Journal of Agricultural Chemistry and Environment
Vol.03 No.03(2014), Article ID:48770,6 pages

Antibacterial Activity of Acylglucinol Derivatives against Flavobacterium columnare

Kumudini M. Meepagala*, Kevin K. Schrader

USDA-ARS, Natural Products Utilization Research Unit, University, MS, USA

Email: *

Copyright © 2014 by authors and Scientific Research Publishing Inc.

This work is licensed under the Creative Commons Attribution International License (CC BY).

Received 6 May 2014; revised 9 June 2014; accepted 27 June 2014


Columnaris (caused by Flavobacterium columnare) is one of the most common bacterial diseases affecting the pond-raised channel catfish (Ictalurus punctatus) in the southeastern United States of America resulting in annual losses of millions of dollars. As part of our continuing effort to discover environmentally benign compounds for the control of columnaris disease, acyl derivatives of phloroglucinol were synthesized and tested against F. columnare using a rapid bioassay. Among the analogs that were tested, diacyl analogs showed very high antibacterial activity against F. columnare in the laboratory bioassay. Diisovaleryl and diisobutyryl analogs were found to have the strongest activity against F. columnare (isolate ALM-00-173) based on 24-h 50% inhibitory concentration (IC50) and minimum inhibitory concentration (MIC). Diisovaleryl and diisobutyryl analogs had IC50 values 0.82 mg/L and 0.80 mg/L, respectively, whereas the drug control florfenicol had an IC50 value of 0.81 mg/L. Diisovaleryl and diisobutyryl analogs also had 24-h relative-to- drug-control IC50 values around 1.0 indicating activities similar to florfenicol, which is included in medicated feed and is one of the current management approaches for columnaris.


Acylphloroglucinol, Columnaris Disease, Flavobacterium columnare, Antibacterial, Aquaculture

1. Introduction

Columnaris disease caused by Flavobacterium columnare is one of the most common bacterial diseases of pond-raised channel catfish (Ictalurus punctatus) in the southeastern United States of America (USA) with mortalities as high as 50% - 60% in pond populations of catfish [1] . Direct economic losses to catfish producers are likely millions of dollars annually from single or mixed infections involving F. columnare [2] . The Gram-nega- tive, rod-shaped bacterium Flavobacterium columnare is the cause of columnaris disease, and two genomovars of F. columnare have been identified, with genomovar II isolates attributed as being more pathogenic towards channel catfish [3] . The U.S. Food and Drug Administration (FDA) has granted conditional approval of Aquaflor® (50% florfenicol active ingredient) for treating columnaris disease in channel catfish [4] .

There are numerous reports about antibacterial activity of phloroglucinol derivatives [5] -[8] . Acylphloroglu- cinols isolated from Hypericum olympicum have shown antibacterial activity against methicillin-resistant Sta- phylococcus aureus (MRSA), multidrug resistant (MDR) strains of Staphylococcus aureus, Mycobacterium spe- cies and Salmonella enterica [9] . Monomeric and polymeric phloroglucinols are secondary metabolites that oc- cur in certain plant species. These derivatives occur in other organisms such as brown algae or bacteria Ecklonia stolonifera, Eisenia bicyclis [10] or species in the genus Zonaria [11] , which produce phloroglucinol and phlo- roglucinol derivatives. The bacterium Pseudomonas fluorescens produces phloroglucinol, phloroglucinolcar- boxylic acid, and diacetylphloroglucinol [12] . Acyl phloroglucinols have been isolated from coastal woodfern Dryopteris crassirhizoma [13] . Some phloroglucinol analogs have been isolated from Eucalyptus species [14] . The pharmacological activity of St. John’s wort (Hypericum perforatum) is due to phloroglucinol derivatives hyperforin and adhyperforin [15] [16] . Humulone is a phloroglucinol derivative with two prenyl groups and one isovaleryl group found in the resin of mature hops (Humulus lupulus) used as flavoring and stability agents in manufacture of beer to provide a bitter, tangy flavor [17] . Diacylphloroglucinols have been shown to possess antiviral and antibacterial activity [18] . Diisobutyrylphloroglucinol has been shown to have a strong anti-bacterial ac- tivity against Bacillus subtilis (IFO 3734) and a moderate activity against Escherichia coli (IFO 3301) [18] .

