GB2509159A

GB2509159A - Triclosan derivative for use in a selective medium for cultivating microbes in a mixed population of cells

Info

Publication number: GB2509159A
Application number: GB1223309.4A
Authority: GB
Inventors: Richard Bovill; Gemma Howse
Original assignee: Oxoid Ltd
Current assignee: Oxoid Ltd
Priority date: 2012-12-21
Filing date: 2012-12-21
Publication date: 2014-06-25
Anticipated expiration: 2032-12-21
Also published as: FR3000104A1; CN103882091B; AU2013260711B2; GB2509159B; DE102013018117A1; US20140178923A1; US9908912B2; FR3000104B1; CN103882091A; AU2013260711A1; GB201223309D0; JP2014121319A; CA2835487A1; JP6368483B2; ITTO20131047A1

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. The target cells may be Salmonella or Campylobacter spp. whilst the non target cells may be Escherichia coli or other coliform bacteria. The microbial target cells may be bacteria, fungal or yeast cells. Methods, kits and composition comprising the triclosan derivatives are claimed.

Description

Triclosan Derivatives And Uses Thereof The present invention relates to triclosan derivatives and uses thereof, in particular selective agents comprising triclosan derivatives and selective media containing triclosan derivatives. The invention also concerns methods for selectively inhibiting the growth of certain cells in a mixed population using a selective agent comprising a triclosan derivative and kits for performing such methods.

Background

The broad spectrum antimicrobial agent, 5-chloro-2-(2,4-dichlorophenoxy)phenol (also referred to as "triclosan" or "Irgasan®", which is Ciba Specialty Chemical's brand name for triclosan), has been commonly used since the early 1970's for personal hygiene products, including soap, toothpaste, deodorant, and for household and industrial cleaning products. Although at high concentrations triclosan is a biocide, at lower levels it functions as a bacteriostatic agent.

Despite the long history of antimicrobial use, triclosan alone has not been routinely used as a selective agent in differentiation media for preferential growth of particular bacterial species. US Patent 5447849 to Toora teaches the use of a combination of cefsulodin and triclosan for selective growth of Yersinia enterocolitica. US Patent 5741663 to Russell teaches the use of triclosan in combination with carbenicillin and nitrofurantoin for selective growth of Pseudomonas fluorescens. US Patent Application Publication 2010/0278847 to Good etal. teaches the addition of triclosan in the range of 100 nM to 10 pM to culture medium (which was also supplemented with ampicillin) for distinguishing genetically modified (transformed) E. co/i showing vector-mediated expression of the gene for enoyl-ACP reductase (fabi). There is no disclosure in Good et al. relating to culture of naturally-occurring bacteria and/or fungi of the type typically found in environmental, industrial or medical samples.

Summary of the Invention

According to a first aspect of the invention there is provided 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.

The mixed populations are of the type found in environmental, industrial and/or clinical samples. The disclosed triclosan derivatives may be used without other antibiotics, inhibitory dyes, biocides or bacteriostatic agents. The conditions within which the microorganisms were existing at the time of collection as well as the conditions of transport of environmental, industrial and/or medical samples is generally stressful and potentially damaging. As a consequence, the target microorganisms within a sample may have weakened resistance to antibiotics and other inhibitory agents and often require an extended lag phase before resuming normal growth. The standard practice in many laboratories is to subject samples to pre-enrichment culture in a nutrient growth medium without antibiotics for a period of time sufficient to facilitate recovery of the target microorganisms, for example, approximately 16 hours. However, target cells are often present in lower numbers than non-target microorganisms and pre-enrichment culture can exacerbate this imbalance leading to overgrowth of non-target microorganisms and masking of target microorganisms.

Because the triclosan derivatives of the present invention and compositions containing such triclosan derivatives permit culture without antibiotics or other biocides, the need for pre-enrichment culture is eliminated for most samples, saving time and decreasing the risks associated with additional handling of samples to perform pre-enrichment recovery culture.

The invention further provides use of a triclosan derivative as a selective agent for selective inhibition of non-target cells in a mixed population of target and non-target cells.

