Antibacterial Activities of Metabolites from Platanus occidentalis (American sycamore) against Fish Pathogenic Bacteria

One approach to the management of common fish diseases in aquaculture is the use of antibiotic-laden feed. However, there are public concerns about the use of antibiotics in agriculture and the potential development of antibiotic resistant bacteria. Therefore, the discovery of other environmentally safe natural compounds as alternatives to antibiotics would benefit the aquaculture industries. Four natural compounds, commonly called platanosides, [kaempferol 3-O-α-L-(2″,3″-di-E-p-coumaroyl)rhamnoside (1), kaempferol 3-O-α-L-(2″-E-p-coumaroyl-3″-Z-p-coumaroyl)rhamnoside (2), kaempferol 3-O-α-L-(2″-Z-p-coumaroyl-3″-E-p-coumaroyl)rhamnoside (3), and kaempferol 3-O-α-L-(2″,3″-di-Z-p-coumaroyl)rhamnoside (4)] isolated from the leaves of the American sycamore (Platanus occidentalis) tree were evaluated using a rapid bioassay for their antibacterial activities against common fish pathogenic bacteria including Flavobacterium columnare, Edwardsiella ictaluri, Aeromonas hydrophila, and Streptococcus iniae. The four isomers and a mixture of all four isomers were strongly antibacterial against isolates of F. columnare and S. iniae. Against F. columnare ALM-00-173, 3 and 4 showed the strongest antibacterial activities, with 24-h 50% inhibition concentration (IC50) values of 2.13 ± 0.11 and 2.62 ± 0.23 mg/L, respectively. Against S. iniae LA94-426, 4 had the strongest antibacterial activity, with 24-h IC50 of 1.87 ± 0.23 mg/L. Neither a mixture of the isomers nor any of the individual isomers were antibacterial against isolates of E. ictaluri and A. hydrophila at the test concentrations used in the study. Several of the isomers appear promising for the potential management of columnaris disease and streptococcosis in fish.


