Seven New Alkaloids Isolated from Marine Flavobacterium Tenacibaculum discolor sv11

Marine flavobacterium Tenacibaculum discolor sv11 has been proven to be a promising producer of bioactive nitrogen-containing heterocycles. A chemical investigation of T. discolor sv11 revealed seven new heterocycles, including the six new imidazolium-containing alkaloids discolins C-H (1–6) and one pyridinium-containing alkaloid dispyridine A (7). The molecular structure of each compound was elucidated by analysis of NMR and HR-ESI-MS data. Furthermore, enzymatic decarboxylation of tryptophan and tyrosine to tryptamine and tyramine catalyzed by the decarboxylase DisA was investigated using in vivo and in vitro experiments. The antimicrobial activity of the isolated compounds (1–7) was evaluated. Discolin C and E (1 and 3) exhibited moderate activity against Gram-positive Bacillus subtilis DSM10, Mycobacterium smegmatis ATCC607, Listeria monocytogenes DSM20600 and Staphylococcus aureus ATCC25923, with MIC values ranging from 4 μg/mL to 32 μg/mL.

As a member of the family Flavobacteriaceae within the phylum Bacteroidetes, isolates of the genus Tenacibaculum have been mainly obtained from marine environments, such as sea water, tidal flat, and aquaculture systems, as well as marine organisms like bryozoan, sea anemone, oyster, sponge and green algae [15][16][17][18][19][20][21][22]. Bacteria of this genus are the etiological agent of an ulcerative disease known as tenacibaculosis, which affects a large number of marine fish species in the world [23]. Up to now, the natural products isolated from Tenacibaculum strains comprise only siderophores that showed beside their chelating activity also cytotoxicity [24][25][26], and phenethylamine-containing heterocycles. The latter include two imidazole alkaloids identified in our previous search for antimicrobial metabolites from marine flavobacteria. It was shown that they could be synthesized by decarboxylation of phenylalanine, catalyzed by the enzyme DisA [27]. Likewise, the tryptamine and phenethylamine moieties of imidazole alkaloids isolated from a marine sponge-associated Bacillus strain were proposed to be formed by an aromatic amino acid

Results
In our continuous search for new bioactive molecules, the six new imidazoliumcontaining alkaloids discolins C-H (1-6) and one pyridinium-containing alkaloid dispyridine A (7) were isolated from the marine-derived bacterium T. discolor sv11 (Figure 1). The antimicrobial activity of these new compounds was investigated, among which, compounds 1 and 3 exhibited moderate activity against Gram-positive Bacillus subtilis DSM10, Mycobacterium smegmatis ATCC607, Listeria monocytogenes DSM20600 and Staphylococcus aureus ATCC25923. In vivo and in vitro experiments indicated that phenethylamine, tryptamine and tyramine residues of the new alkaloids are derived from an enzymatic decarboxylation.
tabolites from marine flavobacteria. It was shown that they could be synthesized by decarboxylation of phenylalanine, catalyzed by the enzyme DisA [27]. Likewise, the tryptamine and phenethylamine moieties of imidazole alkaloids isolated from a marine spongeassociated Bacillus strain were proposed to be formed by an aromatic amino acid decarboxylase-dependent reaction [28]. In order to further expand the array of available nitrogen-containing heterocycles, the metabolome of T. discolor sv11 was further investigated. Herein, we present the isolation, structure elucidation and biological activity of new alkaloids from the bacterium, and link the enzymatic activity of DisA to their biosynthesis using both, in vivo and in vitro assays.