As part of our continuing effort to discover environmentally benign compounds for the control of columnaris disease, acyl derivatives of phloroglucinol were synthesized and tested against F. columnare in a rapid bioassay developed by Schrader and Harries [2] . As a result of our investigation, we were able to identify several potent derivatives with activities similar to the drug control florfenicol.

2. Materials and Methods

2.1. General Experimental Procedures

All solvents were reagent grade and used without further purification. Progress of synthesis and chromatographic profile were monitored by silica gel TLC plates GF with fluorescent indicator (250 micron, Analtech, Newark, DE, USA). Iodine vapor, UV light (at 254 and 365 nm), and anisaldehyde spray reagents were used for the detection of compounds. Flash column chromatography was performed on Biotage Isolera Four (Biotage, Charlotte, NC, USA) using FLASH + silica gel cartridges with ultra violet detection at 254 and 280 nm. 1H and 13C NMR spectra were recorded on a Varian Mercury AS400 spectrometer operating at 400 MHz for 1H NMR and at 100 MHz for 13C NMR. The HR-ESIMS was measured using Jeol ACCU TOF JMS-T1000 mass spectrometer.

2.2. Materials

Phloroglucinol, POCl3, and isovaleric acid were purchased from Sigma Aldrich (St. Louis, MO, USA). AlCl3 was obtained from Lancaster Chemical (Ward Hill, MA, USA). Isobutyric acid, 3-phenylpropionic acid, and 2-methylbutyric acid were supplied by Alfa Aesar (Ward Hill, MA, USA).

2.3. Synthesis

The general synthetic procedure involved reaction of the appropriate acid with POCl3 in the presence of AlCl3 under N2 for 24 h (4 h at 0˚C and then allowed to come to normal room temperature gradually) (Figure 1). The products were purified by biotage flash column chromatography using increasing amounts of amounts of ethyl acetate in hexane (0% to 80%). The purity of these compounds was confirmed by thin layer chromatography, high resolution ESI mass spectrometry and NMR.

triisovalerylphloroglucinol (3a) 1H NMR δ (CDCl3) 0.99 (18H, d, J = 8 Hz), 2.25 (3H, m), 2.98 (6H, d, J = 8 Hz), 13C NMR 22.7, 25.4, 52.7, 103.1, 175.8, 207.3 HR-ESI-MS, [M-H-]377.19357 (calcd for C21H29O6, 377.19641)


Figure 1. General synthetic procedure of phloroglucinol analogs.

diisovalerylphloroglucinol (2a) 1H NMR δ (CDCl3) 0.99 (12H, d, J = 8 Hz), 2.25 (2H, m), 2.98 (4H, d, J = 8Hz), 5.90 (1H, s), 13C NMR δ 22.7, 25.4, 52.7, 103.1 125.9, 128.7, 141.7, 168.6, 205.3 HR-ESI-MS, [M-H-]293.13564 (calcd for C16H21O5, 293.13890)

monoisovaleryl phloroglucinol (1a) 1H NMR δ (CDCl3) 0.91 (6H, d, J = 8 Hz), 2.29 (1H, m), 2.88 (2H, d, J = 8Hz), 5.83 (2H, s), 13C NMR δ 22.7, 24.8, 53.3, 96.1, 104.3, 164.5, 165.7, 205.8 HR-ESI-MS, [M-H-] 209.07698 (calcd for C11H13O4, 209.08138)