The term "derivative" as used herein in relation to triclosan generally refers to a chemical substance derived from triclosan either directly or by modification or partial The cells may be eukaryotic cells (e. g. mammalian cells, fungal cells or yeast cells) but more typically will be bacterial cells. In particular, the target and non-target cells will normally both comprise bacteria.

The term "inhibition" as used herein generally refers to inhibition of the growth of cells by decreasing, slowing or stopping growth of cells. As used herein, "growth" means increase in size or proliferation or both. Thus, a compound of this invention can inhibit cells by killing, inhibiting them from becoming larger, and/or can prevent cells from dividing and replicating and increasing in number. Overall, such inhibition prevents any net increase in viable cell numbers.

In preferred embodiments the selective agent is a composition for inclusion in a bacterial growth medium.

Preferably the triclosan derivative is a glycoside derivative of triclosan. In other words, the selective agent is a glycoside wherein the aglycone is triclosan. The sugar moiety is bonded to the triclosan moiety via an 0-linkage. The triclosan is bonded to the anomeric carbon of a carbohydrate moiety.

Preferably the glycoside derivative of triclosan is a pyranoside derivative (i.e. a glycopyranoside of triclosan). In other words, the glycone moiety (i.e. the sugar moiety) of the glycoside includes a pyranose ring.

In preferred embodiments the triclosan glycoside derivative is selected from: triclosan-a-D-arabinopyranoside, triclosan-R-D-arabinopyranoside, triclosan-ct-D-galactopyranoside, triclosan-R-D-galactopyranoside, triclosan-a-D-glucopyranoside, triclosan-R-D-glucopyranoside, and triclosan-ci-D-mannopyranoside.

In preferred embodiments, the selective agent has the general formula (I): 30:I"I'II_

R OTI t ci

wherein R1 is a glycone (i.e. R1 is a sugar moiety). Preferably the R1 group includes a pyranose ring.

In preferred embodiments a selective agent can be used, wherein the selective agent exerts a toxic effect on non-target cells when contacted with non-target cells, whereas the selective agent does not exert a toxic effect on target cells when contacted with target cells. The selective agent of the present invention may be used in any manner of situations where it is desired to cause inhibition of part of a mixed population of cells.

The selective agent suitably inhibits growth of non-target cells when contacted with non-target cells but is essentially non-inhibitory to target cells, whether stressed or unstressed, when contacted with target cells. When bacterial cells are placed in a suitable growth medium there is a lag phase' during which the net number of viable bacterial cells does not increase, or increases only slowly. After the lag phase, the culture enters an exponential growth phase in which the mean "generation time" (that is, the mean time taken for a number of cells to proceed from formation to fission) is at its shortest. As an illustration of what is considered essentially non-inhibitory', a selective agent will normally be considered essentially non-inhibitory to target cells at a particular concentration if it causes an increase in the lag phase of less than 25%, preferably less than 20% and more preferably less than 15% and if it causes an increase in the mean generation time, during the exponential growth phase, of less than 20%, preferably less than 10%, and more preferably less than 5%.

In a particular preferred embodiment, the selective agent is used in situations wherein the target cells are Salmonella spp. and the non-target cells are E. co/i and/or other coliform bacteria. The selective agent allows Salmonella strains to grow, whilst inhibiting (i.e. preventing any net increase in viable cell numbers) competitor coliform organisms. The selective agent is substantially non-inhibitory to Salmonella strains, even those in a stressed state. Accordingly it is possible to reduce the overall culture time required for Salmonella strains (if present in the original sample) to attain the cell density required to give a positive result in any assay for the presence (e.g. ELISA, PCR etc), since their growth is not inhibited.

In an alternative embodiment the selective agent is used in situations wherein the target cells are Campylobacter spp. In other words, the selective agent is selective for Campylobacter spp.

Also provided is a medium for selective inhibition of non-target cells in a mixed population of target and non-target cells, the medium comprising a selective agent as defined above. The selective medium is typically a culture medium which provides inhibition of non-target cells in a mixed population of non-target and target cells. The medium may be liquid or solid and may comprise any of the components which may conventionally and suitably be included in media, such as a nutrient base, peptones, yeast extract, agar (or other solidifying agent), salts, buffers, indicator dyes and the like. Preferably the selective agent is provided in an effective amount to inhibit non-target cells.