Results
In our continuous search for new bioactive molecules, the six new imidazolium-containing alkaloids discolins C-H (1-6) and one pyridinium-containing alkaloid dispyridine A (7) were isolated from the marine-derived bacterium T. discolor sv11 (Figure 1). The antimicrobial activity of these new compounds was investigated, among which, compounds 1 and 3 exhibited moderate activity against Gram-positive Bacillus subtilis DSM10, Mycobacterium smegmatis ATCC607, Listeria monocytogenes DSM20600 and Staphylococcus aureus ATCC25923. In vivo and in vitro experiments indicated that phenethylamine, tryptamine and tyramine residues of the new alkaloids are derived from an enzymatic decarboxylation. Compound 1 was obtained as a yellowish oil. The HR-ESI-MS spectrum of 1 showed a molecular formula of C26H32N3 + based on the prominent peak [M] + at m/z 386.2606 (calculated 386.2591, Figure S1). The analysis of 1 H NMR and HSQC spectra of 1 revealed three methyl groups at δH 0.80 (H-8), δH 2.15 (H-9) and δH 2.20 (H-10), six methylene groups at δH 1.34 (H-7), δH 2.41 (H-6), δH 2.80 (H-7′'), δH 3.08 (H-10′), δH 4.22 (H-8′') and δH 4.30 (H-11′), as well as ten aromatic protons that resonated from δH 7.00 to δH 7.39 (Table 1). These NMR data exhibited a high similarity with the previously reported compound discolin A that was also isolated from T. discolor sv11 [27]. Therefore, a core structure of the 4,5-dimethyl-2-propyl imidazolium skeleton of 1 was elucidated based on the COSY spin system from H-6 to H-7 to H-8, as well as on HMBC correlations from both H-6 and H-7 to C-2, and from both, H-9 and H-10 to C-4 and C-5. The same phenylethyl moiety as present in discolin A was deducted from compound 1 based on the COSY spin system between H-7′' and H-8′', as well as between five benzene ring protons 7.16 (2H, H-2′' and H-6′'), 7.28 (H-4′') and δH 7.32 (2H, H-3′' and H-5′'), together with the core HMBC correlations from H-7′' to C-1′' and C-2′', and from H-8′' to C-1′' (Figure 2). The significant difference between compound 1 and discolin A are the HMBC correlations from a singlet aromatic proton resonating at δH 7.17 (H-2′) to C-3′, C-4′ and C-9′, from H-5′ to C-3′, C-4′, C-7′ and C-9′ (Figure 2), as well as the COSY spin system from H-5′ to H-6′, to H-7′ to H-8′. These results suggested an indole moiety instead of a phenyl residue in compound 1. Together  Figure S1).  -11 ), as well as ten aromatic protons that resonated from δ H 7.00 to δ H 7.39 (Table 1). These NMR data exhibited a high similarity with the previously reported compound discolin A that was also isolated from T. discolor sv11 [27]. Therefore, a core structure of the 4,5-dimethyl-2-propyl imidazolium skeleton of 1 was elucidated based on the COSY spin system from H-6 to H-7 to H-8, as well as on HMBC correlations from both H-6 and H-7 to C-2, and from both, H-9 and H-10 to C-4 and C-5. The same phenylethyl moiety as present in discolin A was deducted from compound 1 based on the COSY spin system between H-7 and H-8 , as well as between five benzene ring protons 7.16 (2H, H-2 and H-6 ), 7.28 (H-4 ) and δ H 7.32 (2H, H-3 and H-5 ), together with the core HMBC correlations from H-7 to C-1 and C-2 , and from H-8 to C-1 ( Figure 2). The significant difference between compound 1 and discolin A are the HMBC correlations from a singlet aromatic proton resonating at δ H 7.17 (H-2 ) to C-3 , C-4 and C-9 , from H-5 to C-3 , C-4 , C-7 and C-9 ( Figure 2), as well as the COSY spin system from H-5 to H-6 , to H-7 to H-8 . These results suggested an indole moiety instead of a phenyl residue in compound 1. Together with the remaining COSY spin system between the two methylene groups H-10 and H-11 and the HMBC correlations from H-10 to C-2 , C-3 , C-4 and C-11 , a 3-ethylindole moiety (C 10 H 10 N) was elucidated from compound 1, which is further supported by the MS/MS fragment [C 10 H 10 N] + detected at m/z 144.0813 (calculated 144.0813, Figure S1). With the HMBC correlations from H-8 to C-2 and C-4, and from H-11 to C-2 and C-5, the above mentioned phenylethyl moiety and 3-ethylindole were supposed to be located at position 3 and 1 of the imidazolium skeleton ( Figure 2). This assumption was proven by 1 H-15 N HMBC correlations from H-6, H-9, H-7 and H-8 to N-3 and from H-6, H-10, H-10 and H-11 