triisobutyryl phloroglucinol (3b) 1H NMR δ (CDCl3) 1.2 (18H, d, J = 8 Hz), 3.99 (3H, septet, J = 8 Hz ), 13C NMR HR-ESI-MS, [M-H-]347.14841 (calcd for C19H23O6, 347.14946)

diisobutyrylphloroglucinol (2b) 1H NMR δ (CDCl3) 1.1 (12H, d, J = 8 Hz), 3.96 (2H, septet, J = 8 Hz), 5.8 (1H, s), 13C NMR δ 18.2, 38.6, 94.4, 164.4, 210.2 HR-ESI-MS, [M-H-]265.11040 (calcd for C14H17O5, 265.10760)

monoisobutyrylphloroglucinol (1b) 1H NMRδ(CDCl3) 1.2 (6H, d, J = 8 Hz), 3.96 (1H, septet, J = 8 Hz), 5.9 (3H, s), 13C NMR δ 19.1, 39.2, 95.6, 172.2, 172.9, 210.8 HR-ESI-MS, [M-H-]195.06472 (calcd for C10H11O4, 195.06573)

tri-2-methylbutyrylphloroglucinol (3c) 1H NMR δ (CDCl3) 0.9 (9H, t, J = 8 Hz), 1.2 (9H, d, J = 4 Hz), 1.4 (6H, m) 3.8 (3H, m), 13C NMR HR-ESI-MS, [M-H-]377.19540 (calcd for C21H29O6, 377.19641)

di-2-methylbutyrylphloroglucinol (2c) 1H NMR δ (CDCl3) 0.9 (6H, t, J = 8 Hz), 1.2 (6,H, d, J = 6 Hz), 1.4 (4H, m) 3.8 (2H, m), 5.9 (1H, s) 13C NMR HR-ESI-MS, [M-H-]293.13671 (calcd for C16H21O5, 293.13890)

mono-2-methylbutyrylphloroglucinol (1c) 1H NMR δ (CDCl3) 0.9 (3H, t, J = 8 Hz), 1.1 (3,H, d, J = 8 Hz), 1.7 (2H, m) 3.8 (1H, m ), 5.8 (2H, s) 13C NMR HR-ESI-MS, [M-H-]219.15953 (calcd for C11H23O4, 219.159635)

tri-3 phenylpropionylphloroglucinol (3d) 1H NMR δ (CDCl3) 2.8 (6H, t, J = 8 Hz), 3.0 (6H, t, J = 8 Hz), 6.69 (3OH, s), 7.2-7.34 (9H, m), 7.28 - 7.32 (6H, m), 13C NMR δ 30.8, 35.9, 112.7, 126.5, 128.4, 128.6, 139.9, 151.0, 170.5 HR-ESI-MS, [M-H-]521.19659 (calcd for C33H29O6, 521.19641)

di-3-phenylpropionylphloroglucinol (2d) 1H NMR δ (CD3OD) 2.9 (4H, t, J = 8 Hz), 3.34 (4H, t, J = 8 Hz), 5.91 (1H, s), 7.18 - 7.3 (10H, m),13C NMR δ 30.3, 45.6, 95.26, 103.9, 125.9, 128.7, 141.7, 168.6, 205.3 HR- ESI-MS, [M-H-]389.13176 (calcd for C24H21O5, 389.13890)

mono-3-phenylpropionylphloroglucinol (1d) 1H NMR δ (CD3OD) 2.92 (2H, t, J = 8 Hz), 3.31 (2H, t, J = 8 Hz), 5.83 (2H, s), 7.10 - 7.24 (5H, m),13C NMR δ 30.3, 45.6, 95.26, 103.9, 125.9, 128.7, 141.7, 168.6, 205.3 HR-ESI-MS, [M-H-]257.08098 (calcd for C15H13O4, 257.08138)