The medium may include a suitable inducer. Use of a suitable inducer increases the inhibitory activity of the selective agent. Suitable inducers include methyl glycoside, isopropyl--D-thiogalactopyranoside, p-nitrophenyl-ci-L-arabinopyranoside or p-nitrophenyl-3-D-xylopyranoside.

The selective agent is suitably provided in the medium at a suitable concentration which allows target cells to grow, whilst inhibiting non-target cells.

Suitably the invention also provides a medium, as defined above, in contact with a mixed population of target and non-target cells.

In a particularly preferred embodiment a culture medium is provided for differentiation of Salmonella, the culture medium comprising a selective agent as defined above. This provides a medium for differentiation of Salmonella, for example from coliform bacteria such as E. co/i. The target cells are Salmonella spp and the non-target cells are coliforms, preferably E. coIL In this embodiment the selective agent is preferably triclosan R-D-galactopyranoside.

In alternative embodiments a culture medium for enumeration of Campylobacterspp is provided, the culture medium comprising a selective agent as defined above.

This provides a culture medium for growth and enumeration of Campylobacter colonies. The applicant has found that the culture medium of the invention reduces or prevents false positives from the presence of multi-drug resistant Gram negative species. In this embodiment the selective agent is preferably triclosan R-D-galactopyranoside or triclosan cx-D-arabinopyranoside. The invention also provides use of a selective agent or medium as defined above for the growth and enumeration of Carnpy/obacter.

The invention further provides use of a triclosan derivative having any of the preferred features as hereinbefore mentioned as a selective agent or in a selective medium for selective inhibition of non-target cells in a mixed population of target and non-target cells.

In a further aspect, the invention provides 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 with a selective agent as defined above, 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.

The method may utilise a medium as defined above.

The method may be performed without contacting the sample with a pre-enrichment medium that lacks the selective agent.

Prior to the culturing step, the number of non-target cells in the mixed population may be greater than the number of target cells in the mixed population.

It will be understood that performance of methods according to the invention may allow conclusions to be made regarding the identity of organisms which are able to grow successfully in the selective growth conditions. Thus, in some embodiments, the invention may comprise the further step of identifying target cell organisms which are able to grow in a culture comprising the selective agent. Alternatively, or additionally, the method may comprise the step of isolating colonies of the target cell organisms which are able to grow in a culture comprising the selective agent. Such methods of identification and/or isolation are routine for those skilled in the art.

According to the present invention there is further provided a kit for use with a method as defined above, comprising a medium containing a selective agent as defined above or components for preparing the same. The kit may further comprise instiuctions foi performing the method accoiding to the invention.

According to the present invention there is also piovided a composition comprising a glycoside derivative of triclosan. In preferred embodiments the glycoside derivative of triclosan is a pyranoside derivative. In particularly preferred embodiments the pyranoside derivative of triclosan is selected from: tiiclosan-ci-D-aiabinopyianoside, triclosan-R-D-arabinopyranoside, triclosan-a-D-galactopyranoside, tiiclosan-R-D-galactopyranoside, tiiclosan-o-D-glucopyianoside, triclosan-R-D-glucopyranoside, and triclosan-a-D-mannopyranoside.

The terms arabinoside, galactoside, glucoside and mannoside are used herein as shorthand to refer to the relevant glycone moiety.

In preferred embodiments, the selective agent has the geneial formula (I): 2:n° R1

CI

wheiein R1 is a glycone (i.e. S1 is a sugai moiety). Preferably the R1 group includes a pyranose ring.

Description of Figures

Pieferied embodiments of the piesent invention will now be more particularly described by way of example only with reference to the accompanying drawings, wherein: FIG. 1 shows a graph of the growth of a mixed culture of E. co/i 607 and Salmonella typhimurium 722 in Nutrient Broth No. 2 containing triclosan-13-D-galactoside (2 (pg/mi) at 37°C.

Detailed Description of the Invention

Triclosan Derivatives Triclosan derivatives of the present invention comprise glycoside derivatives of the biocide wherein the phenohc 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, a-D- arabinopyranoside, R-D-arabinopyianoside, o-D-galactopyranoside, R-D- galactopyranoside, c-D-glucopyranoside, R-D-glucopyranoside, and Q-D-mannopyranoside.

