Triclosan derivatives and uses thereof

Abstract

A selective agent comprising a triclosan derivative for use in selective inhibition of non-target cells in a mixed population of target and non-target cells. Preferably the triclosan derivative is a glycoside derivative, more preferably a pyranoside derivative. Suitably a selective medium comprising said selective agent and methods of culturing cells using the selective agent are provided.

Claims

1. A bacterial culture medium comprising nutrients supporting growth of the target bacteria cells and a selective agent comprising a glycoside derivative of triclosan, wherein the triclosan glycoside derivative is selected from the group consisting of: triclosan--D-arabinopyranoside, triclosan--D-arabinopyranoside, triclosan--D-galactopyranoside, triclosan--D-galactopyranoside, triclosan--D-glucopyranoside, and triclosan--D-mannopyranoside, the bacterial culture medium selectively inhibiting non-target bacteria cells in a mixed population of target and non-target bacteria cells.

2. The medium according to claim 1, in contact with a mixed population of target and non-target cells.

3. A bacterial culture medium comprising nutrients supporting the growth of Salmonella spp and a selective agent comprising a glycoside derivative of triclosan, wherein the triclosan glycoside derivative is selected from the group consisting of: triclosan--D-arabinopyranoside, triclosan--D-arabinopyranoside, triclosan--D-galactopyranoside, triclosan--D-galactopyranoside, triclosan--D-glucopyranoside, and triclosan--D-mannopyranoside the bacterial culture medium differentiating Salmonella spp.

4. A bacterial culture medium comprising nutrients supporting the growth of Campylobacter spp and a selective agent comprising a glycoside derivative of triclosan, wherein the triclosan glycoside derivative is selected from the group consisting of: triclosan--D-arabinopyranoside, triclosan--D-arabinopyranoside, triclosan--D-galactopyranoside, triclosan--D-galactopyranoside, triclosan--D-glucopyranoside, and triclosan--D-mannopyranoside the bacterial culture medium enumerating Campylobacter spp.

5. A method of culturing bacteria, fungal or yeast cells in a sample suspected to contain a mixed population of target and non-target cells, the method comprising the steps of contacting the sample cells in the bacterial culture medium of claim 1 with the selective agent, wherein the selective agent is inhibitory to non-target cells but is essentially non-inhibitory to target cells, and culturing the cells in conditions which allow for growth of target cells.

6. The method according to claim 5, wherein the method is performed without contacting the sample with a pre-enrichment medium that lacks the selective agent.

7. The method according to claim 5 or 6, wherein prior to the culturing step, the number of non-target cells in the mixed population is greater than the number of target cells in the mixed population.

Description

DESCRIPTION OF FIGURES

(1) Preferred embodiments of the present invention will now be more particularly described by way of example only with reference to the accompanying drawings, wherein:

(2) FIG. 1 shows a graph of the growth of a mixed culture of E. coli 607 and Salmonella typhimurium 722 in Nutrient Broth No. 2 containing triclosan--D-galactoside (2 (g/ml) at 37 C.

DETAILED DESCRIPTION OF THE INVENTION

(3) Triclosan Derivatives

(4) Triclosan derivatives of the present invention comprise glycoside derivatives of the biocide wherein the phenolic group of triclosan is coupled to the anomeric sugar hydroxyl. Exemplary methods of making triclosan glycosides are provided in Examples 1-4. Examples of glycosides include, without limitation, -D-arabinopyranoside, -D-arabinopyranoside, -D-galactopyranoside, -D-galactopyranoside, -D-glucopyranoside, -D-glucopyranoside, and -D-mannopyranoside.

(5) As shown in Table 1, virtually all microorganisms showed a greater minimum inhibitory concentration (MIC) for triclosan glycoside derivatives than for free triclosan. Triclosan (Irgasan, Ciba Specialty Chemicals) or Triclosan--D-galactoside was added in varying concentrations to Nutrient Broth No. 2 (Oxoid CM0067, Thermofisher Scientific). Bacteria species as indicated in Table 1 were incubated for approximately 24 hours at 37 C. Minimum inhibitory concentrations (MIC) of each selective agent were determined as the lowest concentration (g/ml) required to completely inhibit growth (as determined by measuring absorption at 600 nm using a Bioscreen instrument; Oy Growth Curves Ab Ltd) during a 24-hour incubation time.

(6) All of the organisms that were tested were inhibited by triclosan but several were totally resistant to its glycosides (Table 1).