dioctanoylphloroglucinol (2e) 1H NMR δ (CD3OD) 0.9 (6H, t, J = 8 Hz), 1.2 (12H, br S), 1.3-1.4 (8H, m), 2.3 (4H, t, J = 8 Hz ), 5.9 (1H, s),13C NMR δ 14.1, 22.7, 29.2, 29.4, 31.8, 44.2, 95.15, 172.1, 219.2 HR-ESI-MS, [M-H-]377.2391 (calcd for C22H33O5, 377.23280)

dipalmitylphloroglucinol (2f) 1H NMR δ (CD3OD) 0.9 (6H, t, J = 8 Hz), 1.2-1.3 (24H, br S), 1.4 (4H, m), 2.9 (4H, t, J = 8 Hz ), 5.9 (1H, s),13C NMR δ 14.1, 22.7, 29.2, 29.4, 31.8, 44.2, 95.15, 172.1, 219.2 HR-ESI-MS, [M-H-]601.48343 (calcd for C38H65O5, 601.48320)

3. Antibacterial Bioassay

An isolate of F. columnare [isolate ALM-00-173 (genomovar II)] was obtained from Dr. Covadonga Arias (Department of Fisheries and Allied Aquacultures, Auburn University, Alabama, USA). Cultures of the F. columnare isolate were maintained separately on modified Shieh (MS) agar plates (pH 7.2 - 7.4) in order to assure purity. Prior to bioassay, single colony of F. columnare was used to prepare the assay culture material by culturing in 75 mL of MS broth (24 h) at 29˚C ± 1˚C at 150 rpm on a rotary shaker (model C24KC; New Brunswick Scientific, Edison, New Jersey, USA). After overnight incubation, a 0.5 McFarland standard of F. columnare assay material was prepared by transferring cells from the broth culture to fresh MS broth [2] .

Pure compounds (>98% pure) were evaluated for antibacterial activity using a rapid 96-well microplate bioassay and following the procedures of Schrader and Harries [2] . Florfenicol was included as a positive drug control. In addition, control wells (no test compound) were included in each assay. All pure compounds were dissolved separately in technical grade 100% methanol. Drug controls were dissolved in technical grade ethanol. Final concentrations of test compounds and drug controls were 0.01 × 104 µM. Three replications were used for each dilution of each test compound and controls. In order to determine the 24-h 50% inhibitory concentration (IC50) and minimum inhibitory concentration (MIC), sterile 96-well polystyrene microplates (Corning Costar Corp., Acton, Massachusetts, USA) with flat-bottom wells were used. Initially, reconstituted test compounds or drug control were aspirated separately into individual microplate wells (10 µL/well), and solvent was allowed to completely evaporate at room temperature before 0.5 MacFarl and bacterial culture (prepared as described by Schrader and Harries [2] ) was added to the microplate wells (200 µL/well). Microplates were incubated at 29˚C ± 1˚C (VWR model 2005 incubator; Sheldon Manufacturing, Inc., Cornelius, Oregon, USA). A Packard model Spectra Count microplate photometer (Packard Instrument Company, Meriden, Connecticut, USA) was used to measure the absorbance (630 nm) of the microplate wells at time 0 and 24 h.

The means and standard errors of absorbance measurements were calculated, graphed, and compared to controls to determine the 24-h IC50 and MIC of each test compound [2] . The 24-h IC50 and MIC results were divided by the respective 24-h IC50 and MIC results (in ppm) obtained for the positive control, to determine the relative-to-drug-control florfenicol (RDCF) values.

4. Results and Discussion

According to results of the antibacterial bioassay of the phloroglucinol analogs diisovalerylphloroglucinol (2a), dioctanoylphloroglucinol (2e), and dipalmityl phloroglucinol (2f), 2a had similar activity against F. columnare ALM-00-173 as the drug control florfenicol, with a 24-h IC50 of 0.82 µg/mL (Table 1). Florfenicol had a 24-h IC50 of 0.81 µg/mL. While the minimum inhibitory concentrations (MIC) of diisovalerylphloroglucinol and florfenicol were 2.94 µg/mL and 0.36 µg/mL, respectively, and would indicate an approximate order of magnitude less activity compared to florfenicol, the MIC test concentrations were in a ten-fold dilution pattern (1000, 100, 10, 1, 0.1, 0.01 µM), and, therefore, it is possible that the actual MIC may be between two dilution values 0.29 µg/mL and 2.94 µg/mL. Therefore, in bioassay, the 24-h IC50 values provide a more comprehensive evaluation of the test compounds to the drug control, while the MIC values provide a more general guide for the evaluation of antibacterial activities.

