore, Chebba seawater coastal site present only 24% of diversity with other sites.
The principal component analysis (PCA) based on the relative intensities of bands in DGGE pro les provided further information about the microbial community structure existed in ve seawater coastal sites (Figure 4). The PCA analysis results con rmed the differences observed visually by showing a variation of 71.34% and 12.38% according to the rst and second principal axes, respectively. All seawater coastal samples (with three replicates of each site) appeared to be randomly distributed and were relatively adjacent.
However, we could see four groups: the rst group of seawater coastal samples (S1-3, S2-1, S2-3 and s3-2) distributed on the left side, according to the negative values of the rst principal axis F1 and positive values of the secondary axis F2. The second group is represented by the samples S3-1 S4-2 S4-3 S5-1 and the third group distributed on the right side that contained the samples S1-2, S2-2, S3-3, S4-1, S5-2 and S5-3. The last group could be distinguished from the other samples, is the S1-1 sample which corresponds to the one of three replicates samples of Mahdia seawater coastal site. These results indicated that the bacterial community structure was variable even in replicates from the same site ( Figure 4).

2-Isolation and selection of multi-antibiotic resistant epi-endophytic bacteria
Posidonia oceanica leaves and their epiphytes, collected from different seawater coastal sites, were enumerated by cultivating on marine agar media. The obtained results showed that the highest levels of cultivable bacteria in the epiphytes and leaves of Posidonia oceanica were detected in Rejiche seawater coastal sample (Table 2). In addition, we have noticed that the count of cultivable bacteria is higher in the epiphytes of Posidonia oceanica than in the Posidonia leaves recovered from Rejiche seawater coastal sample (2.70 10 10 , 2.32 10 10 ), Salakta seawater coastal sample (5.95 10 9 , 3.72 10 9 ) and Melloulech seawater coastal sample (5.27 10 8 , 9.14 10 7 ). These results con rm that epiphytes and Posidonia leaves are considered to be a favorable matrix for bacterial accumulation and development (Borowitzka et al., 2006). In accordance with these results, Tarquinio and colleagues (2019) has also observed a high population density of epi-and endophytic bacteria associated with Posidonia oceanica seagrass. Furthermore, they have indicated that the presence of a different set of microorganisms can have a bene cial role for plant growth, health and productivity (Tarquinio et al., 2019). Fahimipour et al., (2017) showed that bacterial composition of leaves seagrass was highly similar to the surrounding seawater communities. However, they also found contrasting results related to the seagrass roots communities whereby bacterial composition of roots differed from sediment communities.
As mentioned in previous studies, increased consumption of antibiotics contributes to an increase in environmental waste  Figure 5. A high level of resistance was detected to Aztreonam (72.1%) and Ceftazidime (60.5%). A medium level of resistance was observed to Amoxicillin (56%), Rifampicin (51.2%), Trimethoprim (44.2%), Cefotaxime (35%) and Chloramphenicol (30.2%). Lower levels were detected to Azithromycin (7%), Cipro oxacin (5%) and to Amikacin (2.3%). All the studied isolates showed susceptibility to Imipenem and Tetracycline. An antibiotic multi-resistance phenotype was observed. Seven dominated multi-resistance pro les were detected for all the epi-and endophytic bacterial isolates against to 6 families of antibiotics (β-lactams, Phenicol, Macrolide, Sulphonamides, Rifamycin and Quinolones) ( Table 3). The ecological justi cation of antibiotic resistance is the adaptation of bacteria in the polluted environment. Bacteria in these polluted aquatic matrices share or exchange transferable elements of DNA with other bacteria, this can occur between different bacterial species (Tahrani et al., 2017).
These multi-antibiotic resistant epi-endophytic bacterial strains were identi ed by ampli cation of the ribosomal intergenic transcribed spacer between the 16S and the 23S rRNA genes (ITS-PCR) and by 16S rRNA sequencing. ITS-PCR ngerprinting was applied to assess bacterial diversity of the selected isolates. The ITS pro les showed reproducible patterns consisting of 1 to 7 bands with sizes ranging from 200 to about 400 bp ( Figure 6). Four different ITS haplotypes were produced. H1 was represented by one isolate (S1-L1) and composed by 4 reproducible bands (250, 290, 310 and 350 bp). The other ITS pro les were found in two isolates. Partial 16S rRNA gene sequencing was carried out for these 7 isolates and was analyzed by BLAST algorithm (Table  3). Phylogenetic analysis revealed that the selected isolates were assigned to the phylum Firmicutes with low G+C% content gram positive and represented by Bacillus genus (Figure 7), exhibiting 98-100% of identity to published species sequences. These strains were classi ed in ve spices including B. paranthracis (S1-L1), B. wiedmannii (S2-E2; S3-E1; S3-L3), B. oceanisediminis (S3-E2), B. rmus (S4-L7), and B. paramycoides (S5-L13  (Liu et al., 2017;Bibi et al., 2018) and demonstrated that epiphytic bacteria colonizing the seagrass surface must be able to resist interactions with the host defences processes and competition with other microorganisms (Boontanom and Chantarasiri, 2020).

Conclusion
Only a few studies focused on bacterial community of Posidonia oceanica seagrass. Here, we studied the epi-endophytic bacterial community of Mahdia coast focusing on multi-resistant bacteria to antibiotics as bio-indicators of pollution. The seawater coastal sites revealed a high bacterial diversity assessed by DGGE approach. From the 43 epi-endophytic bacterial strains isolated from Posidonia leaves and their epiphytes, seven strains were the most dominant multi-resistance pro les. These selected isolates were subjected to identi cation, characterization and phylogenetic analysis. Firmicute phylum was the single bacterial group distinguished and represented by Bacillus genus. This collection may be considered as bioindicator for biomonitoring the degree of contamination of pollution in Mediterranean seawater. Leaves (CFU/g) 5.25 10 9 2.32 10 10 3.72 10 9 5.14 10 9 9.14 10 7 Epiphytes (CFU/g) 2.79 10 9 2.70 10 10 5.95 10 9 4.03 10 7 5.27 10 8   Denaturing gradient gel electrophoresis (DGGE) gel pro les from triplicate of ve seawater samples showing the bacterial community structure based on the variable region V3-V5 of 16S rRNA. Urea and formamide gradient ranged from 40% to 60%. Three kinds of bands were distinct with correlation to the running level, as short (A), medium (B) and long migration bands (C).
Species richness index (R) and Simpson diversity index (D) were indicated in the lower part of the gel.

Figure 3
Clustering of DGGE patterns by software analysis using the Jaccard's coe cient with the UPGMA algorithm.    Phylogenetic tree of seven partial 16S rRNA sequences of multidrug-resistant endophytic strains isolated from Posidonia oceanica seagrass. Bootstrap values obtained with 1000 repetitions were indicated as percentages at all branches. GenBank accession numbers are given in parentheses. Staphylococcus arlettae (NR_024664.1) was chosen as the out-group to root the tree.