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-13-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 (pg/mi) required to completely inhibit growth (as determined by measuring absorption at 600nm using a Bioscreen instrument; Oy Growth Curves Ab Ltd) during a 24-hour incubation time.

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

Table 1. MICs of Triclosan-glycosides in Nutrient Broth No.2 c a-D-Glucoside a-D-Galactoside a-D-Arabinoside a-D-Mannoside Strains. . 9 -without with without with without with without with z inducer inducer inducer inducer inducer inducer inducer inducer Gram-negative -____ _______ _______ _______ _______ _______ _______ _______ _______ organisms -____ ______ ______ ______ ______ ______ ______ _______ ______ Aeromonas hydroph/la 0CC 778 1 8 256 256 >256 >256 128 256 >256 >256 Cit roba cter -_____ _______ _______ _______ _______ _______ _______ ________ _______ freundii 0CC 851 10.5 4 4 64 128 8 8 64 64 Crono.sakazak// -____ _______ _______ _______ _______ _______ _______ _______ _______ ATOC 29544 1 0.5 8 4 128 64 8 16 128 32 Enterobacter aero genes ATCC 13048 1 0.5 8 4 128 64 8 16 32 32 Ent. cloacae -____ _______ _______ _______ _______ _______ _______ _______ _______ ATCC 13047 1 0.5 1 1 32 16 0.5 0.5 8 8 0.1 Escherichia co/i to 16 to 0.5 to 0.5 to ____________ 8 0.5 2to8 1 to4 64 8to64 4 8 4to64 4to32 E. herman/i ATCC33650 1 0.5 1 1 32 32 1 2 16 8 Hafnia alvel -____ _______ _______ _______ _______ _______ _______ _______ _______ ATCC13337 1 0.1 1 1 16 8 0.5 0.5 4 4 kIeb. aero genes -____ _______ _______ _______ _______ _______ _______ _______ _______ NCTC88167 1 0.5 2 4 32 64 4 4 32 32 Kieb.

pneumoniae ATCC10031 1 0.1 1 1 1 1 0.5 0.5 4 2 Proteus mire b/Ifs ATCC12453 1 0.5 2 2 32 32 4 2 16 8 Proteus vulgar/s -____ _______ _______ _______ _______ _______ _______ _______ _______ 0CC195 1 0.5 4 4 64 64 16 8 32 32 Ps. Aerugi nose -____ _______ _______ _______ _______ _______ _______ _______ _______ ATCC 27853 1 32 >256 >256 >256 >256 >256 >256 >256 >256 1 0.5 32to 32to lGto Salmonella 1 tol 2to8 1 to4 64 128 2to8 2to8 64 8to32 Serratia marcescens 0CC 217 1 64 >256 >256 >256 >256 >256 >256 >256 >256 Table 1. (cont.) MICs of Triclosan-glycosides in Nutrient Broth No.2 (fl . c 3-D-Glucoside 3-D-Galactoside 3-D-Arabinoside -Ct -(UU) Strains ° »= 2 9. without with without with without with z g inducer inducer inducer inducer inducer inducer Gram-negative organisms ______ _______ _______ _______ _______ _______ _______ Aeromonas hydrophifa 0CC 778 1 8 >256 256 256 256 >256 >256 Cit roba cter freundil 0CC 851 1 0.5 64 8 8 8 64 64 Crono. sakazakil ATOC 29544 1 0.5 64 B 8 8 128 64 Enterobacter aero genes ATCC 13048 1 0.5 64 16 8 16 128 64 Ent. cloacae ATCC 13047 1 0.5 16 1 0.5 0.1 16 16 ____________ -0.1 to 0.5 to Escherichia coIl 8 0.5 8 to 64 ito 8 32 0.5 to 4 8 to 32 4 to 32 E. hermanil ATCC33650 1 0.5 16 2 2 2 32 16 Hafnia a/va! ATCC 13337 1 0.1 4 0.5 0.5 0.5 4 4 kIeb. aerogenes NCTC88i67 1 0.5 32 4 4 4 32 32 kieb.