to N-1 ( Figure 2). The N-atom at position 1 was considered to be positively charged based on the detected chemical shift at δ N 178.5, while N-3 was at δ N 177.3 ( Figures S7 and S8) [29][30][31]. An additional NMR measurement with added trifluoracetic acid (TFA) in DMSO-d 6 (ratio 1:3) was carried out to further prove this conclusion ( Figures S9-S13). The methylene groups of H-8 and H-7 shifted to up-field with a deviation ∆δ H-8 value of 0.13 and ∆δ H-7 value of 0.10 ppm, while the deviation ∆δ H-11 and ∆δ H-10 values were 0.09 and 0.05 ppm, respectively. The addition of TFA lead to the protonation of the tertiary N-atom at position 3, which gives a higher influence on the chemical shift [32]. The detected different chemical shift deviations ∆δ N-3 (155.1) and ∆δ N-1 (154.1) further support this result ( Figures S7, S8 and S13). Thus, the structure of compound 1 was elucidated as shown in Figure 1 and named discolin C. DMSO-d6 (ratio 1:3) was carried out to further prove this conclusion ( Figure S9-S13). The methylene groups of H-8′' and H-7′' shifted to up-field with a deviation ΔδH-8′' value of 0.13 and ΔδH-7′' value of 0.10 ppm, while the deviation ΔδH-11′ and ΔδH-10′ values were 0.09 and 0.05 ppm, respectively. The addition of TFA lead to the protonation of the tertiary Natom at position 3, which gives a higher influence on the chemical shift [32]. The detected different chemical shift deviations ΔδN-3 (155.1) and ΔδN-1 (154.1) further support this result ( Figure S7-S8 and S13). Thus, the structure of compound 1 was elucidated as shown in Figure 1 and named discolin C. Compound 2 was obtained as a yellowish oil. The molecular formula of 2 was determined to be C26H32ON3 + (m/z = 402.2543, [M] + , calcd. 402.2540, Figure S14) based on the HR-ESI-MS spectrum. Comprehensive comparison of NMR data of compounds 1 and 2 revealed the high similarity except for the chemical shift of C-4′', which was shifted from 126.96 to 156.48, and one missing aromatic proton. Together with the detected 16 Da increase in the HR-ESI-MS spectrum of compound 2, this suggested the presence of a 4hydroxyphenylethyl moiety located at position 3 instead of a phenylethyl moiety (Table  1). This assumption was confirmed by the upfield chemical shifts of the benzene ring protons at δH 6.70 (2H, H-3′' and H-5′') and δH 6.91 (2H, H-2′' and H-6′'), which showed similar behavior as the reported 4-hydroxyphenylethyl-containing compound N-Acetyltyramine [33]. This effect is explained by the fact that the hydroxyl group is an electron donor, which shields the protons of the benzene nucleus more strongly and leads to an upfield shift of Compound 2 was obtained as a yellowish oil. The molecular formula of 2 was determined to be C 26 Figure S14) based on the HR-ESI-MS spectrum. Comprehensive comparison of NMR data of compounds 1 and 2 revealed the high similarity except for the chemical shift of C-4 , which was shifted from 126.96 to 156.48, and one missing aromatic proton. Together with the detected 16 Da increase in the HR-ESI-MS spectrum of compound 2, this suggested the presence of a 4-hydroxyphenylethyl moiety located at position 3 instead of a phenylethyl moiety (Table 1). This assumption was confirmed by the upfield chemical shifts of the benzene ring protons at δ H 6.70 (2H, H-3 and H-5 ) and δ H 6.91 (2H, H-2 and H-6 ), which showed similar behavior as the reported 4-hydroxyphenylethyl-containing compound N-Acetyltyramine [33]. This effect is explained by the fact that the hydroxyl group is an electron donor, which shields the protons of the benzene nucleus more strongly and leads to an upfield shift of the corresponding signals. Second, the hydroxyl group that appeared at C-4 in compound 2 changes the spin system of this radical, and therefore influenced the shape of the proton multiplets of the benzene nucleus. Furthermore, the COSY spin system between H-2 and H-3 , as well as the HMBC correlations from H-7 to C-1 , C-2 , from H-3 to C-1 , C-4 , and from H-8 to C-2, C-4 and C-1 proved the 4-hydroxyphenylethyl group in compound 2.  Figure S14) of compound 2, which lost the 4-hydroxyphenylethyl group (-C 8 H 9 O), strongly indicated the above mentioned assumption. Thus, the structure of compound 2 was elucidated as shown in Figure 1 and named discolin D.