(7) TABLE-US-00001 TABLE 1 MICs of Triclosan-glycosides in Nutrient Broth No. 2 -D-Glucoside -D-Galactoside -D-Arabinoside -D-Mannoside Underivatised without with without with without with without with Strains No. of Strains Triclosan inducer inducer inducer inducer inducer inducer inducer inducer Gram-negative organisms Aeromonas 1 8 256 256 >256 >256 128 256 >256 >256 hydrophila OCC 778 Citrobacter 1 0.5 4 4 64 128 8 8 64 64 freundii OCC 851 Crono. sakazakii 1 0.5 8 4 128 64 8 16 128 32 ATCC 29544 Enterobacter 1 0.5 8 4 128 64 8 16 32 32 aerogenes ATCC 13048 Ent. cloacae 1 0.5 1 1 32 16 0.5 0.5 8 8 ATCC 13047 Escherichia coli 8 0.1 to 0.5 2 to 8 1 to 4 16 to 64 8 to 64 0.5 to 4 0.5 to 8 4 to 64 4 to 32 E. hermanii 1 0.5 1 1 32 32 1 2 16 8 ATCC 33650 Hafnia alvei 1 0.1 1 1 16 8 0.5 0.5 4 4 ATCC 13337 Kleb. aerogenes 1 0.5 2 4 32 64 4 4 32 32 NCTC 88167 Kleb. 1 0.1 1 1 1 1 0.5 0.5 4 2 pneumoniae ATCC 10031 Proteus mirabilis 1 0.5 2 2 32 32 4 2 16 8 ATCC 12453 Proteus vulgaris 1 0.5 4 4 64 64 16 8 32 32 OCC 195 Ps. Aeruginosa 1 32 >256 >256 >256 >256 >256 >256 >256 >256 ATCC 27853 Salmonella 11 0.5 to 1 2 to 8 1 to 4 32 to 64 32 to 128 2 to 8 2 to 8 16 to 64 8 to 32 Serratia 1 64 >256 >256 >256 >256 >256 >256 >256 >256 marcescens OCC 217 -D-Glucoside -D-Galactoside -D-Arabinoside Underivatised without with without with without with Strains No. of Strains Triclosan inducer inducer inducer inducer inducer inducer Gram-negative organisms Aeromonas 1 8 >256 256 256 256 >256 >256 hydrophila OCC 778 Citrobacter 1 0.5 64 8 8 8 64 64 freundii OCC 851 Crono. sakazakii 1 0.5 64 8 8 8 128 64 ATCC 29544 Enterobacter 1 0.5 64 16 8 16 128 64 aerogenes ATCC 13048 Ent. cloacae 1 0.5 16 1 0.5 0.1 16 16 ATCC 13047 Escherichia coli 8 0.1 to 0.5 8 to 64 1 to 8 0.5 to 32 0.5 to 4 8 to 32 4 to 32 E. hermanii 1 0.5 16 2 2 2 32 16 ATCC 33650 Hafnia alvei 1 0.1 4 0.5 0.5 0.5 4 4 ATCC 13337 Kleb. aerogenes 1 0.5 32 4 4 4 32 32 NCTC 88167 Kleb. 1 0.1 0.5 0.5 0.5 0.1 4 8 pneumoniae ATCC 10031 Proteus 1 0.5 16 2 4 2 32 16 mirabilis ATCC 12453 Proteus vulgaris 1 0.5 64 8 8 4 128 64 OCC 195 Ps. Aeruginosa 1 32 >256 >256 >256 >256 >256 >256 ATCC 27853 Salmonella 11 0.5 to 1 16 to 32 2 to 8 2 to 16 2 to 8 32 to 64 32 to 64 Serratia 1 64 >256 >256 >256 >256 >256 >256 marcescens OCC 217 -D-Glucoside -D-Galactoside -D-Arabinoside -D-Mannoside Underivatized without with without with without with without with Strains No. of Strains Triclosan inducer inducer inducer inducer inducer inducer inducer inducer Gram-positive organisms Bacillus 1 2 128 128 >256 >256 128 128 256 256 cereus ATCC 14579 Bacillus 1 1 32 32 256 >256 32 32 128 256 subtilis NCTC 10073 Enterococcus 1 8 128 128 >256 >256 128 128 256 256 faecalis ATCC 29212 Enterococcus 1 4 256 256 >256 >256 256 256 256 256 faecium ATCC 19434 Staph. 8 0.01 1 1 8 to 16 8 to 16 0.5 0.5 2 to 8 2 to 8 aureus Staph. 3 0.01 1 1 8 to 32 4 to 256 0.5 0.5 4 to 8 4 to 8 epidermidis Staph. 1 0.01 1 1 16 32 0.5 0.5 8 8 haemolyticus OCC 2223 Staph. 1 0.01 4 4 64 64 8 8 32 32 saprophyticus ATCC 15305 Strep. 1 4 128 128 >256 >256 128 128 128 128 agalactiae OCC 182 Strep. 1 1 32 16 128 256 64 32 64 128 pneumoniae ATCC 6305 Strep. 1 1 32 16 256 256 16 8 32 32 pyogenes ATCC 19615 Strep. 1 4 128 64 >256 >256 128 128 128 128 viridans OCC 234 -D- -D- -D- Glucoside Galactoside Arabinoside Underivatized without with without with without with Strains No. of Strains Triclosan inducer inducer inducer inducer inducer inducer Gram-positive organisms Bacillus cereus 1 2 256 128 256 256 128 128 ATCC 14579 Bacillus subtilis 1 1 256 32 32 32 128 128 NCTC 10073 Enterococcus 1 8 16 128 128 128 256 256 faecalis ATCC 29212 Enterococcus 1 4 >256 256 >256 >256 256 256 faecium ATCC 19434 Staph. aureus 8 0.01 4 to 8 0.5 to 2 0.5 0.5 4 to 8 4 to 8 Staph. 3 0.01 8 to 16 0.5 to 1 0.5 0.5 8 to 16 4 to 64 epidermidis Staph. 1 0.01 16 0.5 0.5 0.5 128 16 haemolyticus OCC 2223 Staph. 1 0.01 64 8 8 8 64 64 saprophyticus ATCC 15305 Strep. 1 4 256 128 256 256 128 128 agalactiae OCC Strep. 1 1 128 64 128 128 64 64 pneumoniae ATCC 6305 Strep. 1 1 32 16 128 128 32 32 pyogenes Strep. viridans 1 4 256 128 256 >256 128 128 OCC 234

