# Antibiotic Resistance in Seawater Samples from East Coast of Spain
## 3 Results
### Antibiotic Resistance Determination of Bacterial Isolates  
A total of 87 strains were isolated from the seawater samples (57 _E. coli_ strains, and 30 _Enterococcus_ sp. strains) and subjected to the antibiotic sensitivity test. The resistance of the bacteria to antibiotics was determined according to the CLSI standards [39] for antimicrobial susceptibility testing. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Target Gene** & **Sequence** & **Conditions** & **Product (bp)** & **Reference** \\ \hline _blat_TEM & 5′-GCKGGCCAACTTACTICTGCACAACG-3′ & 95 \({}^{\circ}\)C 3 min (1 cycle); 95 \({}^{\circ}\)C 15 s and 5′-CTITATCCGCCTCCTCCTCAGTCTA-3′ & 60 \({}^{\circ}\)C 20 s (40 cycles); 72 \({}^{\circ}\)C 1 min & 247 & [41] \\ \hline _em_B & 5′-GATACCGTTATCAGAAATTGG-3′ & 95 \({}^{\circ}\)  
Among the 57 isolates of _E. coli_ (Table 2), 34 isolates (59.6%) were resistant to at least one antibiotic. Resistance to ampicillin was the most frequent observation (20 isolates) followed by ciprofloxacin (10 isolates), sulfamethoxazole, and trimethoprim with 9 isolates for each and tetracycline and nalidixic acid with 8 isolates (Supplementary Materials Table S1). The MIC values for these antibiotics were 32 - > 64 mg/L, 0.125-0.5 mg/L, >1024 mg/L, >32 mg/L, 32 - > 64 mg/L, and >128 mg/L, respectively. There was no correlation (\(p\) > 0.05) between the number of strains resistant to any antibiotic and the area from which they were isolated. Multiple resistance (\(\geq\)3 antibiotic classes) [45] was observed in 5 out of 34 resistant _E. coli_ strains, (14.7%). Each of them presented a unique multi-resistance pattern. \(\beta\)-lactam resistance was present in all profiles, followed by sulfonamide and tetracycline resistance, present in four profiles, and quinolone resistance, observed in three profiles. Resistance to polymyxins (colistin) and aminoglycos (gentamicin) was only present in one multi-resistance profile for each. Finally, none of the isolates showed resistance to chloramphenicol and tigecycline, with MIC ranges of \(\leq\)8-16 mg/L and \(\leq\)0.25-1 mg/L, respectively. Only seven _Enterococcus_ sp. isolates showed resistance to the tested antibiotics, specifically, six isolates against tetracycline and one against daptomycin (Table 3) (Supplementary Materials Table S4). The MIC ranges for these antibiotics were 64-128 mg/L and 16 mg/L. The statistical analysis showed no correlation (\(p\) > 0.05) between the resistance of the isolates to any antibiotic and the area from which they were isolated. \begin{table}
\begin{tabular}{c c c c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Zone**} & \multicolumn{2}{c}{**No. Tested**} & \multicolumn{10}{c}{**Number of _E. coli_ Resistant Isolates**} \\ \cline{2-13}  & **Isolates** & **SMX** & **TMP** & **CIP** & **TET** & **MER** & **AZI** & **NAL** & **FOT** & **CHL** & **TGC** & **TAZ** & **COL** & **AMP** & **GEN** & **MDR*** \\ \hline NV & 14 & 2 & 3 & 2 & 2 & 0 & 1 & 3 & 0 & 0 & 0 & 1 & 0 & 4 & 1 & 2 \\ SV & 23 & 4 & 4 & 2 & 2 & 1 & 3 & 4 & 2 & 0 & 0 & 4 & 1 & 12 & 0 & 2 \\ A & 20 & 3 & 2 & 4 & 4 & 0 & 1 & 1 & 0 & 0 & 0 & 0 & 4 & 0 & 1 \\ \hline Total & 57 & 9 & 9 & 10 & 8 & 1 & 5 & 8 & 2 & 0 & 0 & 5 & 1 & 20 & 1 & 5 \\ \hline \multicolumn{13}{c}{\% of resistant} & \multicolumn{10}{c}{SMX: sulfamethoxazole; TMP: trimethoprim; CIP: ciprofloxacin; TET: tetracycline; MER: meropenem; AZI: azithromycin; NAL: nalidixic acid; FOT: cefotaxime; CHL: chloramphenicol; TGC: igecycline; TAZ: ceftazoline; COL: colistin; AMP: ampicillin; GEN: gentamicin. NV: North Valencia; SV: South Valencia; A: Alicante. \({}^{a}\): MDR: resistance to \(\geq\)3 antibiotic classes.} \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Incidence resistant _E. coli_ isolates for each antimicrobial compound separately and that of multi-resistance in three different zones of Comunitat Valenciana Coast. \begin{table}
\begin{tabular}{c c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Zone**} & \multicolumn{2}{c}{**No.