Introduction
Common bacterial diseases of pond-raised channel catfish (Ictalurus punctatus) in the southeastern United States include columnaris disease and enteric septicemia of catfish (ESC). Columnaris disease-related problems also occur world-wide in many other species of freshwater fish (e.g., rainbow trout, salmon, and tilapia), and this disease can result in heavy economic losses to these aquaculture industries [1,2]. The etiological agent for columnaris disease is the bacterium Flavobacterium columnare which is a Gram-negative motile rod (2-10 μm in length) in the family Flavobacteriaceae [3]. Columnaris disease can result in severe necrosis of gill tissue, skin ulceration from systemic infection, and high mortalities in fish. The etiological agent for ESC is the bacterium Edwardsiella ictaluri which is a Gramnegative weakly motile rod (1-3 μm in length) and in the family Enterobacteriaceae [2]. The gross lesions of ESC in channel catfish can include pale gills, small depigmented lesions and/or ulcers (1-3 mm) on the backs of infected fish, open lesions along the central skull line between the eyes, and hemorrhage at the base of the fins, under the jaw, and on the belly. High mortality rates of channel catfish and prevention and treatment approaches can cost producers millions of dollars annually [2].
Bacterial diseases also adversely impact the aquaculture of sunshine bass, Morone chrysops female × Morone saxatilis male. Sunshine bass production has been the fastest growing segment of the aquaculture industry in the United States over the past decade and has spread to several countries in Europe and Asia [4,5]. However, sunshine bass production has been plagued by many pathogens including Aeromonas hydrophila and F. columnare [2]. A. hydrophila is a Gram-negative short, motile rod, ubiquitous in the aquatic environment, and the agent responsible for motile Aeromonas septicemia (MAS) in fish. Both A. hydrophila and F. columnare typically act as secondary pathogens causing significant losses in fish populations in the presence of predisposing stressors. These two pathogens can also act as primary pathogens, requiring no predisposing conditions to inflict significant mortalities [2].
Another bacterial disease which can cause losses of farmed freshwater fish is streptococcosis which is a prevalent problem in fish species such as tilapias (Oreochromis spp.) and hybrid striped bass [Morone chrysops female × Morone saxatilis male (Percichthyidae)] [6]. One of several streptococcal bacteria that has been attributed as the cause of streptococcosis in freshwater fish is Streptococcus iniae which is a Gram-positive spherical-shaped cell (0.5-2.0 μm in diameter). In tilapia, gross lesions of streptococcosis include hemorrhaging in the skin and at the base of fins, epidermal lesions and/or bloody ulcers, and opaque corneas [2]. Streptococcosis in a fish population can result in very high mortality rates, and therefore management approaches include prevention as well as treatment once the disease is determined to be present.
Catfish producers may use several currently available management approaches for columnaris disease or ESC including the application of medicated feeds [oxytetracycline dihydrate (Terramycin®; Phibro Animal Health, Teaneck, NJ, USA) for ESC and florfenicol (Aquaflor®; Intervet Inc., Millsboro, DE, USA) for columnaris disease and ESC], live attenuated vaccines [7], and nonantibiotic therapeutants such as 35% Perox-Aid ® for external columnaris [2]. However, Perox-Aid ® is not recommended for use in earthen ponds without water exchange. Additional inorganic agents such as copper sulfate pentahydrate (CuSO 4 .5H 2 O) and potassium permanganate (KMnO 4 ) have been cited as potential treatments for columnaris disease [8], but the efficacy of these compounds can be adversely impacted by some water quality variables. Also, extra care must be used when utilizing these therapeutants due to their broad-spectrum toxicity towards non-target organisms such as channel catfish [9].
At present, there are no approved drugs for the treatment of MAS caused by A. hydrophila in sunshine bass. Recent studies examining the use of CuSO 4 .5H 2 O, KMnO 4 , and Aquaflor® have noted the potential efficacy of using these compounds to manage MAS in fish [10,11]. The use of Aquaflor® with CuSO 4 .5H 2 O to manage mixed infections of A. hydrophila and F. columnare in sunshine bass appears to be especially promising [10], but future studies in ponds are still required to confirm this approach.
Preventive management approaches for streptococcosis include maintaining high water quality, the application of high-quality diets, adequate water exchange and disinfection, and the removal of fecal waste from recirculating water systems [2]. Currently, only florfenicol (Aquaflor®) is approved in the United States for the treatment of streptococcal septicemia caused by S. iniae in warmwater fish. Vaccination appears to be a promising approach for protection against S. iniae infection in Nile tilapia [12].
The discovery of novel, environmentally safe, natural antibacterial compounds would benefit aquaculturists due to the current limitations or the absence of efficacious management approaches available to producers for controlling the bacterial species responsible for columnaris disease, ESC, MAS, and streptococcosis. As part of our discovery process to identify active compounds against isolates of F. columnare, E. ictaluri, A. hydrophila, and S. iniae, natural compounds ( Figure 1) from the American sycamore (Plantus occidentalis L. family Platanaceae) previously identified as antibacterial against methicillin-resistant Staphylococcus aureus (MRSA) [13] were evaluated in a rapid bioassay.

Test compounds
Four isolated compounds obtained from the leaves of the common American sycamore (P. occidentalis) were provided by Ironstone Separations, Inc., Etta, MS, USA. These four compounds were previously identified [13] as the following: (4). A test mixture (Mix) of all four isomers (approximately 33% 1, 23% 2, 26% 3, and 19% 4) was also evaluated. The purity of each of the four test compounds and Mix was >95%.

Microorganisms and preparation of culture material for bioassay
An isolate of F. columnare [isolate ALM-00-173 (genomovar II)] was obtained from Dr.

Antibacterial bioassay
The test compounds were evaluated for antibacterial activity using a rapid 96-well microplate bioassay and following previous procedures [15]. Florfenicol and oxytetracycline HCl were included as positive drug controls. Control wells (no test material added) were also included in each assay. Test compounds and drug controls were dissolved separately in technical grade 100% ethanol. Final concentrations of test compounds and drug controls were 0.01, 0.1, 1.0, 10.0, and 100.0 μM. Three replications were used for each dilution of each test compound and controls. Final results were converted to units of mg/L to allow comparison with previous studies.
The 24-h 50% inhibition concentration (IC 50 ) and minimum inhibitory concentration (MIC) were determined using sterile 96-well polystyrene microplates (Corning Costar Corp., Acton, Massachusetts) with flat-bottom wells. Initially, dissolved test compounds or drug controls were micropippeted separately into individual microplate wells (10 μL/well), and the ethanol was allowed to completely evaporate before 0. The means and standard deviations of absorbance measurements were calculated, graphed, and compared to controls to determine the 24-h IC 50 and MIC for each test compound. The 24-h IC 50 and MIC results for each test compound were divided by the respective 24-h IC 50 and MIC results obtained for the positive controls florfenicol and oxytetracycline to determine the relative-to-drug-control florfenicol (RDCF) and relative-to-drug-control oxytetracycline (RDCO) values.