pneumoniae ATCC 10031 1 0.1 0.5 0.5 0.5 0.1 4 8 Proteus mirabilis ATCC 12453 1 0.5 16 2 4 2 32 16 Proteus vulgaris OCC19S 1 0.5 64 B 8 4 128 64 Ps. Aeruginosa ATCC 27853 1 32 >256 >256 >256 >256 >256 >256 0.Sto i6to 32to 32to Salmonella ii 1 32 2to8 2to 16 2to8 64 64 Serratia marcescens 0CC 217 1 64 >256 >256 >256 >256 >256 >256 Table 1. (cont.) MICs of Triclosan-glycosides in Nutrient Broth No.2 cx-D-Glucoside a-D-Galactoside a-D-Arabinoside cx-D-Mannoside

NC

Strains) .? .2 4-o -o without with without with without with without with z D inducer inducer inducer inducer inducer inducer inducer inducer Gram-positive organisms ____ _______ _______ _______ _______ _______ _______ _______ _______ Bacillus cereus ATCC 14579 1 2 128 128 >256 >256 128 128 256 256 Bacillus subtilis NCTC 10073 1 1 32 32 256 >256 32 32 128 256 Enterococcus faecalis ATC029212 1 8 128 128 >256 >256 128 128 256 256 Enterococcus -_____ _______ _______ _______ _______ _______ _______ ________ _______ faecium ATCC 19434 1 4 256 256 >256 >256 256 256 256 256 Staph.

_________ 8 0.01 1 1 8to16 8to16 0.5 0.5 2to8 2to8 Staph. 4 to epidermidis 3 0.01 1 1 8 to 32 256 0.5 0.5 4 to 8 4 to 8 Staph.

haemolyticus 0CC2223 1 0.01 1 1 16 32 0.5 0.5 8 8 Staph.

saprophyticus A1CC15305 1 0.01 4 4 64 64 8 8 32 32 Strep.

agalactiae 0CC 182 1 4 128 128 >256 >256 128 128 128 128 Strep.

pneumoniae ATCC 6305 1 1 32 16 128 256 64 32 64 128 Strep.

pyogenes ATCC 19615 1 1 32 16 256 256 16 8 32 32 Strep.

viridans 0CC 234 1 4 128 64 >256 >256 128 128 128 128 Table 1. (cont.) MICs of Triclosan-glycosides in Nutrient Broth No.2 3-D-Glucoside 3-D-Galactoside 13-D-Arabinoside

NC cow

Strains Cl) * C j.c2 without with without with without with z j inducer inducer inducer inducer inducer inducer Gram-positive organisms Bacillus cereus ATCC 14579 1 2 256 128 256 256 128 128 Bacillus subtilis NCTC 10073 1 1 256 32 32 32 128 128 Enterococcus faecalis A TOG 29212 1 8 16 128 128 128 256 256 Enterococcus faecium ATCC 19434 1 4 >256 256 >256 >256 256 256 0.5 to Staph. aureus 8 0.01 4to8 2 0.5 0.5 4to8 4to8 Staph. 0.5 to epidermidis 3 0.01 8 to 16 1 0.5 0.5 8 to 16 4 to 64 Staph.

haemolyticus 0CC 2223 1 0.01 16 0.5 0.5 0.5 128 16 Staph.

saprophyticus ATCC 15305 1 0.01 64 8 8 8 64 64 Strep.

agalactiae 0CC 1 4 256 128 256 256 128 128 Strep.

pneumoniae ATCC 6305 1 1 128 64 128 128 64 64 Strep.

pyogenes _j_ 1 32 16 128 128 32 32 Strep. viridans 0CC 234 1 4 256 128 256 >256 128 128

EXAMPLE 1

Acetylation of sugars 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 HCI (2 x 50 ml), saturated aqueous NaHCO3 solution (2 x 50 ml) and brine (2 x 50 ml). The dichloromethane layer was then dried with magnesium sulphate, filtered and concentrated in vacuo to yield the product as a white powder.