Compound 3 was also obtained as a yellowish oil. The molecular formula of 3 was determined to be C 28 Figure S20) based on the HR-ESI-MS spectrum. The core scaffold of compound 3 shared the 4,5-dimethyl-2propyl imidazolium skeleton with compound 1 as deduced from a comparison of both, 1D and 2D NMR data (Table 1), which also indicated compound 3 to be a symmetric structure. Integration of the proton signals in the 1 H NMR spectrum together with the COSY spin system of the aromatic protons and the HMBC correlations from H-2 to C-3 , C-4 and C-9 , from H-10 to C-2 , C-4 and C-11 , as well as from H-11 to C-3 , C-10 , C-2, and C-5 proved that two identical 3-ethylindole moieties were connected to the central imidazolium ring as shown in Figure 2. Hence, compound 3 is a member of the discolin family and was named discolin E.
Compounds 4 and 5 were each obtained as a yellowish oil. The molecula formulae of compounds 4 and 5 were identified as C 24 Figure S26) and m/z = 377.2593 (calculated 377.2587, compound 5, Figure S32), respectively. One phenylethyl moiety, one 4-hydroxyphenylethyl moiety and the 4,5-dimethyl-2-propyl imidazolium skeleton were disclosed as constituents of compound 4 by comparing the 1D and 2D NMR data with those of compounds 1 and 2 (Tables 1 and 2). The phenylethyl moiety and the 4-hydroxyphenylethyl moiety were assigned to be located at positions 1 and 3 of the imidazolium skeleton of compound 4, based on the HMBC correlations from H-8 to C-2 and C-5 and from H-8 to C-2 and C-4 ( Figure 2). In compound 5, identical phenylethyl and 4-hydroxyphenylethyl moieties were assigned to be located at the same positions as in compound 4. Comparing the 1D and 2D NMR data of compounds 4 and 5, the only difference is one ethyl group present in compound 5, while compound 4 carries a methyl group ( Table 2). The presence of an ethyl group in compound 5 is corroborated by the COSY correlation between H-9 and H-10, and the HMBC correlations from both, H-9 and H-10 to C-4. In contrast, in compound 4, the methyl group is directly connected to the unsaturated carbon C-4 ( Figure 2), thus verifying the assumed structural relationship between compounds 4 and 5. The 14 Da molecular weight difference between both compounds further supports their structural relationship. Thus, the structures of compounds 4 and 5 were elucidated as shown in Figure 1, and the names discolin F and discolin G were proposed, respectively. Compound 6 was also obtained as a yellowish oil. The molecular formula of compound 6 was established as C 23 H 29 N 2 + based on the prominent [M] + peak in HR-ESI-MS spectrum at m/z = 333.2329 (calculated 333.2325, Figure S37). Two identical phenylethyl moieties were deduced from the NMR spectra of compound 6 and connected at positions 1 and 3 of the core ring based on the HMBC correlations from H-8 to C-2 and C-5, as well as from H-8 to C-2 and C-4. The remaining signals of 6 were assigned to the 4,5-dimethyl-2-ethyl imidazolium scaffold, which showed an ethyl group rather than a propyl group at position 2. This difference was clarified by the COSY correlation between H-6 and H-7, as well as the HMBC correlations from both, H-6 and H-7 to C-2 ( Figure 2). Therefore, compound 6 proved to be a representative of the discolin family and was named discolin H.