The acyl analogs with long chains 2e and 2f (with C7 and C15, respectively) showed weak or no activities at the tested concentrations. Based on this preliminary data, several acyl analogs of phloroglucinol were synthe- sized to determine if a more active analog of 2a could be produced and also determine the potential structure- activity relationships of the test compounds. According to the 24-h IC50 values (Table 1), diisobutyrylphloroglucinol derivative 2b had the highest activity among the compounds tested with a 24-h IC50 of 0.80 µg/mL. The relative-to-drug-control florfenicol (RDCF) 24-h IC50 and MIC values for 2b 1.0 and 7.39, respectively, which were the lowest among all the analogs were tested. Analogs 2a, and 2c also showed strong activities, with

Table 1. Results of the bioassay evaluation of phloroglucinol and derivatives for toxicity against Flavobacterium columnare ALM-00-173. Numbers in parentheses are the standard error of the mean.

a24-h IC50 = 50% inhibitory concentration in mg/L. bMIC = Minimum inhibitory concentration in mg/L. cRDCF = Relative-to-drug-control florfenicol; numbers below “1” indicate greater activity as compared to the drug control. nd = not determined.

*Corresponding author.

Several additional analogs also had moderate strong activities based on 24-h IC50 results. These include mono-3-phenylpropionylphloroglucinol (2d), monoisobutyryl phloroglucinol (1b) and mono-2-methylbutyryl phloroglucinol (1c) which had 24-h IC50 of 1.37, 0.96, and 1.87 µg/mL, respectively. Monoacyl and triacyl analogs showed very weak activity with IC50 values in the range from 13.65 to >378, except for 1b and 1c, which possess isobutyryl and isovareryl moieties. The parent molecule phloroglucinol was inactive.

According to our bioassay results, diacylphloroglucinol analogs showed strong activity. Triacylphloroglucinol analogs showed the weakest activity and the monoacylanalogs showed a moderate activity. Diacyl analog 2b with isobutyryl moieties showed the highest activity. Diisovaleryl (2a) and di-2-methylbutyryl (2c) analogs also showed comparable activities to 2b. Introduction of a phenyl moiety (2d) significantly reduced the activity so as introduction of longer carbon chains containing acyl moieties. Thus there is an optimum size for the acyl group and lipophilicity for antibacterial activity. The current antibiotic florfenicol that is being used to treat columnaris disease is amide. The diacylphloroglucinols are a different class of molecules that have the potential to replace florfenicol.

This study is the first one to evaluate phloroglucinol derivatives for antibacterial acitivities against F. columnare. In summary, 2a, 2b, and 2c were highly active against F. columnare and as active or nearly as active as the current drug florfenicol used in medicated feed for managing columnaris disease. Similar analogs occur in plants and other organisms as monomers and dimers [5] -[11] . Synthesis of these analogs in bulk is feasible as the reaction involved one-pot synthesis with two stages and inexpensive as the starting materials are inexpensive and available in bulk quantities and they could potentially be incorporated into fish feed as an alternative to florfenicol. More testing is needed regarding toxicity to fish and other non-target organisms in the pond environment before assessing the incorporation of acylphloroglucinols in fish feed as a management approach for columnaris disease. In addition, palatability studies would also need to be conducted before any efficacy studies (challenge studies) are initiated.


The authors wish to thank Jason Martin for technical assistance with synthesis and DewayneHarries and Phaedra Page for technical assistance with bioassays.


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*Corresponding author.