Example 1

(8) Acetylation of Sugars

(9) A suspension of 30.0 mmoles of the sugar in 10 ml (129 mmoles) of anhydrous pyridine under an argon atmosphere was cooled in ice with stirring. Acetic anhydride (10 ml, 0.09 mol) was then added drop-wise and the reaction stirred at room temperature for 18 hours. The solution was then concentrated in vacuo, azeotroping with toluene. The resulting residue was dissolved in dichloromethane (50 ml), and washed with 1M HCl (250 ml), saturated aqueous NaHCO.sub.3 solution (250 ml) and brine (250 ml). The dichloromethane layer was then dried with magnesium sulphate, filtered and concentrated in vacuo to yield the product as a white powder.

(10) ##STR00003##

Example 2

(11) Bromination

(12) To 13.0 mmoles of the acetylated sugar from Example 1, cooled to 0 C., 36.7 mmoles of HBr in glacial acetic acid (45% w/v) was added drop-wise. The solution was stirred at 0 C. for 3 hours, then it was poured onto ice and extracted with CH.sub.2Cl.sub.2 (2100 ml). The combined extracts were washed with saturated aqueous NaHCO.sub.3 solution (2100 ml), then dried with anhydrous magnesium sulphate, filtered and concentrated in vacuo to yield a clear orange syrup. The syrup was dissolved in ethyl acetate and crystallized as a white powder.

(13) ##STR00004##

Example 3

(14) Method 1 for Glycosidation

(15) Use of the Koenigs-Knorr method ensured that only the trans anomeric form of the glycoside was formed.

(16) Triclosan (4.04 g, 14.0 mmoles) was dissolved in 100 ml of water containing 14 ml of a 1M sodium hydroxide solution (14 mmoles) and 40 ml of acetone. To the stirred solution was then added 60 ml of a solution of acetobromogalactose (13.2 mmoles) in acetone in one go. The reaction mixture was stirred at room temperature for 18 hours then solvent was removed in vacuo. The crude product was purified by flash chromatography (silica gel eluted with a 3:2 mixture of hexane/ethyl acetate) to yield the product.

(17) ##STR00005##

Example 4

(18) Method 2 for Glycosidation

(19) As an alternative method, both - and -anomers are formed and may be separated using flash chromatography.

(20) Under argon, the acetylated sugar (17.0 mmoles) was dissolved in 100 ml of anhydrous dichloromethane and triclosan (18.0 mmoles) was then added. To the stirred solution at 0 C. was then added boron trifluoride etherate (51.0 mmoles). The reaction was then allowed to warm to room temperature and stirred for 18 hours. Water (20 ml) was added to quench the reaction, which was then stirred for a further 15 minutes. Then 50 ml of dichloromethane was added and the solution was washed with water (2150 ml) and brine (2150 ml) and dried with magnesium sulphate. After filtering, solvent was removed in vacuo and the crude product was purified by flash chromatography (silica gel eluted with a 3:2 mixture of hexane/ethyl acetate).