HCY Pyr/ argon OAc

EXAMPLE 2

Bromina f/on 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 % wlv) was added drop-wise. The solution was stirred at 0 °C for 3 hours, then it was poured onto ice and extracted with CH2CI2 (2 x 100 ml). The combined extracts were washed with saturated aqueous NaHCO3 solution (2 x 100 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.

AcO HBr/AcOI-t/ 0°C AcO\ OAc QAc Br

EXAMPLE 3

Method I for glycosidation Use of the Koenigs-Knorr method ensured that only the trans anomeric form of the glycoside was formed.

Triclosan (4.04 g, 14.0 mmoles) was dissolved in 100 ml of water containing 14 ml of a 1M sodium hydroxide solution (l4mmoles) and 40 ml of acetone. To the stirred solution was then added 60m1 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.

CI CI

AcO\+ HO acetone!H20/NaOH AcotQ

EXAMPLE 4

Method 2 for glycosidation As an alternative method, both a-and I-anomers are formed and may be separated using flash chromatography.

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 minutes. Then 50 ml of dichloromethane was added and the solution was washed with water (2 x 150 ml) and brine (2 x 150 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 hexanetethyl acetate).

ci ci c017ci BF3IcH2cI2fo°c AcO0 General Procedure: Deprotections Under argon, the protected sugar (leq) was dissolved in anhydrous MeOH (1 ml per mmol). K2C03 (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

FIG. 1 shows the growth of a mixed culture of E. co/i 607 and Salmonella typhimurium 722 in Nutrient Broth No. 2 containing triclosan-3-D-galactoside (2 (pg/mI) at 37° C. Samples were taken approximately every 30 minutes and plated onto Nutrient Agar. Although E. co/i 607 showed a slight increase in number from 1.0 x io to 1.86 x 1o4 after 30 minutes, numbers then decreased to 3.98 x io 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 2.51 x io 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

Multi-drug resistant Gram-negative microorganisms including Acinetobacter baumanu 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 Campy/obacter.

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 Campyfobacter are resistant to glycoside derivatives of triclosan. Triclosan-3-D-galactoside and triclosan-cx-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 suface of the plates. Plates were incubated for 24 hours at 37°C in microaerobic conditions. Both compounds inhibited growth of all of the A. baurnanli strains tested (Table 2). Triclosan-I3-galactoside was particularly active in inhibiting two carbapenemase-producing strains of K/cbs/el/a pneumon/ae when used at 5pg/ml while all Campy/obacter strains were resistant to triclosan-ci-arabinoside at concentrations as high as 5Opg/ml.

Table 2. MIC values for Campylobacter and interfering organisms (pglml) Triclosan-a-Triclosan--Organism _______________________________ Arabinoside galactoside Acinetobacter baumanhi GOXA23 40 40 Acinetobacterbaurnanllstiain 99 40 30 AcinetobacterbaumanllOCC834 30 20 AcinetobacterbaumanusE clone B 40 40 AcinetobacterbaumanllOXA23 30 40 Acinetoba c/er baumanhi E36 OXa23 50 50 Campyloba c/er co/i 0CC776 >50 >50 Campy/obacterjejuniOCCl596 >50 >50 CampylobacterjejuniOCCl26l >50 >50 CampylobacterjejuniOCC234o >50 >50 Campy/obacterIariOCCl598 >50 >50 Campy/obactercoliOCC2774 >50 >50 Klebsietla pneumoniae KPC2 >50 5 Kiebsiella pneumoniae 7KPC3 >50 5 Enterobacter c/oacae CTXm9 1 0 5

EXAMPLE 7

Various glycoside derivatives of triclosan were added to 64 pg/mI 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-a-L-arabinopyranoside and p-nitrophenyl-3-D-xylopyranoside. Concentration of inducer in all cases was lOOpg/ml.

From Table 1 it can be seen that the addition of triclosan-cx-D-mannoside to Nutrient Broth at 64pg/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 coil, Escherichia hermanil, Hafnia a/ye!, Klebsiella, Proteus, Salmonella and Staphylococcus were inhibited by this concentration. In addition Triclosan--D-arabinoside at 16 pglml would allow the selective recovery of Salmonella and triclosan-c-D-glucoside would allow the selective recovery of pathogenic strains of Streptococcus.