Compound 7 was isolated as a colorless powder. Its molecular formula was established as C 22 H 29 N 2 + based on the prominent ion peak [M] + observed at m/z 321.2322 (calcd. 321.2325, Figure S43). Comprehensive analysis of 1D and 2D NMR data of compound 7 revealed one 3-ethylindole moiety as found in compounds 1-3, as well as two ethyl groups and one propyl group ( Table 3). The remaining two aromatic protons at δ H 8.20 (H-4) and δ H 8.40 (H-6) in the 1 H NMR spectrum and five aromatic carbons at δ C 153.07 (C-2), δ C 142.58 (C-3), δ C 144.54 (C-4), δ C 140.61 (C-5) and δ C 142.62 (C-6) in the 13 C NMR spectrum were attributed to a pyridinium ring as apparent from comparison with the data of dispyridine, a pyridinium-containing alkaloid isolated previously [27]. The location of the 3-ethylindole moiety was determined from the HMBC correlations from H-11 to C-2 and C-6, which also confirmed the location of the aromatic proton H-6 resonating at δ H 8.40. The second aromatic proton resonating at δ H 8.20 was attributed to position 4, based on the HMBC correlations from H-4 to C-2, C-3 and C-6. The propyl group and the two ethyl groups attached to the pyridinium ring were located at C-2, C-3 and C-5, as inferred from HMBC correlations from H-7 to C-2 and C-3, from H-10 to C-2, C-3 and C-4, and from H-12 to C-4, C-5 and C-6 ( Figure 2). Therefore, compound 7 was found to be a new member of the dispyridine family and named dispyridine A. Based on previous research, it was known that phenylalanine can be converted to phenethylamine by the catalytic action of the decarboxylase DisA. This molecule could serve as building block to yield different derivatives [27]. Hence, the newly isolated imidazolium-containing alkaloids (1)(2)(3)(4)(5)(6) were also supposed to be produced via the same biosynthetic route, i.e., first an enzymatic decarboxylation of the aromatic-L-amino acid tryptophan or tyrosine yielding tryptamine or tyramine, respectively, followed by a nonenzymatic condensation to form the central imidazolium ring. To confirm this hypothesis, the candidate enzyme DisA from T. discolor sv11 was analyzed in vivo in a heterologous system. Therefore, the previously constructed transgenic host strain E. coli ROSETTA (disA) carrying the respective disA gene and E. coli ROSETTA (pRSF) (negative empty vector control) were cultivated in LB medium, whereby 2 mM tryptophan and tyrosine were added as substrates, respectively. After 24 h incubation, tryptamine and tyramine were only detected in the extract of E. coli ROSETTA (disA), while only the substrates, i.e., tryptophan and tyrosine were detected in the negative control (Figure 3). To further validate these results, a His-tagged version of DisA was purified using affinity chromatography and assayed in vitro. This confirmed that tryptamine and tyramine can be obtained from tryptophan and tyrosine by a DisA-dependent catalytic conversion (Figure 3). tophan or tyrosine yielding tryptamine or tyramine, respectively, followed by a non-enzymatic condensation to form the central imidazolium ring. To confirm this hypothesis, the candidate enzyme DisA from T. discolor sv11 was analyzed in vivo in a heterologous system. Therefore, the previously constructed transgenic host strain E. coli ROSETTA (disA) carrying the respective disA gene and E. coli ROSETTA (pRSF) (negative empty vector control) were cultivated in LB medium, whereby 2 mM tryptophan and tyrosine were added as substrates, respectively. After 24 h incubation, tryptamine and tyramine were only detected in the extract of E. coli ROSETTA (disA), while only the substrates, i.e., tryptophan and tyrosine were detected in the negative control (Figure 3). To further validate these results, a His-tagged version of DisA was purified using affinity chromatography and assayed in vitro. This confirmed that tryptamine and tyramine can be obtained from tryptophan and tyrosine by a DisA-dependent catalytic conversion (Figure 3). All isolated compounds 1-7 were investigated for their bioactivity against bacteria (B. subtilis DSM10, M. smegmatis ATCC607, L. monocytogenes DSM20600, S. aureus ATCC25923, and E. coli ATCC25922) and fungi (Candida albicans FH2173). As shown in Table 4, discolin C (1) showed activity against M. smegmatis ATCC607 and B. subtilis DSM10 with MIC values ranging from 4 µg/mL to 8 µg/mL and moderate to weak activity against S. aureus ATCC25923 and L. monocytogenes DSM20600 with MIC values ranging from 16 µg/mL to 32 µg/mL. Discolin E (3) exhibited activity against four tested Grampositive bacteria with MIC values ranging from 4 µg/mL to 8 µg/mL and moderate activity against C. albicans FH2173 with an MIC value of 16 µg/mL. The other compounds (2, 4-7) were inactive against all the tested microorganisms in the range tested.