(21) ##STR00006##
General Procedure: Deprotections

(22) Under argon, the protected sugar (1 eq) was dissolved in anhydrous MeOH (1 ml per mmol). K.sub.2CO.sub.3 (0.1 eq) was then added. The reaction was then stirred until it was deemed to be complete as evidenced by TLC analysis. Amberlite IR-120 (plus) resin was then added and the reaction was stirred for a further 30 minutes. The resin was then filtered off and the filtrate concentrated in vacuo to yield the desired product.

Example 5

(23) FIG. 1 shows the growth of a mixed culture of E. coli 607 and Salmonella typhimurium 722 in Nutrient Broth No. 2 containing triclosan--D-galactoside (2 (g/ml) at 37 C. Samples were taken approximately every 30 minutes and plated onto Nutrient Agar. Although E. coli 607 showed a slight increase in number from 1.010.sup.4 to 1.8610.sup.4 after 30 minutes, numbers then decreased to 3.9810.sup.3 cfu/ml after 7 hours. In contrast, Salmonella typhimurium 722, after a brief lag period of 1.5 hours, entered a normal logarithmic growth phase (doubling time=28.5 minutes) and after 7 hours had increased to 2.5110.sup.5 cfu/ml. Because in typical samples background microorganisms are present in much greater numbers than the target organism, for this experiment initial numbers of E. coli in the mixed culture were 1.5 log cfu/ml higher than Salmonella typhimurium.

Example 6

(24) Multi-drug resistant Gram-negative microorganisms including Acinetobacter baumanii and species of Enterobacteriaceae are starting to appear in the food chain due to widespread agricultural antibiotic use. These microorganisms can appear as false positives on enumeration agar for Campylobacter.

(25) Campylobacter species are unusual in their nutritional requirements in that they require only amino acids and TCA cycle intermediates for growth and do not utilize carbohydrates. The inventors have discovered that Campylobacter are resistant to glycoside derivatives of triclosan. Triclosan--D-galactoside and triclosan--arabinoside both have low MIC values for organisms of Enterobacteriaceae and were, therefore, chosen for examination. The triclosan compounds were added to Brilliance CampyCount agar (Oxoid) and organisms multi-point inoculated onto the surface of the plates. Plates were incubated for 24 hours at 37 C. in microaerobic conditions. Both compounds inhibited growth of all of the A. baumanii strains tested (Table 2). Triclosan--galactoside was particularly active in inhibiting two carbapenemase-producing strains of Klebsiella pneumoniae when used at 5 g/ml while all Campylobacter strains were resistant to triclosan--arabinoside at concentrations as high as 50 g/ml.

(26) TABLE-US-00002 TABLE 2 MIC values for Campylobacter and interfering organisms (g/ml) Triclosan-- Triclosan-- Organism Arabinoside galactoside Acinetobacter baumanii GOXA23 40 40 Acinetobacter baumanii Strain 99 40 30 Acinetobacter baumanii OCC834 30 20 Acinetobacter baumanii SE clone B 40 40 Acinetobacter baumanii OXA23 30 40 Acinetobacter baumanii E36 OXa23 50 50 Campylobacter coli OCC776 >50 >50 Campylobacter jejuni OCC1596 >50 >50 Campylobacter jejuni OCC1261 >50 >50 Campylobacter jejuni OCC2340 >50 >50 Campylobacter lari OCC1598 >50 >50 Campylobacter coli OCC2774 >50 >50 Klebsiella pneumoniae KPC2 >50 5 Klebsiella pneumoniae 7KPC3 >50 5 Enterobacter cloacae CTXm9 10 5

Example 7

(27) Various glycoside derivatives of triclosan were added to 64 g/ml of Nutrient Broth No. 2 to determine minimum inhibitory concentrations (MIC) of each selective agent and the results are shown in Table 1. The tests were repeated with the addition of inducers. Methyl glycoside was the main inducer used, but other suitable inducers include isopropyl--D-thiogalactopyranoside, p-nitrophenyl--L-arabinopyranoside and p-nitrophenyl--D-xylopyranoside. Concentration of inducer in all cases was 100 g/ml.