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

EXAMPLE 8

Method for the determination of minimum inhibitor-v concentrations in Nutrient Agar 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 lafter 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.

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 pg/mI to 0 pg/mI in doubling dilutions. Inducers were also added at a final concentration of 0.1 mg/mI. The mixtures were swirled then four plates (25m1 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 io 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.

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.

Table 3. MICs of Triclosan-glycopyranosides (pg/mi) in Nutrient Agar containing O.lpg/ml of inducer 0) 0) 0) CD :2:2 (I) C C 0 c c c C * (V * (V * C * (V * (V * * C Organisms oa a. aD. a>' t.E t S o V C --(V --o (V 0) C C Z (V 0) C (V 0) 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 to2 4to8 0.25to 4to8 8to16 2 4to8 S. epidermidis 3 2to64 8 0.5to 1 8 16 4 8to 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. freundll 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 F. coIl 8 2to8 8to32 0.5to4 8to64 8to32 1 to4 16to32 E.hermanü 1 4 32 1 16 32 4 16 Hafniaalvei 1 2 4 0.5 4 8 2 8 K/eli aero genes 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 ito 4 l6to 32to 4to l6to Ser. marcescens 1 >256 >256 >256 >256 >256 >256 >256

Claims

Claims 1. 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.
2. A selective agent according to claim 1, wherein the triclosan derivative is a glycoside derivative of triclosan.
3. A selective agent according to claim 2, wherein the glycoside derivative of triclosan is a pyranoside derivative.
4. A selective agent according to claim 2 or 3, wherein the triclosan glycoside derivative is selected from: triclosan-o-D-arabinopyranoside, triclosan-R-D-arabinopyranoside, triclosan-a-D-galactopyranoside, triclosan-R-D-galactopyranoside, triclosan-o-D-glucopyranoside, triclosan-R-D-glucopyranoside, and triclosan-o-D-mannopyranoside.
5. A selective agent according to any preceding claim, wherein the selective agent exerts a toxic effect on non-target cells when contacted with non-target cells, whereas the selective agent does not exert a toxic effect on target cells when contacted with target cells.
6. A selective agent according to any preceding claim, wherein the selective agent inhibits growth of non-target cells when contacted with non-target cells but is essentially non-inhibitory to target cells, whether stressed or unstressed, when contacted with target cells.
7. A selective agent according to any preceding claim for use in selective inhibition wherein the target cells are Salmonella spp. and the non-target cells are E. co/i and/or other coliform bacteria.
8. A selective agent according to any of claims 1 to 6, wherein the target cells are Campylobacter spp.
9. A medium for selective inhibition of non-target cells in a mixed population of target and non-target cells, the medium comprising a selective agent according to any preceding claim.
10. A medium according to claim 9, in contact with a mixed population of target and non-target cells.
11. A medium for differentiation of Salmonella spp, the medium comprising a selective agent according to any of claims 1 to 7.
12. A medium for enumeration of Campylobacterspp, the medium comprising a selective agent according to any of claims 1 to 6 orB.
13. 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 with a selective agent according to any of claims 1 to 8, 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.
14. A method according to claim 13, wherein the method is performed without contacting the sample with a pre-enrichment medium that lacks the selective agent.
15. A method according to claim 13 or 14, 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.
16. A kit for use with a method according to any of claims 13 to 15, comprising a medium in accordance with any of claims 9 to 12 or components for preparing the same.
17. A kit according to claim 16, wherein the kit further comprises instructions for performing the method of any of claims l3to 15.
18. A composition comprising a glycoside derivative of triclosan.
19. A composition according to claim 18, wherein the glycoside derivative of triclosan is a pyranoside derivative.
20. A composition according to claim 19, wherein the pyranoside derivative of triclosan is selected from: triclosan-o-D-arabinopyranoside, triclosan-R-D-arabinopyranoside, triclosan-o-D-galactopyranoside, triclosan-R-D-galactopyranoside, triclosan-ci-D-glucopyranoside, triclosan-R-D-glucopyranoside, and triclosan-ci-D-mannopyranoside.
21. A selective agent, medium, method, kit, and/or composition, substantially as herein described.