Results and Discussion
Among the four isomers of the sycamore-derived antibacterial metabolite, 3 and 4 were more antibacterial against F. columnare ALM-00-173 based on 24-h IC 50 values of 2.13±0.11 mg/L and 2.62±0.23 mg/L, respectively ( Table 1). The mixture of all four isomers (Mix) also had similar activity, with a 24-h IC 50 of 3.1±0.49 mg/L. The RDCF and RDCO 24-h IC 50 values also indicated the greatest activities of 3, 4, and Mix against F. columnare ALM-00-173, with RDCO values closer to "1.0" for 3 and 4 which indicates similar antibacterial activities of these isomers to oxytetracycline. Essentially, all four isomers and Mix had the same or similar MIC values (7.47 mg/L) when considering the standard error of the mean for 3. The RDCF and RDCO MIC values indicate substantially weaker antibacterial activity for three isomers (1, 2 and 4) and Mix compared to the two drug controls while 3 had approximately one order of magnitude less antibacterial activity than the drug controls.
Evaluation of the four isomers and mixture of the isomers against E. ictaluri S02-1039 revealed no activity at the concentrations tested (Table 2). In addition, the four isomers and the mixture were also not antibacterial towards A. hydrophila HSB-B2 at test concentrations (Table 3). Therefore, the results demonstrate that these isomers do not possess broadspectrum antibacterial activity towards species of Gram-negative, rod-shaped bacteria.
Based on 24-h IC 50 results, Mix was slightly more antibacterial than any of the other individual four isomers against S. iniae LA94-426, with a value of 1.76±0.04 mg/L ( Table   4). The 24-h IC 50 RDCF values indicate that the four isomers and Mix were only slightly less active against S. iniae LA94-426 than florfenicol, with values slightly above "1" and Mix with the lowest value at 1.2±0. Conversely, the four isomers and Mix had 24-h IC 50 RDCO values more than an order of magnitude higher than the RDCF values which indicates substantially less activity than the drug control oxytetracycline. The MIC results show similar activities among the four isomers and Mix against S. iniae LA94-426, with a range of 4.11 ± 3.36 to 7.47±0 mg/L. The MIC RDCF values indicate 3, 4, and Mix to be slightly more antibacterial than 1 and 2, but all four isomers and Mix were substantially less active than the drug controls.
The previous report on the antibacterial activities of the four isolated kaempferol isomers found them to be relatively inactive against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa [13]. However, the current results are the first to report of their antibacterial activities against the Gram-negative bacterium F. columnare. Also, this is the first report on their antibacterial activities against S. iniae. A similar range of antibacterial activities (IC 50 of 0.4 to 2.0 mg/L) for 1, 2, 3, and 4 against MRSA, which is also a Grampositive, coccus, has been previously reported [13]. In addition, 4 was the most antibacterial among the four isomers against MRSA (IC 50 0.4 mg/L) which is similar to our results for S. iniae LA94-426 (Table 4). Apparently, the Z configuration around the double bonds of the pcoumaroyl units as compared to the E configuration plays an important role in the enhancement of the antibacterial activity of these compounds [13].
The bioassay evaluation of such natural compounds is the first step in the discovery of novel natural compounds to potentially be used in the management of columnaris disease and streptococcosis. Future studies of the four isomers include challenge studies in order to more fully evaluate the effectiveness of these compounds for disease management.  Results of the bioassay evaluation of antimicrobial compounds from American sycamore leaves for antibacterial activity against   Table 3 Results of the bioassay evaluation of antimicrobial compounds from American sycamore leaves for antibacterial activity against   Table 4 Results of the bioassay evaluation of antimicrobial compounds from American sycamore leaves for antibacterial activity against