(28) From Table 1 it can be seen that the addition of triclosan--D-mannoside to Nutrient Broth at 64 g/ml may allow the growth and recovery of Cronobacter sakazakii, an important pathogen found in particular in infant formula milk, but would inhibit the growth of many of the organisms that are often isolated with the organism. Thus, all strains of Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Escherichia hermanii, Hafnia alvei, Klebsiella, Proteus, Salmonella and Staphylococcus were inhibited by this concentration. In addition Triclosan--D-arabinoside at 16 g/ml would allow the selective recovery of Salmonella and triclosan--D-glucoside would allow the selective recovery of pathogenic strains of Streptococcus.

(29) It was also observed that the addition of inducers substantially increased the inhibitory activity of the triclosan glycosides.

Example 8

(30) Method for the Determination of Minimum Inhibitory Concentrations in Nutrient Agar

(31) The triclosan-glycosides were added to Nutrient Agar (Oxoid CM0003; Thermofisher Scientific) and organisms were transferred onto the plates surface using a multi-point inoculation device (Oxoid Cathra, Thermofisher Scientific). The latter is essentially a metal plate containing a number of needles. Each needle tip (usually 36 per plate) dips into an organism suspension (in phosphate buffered saline; PBS) and the needles are then moved over the agar plate and lowered onto its surface. In this way the growth of colonies of up to 36 different organisms can be observed on one plate.

(32) Nutrient Agar No. 2 was prepared according to the manufacturers instructions, autoclaved and cooled to 50 C. Test compounds were then added as filter sterilised solutions (50:50 deionised water:ethanol), to give final concentrations from 256 g/ml to 0 g/ml in doubling dilutions. Inducers were also added at a final concentration of 0.1 mg/ml. The mixtures were swirled then four plates (25 ml molten agar) were poured for each concentration. Plates were dried in a laminar flow cabinet then inoculated with overnight cultures of organisms that had been decimally diluted twice in sterile saline solution (approximately 10.sup.7 cfu/ml) using a multi-point inoculator. Plates were then incubated at 37 C. for 24 hours. MICs were determined as the concentration at which no growth was observed.

(33) The results are shown in Table 3. MICs obtained from the plates were similar to those obtained in broth indicating that the surface tension of the agar did not stress cells to any great extent. It also showed that free triclosan released from susceptible organisms did not inhibit more resistant organisms on the plates and the glycosides could, therefore be used for the recovery of mixed cultures.

(34) TABLE-US-00003 TABLE 3 MICs of Triclosan-glycopyranosides (g/ml) in Nutrient Agar containing 0.1 g/ml of inducer -D- No. of -D- -D- -D-gluco- -D- -D- -D- gluco- Organisms Strains arabinopyranoside galactopyranoside pyranoside mannopyranoside arabinopyranoside galactopyranoside pyranoside Bacillus cereus 1 128 >256 64 128 256 256 256 Bacillus subtilis 1 128 128 16 256 256 64 256 Ent. faecalis 1 256 >256 128 256 256 >256 >256 Ent. faecium 1 256 >256 128 256 256 >256 >256 S. aureus 8 1 to 2 4 to 8 0.25 to 1 4 to 8 8 to 16 2 4 to 8 S. epidermidis 3 2 to 64 8 0.5 to 1 8 16 4 8 to 16 S. haemolyticus 1 2 8 1 8 16 4 16 S. saprophyticus 1 128 32 4 32 64 16 32 Str. agalactiae 1 128 >256 128 128 128 256 >256 Str. pneumoniae 1 64 256 32 64 64 64 128 Str. pyogenes 1 128 >256 64 64 64 256 64 Str. viridans 1 128 >256 64 128 128 256 256 Aer. hydrophila 1 128 >256 128 256 256 256 >256 C. freundii 1 16 64 4 64 64 8 64 Cr. sakazakii 1 16 32 4 64 256 64 32 Ent. aerogenes 1 16 64 4 64 64 16 32 Ent. cloacae 1 4 32 1 16 16 1 16 E. coli 8 2 to 8 8 to 32 0.5 to 4 8 to 64 8 to 32 1 to 4 16 to 32 E. hermanii 1 4 32 1 16 32 4 16 Hafnia alvei 1 2 4 0.5 4 8 2 8 Kleb. aerogenes 1 8 32 2 32 64 8 32 Kleb. 1 2 16 0.5 16 8 1 4 pneumoniae Ps. aeruginosa 1 >256 >256 >256 >256 >256 >256 >256 Salmonella 11 4 to 16 8 to 64 1 to 4 16 to 32 32 to 64 4 to 16 16 to 128 Ser. marcescens 1 >256 >256 >256 >256 >256 >256 >256