Conclusions and Discussion
In conclusion, seven new alkaloids were obtained from the crude extract of T. discolor sv11 after fermentation in LB medium. In vivo and in vitro experiments proved the decarboxylase DisA to catalyze the decarboxylation of the aromatic-L-amino acids phenylalanine, tryptophan and tyrosine to phenethylamine, tryptamine and tyramine, respectively. These molecules serve as substrates for the formation of the central imidazolium ring of discolins A-H through a non-enzymatic condensation. Hence, by combining an enzymecatalyzed and a non-enzymatic reaction, the bacterium generates a mix of structurally related molecules. Besides the understanding of the biosynthetic mechanisms of the discolins, some insights into the structure-activity relationship of the antibacterial discolins A-H were also obtained [27]. Discolin A and discolin H feature the same molecular skeleton, except for the length of the carbon chain linked to C-2 of the central imidazolium ring. Both molecules showed the similar moderate bioactivity, which suggests that the substructure at position 2 can be altered without affecting the activity. The structural differences and changes in the bioactivity of discolins C-F and discolin A indicated that the substructures at position 1 and 3 of the central ring instead play an important role concerning antibacterial activity. Earlier evidence indicated that the activity of imidazolium salts is highly dependent upon the substituents on the nitrogen atoms of the imidazolium cation [34], which is in agreement with our observation. In summary, our finding, together with previous reports, clearly indicates that the genus Tenacibaculum exhibits a high potential to produce nitrogen-containing heterocycles with a unique structure and various biological activities. This includes positively charged imidazolium-containing natural products.

Enzymatic Activity of Dis A
To investigate the enzymatic activity of Dis A in vivo, E. coli ROSETTA (disA) was cultured in 30 mL kanamycin-containing (50 µg mL −1 ) LB medium at 30 • C overnight as pre-culture. A volume of 100 µL of this pre-culture was used to inoculate at 37 • C in two 300 mL Erlenmeyer flasks with 100 mL kanamycin-containing (50 µg mL −1 ) LB medium; 0.1 mM IPTG was added into the medium when the cultures reached an OD 600 of 0.5 and were cultured at 30 • C for 3 h. Then, 2 mM tryptophan or tyrosine were added to the medium and cultured at 30 • C overnight. Next, 2 mL medium was harvested, dried in vacuo, re-dissolved in 200 µL DMSO and analyzed by UPLC-HRMS. The E. coli ROSETTA strain harboring the empty vector pRSF without the target disA gene was cultivated under the same conditions and analyzed by UPLC-HRMS as the negative control.
An in vitro enzymatic characterization was carried out after the purification of the His-tagged DisA. An inoculum of 15 mL of same pre-culture prepared for in vivo assay was used to inoculate 1.5 L kanamycin-containing (50 µg mL −1 ) LB medium; 0.1 mM IPTG was added to the medium when the cultures reached an OD 600 of 0.5 and were cultured overnight. Cells were collected by centrifugation at 4 • C with 10,000 rpm and resuspended in lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl and 10 mM imidazole; pH 8.0). The resulting suspensions were sonicated and centrifuged at 4 • C at maximum speed for 30 min. The supernatant was loaded onto a pre-equilibrated 750 µL Qiagen ® Ni-NTA column. After washing with a 3 mL lysis buffer and 3 mL wash buffer (20 mM imidazole lysis buffer), the His-tagged protein DisA was eluted from the column using an elution buffer (250 mM imidazole lysis buffer) ( Figure S49). The protein was resuspended into an imidazole-free buffer (50 mM NaH 2 PO 4 , 300 mM NaCl; pH 8.0) and concentrated using the Amicon ® Ultra-15 centrifugation membrane column.
Enzymatic reactions were performed in 50 mM lysis buffer without imidazole (50 mM NaH 2 PO 4 , 300 mM NaCl, pH 8.0), containing 100 µM tryptophan (or 20 µM tyrosine) and 5 µM DisA in a total volume of 0.5 mL. After incubation at 30 • C overnight, the same volume of MeOH was added to quench the reactions. The reaction mixture was then centrifuged and the supernatant was dried and re-dissolved in 50 µL 50% MeOH and analyzed by analytical HPLC (0-16 min, 5% MeCN; 16-26 min, gradient increased from 5% to 100% MeCN).

Bioactivity Tests
Determination of the minimum inhibitory concentration (MIC) of purified compounds 1-7 was carried out by micro broth dilution assays in 96 well plates as described previously [27]. All compounds were dissolved in dimethyl sulfoxide (DMSO, Carl Roth GmbH + Co., Karlsruhe, Germany) with a concentration of 3.2 mg/mL and tested in triplicate. Dilution series (64−0.03 µg/mL) of rifampicin, tetracycline, and gentamicin (all Sigma-Aldrich, St. Louis, MS, USA) were prepared as positive controls for B. subtilis DSM10, L. monocytogenes DSM20600, S. aureus ATCC25923, and E. coli ATCC25922. Same dilution series of rifampicin, tetracycline, and isoniazid for M. smegmatis ATCC607. For fungi (C. albicans